Antibodies for binding human gp130 and uses thereof

Abstract

The present application relates to an antibody for binding human GP130 and its use, the heavy chain variable region of the antibody comprises:V H CDR1 is selected from the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2;V H CDR2 is selected from the amino acid sequence as shown in SEQ ID NO: 3;V H CDR3 is selected from the 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 the amino acid sequence as shown in SEQ ID NO: 7; the antibody can specifically bind to human GP130 to hinder human IL-6 from binding to human IL-6 receptor. The scheme provided in the present application can target the binding of human GP130, specifically block the binding of IL-6 to its receptor, so as to regulate abnormal immune response and inhibit inflammatory signals in pathological process.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, and in particular to an antibody for binding human GP130 and uses thereof. BACKGROUND

[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 various diseases, such as autoimmune diseases, inflammatory diseases, infectious diseases, and tumors, etc. Human IL6 receptor is a specific receptor for human IL-6, which can activate multiple signaling pathways (such as JAK / STAT, MAPK and PI3K, etc.) by binding with human IL-6, and 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] Human IL6 receptor is generally IL6R, which is composed of an alpha subunit (also known as IL6RA) and a beta subunit (also known as IL6ST, GP130, IL6β). Among them, the alpha subunit is the main functional subunit of the receptor, and the beta subunit is the signal transduction subunit. In the classical signaling pathway of human IL-6, human IL-6 first binds to membrane-bound IL-6RA, then binds to signal transduction molecule GP130, forms a complex and activates the downstream signaling pathway.

[0004] In the field of drug development, intervention strategies targeting 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 human IL6 receptor, which have been used in the clinic for the treatment of rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), and COVID-19 cytokine storm syndrome, etc. In addition, Siltuximab is a monoclonal antibody targeting human IL-6 protein, which has been approved for the treatment of multicentric Castleman disease (MCD), etc. Abnormal activation of IL6 signaling pathway is also associated with the occurrence and development of various diseases, including cancer and autoimmune diseases. Antibody drugs targeting human GP130 can inhibit the pathological process 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 response and inhibiting inflammatory signals in pathological processes. However, there are not many monoclonal antibody drugs targeting IL6ST / gp130.

[0005] Therefore, it is of great therapeutic prospect and necessity to develop monoclonal antibodies capable of specifically regulating human GP130 pathway. SUMMARY

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

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

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

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

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

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

[0012] The antibody can specifically bind to human GP130 to hinder the binding of human IL-6 and 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 as shown in SEQ ID NO: 8.

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

[0016] In some embodiments, the antibody has an affinity to human GP130 at the 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 comprised in the antibody of any one of the first aspect.

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

[0019] The fourth aspect of the present application provides a recombinant cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.

[0020] The fifth aspect of the present application provides a pharmaceutical composition comprising the antibody of any one of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect and / or the recombinant cell of the fourth aspect, and optionally a pharmaceutically acceptable excipient.

[0021] The sixth aspect of the present application provides use of the antibody of any one of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect and / or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for preventing, treating and / or ameliorating a disease or disorder associated with IL-6 expression (including overexpression).

[0022] The technical solutions provided by the present application can include 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, i.e. blocking the signal pathway of IL-6 and its receptor. The antibody of the present application can be used as a potential drug for regulating immune response and inhibiting inflammatory signals in pathological processes by targeting abnormal activation of the IL6-GP130 signal pathway.

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

[0025] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which like reference characters designate like elements throughout the several views.

[0026] Figure 1 is a graph of absorbance after the binding of the antibody to be tested and human GP130 at different concentrations;

[0027] Figure 2 is a graph showing the absorbance of the test antibody binding to human GP130 at different concentrations;

[0028] Figure 3 is a graph showing the absorbance of the test antibody blocking the binding of human GP130 to LIP at different concentrations;

[0029] Figure 4 is a graph showing the absorbance of the test antibody blocking the binding of human GP130 to LIP at different concentrations;

[0030] Figure 5 is a graph showing the absorbance of the test antibody inhibiting human IL-6 induced STAT-3 phosphorylation at different concentrations;

