A nanobody against canine oncostatin m receptor beta and applications thereof

Abstract

This invention provides a nanobody targeting canine OSMRβ receptor M and its applications, belonging to the field of nanobody preparation technology. This invention achieves recombinant expression and purification of the canine OSMRβ receptor, and obtains the targeting nanobody HX016 based on ribosome display technology. Enzyme-linked immunosorbent assay (ELISA) confirms that the nanobody HX016-cFc specifically binds to canine OSMRβ. SPR technology confirms that the nanobody HX016-cFc exhibits good affinity for canine OSMRβ. Cell-level experiments show that the nanobody HX016-cFc can effectively bind to canine OSMRβ and significantly inhibit IL-31-induced downstream signaling pathway activation in a dose-dependent manner, thereby effectively intervening in the IL-31-driven itching and inflammatory response in canine atopic dermatitis at the mechanistic level. A mutated portion of HX016 also possesses the corresponding ability to bind to the canine OSMRβ antigen.

Description

Technical Field

[0001] This invention belongs to the field of nanobody preparation technology, and particularly relates to a nanobody against canine tumor suppressor M receptor β and its application. Background Technology

[0002] Canine atopic dermatitis (CAD) is a hereditary, usually itchy, inflammatory skin disease primarily driven by T cells. Its occurrence involves the interaction of skin barrier dysfunction, allergen sensitization, and microbial imbalance. The age of onset is generally between 6 months and 3 years, and the incidence is not related to sex. The pathogenesis of CAD is complex, involving multiple aspects such as genetic factors, environmental factors, skin barrier dysfunction, immune dysfunction, and microbial dysbiosis. The most typical clinical signs are itching, erythema, hair loss, and lichenification, commonly found in the interdigital spaces, ears, and armpits. CAD pathogenesis mainly consists of four stages: sensitization, IgE production and binding, re-exposure triggering the disease, and a persistent "itch-scratching cycle." The inflammatory response of T helper 2 cells (Th2) is present throughout the entire course of CAD and is its fundamental characteristic. When Th2 cells are activated, they release various cytokines, such as interleukin-31 (IL-31). IL-31 is mainly produced by Th2 T cells. It is a four-helix cytokine belonging to the glycoprotein 130 (gp130) / IL-6 cytokine family and is considered a strong driver of CAD.

[0003] IL-31 exerts its function by binding to its specific receptor complex, which is composed of interleukin-31 receptor α (IL-31RA) and the oncostatin M receptor β subunit (OSMRβ). OSMRβ belongs to the interleukin-6 (L-6) receptor family and is widely expressed in various tissues, including epithelial cells, pancreatic endocrine cells, muscle cells, mesenchymal cells, endothelial cells, microglia, adipocytes, and trophoblast cells. The structure of OSMRβ includes a cytoplasmic domain, a transmembrane domain, and an extracellular binding domain. The extracellular binding domain consists of three fibronectin type III domains (FNⅢ), three cytokine binding domains (CBD), and an immunoglobulin-like domain. IL-31 induces inflammation and itching by binding to its two receptors, interleukin-31 receptor α (IL-31RA) and the heterodimer of oncostatin M receptor β (OSMRβ), activating signaling pathways including the JAK / STAT signaling pathway, the MAPK cascade amplification signaling pathway, and the PI3K / AKT signaling pathway. Activation of downstream transcription factors can further regulate various biological processes, such as inflammatory diseases, neurological diseases, cardiovascular diseases, insulin resistance and fatty degeneration, and the development and progression of tumors and cancer.

