Anti-TNFR2 antibody as well as preparation method and application thereof

Abstract

An antibody or antigen-binding fragment thereof that specifically binds to TNFR2 with high affinity is provided. The invention also provides a nucleic acid molecule for coding the antibody or the antigen binding fragment thereof, an expression vector and a host cell for expressing the antibody or the antigen binding fragment thereof, and a production method of the antibody or the antigen binding fragment thereof. The provided antibodies can mediate proliferation and activation of Treg cells, and can be used to treat autoimmune diseases, asthma, anaphylaxis, graft versus host disease, graft rejection, and a variety of other immune diseases.

Description

An anti-TNFR2 antibody, its preparation method and application

[0001] This invention belongs to the field of biomedicine, specifically relating to an anti-TNFR2 antibody, its preparation method, and its application.

[0002] Recent studies have shown that enhancing the body's own ability to fight disease by modulating the immune response is an effective alternative and / or complement to traditional treatments. For example, research indicates that immune balance can be restored by activating the ability of regulatory T cells (Tregs) to suppress T lymphocyte activity and downregulating the overactivation of immune cells.

[0003] Treg cells (Tregs) are a subset of T cells with significant immunosuppressive activity, characterized by the expression of Foxp3, CD25, and CD4. They suppress the immune responses of other cells and are the main controllers of self-tolerance. Normally, their absence or dysfunction leads to autoimmune diseases. When immune cells such as T cells, B cells, and NK cells face invading bacteria, viruses, and other harmful substances, they generate an immune response to recognize and eliminate these substances, thus protecting the body from attack. After these cells clear the harmful substances, Treg cells send a signal to stop the attack, regulating the body's immune homeostasis and preventing autoimmune diseases. When the autoimmune system overexpresses and attacks normal tissues, it causes damage to the body. Treg cells can negatively regulate the body's immune response through intercellular contact and the secretion of inhibitory cytokines among various immune cell subsets, suppressing the body's immune response and active tolerance to self-antigens, thereby preventing the occurrence of autoimmune diseases.

[0004] Tumor necrosis factor receptor 2 (TNFR2), also known as TNFRSF1B and CD120b, is a co-stimulatory member of the tumor necrosis factor receptor superfamily (TNFRSF), which includes proteins such as GITR, OX40, CD27, CD40, and 4-IBB (CD137). By regulating TRAF2 / 3 and NF-κB signaling, TNFR2 can mediate the transcription of genes that promote Treg cell survival and proliferation. Therefore, developing TNFR2 antibodies targeting Treg cells could be used to treat autoimmune diseases, asthma, allergic reactions, graft-versus-host disease, graft rejection, and various other immune disorders.

[0005]

[0006] To address the existing problems, this invention provides an antibody that specifically binds to TNFR2 with high affinity, which can mediate the proliferation and activation of Treg cells to regulate the immune response.

[0007] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to human tumor necrosis factor receptor 2 (TNFR2), comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises heavy chain complementarity-determining regions CDR1, CDR2, and CDR3, and the variable region of the light chain comprises light chain complementarity-determining regions CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 of the variable region of the heavy chain are composed of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, and the CDR1, CDR2, and CDR3 of the variable region of the light chain are composed of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

[0008] In some embodiments, the heavy chain variable region has the amino acid sequence given in SEQ ID NO: 1, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence given in SEQ ID NO: 1, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 1.

[0009] The light chain variable region has the amino acid sequence given in SEQ ID NO: 5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same as the amino acid sequence given in SEQ ID NO: 5, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 5.

[0010] The present invention also provides a polynucleotide that encodes the above-described antibody or its antigen-binding fragment.

[0011] The present invention also provides a recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector containing the above-mentioned polynucleotides.

[0012] The present invention also provides the application of the recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector described above in the preparation of anti-TNFR2 antibody.

[0013] The present invention also provides an isolated host cell containing the recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector.

[0014] In some implementations, the host cell is a prokaryotic cell.

[0015] In some implementations, the host cell is a eukaryotic cell.

[0016] In some embodiments, the eukaryotic cell is a mammalian cell.

[0017] In some embodiments, the mammalian cell is a CHO cell.

[0018] The present invention also provides an antibody expression method, wherein the antibody protein is expressed in any of the host cells described herein using the recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector.

