Antibodies for binding to human GP130 and uses thereof

Abstract

The invention relates to an antibody for binding with human GP130 and application thereof. In a heavy chain variable region of the antibody, VHCDR1 is selected from an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; the VHCDR2 is selected from an amino acid sequence as shown in SEQ ID NO: 3; the VHCDR3 is selected from an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 7; the antibodies are capable of specifically binding to human GP130 to impede binding of human IL-6 to the human IL-6 receptor. According to the scheme provided by the invention, the human GP130 can be bound in a targeted manner, and the binding of IL-6 and a receptor thereof is specifically blocked, so that abnormal immune response is regulated, and inflammation signals in a pathological process are inhibited.

Description

Technical Field

[0001] The present application relates to the field of antibody technology, and in particular to an antibody for binding to human GP130 and its application. Background Art

[0002] Human IL-6 is a pleiotropic cytokine produced by a variety of cells (such as T lymphocytes, B lymphocytes, monocytes, endothelial cells, etc.) and belongs to the interleukin family. Human IL-6 plays a key role in inflammation and immune response. Abnormal expression of human IL-6 is associated with the occurrence and development of a variety of diseases, such as autoimmune diseases, inflammatory diseases, infectious diseases, and tumors. Human IL6 receptor is a specific receptor for human IL-6. By binding to human IL-6, it activates multiple signaling pathways (such as JAK / STAT, MAPK, and PI3K, etc.). These signaling pathways can affect the gene expression and metabolism of cells, thereby regulating the biological functions of cells. Human IL6 receptor plays a key role in mediating the biological effects of human IL-6.

[0003] The human IL6 receptor is generally IL6R, which is composed of an α subunit (also known as IL6RA) and a β subunit (also known as IL6ST, GP130, IL6β). Among them, the α subunit is the main functional subunit of the receptor, while the β subunit is the signal transduction subunit. In the classical signal transduction pathway of human IL-6, human IL-6 first binds to the membrane-bound IL-6RA, and then binds to the signal transduction molecule GP130 to form a complex and activate the downstream signal transduction pathway.

[0004] In the field of drug development, intervention strategies targeting the IL6-IL6R signaling pathway have been widely studied, especially in the treatment of autoimmune diseases and cancer. For example, Tocilizumab and Sarilumab are monoclonal antibodies targeting IL6RA in the human IL6 receptor, which have been clinically used to treat rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), and COVID-19 cytokine storm syndrome. In addition, Siltuximab is a monoclonal antibody targeting human IL-6 protein that has been approved for the treatment of multicentric Castleman disease (MCD), etc. Abnormal activation of the IL6 signaling pathway is also associated with the occurrence and development of a variety of diseases, including cancer and autoimmune diseases. Antibody drugs targeting human GP130 can inhibit the pathological processes of these diseases by specifically blocking the binding of human IL-6 to its receptor. These antibodies can be used as potential drugs for regulating immune responses and inhibiting inflammatory signals in pathological processes. However, monoclonal antibody drugs targeting IL6ST / gp130 are currently rare.

[0005] Therefore, the development of monoclonal antibodies that can specifically regulate the human GP130 pathway also has important therapeutic prospects and development necessity. Summary of the invention

[0006] In order to solve or partially solve the problems existing in the related art, the present application provides an antibody for binding to human GP130 and its application, which can target and bind to human GP130 and specifically block the binding of IL-6 to its receptor so as to regulate abnormal immune responses and inhibit inflammatory signals in pathological processes.

[0007] In a first aspect, the present application provides an antibody for binding to human GP130, wherein the antibody has a V H CDR1, V H CDR2 and V H The heavy chain variable region and the light chain variable region of CDR3, wherein:

[0008] The V H CDR1 is selected from the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence having at least 90% homology to SEQ ID NO: 1 or SEQ ID NO: 2;

[0009] The V H CDR2 is selected from the amino acid sequence shown in SEQ ID NO: 3 or a sequence having at least 90% homology to SEQ ID NO: 3;

[0010] The V H CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or a sequence having at least 90% homology to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

[0011] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7;

[0012] The antibody can specifically bind to human GP130 to prevent human IL-6 from binding to human IL-6 receptor.

[0013] In some embodiments, the antibody is a monoclonal antibody. Preferably, the antibody comprises a heavy chain constant region and a light chain constant region.

[0014] In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:8.

