Anti-ox40l nanobodies and uses thereof
High-affinity anti-OX40L nanobodies were screened from an alpaca nanobody library using phage display technology, solving the problems of high cost and low screening efficiency in existing technologies. This enabled the efficient preparation of nanobodies for the treatment and detection of autoimmune diseases, demonstrating promising application prospects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BIOINTRON (JIANGSU) BIOLOGICAL INC
- Filing Date
- 2026-03-31
- Publication Date
- 2026-06-23
AI Technical Summary
Existing technologies for developing drugs targeting OX40L suffer from high development costs, dependence on antigen immunogenicity, and difficulty in efficiently screening for nanobodies with high affinity and specificity, which limit their application in tumor treatment and autoimmune diseases.
Phage display technology was used to screen high-affinity and specific anti-OX40L nanobodies from a natural library of alpaca nanobodies. After screening and amplification using phage display technology, nanobodies that can specifically recognize Human OX40L recombinant protein and overexpressing cells were obtained, avoiding the cost and antigen immunogenicity dependence of traditional immune library construction.
The obtained nanobodies have high affinity and stability, and can effectively block the OX40/OX40L signaling pathway. They can be used to prepare drugs for treating autoimmune diseases such as rheumatoid arthritis and multiple sclerosis, and can also be used for in vitro detection of OX40L protein, reducing development costs and improving screening efficiency.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to an anti-OX40L nanobody and its application. Technical Background
[0002] OX40L (OX40 ligand), with a molecular weight of approximately 45 kDa, is a member of the tumor necrosis factor superfamily and is primarily encoded by the CD276 gene. It is a type II glycoprotein expressed in trimer form, mainly on antigen-presenting cells (APCs) such as B cells and dendritic cells. OX40L, by binding to the OX40 receptor, activates signaling pathways such as NF-κB and PI3K / Akt, promoting T cell activation, proliferation, and survival, playing a crucial role in immune responses. In cancer treatment, OX40L can enhance the anti-tumor activity of T cells, but its efficacy as a single agent is limited; combination therapy yields better results. In the fields of autoimmune diseases and inflammation, blocking the OX40L signaling pathway can alleviate disease symptoms. Currently, drug development targeting OX40L is progressing rapidly, with several drugs already in clinical trials, showing promising application prospects.
[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 an anti-OX40L nanobody and its applications. The anti-OX40L nanobody can bind to the Human OX40L recombinant protein with high affinity and specifically recognize HEK293 hOX40L overexpressing cells and CHO-K1cynoOX40L overexpressing cells, exhibiting good species cross-reactivity. Based on its ability to block the OX40L / OX40 signaling pathway, this nanobody can be used to prepare drugs for treating autoimmune diseases, including but not limited to rheumatoid arthritis, multiple sclerosis, type 1 diabetes, Crohn's disease, ulcerative colitis, celiac disease, psoriasis, lupus nephritis, and polymyositis.
[0006] Furthermore, the present invention also discloses a nucleotide molecule encoding the above-mentioned anti-OX40L nanobody;
[0007] This invention also discloses an expression vector and a eukaryotic host cell;
[0008] Finally, the present invention also provides a pharmaceutical composition comprising the nanobody and a pharmaceutically acceptable carrier, and a reagent or kit for using it for in vitro detection of OX40L protein.
[0009] More specifically, a first aspect of the present invention provides an anti-OX40L nanobody, the anti-OX40L nanobody comprising a heavy chain variable region; the heavy chain variable region comprising complementarity-determining regions CDR1, CDR2 and CDR3, wherein 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; and the amino acid sequence of CDR3 is shown in SEQ ID NO:5.
[0010] Preferably, the amino acid sequence of the variable region of the heavy chain antibody 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.
[0011] A second aspect of the present invention provides a nucleic acid molecule that encodes the anti-OX40L nanobody described in the first aspect.
[0012] Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 2.
[0013] A third aspect of the present invention provides a recombinant expression vector containing nucleotide molecules as described in the second aspect.
[0014] A fourth aspect of the present invention provides a host cell containing the expression vector as described in the third aspect. Preferably, the host cell is a CHO-K1 cell.
