WO2023098491A1 - 抗tslp单克隆抗体、其抗原结合片段及其用途 - Google Patents

抗tslp单克隆抗体、其抗原结合片段及其用途 Download PDF

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WO2023098491A1
WO2023098491A1 PCT/CN2022/132848 CN2022132848W WO2023098491A1 WO 2023098491 A1 WO2023098491 A1 WO 2023098491A1 CN 2022132848 W CN2022132848 W CN 2022132848W WO 2023098491 A1 WO2023098491 A1 WO 2023098491A1
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amino acid
acid sequence
seq
heavy chain
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白义
周俊杰
刘思
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北京东方百泰生物科技股份有限公司
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention relates to the technical field of biomedicine, in particular to an anti-TSLP monoclonal antibody, its antigen-binding fragment and its application.
  • Asthma is one of the most common chronic diseases in the world.
  • the incidence of asthma is about 4.3%.
  • the prevalence of asthma in various countries ranges from 1% to 18%. It is difficult to control patients with current treatment methods. These patients consume 50-60% of asthma medical expenses. Due to economic constraints, many patients in developing countries have not been effectively diagnosed and treated, including China.
  • On June 21, 2019, the international authoritative medical journal "The Lancet” published another important result of a large-scale population study [Chinese Adult Lung Health Study] (CPH Study) completed by Chinese professionals, revealing the prevalence of asthma in my country. According to the situation, it is clear that the prevalence of asthma in the population aged 20 and over in my country is 4.2%, and the number of patients reaches 45.7 million.
  • Asthma is a chronic airway inflammatory disease involving a variety of inflammatory cells and mediators. This chronic inflammation is related to airway hyperresponsiveness. The clinical manifestations are recurrent wheezing, shortness of breath, chest tightness, coughing and other symptoms. Onset and exacerbation at night and (or) early morning, most patients can be relieved spontaneously or after treatment; pathologically manifested as chronic inflammatory changes of the airway, and airway remodeling, the latter including airway wall thickening, stroma Subepithelial fibrosis with collagen deposition, smooth muscle hyperplasia and hypertrophy, myofibroblast proliferation and mucus glands, goblet cell metaplasia and hyperplasia, etc., bring great difficulties to treatment. So far, there are many kinds of asthma drugs on the market in the world, most of which are chemical drugs. With the continuous development of biological drugs, the research and development of biological drugs for asthma has been attracting more and more attention.
  • T2 inflammation-driven (T2 high) asthma is present in more than two-thirds of patients with severe asthma and is typically characterized by elevated levels of T2 inflammatory biomarkers, diagnostic and predictive biomarkers including blood eosinophils , serum IgE and exhaled nitric oxide (FeNO), Periostin, etc.) have revolutionized the field of targeted therapy for severe asthma.
  • Monoclonal antibodies that target Th2-driven inflammation are generally safe in adults with moderate-to-severe asthma. The remaining approximately one-third of patients with severe asthma do not have the signature of an activated T2 inflammatory pathway, and their non-T2-driven disease remains uncontrolled with standard clinical guidelines.
  • Research in non-type 2 asthma is inconclusive, and further research is needed to identify biomarkers to guide targeted therapy for different forms of non-type 2 asthma.
  • Thymic stromal lymphopoietin is an epithelial cytokine produced in response to pro-inflammatory stimuli (such as pulmonary allergens, viruses and other pathogens), and has the effect of enhancing thymocyte proliferation.
  • TSLP drives the release of downstream T2 cytokines, including IL-4, IL-5, and IL-13, leading to inflammation and asthma symptoms.
  • TSLP also activates various cell types involved in non-T2-driven inflammation.
  • TSLP's early upstream activity in the inflammatory cascade has been identified as a potential target in a broad population of asthmatic patients.
  • TSLP modulates immunity by activating immature DCs, lymphocytes, mast cells, basophils, and eosinophils.