[0004] With the continuous improvement of veterinary medical standards, the treatment of CAD is evolving from traditional broad-spectrum drugs to precise targeted therapies. Existing conventional treatments, such as corticosteroids, antihistamines, and JAK kinase inhibitors, primarily alleviate symptoms through non-specific immunosuppression. While they have some efficacy, their treatment is relatively weakly targeted, and long-term use is prone to varying degrees of side effects, making it difficult to meet the clinical needs for long-term, safe management of affected dogs. Currently, antibody therapy is gaining popularity due to its advantages such as good targeting, effectiveness, safety, and long-lasting effects, and is expected to become a novel treatment approach. Currently, the expression and function of the IL-31 receptor OSMRβ in humans have been thoroughly studied, and the investigational fully human monoclonal antibody Vixarelimab targeting human OSMRβ has met its primary efficacy endpoint in a phase II clinical trial for the treatment of nodular prurigo. In 2022, the world's first pet-use anti-canine IL-31 monoclonal antibody, Lokivetmab (Cytopoint, Zoetis), targeting canine atopic dermatitis, was launched in China. It rapidly controls inflammation and itching without side effects, demonstrating good therapeutic efficacy and showcasing the enormous potential of targeted therapy. In 2023, Zoetis disclosed patents for monoclonal antibodies targeting the canine and feline OSMRβ receptor, inhibiting itching and inflammation by targeting the OSMRβ receptor. However, traditional monoclonal antibodies also have inherent limitations, such as large molecular size leading to limited tissue penetration, high production costs, and relatively poor stability, which to some extent restrict their wider application. To overcome the shortcomings of traditional antibodies, nanobodies (Nbs), with their unique structure and excellent physicochemical properties, provide a new pathway for antibody drug development. Nbs are heavy chain antibodies derived from camels, naturally lacking the light chain variable region, with a molecular weight of only 15 kDa, one-tenth the molecular weight of conventional antibodies. Nbs have advantages such as small molecular weight, simple structure, easy expression and production, strong tissue penetration ability, short half-life, and avoidance of complement reaction caused by Fc segment. They also have advantages such as low immunogenicity, better heat resistance, reversible unfolding, resistance to protein hydrolysis and high solubility, which can better demonstrate significant application prospects in the fields of biomedical research and development and basic research. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a nanobody against canine tumor suppressor M receptor β and its application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a nanobody against canine tumor suppressor M receptor β, wherein the VHH chain of the nanobody comprises three complementarity-determining regions CDR1, CDR2, and CDR3 / CDR3 variant / CDR3 cyclic peptide / CDR3 derived peptide, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:3; The CDR3 variant is formed by mutating a single amino acid to alanine (A) at positions 3 to 16 of the CDR3 amino acid sequence, where the original alanine (A) is mutated to glycine (G). The nucleotide sequence encoding CDR1 is shown in SEQ ID NO:4, the nucleotide sequence encoding CDR2 is shown in SEQ ID NO:5, and the nucleotide sequence encoding CDR3 is shown in SEQ ID NO:6.

[0007] Preferably, the nanobody VHH chain includes four constant regions FR1, FR2, FR3 and FR4, wherein the amino acid sequence of FR1 is shown in SEQ ID NO: 7, the amino acid sequence of FR2 is shown in SEQ ID NO: 8, the amino acid sequence of FR3 is shown in SEQ ID NO: 9, and the amino acid sequence of FR4 is shown in SEQ ID NO: 10. The nucleotide sequence encoding FR1 is shown in SEQ ID NO: 11, the nucleotide sequence encoding FR2 is shown in SEQ ID NO: 12, the nucleotide sequence encoding FR3 is shown in SEQ ID NO: 13, and the nucleotide sequence encoding FR4 is shown in SEQ ID NO: 14.

[0008] Preferably, the amino acid sequence of the nanobody is shown in SEQ ID NO:15.

[0009] The present invention also provides a gene encoding the nanobody, the nucleotide sequence of which is shown in SEQ ID NO:16.

[0010] The present invention also provides a nanobody fusion protein against canine tumor suppressor M receptor β, comprising the aforementioned nanobody against canine tumor suppressor M receptor β and a canine Fc fragment, wherein the amino acid sequence of the canine Fc fragment is shown in SEQ ID NO:17.

[0011] The present invention also provides a recombinant expression vector, comprising an original expression vector and the aforementioned gene.

[0012] Preferably, the original expression vector comprises pET-28a(+)-His-sumo or pcDNA3.4-hIgG1 Fc.

[0013] The present invention also provides a host cell comprising the recombinant expression vector described above.

