An inhibitory antibody or antigen-binding fragment thereof against human OX40, preparation method and application

Abstract

The application discloses an inhibitory antibody or antigen-binding fragment thereof against human OX40, a preparation method and application, and belongs to the technical field of biological immunity. The human-mouse chimeric antibody Y07-L2 and the humanized antibody Y07-C2 prepared by the application have similar EC50 in combination with human OX40, and are similar to the targeted antibody drug KHK-4083 in inhibiting OX40. The heavy chain variable region of the human-mouse chimeric antibody Y07-L2 is shown as SEQ ID NO. 14, and the light chain variable region is shown as SEQ ID NO. 15; the heavy chain variable region of the humanized antibody Y07-C2 is shown as SEQ ID NO. 20, and the light chain variable region of the humanized antibody Y07-C2 is shown as SEQ ID NO. 21; the CDR region of the humanized antibody Y07-C2 is subjected to mutation, and the mutant antibody Y07-C2MH4 and Y07-C2MH4L3 are obviously superior to other antibodies.

Description

Technical Field

[0001] This invention belongs to the field of bioimmunotechnology, specifically relating to an inhibitory antibody against human OX40 or its antigen-binding fragment, preparation method, and application. Background Technology

[0002] OX40 (also known as CD134 or TNFRSF4) is a member of the tumor necrosis factor receptor superfamily and a key co-stimulatory molecule expressed on the surface of activated T cells. Its ligand, OX40L, is mainly found on the surface of antigen-presenting cells. The binding of OX40 to OX40L activates downstream signaling pathways such as NF-κB, promoting T cell proliferation, survival, and cytokine release, while inhibiting the function of regulatory T cells, thereby enhancing the immune response. Studies have shown that this pathway plays a central role in the pathogenesis of autoimmune and inflammatory diseases. In diseases such as atopic dermatitis, abnormal activation of OX40 / OX40L signaling leads to an exacerbation of Th2-type inflammatory responses, driving skin barrier damage and chronic itching. Atopic dermatitis is a chronic, relapsing inflammatory skin disease affecting approximately 15%-30% of children and 2%-10% of adults worldwide, characterized by intense itching, dry skin, and eczematous lesions. While existing therapies such as corticosteroids and IL-4 inhibitors have some efficacy, some patients experience insufficient response, drug resistance, or side effects. In recent years, regulating abnormal T cell activity by blocking the OX40 / OX40L pathway has become a new strategy for treating Alzheimer's disease (AD).

[0003] Currently, several targeted antibody drugs that inhibit OX40 have entered clinical trials worldwide:

[0004] (1) Rocatinlimab (KHK4083) (Amgen / Kirin): A fully humanized anti-OX40 antibody that can deplete OX40+ activated T cells by enhancing ADCC effect. Phase III clinical trials showed that it can significantly improve the eczema area and severity index score of AD patients.

[0005] (2) Amlitelimab (KY1005) (Sanofi): A non-depleted IgG4 antibody targeting OX40L that restores immune homeostasis by blocking ligand-receptor interactions. The phase II study met its primary endpoint.

[0006] (3) Telazorlimab (GBR830), humanized IgG1, is a blocking antibody. A phase IIb clinical trial has been completed, showing significant efficacy and safety in patients with moderate to severe AD.

[0007] (4) Clinical studies of Rocatinlimab and Telazorlimab showed that their efficacy could last until discontinuation, suggesting that targeting OX40 may have potential long-term immunomodulatory effects. Summary of the Invention

[0008] This invention provides an inhibitory antibody against human OX40 or its antigen-binding fragment, a preparation method, and applications. The antibody or its antigen-binding fragment has more efficient OX40 signaling pathway blocking activity.

[0009] In a first aspect, the present invention provides an inhibitory antibody against human OX40 or an antigen-binding fragment thereof.

[0010] (1) When using Kabat to define CDR, the antibody or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region;

[0011] (a) The heavy chain variable region comprises the following three CDRs:

[0012] HCDR1 consists of the following sequence: SEQ ID NO.1, or has a mutation of one amino acid compared to it;

[0013] HCDR2 consists of the following sequence: SEQ ID NO.2, or has a mutation of one amino acid compared to it;

[0014] HCDR3 consists of the following sequence: SEQ ID NO.3, or has a mutation of one amino acid compared to it;

[0015] (b) The light chain variable region comprises the following three CDRs:

[0016] LCDR1, which consists of the following sequence: SEQ ID NO.4, or has a mutation of one, two or three amino acids compared to it;

[0017] LCDR2, which consists of the following sequence: SEQ ID NO.5, or has a mutation of one, two or three amino acids compared to it;

[0018] LCDR3 consists of the following sequence: SEQ ID NO.6, or has a mutation of one, two or three amino acids compared to it.