  • TSLP initiates intracellular signaling by forming a complex with its specific receptor TSLPR and co-receptor IL-7R ⁇ .
  • TSLP first binds to TSLPR with high affinity and then forms a ternary complex with the extracellular domain of IL-7R ⁇ .
  • the two opposing faces of TSLP interact with TSLPR and IL-7R ⁇ , respectively.
  • Activation of STAT5 is a signal required for TSLP-mediated Th2 responses.
  • a humanized monoclonal antibody against TSLP specifically binds human TSLP and blocks its interaction with the receptor complex, thereby potentially preventing the release of pro-inflammatory cytokines from immune cells targeted by TSLP, thereby preventing asthma attack and improve asthma control.
  • Tezepelumab (also known as AMG157) of AstraZeneca and its partner Amgen is the first monoclonal antibody drug targeting TSLP, but it is still not on the market.
  • Clinical studies have shown that the humanized monoclonal anti-TSLP Antibody acts early upstream in inflammatory cascade for use in a broad range of patients with severe uncontrolled asthma, including non-T2-driven asthma, a proof-of-concept in mild, atopic asthmatic inhaled allergen Confirmed in the challenge study, it demonstrated its ability to suppress early and late asthmatic responses and reduce levels of T2 inflammatory biomarkers.
  • TSLP target drugs in the treatment of asthma, in order to meet the needs of asthma patients at home and abroad, there is an urgent need to develop monoclonal antibody therapy drugs that can be used alone or as adjuvant therapy for asthma.
  • the present invention obtains anti-TSLP monoclonal antibodies that can specifically bind to TSLP and have high biological activity, antigen-binding fragments thereof, and uses thereof by screening immune libraries.
  • the present invention provides an anti-TSLP monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes three heavy chains represented by HCDR1, HCDR2 and HCDR3, respectively.
  • chain complementarity determining region the light chain variable region includes three light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3 respectively, and the monoclonal antibody or antigen-binding fragment thereof is selected from any one of the following:
  • the heavy chain complementarity determining region HCDR1 comprises the amino acid sequence shown in SEQ ID No: 1
  • the heavy chain complementarity determining region HCDR2 comprises the amino acid sequence shown in SEQ ID No: 2
  • the heavy chain The complementarity determining region HCDR3 comprises the amino acid sequence shown in SEQ ID No: 3
  • the light chain complementarity determining region LCDR1 comprises the amino acid sequence shown in SEQ ID No: 4
  • the light chain complementarity determining region LCDR2 comprises the amino acid sequence shown in SEQ ID
  • the light chain complementarity determining region LCDR3 comprises the amino acid sequence shown in SEQ ID No:6;
  • the heavy chain complementarity determining region HCDR1 comprises the amino acid sequence shown in SEQ ID No: 7
  • the heavy chain complementarity determining region HCDR2 comprises the amino acid sequence shown in SEQ ID No: 8
  • the heavy chain The complementarity determining region HCDR3 comprises the amino acid sequence shown in SEQ ID No: 9
  • the light chain complementarity determining region LCDR1 comprises the amino acid sequence shown in SEQ ID No: 4
  • the light chain complementarity determining region LCDR2 comprises the amino acid sequence shown in SEQ ID
  • the light chain complementarity determining region LCDR3 comprises the amino acid sequence shown in SEQ ID No:6;
  • the heavy chain complementarity determining region HCDR1 comprises the amino acid sequence shown in SEQ ID No: 1
  • the heavy chain complementarity determining region HCDR2 comprises the amino acid sequence shown in SEQ ID No: 11
  • the heavy chain The complementarity determining region HCDR3 comprises the amino acid sequence shown in SEQ ID No: 3
  • the light chain complementarity determining region LCDR1 comprises the amino acid sequence shown in SEQ ID No: 12
  • the light chain complementarity determining region LCDR2 comprises the amino acid sequence shown in SEQ ID
  • the light chain complementarity determining region LCDR3 comprises the amino acid sequence shown in SEQ ID No:14;
  • the heavy chain complementarity determining region HCDR1 comprises the amino acid sequence shown in SEQ ID No: 1
  • the heavy chain complementarity determining region HCDR2 comprises the amino acid sequence shown in SEQ ID No: 11
  • the heavy chain The complementarity determining region HCDR3 comprises the amino acid sequence shown in SEQ ID No: 3
  • the light chain complementarity determining region LCDR1 comprises the amino acid sequence shown in SEQ ID No: 12
  • the light chain complementarity determining region LCDR2 comprises the amino acid sequence shown in SEQ ID
  • the light chain complementarity determining region LCDR3 comprises the amino acid sequence shown in SEQ ID No:16.
