Anti-TNFR2 antibody, preparation method therefor, and use thereof

Abstract

Provided is an antibody or an antigen-binding fragment thereof that specifically binds to TNFR2 with high affinity. Further provided are a nucleic acid molecule encoding 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 for the antibody or the antigen-binding fragment thereof. The antibody provided can mediate proliferation and activation for Treg cells, and can be used to treat autoimmune diseases, asthma, allergic reactions, graft-versus-host disease, transplant rejection, and various other immune diseases.

Description

An anti-TNFR2 antibody and its preparation method and application Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an anti-TNFR2 antibody and a preparation method and application thereof. Background Art

[0002] Recent research indicates that enhancing the body's ability to fight disease by regulating the immune response is an effective alternative and / or supplement to traditional treatments. For example, studies have shown that activating regulatory T cells (Tregs) can suppress T lymphocyte activity, downregulating excessive immune cell activation and thereby restoring immune balance.

[0003] Tregs are a subset of T cells with significant immunosuppressive properties, characterized by the expression of Foxp3, CD25, and CD4. They suppress the immune responses of other cells and are the primary regulators of self-tolerance. Their absence or dysfunction often leads to the development of autoimmune diseases. When immune cells such as T cells, B cells, and NK cells are challenged with invading bacteria, viruses, and other harmful substances, they mount an immune response to identify and eliminate them, thereby protecting the body from attack. After these cells eliminate the harmful substances, Tregs signal a halt to the attack, regulating immune homeostasis and preventing autoimmune diseases. When autoimmunity becomes excessive, normal tissues are targeted, causing damage. Tregs, among various immune cell subsets, negatively regulate the body's immune response through intercellular contact and secretion of inhibitory cytokines, suppressing immune responses and fostering active tolerance to self-antigens, thereby preventing the development 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-kB signaling, TNFR2 can mediate the transcription of genes that promote the survival and proliferation of Treg cells. Therefore, the development of TNFR2 antibodies targeting Treg cells can be used to treat autoimmune diseases, asthma, allergic reactions, graft-versus-host disease, transplant rejection, and various other immune diseases.

[0005] Summary of the Invention

[0006] In response to the existing problems, the present invention provides an antibody that specifically binds to TNFR2 with high affinity, and the antibody 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 that can specifically bind to human tumor necrosis factor receptor 2 (TNFR2), comprising a heavy chain and a light chain, wherein the heavy chain variable region comprises heavy chain complementary determining regions CDR1, CDR2 and CDR3, and the light chain variable region comprises light chain complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 of the heavy chain variable region consist of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and CDR1, CDR2 and CDR3 of the light chain variable region consist 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 set forth in SEQ ID NO: 1, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence that has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative 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 identity to 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) conservative 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 encoding the above-mentioned antibody or antigen-binding fragment thereof.

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

[0012] The present invention also provides a use of the recombinant vector, transgenic cell line, phage, recombinant bacteria or viral vector in preparing anti-TNFR2 antibodies.

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

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

[0015] In some embodiments, 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, which uses the recombinant vector, transgenic cell line, phage, recombinant bacteria or viral vector to express the antibody protein in any one of the host cells.

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

[0020] In some embodiments, 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 a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a bispecific antibody, a trispecific antibody, a nanobody, an antibody-like protein scaffold, a domain antibody, an Fv fragment, a Fab fragment, a F(ab')2 molecule, and a tandem scFv.

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

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

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

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

[0026] In some embodiments, 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 drugs for inducing Treg cell proliferation or treating autoimmune diseases, asthma, allergic reactions, graft-versus-host disease, transplant rejection and various other immune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a schematic diagram of FACS binding of candidate antibodies to hTNFR2.

[0029] FIG2 is a schematic diagram of a ligand blocking ELISA assay for binding of candidate antibodies to hTNFR2.

[0030] FIG3 is a schematic diagram of the Treg proliferation test results.

[0031] FIG4 is a schematic diagram of the Treg proliferation test results. DETAILED DESCRIPTION

[0032] The following definitions are provided to aid in understanding the invention set forth herein.

[0033] As used herein, the term "antibody" refers to a natural 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 synthesized partially or completely using techniques such as genetic recombination. Preferred antibodies include, for example, antibodies of the isotype of an immunoglobulin or subclasses of these isotypes. Known human immunoglobulins include 9 categories (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Among these isotypes, the antibodies of the present invention can include IgG1, IgG2, IgG3, and IgG4.