[0019] The present invention also provides an antibody or antigen-binding fragment thereof obtained by humanization of the antibody or antigen-binding fragment thereof.

[0020] In some implementations, the antibody is a murine antibody, a human-mouse chimeric antibody, a humanized antibody, or a fully human antibody.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: monoclonal antibodies or antigen-binding fragments thereof, polyclonal antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, primate-like antibodies or antigen-binding fragments thereof, bispecific antibodies or antigen-binding fragments thereof, multispecific antibodies or antigen-binding fragments thereof, dual variable immunoglobulin domains, monovalent antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, single-chain Fv molecules (scFv), bispecific antibodies, trispecific antibodies, nanobodies, antibody-like protein scaffolds, domain antibodies, Fv fragments, Fab fragments, F(ab')2 molecules, and tandem scFv.

[0022] In some implementations, the antibody is a monoclonal antibody.

[0023] The present invention also provides an antibody-drug conjugate, which is optionally conjugated to a therapeutic agent via a linker based on the antibody or its antigen-binding fragment.

[0024] The present invention also provides a pharmaceutical composition comprising the antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier, diluent or excipient.

[0025] In some embodiments, the composition further comprises an additional therapeutic agent.

[0026] In some implementations, the additional therapeutic agent is an immunotherapeutic agent.

[0027] The present invention also provides the use of the antibody or its antigen-binding fragment in the preparation of medicaments for inducing Treg cell proliferation or for treating autoimmune diseases, asthma, allergic reactions, graft-versus-host disease, graft rejection and various other immune diseases.

[0028] Figure 1 is a schematic diagram of the FACS binding of the candidate antibody to hTNFR2.

[0029] Figure 2 is a schematic diagram of ligand blocking ELISA detection of candidate antibody binding to hTNFR2.

[0030] Figure 3 is a schematic diagram of the Treg proliferation experiment results.

[0031] Figure 4 is a schematic diagram of the Treg proliferation experiment results.

[0032] The following definitions are provided to help understand the invention described herein.

[0033] As used herein, the term "antibody" refers to a naturally occurring immunoglobulin or an immunoglobulin prepared by partial or complete synthesis. Antibodies can be isolated from natural resources such as plasma or serum in which the antibody is naturally present, or from the culture supernatant of hybridoma cells that produce the antibody. Alternatively, they can be partially or completely synthesized using techniques such as gene recombination. Preferred antibodies include, for example, antibodies to isotypes of immunoglobulins or subtypes of these isotypes. Human immunoglobulins are known to include nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgG1, IgA2, IgD, IgE, and IgM. Among these isotypes, the antibodies of the present invention may include IgG1, IgG2, IgG3, and IgG4.

[0034] Various methods / systems exist in this field for defining and describing CDRs. These systems and / or definitions have been developed and refined over many years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used, defining CDRs based on sequence variability; Chothia defines CDRs based on the position of structural loop regions; the IMGT system defines CDRs based on sequence variability and position within variable domain structures; AbM is defined using Oxford Molecular's AbM antibody modeling software, representing a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures, and is similar to Chothia in several ways. In this invention, the numbering of amino acid positions (e.g., amino acid residues in the Fc region) and target regions (e.g., CDRs) uses the Kabat system and has not been artificially modified. It should be noted that CDRs using the Kabat system and with artificial modifications also fall within the scope of this patent. For example, the heavy chain CDR1 can be adjusted, such as by adding five amino acids before the heavy chain CDR1. For instance, the heavy chain CDR1 in this patent can be artificially adjusted to GFTFSNYDMA, and such adjustments also fall within the scope of this patent.

[0035] As used herein, the term "humanized antibody" refers to an antibody comprising at least one humanized antibody chain (i.e., at least one humanized light or heavy chain). The term "humanized antibody chain" (i.e., "humanized immunoglobulin chain") refers to an antibody chain having variable regions (i.e., light or heavy chains, respectively), said variable regions comprising substantially variable framework regions and complementarity-determining regions substantially derived from non-human antibodies (e.g., at least one CDR, two CDRs, or three CDRs). In some embodiments, the humanized antibody chain also includes constant regions (e.g., in the case of a light chain, one constant region or a portion thereof; in the case of a heavy chain, preferably three constant regions).