  • the present invention obtains the above four monoclonal antibody molecules capable of binding to TSLP antigens with high affinity by screening the immune library, and has better binding activity, thereby blocking its interaction with receptor complexes, thereby preventing the TSLP-targeted
  • the immune cells released by the immune cells release pro-inflammatory cytokines to prevent asthma attacks and improve asthma control.
  • the monoclonal antibody molecules screened by the present invention also have high thermal stability and meet the requirements for drug preparation.
  • the monoclonal antibody or its antigen-binding fragment is a murine antibody molecule, and the murine antibody molecule is selected from any of the following:
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 18;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 19, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 20;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 21, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 22;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 24;
  • the mouse antibody molecule is MA-I.
  • the present invention immunizes mice with TSLP antigen, optimizes the immunization method, creates a phage display library, and screens out the above-mentioned murine antibody molecules with high affinity, good activity and stability. Through a large number of cell-level experiments, it is found that Of the three murine antibody molecules, MA-I has higher biological activity, so MA-I is preferably selected in the present invention.
  • the murine antibody molecule further includes a murine antibody heavy chain constant region and a murine antibody light chain constant region, and the murine antibody heavy chain constant region is selected from murine IgG1, IgG2a, IgG2b or IgG3
  • the heavy chain constant region of type IgG1 wherein, the amino acid sequence of the heavy chain constant region of IgG1 type is shown in SEQ ID No: 26, and the amino acid sequence of the heavy chain constant region of IgG2a type is shown in SEQ ID No: 27, the The amino acid sequence of the heavy chain constant region of the IgG2b type is shown in SEQ ID No: 28, and the amino acid sequence of the heavy chain constant region of the IgG3 type is shown in SEQ ID No: 29; the light chain constant region of the murine antibody is mouse The light chain constant region of C kappa type, and its amino acid sequence is as shown in SEQ ID No:25;
  • the murine antibody molecule includes a heavy chain constant region of a murine IgG1 type and a light chain constant region of a murine C kappa type.
  • the monoclonal antibody or its antigen-binding fragment is a chimeric antibody molecule, and the chimeric antibody molecule includes the heavy chain variable region of the murine antibody molecule, the light chain variable region of the murine antibody molecule region and the constant region of human antibodies.
  • the chimeric antibody molecule includes the variable region sequence of the murine antibody molecule and the constant region of the human antibody.
  • the design of the chimeric antibody molecule is used to verify that the specific function of the CDR has not been changed after the humanization of the constant region of the present invention, and it is a humanized antibody molecule.
  • the research provides the basis for further research and development.
  • the monoclonal antibody or its antigen-binding fragment is a humanized antibody molecule, and the humanized antibody molecule is selected from any of the following:
  • HA-I the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 34, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 35;
  • HA-II the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 34, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 36;
  • HA-III the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 38;
  • HA-IV the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 36;
  • the humanized antibody molecule is HA-I.
  • humanized antibody molecules are screened after humanized design of mouse antibody molecules. Through in vivo and in vitro experimental verification, it is found that among the four humanized antibody molecules provided by the present invention, HA-I has the highest biological activity , the drug effect is the most significant, so HA-I is preferred in the present invention.
