Method for identifying anti-human TSLP bispecific antibodies

Abstract

The present application provides a method for identifying an anti-human thymic stromal lymphopoietin (TSLP) bispecific antibody having a function capable of blocking the binding of human thymic stromal lymphopoietin (TSLP) to human TSLP receptor (TSLPR) and capable of blocking the binding of human IL-7 receptor alpha chain (IL-7R alpha) to human TSLP: TSLPR complex.

Description

Field of the Invention

[0001] The present invention generally relates to the fields of biochemistry, biophysics and genetic engineering; in particular, the present application relates to a method for identifying anti-human TSLP bispecific antibodies that have the function of blocking the binding of human thymic stromal lymphopoietin (TSLP) to the human TSLP receptor (TSLPR) and blocking the binding of the human IL-7 receptor α chain (IL-7Rα) to the human TSLP:TSLPR complex. Background of the Invention

[0003] Human thymic stromal lymphopoietin (TSLP), also known as an IL-7-like cytokine, is a member of the IL-2 cytokine family. TSLP is primarily secreted by epithelial cells of the thymus, lungs, intestine, and skin, and to a lesser extent by some fibroblasts, airway smooth muscle endothelial cells, mast cells, monocytes, granulocytes, and dendritic cells (DCs). TSLP expression is regulated by factors such as allergens, proinflammatory cytokines (IL-1β, TNF-α, TGF-β, IL-4, IL-13), Th2 cytokines, trauma, mechanical injury, and bacteria.

[0004] TSLP's target cells co-express the TSLP receptor (thymic stromal lymphopoietin receptor, TSLPR) and the IL-7 receptor α chain (IL-7Rα). TSLPR binds to TSLP with high affinity, allosterically activating TSLP, which in turn recruits IL-7Rα to form a ternary complex of TSLP-TSLPR-IL-7Rα, which transmits the signal. IL-7Rα binds to TSLP-TSLPR with high affinity but binds very weakly to TSLP.

[0005] Numerous studies have demonstrated that approximately two-thirds of severe asthma cases manifest as overexpression of Th2 cytokines, and TSLP is a key factor in this overexpression. Researchers have extensively explored and studied drugs (such as antibodies) targeting TSLP. The only anti-TSLP monoclonal antibody currently under clinical investigation has demonstrated a good objective response rate in early clinical trials, effectively alleviating symptoms in trial patients. Given the broad applicability of anti-TSLP antibodies and their clinical potential, identifying anti-TSLP antibodies is of great biological and medical significance. SUMMARY OF THE INVENTION

[0007] The present application provides a method for identifying an anti-human TSLP bispecific antibody capable of blocking the binding of human thymic stromal lymphopoietin (TSLP) to a human TSLP receptor (TSLPR) and capable of blocking the binding of a human IL-7 receptor α chain (IL-7Rα) to a human TSLP:TSLPR complex, wherein the bispecific antibody has a first arm and a second arm that bind to different epitopes of TSLP, the method comprising:

[0008] binding the candidate anti-human TSLP bispecific antibody to TSLP shown in SEQ ID NO: 1;

[0009] Analyzing the binding epitope of the candidate anti-human TSLP bispecific antibody to TSLP shown in SEQ ID NO: 1; and

[0010] The candidate anti-human TSLP bispecific antibody that is determined to bind to one or more of the α-A, BC loop, α-C, α-D and CD loop regions of TSLP is identified as an anti-human TSLP bispecific antibody that has the function of blocking the binding of human TSLP to the human TSLP receptor (TSLPR) and blocking the binding of the human IL-7 receptor α chain (IL-7Rα) to the human TSLP:TSLPR complex.

[0011] In some embodiments, the first arm of the anti-human TSLP bispecific antibody binds to the α-A, BC loop, α-C, and CD loop regions of TSLP.

[0012] In some embodiments, the second arm of the anti-human TSLP bispecific antibody binds to the α-A and α-D regions of TSLP.

[0013] In some embodiments, the method includes determining that the binding epitope of the first arm of the candidate anti-human TSLP bispecific antibody to TSLP includes positions 42, 45, 46, 50, 95, 97, 100, 101, 103, 104, 105, 107, 108, and 114 as shown in SEQ ID NO:1.