[0034] There are a variety of methods / systems in the art to define and describe CDRs, and these systems and / or definitions have been developed and refined over the 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 sequence variability based on the position of the structural loop region; the IMGT system defines CDRs based on sequence variability and position within the variable domain structure; AbM is based on Oxford Molecular's AbM antibody modeling software and is a compromise between Kabat and Chothia; Contact defines CDRs based on analysis of complex crystal structures and is similar to Chothia in many aspects. The numbering of amino acid positions (e.g., amino acid residues in the Fc region) and target regions (e.g., CDRs) in the present invention uses the Kabat system and has not been artificially adjusted. It should be noted that CDRs that use the Kabat system and have been artificially adjusted also fall within the scope of this patent. For example, the heavy chain CDR1 can be adjusted, such as adding five amino acids before the heavy chain CDR1. For example, the heavy chain CDR1 of this patent can be artificially adjusted to GFTFSNYDMA, and such an adjustment also falls 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 chain or heavy chain). The term "humanized antibody chain" (i.e., "humanized immunoglobulin chain") refers to an antibody chain with a variable region (i.e., a light chain or a heavy chain, respectively), which includes a variable framework region and complementarity determination that is substantial for a human antibody. Regions (CDRs) substantially derived from non-human antibodies (e.g., at least one CDR, two CDRs, or three CDRs). In some embodiments, the humanized antibody chain further comprises a constant region (e.g., in the case of a light chain, a 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" is an artificial hybrid antibody with multiple different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusing 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 having different antigenic specificities. Additionally, an isolated antibody is typically substantially free of other cellular material and / or chemicals.

[0038] As used herein, the term "Fc region," "Fc domain," or "Fc" refers to the C-terminal region of an antibody heavy chain. Thus, the Fc region comprises the constant region of an antibody, excluding the first constant region immunoglobulin domain (e.g., CH1 or CL).

[0039] As used herein, the term "antigen" is an entity (eg, a proteinaceous entity or a peptide) to which an antibody binds, such as TNFR2.

[0040] As used herein, the term "specific binding" refers to an antibody that exhibits significant affinity for a particular antigen or epitope and generally does not exhibit significant cross-reactivity with other antigens or epitopes. "Appreciable" or preferred binding includes binding with a specific affinity of 10 -7 , 10 -8 , 10 -9 or 10 -10 M or more preferably KD binding. The KD (affinity constant) of an antibody-antigen interaction represents the antibody concentration at which 50% of the antibody and antigen molecules bind together. Thus, at an appropriate fixed antigen concentration, 50% of an antibody with a higher affinity (i.e., stronger) binds to the antigen molecule at a lower antibody concentration than the antibody concentration required to achieve the same percentage binding with a lower affinity antibody. Thus, a lower KD value indicates a higher (stronger) affinity. As used herein, a "better" affinity is stronger than an affinity with a lower value, and its 10 -7 The KD value of M is lower, so it is the same as 10 -6 The affinity is better than that of KD of M. It is usually preferred to have a KD of less than 10 -7 M, therefore preferably greater than 10 -8 KD values ​​of M, intermediate values ​​as described herein, may also be considered, and preferred binding affinities may be expressed as a range of affinities, for example, 10 for the anti-TNFR2 antibodies disclosed herein. -7 to 10 -12 M, more preferably 10 -8 to 10 -12 M. An antibody that "does not exhibit significant cross-reactivity" or "does not bind with physiologically relevant affinity" is one that does not significantly bind to an off-target antigen (e.g., a non-TNFR2 protein) or epitope. Specific or selective binding can be determined according to any technique in the art. Recognized methods for determining such binding include, for example, Scatchard analysis, biomacromolecular interaction analysis, biofilm interferometry techniques, and / or 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 is composed of a pair of antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH). In 1988, Skerra and Pluckthun discovered that by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli, homologous and active antibodies can be prepared from the periplasmic fraction of E. coli. In Fv prepared from the periplasmic fraction, VH binds to VL in the same manner as antigen binding.

[0042] As used herein, the terms "scFv," "single-chain antibody," and "sc(Fv)2" all refer to antibody fragments of a single polypeptide chain that contain variable regions derived from both heavy and light chains, but lack constant regions. Typically, single-chain antibodies also contain a polypeptide linker between the VH and VL domains, which enables the formation of the desired structure believed to permit antigen binding.