  • humanized antibody molecule also includes a human antibody constant region.
  • the humanized antibody molecule is a full-length antibody or an antibody fragment, and the humanized antibody molecule includes one or a combination of Fab, F(ab)2, Fv or ScFv.
  • the human antibody constant region includes a human antibody heavy chain constant region and a human antibody light chain constant region
  • the human antibody heavy chain constant region is selected from human IgG1, IgG2 or IgG4 heavy chains chain constant region
  • the heavy chain constant region amino acid sequence of the IgG1 type is shown in SEQ ID No: 30
  • the heavy chain constant region amino acid sequence of the IgG2 type is shown in SEQ ID No: 31
  • the IgG4 type heavy chain constant region amino acid sequence is shown in SEQ ID No: 31.
  • the amino acid sequence of the chain constant region is shown in SEQ ID No:32
  • the light chain constant region of the human antibody is a light chain constant region of human C kappa type, and its amino acid sequence is shown in SEQ ID No:33;
  • the human antibody constant region includes the heavy chain constant region of human IgG1 type and the light chain constant region of human C kappa type.
  • the present invention also provides a protein comprising the anti-TSLP monoclonal antibody or an antigen-binding fragment thereof.
  • the present invention also provides a polynucleotide molecule encoding the anti-TSLP monoclonal antibody or an antigen-binding fragment thereof.
  • the present invention also provides a recombinant DNA expression vector, which contains the polynucleotide molecule.
  • the present invention also provides a host cell transfected with the recombinant DNA expression vector, and the host cell includes prokaryotic cells, yeast cells, insect cells or mammalian cells;
  • the host cell is a mammalian cell
  • the mammalian cell is HEK293 cell, CHO cell or NSO cell.
  • the present invention also provides a medicine, which comprises the anti-TSLP monoclonal antibody or an antigen-binding fragment thereof.
  • the present invention also provides the use of the anti-TSLP monoclonal antibody or its antigen-binding fragment in the preparation of drugs for treating immune diseases or cancer;
  • the immune disease includes asthma, chronic obstructive pulmonary disease, chronic eosinophilic pneumonia, idiopathic pulmonary fibrosis, allergic dermatitis;
  • the asthma includes severe asthma, eosinophilic or non-eosinophilic Cellular asthma and hypoeosinophilic asthma;
  • the cancer includes pancreatic cancer, non-small cell lung cancer, melanoma, prostate cancer, kidney cancer, colorectal cancer or breast cancer.
  • the present invention also provides a method for treating or preventing TSLP-mediated diseases, the method comprising administering a therapeutically effective amount of the anti-TSLP monoclonal antibody to an individual in need, the diseases including immune diseases or cancer;
  • the immune diseases include asthma, chronic obstructive pulmonary disease, chronic eosinophilic pneumonia, idiopathic pulmonary fibrosis, atopic dermatitis; the asthma includes severe asthma, eosinophilic or non-eosinophilic asthma and hypoeosinophilic asthma;
  • the cancer includes pancreatic cancer, non-small cell lung cancer, melanoma, prostate cancer, kidney cancer, colorectal cancer or breast cancer.
  • the beneficial effects of the present invention include at least: the anti-TSLP monoclonal antibody or its antigen-binding fragment provided by the present invention has a high affinity with the TSLP antigen, can effectively inhibit the binding of the TSLP antigen to its receptor complex, and then prevent the TSLP-targeted
  • the immune cells released pro-inflammatory cytokines can prevent asthma attacks and improve asthma control.
  • the monoclonal antibody molecules screened by the present invention also have high thermal stability and good safety.