[0036] As used herein, the term "multispecific antibody" refers to an artificial hybrid antibody having multiple distinct binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion hybridomas or linking Fab fragments.

[0037] As used herein, the term "isolated" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities. Furthermore, isolated antibodies are generally substantially free of other cellular material and / or chemicals.

[0038] As used herein, the terms “Fc region,” “Fc domain,” or “Fc” refer to the C-terminal region of the antibody heavy chain. Therefore, the Fc region contains the constant region of the antibody but does not include the first constant region, the immunoglobulin domain (e.g., CH1 or CL).

[0039] As used herein, the term “antigen” refers to an entity that an antibody binds to (e.g., a protein entity or peptide), such as TNFR2.

[0040] As used herein, the term "specific binding" refers to an antibody exhibiting significant affinity for a specific antigen or epitope and typically not showing significant cross-reactivity with other antigens or epitopes. "Observable" or preferred binding includes binding with a concentration of 10... -7 10 -8 10 -9 Or 10 -10 M or more preferred KD binding. The KD (affinity constant) of antibody-antigen interaction represents the antibody concentration at which 50% of the antibody and antigen molecules bind together. Therefore, at a suitable fixed antigen concentration, a antibody with higher affinity (i.e., stronger) binds the antigen molecule at a lower antibody concentration than an antibody with lower affinity would require to achieve the same percentage binding. Therefore, a lower KD value indicates higher (stronger) affinity. As used herein, "better" affinity is stronger than its affinity and has a lower numerical value, typically 10-1. -7 M has a lower KD value, therefore it is lower than 10. -6 Compared to KD, M has better affinity. Generally, a affinity of less than 10 is preferred. -7 M, therefore preferably greater than 10 -8 The KD value of M can also be considered within the intermediate values ​​described herein, and the preferred binding affinity can be expressed as a range of affinity, for example, 10 for the anti-TNFR2 antibody disclosed herein. -7 Up to 10 -12 M, more preferably 10 -8 Up to 10 -12 M. Antibodies that “do not exhibit significant cross-reactivity” or “do not bind with physiologically relevant affinity” are antibodies that do not bind significantly. Off-target antigens (e.g., non-TNFR2 proteins) or epitopes. Specific or selective binding can be determined according to any technique in the art. Recognized methods for determining such binding include, for example, based on Scatcherd analysis, biomolecular interaction analysis, biomembrane interferometry and / or competitive (competitive) binding assays.

[0041] As used herein, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding domain, which consists of a pair of antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH). In 1988, Skerra and Pluckthun discovered that homologous and active antibodies could be prepared from the periplasmic fraction of *E. coli* by inserting an antibody gene downstream of a bacterial signal sequence and inducing its expression in *E. coli*. In Fv prepared from the periplasmic fraction, the VH binds to the VL in a manner that binds to the antigen.

[0042] As used herein, the terms "scFv," "single-chain antibody," and "sc(Fv)2" all refer to antibody fragments consisting of a single polypeptide chain containing variable regions derived from the heavy and light chains, but without constant regions. Typically, single-chain antibodies also contain a polypeptide linker between the VH and VL domains, which allows for the formation of desired structures that are thought to permit antigen binding.

[0043] As used herein, the term "CHOK1-hTNFR2" refers to a cell constructed using specific techniques. Specific construction methods include, but are not limited to, the following methods, which can be found in WO2022267926A1.

[0044] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0045] Example 1: Animal Immunization

[0046] Recombinant human TNFR2-His Tag protein (Sino, Cat: 10417-H08H) was used as an immunogen to immunize SD rats. To avoid poor rat response or apoptosis during immunization, 3-4 rats could be immunized simultaneously. One day prior to immunization, negative serum was collected for the first immunization. 60 μg of fully emulsified recombinant human TNFR2-His Tag protein, fully emulsified with Freund's complete adjuvant, was injected at multiple sites via subcutaneous, intraperitoneal, and plantar immunization. On day 23, 40 μg of fully emulsified recombinant human TNFR2-His Tag protein was injected at multiple sites via subcutaneous and plantar immunization for a second immunization. The third, fourth, fifth, sixth, and seventh immunizations were performed every two weeks using the same method as the second immunization. Thirty days later, the antiserum titer was assessed by testing serum collected from tail bleeding in ELISA plates coated with recombinant human TNFR2-His Tag protein at different dilutions from 1:100 to 1:1,000,000. When the titer results meet the requirements and anti-human TNFR2 antibody is detected at a dilution of >1:1,000,000, rat spleen and lymph nodes can be harvested for cell fusion.