[0014] The present invention also provides the application of the nanobody in the preparation of formulations for the prevention / treatment of canine atopic dermatitis.

[0015] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the nanobody described herein.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention successfully achieved the recombinant expression and purification of the canine OSMRβ receptor, and obtained the targeted nanobody HX016 based on ribosome display technology. The HX016-cFc nanobody, modified for canine origin, showed binding activity to canine OSMRβ via enzyme-linked immunosorbent assay (ELISA). SPR technology further confirmed that the HX016-cFc nanobody exhibited good affinity for canine OSMRβ. Cellular level experiments further demonstrated that the HX016 nanobody effectively bound to canine OSMRβ and significantly inhibited IL-31-induced downstream signaling pathway activation in a dose-dependent manner, thereby effectively intervening in the IL-31-driven itching and inflammatory response in canine atopic dermatitis at the mechanistic level.

[0017] Compared to traditional monoclonal antibodies, the nanobody HX016-cFc of this invention possesses comprehensive advantages such as small molecular weight, strong tissue penetration, high stability, low immunogenicity, and ease of large-scale production, and is expected to provide a more promising precision-targeted drug option for the treatment of canine atopic dermatitis. It provides a promising therapeutic candidate for the field and lays an important foundation for the development of nanobody drugs for canine atopic dermatitis. Attached Figure Description

[0018] Figure 1 This is an SDS-PAGE image of the purified recombinant proteins cIL-31, cOSMRβ-1, 2, and 3. Figure 2 This is the result of VHH potency measurement (relative to background value); Figure 3 These are agarose gel electrophoresis images of total RNA from alpaca PBMCs in the 4th, 5th, and 6th tests. Figure 4 This is an agarose gel electrophoresis image of the PCR amplification product of the VHH gene fragment before screening; Figure 5 This is an SDS-PAGE image of the purified recombinant nanobody HX016-cFc. Figure 6 This is a verification of the binding ability between rcOSMRβ and HX016-cFc; Figure 7 This is an affinity assay for rcOSMRβ-2 antigen and HX016-cFc antibody protein; Figure 8 It is an affinity assay for rcIL-31 antigen and Lokivetmab antibody protein; Figure 9 It is an affinity assay for rcIL-31 antigen and HX016-cFc antibody protein; Figure 10 Phospho-STAT3 levels in DH-82 cells after treatment with different concentrations of rcIL-31; Figure 11 The dose-dependent inhibitory effects of different concentrations of HX016-cFc and Lokivetmab on rcIL-31-induced STAT3 phosphorylation in DH-82 cells and their IC50 values. 50 curve; Figure 12 This is an SDS-PAGE image of the purified recombinant nanobody HX016-TB-1-6 after mutation. Figure 13 This is an SDS-PAGE image of the purified recombinant nanobody HX016-TB-7-14 after mutation. Detailed Implementation

[0019] This invention provides a nanobody against canine tumor suppressor M receptor β. The VHH chain of the nanobody includes three complementarity-determining regions: CDR1, CDR2, and CDR3 / CDR3 variant / CDR3 cyclic peptide / CDR3 derived peptide. The amino acid sequence of CDR1 is shown in SEQ ID NO:1, specifically GRTFSTHA; the amino acid sequence of CDR2 is shown in SEQ ID NO:2, specifically ITWSDGST; and the amino acid sequence of CDR3 is shown in SEQ ID NO:3, specifically AAREQIRVGLVGDFAF. The CDR3 variant is formed by mutating a single amino acid to alanine (A) at positions 3 to 16 of the CDR3 amino acid sequence, where the original alanine (A) is mutated to glycine (G). The nucleotide sequence encoding CDR1 is shown in SEQ ID NO:4, specifically GGCCGCACTTTCTCCACACATGCA; the nucleotide sequence encoding CDR2 is shown in SEQ ID NO:5, specifically ATCACATGGAGTGACGGGAGCACC; and the nucleotide sequence encoding CDR3 is shown in SEQ ID NO:6, specifically GCCGCAAGAGAGCAGATTAGAGTGGGTCTCGTGGGCGACTTTGCGTTC.