[0031] Figure 6 is an electron microscope image of the complex structure of the positive reference 2 binding to human GP130;

[0032] Figure 7 is an electron microscope image of the complex structure of the target antibody C082 binding to human GP130;

[0033] Figure 8 is an electron microscope image of the complex structure of the target antibody A084 binding to human GP130;

[0034] Figure 9 is an electron microscope image of the complex structure of the target antibody A088 binding to human GP130;

[0035] Figure 10 is an electron microscope image of the complex structure of the target antibody C082 binding to human GP130 after freezing. DETAILED DESCRIPTION

[0036] In order that the application can be readily understood, the application will be described in detail below with reference to the following illustrative embodiments. It should be appreciated that the application is not limited in scope to the particular embodiments described, which are intended as illustrations of single aspects of the application. The application is instead intended to cover any and all alternatives, modifications, adaptations and variations that come within the scope and equivalents of the appended claims.

[0037] Where a range of values is provided, it is understood that each intervening value, to the extent that there are intervening values between the upper and lower limit, between the lower and upper limit, and between each intervening value are also specifically included within the scope of the application. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges and are also encompassed within the scope of, and are meant to be included in, the application, subject to any specifically excluded limit in the stated limitation. Where the stated limit excludes one or both of the limits, ranges excluding either or both of the excluded limits are also included in the application.

[0038] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials, or equivalents thereof, may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0039] Terminology Explanation:

[0040] The term "antibody" is synonymous with immunoglobulin and is understood in the sense commonly understood in this field. The basic structural unit of an antibody is a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminal portion of each chain contains a variable region of approximately 100 to 120 or more amino acids. The carboxyl-terminal portion of each chain defines the constant region primarily responsible for effector function. Human light chains are classified as kappa (κ) type and lambda (λ) type. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain contains the heavy chain constant region (C). H ) and heavy chain variable region (V H Light chains contain light chain constant regions (C). L ) and light chain variable region (V L V H and V L Each region contains highly variable complementary determinant regions (CDRs) and structural regions (FRs) scattered among them, each V H and V L It contains FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 sequentially from the amino terminus to the carboxyl terminus.

[0041] Variable region (V) of each heavy chain / light chain pair H and V L Each variable domain forms an antigen-binding site; therefore, a complete IgG antibody, for example, has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are identical. The term "variable" refers to the fact that certain portions of the variable domain differ significantly in sequence between antibodies and are used for the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the variable domain of an antibody; it is concentrated in the CDR region.

[0042] This application provides an antibody for binding 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 as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence having at least 90% homology with SEQ ID NO: 1 or SEQ ID NO: 2; V H CDR2 is selected from the amino acid sequence as shown in SEQ ID NO: 3 or a sequence having at least 90% homology with SEQ ID NO: 3; V H CDR3 is selected from the amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or a sequence having at least 90% homology with SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 7. Wherein, the amino acid sequence of the light chain variable region is a human sequence.

[0044] As shown in Table 1 below, V H CDR1, V H CDR2 and V H CDR3 of the heavy chain variable region and the amino acid sequence of the light chain variable region are shown in Table 1 below. It can be understood that, V H CDR1, V H CDR2 and V H CDR3 of the heavy chain variable region and the amino acid sequence of the light chain variable region can also be a sequence having at least 90% homology with the corresponding sequence in Table 1.

[0045] Table 1

[0046]

[0047]

[0048] In the amino acid sequence corresponding to the V L variable region shown in Table 1, the corresponding sequence number of FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4 is contained in turn. The light chain variable region of the present application is selected from a common light chain variable region of human, i.e. different antibodies can adopt the light chain variable region with the same amino acid sequence, through the known human amino acid sequence, which is more conducive to the design of the double antibody while providing a stable antibody structure.