[0015] In some embodiments, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:9.

[0016] In some embodiments, the affinity of the antibody to human GP130 reaches nM level or sub-nM level.

[0017] The second aspect of the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain variable region and the light chain variable region contained in the antibody described in any one of the first aspect.

[0018] The third aspect of the present application provides an expression vector comprising the nucleic acid molecule described in the second aspect.

[0019] The fourth aspect of the present application provides a recombinant cell, which comprises the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect.

[0020] The fifth aspect of the present application provides a pharmaceutical composition, which comprises the antibody described in any one of the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect and / or the recombinant cell described in the fourth aspect, and optional pharmaceutically acceptable excipients.

[0021] In the sixth aspect of the present application, there is provided a use of the antibody described in any one of the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect and / or the pharmaceutical composition described in the fifth aspect in the preparation of a medicament for preventing, treating and / or ameliorating a disease or condition, wherein the disease or condition is related to IL-6 expression (including overexpression).

[0022] The technical solution provided by this application may have the following beneficial effects:

[0023] The antibody of the present application can specifically bind to human GP130, thereby occupying the binding site of human GP130 and IL-6, hindering the binding of IL-6 to human GP130, and thus blocking the signaling pathway of IL-6 and its receptor. The antibody of the present application can be used as a potential drug for abnormal activation of the IL6-GP130 signaling pathway, regulating immune responses and inhibiting inflammatory signals in pathological processes.

[0024] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and other objects, features and advantages of the present application will become more apparent by describing in more detail exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0026] Figure 1 The figure is an absorbance curve of the antibody to be tested after binding to human GP130 at different concentrations;

[0027] Figure 2 The figure is an absorbance curve of the antibody to be tested after binding to human GP130 at different concentrations;

[0028] Figure 3 The figure is an absorbance curve diagram of the blocking effect of the tested antibody on the binding of human GP130 to LIP at different concentrations;

[0029] Figure 4 The figure is an absorbance curve diagram of the blocking effect of the tested antibody on the binding of human GP130 to LIP at different concentrations;

[0030] Figure 5 The figure is an absorbance curve diagram of the tested antibody after inhibiting the phosphorylation of STAT-3 induced by human IL-6 at different concentrations;

[0031] Figure 6 This is the electron microscopy image of the complex structure after the positive reference 2 binds to human GP130;

[0032] Figure 7 This is the electron micrograph of the complex structure after the target antibody C082 binds to human GP130;

[0033] Figure 8 This is an electron micrograph of the complex structure of the target antibody A084 and human GP130;

[0034] Fig. 9 This is the electron micrograph of the complex structure after the target antibody A088 binds to human GP130;

[0035] Fig.10 This is an electron micrograph of the complex of target antibody C082 and human GP130 after freezing. DETAILED DESCRIPTION

[0036] To make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0037] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any explicitly excluded limits in the specified ranges. Where a specified range includes one or two limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0038] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials or equivalent methods and materials described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0039] Terminology explanation:

[0040] "Antibody" is synonymous with immunoglobulin and is understood as generally understood in the art. The basic antibody structural unit is a tetramer. Each tetramer is composed of 2 identical pairs of polypeptide chains, each pair having a light chain and a heavy chain. The amino terminal portion of each chain contains a variable region of about 100 to 120 or more amino acids. The carboxyl terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa (κ) and lambda (λ). Heavy chains are classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. The heavy chain contains a heavy chain constant region (C H ) and the heavy chain variable region (V H The light chain contains a light chain constant region (C L ) and light chain variable region (V L ). H and V L Each of them contains highly variable complementarity determining regions (CDRs) and interspersed framework regions (FRs). H and V L From the amino terminus to the carboxyl terminus, it includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in sequence.

[0041] The variable region (V H and V L ) form antigen binding sites respectively; therefore, a complete IgG antibody, for example, has 2 binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are identical. The term "variable" means that some parts of the variable domains vary greatly in sequence between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed throughout the variable domain of an antibody, but is concentrated in the CDR region.

[0042] The present application provides an antibody for binding to human GP130, the antibody having a V H CDR1, V H CDR2 and V H The heavy chain variable region and the light chain variable region of CDR3, wherein:

[0043] V HCDR1 is selected from the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence having at least 90% homology to SEQ ID NO: 1 or SEQ ID NO: 2; V H CDR2 is selected from the amino acid sequence shown in SEQ ID NO: 3 or a sequence having at least 90% homology to SEQ ID NO: 3; V H The CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or a sequence having at least 90% homology to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7. The amino acid sequence of the light chain variable region is a human sequence.