[0020] In this invention, the nanobody VHH chain comprises four constant regions FR1, FR2, FR3 and FR4. The amino acid sequence of FR1 is shown in SEQ ID NO: 7, specifically DVQLQESGGGLVQAGGSLRLSCAAS. The amino acid sequence of FR2 is shown in SEQ ID NO: 8, specifically MGWFRQAPGKEREFVAA. The amino acid sequence of FR3 is shown in SEQ ID NO: 9, specifically YYAQSVKGRFTIYKDNAKDTVYLQMNSLKPEDTAVYYC. The amino acid sequence of FR4 is shown in SEQ ID NO: 10, specifically WGQGTQVTVSS. The nucleotide sequence encoding FR1 is shown in SEQ ID NO: 11, specifically GAGTGCAACTCCAAGAATCTGGTGGTGGTCTTGTGCAGGCGGGGGGCTCACTGAGACTTTCTTGCGCTGCATCC; the nucleotide sequence encoding FR2 is shown in SEQ ID NO: 12, specifically ATGGGATGGTTTCGCCAGGCACCAGGGAAAGAAAGGGAGTTCGTCGCTGCA; the nucleotide sequence encoding FR3 is shown in SEQ ID NO: 13, specifically TACTACGCCCAGAGTGTCAAGGGTCGCTTCACCATCTACAAGGACAATGCCAAAGACACAGTGTACCTGCAGATGAATAGCCTCAAGCCTGAGGACACAGCCGTATACTACTGT; and the nucleotide sequence encoding FR4 is shown in SEQ ID NO: 14, specifically TGGGGTCAGGGAACACAGGTGACCGTGTCAAGC.

[0021] In this invention, the amino acid sequence of the nanobody is shown in SEQ ID NO:15, specifically DVQLQESGGGLVQAGGSLRLSCAASGRTFSTHAMGWFRQAPGKEREFVAAITWSDGSTYYAQSVKGRFTIYKDNAKDTVYLQMNSLKPEDTAVYYCAAREQIRVGLVGDFAFWGQGTQVTVSS.

[0022] The present invention also provides a gene encoding the nanobody, the nucleotide sequence of which is shown in SEQ ID NO:16, specifically GACGTGCAACTCCAAGAATCTGGTGGTGGTCTTGTGCAGGCGGGGGGCTCACTGAGACTTTCTTGCGCTGCATCCGGCCGCACTTTCTCCACACATGCAATGGGATGGTTTCGCCAGGCACCAGGGAAAGAAAGGGAGTTCGTCGCTGCAATCACATGGAGTGACGGGAGCACCTACTACGCCCAGAGTGTCAAGGGTCGCTTCACCATCTACAAGGACAATGCCAAAGACACAGTGTACCTGCAGATGAATAGCCTCAAGCCTGAGGACACAGCCGTATACTACTGTGCCGCAAGAGAGCAGATTAGAGTGGGTCTCGTGGGCGACTTTGCGTTCTGGGGTCAGGGAACACAGGTGACCGTGTCAAGC.

[0023] This invention also provides a nanobody fusion protein against canine tumor suppressor M receptor β, comprising the aforementioned nanobody against canine tumor suppressor M receptor β and a canine Fc fragment. The amino acid sequence of the canine Fc fragment is shown in SEQ ID NO:17, specifically: PRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG.

[0024] The present invention also provides a recombinant expression vector, comprising an original expression vector and the aforementioned gene. In the present invention, the original expression vector comprises pET-28a(+)-His-sumo or pcDNA3.4-hIgG1 Fc.

[0025] The present invention also provides a host cell comprising the recombinant expression vector described above.

[0026] The present invention also provides the application of the nanobody in the preparation of formulations for the prevention / treatment of canine atopic dermatitis.