[0014] In some embodiments, the method includes determining that the binding epitope of the second arm of the candidate anti-human TSLP bispecific antibody to TSLP includes positions 37, 40, 49, 149, 150, 153, and 154 as shown in SEQ ID NO:1.

[0015] In some embodiments, the bispecific antibody is a Doubody, a Diabody, a CrossMab, a DVD-ig, a Knob-in-Hole, a BiTE, a DART, a YBODY, a FIT-Ig, or a F(ab')2. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The ELISA analysis of the inhibitory effect of GR2002 antibody on the binding of TSLP to TSLPR is shown.

[0018] Figure 2 The results show that GR2002 antibody inhibits TSLP-promoted BAF3-TSLPR / IL-7Rα cell proliferation.

[0019] Figure 3 ELISA analysis of the ability of GR2002 antibody to inhibit TARC secretion from PBMCs stimulated by TSLP is shown.

[0020] Figure 4 The SEC detection results of the incubation product of GR2002-F(ab')2 / TSLP at a molar ratio of 1:1 are shown.

[0021] Figure 5 The SEC detection results of the GR2002-F(ab')2 / TSLP complex are shown.

[0022] Figure 6a-6c The electron microscopy data processing results of the GR2002-F(ab')2 / TSLP complex are shown. Figure 6a Representative steps for electron microscopy data processing of GR2002-F(ab')2 / TSLP complex, Figure 6b Fourier shell correlation function curve (FSC curve) representing the semicircular structure of GR2002-F(ab')2 / TSLP complex, and Figure 6c Fourier shell correlation function curve (FSC curve) representing the pancake structure of GR2002-F(ab')2 / TSLP complex.

[0023] Figure 7 The crystal structure of the GR2002-F(ab')2 / TSLP complex is shown.

[0024] Sequence Description

[0025] SEQ ID NO: 1 shows the amino acid sequence of TSLP.

[0026] SEQ ID NO: 2 shows the amino acid sequence of the heavy chain variable region of H3B10-L3E8-M7 in the GR2002 antibody.

[0027] SEQ ID NO: 3 shows the amino acid sequence of the heavy chain variable region of H1A10-L3E8-M7 in the GR2002 antibody.

[0028] SEQ ID NO: 4 shows the amino acid sequence of the light chain variable region of L3E8-M7 in the GR2002 antibody. Detailed Description of the Invention

[0030] The inventors of this application have conducted in-depth research on the binding epitope of anti-human TSLP bispecific antibodies to TSLP. Using model construction and cryo-electron microscopy, they have identified the amino acid residues of the epitope where the anti-human TSLP bispecific antibodies bind to TSLP. The identified amino acid residues can be used to screen for anti-human TSLP bispecific antibodies for clinical use in preventing or treating TSLP-mediated diseases.

[0031] The practice of this application employs, unless otherwise indicated, conventional techniques of biochemistry, biophysics, genetic engineering, molecular biology, microbiology, cell biology, and immunology within the skill of the art.

[0032] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0033] definition

[0034] The term "antibody" as used herein refers to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. "Antibodies" as used herein include not only complete (i.e., full-length) antibodies, but also binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, VHH antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0035] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and the first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). A full-length antibody can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or any of the above subclasses), but the antibody does not need to belong to any specific class. Based on the antibody amino acid sequence of the constant region of the heavy chain, immunoglobulins can be assigned to different classes. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional structure of different classes of immunoglobulins are well known.

[0036] As used herein, the term "bispecific antibody" refers to an antibody that has the ability to bind to two antigenic epitopes at the same time. The two antigenic epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have a variety of structural configurations. For example, a bispecific antibody can be composed of two Fc fragments and two binding parts fused thereto (similar to natural antibodies, the difference is that the two arms bind to different antigenic targets or epitopes), and the antigen binding portion can be a single-chain antibody (scFv) or a Fab fragment. When targeting two non-overlapping epitopes of a given same antigen, the two different binding portions of the bispecific antibody each bind to the N-terminus of an Fc fragment, and the antigen binding portion configuration of the two arms can have four combinations: scFv+Fab fragment, Fab fragment+scFv, scFv+scFv, and Fab fragment+Fab fragment. The Fc fragment can contain mutations that can ensure heavy chain heteromerization. KIH technology (knob-in-hole, KIH) is a strategy to address heavy chain heteromerization. Generally, KIH technology involves modifying the amino acid sequence of the CH3 region to create a structure that facilitates the pairing of heterologous half-antibodies, thereby allowing the formation of bispecific antibodies while maintaining the normal antibody structure as much as possible. For guidance on KIH technology, see, for example, "An efficient route to human bispecific IgG," A. Margaret Merchant et al., Nature Biotechnology, Volume 16, 1998. [1], which is incorporated herein by reference in its entirety. Furthermore, the structure of a bispecific antibody can be an antibody (e.g., a native antibody form) that binds to a first epitope of an antigen, with an antigen-binding portion extending from the C-terminus of the CH3 region (possibly via a flexible linker) that binds to a second epitope of the antigen.