[0019] (2) When CDR is defined using IMGT, the antibody or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region;

[0020] (a) The heavy chain variable region comprises the following three CDRs:

[0021] HCDR1 consists of the following sequence: SEQ ID NO.7, or has a mutation of one amino acid compared to it;

[0022] HCDR2 consists of the following sequence: SEQ ID NO.8, or has a mutation of one amino acid compared to it;

[0023] HCDR3 consists of the following sequence: SEQ ID NO.9, or has a mutation of one amino acid compared to it;

[0024] (b) The light chain variable region comprises the following three CDRs:

[0025] LCDR1, which consists of the following sequence: SEQ ID NO.10, or has a mutation of one, two or three amino acids compared to it;

[0026] LCDR2, which consists of the following sequence: SEQ ID NO.35, or has a mutation of one, two or three amino acids compared to it;

[0027] LCDR3 consists of the following sequence: SEQ ID NO.12, or has a mutation of one, two or three amino acids compared to it.

[0028] Furthermore, the antibody or its antigen-binding fragment comprises the heavy chain variable region sequence (mouse antibody heavy chain variable region amino acid sequence) shown in SEQ ID NO.14 and the light chain variable region sequence (mouse antibody light chain variable region amino acid sequence) shown in SEQ ID NO.15.

[0029] When using Kabat to define CDRs, the three CDRs of the mouse antibody heavy chain variable region are shown in SEQ ID NO.1-3, and the three CDRs of the mouse antibody light chain variable region are shown in SEQ ID NO.4-5.

[0030] When using IMGT to define CDRs, the three CDRs of the mouse antibody heavy chain variable region are shown in SEQ ID NO.7-9, and the three CDRs of the mouse antibody light chain variable region are shown in SEQ ID NO.10, 12, and 35.

[0031] Furthermore, the antibody is a human-mouse chimeric antibody Y07-L2, the heavy chain amino acid sequence of which is shown in SEQ ID NO.18 and the light chain amino acid sequence of which is shown in SEQ ID NO.19.

[0032] The preparation method of human-mouse chimeric antibody Y07-L2 is as follows:

[0033] The heavy chain of the human-mouse chimeric antibody Y07-L2 is formed by linking the variable region of the mouse antibody heavy chain (SEQ ID NO.14) with the constant region of the human IgG1 heavy chain (SEQ ID NO.16), and the amino acid sequence of the heavy chain is SEQ ID NO.18;

[0034] The light chain of the human-mouse chimeric antibody Y07-L2 is formed by linking the variable region of the mouse antibody light chain (SEQ ID NO.15) with the constant region of the human κ chain (SEQ ID NO.17).

[0035] Furthermore, the antibody is a humanized antibody Y07-C2, the heavy chain variable region sequence of which is shown in SEQ ID NO.20, and the light chain variable region sequence of which is shown in SEQ ID NO.21.

[0036] Furthermore, the preparation method of the humanized antibody Y07-C2 is as follows:

[0037] The heavy chain variable region and light chain variable region of the humanized antibody Y07-C2 were obtained by transplanting the CDR of mouse antibody (SEQ ID NO.1-6 as defined by Kabat, or SEQ ID NO.7-10, 12, 35 as defined by IMGT) into the human germline antibody framework region and then performing partial reversion mutations.

[0038] Furthermore, the antibody is a mutant antibody of humanized antibody Y07-C2, prepared by: mutating at least one amino acid in the heavy chain variable region CDR and / or light chain variable region CDR of humanized antibody Y07-C2 to obtain the mutant antibody.

[0039] Mutant antibodies contain at least one of the following mutant heavy chain variable regions or one mutant light chain variable region:

[0040] (a) Mutant heavy chain variable region sequences: SEQ ID NO.25 (MH1), SEQ ID NO.26 (MH2), SEQ ID NO.27 (MH3), SEQ ID NO.28 (MH4), SEQ ID NO.29 (MH5);

[0041] (b) Mutant light chain variable region sequences: SEQ ID NO.30 (ML1), SEQ ID NO.31 (ML2), SEQ ID NO.32 (ML3), SEQ ID NO.33 (ML1 / 3), SEQ ID NO.34 (ML2 / 3), SEQ ID NO.11 (ML1 / 2 / 3).

[0042] Preferably, the mutant heavy chain variable region sequence is shown in SEQ ID NO.28, and the mutant light chain variable region sequence is shown in SEQ ID NO.32 (Y07-C2MH4L3).

[0043] Preferably, the mutant heavy chain variable region sequence is shown in SEQ ID NO.28, and the mutant light chain variable region sequence is shown in SEQ ID NO.21 (Y07-C2MH4).

[0044] Furthermore, the amino acid sequences of the heavy chain constant region of both humanized antibody Y07-C2 and its mutant antibody are shown in SEQ ID NO.16; the amino acid sequences of the light chain constant region are shown in SEQ ID NO.17.