[0015] A fifth aspect of the present invention provides a pharmaceutical composition comprising an anti-OX40L nanobody as described in the first aspect of the invention, and a pharmaceutically acceptable carrier.
[0016] A sixth aspect of the present invention provides an OX40L binding molecule, wherein the OX40L binding molecule comprises a nanobody as described in claim 1 or 2; preferably, the OX40L binding molecule is a monovalent or multivalent nanobody, bispecific antibody, multispecific antibody, heavy chain antibody or antigen-binding fragment thereof comprising one, two or more nanobodies as described in any one of claims 1 or 2.
[0017] The seventh aspect of the present invention provides the use of the anti-OX40L nanobody described in the first aspect in the preparation of products for in vitro detection of OX40L protein, wherein the products include reagents or kits.
[0018] The eighth aspect of the present invention provides the use of the anti-OX40L nanobody as described in the first aspect in the preparation of a medicament for treating autoimmune diseases, wherein the autoimmune diseases are selected from: rheumatoid arthritis, multiple sclerosis, type 1 diabetes, Crohn's disease, ulcerative colitis, celiac disease, psoriasis, lupus nephritis and polymyositis.
[0019] Compared with the prior art, the present invention has the following technical effects:
[0020] This invention utilizes phage display technology to screen and obtain a nanobody targeting OX40L from a natural library of alpaca nanobodies. This antibody specifically recognizes Human OX40L recombinant protein, HEK293 hOX40L overexpressing cells, and CHO-K1 cynoOX40L overexpressing cells, exhibiting good binding activity and cross-species reactivity.
[0021] Compared to traditional methods for constructing immune libraries, this invention utilizes a natural library of alpaca nanobodies for screening, eliminating the need for animal immunization. This significantly reduces development costs and avoids dependence on antigen immunogenicity, enabling more efficient enrichment of high-affinity and high-specificity candidate molecules. The resulting nanobodies possess advantages such as small molecular weight, high stability, strong tissue penetration, and ease of engineering. They demonstrate good potential in blocking the OX40 / OX40L signaling pathway and can be used to prepare drugs for treating various autoimmune and inflammatory diseases, including rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease. They can also be applied to the in vitro detection of OX40L protein and related mechanism studies, providing new technical means and broad application prospects for the treatment of autoimmune diseases and the development of biomedical tools. Attached Figure Description
[0022] Figure 1 : Screening results of anti-OX40L nanobody monoclonal antibodies; where (A) is the ELISA screening result based on human OX40L / His recombinant protein, and (B) is the FACS binding verification result based on HEK293 hOX40L overexpressing cells.
[0023] Figure 2 : OX40L-LP1R3-C9 nanobody expression vector map for lactation system;
[0024] Figure 3 SDS-PAGE analysis of purified OX40L-LP1R3-C9 nanobody (R represents reduction conditions, NR represents non-reduction conditions, and M represents marker).
[0025] Figure 4 SEC-HPLC analysis chromatograms of purity and homogeneity of OX40L-LP1R3-C9 nanobody; (A: detection wavelength 214 nM, B: detection wavelength 280 nM);
[0026] Figure 5 Figure: ELISA results of the binding ability of OX40L-LP1R3-C9 nanobody to hOX40L / His protein;
[0027] Figure 6 FACS analysis results of the binding ability of OX40L-LP1R3-C9 nanobody to HEK293 hOX40L overexpressing cells;
[0028] Figure 7 FACS analysis results of the binding ability of OX40L-LP1R3-C9 nanobody to CHO-K1 cynoOX40L overexpressing cells;
[0029] Figure 8 Figure 1 shows the SPR affinity analysis results of the OX40L-LP1R3-C9 nanobody with the hOX40L / His protein; where A is the nanobody OX40L-LP1R3-C9 of this invention; B is the positive control antibody anti-OX40L-PC1; and C is the negative control antibody Anti-HEL VHH-Human IgG1 Fc Isotype Control. Detailed Implementation
[0030] 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.