[0047] Example 2: Cell Fusion

[0048] Myeloma cells SP2 / 0 (ATCC) were passaged one day before fusion to ensure they were in the logarithmic growth phase at the time of the experiment. Before fusion, cells were collected in centrifuge tubes, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and 10 mL of DMEM complete medium was added to mix the cells thoroughly. Spleen cells and lymph node cells used in the experiment were obtained from SD rats immunized seven times with recombinant human TNFR2-His Tag protein. Before fusion, the rats were sacrificed, and the spleen and lymph nodes were removed. 10 mL of DMEM complete medium was added, and the cells were compacted and passed through a mesh. The resulting cell suspension was centrifuged at 2000 rpm for 8 minutes, the supernatant was discarded, and DMEM complete medium was added to mix the cells thoroughly. The feeder cells used in the selection culture of the fusion were macrophages from the peritoneal cavity of unimmunized animals. Before fusion, the collected macrophage suspension was centrifuged at 100 rpm for 8 minutes, the supernatant was discarded, and 25 mL of HAT selection medium was added and mixed. The mixture was then dispensed into two 24-well culture plates to assist the growth of new hybrid B lymphocyte hybridomas.

[0049] B lymphocytes and myeloma cells were mixed at a 1:2 ratio. The suspension was centrifuged at 1000 rpm for 8 minutes, and the pellet was collected and washed twice with electrofusion buffer. Electrofusion buffer was then added to a final volume of 1.2 mL, and 0.4 mL of cell suspension was injected into each of the three multi-electrode cells. The following electric field conditions were applied sequentially to each cell: a sinusoidal AC electric field signal with a frequency of 1 MHz and an amplitude of 250 V / cm for 30 seconds during dielectric electrophoresis; immediately followed by a perforated RC pulse with an amplitude of 5 kV / cm, a pulse width of 20 μs, a pulse count of 3, and a pulse interval of 1 second. The mixture was allowed to stand at room temperature for 10 minutes, then rinsed with 5 mL of PFM buffer and incubated at 37°C for 30 minutes. After centrifugation at 100 rpm for 8 minutes, the pellet was collected, mixed with 500 mL of HAT, and aliquoted into 96-well plates at a cell count of 1 × 10⁶ cells / well. 4 Cells / well (1.2*10⁻⁶ feeder cells) 4 (each well contains 100 cells) and incubated in a CO2 incubator at 37°C.

[0050] Example 3: Screening positive clones using an indirect ELISA method

[0051] Positive cell clones were screened using an indirect ELISA method. A checkerboard assay was used to determine the optimal coating concentration for detecting recombinant human TNFR2-His Tag protein (Sino; Cat: 10417-H08H). The antigen coating buffer was serially diluted horizontally, with 50 μL per well, and incubated overnight at 4°C. After washing three times with PBST, 200 μL of blocking buffer was added to each well, and the plate was incubated overnight at 4°C. Immune mouse serum was serially diluted vertically, with 50 μL per well, and normal rat serum was diluted similarly as a negative control. The plates were incubated at 37°C for 2 h. After a third wash with PBST, 50 μL of the working concentration of enzyme-labeled secondary antibody was added to each well, and the plate was incubated at 37°C for 1.5 h. After washing with PBST, TMB was used for color development, and the OD450 value was measured using an ELISA reader to determine the optimal coating concentration of the antigen.

[0052] The established indirect ELISA method was used to detect the antibody secretion of hybridoma cells. The specific method is as follows: Hybridoma cell culture supernatant was added to an ELISA plate pre-coated with recombinant human TNFR2-His Tag protein (Sino; Cat: 10417-H08H), 50 μL / well. SP2 / 0 cell supernatant was used as a negative control, and immunoglobulin was used as a positive control. The plate was incubated at 37°C for 2 h. The plate was washed three times with PBST. Working concentrations of HRP-labeled goat anti-mouse IgG and IgM antibodies were added, 50 μL / well, and the plate was incubated at 37°C for 1.5 h. After washing, TMB was used for color development for 10 min. After the display was terminated, the OD450 reading was measured using a microplate reader. A positive result was defined as an OD450 reading greater than twice that of the negative control.