[0045] Secondly, the present invention provides a method for preparing an inhibitory antibody against human OX40 and its antigen-binding fragment:

[0046] The nucleotide sequences of the heavy chain and light chain encoding the antibody or its antigen-binding fragment were cloned into mammalian cell expression vectors to construct heavy chain expression plasmids and light chain expression plasmids, respectively. The heavy chain expression plasmid and light chain expression plasmid were transfected into HEK293 cells at a molar ratio of 2:1 using a transfection reagent. After culturing for a period of time, the antibody or its antigen-binding fragment was recovered from the culture supernatant.

[0047] Nucleic acid molecules encoding the antibody or its antigen-binding fragment are also within the scope of protection of this invention.

[0048] Thirdly, the present invention provides an inhibitory antibody against human OX40 or its antigen-binding fragment for use in the preparation of targeted antibody drugs that inhibit OX40, such as drugs for atopic dermatitis.

[0049] Beneficial effects:

[0050] 1) The EC50 of the human-mouse chimeric antibody Y07-L2 bound to human OX40 prepared in this invention is 152.3 ng / mL, and the EC50 of the humanized antibody Y07-C2 bound to human OX40 is 90.72 ng / mL, both similar to the target antibody drug KHK-4083 that inhibits OX40. The IC50 of both Y07-L2 and Y07-C2 inhibiting the NFκB-luciferase reporter gene are similar to those of KHK-4083.

[0051] 2) ELISA competition assays showed that single heavy chain mutations MH2 and MH4 and single light chain mutations ML1 and ML3 of humanized antibody Y07-C2 could improve the antigen competitive binding ability of mutant antibodies, with MH4, ML1 and ML2 showing the greatest improvement; different combinations of heavy chain mutation MH4 and light chain mutations ML1, ML2 and ML3 could all improve the antigen competitive binding ability of mutant antibodies.

[0052] 3) The signaling pathway blocking activities of the control antibody KHK-4083, the maternal antibody Y07-C2, and the mutant antibodies Y07-C2MH4 and Y07-C2MH4L3 were evaluated. The results showed that the OX40 signaling pathway blocking activity of Y07-C2MH4L3 was significantly stronger than that of the other three antibodies.

[0053] 4) The ligand binding blocking experiment showed that Y07-C2MH4 and Y07-C2MH4L3 had significantly stronger blocking activity against the binding of OX40L to OX40 than the other three antibodies (antibodies KHK-4083, GBR830, and Y07-C2MH4L2 / 3).

[0054] 5) Antibody ADCC experiments showed that the ADCC activity of mutant antibodies Y07-C2MH4, Y07-C2MH4L3 and Y07-C2MH4L2 / 3 was significantly stronger than that of KHK-4083 and GBR830.

[0055] 6) Antigen-antibody affinity assays showed that, compared with Y07-C2, Y07-C2MH4 and Y07-C2MH4L3 had increased affinity for human OX40. However, the increase in affinity did not correspond one-to-one with the antibody’s signaling pathway blocking activity. Attached Figure Description

[0056] Figure 1 The diagram shows the binding of the human-mouse chimeric antibody and humanized antibody of the present invention to human OX40 protein using ELISA; wherein, A is the binding of the human-mouse chimeric antibody to human OX40 protein, and B is the binding of the humanized antibody to human OX40 protein.

[0057] Figure 2 To demonstrate the inhibitory effect of the human-mouse chimeric and humanized antibodies of the present invention on the OX40 signaling pathway using the reporter gene method; wherein, A is the inhibitory effect of the human-mouse chimeric antibody on the OX40 signaling pathway, and B is the inhibitory effect of the humanized antibody on the OX40 signaling pathway.

[0058] Figure 3 This is a schematic diagram of the plasmid map of the expression vector pUYD1-Y07C2 (LH) for yeast protein display designed in this invention.

[0059] Figure 4This is a flow cytometry curve showing the concentration of antigen binding to monoclonal yeast cells.

[0060] Figure 5 To demonstrate the competitive binding of mutant antibodies to maternal antibodies using ELISA; where A represents the effect of mutations at different sites on the heavy chain on competitive binding with maternal antibodies, and B represents the effect of mutations at different sites on the light chain on competitive binding with maternal antibodies.

[0061] Figure 6 To demonstrate the competitive binding of combined mutant antibodies to the parent antibody using ELISA; where A represents the effect of a combination of one mutation site in the heavy chain and one different mutation site in the light chain on the competitive binding of the parent antibody, and B represents the effect of a combination of one mutation site in the heavy chain and two different mutation sites in the light chain on the competitive binding of the parent antibody.

[0062] Figure 7 To demonstrate the inhibitory effect of the mutant antibody of the present invention on the OX40 signaling pathway using the reporter gene method.

[0063] Figure 8 To detect the blocking effect of mutant antibodies on ligand-antigen binding by flow cytometry.

[0064] Figure 9 To demonstrate the difference in ADCC activity of the mutant antibodies of the present invention through reporter gene expression. Detailed Implementation

[0065] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described. The reagents, experimental mice, cell lines, human OX40 protein, etc., used in the embodiments of the present invention can be purchased from the market or prepared according to existing techniques in the art.

