Anti-il-31ra nanobodies and uses thereof

Abstract

The application relates to an anti-IL-31Ra nanobody and application thereof, and belongs to the technical field of biology. The anti-IL-31Ra nanobody provided by the application comprises a framework region (FR) and a complementarity determining region (CDR); the complementarity determining region comprises CDR1, CDR2 and CDR3, and the amino acid sequences of CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The nanobody targeting IL-31Ra is obtained by screening a llama nanobody natural library through phage display technology, and the nanobody can specifically recognize Human IL-31Ra and Cynomolgus IL-31Ra recombinant proteins and HEK293 hIL-31Ra overexpression cells. The antibody has high specificity and good species cross-reactivity, and has a wide application prospect in treating inflammatory and autoimmune diseases mediated by the IL-31 / IL-31Ra pathway.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-IL-31Ra nanobody and its application. Technical Background

[0002] IL-31Ra is the α subunit of the interleukin-31 receptor. The theoretical molecular weight of human IL-31Ra is 83.8 kDa, but due to glycosylation, its apparent molecular weight is approximately 110-135 kDa. It forms a heterodimeric receptor complex with OSMRβ, participating in inflammatory responses, regulating airway hyperresponsiveness, and physiological functions such as skin physiology and immunity by activating signaling pathways such as JAK-STAT, PI3K / AKT, and MAPK. It plays a crucial role in chronic inflammatory diseases such as atopic dermatitis and allergic asthma. Due to its key role in these diseases, drugs targeting IL-31Ra, such as Nemolizumab, show promising potential in treating related diseases and may become novel therapeutic approaches. Simultaneously, IL-31Ra and its related products can also serve as research tools to help explore its potential functions in other diseases.

[0003] Phage display technology involves fusing a foreign protein gene with a phage coat protein gene, allowing the foreign protein to be expressed and displayed on the phage surface along with the phage coat protein. After incubation with a specific target molecule (such as an antibody), the bound phage is eluted and amplified. Through multiple rounds of screening, high-affinity phage clones can be enriched. This technology offers high screening efficiency, enabling the screening of a large number of clones in a short time; it is simple to operate, relatively low in cost, and directly provides the gene corresponding to the displayed protein, facilitating subsequent research and applications.

[0004] Nanobodies (single-domain antibodies) are antigen-binding fragments of heavy-chain antibodies, possessing unique advantages. Their small molecular weight (approximately 15 kDa) results in high affinity and specificity, enabling them to bind deeply to antigen sites that are difficult for conventional antibodies to reach. Nanobodies exhibit high stability, maintaining activity even under extreme conditions, and are easily produced through prokaryotic expression systems. Furthermore, nanobodies can display multiple valences, enhancing antigen-binding capabilities, and are widely used in diagnostics, therapy, and research, providing a powerful new tool for biomedical research and clinical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a nanobody capable of binding to IL-31Ra with high affinity and high specificity. This invention also provides the encoding gene of this antibody, the recombinant expression vector, the host cell, and related applications. The anti-IL-31Ra nanobody obtained by this invention has good application value in basic research, drug screening or preparation, and in vitro diagnostics.

[0006] The technical solution of this invention to solve its technical problem is as follows:

[0007] In a first aspect of the invention, an anti-IL-31Ra nanobody is provided, the nanobody comprising a framework region (FR) and a complementarity-determining region (CDR); the complementarity-determining region comprises CDR1, CDR2 and CDR3, the amino acid sequence of the complementarity-determining region CDR1 is shown in SEQ ID NO:3; the amino acid sequence of the complementarity-determining region CDR2 is shown in SEQ ID NO:4; and the amino acid sequence of the complementarity-determining region CDR3 is shown in SEQ ID NO:5.

[0008] Furthermore, the amino acid sequence of the nanobody is as shown in SEQ ID NO:1, or has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1.

[0009] In a second aspect of the invention, an isolated nucleic acid molecule is provided that encodes the anti-IL-31Ra nanobody described in the first aspect.

[0010] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0011] In a third aspect of the invention, a recombinant expression vector is provided, comprising the nucleic acid molecule described in the second aspect.