[0044] As shown in Table 1, V H CDR1, V H CDR2 and V H The amino acid sequences of the heavy chain variable region and light chain variable region of CDR3 are shown in Table 1. H CDR1, V H CDR2 and V H The amino acid sequences of the heavy chain variable region and the light chain variable region of CDR3 may also be sequences having at least 90% homology with the corresponding sequences in Table 1.

[0045] Table 1

[0046]

[0047]

[0048] In Table 1, V L The amino acid sequence corresponding to the variable region includes FR1- CDR1 -FR2- CDR2 -FR3- CDR3 The light chain variable region of the present application is selected from the common light chain variable region of humans, that is, different antibodies can use light chain variable regions with the same amino acid sequence. The known human amino acid sequence provides a stable antibody structure and is more conducive to the design of dual antibodies.

[0049] The antibody of the present application can specifically bind to human GP130 through the above-mentioned heavy chain variable region and light chain variable region.

[0050] In some embodiments, the heavy chain variable region of the present application also includes V H FR1, V H FR2 and V HFR3 and V H FR4. V H FR1 is selected from the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9 or a sequence having at least 90% homology to SEQ ID NO: 8 or SEQ ID NO: 9. H FR2 is selected from SEQ ID NO: 10 or a sequence having at least 90% homology to SEQ ID NO: 10. H FR3 is selected from SEQ ID NO: 11 or a sequence having at least 90% homology to SEQ ID NO: 11. H FR4 is selected from SEQ ID NO: 12 or a sequence having at least 90% homology to SEQ ID NO: 12. In some embodiments, V H FR1, V H FR2 and V H FR3 and V H The amino acid sequence of FR4 is shown in Table 2 below. H FR1, V H FR2 and V H FR3 and V H FR4 may also be a sequence having at least 90% homology with the corresponding sequence in Table 2.

[0051] Table 2

[0052]

[0053] In some embodiments, the heavy chain variable region of the antibody of the present application comprises an amino acid sequence as shown in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, or a sequence having at least 90% homology with SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, as shown in Table 3 below.

[0054] Table 3

[0055]

[0056] In the amino acid sequence corresponding to the heavy chain variable region shown in Table 3, FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -The amino acid sequence number corresponding to FR4.

[0057] Furthermore, the antibody of the present application is a monoclonal antibody. In some embodiments, the antibody of the present application further comprises a heavy chain constant region and a light chain constant region. In some specific embodiments, the heavy chain constant region is human IgG1 type or human IgG2 type; the light chain constant region is kappa type.

[0058] As shown in Table 4 below, in some embodiments, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 16, or a sequence having at least 90% homology to SEQ ID NO: 16. The light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 17, or a sequence having at least 90% homology to SEQ ID NO: 17.

[0059] Table 4

[0060]

[0061]

[0062] To make the present invention easier to understand, the preparation of the antibodies of the present application and related performance tests are further described in detail below in conjunction with the examples. These examples are only illustrative and are not limited to the scope of application of the present application. The raw materials or components used in the present application can be obtained by commercial channels or conventional methods unless otherwise specified.

[0063] Example 1 Binding human GP130 and using AI to calculate and design antibodies and phage display screening

[0064] The traditional method of antibody design is to immunize and isolate in animals, and there is currently no antibody designed for a specific epitope that binds to human GP130 (Uniport ID: P40189) and human IL-6 (Uniport ID: P05231). In this embodiment, fully human co-antibody light chain mice are used to immunize various antigens to obtain an antibody library composed of fully human and co-light chain antibody sequences with a diversity of millions. Combined with the target epitope corresponding to human GP130, representative antibody sequences are screened out in the antibody library, and the binding probability of the CDR region of the antibody sequence to the target epitope is predicted, and the antibody sequence with a higher potential for binding to the target epitope is obtained by binding probability screening as a template antibody sequence.

[0065] Based on the masking mechanism, the residues of all or part of the heavy chain variable region of the template antibody sequence are masked, and the types of the masked residues at each site are predicted based on the human antibody sequence to generate candidate antibody sequences. According to the preset antigen-antibody affinity prediction model, the affinity between human GP130 and the candidate antibody sequence is predicted, and the antibody sequence whose affinity prediction score meets the preset conditions is selected as the target antibody sequence.