[0066] Example 1: Obtaining mouse anti-human OX40 monoclonal antibody

[0067] (1) Immunize mice with human OX40 protein to obtain mouse antibodies.

[0068] The human OX40 protein sequence (SEQ ID NO.13) is as follows:

[0069] MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDN QACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0070] Based on the above sequences, a CHO cell line overexpressing human OX40 protein was constructed and used to immunize Balb / c mice. Spleen cells from immunized mice were fused with SP2 / 0-AG14 cells to form hybridoma cells, and an appropriate amount of the fused cells were seeded into 96-well plates. On day 10 after fusion, the supernatant from each well was collected, and the binding activity of mouse antibodies secreted by the hybridoma cells to human OX40 was detected by ELISA (see Example 4 for the method) and their inhibitory effect on the OX40 signaling pathway (see Example 5 for the method). A series of hybridoma cells with high activity were obtained. The hybridoma cells with the best activity were selected, and the heavy chain variable region cDNA sequence and light chain variable region cDNA sequence corresponding to their secreted antibodies were obtained by sequencing. The encoded amino acid sequences of the heavy chain and light chain variable regions are shown below:

[0071] Amino acid sequence of the variable region of the mouse antibody heavy chain (SEQ ID NO.14):

[0072] QVQLKESGPGLVAPSQSLSITCTVS GFSLT SYG VH WVRQPPGKGLEWLG V IWAGGDT NLNSALMS RLGISKDNSKSQVFLKMNSLQTDDTAMYYC AS FDGYYGWFPY WGQGTLVTVSA

[0073] Amino acid sequence of the variable region of the mouse antibody light chain (SEQ ID NO.15):

[0074] DIQLTQTTSSLSASLGDRVTISC RAS QDISYY LN WYQQRPDGTVKLLIF YAS RLHSGVPSRFSGSGSGTDYSLTISNLEQDDIAAYFC QQGHTLPWT FGGGTKLEIK

[0075] In this context, the CDR area defined by kabat is represented in italics; the CDR area defined by IMGT is represented by underscores.

[0076] (2) The variable regions of the heavy chain and light chain of the mouse antibody were linked to the constant regions of the heavy chain and κ chain of human IgG1, respectively, and the resulting human-mouse chimeric antibody was named Y07-L2.

[0077] The IgG1 heavy chain constant region sequence (SEQ ID NO.16) is as follows:

[0078] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0079] The constant region sequence of the κ chain (SEQ ID NO.17) is as follows:

[0080] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0081] The heavy chain amino acid sequence (SEQ ID NO.18) of Y07-L2 is as follows:

[0082] QVQLKESGPGLVAPSQSLSITCTVS GFSLT SYG VH WVRQPPGKGLEWLG V IWAGGDT NLNSALMSRLGISKDNSKSQVFLKMNSLQTDDTAMYYC AS FDGYYGWFPY WGQGTLVTVSA

[0083] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0084] The light chain amino acid sequence of Y07-L2 (SEQ ID NO.19) is as follows:

[0085] DIQLTQTTSSLSASLGDRVTISC RAS QDISYY LN WYQQRPDGTVKLLIF YAS RLHS GVPSRFSGSGSGTDYSLTISNLEQDDIAAYFC QQGHTLPWT FGGGTKLEIK

[0086] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0087] In the Y07-L2 heavy chain amino acid sequence and light chain amino acid sequence, italics indicate the CDR region defined by kabat; underlined text indicates the CDR region defined by IMGT.

[0088] Example 2: Humanization of Antibodies

[0089] (1) The sequences of the heavy chain variable region and light chain variable region of the mouse antibody obtained in Example 1 were analyzed, and the complementarity determination regions (CDRs) of the heavy chain and light chain (defined by Kabat and IMGT) were obtained as follows:

[0090] Table 1. Mouse antibody heavy chain CDR sequences and light chain CDR sequences

[0091]

[0092] (2) Search the Human Germline Antibody Sequence Database (IMGT) to obtain human germline antibody sequences with high homology to the heavy / light chain variable regions of mouse antibodies. Combine their frame regions with the CDRs of the aforementioned mouse antibodies, i.e., CDR grafting. At the same time, some amino acids in the frame regions were reversed to obtain the humanized antibody Y07-C2. Its heavy chain variable region sequence and light chain variable region sequence are shown below:

[0093] Y07-C2 heavy chain variable region sequence (SEQ ID NO.20):

[0094] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGDTNLNSALMSRVGISKDTSKNQFSLKLSSVTAADTAVYYCASFDGYYGWFPYWGQGTLVTVSS

[0095] Y07-C2 light chain variable region sequence (SEQ ID NO.21):

[0096] DIQLTQSPSSSLSASVGDRVTISCRASQDISYYLNWYQQKPGKAVKLLIFYASRLHSGVPSRFSGSGSGTDYSLTISSLQPEDFATYFCQQGHTLPWTFGGGTKLEIK

[0097] Example 3: Antibody Preparation

[0098] The heavy and light chain cDNAs encoding antibodies Y07-L2 and Y07-C2 were cloned into the mammalian cell expression vector pcDNA3.4, respectively. HEK293 cells were transfected with the heavy and light chain expression plasmids at a 2:1 molar ratio using Lipofectamine 2000 transfection reagent (Invitrogen) and cultured at 37°C and 5% CO2 for 7 days. The culture supernatant was collected, and the antibody was purified from the supernatant using Protein A affinity chromatography. The purified antibody was dialyzed against PBS and then freeze-dried for concentration before being stored at -20°C.