[0043] As used herein, the term "CHOK1-hTNFR2" refers to a cell constructed by a certain technique. 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 facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0045] Example 1: Animal Immunization

[0046] SD rats were immunized with recombinant human TNFR2-His Tag protein (Sino, Cat: 10417-H08H) as the immunogen. To avoid poor response or apoptosis during immunization, 3-4 rats were immunized simultaneously. Negative serum was collected one day in advance. For the first immunization, 60 μg of recombinant human TNFR2-His Tag protein, fully emulsified in Freund's complete adjuvant, was injected subcutaneously, intraperitoneally, and intrapedicularly. On day 23, a second immunization was performed with 40 μg of recombinant human TNFR2-His Tag protein, fully emulsified in Freund's complete adjuvant, injected subcutaneously and intrapedicularly. The third, fourth, fifth, sixth, and seventh immunizations were performed two weeks apart, using the same protocol. Thirty days later, antiserum titers were assessed by testing sera collected from tail bleeds at dilutions ranging from 1:100 to 1:1,000,000 on ELISA plates coated with recombinant human TNFR2-His Tag protein. When the titer results meet the requirements and anti-human TNFR2 antibodies are detected at a dilution of >1:1000000, the rat spleen and lymph nodes can be harvested and cell fusion can be performed.

[0047] Example 2: Cell fusion

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

[0049] B lymphocytes and myeloma cells were mixed in a ratio of 1:2, and the suspension was centrifuged at 1000 rpm for 8 minutes. The precipitate was collected and washed twice with electrofusion solution. The precipitate was then taken and added with electrofusion solution to 1.2 mL, and 0.4 mL of the cell suspension was injected into each of the three multi-electrode pools. The following electric field conditions were applied to each electrode pool in turn: the AC electric field sinusoidal signal frequency of dielectric electrophoresis was 1 MHz, the amplitude was 250 V / cm, and it was applied for 30 seconds; then, a perforation RC electric pulse amplitude of 5 kV / cm, a pulse width of 20 μs, 3 pulses, and a time interval of 1 second was immediately applied. After standing at room temperature for 10 minutes, the fusion was washed out with a total of 5 mL of PFM solution and incubated at 37°C for 30 minutes. Then, it was centrifuged at 100 rpm for 8 minutes, the precipitate was collected, 500 mL of HAT was added to mix, and the mixture was dispensed into a 96-well culture plate with a cell number of 1×10 4 cells / well (with feeder cells 1.2*10 4 / well), and cultured in a CO2 incubator at 37°C.

[0050] Example 3: Screening of positive clones by indirect ELISA

[0051] Positive cell clones were screened using an indirect ELISA method. The coating concentration for detecting recombinant human TNFR2-His Tag protein (Sino; Cat: 10417-H08H) was determined by array assay. The plate was coated with 50 μL of the detection antigen coating buffer in a horizontal series dilution format and incubated at 4°C overnight. The plate was washed three times with PBST, and 200 μL of blocking buffer was added to each well and incubated at 4°C overnight. The plate was then diluted serially with immune mouse serum and incubated at 37°C for 2 hours. Normal rat serum was diluted in the same series as a negative control and incubated at 37°C for 2 hours. The plate was washed a third time with PBST, and the working concentration of enzyme-linked secondary antibody was added to 50 μL of the plate per well. The plate was incubated at 37°C for 1.5 hours. After washing with PBST, TMB was used for color development, and the OD450 value was measured with an enzyme-linked immunosorbent assay (ELISA) to determine the optimal coating concentration for the detection antigen.

[0052] Antibody secretion by hybridoma cells was detected using a pre-established indirect ELISA method. 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) at a rate of 50 μL / well. SP2 / 0 cell supernatant was used as a negative control, and immune polyclonal antibody serum was used as a positive control. The plates were incubated at 37°C in a water bath for 2 hours. After washing three times with PBST, HRP-labeled goat anti-mouse IgG and IgM antibodies were added at a working concentration at a rate of 50 μL / well and incubated at 37°C in a water bath for 1.5 hours. After washing, TMB color development was performed for 10 minutes. After display was stopped, the OD450 reading was measured on a microplate reader. Wells were considered positive if the OD450 reading was at least twice that of the negative control.