[0027] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the nanobody described herein.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] Preparation of recombinant canine IL-31 and OSMRβ proteins

[0031] The amino acid sequence of canine IL-31 (NP_001159386.1) was searched in NCBI, and amino acids 28-159 were selected for gene synthesis. The amino acid sequence of canine OSMRβ (XP_005619431.1) was searched in NCBI, and the signal peptide region of amino acids 1-27 was removed. The remaining amino acids 28-968 were selected and sent to Nanjing GenScript Biotech Co., Ltd. for gene synthesis. The modified mammalian expression vector pcDNA3.4-hIgG1 Fc (with a mouse IgG1 heavy chain signal peptide introduced at the N-terminus and a human IgG1-Fc tag introduced at the C-terminus, College of Veterinary Medicine, China Agricultural University) was used as the vector for cOSMRβ. It was transiently transfected into 293F cells (College of Veterinary Medicine, China Agricultural University) using polyethyleneimine (PEI) transfection reagent. On the third day after transfection, the expression supernatant was collected and the recombinant protein was obtained by affinity purification using rProtein A Beads.

[0032] The synthesized cIL-31 protein, from amino acids 28 to 159, was used for recombinant protein expression and purification. Because the entire segment was difficult to express, the synthesized cOSMRβ was divided into three segments: amino acids 28-327 (cOSMRβ-1), 328-618 (cOSMRβ-2), and 619-730 (cOSMRβ-3), which were then separately expressed and purified as recombinant proteins. The purified recombinant proteins cIL-31 and cOSMRβ-1, 2, and 3 were analyzed by SDS-PAGE to verify the purification effect.

[0033] Experimental results: such as Figure 1 As shown, three segments of the extracellular domain of canine IL-31 and canine OSMRβ were successfully expressed, and recombinant proteins with high purity were obtained, providing a foundation for subsequent studies on their interactions, immunization, and antibody screening.

[0034] Example 2

[0035] Screening, expression and purification of nanobodies

[0036] cOSMRβ-1, 2, and 3 were mixed at 1 mg each and then combined with MnJ(β) colloidal manganese adjuvant (Qimeng Biotechnology (Jiangsu) Co., Ltd.) and delivered to Nanchang Dajia Technology Co., Ltd. for immunization of alpacas. Specifically, a healthy adult (2-3 years old) female alpaca was selected. Each time, 2 mg of the protein mixture was mixed with 0.8 mL of MnJ(β) colloidal manganese adjuvant, allowed to stand at room temperature for 3 minutes, and then 1.2 mL of physiological saline was added for subcutaneous injection. A total of 6 immunizations were performed. Starting from the first immunization, blood was collected 7 days after each immunization to test the antiserum titer. The potency assay was performed as follows: 2 μg / mL of the target antigen was coated and incubated overnight at 4°C; 4% skim milk was used for blocking for 1 h; 100 μL of serially diluted alpaca antiserum was added and incubated at 37°C for 30 min; the strips were washed with PBST; 100 μL of rabbit anti-camelid VHH Cocktail [HRP] was added and incubated at 37°C for 30 min; after washing the strips with PBST, 100 μL of TMB substrate solution was added for color development, and the color development was stopped after 5 min.

[0037] Experimental results: such as Figure 2 As shown, the titer of alpaca heavy chain antibodies gradually increases with immunization, rising from 16K in the second immunization to 256K, peaking in the fifth immunization, and then showing a downward trend.

[0038] Total RNA was extracted from PBMCs after the 4th, 5th, and 6th alpaca immunizations, such as... Figure 3 As shown. Next, cDNA was obtained through reverse transcription, and the gene fragment of the nanobody was specifically amplified by PCR, as shown. Figure 4 As shown in the figure. The mRNA was cloned into a ribosomal vector and synthesized using the PUREfrex SS in vitro transcription system, forming a non-covalent polypeptide-ribosome-mRNA ternary complex to construct a ribosomal display library. A mixture of rcOSMRβ-1, 2, and 3 proteins was added for incubation, followed by washing with beads to remove unbound antibodies and ensure high-specificity binding. Finally, lysis buffer was added to dissociate the mRNA, which was then reverse transcribed into cDNA. Subsequently, PCR amplification successfully obtained the gene sequence of the candidate nanobody HX016 (as shown in SEQ ID NO:16).