[0099] Example 4: Binding of antibody to human OX40 protein

[0100] Coat 96-well high-affinity plates with 100 μL / well of 1 μg / mL human OX40 protein solution and incubate overnight at 4°C with shaking. The next day, wash three times with 300 μL PBST (Tween 20: 0.5‰), then block with 100 μL / well of 5% BSA / PBS for 2 hours with shaking at room temperature. Wash three times with 300 μL PBST. Prepare serial dilutions of antibody samples using PBS. Add 100 μL / well to each well of the 96-well plate and incubate for 1 hour with shaking at room temperature. Wash three times with 300 μL PBST. Prepare secondary antibody goat anti-human IgGHRP solution and add 100 μL / well to each well of the 96-well plate. Incubate for 1 hour with shaking at room temperature. Wash four times with 300 μL PBST. Add 100 μL / well of TMB and incubate for 20 min. Add 100 μL / well of 0.6N H2SO4 to stop the color development and measure OD. 450 nm.

[0101] The test results are as follows: Figure 1 As shown, the EC50 of human-mouse chimeric antibody Y07-L2 binding to human OX40 was 152.3 ng / mL, and the EC50 of humanized antibody Y07-C2 binding to human OX40 was 90.72 ng / mL, both of which were similar to the positive control KHK-4083 (KHK-4083 sequence from the IMGT database).

[0102] Heavy chain of KHK-4083 (SEQ ID NO.22):

[0103] QITLKESGPTLVKPKQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAVIYWDDHQLYSPSLKSRLTITKDTSKNQVVLTTMTNMDPVDTATYYCAHRRGAFQHWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0104] Light chain of KHK-4083 (SEQ ID NO.23):

[0105] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDSSLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0106] Example 5: Inhibitory effect of antibody on OX40 signaling pathway

[0107] This embodiment constructs a cell experimental system for detecting the function of OX40 inhibitory antibodies. Specifically, a stable transgenic cell line, HEK293T-OX40-NFκB-luciferase reporter, was constructed. When the OX40 inhibitory antibody was premixed with this stable transgenic cell line, the activation effect of OX40L on the NFκB-luciferase reporter gene on the cell line could be blocked.

[0108] Prepare an OX40 antibody gradient using PBS to achieve a final concentration of 2× working solution, and operate on ice. Collect HEK293T-OX40-NFκB-luciferase reporter cells, centrifuge, and resuspend in culture medium. Add OX40Ab and an appropriate amount of cell suspension to 384-well plates, pre-incubate for 0.5 hours, then add an appropriate concentration of OX40L and incubate in a cell culture incubator. After 5 hours, add One-Glo (Promega) assay reagent, mix well, and detect fluorescence signal using Pherastar.

[0109] like Figure 2 As shown, the IC50 values ​​of Y07-L2 and Y07-C2 in inhibiting the NFκB-luciferase reporter gene in the above experimental system were similar to those of KHK-4083.

[0110] Example 6: Yeast display and antibody affinity maturation

[0111] (1) Construction of mutant libraries

[0112] To obtain antibodies with stronger OX40 blocking function, yeast display technology was used to screen for functional enhancement mutations in the Y07-C2 antibody. Specifically, the variable region of the parent antibody Y07-C2 was converted into an scFv form and fused with the yeast Aga2 protein. The Aga2 protein can bind to the yeast surface protein Aga1, thereby displaying the scFv on the yeast cell surface.

[0113] Based on the amino acid sequence (variable region sequence of Y07-C2), a nucleotide sequence suitable for expression in yeast cells was designed, and its translated amino acid sequence is VL-(GGGGS)3-VH, as shown below (SEQ ID NO.24):

[0114] DIQLTQSPSSLSASVGDRVTISCRAS Q DISYY LNWYQQKPGKAVKLLIF YAS RLHSGVPSRFSGSGSGTDYSLTISSLQPEDFATYFC QQGHTLPWT FGGGTKLEIK

[0115] GGGGSGGGGSGGGGSGGGGS

[0116] QVQLQESGPGLVKPSETLSLTCTVS GFSLT SYG V HWVRQPPGKGLEWLG V IWAGGDT NLNSALMSRVGISKDTSKNQFSLKLSSVTAADTAVYYC ASFDGYYGWFPY WGQGTLVTVSS

[0117] In this context, the CDR region defined by kabat is indicated in italics; the CDR region defined by IMGT is indicated by underscores; and bold letters indicate amino acids for subsequent mutation screening.