[0066] In this embodiment, based on the specific epitope of human GP130, an antibody sequence with high affinity and high specificity is designed from scratch by relying on a computer model, which provides important support for the subsequent development of antibody drugs. It not only greatly saves experimental costs, but is also not limited by experimental conditions. During the generation process, it can explore all possible sequence conformations, set parameters, and artificially control the direction and goal of antibody generation, thereby improving the efficiency and quality of antibody engineering and providing more drug-making possibilities for antibody molecules.

[0067] In this embodiment, the amino acid sequences of the heavy chain variable regions of multiple target antibody sequences were synthesized and spliced ​​to construct a single-chain variable fragment (scFv) antibody library of a common light chain. That is, each antibody has the same light chain variable region (SEQ ID NO: 7) and the corresponding heavy chain variable region. Then, an antibody phage display library was constructed, and positive phage clones with binding activity were screened using human GP130 protein, and single-chain variable fragment (scFv) antibodies as shown in Table 3 were screened.

[0068] Example 2 Expression and purification of antibodies binding to human GP130

[0069] After obtaining the antibody heavy chain sequences of the positive clones in Table 3 above according to Example 1, the heavy chain sequences and co-light chain sequences of each antibody were cloned into the pcDNA3.4 expression vector containing the human IgG1 subtype constant region and the Kappa light chain constant region (as shown in Table 4 above) in this example. Subsequently, these recombinant expression plasmids were transferred to CHO cells by transfection. After the expression process was completed, the culture supernatant was treated by Protein A affinity chromatography technology to separate and purify the three corresponding target antibodies A084, A088 and C082.

[0070] The target antibody of this embodiment has the heavy chain variable region shown in Table 3, the light chain variable region shown in Table 1, and the IgG1 heavy constant region and Kappa light chain constant region shown in Table 4. It can be understood that the target antibody of the present application has a human light chain variable region, a human IgG1 heavy constant region and a Kappa light chain constant region, and a heavy chain variable region predicted based on the human antibody sequence, so that the target antibody as a whole is humanized.

[0071] Example 3 Detection of Antibody Binding Activity to IL6ST / gp130

[0072] In this example, the indirect ELISA method was used to evaluate the binding activity of the target antibody to the human gp130 protein.

[0073] The three target antibodies of Example 2 were used as test antibodies and were tested for binding activity with human gp130 protein. In addition, a known mouse antibody capable of binding to human GP130 was selected as positive reference 1 (benchmark 1), which was selected from patent JP2019147786A, and its heavy chain variable region amino acid sequence and light chain variable region amino acid sequence and the corresponding SEQ ID NO 15 and SEQ ID NO 20 in the disclosed patent are shown in Table 5 below. A 1% PBSM solution was used as a blank reference (BC). IPI antibody was used as a negative reference (NC).

[0074] Table 5

[0075]

[0076] The above three antibodies to be tested, positive reference 1, blank reference, and negative reference were respectively used to complete the experiment according to the following steps, and the corresponding OD values ​​and EC50 values ​​were obtained. The specific steps are as follows:

[0077] 3.1 Human GP130 (Lot: ILT-H52H2, Biopsies Biotech) protein was selected and added to the ELISA microplate at a concentration of 2 μg / mL, with 30 μL per well for coating and fixation.

[0078] 3.2 Use 100 μL of 5% PBSM solution to block to prevent non-specific binding of antibodies.

[0079] 3.3 Use 1% PBSM solution to dilute the same test antibody in a gradient manner to obtain test antibodies with concentrations of 0.00015μg / mL, 0.00137μg / mL, 0.00412μg / mL, 0.01235μg / mL, 0.03704μg / mL, 0.1111μg / mL, 0.3333μg / mL and 3μg / mL, respectively, and add the test antibodies of various concentrations to different microplates to allow them to bind to the coated antigen.

[0080] 3.4 After washing three times with PBST to remove unbound test antibodies, add 30 μL of Goat-Anti-human-IgG-Fc-HRP (Lot: ab97225 Abcam) diluted 1:8000 with 1% PBSM solution and react at room temperature for 50 minutes.

[0081] 3.5 Wash with PBST for 6 times, add TMB colorimetric solution and 2M stop solution.