[0053] Example 4: Preparation of antibodies

[0054] After obtaining a stable hybridoma cell line, monoclonal antibodies are mainly obtained by in vitro culture. The cell line is expanded in a T75 culture flask and cultured until the cell confluence is 80-90%. The cell supernatant is discarded and 30 mL of hybridoma production medium (hybridoma-SFM, Gibco) is added and cultured at 37°C and 5% CO2. After culturing for 2-3 days, hybridoma production medium (hybridoma-SFM, Gibco) is added. If the cell viability is less than 30%, fresh live cells can be added. After culturing for 6-7 days, when the cell viability is less than 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 affinity chromatography column (GE Biosciences): ① Column loading: Load an appropriate amount of purchased Protein A filler into a gravity chromatography column and rinse with equilibration buffer (PBS, pH 7.4) until equilibrium is reached. ② Sample loading: Add hybridoma cell culture supernatant filtered through a 0.22 μm filter to the loaded column at a flow rate of 1 drop / second. ③ Equilibration: After sample loading, rinse with equilibration buffer until equilibrium is reached. ④ Elution: Rinse the column with elution buffer (50 mM acetic acid / sodium acetate, pH 3.5) and collect the eluate. ⑤ Regeneration: After elution, rinse the column with two column volumes of 0.1 M NaOH, followed by two column volumes of 20% ethanol, and store at 4°C. Antibody purity was assessed by SDS-PAGE and SEC-HPLC, and antibody concentration was determined by UV microspectrophotometry.

[0056] Example 5: FACS Binding Activity of Anti-TNFR2 Antibodies to Human TNFR2

[0057] CHOK1-hTNFR2 cells were cultured at 1×10 5 / well for plating (containing 1% BSA). Add 100 μL of isotype control and candidate antibodies at a concentration of 100 nm, 4-fold dilution of 11 gradients (containing 1% BSA). The cells were incubated at 4 ° C for 1 hour and then washed twice with excess FACS buffer. The cells were resuspended in 100 μL FACS buffer, goat anti-rat IgG Fc-AF647 (1:500) was added, 100 μL was incubated at 4 ° C in the dark for 30 minutes and washed twice with excess FACS buffer. The cells were fixed in a fixation buffer and then analyzed by flow cytometry. The FACS method screened out candidate antibodies with high specific binding activity to human TNFR2. As shown in Table 1 and Figure 1, compared with BMK2 (sequence from SEQ ID NO: 3 and SEQ ID NO: 4 in WO2017083525A1), the candidate antibodies showed comparable binding ability to hTNFR2.

[0058] Table 1

[0059] Example 6: Ligand blocking experiment

[0060] Coat with anti-His antibody (1 μg / mL) at 50 μL / well, incubate overnight at 4°C, and wash three times with PBST. Add 2% BSA at 200 μL / well, incubate at room temperature for 1 hour, and wash three times with PBST. Add recombinant human TNFR2-His Tag protein (Sino; Cat: 10417-H08H) at 0.25 μg / mL (containing 2% BSA) at 50 μL / well, incubate at room temperature for 1 hour, and wash three times with PBST. Add primary antibody (isotype control or candidate antibody) at a concentration of 100 nM and dilute 11 times in a 4-fold dilution series containing 1.6 nM recombinant human TNF-alpha-biotin protein (Sino; Cat: 10602-HANE-B) and 2% BSA. Incubate at room temperature for 2 hours and wash three times with PBST. Secondary antibody, SA-HRP (1:5000), 50 μL / well, containing 2% BSA, was added, incubated at room temperature for 1 hour, and washed three times with PBST.

[0061] After washing, TMB color development was performed for 10 minutes. After display was terminated, the OD450 reading was measured using a microplate reader. The results were used to determine whether the candidate antibody had ligand-blocking activity. As shown in Table 2 and Figure 2, the candidate antibody did not exhibit ligand-blocking activity and retained endogenous ligand activity.

[0062] Table 2

[0063] Example 7: Affinity detection of anti-TNFR2 antibodies to human TNFR2

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

[0065] (1) Fixed:

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

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

[0068] (2) Sample capture:

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

[0070] (3) Regeneration:

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

[0072] (4) Data analysis:

[0073] Subtract the Fc1 and buffer channel results from the test results. The experimental data fit a 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 SPR affinity between the candidate antibodies and human TNFR2 is very high.

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

[0076] Example 8: Treg proliferation assay

[0077] Cryopreserved PBMCs (donor #190155) purchased from Saili Bio were thawed and washed twice with RPMI1640 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 a cell density of 5 × 10 7 cells / mL. To isolate CD4 + T cells, PBMCs resuspended in EasySep buffer were stained with EasySep human CD4 + T cell enrichment kit (STEMCELL Technology, 19052) was used to negatively isolate CD4 + In brief, CD4 T cells were added to PBMCs +T cell enrichment cocktail. In 1 mL EasySep buffer, the ratio is 50 μL cocktail / 5x10 7 PBMCs. Place PBMCs at room temperature for 10 minutes. Add magnetic beads to PBMCs. In 1 mL of EasySep buffer, the ratio is 100 μL magnetic beads / 5 x 10 7 PBMCs were stored at room temperature for 5 minutes. 5 mL of EasySep buffer was added and incubated in the EasySep magnetic field for 5 minutes. + Pour the supernatant of T cells into a new tube. + The T cells were counted and then resuspended in RPMI1640 complete medium (RPMI1640 medium containing 10% FBS) for use.