[0031] Example 1: Screening anti-OX40L nanobodies from a natural library of alpaca nanobodies
[0032] 1.1 Phage display technology was used to screen anti-OX40L nanobodies from a natural library of alpaca nanobodies.
[0033] 1.2 Screening process for anti-OX40L nanobodies:
[0034] 1.2.1 All three rounds of screening employed liquid phase panning, with the specific operation as follows:
[0035] (1) Take 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°C for 1 h to obtain blocked magnetic beads.
[0036] (2) Take a portion of sealed magnetic beads, add 5% NON-fat Powdered Milk (Sangon Biotech, A600669-0250) and phage, and incubate at 25°C for 1 h.
[0037] (3) Take another batch of sealed magnetic beads, add 100 nM biotin-labeled hOX40L / His recombinant protein (Biointron, B22727607) in the first round, add 30 nM biotin-labeled hOX40L / His recombinant protein in the second round, and add 10 nM biotin-labeled hOX40L / His recombinant protein in the third round. Incubate at 25°C for 1 h to perform antigen conjugation.
[0038] (4) Take the magnetic beads that have been bound to the positive screening antigen in step (3), add the phage supernatant that has been removed from the background in step (2), and incubate at 25°C for 1 h.
[0039] (5) Remove the phage supernatant that did not bind to the magnetic beads, and use 0.05% PBST and PBS to wash the remaining magnetic beads and infect logarithmic TG1 bacterial culture for amplification. The resulting phages are used for the next round of panning.
[0040] 1.2.2 After three rounds of pressure screening, the bacterial culture after the third round of amplification was plated. The next day, 94 clones were selected from one plate for screening. The sample preparation process was as follows: Single colonies were inoculated into 2×YT medium containing carbenicillin and cultured at 37°C with shaking until the logarithmic growth phase; a portion of the bacterial culture was retained for preservation and sequencing, and the remaining bacterial culture was infected with helper phage M13KO7; after infection, 2×YT medium containing carbenicillin, kanamycin, and IPTG was added, and expression and phage assembly were induced at 30°C; after culture, the supernatant sample was collected by centrifugation for subsequent screening or verification.
[0041] 1.3 The ELISA screening and detection process for monoclonal antibodies is as follows:
[0042] (1) HOX40L / His protein and blocking buffer (3% NON-fat Powdered Milk in PBS) 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 then washed with 0.1% PBST.
[0043] (2) Add the test sample and control antibody (positive control: Anti-OX40L-PC1, negative control: Anti-HEL VHH-Human IgG1 Fc Isotype Control) prepared in advance in step 1.2.2, and incubate at 25℃ for 1 h.
[0044] (3) After washing with 0.1% PBST, Mouse anti-M13 mAb HRP (Sino Biolo, 11973-MM05T-H) was added and incubated at 25°C for 1 h. The control antibody secondary antibody was Goat Anti-Human IgG-Fc, HRP (Sigma, A0170).
[0045] (4) After washing with 0.1% PBST, add ABTS (Thermo, 002024) and develop color at room temperature in the dark. Read the absorbance at 415 nm wavelength using an ELISA reader.
[0046] Criteria for determining a positive clone: OX40L > 3 × NC and Milk < 3 × NC, OX40L / Milk > 2.5. Test results are as follows: Figure 1 As shown in Figure A, 79 out of 94 monoclonal antibodies were positive clones that bound the hOX40L / His recombinant protein.
[0047] 1.4 The FACS screening and detection process for monoclonal phages is as follows: HEK293 hOX40L cells and HEK293 GFP cells were mixed at a 1:1 ratio and seeded into 96-well V plates. After centrifugation, the supernatant was discarded. The monoclonal phage supernatant prepared in step 1.2.2 and control antibodies (positive control: Anti-OX40L-PC1; negative control: Anti-HEL VHH-Human IgG1 FcIsotype Control) were added and incubated at 4℃ for 0.5 h. After centrifugation, the supernatant was discarded. THE™ DYKDDDDK TagAntibody (Thermo, A-21281) was added to the sample wells, and Goat Anti-hIgG (Fcγ Specific) pAb [Alexa Fluor 647] (Jackson, 109-605-190) was added to the control wells. After incubation at 4℃ for 0.5 h, the supernatant was discarded. The cells were washed with PBS buffer and resuspended for flow cytometry analysis.