[0082] 3.6 Read the OD value of the microplate using a spectrophotometer, and use multi-parameter fitting to obtain the corresponding EC50 value of the antibody to be tested.

[0083] like Figure 1 and Figure 2 From the coordinate system, it can be seen that based on the fixed amount of human GP130, as the concentration of each analyte added increases, the binding signal (OD450 value) becomes stronger, but after reaching the saturation amount of antigen binding, the signal will not increase with the increase of concentration. However, at the same concentration of each analyte, the faster the saturation amount of antigen binding is reached, that is, the smaller the EC50, the better the binding effect of the corresponding analyte with human GP130.

[0084] Example 4 Detection of affinity between antibody and human GP130

[0085] In this example, the biofilm interferometry (BLI) technology of the Fortebio Octet system was used to detect the affinity of the three target antibodies in Example 2 and the above-mentioned positive reference 1 (benchmark 1) to the human GP130 protein. The specific method is as follows:

[0086] In the Octet system, the AHC chip is used to capture the antibody to be tested as the stationary phase, and the analyte is human GP130 protein. The binding and dissociation process of the antibody to be tested and the GP130 protein is recorded in real time, and the affinity constant KD value, ka value, kd value, and R2 value are calculated based on the kinetic curve fitting. The calculation results are shown in Table 6 below.

[0087] Table 6

[0088] Antibody KD(M) ka(1 / Ms) kd(1 / s) R2 Benchmark 1 8.56E-07 1.45E+04 1.24E-02 0.991 A084 5.87E-09 9.68E+04 5.68E-04 0.9589 A088 3.97E-09 1.70E+05 6.75E-04 0.9334 C082 2.82E-08 2.09E+05 5.91E-03 0.9207

[0089] The affinity constant KD value refers to the equilibrium dissociation constant, which is a parameter that measures the strength of intermolecular interactions. The smaller the KD value, the stronger the binding affinity between molecules, and the larger the KD value, the weaker the binding. The Ka value refers to the binding constant. The larger the Ka value, the stronger the binding ability between molecules. The kd value refers to the dissociation rate constant, which describes the rate of intermolecular dissociation. The larger the kd value, the faster the dissociation process. The R2 value represents the degree of fitting of the data. The range of the R2 value is between 0 and 1. The closer the R2 value is to 1, the better the data fitting effect. After testing, the affinity KD values ​​of the three antibodies of the present application can reach the nM level or sub-nM level, which is higher than the positive reference 1 antibody of the control.

[0090] Example 5 Antibodies blocking the activity of IL6ST / gp130 and its ligand LIF protein

[0091] In this embodiment, the competitive ELISA method is used to evaluate three target antibodies, positive reference 1 (benchmark 1) and positive reference 2 (benchmark 2), as the test antibodies, to block the activity of human GP130 protein and its ligand LIF. A 1% PBSM solution is used as a blank reference (BC). IPI antibody is used as a negative reference (NC). Among them, LIF and IL-6 can both bind to GP130 and the binding epitopes are close, and the test results have reference value, so LIF is used instead of IL-P for testing.

[0092] The positive reference 2 is selected from patent JP2018153172A, and its heavy chain variable region amino acid sequence and light chain variable region amino acid sequence and the corresponding SEQ ID NO 16 and SEQ ID NO 21 in the disclosed patent are shown in Table 7 below.

[0093] Table 7

[0094]

[0095] The specific steps are as follows:

[0096] 5.1 Add human GP130 protein at a concentration of 2 μg / mL into the ELISA microplate for coating and fixation.

[0097] 5.2 Use 5% PBSM solution to block to prevent non-specific binding of the antibody to be tested.

[0098] 5.3 After the same antibody to be tested was diluted into various concentrations in a gradient manner, the antibody to be tested with concentrations of 0.00457μg / mL, 0.01372μg / mL, 0.04115μg / mL, 0.12346μg / mL, 0.37037μg / mL, 1.11111μg / mL, 3.33333μg / mL and 10μg / mL and 1μg / mL biotin-labeled LIF (Lot: LIF-H52H3, Biopsies Biotech) protein were added to the microplate to compete with the coated antigen for binding.

[0099] 5.4 After washing to remove unbound test antibodies, add 30 μL of NeutrAvidin-HRP (Lot: 31001, Thermo Fisher) diluted 1:2000 with 1XPBS solution and react at room temperature for 50 minutes.