[0012] In a preferred embodiment, the recombinant expression vector is a vector suitable for mammalian cell expression systems, such as pcDNA3.4.

[0013] In a fourth aspect of the invention, a host cell is provided comprising the recombinant expression vector described in the third aspect.

[0014] In a preferred embodiment, the host cell is a mammalian cell line, such as CHO-K1 cells.

[0015] In a fifth aspect of the invention, an antibody derivative is provided; said antibody derivative comprises at least one anti-IL-31Ra nanobody as described in the first aspect; said antibody derivative is selected from any of the following forms:

[0016] (a) A multivalent nanobody comprising two or more identical or different anti-IL-31Ra nanobodies;

[0017] (b) A bispecific or multispecific antibody, wherein at least one binding domain is the anti-IL-31Ra nanobody and the other binding domain targets a second antigen;

[0018] (c) Antibody conjugate, wherein the anti-IL-31Ra nanobody is covalently linked to a drug, toxin, radioisotope or detectable marker;

[0019] (d) A heavy chain antibody or its antigen-binding fragment, wherein the variable region contains the anti-IL-31Ra nanobody sequence.

[0020] In a sixth aspect of the invention, a pharmaceutical composition is provided comprising the anti-IL-31Ra nanobody described in the first aspect, or the antibody derivative described in the fifth aspect, and a pharmaceutically acceptable carrier.

[0021] In a seventh aspect of the invention, a kit for detecting IL-31Ra protein in a sample is provided, comprising the anti-IL-31Ra nanobody described in the first aspect.

[0022] In an eighth aspect of the present invention, a method for detecting IL-31Ra is provided, the method comprising the following steps:

[0023] (1) Contact the sample with the anti-IL-31Ra nanobody as described in the first aspect or the kit as described in the seventh aspect;

[0024] (2) Quantitatively or qualitatively determine the IL-31Ra in the sample based on the binding of the anti-IL-31Ra nanobody or the kit described in the seventh aspect to the sample;

[0025] The method described is not for diagnostic or therapeutic purposes.

[0026] In a ninth aspect of the invention, the use of the anti-IL-31Ra nanobody described in the first aspect is provided in the preparation of a medicament for treating IL-31Ra-mediated chronic inflammation, including atopic dermatitis or allergic asthma.

[0027] Compared with the prior art, the present invention has the following technical effects:

[0028] This invention successfully screened and obtained nanobodies targeting IL-31Ra from a natural library of alpaca nanobodies using phage display technology. Compared to screening via immune libraries, this method offers advantages such as lower cost, no antigenicity limitations, and the ability to enrich specific nanobodies without animal immunization. The antibody can be efficiently expressed in mammalian cells, exhibits good physicochemical homogeneity and stability, and meets the basic requirements for drug development.

[0029] Functional validation showed that the nanobody of the present invention can specifically recognize recombinant Human IL-31Ra protein and Cynomolgus IL-31Ra protein, exhibiting excellent cross-species binding ability. Furthermore, it can also specifically recognize cell surface targets stably expressing human IL-31Ra, confirming its excellent affinity and high specificity in its native conformation. Based on these superior molecular properties and its potential blocking effect on the IL-31 / IL-31Ra signaling pathway, this nanobody has broad application prospects in the treatment or detection of atopic dermatitis, allergic asthma, and other IL-31Ra-mediated inflammatory and autoimmune diseases. In addition, thanks to its high specificity and stable binding ability, the nanobody of the present invention can be used to construct ELISA or flow cytometry detection systems to achieve quantitative or qualitative detection of IL-31Ra protein on cell surfaces or in solution, demonstrating good application value in basic research, drug screening, and in vitro diagnostics. Attached Figure Description

[0030] Figure 1 The results are from an ELISA screening of monoclonal nanobodies for cross-reactivity. A: Screening results using Human IL-31Ra / His protein as the antigen; B: Screening results of the same group of monoclonal nanobodies using Cynomolgus IL-31Ra / His protein as the antigen.

[0031] Figure 2 Map of the mammalian system expression vector for the anti-IL-31Ra nanobody.

[0032] Figure 3 SDS-PAGE analysis of purified nanobody (IL-31Ra-LP1R3-F9). R: reducing conditions; NR: non-reducing conditions; M: marker.