[0053] Example 4: Antibody Preparation

[0054] After obtaining a stable hybridoma cell line, monoclonal antibodies are mainly obtained through in vitro culture. The cell line is expanded into T75 culture flasks and cultured until the cell coverage reaches 80-90%. The cell supernatant is discarded, and 30 mL of hybridoma production medium (hybridoma-SFM, Gibco) is added. The culture is then incubated at 37°C with 5% CO2. After 2-3 days of culture, more hybridoma production medium (hybridoma-SFM, Gibco) is added. If the cell viability is below 30%, fresh live cells can be added. After 6-7 days of culture, when the cell viability is below 20%, the culture supernatant is collected after low-speed centrifugation and stored at 4°C for later use.

[0055] Antibodies were purified using a MabSelect SuRe-LX (GE) affinity chromatography column: ① Column packing: An appropriate amount of purchased Protein A packing material was packed into the gravity chromatography column and washed with equilibration buffer (PBS, pH 7.4) until equilibration. ② Sample loading: Hybridoma cell culture supernatant filtered through a 0.22 μm filter was added to the packed column at a flow rate of 1 drop / second. ③ Equilibration: After sample loading, the column was washed with equilibration buffer until equilibration. ④ Elution: The column was washed with elution buffer (50 mM acetic acid / sodium acetate, pH 3.5) and the eluent was collected. ⑤ Regeneration: After elution, the column was washed with 2 column volumes of 0.1 M NaOH and 2 column volumes of 20% ethanol, and then stored at 4°C. Finally, antibody purity was identified by SDS-PAGE and SEC-HPLC, and antibody concentration was determined by UV microspectrophotometry.

[0056] Example 5: FACS binding activity of anti-TNFR2 antibody to human TNFR2

[0057] CHOK1-hTNFR2 cells were fed at a dose of 1×10 5 Cells were plated per well (containing 1% BSA). 100 μL of isotype control and candidate antibody were added at a concentration of 100 nm, diluted 4-fold in 11 gradients (containing 1% BSA). Cells were incubated at 4°C for 1 hour, then washed twice with excess FACS buffer. Cells were resuspended in 100 μL of FACS buffer, and goat anti-rat IgG Fc-AF647 (1:500) and 100 μL of 1% BSA were added. Cells were incubated at 4°C in the dark for 30 minutes and washed twice with excess FACS buffer. Cells were fixed in fixation buffer and then analyzed by flow cytometry. FACS screening identified candidate antibodies with high specific binding activity to human TNFR2. As shown in Table 1 and Figure 1, compared with BMK2 (sequences from SEQ ID NO:3 and SEQ ID NO:4 in WO2017083525A1), the candidate antibodies showed binding capacity comparable to hTNFR2.

[0058] Table 1

[0059] Example 6: Ligand Blocking Experiment

[0060] Coat wells with anti-His antibody (1 μg / mL), 50 μL / well, incubate overnight at 4°C, then wash three times with PBST. Add 2% BSA, 200 μL / well, incubate at room temperature for 1 hour, then wash three times with PBST. Add recombinant human TNFR2-His Tag protein (Sino; Cat: 10417-H08H), 0.25 μg / mL (containing 2% BSA), 50 μL / well, incubate at room temperature for 1 hour, then wash three times with PBST. Add primary antibody (isotype control or candidate antibody), 100 nM, diluted 4-fold in 11 gradients with 1.6 nM recombinant human TNF-alpha-Biotin protein (Sino; Cat: 10602-HANE-B) and 2% BSA solution, incubate at room temperature for 2 hours, then wash three times with PBST. Add secondary antibody, SA-HRP (1:5000), 50 μL / well, containing 2% BSA, incubate at room temperature for 1 hour, and wash 3 times with PBST.

[0061] After washing, TMB was used for color development for 10 minutes, and the OD450 reading was measured by an ELISA reader after the process was terminated. The experimental results were used to determine whether the candidate antibody possessed ligand blocking activity. As shown in Table 2 and Figure 2, the candidate antibody did not possess ligand blocking ability but retained endogenous ligand activity.