[0037] As used herein, the terms "binding portion" or "binding fragment" are used interchangeably and refer to a portion or region of an intact antibody molecule that is responsible for binding to an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically contains three complementary determining regions (CDR1, CDR2, and CDR3).

[0038] It is well known to those skilled in the art that the complementarity determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of an antibody. There are two common definitions of VH or VL CDR sequences: the Kabat definition and the Chothia definition. (See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)) [2] ;A1-Lazikani et al., J.Mol.Biol.273:927-948(1997) [3] ; and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989) [4] For the variable region sequence of a given antibody, the CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In the embodiments of the present application, the Kabat definition of CDR sequences is used.

[0039] For a given antibody variable region sequence, the CDR region sequence in the variable region sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0040] For general antibodies, examples of antigen-binding fragments include, but are not limited to: (1) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment, which can be a bivalent fragment having two Fab' fragments connected by a disulfide bridge at the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having the VL and VH domains of a single arm of an antibody; (4) a single-chain Fv (scFv), which can be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; (5) (scFv)2, which can comprise two VH domains connected by a peptide linker and two VL domains, the two VL domains being combined with the two VH domains via a disulfide bridge; and (6) single-domain antibody formats.

[0041] In bispecific antibody constructions, the "binding moiety" includes, but is not limited to, in the form of a Fab fragment or a single-chain antibody (scFv).

[0042] As used herein, the term "single-chain antibody (scFv)" refers to an antibody with a single-chain structure, typically constructed using genetic engineering techniques, comprising a single polypeptide chain consisting of a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker is typically designed between the heavy and light chain variable regions to facilitate folding of the heavy and light chain variable regions into the correct conformation for antigen binding.

[0043] As used herein, the term "Fab (fragment antigen binding) fragment", "Fab portion" or similar terms refers to an antibody fragment capable of binding to an antigen produced by treating an intact antibody with papain, comprising a complete light chain (VL-CL), a heavy chain variable region and a CH1 fragment (VH-CH1).

[0044] The present application provides a method for identifying an anti-human TSLP bispecific antibody capable of blocking the binding of human thymic stromal lymphopoietin (TSLP) to a human TSLP receptor (TSLPR) and capable of blocking the binding of a human IL-7 receptor α chain (IL-7Rα) to a human TSLP:TSLPR complex, wherein the bispecific antibody has a first arm and a second arm that bind to different epitopes of TSLP, the method comprising:

[0045] binding the candidate anti-human TSLP bispecific antibody to TSLP shown in SEQ ID NO: 1;

[0046] Analyzing the binding epitope of the candidate anti-human TSLP bispecific antibody to TSLP shown in SEQ ID NO: 1; and

[0047] The candidate anti-human TSLP bispecific antibody that is determined to bind to one or more of the α-A, BC loop, α-C, α-D and CD loop regions of TSLP is identified as an anti-human TSLP bispecific antibody that has the function of blocking the binding of human TSLP to the human TSLP receptor (TSLPR) and blocking the binding of the human IL-7 receptor α chain (IL-7Rα) to the human TSLP:TSLPR complex.

[0048] In some embodiments, the first arm of the anti-human TSLP bispecific antibody binds to the α-A, BC loop, α-C, and CD loop regions of TSLP.

[0049] In some embodiments, the second arm of the anti-human TSLP bispecific antibody binds to the α-A and α-D regions of TSLP.