  • TSLP monoclonal antibodies or antigen-binding fragments thereof can be used to treat immune diseases or cancers, including but not limited to asthma, chronic obstructive pulmonary disease, chronic eosinophilic pneumonia, idiopathic pulmonary fibrosis, allergic Dermatitis; asthma including but not limited to severe asthma, eosinophilic or non-eosinophilic asthma, and hypoeosinophilic asthma; cancer including but not limited to pancreatic cancer, non-small cell lung cancer, melanoma, prostate cancer, kidney cancer, Colorectal or breast cancer.
  • asthma chronic obstructive pulmonary disease
  • chronic eosinophilic pneumonia chronic eosinophilic pneumonia
  • idiopathic pulmonary fibrosis allergic Dermatitis
  • asthma including but not limited to severe asthma, eosinophilic or non-eosinophilic asthma, and hypoeosinophilic asthma
  • cancer including but not limited to pancreatic cancer, non-small cell lung cancer,
  • Fig. 1 is the plasmid map of the pScFv-Disb-HS vector in Example 2 of the present invention
  • Fig. 2 is the comparative figure of the relative affinity of the anti-TSLP phage monoclonal antibody of gradient dilution ELISA in the embodiment 3 of the present invention
  • Fig. 3 is the collection of maps of carrier pTSE in the embodiment 5 of the present invention.
  • Fig. 4 is the denatured polyacrylamide gel electrophoresis picture of the murine antibody molecule in Example 5 of the present invention.
  • Fig. 5 is a comparison diagram of the binding ability between murine antibody and TSLP in Example 6 of the present invention.
  • Fig. 6 is a comparison diagram of the competition inhibition experiment between the murine antibody and the TSLP receptor protein CRLF2 in Example 7 of the present invention.
  • Figure 7 is a denatured polyacrylamide gel electrophoresis image of the humanized antibody molecule in Example 12 of the present invention.
  • Fig. 8 is a comparison diagram of the binding ability between the humanized antibody molecule and TSLP in Example 15 of the present invention.
  • Fig. 9 is a comparison diagram of the competition inhibition experiment between the humanized antibody and the control antibody in Example 16 of the present invention.
  • Fig. 10 is a cross-binding experiment diagram of humanized antibody and TSLP of different species in Example 17 of the present invention.
  • Fig. 11 is a comparison diagram of an anti-TSLP monoclonal antibody inhibiting the binding of TSLP to cell surface receptors in Example 18 of the present invention.
  • Fig. 12 is a comparison chart of biological activity detection (reporter gene method) of anti-TSLP monoclonal antibody in Example 19 of the present invention.
  • Figure 13 is a comparative diagram of anti-TSLP monoclonal antibodies blocking TSLP-induced release of chemokines from mDC cells in Example 20 of the present invention
  • Fig. 14 is a thermal stability evaluation graph of the anti-TSLP monoclonal antibody HA-1 in Example 21 of the present invention.
  • antibody used herein includes whole antibodies and any antigen-binding fragments thereof.
  • Antibodies include murine antibodies, humanized antibodies, bispecific antibodies or chimeric antibodies.
  • Antibodies can also be Fab, F(ab)2 , Fv or ScFv (single-chain antibody), the antibody can be a naturally occurring antibody or an antibody that has been modified (eg, mutation, deletion, substitution, etc.).
  • variable region and “constant region” used herein mean that the sequence region of the antibody heavy chain and light chain near the N segment is the variable region (V region), and the rest of the amino acid sequence near the C segment is relatively stable, which is
  • the constant region (C region) the variable region includes 3 complementarity determining regions (CDR) and 4 framework regions (FR), each light chain variable region and heavy chain variable region have 3 CDR regions and 4
  • the three CDR regions of the heavy chain are represented by HCDR1, HCDR2 and HCDR3
  • the three CDR regions of the light chain are represented by LCDR1, LCDR2 and LCDR3, respectively.
  • murine antibody molecule as used herein is derived from the antibody obtained after immunizing mice with TSLP antigen.
  • chimeric antibody molecule refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody in the human body.