[0062] Table 2

[0063] Example 7: Detection of the affinity between anti-TNFR2 antibody and human TNFR2

[0064] Equipment: Biacore 8K (GE). Sensor chip: CM5 chip (GE).

[0065] (1) Fixed:

[0066] Activator preparation: Prepare by immediately mixing 400mM EDC and 100mM NHS (GE) before use.

[0067] The CM5 sensor chip was activated at a flow rate of 10 μL / min for 420 s. Then, 30 μg / mL of anti-rat Fc IgG was injected into the channel in 10 mM NaAc (pH 4.5) at a flow rate of 10 μL / min. The chip was inactivated with 1 M ethanolamine-hydrochloric acid (GE) at a flow rate of 10 μL / min for 420 s.

[0068] (2) Sample capture:

[0069] Candidate antibodies in running buffer 1×HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween 20, pH 7.4) were captured onto Fc2 with anti-rat Fc IgG at a flow rate of 10 μL / min. 10 nm of recombinant human TNFR2-His Tag protein (Sino; Cat: 10417-H08H) and running buffer were sequentially injected into Fc1-Fc2 at a flow rate of 30 min, binding for 180 s, followed by dissociation for 3600 s. After each dissociation, 10 mM glycine (pH 1.5) was injected as a regeneration buffer.

[0070] (3) Regeneration:

[0071] The chip was regenerated using 10 mM glycine (pH 1.5).

[0072] (4) Data Analysis:

[0073] The result is obtained by subtracting the reference channel Fc1 and the buffer channel from the test results graph. The experimental data conform to the 1:1 binding model. The molar concentration of recombinant human TNFR2-His Tag protein was calculated using a molecular weight of 26.6 kDa.

[0074] The results are shown in Table 3. The candidate antibody showed very high SPR affinity to human TNFR2.

[0075] Table 3. SPR affinity between candidate antibodies and human TNFR2

[0076] Example 8: Treg proliferation assay

[0077] PBMCs (donor #190155) purchased from Saili Bio and cryopreserved were thawed and washed twice with RPMI 1640 medium (Gibco, 22400-089) at 1500 rpm / min for 5 min at room temperature. PBMCs were counted using ViCell and resuspended in EasySep buffer (STEMCELL Technology, 20144) to achieve a cell density of 5 x 10⁻⁶ cells / mL. 7 Cells / mL. For the isolation of CD4 + T cells, PBMCs resuspended in EasySep buffer were used with EasySep human CD4 + T-cell enrichment kit (STEMCELL Technology, 19052) was used for negative CD4 separation according to the manufacturer's instructions. + T cells. In short, adding CD4 to PBMCs. +T-cell enrichment cocktail. In 1 mL EasySep buffer, the ratio is 50 μL of the cocktail / 5 x 10- 7 PBMC. Incubate PBMC at room temperature for 10 minutes. Add magnetic beads to the PBMC. The ratio is 100 μL magnetic beads / 5 x 10⁶ beads in 1 mL EasySep buffer. 7 One PBMC. Incubate the PBMCs at room temperature for 5 min. Add 5 mL of EasySep buffer and incubate in an EasySep magnetic field for 5 min. Add CD4+ with negative separation. + The supernatant of T cells was poured into a new test tube. CD4 cells isolated from negative samples were processed using ViCell. + T cells were counted and then resuspended in RPMI 1640 complete medium (RPMI 1640 medium containing 10% FBS) for later use.

[0078] For antibody (Abs) dilution preparation, TNFα was diluted to 40 ng / mL (twice the final concentration in cell culture) in RPMI 1640 complete medium (containing 400 U / mL IL-2 (R&D System, 202-IL-050)). BMK2 was diluted in RPMI 1640 complete medium (containing 400 U / mL IL-2, with or without 40 ng / mL TNFα): starting at 400 nM (twice the final concentration in cell culture), with 5-fold serial dilutions, resulting in 9 concentrations. The concentrations of IL-2, TNFα, and BMK2 listed here are twice the final concentration in cell culture. For example, the final concentration of TNFα in cell culture is 20 ng / mL.

[0079] CD4 + T cells were seeded into 96-well plates (Corning Costar, 3370): 100 μL, 2 x 10⁻⁶ cells / well. 5 Cells / well. Add Abs dilution buffer: 100 μL / well to the same 96-well plate. Add CD4 + T cells were cultured with the specified Abs dilution in a cell culture incubator for 72 hours: 37°C, 5% CO2. The total cell culture volume was 200 μL.