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

[0050] In some embodiments, the heavy chain variable region of the present application further comprises V H FR1, V H FR2 and V HFR3 and V H FR4 and V H FR1 is selected from the amino acid sequence as shown in SEQ ID NO: 8 or SEQ ID NO: 9 or a sequence having at least 90% homology with SEQ ID NO: 8 or SEQ ID NO: 9. H FR2 is selected from the amino acid sequence as shown in SEQ ID NO: 10 or a sequence having at least 90% homology with SEQ ID NO: 10. H FR3 is selected from the amino acid sequence as shown in SEQ ID NO: 11 or a sequence having at least 90% homology with SEQ ID NO: 11. H FR4 is selected from the amino acid sequence as shown in SEQ ID NO: 12 or a sequence having at least 90% homology with SEQ ID NO: 12. In some embodiments, H FR1, and V H FR2, and V H FR3, and V H FR4 are as shown in Table 2. It can be understood that, H FR1, and V H FR2, and V H FR3, and V H FR4 can 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. The corresponding amino acid sequences are shown in Table 3 as follows.

[0054] Table 3

[0055]

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

[0057] Further, 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 of human IgGl type or human IgG2 type; and the light chain constant region is of 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 with 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 with SEQ ID NO: 17.

[0059] Table 4

[0060]

[0061]

[0062] In order to make the present application more easily understood, the preparation of the antibody of the present application and the related performance test will be further described in detail below in conjunction with examples, which are only illustrative and do not limit the scope of application of the present application. The raw materials or components used in the present application can be prepared by commercial means or conventional methods if not specifically stated.

[0063] Example 1 Antibody designed by combining human GP130 and AI calculation and phage display screening

[0064] The traditional antibody design method is to immunize in animals and separate, and there is no antibody designed for the specific epitope of human GP130 (Uniport ID: P40189) and human IL-6 (Uniport ID: P05231) binding. In this embodiment, a variety of antigens are immunized using a full human co-antibody light chain mouse to obtain a full human co-light chain antibody sequence library with a million-level diversity. The representative antibody sequence is screened from the antibody library by combining the target epitope of human GP130, and the binding probability of the CDR region of the antibody sequence and the target epitope is predicted. The antibody sequence with high potential to bind to the target epitope is selected as a template antibody sequence by screening based on the binding probability.

[0065] The residues of all or part of the sites in the heavy chain variable region of the template antibody sequence are masked based on the masking mechanism, and the type of each site residue masked is predicted based on the human antibody sequence to generate a candidate antibody sequence. According to a preset antigen-antibody affinity prediction model, the affinity between human GP130 and the candidate antibody sequence is predicted, and the antibody sequence with an affinity prediction score reaching a preset condition is selected as a target antibody sequence.

[0066] In this embodiment, based on the epitope specific to human GP130, the antibody sequence with high affinity and high specificity is designed from scratch by computer model, which provides important support for the subsequent development of antibody drugs. Not only can it greatly save experimental costs, but also it is not limited by experimental conditions. It can control the direction and target of antibody generation by exploring all possible sequence conformations and setting parameters during the generation process, improving the efficiency and quality of antibody engineering, and providing more drug possibilities for antibody molecules.

[0067] In this embodiment, the heavy chain variable region amino acid sequences of multiple target antibody sequences are constructed into a single-chain variable fragment (scFv) antibody library by gene synthesis and splicing. That is, each antibody has the same light chain variable region (SEQ ID NO: 7) and the corresponding heavy chain variable region. Then the antibody phage display library is constructed, and the human GP130 protein is used to screen positive phage clones with binding activity, and the single-chain variable fragment (scFv) antibodies shown in Table 3 are screened out.

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

[0069] After obtaining the antibody heavy chain sequences of the positive clones in Table 3 according to Embodiment 1, the heavy chain sequence of each antibody is 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) in this embodiment. Subsequently, these recombinant expression plasmids are transferred to CHO cells by transfection, and after the expression process is completed, the culture supernatant is treated using Protein A affinity chromatography technology, thereby isolating and purifying three corresponding target antibodies A084, A088 and C082.

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

[0071] Detection of binding activity of antibodies to IL6ST / gp130

[0072] In this embodiment, the indirect ELISA method is used to evaluate the binding activity of the target antibodies to human gp130 protein.

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

[0074] Table 5

[0075]

[0076] The above three test antibodies, positive reference 1, blank reference, and negative reference were tested 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, Biotech) protein was selected and added to the ELISA microplate at a concentration of 2 μg / mL, 30 μL per well for coating and fixation.