  • Chimeric antibodies use DNA recombination technology to insert the light and heavy chain variable region genes of mouse monoclonal antibodies into expression vectors containing human antibody constant regions, so that the variable regions of the light and heavy chains in the expressed antibody molecules are of mouse origin , while the constant region is of human origin, nearly 2/3 of the entire antibody molecule is of human origin.
  • the antibodies thus generated have reduced immunogenicity of the murine antibodies while retaining the ability of the parental antibody to specifically bind the antigen.
  • humanized antibody molecule refers to grafting the CDRs of the mouse monoclonal antibody to the variable region of the human antibody to replace the CDRs of the human antibody, so that the human antibody obtains the antigen-binding specificity of the mouse monoclonal antibody. sex while reducing its heterogeneity.
  • CHO cell refers to Chinese hamster ovary cell
  • HEK293 cell refers to human embryonic kidney 293 cell (human embryonic kidney 293 cell)
  • NS0 cell refers to mouse NS0 thymoma cell.
  • Example 1 of the present invention provides an anti-TSLP monoclonal antibody or an antigen-binding fragment thereof, which specifically includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes three regions represented by HCDR1, HCDR2 and HCDR3 respectively
  • the heavy chain complementarity determining region, the light chain variable region includes three light chain complementarity determining regions respectively represented by LCDR1, LCDR2 and LCDR3, and the monoclonal antibody or its antigen-binding fragment is selected from any one of the following.
  • the following CDRs are identified based on the Kabat numbering system.
  • the present invention immunizes mice with TSLP antigen, optimizes the immunization method, creates a phage display library and establishes an antigen site screening method.
  • the construction and screening identification of the specific phage display library are as follows:
  • Step 1 Immunization of mice with TSLP antigen
  • mice were immunized, and the immune antigen was human TSLP (a gene synthesized by Nanjing KingScript Biotechnology Co., Ltd., the company constructed a vector and expressed and purified it).
  • human TSLP a gene synthesized by Nanjing KingScript Biotechnology Co., Ltd., the company constructed a vector and expressed and purified it).
  • Step 2 Construction of phage antibody library
  • mice splenocytes with higher titer Take mouse splenocytes with higher titer, use Trizol reagent (purchased from Ambion, article number: 15596026), extract total RNA in mouse splenocytes, obtain cDNA by RT-PCR, use cDNA as a template, and use degenerate primers ( The degenerate primer reference used: Journal of Immunological Methods 233 (2000) 167-177) for PCR amplification to obtain the heavy chain variable region gene library (VH) and light chain variable region gene library (VL ), the light and heavy chains were double-enzyme-digested respectively, and connected to the same step-by-step enzyme-digested vector to construct the pScFv-Disb-HS-VH-VL gene library.
  • VH heavy chain variable region gene library
  • VL light chain variable region gene library
  • the PscFv-DisB-HS vector was cloned using a series of gene cloning methods
  • the vector pComb3 vector purchased from China Plasmid Vector Strain Cell Strain Gene Collection Center
  • the transformed vector was named pScFv-Disb-HS vector, and its plasmid map was obtained as shown in Figure 1. Based on this vector, a mouse immune phage antibody library was constructed.
  • Step 3 Coat the immune tube with TSLP as the antigen, the amount of antigen coating is 5 ⁇ g/500 ⁇ l/tube, coat overnight at 4°C, then seal the immune tube and immune phage antibody library with 4% skimmed milk powder/PBST respectively, and block at room temperature for 1h .
  • the blocked immune phage antibody library was added to the immune tube for antigen-antibody binding.
  • the amount of phage input was about 10 9 to 10 12 .
  • Step 4 Infect 10ml of the TG1 bacterial solution grown to the logarithmic phase with the above-mentioned neutralized phage, let it stand in a 37°C incubator for 30 minutes, take out part of the bacterial solution for gradient dilution, and spread it on a 2YTAG plate for the calculation of phage output. The remaining bacterial liquid was centrifuged to discard the supernatant, resuspend the bacterial cell pellet in a small amount of culture medium, suck it out and spread it on a large 2YTAG plate to prepare for the next round of screening.