[0033] Figure 4 SEC-HPLC chromatogram of purified nanobody (IL-31Ra-LP1R3-F9). A: Detection result at 214 nM; B: Detection result at 280 nM.

[0034] Figure 5 ELISA binding curves of purified nanobody (IL-31Ra-LP1R3-F9) with Human IL-31Ra / His protein.

[0035] Figure 6 ELISA binding curves of purified nanobody (IL-31Ra-LP1R3-F9) and Cynomolgus IL-31Ra / His protein.

[0036] Figure 7 The results of FACS binding analysis were obtained for the purified nanobody (IL-31Ra-LP1R3-F9) and HEK293 hIL-31Ra overexpressing cells. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments. However, the present invention is not limited to the examples given. Unless otherwise specified, all methods used are conventional methods, and all reagents and materials used are commercially available unless otherwise specified.

[0038] Example 1: Screening anti-IL-31Ra nanobodies from a natural library of alpaca nanobodies

[0039] 1. Phage display technology was used to pan for anti-IL-31Ra nanobodies from a natural library of alpaca nanobodies. All three rounds of panning were performed using liquid chromatography.

[0040] 1.1 Take 200 μL of streptavidin magnetic beads (Thermo Fisher Scientific, 11206D), wash with PBS, add 5% NON-fat Powdered Milk (Sangon Biotech, A600669-0250), mix well, and incubate at 25℃ for 1 h to block the magnetic beads.

[0041] 1.2 Take 100 μL of blocked magnetic beads, add 900 μL of 5% NON-fat Powdered Milk (Sangon Biotech, A600669-0250), and then add 100 μL of phage to a total volume of 1 mL. Incubate at 25°C for 1 h by rotation. Separate using a magnetic rack, discard the phage bound to the magnetic beads, and take the supernatant.

[0042] 1.3 Take another 100 μL of blocked magnetic beads, add 100 nM biotin-labeled hIL-31Ra / His recombinant protein in the first round, and add 30 nM biotin-labeled hIL-31Ra / His recombinant protein and 10 nM biotin-labeled cynoIL-31Ra / His recombinant protein in the second and third rounds, respectively, and incubate at 25°C for 1 h.

[0043] 1.4 Take the magnetic beads that have bound the positive screening antigen from step 1.3, add them to the phage supernatant (after removing the background) from step 1.2, and incubate at 25°C for 1 h. Remove the phage supernatant that has not bound to the magnetic beads. For the first round, wash the remaining magnetic beads twice each with 1 mL of 0.05% PBST and 1 mL of PBS. For the second and third rounds, wash them four times each with 1 mL of 0.05% PBST and 1 mL of PBS. After resuspending, add them to the logarithmic phase bacterial culture of TG1 for amplification and use in the next round of selection.

[0044] 2. After three rounds of pressure screening, the bacterial culture from the third round of amplification was plated. The next day, one plate of 94 clones was selected for further screening. The sample preparation process was as follows: 2×YT carbenicillin medium was added to a 96-well deep-well plate, a single clone was inoculated, and the plate was incubated at 37°C with shaking for 3 hours (OD of bacterial culture was 0.05%). 600 The value is approximately 0.5-0.6). Pipette 100 μL of bacterial culture from each well into a new plate and incubate at 4°C for testing. Add helper phage M13KO7 (1×10⁻⁶) to each well of the remaining bacterial culture. 10 Superinfection was performed using CFU / Well medium, and the plates were cultured at 37°C with shaking for 1 h. Subsequently, 2×YT medium containing carbenicillin, kanamycin, and IPTG was added to each well, and the plates were sealed with a breathable membrane and incubated at 30°C with shaking for 16 h. After incubation, the supernatant was collected by centrifugation and stored at 4°C to obtain antibody samples.