[0048] Positive clone determination: Sample / negative control Median APC-H (≥3). Results as follows Figure 1 As shown in B, 69 out of 94 clones bound to HEK293 hOX40L overexpressing cells.
[0049] Based on the combined results of screening with two antigens, 68 positive clones were found to bind to both hOX40L / His recombinant protein and HEK293 hOX40L cells. After sequencing analysis and comparative screening, the dominant single clone was finally obtained as OX40L-LP1R3-C9. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 1, and its corresponding nucleotide sequence is shown in SEQ ID NO: 2. In the amino acid sequence SEQ ID NO: 1, amino acid residues 31-35 (i.e., SEQ ID NO: 3) are heavy chain CDR1, amino acid residues 50-66 (i.e., SEQ ID NO: 4) are heavy chain CDR2, and amino acid residues 99-116 (i.e., SEQ ID NO: 5) are heavy chain CDR3.
[0050] Example 2: Expression and purification of anti-OX40L nanobody in a lactating system
[0051] The mammalian system expression vector pcDNA3.4 for the OX40L-LP1R3-C9 nanobody in Example 1 was constructed (see figure). Figure 2 Then, plasmids were prepared using this method. CHO-K1 cells were selected as the host cells for antibody expression, with an expression volume of 50 mL. The supernatant after expression was purified using a Protein A affinity chromatography column.
[0052] The specific steps are as follows: Mix CHO-K1 cells with the constructed plasmid, transfect them, and then seed them in a shake flask containing DMEM / F12 medium. After standing, culture the cells at 37°C with shaking at 5% CO2. Collect the cell culture supernatant and purify it using a Protein A affinity chromatography column to obtain a high-purity expression antibody.
[0053] 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.
[0054] Example 3: Detection of the binding ability of the antibody from Example 2 to the hOX40L antigen.
[0055] The ELISA detection procedure for antibody binding to hOX40L / His protein is as follows:
[0056] (1) Coat the hOX40L / His recombinant protein onto an ELISA plate (Corning, 3590) and incubate overnight at 4°C; the next day, wash with 0.1% PBST and block with blocking buffer (3% NON-fat Powdered Milk in PBS) for 1 h;
[0057] (2) The purified antibody OX40L-LP1R3-C9 and control antibody (positive control: Anti-OX40L-PC1, negative control Anti-HEL VHH-Human IgG1 Fc Isotype Control) from Example 2 were serially diluted 3-fold with 100 nM as the initial concentration and the last well was blank. They were incubated at 25°C for 1 h and then washed with 0.1% PBST.
[0058] (3) Add secondary antibody Goat Anti-Human IgG-Fc, HRP (Sigma, A0170), dilute 1:10000, 100 μL / well, incubate at 25℃ for 1 h; wash with 0.1% PBST and add ABTS (Thermo, 002024) for color development at room temperature in the dark, and read the absorbance at 415 nm wavelength using an ELISA reader.
[0059] Test results as follows Figure 5 As shown, OX40L-LP1R3-C9 binds to the hOX40L / His protein with an EC50 value of 1.177 nM.
[0060] Example 4: Detection of the binding affinity of the antibody from Example 2 to HEK293 hOX40L and CHO-K1 CynoOX40L.
[0061] The FACS detection procedure for antibody binding to HEK293 hOX40L and CHO-K1 CynoOX40L cells is as follows:
[0062] (1) HEK293 hOX40L and HEK293 GFP were mixed in a 1:1 ratio, and CHO-K1 CynoOX40L and CHO-K1GFP were also mixed in a 1:1 ratio; the two mixtures were inoculated into 96-well V plates, and the supernatant was discarded after centrifugation;
[0063] (2) The purified antibody OX40L-LP1R3-C9 and the control antibody (positive control is Anti-OX40L-PC1, negative control is Anti-HEL VHH-Human IgG1 Fc Isotype Control) in Example 2 were diluted to 100 nM as the initial concentration, serially diluted 3 times, and the last well was blank. 100 μL / well, incubated at 4℃ for 0.5 h, and the supernatant was discarded by centrifugation.