[0050] In some embodiments, the method comprises binding an epitope of the first arm of the candidate anti-human TSLP bispecific antibody to TSLP comprising amino acid residues 42, 45, 46, 50, 95, 97, 100, 101, 103, 104, 105, 107, 108, and 114 as set forth in SEQ ID NO: 1. In some embodiments, amino acid residues 42, 45, 46, and 50 are located in the α-A region of TSLP as set forth in SEQ ID NO: 1. In some embodiments, amino acid residues 95 and 97 are located in the BC loop region of TSLP as set forth in SEQ ID NO: 1. In some embodiments, amino acid residues 100, 101, 103, 104, 105, 107, and 108 are located in the α-C region of TSLP as set forth in SEQ ID NO: 1. In some embodiments, amino acid residue 114 is located in the CD loop region of TSLP as set forth in SEQ ID NO: 1.

[0051] In some embodiments, the method determines the binding epitope of the second arm of the candidate anti-human TSLP bispecific antibody to TSLP with reference to positions 37, 40, 49, 149, 150, 153, and 154 as set forth in SEQ ID NO: 1. In some embodiments, amino acid residues 37, 40, and 49 are located in the α-A region of TSLP with reference to SEQ ID NO: 1. In some embodiments, amino acid residues 149, 150, 153, and 154 are located in the α-D region of TSLP with reference to SEQ ID NO: 1.

[0052] In some embodiments of the first aspect, the anti-human TSLP bispecific antibody is in the form of a Doubody, a Diabody, a CrossMab, a DVD-ig, a Knob-in-Hole, a BiTE, a DART, a YBODY, a FIT-Ig, or a F(ab')2. In some embodiments, the anti-human TSLP bispecific antibody is in the form of a F(ab')2. For a description of bispecific antibody formats, see AF ​​Labrijn, ML Janmaat, JM Reichert, PWHI Parren, Bispecific antibodies: a mechanistic review of the pipeline, Nat Rev Drug Discov. 2019 Aug; 18(8): 585-608 [5] .

[0053] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application and is not intended to limit the present invention in any respect. Those skilled in the art can make various modifications and variations to the embodiments described.

[0054] The following examples are for illustration purposes only and are not intended to limit the scope of this application. Example

[0055] Example 1: Preparation of GR2002 antibody

[0056] The present application aims to identify an anti-human TSLP bispecific antibody capable of blocking the binding of human thymic stromal lymphopoietin (TSLP) to the human TSLP receptor (TSLPR) and the binding of human IL-7 receptor α chain (IL-7Rα) to the human TSLP:TSLPR complex. The inventors of the present application have obtained an anti-human TSLP bispecific antibody GR2002 (patent number 202110147687.1) with excellent properties through extensive research and experiments. [6] The bispecific antibody H1A10×H3B10-M1+L3E8-M7 BsAb is a Chinese patent application, which is incorporated herein by reference in its entirety).

[0057] Example 2: Functional identification of GR2002 antibody

[0058] The GR2002 antibody has equivalent or superior functions compared to the positive control antibody Tezepelumab. Please refer to the following subsections for details.

[0059] 2.1 Affinity Analysis of GR2002 Antibody

[0060] The affinity of the GR2002 antibody for hTSLP was determined by surface plasmon resonance using a Biacore X100. Reagents and consumables, including the amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), CM5 chip (BR100012), and 10× HBS-EP, pH 7.4 (BR100669), were purchased from GE Healthcare. Following the kit instructions, the carboxylated CM5 chip surface was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Anti-human IgG (Fc) antibody (capture antibody) was diluted to 25 μg / mL with 10 mM sodium acetate, pH 5.0, and injected at a flow rate of 10 μL / min to achieve a coupling capacity of approximately 10,000 response units (RU). Following injection of the capture antibody, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, GR2002 antibody and tezepelumab were diluted to 0.5-1 μg / mL and injected at a flow rate of 10 μL / min to ensure that approximately 100 RU of antibody were captured by the anti-human Fc antibody. hTSLP-His was then set up in a series of concentration gradients (e.g., 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, and 10 nM) and injected from low to high concentrations at a flow rate of 30 μL / min at 25°C. The association time was 120 s and the dissociation time was 3600 s. 3 M MgCl2 was injected at a flow rate of 10 μL / min for 30 s to regenerate the chip surface. The association rate (K) was calculated using Biacore X100 Evaluation Software version 2.0.1 by fitting the association and dissociation sensorgrams with a 1:1 binding model. a ) and dissociation rate (K d ). With ratio K d / K a Calculate the dissociation equilibrium constant (K D The fitting results are shown in Table 1.