[0100] 5.5 Wash with PBST for 6 times, add TMB colorimetric solution and 2M stop solution.

[0101] 5.6 Read the OD value of the microplate using a spectrophotometer and use multi-parameter fitting to obtain the IC50 value of the antibody to be tested. The distribution curve of concentration and absorbance OD value and IC50 value are shown in Figure 3 and Figure 4 shown.

[0102] Competitive ELISA assay showed that antibodies A084, A088 and C082 could block the binding of human GP130 and its ligand LIF, with the corresponding IC50 reaching 0.21μg / mL, 0.18μg / mL and 0.06μg / mL, respectively.

[0103] Example 6 Antibodies inhibit IL6-induced STAT-3 phosphorylation activity in NCI-H226 cells

[0104] In this example, three target antibodies and positive reference 2 were used as test antibodies, and IgG1 was used as a negative reference antibody to test the inhibition of human IL6-induced STAT-3 phosphorylation activity, and the cellular functional activity of the antibody was judged to determine whether the antibody could inhibit IL-6-induced STAT3 phosphorylation.

[0105] The specific steps are as follows:

[0106] 6.1 NCI-H226 cells were plated and cultured at a density of 2E4 / well.

[0107] 6.2 0.137nM, 1.372nM, 4.115nM, 12.346nM, 37.037nM, 111.111nM, 333.333nM and 1000nM of the test antibody and recombinant 25nM hyper-IL6 (CHO expression, Sanyou Biopharmaceuticals) protein 5 were added to the microplate for culture treatment, and cells treated with only 25nM Hyper-IL6 and untreated cells were used as controls, i.e. Figure 5 Hyper-IL6 and cell only as shown.

[0108] 6.3 Lyse cells and harvest supernatant.

[0109] 6.4 Use STAT-3phosphorylation kit (Lot: DYC4607B-5, R&D Systems) to detect the phosphorylation level of STAT-3 and obtain the inhibitory activity IC50 value of the antibody to be tested.

[0110] After testing, combined Figure 5As shown, the STAT-3 phosphorylation level of untreated NCI-H226 cells is low (cellonly), and Hyper-IL6 induction can cause the phosphorylation level of NCI-H226 cells to increase (hyper-IL6). The tested antibodies C082, A084, A088 and Benchmark 2 can inhibit the STAT-3 phosphorylation activity induced by IL6, among which the inhibitory activity IC50 values ​​of A084 and A088 are 159.3nM and 68.3nM, respectively.

[0111] Example 7 Negative staining electron microscopy of target antibody and human GP130

[0112] In this example, negative staining electron microscopy was used to observe the binding modes and positions of three target antibodies and positive reference 2 as the test antibody with human GP130 protein.

[0113] 7.1 Prepare a mixed sample of human GP130 protein and the Fab fragment of the antibody to be tested at a molar ratio of 2:1 or 4:1.

[0114] 7.2 Discharge the copper grid carrying the carbon film at 15 mA for 90 seconds.

[0115] 7.3 Add the mixed sample prepared in 7.1 onto the grid, rinse it quickly with ultrapure water to remove impurities, and remove excess water by blotting.

[0116] 7.4 Use 0.75% uranyl formate staining solution to stain the mixed sample in 7.3 to enhance the contrast of the image.

[0117] 7.5 The grid was observed using a Talos L120C electron microscope and images were collected using high-resolution imaging techniques with a pixel size of To ensure a clear structural image of the antibody to be tested-human GP130 protein complex.

[0118] Table 8

[0119]

[0120] As shown in Table 8 above, and in combination with the attached Figure 6-9 It can be seen that the 2D schematic diagram of the test antibody-human GP130 binding was selected from each electron micrograph and 3D reconstruction was performed. It can be seen that antibodies C082, A084 and A088 all bind to the middle position of the human GP130 protein, which overlaps with the epitope of human IL-6 binding to human GP130, further illustrating that antibodies C082, A084 and A088 bind to the target epitope, and can then be used to hinder the binding of human IL-6 to human GP130.

[0121] Example 8 Cryo-EM analysis of target antibody and human GP130

[0122] In this example, cryo-electron microscopy was used to analyze the fine structure of the complex between antibody C082 and human GP130 protein.

[0123] 8.1, mixing the Fab fragment of the target antibody and the human GP130 protein at a molar ratio of 4:1 to prepare a mixed sample required for cryo-electron microscopy.