[0078] 3.2 100 μL of 5% PBSM solution was used for blocking to prevent non-specific binding of antibodies.

[0079] 3.3 The same test antibody was diluted in 1% PBSM solution according to the gradient to obtain test antibodies with concentrations of 0.00015 μg / mL, 0.00137 μg / mL, 0.00412 μg / mL, 0.01235 μg / mL, 0.03704 μg / mL, 0.1111 μg / mL, 0.3333 μg / mL, and 3 μg / mL, respectively, and the test antibodies at each concentration were added to different microplates to bind with the coated antigen.

[0080] 3.4 After washing 3 times with PBST to remove unbound test antibodies, 30 μL of Goat-Anti-human-IgG-Fc-HRP (Lot: ab97225 Abeam) diluted 1:8000 with 1% PBSM solution was added and reacted at room temperature for 50 minutes.

[0081] 3.5 PBST was used to wash 6 times, and color developing solution TMB and 2M stop solution were added.

[0082] 3.6 The OD value of the microplate was read by spectrophotometer, and the corresponding EC50 value of the test antibody was obtained by multi-parameter fitting.

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

[0084] Example 4 Detection of affinity of antibodies to human GP130

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

[0086] In the Octet system, AHC chips were used to capture the test antibody as the stationary phase, and the analyte was human GP130 protein. The binding and dissociation processes of the test antibody and GP130 protein were recorded in real time, and the affinity constant KD value, ka value, kd value and R2 value were calculated according to 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 for measuring the strength of the interaction between molecules. The smaller the KD value, the stronger the binding affinity between molecules. The larger the KD value, the weaker the binding. The ka value refers to the association 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 dissociation between molecules. The larger the kd value, the faster the dissociation process. The R2 value is the fitting degree of the data. The R2 value ranges from 0 to 1. The closer the R2 value to 1, the better the data fitting effect. Through detection, the affinity KD value of the three antibodies of the present application can reach nM level or sub-nM level, which is higher than that of the control positive reference 1 antibody.

[0090] Example 5 Antibody blocking IL6ST / gp130 and its ligand LIF protein activity

[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 antibody to be tested, which can block the activity of human GP130 protein and its ligand LIF. The concentration of 1% PBSM solution is used as a blank reference (BC). The IPI antibody is used as a negative reference (NC). Among them, LIF and IL-6 can bind to GP130 and the binding epitopes are close, so the test results have reference value, so LIF is used instead of IL-P for testing.

[0092] The positive reference 2 is selected from the patent JP2018153172A, and the heavy chain variable region amino acid sequence and the 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 as follows.

[0093] Table 7

[0094]

[0095] The specific steps are as follows:

[0096] 5.1 Human GP130 protein is added to the ELISA microplate at a concentration of 2 μg / mL for coating and fixing.

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

[0098] 5.3 The same antibody to be tested is diluted to multiple concentrations, and then the antibody to be tested with a concentration of 0.00457 μg / mL, 0.01372 μg / mL, 0.04115 μg / mL, 0.12346 μg / mL, 0.37037 μg / mL, 1.11111 μg / mL, 3.33333 μg / mL and 10 μg / mL and 1 μg / mL biotin-labeled LIF (Lot: LIF-H52H3, Biolegend) protein are added to the microplate to compete with the coated antigen for binding.

[0099] 5.4 After washing to remove the unbound antibody to be tested, 30 μL of NeutrAvidin-HRP (Lot: 31001, Thermo Fisher) diluted 1:2000 with 1X PBS solution is added, and the reaction is carried out at room temperature for 50 minutes.

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

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

[0102] Through the competition ELISA detection, the antibodies A084, A088 and C082 can all block the binding of human GP130 and ligand LIF, and the corresponding IC50 values are 0.21 μg / mL, 0.18 μg / mL and 0.06 μg / mL respectively.

[0103] Example 6 Inhibition of IL6-induced STAT-3 phosphorylation activity by antibodies in NCI-H226 cells

[0104] In this example, three target antibodies and positive reference 2 are used as the antibodies to be tested, IgG1 is used as the negative reference antibody, and the inhibition of human IL6-induced STAT-3 phosphorylation activity is tested to determine the cell function activity of the antibodies to determine whether the antibodies can inhibit IL-6-induced STAT3 phosphorylation.