[0061] Table 1. Affinity constants of GR2002 antibody binding to hTSLP

[0062] Antibody <![CDATA[K a ]]> <![CDATA[K d ]]> <![CDATA[K D ]]> GR2002 1.741E+7 1.118E-4 6.421E-12 Tezepelumab 8.633E+6 1.278E-5 1.48E-12

[0063] 2.2 GR2002 antibody inhibits the binding of TSLP to TSLPR

[0064] A 96-well plate was coated with the recombinant antigen TSLPR-Fc (3 μg / mL, 100 μL / well) overnight at 4°C. Blocking was performed with a blocking buffer of PBS-0.1% Tween 20-3% milk at 37°C for 1 hour. GR2002 antibody and the positive control antibody, tezepelumab, were serially diluted with a fixed concentration of hTSLP1-m-his (0.2 μg / mL) starting at 100 μg / mL and then diluted 3-fold over a total of 10 concentrations. 100 μL / well of the blocked 96-well ELISA plate was added and incubated at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and then 100 μL / well of a 1:5000 dilution of HRP-anti-His tag mouse monoclonal antibody (Beijing Kangwei Century Biotechnology Co., Ltd., CW0285M) was added and incubated at 37°C for 1 hour. Wash the ELISA plate with PBS-0.1% Tween 20, add OPD substrate colorimetric solution, stop colorimetric development with 1M H2SO4 after 5-10 minutes, and measure the optical density at 492nm / 630nm using a microplate reader. ELISA analysis results ( Figure 1 ) showed that the ability of GR2002 antibody to inhibit the binding of TSLP to TSLPR was basically equivalent to that of Tezepelumab, and IC 50 See Table 2 for values.

[0065] Table 2. IC of GR2002 antibody inhibiting the binding of TSLP to TSLPR 50 value

[0066] Antibody GR2002 antibody Tezepelumab <![CDATA[IC 50 (nM)]]> 1.906 1.724

[0067] 2.3GR2002 antibody inhibits TSLP-promoting proliferation of BAF3-TSLPR / IL-7Ra cells

[0068] BAF3-TSLPR / IL-7Rα is a stably transfected cell line that overexpresses the TSLP receptor and relies on mIL-3 for proliferation. In the absence of mIL-3, TSLP can effectively promote the proliferation of BA / F3-TSLPR / IL-7Rα cells. This method can effectively evaluate the inhibitory effect of GR2002 antibody on TSLP. In the experiment, BAF3-TSLPR / IL-7Rα cells were pre-cultured for two days using complete medium without mIL-3. On the day of the experiment, the cells were centrifuged and resuspended to 1*10 6 / mL, add 50μL / well to a 96-well flat-bottom cell culture plate; GR2002 antibody and positive control antibody Tezepelumab start at a final concentration of 60μg / mL, the second concentration is 30μg / mL, and the next 8 points are all 5-fold serial dilutions. The diluted GR2002 antibody is premixed with an equal volume of TSLP with a final concentration of 0.4ng / mL for 30min, and then mixed with BAF3-TSLPR / IL-7Rα cells and incubated in a total volume of 150μL / well. After 48h, use Cell Luminescent cell viability assay kit (Promega, Cat#G7570) was used to analyze the cells using a multifunctional microplate reader ( I3X) to detect cell proliferation. The chemiluminescence readings were used for fitting analysis. The results ( Figure 2 ) showed that GR2002 antibody had a more significant inhibitory effect on the proliferation-promoting effect of TSLP on BAF3-TSLPR / IL-7Rα cells than the positive control antibody Tezepelumab.

[0069] 2.4GR2002 antibody inhibits TSLP-stimulated TARC secretion by PBMC

[0070] Blood was collected from normal volunteers (50 mL each). The blood collected was provided by the inventors and their colleagues as volunteers, and all volunteers signed informed consent. The inclusion criteria for volunteers were:

[0071] 1. Aged 18 and above;

[0072] 2. No HIV or HBV infection;

[0073] 3. Routine blood test results are normal;

[0074] 4. Non-pregnant or breastfeeding women.

[0075] Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy human peripheral blood using Ficoll density gradient centrifugation.