[0118] The nucleotide sequence encoding the amino acid sequence VL-(GGGGS)3-VH was cloned into the laboratory-designed yeast protein display expression vector pUYD1, and the resulting plasmid was named pUYD1-Y07C2(LH). A schematic diagram of this plasmid is shown below. Figure 3 As shown in the figure. Verification showed that after transforming EBY100 Saccharomyces cerevisiae cells with this plasmid, high levels of scFv expression could be detected on the yeast surface.

[0119] The next step is to mutate the nucleotide sequence encoding VH (SEQ ID NO.20). Specifically, we will select some amino acids in the CDR region defined by Kabat or IMGT and perform point mutations to create 18 amino acids other than Cystein and Cystein itself. These will be combined with the parent VH sequence to form a VH sublibrary. Similarly, we will also construct a VL sublibrary.

[0120] (2) Yeast display and screening

[0121] Mix 10 μg of the library plasmid or the parent plasmid pUYD1-Y07C2(LH), carrier DNA, and 600 μL of competent EBY100 cells. Add PEG / LiAc solution and incubate at 30°C for 45 minutes. Then add 160 μL of DMSO solution and incubate at 42°C for another 20 minutes. Centrifuge to remove the supernatant, add 3 mL of YPD Plus resuscitation medium (Y1003, ZymoResearch), and incubate at 30°C for 90 minutes. Resuspend in 25 mL of YNB-CAA medium (B540132, A603060, Sangon Biotech (Shanghai) Co., Ltd.) containing 2% glucose in a 100 mL Erlenmeyer flask and amplify at 30°C for 3 days. OD values ​​should be between 2 and 5. Dilute to 0.5 with 25 mL of YNB-CAA medium containing 2% glucose and incubate at 20°C for 20 hours.

[0122] Yeast samples with high binding activity were screened using an antibody competition method. Specifically, 5 × 10⁻⁶ samples were taken. 6Yeast library cells induced to express antibodies were incubated with human biotin-OX40 protein at 4°C for 20 minutes, followed by centrifugation to remove the supernatant and washing twice with PBS. The cells were then competitively incubated with human OX40 protein for 72 hours, followed by centrifugation to remove the supernatant, washing twice with MACS buffer, and then incubated with Anti-Biotin MicroBeads (130-097-046, Miltenyi Biotec) at 4°C for 15 minutes. After washing twice with MACS buffer, the cells were then sorted using magnetic beads according to the MicroBeads sorting method, selecting yeast cells with strong antigen binding and good expression. The resulting high-affinity expression cells were cultured and expanded, then induced to express antibodies again, and the second round of cell magnetic bead screening was performed using the same steps.

[0123] (3) Yeast monoclonal validation

[0124] After the second round of sorting, the cells were spread on medium-agar plates (amplification medium + 1.5% Agar). After 3 days of culture, 48 clones were selected and amplified in 96-well deep-well plates. After induction of expression, they were confirmed by FACS.

[0125] Specific procedure: Take the cell lysate from each clone and add 4 × 10⁻⁶ cells. 5 Cells were seeded per well into 96-well round-bottom plates. Human His-OX40 protein was diluted to an initial concentration of 1 μM. Serial dilutions were prepared in 5-fold increments and added to each well. The plates were incubated at 4°C on a shaker for 1 hour. After washing twice with PBS, the anti-His APC antibody was added and the plates were incubated at 4°C on a shaker for 30 minutes. After washing twice with PBS, the anti-His APC reading was measured, and the EC50 of each clone binding to human protein was calculated.

[0126] All selected clones showed significantly higher upper plateaus for binding to human protein compared to the control clone (maternal scFv control), while their EC50 values ​​were significantly lower than those of the control clone, confirming the reliability of the selection results. Typical results of FACS validation experiments for monoclonal yeast cells are shown below. Figure 4 As shown.

[0127] (4) Sequencing

[0128] Specific PCR primers were designed to amplify the scFv region of the transformed plasmid in yeast, and the amplified fragment was sequenced. The mutation sites were obtained by comparing it with the scFv sequence of the maternal antibody. The results are summarized in Table 2.

[0129] Table 2 Summary of yeast scFv mutation sites

[0130]

[0131] Note: The mutation locations in Table 2 refer to the locations defined using the Kabat rules. The bolded and underlined parts in the table are mutations.