[0039] The HX016 gene sequence was followed by a canine Fc fragment PRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (as shown in SEQ ID NO:17) and constructed into the promoter-optimized eukaryotic expression vector pCMV3 (Beijing Yiqiao Shenzhou Technology Co., Ltd.). The cells were transiently transfected into 293F cells using TF1 transfection reagent. Serum-free 293 feed medium was added on days 1, 3, and 5 after transfection. After 5-7 days of cell culture, Protein At Beads was used for expression. Recombinant protein was obtained by 4FF affinity purification.

[0040] Experimental results: such as Figure 5 As shown, the HX016-cFc chimeric nanobody was successfully expressed, and a high-purity recombinant protein was obtained, laying the foundation for subsequent functional research and application development of nanobodies.

[0041] Example 3

[0042] Enzyme-linked immunosorbent assay (ELISA) was used to verify the binding ability of nanobodies to antigens.

[0043] The antigen rcOSMRβ-his (Shanghai Unimicron Biotechnology Co., Ltd.) was prepared at a concentration of 5 μg / mL, with the negative control being the coating buffer, 100 μL / well, and coated overnight at 4℃. After removing the liquid from the plate and patting it dry, 300 μL / well of 2% BSA was added, sealed, and incubated at room temperature for 1 h. Then, 300 μL / well of washing buffer was added, and the plate was washed twice, with the last wash patting dry. The HX016-cFc antibody prepared in Example 2 was diluted to 20000 ng / mL with sample diluent, and then serially diluted five times for 7 spots. 100 μL of sample diluent was added to each well for the negative control, mixed thoroughly, and reacted at room temperature for 2 h. 300 μL / well of washing buffer was added, and the plate was washed three times, with the last wash patting dry. The secondary antibody anti-his / HRP was diluted to the working concentration, 100 μL / well, mixed thoroughly, and incubated at room temperature for 1 h. 300 μL / well of washing buffer was added, and the plate was washed three times, with the last wash patting dry. Mix colorimetric solutions A and B at a 1:1 ratio, add 200 μL to each well, and incubate at room temperature in the dark for 20 min. Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0044] Experimental results: such as Figure 6 As shown, HX016-cFc possesses biological activity and exhibits a high affinity for the antigen protein rcOSMR-his, with a binding constant EC50. 50 It reached 29.75 ng / mL.

[0045] Example 4

[0046] A method for determining the affinity of HX016-cFc nanobody for cOSMRβ using surface plasmon resonance.

[0047] The affinity of candidate anti-cOSMRβ nanobody HX016-cFc for cOSMRβ was determined using surface plasmon resonance (SPR) on the WeSPR™ One Auto system, using the LifeDisc™ carboxylation chip card kit.

[0048] First, the purified antibody and antigen protein buffer were replaced with 1×PBS, and rcOSMR-his was immobilized on a LifeDisc™ carboxylation chip as a ligand. The nanobody HX016-cFc was used as the analyte, and a multi-cycle kinetic method was employed for detection. Injections were performed at concentrations of 0, 2.5, 5, 10, 20, and 40 μg / mL. Finally, a 1:1 binding formula was used for data analysis.

[0049] Experimental results: such as Figure 7 As shown, HX016-cFc exhibits good binding activity with rcOSMRβ-his, with a KD value of 2.47 × 10⁻⁶. -9 M has a high binding affinity.

[0050] Additionally, cIL-31 was selected as a ligand and immobilized on a LifeDisc™ carboxylation chip. The anti-cIL-31 monoclonal antibody Lokivetmab (MEDCHEMEXPRESS LLC) and the nanobody HX016-cFc (negative control) were used as analytes, and a multi-cycle kinetic method was employed for detection. Injections were performed at concentrations of 0, 2.5, 5, 10, 20, and 40 μg / mL, and data analysis was finally performed using a 1:1 binding formula.

[0051] Experimental results: such as Figure 8 and 9 As shown, Lokivetmab exhibits good binding activity with cIL-31, with a KD value of 1.8 × 10⁻⁶. -9 M has a high binding affinity. The nanobody HX016-cFc does not bind to rcIL-31.