[0078] For antibody (Ab) dilution preparation, TNFα was diluted to 40 ng / mL (twice the final concentration in cell culture) in RPMI1640 complete medium (containing 400 U / mL IL-2 (R&D System, 202-IL-050)). BMK2 was diluted in RPMI1640 complete medium (containing 400 U / mL IL-2, with or without 40 ng / mL TNFα) in a five-fold serial dilution series starting at 400 nM (twice the final concentration in cell culture) for nine concentrations. The listed concentrations of IL-2, TNFα, and BMK2 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, 2×10 5 cells / well. Add Abs dilution solution to the same 96-well plate: 100 μL / well. + T cells were cultured with the indicated Ab dilutions for 72 hours in a cell culture incubator at 37°C, 5% CO2. The total cell culture volume was 200 μL.

[0080] For staining of Treg populations, cells were stained using the True-Nuclear Human Treg Flow Kit (BioLegend, 320403) according to the manufacturer's protocol. First, cell surface markers were stained with PE-anti CD25 / PE-CY5anti-CD4 at a ratio of 1:50 in 1% BSA buffer. Then, the cells were fixed and permeabilized. Finally, cells were stained with Alexa Fluor 488anti-Foxp3 at a ratio of 1:50 in 1xPerm buffer. The cells were resuspended in 1% BSA, 100 μL / well, and then filtered into another 96-well plate for FACS analysis. CD4 + CD25 in cell populations + Foxp3 + To distinguish 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 antibodies showed excellent agonistic activity on Treg cell survival.

[0082] Example 9: Sequencing of monoclonal antibodies

[0083] The candidate antibodies were sequenced and the amino acid sequences of the candidate antibodies 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

1. An antibody or an antigen-binding fragment thereof that can specifically bind 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 complementary determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and LCDR1, LCDR2 and LCDR3 of the light chain variable region are composed of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, 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) conservative 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 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) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO:

5.

3. A polynucleotide, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-2.

4. A recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector, characterized in that: It contains the polynucleotide according to claim 3.

5. Use of the recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector according to claim 4 in the preparation of anti-TNFR2 antibodies.

6. An isolated host cell, characterized in that It contains the recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector according to claim 4.

7. The host cell according to claim 6, wherein the host cell is a prokaryotic cell.

8. The host cell of claim 6, wherein the host cell is a eukaryotic cell.

9. The host cell of claim 8, wherein the eukaryotic cell is a mammalian cell.

10. The host cell according to claim 9, wherein the mammalian cell is a CHO cell.

11. A method for expressing an antibody, characterized in that: The antibody protein is expressed in a host cell according to any one of claims 6 to 10 using the recombinant vector, transgenic cell line, bacteriophage, recombinant bacteria or viral vector according to claim 4.

12. A humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 2.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein The antibody is a mouse antibody, a human-mouse chimeric antibody, a humanized antibody or a fully human antibody.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein The antibody or antigen binding fragment thereof is selected from the group consisting of: a monoclonal antibody or an antigen binding fragment thereof, a polyclonal antibody or an antigen binding fragment thereof, a humanized antibody or an antigen binding fragment thereof, a primatized antibody or an antigen binding fragment thereof, a bispecific antibody or an antigen binding fragment thereof, a multispecific antibody or an antigen binding fragment thereof, a dual variable immunoglobulin domain, a monovalent antibody or an antigen binding fragment thereof, a chimeric antibody or an antigen binding fragment thereof, a single-chain Fv molecule (scFv), a bispecific antibody, a trispecific antibody, a nanobody, an antibody-like protein scaffold, a domain antibody, an Fv fragment, a Fab fragment, a F(ab')2 molecule, and a tandem scFv.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein The antibody is a monoclonal antibody.

16. An antibody-drug conjugate, characterized in that: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 is optionally coupled to a therapeutic agent via a linker.

17. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier, diluent or excipient.

18. The pharmaceutical composition of claim 17, wherein the composition further comprises an additional therapeutic agent.

19. The pharmaceutical composition of claim 18, wherein the additional therapeutic agent is an immunotherapeutic agent.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 in the preparation of a drug for inducing Treg cell proliferation or treating autoimmune diseases, asthma, allergic reactions, graft-versus-host disease, transplant rejection and various other immune diseases.

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