[0132] Example 7: ELISA Competition Experiment

[0133] Since conventional ELISA binding assays cannot effectively distinguish differences in antigen-binding ability between high-affinity antibodies, this embodiment designs an ELISA competition assay to compare the differences in antigen-binding ability between mutant antibodies and maternal antibodies. The specific method is as follows: A 1 μg / mL human OX40 protein solution was coated onto a 96-well high-affinity plate at 100 μL / well and incubated overnight at 4°C with shaking. The next day, the plate was washed three times with 300 μL PBST (Tween 20: 0.5‰), then blocked with 100 μL / well of 5% BSA / PBS for 1 hour with shaking at room temperature. The plate was washed three times with 300 μL PBST. A serial dilution solution of the antibody sample was prepared using PBS. This solution was added to a 96-well plate at 100 μL / well and incubated for 1 hour with shaking at room temperature. The plate was washed three times with 300 μL PBST. The biotin-labeled Y07-C2 was diluted to 0.5 μg / mL with PBS, and then added to a 96-well plate at 100 μL / well with shaking at room temperature for 1 hour. Wash three times with 300 μL PBST. Prepare secondary antibody HRP-labeled streptavidin solution and add 100 μL / well to a 96-well plate, incubate at room temperature with shaking for 30 minutes. Wash four times with 300 μL PBST. Add 100 μL / well TMB and develop color for 3 minutes. Add 100 μL / well 0.6N H2SO4 to stop the color development and detect OD at 450 nm.

[0134] (1) Expression and evaluation of single mutant antibodies

[0135] First, antibodies containing only one mutation in either the heavy or light chain were expressed. Based on the sequencing results in Example 6, a total of eight different mutant antibodies (Y07-C2MH1~5, Y07-C2ML1~3) were designed, and their variable region sequence combinations and parent antibody sequences are shown in Table 3. Antibody expression was performed using the method described in Example 3.

[0136] Table 3. Light and heavy chain sequences of mutant antibodies

[0137]

[0138] The constant region sequences of all antibodies are as follows:

[0139] Heavy chain constant region (SEQ ID NO:16):ASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0140] Light chain constant region (SEQ ID NO:17):

[0141] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0142] The mutant antibody and the maternal antibody were subjected to an ELISA competition assay to compare their binding affinity to human OX40. The results are as follows: Figure 5 As shown, different mutations can significantly affect the competitive ability of antibodies to bind to the parent antibody Y07-C2 and human OX40 protein. Under the experimental conditions, if the competitive ability of Biotin-Y07-C2 to bind to itself (Y07-C2) and human OX40 at a concentration of 10 µg / mL is defined as 100%, we found that heavy chain mutations MH2 and MH4 and light chain mutations ML1 and ML3 can all improve the antigen competitive binding ability of mutant antibodies, with MH4, ML1, and ML2 showing the largest increases.

[0143] (2) Expression and evaluation of combined mutant antibodies

[0144] Furthermore, MH4 was combined with ML1, ML2, and ML3 to explore whether different mutations could synergistically enhance the competitive binding ability of the antibody to OX40. A total of six different mutant antibodies were designed, and their variable region sequence combinations and parent antibody sequences are shown in Table 4.

[0145] Table 4. Light and heavy chain sequences of combined mutant antibodies

[0146]

[0147] Experimental results are as follows Figure 6 As shown, under the experimental conditions, the ability of Biotin-Y07-C2 to compete with itself (Y07-C2) for binding to human OX40 at a concentration of 10 µg / mL was defined as 100%. Different combinations of heavy chain mutation MH4 and light chain mutations ML1, ML2, and ML3 can all improve the antigen competitive binding ability of mutant antibodies.

[0148] Example 8: Evaluation of the inhibitory activity of some combined mutant antibodies on the OX40 signaling pathway

[0149] (1) OX40 reporter gene experiment

[0150] Following the method described in Example 5, the signaling pathway blocking activities of the control antibody KHK-4083, the maternal antibody Y07-C2, and the mutant antibodies Y07-C2MH4 and Y07-C2MH4L3 were evaluated.

[0151] The results are as follows Figure 7 As shown, under the experimental conditions, Y07-C2MH4L3 exhibits significantly stronger OX40 signaling pathway blocking activity than the other three antibodies.

[0152] (2) Ligand binding blocking experiment

[0153] First, a stable CHO cell line overexpressing OX40 was constructed. CHO-OX40 cells were collected by digestion, centrifuged, and resuspended in PBS. Antibody was serially diluted with PBS, and 50 μL of cell suspension was added to each well of a 96-well round-bottom plate, followed by 50 μL of serially diluted antibody solution. The plates were incubated at 4°C with shaking for 2 h. Cells were then washed three times with PBS, and 100 μL of 0.5 μg / mL human OX40L-his protein was added to each well. The plates were incubated at 4°C with shaking for 30 min. Cells were then washed three times with PBS, and 100 μL of APC anti-his antibody was added. The plates were incubated at 4°C with shaking for 30 min. Cells were washed three times with PBS, resuspended in 400 μL of FACS buffer, and the proportion and signal intensity of APC-labeled cells were detected by flow cytometry.