[0052] Example 5

[0053] Phosphorylation activation and inhibition experiments of the IL-31 signaling pathway

[0054] Canine macrophages (DH-82 cells) express cOSMRβ and cIL-31RA on their surface. Upon stimulation with cIL-31, cIL-31 can mediate Janus phosphorylation and activate the STAT signaling cascade. The experiments included rcIL-31-induced phosphorylation to verify whether rcIL-31 possesses the biological activity to activate the downstream JAK-STAT pathway, and whether the nanobody HX016-cFc could block rcIL-31-induced phosphorylation and downstream signaling pathway transduction.

[0055] 1. rcIL-31-induced phosphorylation experiment

[0056] Canine DH-82 cells (College of Veterinary Medicine, China Agricultural University) were cultured at 37°C until they reached a good condition, and then seeded at a density of 10-1. 5 Seed cells / well into 96-well plates, with Canine Gamma Interferon (cIFN-γ) added to a final concentration of 10 ng / mL, and incubated at 37°C for 24 h. After induction, the cells were starved in serum-free DMEM medium for 2 h, 100 μL / well. The medium was then removed, and the rcIL-31 concentration was adjusted from 10 ng / mL to 10 ng / mL. 4 Starting with ng / mL, the solution was serially diluted 10-fold to obtain 7 concentrations. 100 μL of each concentration was added to a 96-well plate and the plate was stimulated for 10 min. Following the procedure of the STAT3 phosphorylation assay kit HTRF PHOSPHO-STAT3 (TYR705), HTRF data were measured, and the receptor and donor emission signals for each individual well were calculated.

[0057] Experimental results: such as Figure 10 As shown, rcIL-31 has biological activity and activates STAT3 phosphorylation in canine DH-82 cells.

[0058] 2. Antibody inhibition of rcIL-31-induced phosphorylation experiment

[0059] Canine DH-82 cells were cultured at 37°C until they reached a good condition, and then seeded at a density of 10-1. 5 Seed cells / well into 96-well plates, and cIFN-γ was added for induction to a final concentration of 10 ng / mL. Incubate at 37°C for 24 h. After induction, starve the cells with serum-free DMEM for 2 h, 100 μL / well. Remove the culture medium and adjust the concentration of the HX016-cFc nanobody from 10 ng / mL to 10 ng / mL. 5Starting with ng / mL, the solution was serially diluted 10-fold to obtain 8 concentrations. 50 μL of each concentration was added to a 96-well plate and incubated for 1 h. Then, 50 μL of rcIL-31 was added to a final concentration of 1 μg / mL to induce phosphorylation, and the incubation period was 10 min. The concentration of the anti-cIL-31 monoclonal antibody Lokivetmab was increased from 10... 5 Starting with ng / mL, the solution was serially diluted 10-fold to obtain eight concentrations, each 50 μL. Each concentration was mixed with 50 μL of rcIL-31 to a final concentration of 1 μg / mL and reacted for 1 h. The mixture was then added to a 96-well plate and incubated for 10 min. Samples were lysed using the HTRF PHOSPHO-STAT3 (TYR705) STAT3 phosphorylation assay kit, and data were collected. The acceptor and donor emission signals for each individual well were calculated.

[0060] Experimental results: such as Figure 11 As shown in the figure, the HX016-cFc nanobody inhibits STAT3 phosphorylation in canine DH-82 cells, with an IC50 value of [missing information]. 50 The concentration was 0.1774 μg / mL. The anti-cIL-31 monoclonal antibody Lokivetmab inhibited STAT3 phosphorylation in canine DH-82 cells, with an IC50 concentration of 0.1774 μg / mL. 50 It was 0.1132 μg / mL.