[0105] The specific steps are as follows:

[0106] 6.1 Plate and culture NCI-H226 cells at a density of 2E4 / well.

[0107] 6.2 Add 0.137 nM, 1.372 nM, 4.115 nM, 12.346 nM, 37.037 nM, 111.111 nM, 333.333 nM and 1000 nM of the antibody to be tested and 25 nM of recombinant hyper-IL6 (CHO expression, Sunny Biopharma) protein 5 to the microplate for culture and treatment, and use cells treated with only 25 nM of Hyper-IL6 and untreated cells as controls, i.e. Figure 5 Hyper-IL6 and cell only shown in

[0108] 6.3 Lyse the cells and harvest the supernatant.

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

[0110] Through the detection, the binding Figure 5 ​As shown, the STAT-3 phosphorylation level of untreated NCI-H226 cells is low (cell only), and the induction of Hyper-IL6 can cause the phosphorylation level of NCI-H226 cells to increase (hyper-IL6), while the antibodies C082, A084, A088 and Benchmark 2 can all inhibit the STAT-3 phosphorylation activity induced by IL6, among which the inhibition activities of A084 and A088 have IC50 values of 159.3 nM and 68.3 nM, respectively.

[0111] Example 7 Electron Microscopy of Target Antibodies Binding to Human GP130

[0112] In this example, the negative staining electron microscopy technique was used to observe the binding mode and position of three target antibodies and positive reference 2 as test antibodies to human GP130 protein, respectively.

[0113] 7.1 Prepare a mixed sample of human GP130 protein and test antibody Fab fragments in a molar ratio of 2:1 or 4:1.

[0114] 7.2 Discharge the carbon film-mounted copper support grid at a current of 15 mA for 90 seconds.

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

[0116] 7.4 Stain the mixed sample of 7.3 with 0.75% uranium formate solution to enhance the contrast of the image.

[0117] 7.5 Observe the support grid using a Talos L120C electron microscope and collect images through high-resolution imaging technology with a pixel size of to ensure that clear structure images of the test antibody-human GP130 protein complex are obtained.

[0118] Table 8

[0119]

[0120] As shown in Table 8 above, and in combination with the Figure 6-9 It can be seen that the 2D schematic diagram of the binding of the test antibody to human GP130 is selected from each electron microscopy image and reconstructed in 3D, and it can be seen that antibodies C082, A084 and A088 are all bound to the middle position of human GP130 protein, which overlaps with the epitope when human IL-6 binds to human GP130, further indicating that antibodies C082, A084 and A088 are bound 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 resolution of target antibody with human GP130

[0122] In this example, the fine structure of the antibody C082 and human GP130 protein complex was resolved using cryo-EM technology.

[0123] 8.1 The Fab fragment of the target antibody and the human GP130 protein were mixed in a molar ratio of 4:1 to prepare the mixed sample required for cryo-EM.

[0124] 8.2 A multi-well carbon film grid was selected for sample support, and the mixed sample was dropped onto the grid.

[0125] 8.3 Using Virtobot, the sample was quickly frozen in liquid ethane to maintain its natural structure under the condition of 100% humidity and 4°C by quickly absorbing the excess sample through filter paper.

[0126] 8.4 The frozen sample was initially observed using a Glacios electron microscope to ensure the quality and frozen state of the sample.

[0127] 8.5 Data was collected using a Krios electron microscope at a magnification of 165k, with the pixel size of the image reaching

[0128] 8.6 The binding site of the target antibody C082 and human GP130 protein complex (the area covered by the red circle on the top right) was intercepted, and the high-resolution three-dimensional structure of the target antibody C082 and human GP130 protein complex was obtained through data processing and reconstruction, with a resolution of 3.63 angstroms (the three-dimensional structure shown below). Figure 10 Figure 10 As shown, the binding epitope of the antibody C082 has a significant overlapping region with the known IL6-GP130 complex (PDB ID: 1I1R), indicating that C082 has potential activity in inhibiting IL6 signaling. This structural overlap provides direct evidence for understanding the inhibition mechanism of antibody C082. Figure 10