[0154] The results are as follows Figure 8 As shown, under the experimental conditions, Y07-C2MH4 and Y07-C2MH4L3 exhibited significantly stronger activity in blocking the binding of OX40L to OX40 than the other three antibodies (antibodies KHK-4083, GBR830, and Y07-C2MH4L2 / 3).

[0155] Example 9: Antibody ADCC Experiment

[0156] ADCC activity may be an important mechanism of action for antibodies targeting the OX40 protein. KHK-4083, used in clinical trials, employs a special defucosylation process, which significantly enhances its ADCC activity. Therefore, the ADCC activity of the partially mutant antibody was evaluated using a Jurkat cell reporter gene assay and compared with clinical antibodies KHK-4083 and GBR830.

[0157] One day in advance, CHO-OX40 cells were seeded onto 96-well plates at a density of 5000 cells per well and cultured overnight. The next day, antibody was serially diluted using 1640 medium to prepare a final concentration of 2× working solution. After removing the original culture medium, 15 μl of the prepared antibody working solution was added to each well of the 96-well plate and incubated in a cell culture incubator for 1 hour. Jurkat-NFAT-CD16A effector cells were prepared by adding a cell suspension at a density of 50,000 cells / well / 15 μl to each well of the 96-well plate and incubated in a cell culture incubator for 1 hour. The cells were then detected using the One-Glo assay kit.

[0158] like Figure 9 As shown, KHK-4083 had a maximum ADCC activation activity of 100% in this experiment. The ADCC activities of the mutant antibodies Y07-C2MH4, Y07-C2MH4L3 and Y07-C2MH4L2 / 3 were significantly stronger than those of KHK-4083 and GBR830, with the maximum activation plateau reaching 196.0%, 190.7% and 187.7%, respectively.

[0159] Example 10: Antigen-antibody affinity detection

[0160] The affinity of antigen-antibody binding was detected using single-concentration SPR (Surface Plasmon Resonance) technology. In simple terms, antibody was captured by incubating a 2 μg / mL antibody with a protein A sensor chip (Cytiva, Cat#29127556).

[0161] During the antigen binding phase, 200 nM human OX40 protein was used as the mobile phase to bind with the antibody captured on the sensor chip until the relative response (RU) reading reached 330-750 RU. During the dissociation phase, elution was performed continuously with HBS-EP+Buffer for approximately 120 seconds.

[0162] The binding of human OX40 to antibodies on the sensor chip was quantitatively detected using a Biacore 8K (GE Healthcare), and the results are shown in Table 5.

[0163] Table 5 Affinity and kinetic data between Y07-C2 and OX40

[0164]

[0165] The data above show that compared to Y07-C2, Y07-C2MH4 and Y07-C2MH4L3 exhibit increased affinity for human OX40. However, this increase in affinity does not necessarily correlate one-to-one with the antibody's signaling pathway blocking activity. For example, although Y07-C2MH4 showed stronger antigen-binding ability in the ELISA competition assay, consistent with the affinity test results, its signaling pathway blocking activity in Example 9 was not stronger than that of the parent molecule Y07-C2. This suggests that the stronger inhibitory effect of the mutant antibody Y07-C2MH4L3 may be related to the conformation of antibody-antigen binding, rather than a simple increase in affinity.

[0166] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. An inhibitory antibody against human OX40 or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment comprises: a heavy chain variable region sequence as shown in SEQ ID NO.14, and a light chain variable region sequence as shown in SEQ ID NO.15; Alternatively, the antibody or its antigen-binding fragment may comprise: a heavy chain variable region sequence as shown in SEQ ID NO.20, or a light chain variable region sequence as shown in SEQ ID NO.21; Alternatively, the antibody or its antigen-binding fragment may comprise any one of the heavy chain variable region sequences shown in SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, or SEQ ID NO.29, or the light chain variable region sequence shown in SEQ ID NO.

21. Alternatively, the antibody or its antigen-binding fragment may comprise: a heavy chain variable region sequence as shown in SEQ ID NO. 20, or any one of a light chain variable region sequences as shown in SEQ ID NO. 30, SEQ ID NO. 31, or SEQ ID NO. 32; Alternatively, the antibody or its antigen-binding fragment may comprise: a heavy chain variable region sequence as shown in SEQ ID NO.28, or any one of the light chain variable region sequences shown in SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, or SEQ ID NO.

11.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further comprises a heavy chain constant region as shown in SEQ ID NO. 16 and a light chain constant region as shown in SEQ ID NO.

17.

3. A nucleic acid molecule, characterized in that, Encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-2.

4. A method for preparing the antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, include: The nucleotide sequences encoding the heavy chain and light chain of the antibody or its antigen-binding fragment as described in claim 1 or 2 are cloned into mammalian cell expression vectors to construct heavy chain expression plasmids and light chain expression plasmids, respectively. The heavy chain expression plasmid and the light chain expression plasmid were transfected into HEK293 cells at a molar ratio of 2:1 using a transfection reagent. After culturing for a period of time, the antibody or its antigen-binding fragment was recovered from the culture supernatant.

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