[0061] Example 6

[0062] Validation of the efficacy of the HX016 mutant nanobody sequence

[0063] 1. Mutant antibody

[0064] Single-point mutations were performed on certain amino acids in the CDR3 region of the nanobody HX016. A total of 14 amino acids, REQIRVGLVGDFAF, from positions 3 to 16 of the CDR3 region, were individually mutated to alanine (A), with the original alanine (A) mutated to glycine (G). The mutated nanobody HX016-TB-1-14 was constructed into the prokaryotic expression vector pET-28a(+)-His-sumo (College of Veterinary Medicine, China Agricultural University). Expression was induced at 16℃ from 18:00 on the first day to 8:00 on the second day using 0.1 mM IPTG. The nanobody was purified using TALON Superflow affinity chromatography. The purification effect of the purified recombinant single-point mutated nanobody HX016-TB-1-14 was verified by SDS-PAGE analysis.

[0065] Experimental results: such as Figures 12-13 As shown, the nanobody HX016-TB-1-14 was successfully expressed, and a recombinant protein with high purity was obtained.

[0066] 2. Effect Verification

[0067] The above antibodies were validated by performing affinity tests on the purified mutant antibodies. The affinity test steps were as described in Example 4. The specific affinities of different mutant antibodies are shown in Table 1 below.

[0068] Table 1. Affinity determination of rcOSMRβ-His antigen and mutated recombinant nanobody HX016-TB-1-14 antibody protein.

[0069] The results above show that the mutation modification of nanobody HX016 resulted in a decrease in antibody affinity for some of the mutated antibodies, but they still bound.

[0070] As can be seen from the above embodiments, the present invention provides a nanobody targeting canine OSMRβ, which can specifically bind to canine OSMRβ and competitively inhibit the downstream signaling pathway mediated by the cytokine IL-31, thereby effectively intervening in the IL-31-driven itching and inflammatory response in canine atopic dermatitis at the mechanistic level.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nanobody against canine tumor suppressor M receptor β, characterized in that, The nanobody VHH chain includes three complementarity-determining regions: CDR1, CDR2, and CDR3 / CDR3 variant / CDR3 cyclic peptide / CDR3 derived peptide. The amino acid sequence of CDR1 is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

3. The CDR3 variant is formed by mutating a single amino acid to alanine at positions 3 to 16 of the CDR3 amino acid sequence, where the original alanine is mutated to glycine. The nucleotide sequence encoding CDR1 is shown in SEQ ID NO:4, the nucleotide sequence encoding CDR2 is shown in SEQ ID NO:5, and the nucleotide sequence encoding CDR3 is shown in SEQ ID NO:

6.

2. The nanobody according to claim 1, characterized in that, The nanobody VHH chain comprises four constant regions FR1, FR2, FR3 and FR4, wherein the amino acid sequence of FR1 is shown in SEQ ID NO: 7, the amino acid sequence of FR2 is shown in SEQ ID NO: 8, the amino acid sequence of FR3 is shown in SEQ ID NO: 9, and the amino acid sequence of FR4 is shown in SEQ ID NO:

10. The nucleotide sequence encoding FR1 is shown in SEQ ID NO: 11, the nucleotide sequence encoding FR2 is shown in SEQ ID NO: 12, the nucleotide sequence encoding FR3 is shown in SEQ ID NO: 13, and the nucleotide sequence encoding FR4 is shown in SEQ ID NO:

14.

3. The nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO:

15.

4. The gene encoding the nanobody of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

16.

5. A nanobody fusion protein targeting canine tumor suppressor M receptor β, characterized in that, The nanobody comprising the anti-canine tumor suppressor M receptor β as described in claim 1 and a canine Fc fragment, wherein the amino acid sequence of the canine Fc fragment is shown in SEQ ID NO:

17.

6. A recombinant expression vector, characterized in that, It includes the original expression vector and the gene as described in claim 4.

7. The recombinant expression vector according to claim 6, characterized in that, The original expression vectors include pET-28a(+)-His-sumo or pcDNA3.4-hIgG1 Fc.

8. A host cell, characterized in that, Includes the recombinant expression vector as described in claim 6 or 7.

9. The use of the nanobody according to any one of claims 1 to 3 in the preparation of a formulation for the prevention / treatment of canine atopic dermatitis.

10. A pharmaceutical composition, characterized in that, Includes pharmaceutically acceptable carriers and nanobodies as described in any one of claims 1 to 3.

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