[0129] As shown, the binding epitope of the antibody C082 has a significant overlapping region with the known IL6-GP130 complex (PDB ID: 1I1R), indicating that C082 has potential activity in inhibiting IL6 signaling. This structural overlap provides direct evidence for understanding the inhibition mechanism of antibody C082. Figure 10

[0130] ​​​Based on the above Examples 1 to 8, the target antibody of the present application, whose antibody sequence is human-derived, does not need to be humanized, and experiments have proved that it has the binding performance and excellent affinity to human GP130, its binding epitope is on the binding interface of IL6-GP130, can block the binding of human GP130 to ligand LIP, and inhibit IL6-induced STAT-3 phosphorylation in 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 signal pathway of human IL-6, and achieving the effect of regulating abnormal immune response and inhibiting inflammatory signals in pathological processes.

[0131] The technical features of the above-described embodiments can be combined in any manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments are described, however, as long as the combination of the technical features does not exist contradictory, it should be considered as the scope of the present disclosure.

[0132] It should be noted that the above-described embodiments are only used to explain the present application and do not constitute any limitation on the present application. The present application is described by referring to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words. The present application can be modified within the scope of the claims, and the present application can 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 examples, 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 function.

Claims

1. An antibody for binding human GP130, characterized in that, The antibody has 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 the amino acid sequence shown in SEQ ID NO: 2, and the V H CDR2 is the amino acid sequence shown in SEQ ID NO: 3, and the V H CDR3 is the amino acid sequence shown in SEQ ID NO: 5; or The V H CDR1 is the amino acid sequence shown in SEQ ID NO: 1, and the V H CDR2 is the amino acid sequence shown in SEQ ID NO: 3, and the V H CDR3 is the amino acid sequence shown in SEQ ID NO: 4; or The V H CDR1 is the amino acid sequence shown in SEQ ID NO: 2, and the V H CDR2 is the amino acid sequence shown in SEQ ID NO: 3, and the V H CDR3 is the amino acid sequence shown in SEQ ID NO: 6; The light chain variable region is an amino acid sequence as shown in SEQ ID NO: 7; The antibody can specifically bind to human GP130 to prevent human IL-6 from binding to the human IL-6 receptor.

2. The antibody according to claim 1, characterized in that, The antibody is a monoclonal antibody.

3. The antibody according to claim 1, characterized in that, The antibody comprises a heavy chain constant region and a light chain constant region.

4. The antibody according to claim 3, characterized in that, The heavy chain constant region is human IgG type 1 or human IgG type 2; the light chain constant region is kappa type.

5. The antibody according to any one of claims 3 to 4, characterized in that, The heavy chain constant region is an amino acid sequence as shown in SEQ ID NO: 16, or a sequence having at least 90% homology with SEQ ID NO: 16; and / or The light chain constant region is an amino acid sequence as shown in SEQ ID NO: 17, or a sequence having at least 90% homology with SEQ ID NO:

17.

6. 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 with SEQ ID NO: 8 or SEQ ID NO: 9; and / or The V H FR2 is selected from sequences such as SEQ ID NO: 10 or sequences having at least 90% homology with SEQ ID NO: 10; and / or The V H FR3 is selected from sequences such as SEQ ID NO: 11 or sequences having at least 90% homology with SEQ ID NO: 11; and / or The V H FR4 is selected from sequences such as SEQ ID NO: 12 or sequences that have at least 90% homology with SEQ ID NO:

12.

7. The antibody according to claim 1, characterized in that: The antibody has an affinity for human GP130 at the nM or sub-nM level.

8. A nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain variable region and the light chain variable region contained in the antibody of any one of claims 1 to 7.

9. An expression vector comprising the nucleic acid molecule of claim 8.

10. A recombinant cell comprising the nucleic acid molecule of claim 8 or the expression vector of claim 9.

11. A pharmaceutical composition comprising the antibody of any one of claims 1 to 7 and / or the recombinant cells of claim 10, and pharmaceutically acceptable excipients.

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