[0076] On the day of the experiment, PBMCs were resuspended in R1640 medium containing 10% (inactivated) serum to a concentration of 2 × 10 7ng / mL and added to a 96-well flat-bottom cell culture plate (50 μL / well). Both the GR2002 antibody and the positive control antibody, tezepelumab, were initially diluted 4-fold at the first three sites, 3-fold at sites 4-6, and 5-fold at sites 7-9. An isotype control antibody, IgG1m3, was used. The diluted anti-human TSLP antibody was premixed with an equal volume of hTSLP-m-his at a final concentration of 1 ng / mL for 30 minutes, then added to the cells and mixed in a total volume of 150 μL / well. The cells were incubated at 37°C, 5% CO₂ for 24 hours. Then, the supernatant was collected and TARC was detected using an ELISA antibody pairing set (Beijing Sino Biological Technology Co., Ltd., Cat#SEK10233) that matched the human antibody TARC. The color development solution was TMB high-sensitivity substrate solution (Biolegend, Cat#421501). The OD450 optical density value was measured using a microplate reader (Biotek, Cat#ELX800). The reading results were used for fitting analysis, and the results ( Figure 3 ) showed that the dual GR2002 antibody significantly inhibited the effect of TSLP on PBMC secretion of TARC more than the positive control antibody Tezepelumab.

[0077] Example 3: Preparation of GR2002-F(ab')2

[0078] GR2002 antibody was digested with pepsin (P6887, Sigma) to prepare GR2002-F(ab')2 fragment. GR2002 antibody was digested with digestion buffer (100 mM citric acid, 20 mM NaCl, pH 2.0) at 37°C for 30 min, and then digestion was terminated with 0.5 M NaOH.

[0079] Connect the molecular sieve chromatography column Superdex 200 with 10 / 300GL (28990944, Cytiva) to the BIO-RADNGC Quest TM 10Plus chromatography system, and the above mixture was manually injected through a sample loop, with 20mM Tris-HCl (pH 7.2), 50mM NaCl as the mobile phase, and elution was performed at a flow rate of 0.6min / mL. The elution peak was segmented and collected to obtain GR2002-F(ab')2.

[0080] Example 4: Incubation, Analysis and Purification of GR2002-F(ab')2 / TSLP Complex

[0081] The antigen TSLP was incubated with GR2002-F(ab')2 at a molar ratio of 1:1 at 4°C overnight, and the product was subjected to SEC detection. Molecular exclusion chromatography column: TSKgel G3000SWXL (0008541) was purchased from Tosoh (Shanghai) Biotechnology Co., Ltd.; mobile phase: 50nM PB / 300mM NaCl, pH7.0, and related reagents for preparing the mobile phase were purchased from China National Pharmaceutical Group Co., Ltd. Before injection, the chromatographic column was equilibrated at a flow rate of 0.8mL / min, 10-100μg of protein sample was injected, the detection wavelength was 280nm, the column temperature was room temperature, and the isocratic elution was 20min. The SEC detection results of the GR2002-F(ab')2 / TSLP complex are shown in Figure 2. Figure 4 As shown: the purity of the complex after GR2002-F(ab')2 / TSLP incubation was 73.98%.

[0082] Referring to Example 3, the product after incubation of GR2002-F(ab')2 / TSLP was purified by molecular sieve to obtain GR2002-F(ab')2 / TSLP complex protein. The SEC detection results of the purified GR2002-F(ab')2 / TSLP complex were as follows: Figure 5 As shown: its purity is 88.83%.

[0083] Example 5: Structural analysis of the GR2002-F(ab')2 / TSLP complex

[0084] 5.1 Image processing, sample preparation, and cryo-EM data collection

[0085] 3.0 μL of purified protein sample of GR2002-F(ab')2 / TSLP complex with a concentration of approximately 5 mg / mL was added dropwise to a Quantifoil gold grid (R1.2 / 1.3, 300 mesh) treated with glow discharge. The Vitrobot (FEI) was set to a blot force of 4, a relative humidity of 100%, and a temperature of 4°C. Under these environmental conditions, the gold grid was adsorbed with filter paper for 3.5 seconds, and then the gold grid was quickly inserted into liquid ethane to complete the frozen sample preparation. The frozen grid was placed under a 300keV Titan Krios (Thermo Fisher) electron microscope (equipped with a Falcon4 camera) for imaging. The automatic data collection program EPU (Thermo Fisher) was used, the image magnification was 130k×, and the image pixel size was . The defocus range was set between -1.0 and -2.0 μm. The exposure time for each micrograph was 6 s, the dose rate was 7.5 e- / pixel / s, and the total dose was approximately

[0086] 5.2 Image Processing

[0087] Using Relion [7] Correction for electron beam-induced sample drift. Then use Gctf [8] The contrast transfer function parameters of these drift-corrected images were determined. After particle selection using Gautomatch, four rounds of three-dimensional (3D) classification were performed in cryoSPARC, followed by non-uniform 3D refinement in cryoSPARC. Details of data processing are provided in the Extended Data. Figure 6a shown.