[0045] 3. The ELISA screening procedure for monoclonal antibodies is as follows: hIL-31Ra / His protein and cynoIL-31Ra / His protein were coated separately onto an ELISA plate (Corning, 3590), blocked with blocking buffer (3% non-fat powdered milk in PBS), and incubated overnight at 4°C. The next day, after washing with 0.1% PBST, the plate was blocked with blocking buffer (3% non-fat powdered milk in PBS) for 1 h, followed by washing again with 0.1% PBST. The sample prepared in step 2 was added, with purified antibody Nemolizumab as a positive control and Anti-HEL IgG1 hFc (Biointron, B117901) as a negative control, and incubated at 25°C for 1 h. After washing with 0.1% PBST, Mouse anti-M13 mAb HRP (Sino Biolo, 11973-MM05T-H) was added, and the plate was incubated at 25°C for 1 h. The control antibody secondary antibody used was Goat Anti-Human IgG-Fc, HRP (Sigma, A0170). After washing with 0.1% PBST, ABTS (Thermo, 002024) was added and the mixture was incubated at room temperature in the dark. The absorbance was read at 415 nm using a microplate reader. The criteria for positive clones were: IL-31Ra > 3 × NC and Milk < 3 × NC, IL-31Ra / Milk > 3. The detection results are as follows. Figure 1 As shown (where Figure 1 A represents the results of ELISA screening using Human IL-31Ra / His protein as the antigen. Figure 1(B represents the results of ELISA screening of the same monoclonal protein using Cynomolgus IL-31Ra / His protein as the antigen). Among the 94 monoclonal proteins, there were 2 positive clones that bound the hIL-31Ra / His recombinant protein and 1 positive clone that bound the cynoIL-31Ra / His recombinant protein.

[0046] Based on the combined results of screening with two antigens, one clone that simultaneously binds to both Huamn IL-31Ra recombinant protein and Cynomolgus IL-31Ra was sequenced. The single clone was named IL-31Ra-LP1R3-F9, and its heavy chain variable region amino acid sequence is shown in SEQ ID NO:1; the amino acid sequence of CDR1 is shown in SEQ ID NO:3; the amino acid sequence of CDR2 is shown in SEQ ID NO:4; the amino acid sequence of CDR3 is shown in SEQ ID NO:5; and their corresponding nucleotide sequences are shown in SEQ ID NO:2.

[0047] Example 2: Expression and purification of anti-IL-31Ra nanobodies in a lactational system

[0048] The mammalian system expression vector pcDNA3.4 for constructing the IL-31Ra-LP1R3-F9 nanobody in Example 1 is shown in the figure. Figure 2 Then, plasmids were prepared. CHO-K1 cells were selected as the host cells for antibody expression. The constructed plasmid was transfected into CHO-K1 cells for antibody expression, with an expression volume of 40 mL. The supernatant was collected after expression, and the target nanobody was purified using a Protein A affinity chromatography column.

[0049] The specific procedures are as follows: Take CHO-K1 cells (7.2E+6 cells / mL, passaged 4 times), centrifuge, discard the supernatant, and collect the cell pellet; add electroporation buffer, mix well, and then add the constructed plasmid; after mixing, transfer to an electroporation cuvette and electroporate at 540V-580V; transfer the electroporated cells to a shake flask containing 20 mL of DMEM medium and incubate at room temperature for 30 min; place the shake flask in a CO2 incubator at 37°C, 250 rpm, and a CO2 concentration of 5.0% for 24 h; add 2 mL of feedstock and continue culturing for 4 days; equilibrate the Protein A affinity chromatography column with 1×PBS; after equilibration, prepare for sample loading (i.e., cell culture supernatant), setting the loading flow rate to 1.0. After loading the sample at a rate of mL / min, impurities are washed with 1×PBS, and then the antibody bound to the affinity column is eluted with sodium acetate buffer (pH=3.4). Finally, the high-concentration protein is aspirated into a dialysis bag and placed in a beaker containing 1×PBS for dialysis to obtain high-purity expression antibody.

[0050] Its purity was determined by SDS-PAGE and SEC-HPLC, and the results are as follows: Figure 3 and Figure 4 As shown, the antibody purity all reached over 95%, indicating high purity.

[0051] Example 3: Detection of the binding ability of the antibody from Example 2 to two antigens.