  • Step 5 Scrape off the plated bacteria after the above infection from the large plate, inoculate into 2YTAG liquid medium, shake to the logarithmic phase, add M13KO7 helper phage for superinfection, and culture overnight at 28°C and 220rpm to prepare Phage, PEG/NaCl precipitation purified phage for the next round of screening, a total of phage library enrichment screening.
  • Step 6 Screening of TSLP phage single-chain antibody-positive clones: After one round of screening, pick well-separated monoclonal colonies and inoculate them in a 96-well deep-well plate supplemented with 2YTAG liquid medium, at 37°C, 220rpm Cultivate to its logarithmic growth phase under the condition of , add about 10 10 helper phage M13KO7 to each well, and infect statically at 37°C for 30min. Centrifuge at 4000rpm for 15min, discard the supernatant, resuspend the pellet with 2YTAK, and culture overnight at 28°C and 220rpm.
  • Mouse Antibody Molecules heavy chain variable region sequence light chain variable region sequence MA-I SEQ ID No:17 SEQ ID No:18 MA-II SEQ ID No:19 SEQ ID No: 20 MA-III SEQ ID No: 21 SEQ ID No:22 MA-IV SEQ ID No:23 SEQ ID No:24
  • SEQ ID No:17 amino acid sequence of the heavy chain variable region of MA-I:
  • SEQ ID No: 18 amino acid sequence of light chain variable region of MA-I:
  • SEQ ID No: 19 amino acid sequence of heavy chain variable region of MA-II:
  • SEQ ID No:20 amino acid sequence of the light chain variable region of MA-II:
  • SEQ ID No:21 amino acid sequence of heavy chain variable region of MA-III:
  • SEQ ID No:22 amino acid sequence of light chain variable region of MA-III:
  • SEQ ID No:23 amino acid sequence of heavy chain variable region of MA-IV:
  • SEQ ID No:24 amino acid sequence of light chain variable region of MA-IV:
  • the present invention also relates to an anti-TSLP monoclonal antibody or an antigen-binding fragment thereof, which specifically includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes three regions that are respectively identified by HCDR1,
  • the heavy chain complementarity-determining region represented by HCDR2 and HCDR3 the light chain variable region includes 3 respectively LCDR1, LCDR2 and LCDR3, wherein, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID No: 17, 19, 21 or 23 respectively HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; LCDR1, LCDR2 and LCDR3 are LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID No: 18, 20, 22 or 24, respectively.
  • CDR sequence of the heavy chain variable region or light chain variable region with known amino acid sequence based on the common numbering system (such as Kabat, AbM, Chothia, Contact or IMGT, etc.).
  • the CDR sequences of the heavy chain variable region and the light chain variable region are shown in Example 1 when the Kabat numbering system is used to determine the CDRs.
  • Example 3 Gradient dilution ELISA compares the affinity of anti-TSLP phage monoclonal antibody
  • the four murine antibody molecules (MA-I, MA-II, MA-III and MA-IV) obtained in Example 2 were displayed and purified by monoclonal phage, and then the phage gradient dilution ELISA experiment was carried out to identify the affinity.
  • the antibody was selected from Amgen’s anti-TSLP monoclonal antibody Tezepelumab (also known as AMG157, the patent application number is CN201880026131.3, and the patent name is treating asthma with anti-TSLP antibody).
  • the specific method is as follows:
  • Coat TSLP with pH 9.6 carbonate buffer solution 100ng/well/100 ⁇ l, coat overnight at 4°C, wash three times with PBST, and separate the four phage monoclonal antibodies screened in Example 2
  • Four-fold gradient dilution was used with PBST, and 100 ⁇ l of the diluted sample was added to each well, and allowed to stand at room temperature for 1 hour.