[0064] (3) Add secondary antibody Goat Anti-hIgG (Fcγ Specific) pAb [Alexa Fluor 647], 1:800, 100 μL / well, 4℃, 0.5 h, centrifuge and discard the supernatant; add PBS buffer, wash and resuspend the cells, and then detect them by flow cytometry.
[0065] The results of the binding of OX40L-LP1R3-C9 with HEK293 hOX40L cells and CHO-K1 CynoOX40L cells are as follows: Figure 6-7 As shown, OX40L-LP1R3-C9 can bind to HEK293 hOX40L and CHO-K1 CynoOX40L, with EC50 values of 8.228 nM and 3.824 nM, respectively, but does not bind to control cells HEK 293 GFP and CHO-K1 GFP.
[0066] Example 5: SPR affinity assay of the antibody from Example 2 with hOX40L (TNFSF4) / Trimer / His
[0067] (1) The purified antibody OX40L-LP1R3-C9 and the control antibody anti-OX40L-PC1 were injected into the experimental channel at a flow rate of 10 μL / min for 60 s, and the capture volume was approximately 145-2000 RU.
[0068] (2) The recombinant hOX40L(TNFSF4) / Trimer / His (Biointron, B22727607) protein was diluted 2-fold starting from 200 nM using HBS-EP+Buffer. The diluted hOX40L / His recombinant protein was then injected sequentially into the experimental and reference channels at a flow rate of 30 μL / min for the corresponding binding and dissociation times. Both binding and dissociation steps were performed in the running buffer. Protein A Chip (Cytiva, 29127556) required regeneration with 10 mM Gly-HCl (pH=1.5) at a flow rate of 30 μL / min for 30 s to wash away any undissociated analytes.
[0069] (3) Use Biacore 8K analysis software to calculate the KD value of the sample.
[0070] The reference channel (Fc1) is used for background subtraction, with a 1:1 fitting model. The results are as follows: Figure 8 As shown, the OX40L-LP1R3-C9 antibody exhibits good binding activity to the hOX40L / His recombinant protein, with a KD value of 2.70E-09M. The KD is in the nanomolar range (~2.7 nM), which falls within the high affinity range and is suitable for use in drug development or diagnostic reagents.
[0071] 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-OX40L nanobody, characterized in that, The anti-OX40L nanobody includes a heavy chain variable region; the heavy chain variable region includes complementarity-determining regions CDR1, CDR2 and CDR3, wherein 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; and the amino acid sequence of CDR3 is shown in SEQ ID NO:
5.
2. The anti-OX40L nanobody according to claim 1, characterized in that, The amino acid sequence of the variable region of the heavy chain of the anti-OX40L nanobody is 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. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-OX40L nanobody as described in claim 1 or 2.
4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:
2.
5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 3 or 4.
6. A host cell, characterized in that, The host cell contains the expression vector as described in claim 5.
7. A pharmaceutical composition, characterized in that, It comprises the anti-OX40L nanobody as described in claim 1 or 2, and a pharmaceutically acceptable carrier.
8. An OX40L-bound molecule, wherein, The OX40L binding molecule comprises the nanobody as described in claim 1 or 2; preferably, the OX40L binding molecule is a monovalent or multivalent nanobody, bispecific antibody, multispecific antibody, heavy chain antibody or antigen-binding fragment thereof comprising one, two or more nanobodies as described in any one of claims 1 or 2.
9. Use of the anti-OX40L nanobody as described in claim 1 or 2 in the preparation of products for in vitro detection of OX40L protein, wherein said products include reagents or kits.
10. Use of the anti-OX40L nanobody as described in claim 1 or 2 in the preparation of a medicament for treating autoimmune diseases, wherein the autoimmune diseases are selected from: rheumatoid arthritis, multiple sclerosis, type 1 diabetes, Crohn's disease, ulcerative colitis, celiac disease, psoriasis, lupus nephritis, and polymyositis.