[0088] 5.3 Model construction and modification

[0089] The structural model was constructed based on 5J13.pdb using UCSF Chimera

[10] The model was fitted to the cryo-EM electron density map. All models were in Coot

[11] Manual adjustment and real space correction were performed in PHENIX

[12] The structure of the GR2002-F(ab')2 / TSLP complex crystal and the details of GR2002's action are as follows. Figure 7 shown.

[0090] The interaction area between GR2002-F(ab')2 / TSLP complex and TSLP (left) and heavy chain H1A10 is approximately The interaction area with light chain L3E8-M7 is approximately The HCDR1, HCDR2, and HCDR3 of the heavy chain H1A10 variable region and the LCDR1 and LCDR3 of the light chain variable region L3E8-M7 are involved in the recognition of TSLP. The amino acid residues that bind to H1A10-L3E8-M7 and TSLP (left) are shown in Table 3. The interaction area between TSLP (left) and heavy chain H3B10 is approximately The interaction area with light chain L3E8-M7 is approximately HCDR1, HCDR2, and HCDR3 of the heavy chain H3B10 variable region and LCDR1 and LCDR3 of the light chain L3E8-M7 variable region are involved in the recognition of TSLP. The amino acid residues that bind to H3B10-L3E8-M7 and TSLP (left) are shown in Table 4.

[0091] Table 3. Amino acid residues of H1A10-L3E8-M7 binding to TSLP (left)

[0092]

[0093] Table 4. Amino acid residues of H3B10-L3E8-M7 binding to TSLP (left)

[0094]

[0095]

[0096] The amino acids that interact with TSLP (right) and H1A10-L3E8-M7 and H3B10-L3E8-M7 are completely consistent with those of TSLP (left), as shown in Tables 5 and 6 for details.

[0097] Table 5. Amino acid residues that bind to H1A10-L3E8-M7 and TSLP (right)

[0098]

[0099] Table 6. Amino acid residues of H3B10-L3E8-M7 binding to TSLP (right)

[0100]

[0101]

[0102] The binding epitope of Tezepelumab antibody to TSLP includes positions 65-68, 70-72, 74-75, 78, 150 and 153 shown in SEQ ID NO: 1 (see Table 4 in the supplementary information of Kenneth Verstraete, et al., Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma, Nature Communications, 2017, 8: 14937).

[13] ), which has a different binding epitope to TSLP from the anti-human TSLP bispecific antibody GR2002 of the present application, which gives the GR2002 antibody excellent function.

[0103] The above describes exemplary embodiments of the various inventions of the present application. However, without departing from the essence and scope of the present application, those skilled in the art will be able to modify or improve the exemplary embodiments described in the present application, and the resulting variations or equivalents also fall within the scope of the present application.

[0104] References

[0105] [1]A.Margaret Merchant et al.,An efficient route to human bispecificIgG,Nature Biotechnology,Volume 16,1998.

[0106] [2]Kabat,“Sequences of Proteins of Immunological Interest”,NationalInstitutes of Health,Bethesda,Md.(1991).

[0107] [3]A1-Lazikani et al.,J.Mol.Biol.273:927-948(1997).

[0108] [4]Martin et al.,Proc.Natl.Acad.Sci.USA86:9268-9272(1989).

[0109] [5]AF Labrijn,ML Janmaat,JM Reichert,PWHIParren,Bispecificantibodies:amechanistic review of the pipeline,Nat Rev Drug Discov.2019Aug;18(8):585-608.

[0110] [6]CN 202110147687.1.

[0111] [7]Fernandez-Leiro R,Scheres S H W,A pipeline approach to single-particle processing in RELION[J].Acta Crystallographica Section D-StructuralBiology,2017,73(496-502.

[0112] [8]Zhang K,Gctf:Real-time CTF determination and correction[J].Journalof Structural Biology,2016,193(1):1-12.