[0124] 8.2 Use a porous carbon film grid to support the sample and drop the mixed sample onto the grid.

[0125] 8.3 Using Virtobot, at 100% humidity and 4°C, the excess sample was quickly blotted with filter paper and then placed in liquid ethane to achieve rapid freezing to preserve its native structure.

[0126] 8.4 Use Glacios electron microscope to conduct preliminary observation of frozen samples to ensure the quality and freezing state of the samples.

[0127] 8.5 The data were collected using a Krios electron microscope at a magnification of 165k, with an image pixel size of

[0128] 8.6 Interception Fig.10 The binding site of the target antibody C082 and the human GP130 protein complex ( Fig.10 The area covered by the red circle in the upper right corner) was obtained after data processing and reconstruction, and the high-resolution three-dimensional structure of the target antibody C082 and human GP130 protein complex was obtained with a resolution of 3.63 angstroms ( Fig.10 The three-dimensional structure shown below).

[0129] like Fig.10 The results showed that the binding epitope of antibody C082 had a significant overlap with the known IL6-GP130 complex (PDB ID: 1I1R), indicating that C082 has the potential to inhibit IL6 signaling. This structural overlap provides direct evidence for understanding the inhibitory mechanism of antibody C082.

[0130] Based on the above examples 1 to 8, it can be seen that the target antibody of the present application has a human antibody sequence and does not need to be humanized, and experiments have shown that it has binding performance and excellent affinity with human GP130, and its binding epitope is on the binding interface of IL6-GP130, which can block the binding of human GP130 with the ligand LIP, and inhibit IL6 from inducing STAT-3 phosphorylation on NCI-H226 cells. In summary, the target antibody of the present application can be used as a potential drug in the human body, thereby binding to GP130 in the human body, specifically preventing human IL-6 from binding to human GP130 in vivo, blocking the signaling pathway of human IL-6, and achieving the effect of regulating abnormal immune responses and inhibiting inflammatory signals in pathological processes.

[0131] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0132] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application is described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the present application may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same functions.

Claims

1. An antibody for binding to human GP130, characterized in that The antibody has a V H CDR1, V H CDR2 and V H The heavy chain variable region and the light chain variable region of CDR3, wherein: The V H CDR1 is selected from the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence having at least 90% homology to SEQ ID NO: 1 or SEQ ID NO: 2; The V H CDR2 is selected from the amino acid sequence shown in SEQ ID NO: 3 or a sequence having at least 90% homology to SEQ ID NO: 3; The V H CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or a sequence having at least 90% homology to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7; The antibody can specifically bind to human GP130 to prevent human IL-6 from binding to human IL-6 receptor.

2. The antibody according to claim 1, characterized in that The antibody is a monoclonal antibody; preferably, the antibody comprises a heavy chain constant region and a light chain constant region; More preferably, the heavy chain constant region is human IgG 1 type or human IgG 2 type; and the light chain constant region is kappa type.

3. The antibody according to claim 2, characterized in that The heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 16, or a sequence having at least 90% homology to SEQ ID NO: 16; and / or The light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 17, or a sequence having at least 90% homology to SEQ ID NO:

17.

4. The antibody according to claim 1, characterized in that The heavy chain variable region also includes VHFR1, VHFR2 and V H FR3 and V H FR4; The V H FR1 is selected from the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9 or a sequence having at least 90% homology to SEQ ID NO: 8 or SEQ ID NO: 9; and / or The V H FR2 is selected from SEQ ID NO: 10 or a sequence having at least 90% homology to SEQ ID NO: 10; and / or The V H FR3 is selected from SEQ ID NO:11 or a sequence having at least 90% homology to SEQ ID NO:

11.

5. The antibody according to claim 1, characterized in that: The affinity of the antibody to human GP130 reaches nM level or sub-nM level. 6 . A nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain variable region and the light chain variable region contained in the antibody according to any one of claims 1 to 5 . An expression vector comprising the nucleic acid molecule according to claim 6 . A recombinant cell comprising the nucleic acid molecule of claim 6 or the expression vector of claim 7.

9. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7 and / or the recombinant cell according to claim 8, and optional pharmaceutically acceptable excipients.

10. Use of the antibody according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7, the recombinant cell according to claim 8 and / or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing, treating and / or ameliorating a disease or condition, wherein the disease or condition is related to human IL-6 expression (including overexpression).

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