[0080] For staining of the Treg population, cells were stained using the True-Nuclear Human Treg Flow Kit (BioLegend, 320403) according to the manufacturing protocol. First, cell surface markers were stained in 1% BSA buffer at a 1:50 ratio with PE-antiCD25 / PE-CY5anti-CD4. Then, cells were fixed and permeated. Finally, cells were stained in 1xPerm buffer at a 1:50 ratio with Alexa Fluor 488 anti-Foxp3. Cells were resuspended in 1% BSA, 100 μL / well, and then filtered into another 96-well plate for FACS analysis. (CD4+) + CD25 in cell population + FoxP3 + To differentiate Treg cells, and then analyze the Treg cell population in CD4 + Changes in the percentage of T cells.

[0081] As shown in Figures 3 and 4, the candidate antibody exhibited excellent agonistic activity against Treg cell survival.

[0082] Example 9: Sequencing of monoclonal antibodies

[0083] The candidate antibodies were sequenced, and their amino acid sequences were finally obtained. The correspondence between the light chain variable region sequence and the heavy chain variable region sequence and the sequence listing is shown in Table 4.

[0084] Table 4. Correspondence between Numbers and Sequences

Claims

An antibody or its antigen-binding fragment that specifically binds to human tumor necrosis factor receptor 2 (TNFR2), wherein, The antibody or its antigen-binding fragment comprises a heavy chain and a light chain, characterized in that the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 are composed of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, and the light chain variable regions LCDR1, LCDR2, and LCDR3 are composed of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. The antibody or its antigen-binding fragment according to claim 1 is characterized in that, The heavy chain variable region has the amino acid sequence given in SEQ ID NO: 1, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence given in SEQ ID NO: 1, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 1, and / or the light chain variable region has the amino acid sequence given in SEQ ID NO: 5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with ...1, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, NO:5 has an amino acid sequence with one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence. A polynucleotide, characterized in that, It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-2. A recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria, or viral vector, characterized in that, It contains the polynucleotide as described in claim 3. The application of the recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector according to claim 4 in the preparation of anti-TNFR2 antibody. An isolated host cell, characterized in that, It contains the recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector as described in claim 4. The host cell according to claim 6, wherein the host cell is a prokaryotic cell. The host cell according to claim 6, wherein the host cell is a eukaryotic cell. The host cell according to claim 8, wherein the eukaryotic cell is a mammalian cell. The host cell according to claim 9, wherein the mammalian cell is a CHO cell. An antibody expression method, characterized in that, The antibody protein is expressed in the host cells according to any one of claims 6-10 using the recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector according to claim 4. An antibody or antigen-binding fragment thereof according to any one of claims 1-2, or an antibody or antigen-binding fragment thereof that has been humanized. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein, The antibody is a murine antibody, a human-mouse chimeric antibody, a humanized antibody, or a fully human antibody. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein, The antibody or its antigen-binding fragment is selected from the group consisting of: monoclonal antibody or its antigen-binding fragment, polyclonal antibody or its antigen-binding fragment, humanized antibody or its antigen-binding fragment, primate-like antibody or its antigen-binding fragment, bispecific antibody or its antigen-binding fragment, multispecific antibody or its antigen-binding fragment, dual variable immunoglobulin domain, monovalent antibody or its antigen-binding fragment, chimeric antibody or its antigen-binding fragment, single-chain Fv molecule (scFv), bispecific antibody, trispecific antibody, nanobody, antibody-like protein scaffold, domain antibody, Fv fragment, Fab fragment, F(ab')2 molecule, and tandem scFv. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein, The antibody is a monoclonal antibody. An antibody-drug conjugate, characterized in that, The antibody or its antigen-binding fragment according to any one of claims 1-2 may optionally be conjugated to a therapeutic agent via a linker. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-2 and a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition of claim 17, wherein the composition further comprises an additional therapeutic agent. The pharmaceutical composition of claim 18, wherein the additional therapeutic agent is an immunotherapeutic agent. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2 in the preparation of drugs for inducing Treg cell proliferation or for treating autoimmune diseases, asthma, allergic reactions, graft-versus-host disease, graft rejection and various other immune diseases.

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