[0113] [9]Punjani A,Rubinstein J L,Fleet D J,et al.,cryoSPARC:algorithms for rapid unsupervised cryo-EM structure determination[J].Nature Methods,2017,14(3):290-+.

[0114]

[10] Pettersen E F,Goddard T D,Huang C C,et al.,UCSF chimera-A visualization system for exploratory research and analysis[J].Journal of Computational Chemistry,2004,25(13):1605-1612.

[0115]

[11] Emsley P,Lohkamp B,Scott W G,et al.,Features and development of Coot[J].Acta Crystallographica Section D-Biological Crystallography,2010,66(486-501.

[0116]

[12] Adams P D,Afonine P V,Bunkoczi G,et al.,PHENIX:a comprehensive Python-based system for macromolecular structure solution[J].Acta Crystallographica Section D-Structural Biology,2010,66(213-221.

[0117]

[13] Kenneth Verstraete,et al.,Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma,Nature Communications,2017,8:14937.

[0118] Sequence information

[0119] SEQ ID NO:1

[0120] MFPFALLYVL SVSFRKIFIL QLVGLVLTYD FTNCDFEKIK AAYLSTISKD LITYMSGTKSTEFNNTVSCS NRPHCLTEIQ SLTFNPTAGC ASLAKEMFAM KTKAALAIWC PGYSETQINA TQAMKKRRKRKVTTNKCLEQ VSQLQGLWRR FNRPLLKQQ

[0121] SEQ ID NO:2

[0122] QVTLKESGPVLVKPTETLTLTCTVSGFSLSSYDISWIRQPPGKALEWLGVIWSEGGTSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARDSDDGYLNWGQGTLVTVSS

[0123] SEQ ID NO:3

[0124] QVQLVQSGAEVKKPGASVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGQIFPGDSDTDYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAGYYRGYYAMDYWGQGTTVTVSS

[0125] SEQ ID NO:4

[0126] EIVMTQSPATLSVSPGERATLSCSVSSSISSSNLHWYQQKPGQAPRPWIYGTSNLASGIPARFSGSGSGTEFTLTISSvQaEDFAVYYCQQWSSYPLTFGQGTKLEIK。

Claims

1. A method for identifying an anti-human TSLP bispecific antibody capable of blocking the binding of human thymic stromal lymphopoietin (TSLP) to the human TSLP receptor (TSLPR) and the binding of human IL-7 receptor α chain (IL-7Rα) to the human TSLP:TSLPR complex, wherein the bispecific antibody has a first arm and a second arm that bind to different epitopes of TSLP, the method comprising: binding the candidate anti-human TSLP bispecific antibody to TSLP shown in SEQ ID NO: 1; analyzing the binding epitope of the candidate anti-human TSLP bispecific antibody to TSLP shown in SEQ ID NO: 1; as well as The candidate anti-human TSLP bispecific antibody that is determined to bind to one or more of the α-A, BC loop, α-C, α-D and CD loop regions of TSLP is identified as an anti-human TSLP bispecific antibody that has the function of blocking the binding of human TSLP to the human TSLP receptor (TSLPR) and blocking the binding of the human IL-7 receptor α chain (IL-7Rα) to the human TSLP:TSLPR complex.

2. The method of claim 1, wherein the first arm of the anti-human TSLP bispecific antibody binds to the α-A, BC loop, α-C, and CD loop regions of TSLP.

3. The method of claim 1, wherein the second arm of the anti-human TSLP bispecific antibody binds to the α-A and α-D regions of TSLP.

4. The method of claim 1 or 2, comprising determining that the binding epitope of the first arm of the candidate anti-human TSLP bispecific antibody to TSLP includes positions 42, 45, 46, 50, 95, 97, 100, 101, 103, 104, 105, 107, 108, and 114 as shown in SEQ ID NO:

1.

5. The method of claim 1 or 3, comprising determining that the binding epitope of the second arm of the candidate anti-human TSLP bispecific antibody to TSLP comprises positions 37, 40, 49, 149, 150, 153, and 154 as shown in SEQ ID NO:

1.

6. The method of any one of claims 1 to 5, wherein the anti-human TSLP bispecific antibody is in the form of Doubody, Diabody, CrossMab, DVD-ig, Knob-in-Hole, BiTE, DART, YBODY, FIT-Ig or F(ab')2; preferably, the anti-human TSLP bispecific antibody is in the form of F(ab')2.

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