[0052] The ELISA detection procedure for antibody binding to IL-31Ra / His protein is as follows: Recombinant hIL-31Ra / His protein and recombinant CynoIL-31Ra / His protein were coated onto microplates (Corning, 3590) and incubated overnight at 4°C. The next day, the plates were washed with 0.1% PBST, blocked with blocking buffer (3% non-fat powdered milk in PBS) for 1 h, and washed again with 0.1% PBST. The purified IL-31Ra-LP1R3-F9 antibody and control antibody (positive control: Nemolizumab; negative control: Anti-HEL IgG1 hFc (Biointron, B117901)) from Example 2 were diluted to 100 nM as the initial concentration for each well. A 3-fold serial dilution was performed, with the last well being a blank. 100 μL / well was incubated at 25°C for 1 h, followed by washing with 0.1% PBST. Secondary antibody was Goat Anti-Human IgG-Fc, HRP (Sigma, A0170), incubated at 25°C for 1 h. After washing with 0.1% PBST, ABTS (Thermo, 002024) was added and incubated at room temperature in the dark. The absorbance was read at 415 nm using a microplate reader. The detection results are as follows: Figure 5-6 As shown, both IL-31Ra-LP1R3-F9 and IL-31Ra / His protein can bind to CynomolgusIL-31Ra / His protein, with EC50 values ​​of 0.7068 nM and 0.6147 nM, respectively.

[0053] Example 4: Detection of the binding ability of the antibody from Example 2 to HEK293 hIL-31Ra overexpressing cells.

[0054] The FACS assay for antibody binding to HEK293 hIL-31Ra overexpressing cells was performed as follows: HEK293 hIL-31Ra and HEK293 GFP were mixed 1:1, and 1.5E+ cells / well were seeded into 96-well V plates. The supernatant was discarded after centrifugation. The purified IL-31Ra-LP1R3-F9 antibody and control antibody from Example 2 were diluted to 100 nM as the initial concentration, serially diluted 3-fold, with the last well being a blank. 100 μL / well was incubated at 4°C for 0.5 h, centrifuged at 400 g for 5 min, and the supernatant was discarded. Goat Anti-hIgG (FcγSpecific) pAb [Alexa Fluor 647] was added, and the cells were incubated at 4°C for 0.5 h. The supernatant was discarded after centrifugation. 200 μL / well of PBS buffer was added, mixed thoroughly, centrifuged, and the supernatant was discarded. The cells were resuspended in PBS buffer and analyzed by flow cytometry. The positive control was Nemolizumab, and the negative control was Anti-HEL IgG1 hFc (Biointron, B117901).

[0055] The results of IL-31Ra-LP1R3-F9 binding to HEK293 hIL-31Ra cells are as follows: Figure 7 As shown, its EC50 is 1.912 nM.

[0056] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. An anti-IL-31Ra nanobody, said nanobody comprising a framework region (FR) and a complementarity-determining region (CDR); said complementarity-determining region comprising CDR1, CDR2, and CDR3, characterized in that, The amino acid sequence of the complementarity-determining region CDR1 is shown in SEQ ID NO:3; the amino acid sequence of the complementarity-determining region CDR2 is shown in SEQ ID NO:4; and the amino acid sequence of the complementarity-determining region CDR3 is shown in SEQ ID NO:

5.

2. The anti-IL-31Ra nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is as shown in SEQ ID NO:1, or has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

1.

3. An isolated nucleic acid molecule, characterized in that, It encodes the anti-IL-31Ra nanobody as described in claim 1 or 2.

4. A recombinant expression vector, characterized in that, It comprises the nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, It comprises the recombinant expression vector as described in claim 4.

6. An antibody derivative, characterized in that, The antibody derivative comprises the anti-IL-31Ra nanobody as described in claim 1 or 2.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the anti-IL-31Ra nanobody as described in claim 1 or 2; and (ii) Pharmaceutically acceptable carriers.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the antibody derivative as described in claim 6; and (ii) Pharmaceutically acceptable carriers.

9. A kit for detecting IL-31Ra protein in a sample, comprising the anti-IL-31Ra nanobody as described in claim 1 or 2.

10. The use of the anti-IL-31Ra nanobody according to claim 1 or 2 in the preparation of a medicament for treating IL-31Ra-mediated chronic inflammation; said chronic inflammation includes atopic dermatitis and allergic asthma.

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