  • the ELISA plate was washed with PBST, and the HRP-anti-M13 (purchased from Bio-viewshine, product number: GE27-9421-01) monoclonal antibody diluted in PBST was added to the ELISA plate and left at room temperature for 1 h.
  • TMB color development kit develops color, develops color at room temperature for 10 minutes, stops with 2M H 2 SO 4 , reads at 450nm/630nm with microplate reader, and calculates the corresponding EC50 value, the specific data is as follows:
  • the four different murine antibody molecules screened out in Example 2 can all bind to TSLP, but compared with the other three murine antibody molecules and the control antibody, the single murine antibody molecules provided by the present invention
  • the cloned antibody MA-I has a higher affinity to TSLP.
  • Example 4 of the present invention further defines on the basis of Example 2 that the murine antibody molecule further includes a murine antibody heavy chain constant region and a murine antibody light chain constant region, and the murine antibody heavy chain constant region is selected from mouse The heavy chain constant region of IgG1 type, IgG2a type, IgG2b type or IgG3 type; wherein, the amino acid sequence of the heavy chain constant region of IgG1 type is shown in SEQ ID No: 26, and the amino acid sequence of the heavy chain constant region of IgG2a type is shown in SEQ ID No Shown in: 27, the heavy chain constant region amino acid sequence of IgG2b type is shown in SEQ ID No: 28, the heavy chain constant region amino acid sequence of IgG3 type is shown in SEQ ID No: 29;
  • the murine antibody light chain constant region is mouse The light chain constant region of C kappa type, and its amino acid sequence is as shown in SEQ ID No:25; Concrete sequence is as follows:
  • SEQ ID No:25 (light chain constant region amino acid sequence of mouse C kappa type):
  • SEQ ID No:26 (the heavy chain constant region amino acid sequence of the IgG1 type of the mouse):
  • SEQ ID No:27 (the heavy chain constant region amino acid sequence of the IgG2a type of mouse):
  • SEQ ID No:28 (the heavy chain constant region amino acid sequence of the IgG2b type of the mouse):
  • SEQ ID No:29 (the amino acid sequence of the heavy chain constant region of the IgG3 type of mouse):
  • Example 5 of the present invention on the basis of Example 4, preferably defines that the murine antibody molecule includes the heavy chain constant region of the murine IgG1 type (the amino acid sequence of which is shown in SEQ ID No: 26) and the light weight chain of the murine Ck type. Chain constant region (its amino acid sequence is shown in SEQ ID No: 25).
  • the antibody preparation method is as follows:
  • the preferred heavy chain constant region is a murine IgG1 type constant region (the amino acid sequence is shown in SEQ ID No: 26), the light chain constant region is the mouse Ck chain (the amino acid sequence is shown in SEQ ID No: 25), pTSE
  • the vector structure is shown in Figure 3 (see paragraph [0019] on page 3 of the specification of CN103525868A for the preparation process of the pTSE vector).
  • HEK293E cells purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, the article number is GNHu43
  • AKTA instrument to obtain 4 monoclonal antibodies through protein A affinity column purification
  • BCA kit Purchased from: Beijing Huitian Oriental Technology Co., Ltd., article number: BCA0020
  • Coat TSLP with pH 9.6 carbonate buffer 100 ng/well/100 ⁇ l, and coat overnight at 4° C. Wash five times with 300 ⁇ l/well PBST, then add 1% BSA-PBST to block at 37°C for 1 hour, add different dilution concentrations of MA-I, MA-II, MA-III and MA-IV mouse antibodies, 4 The initial maximum concentration of each antibody was 1 ⁇ g/ml, and each antibody was diluted 8 times in 5-fold gradients, and incubated at 37°C for 1 hour.
  • the four different murine antibodies screened out can all bind to TSLP.
  • the EC50 value of MA-I among the four murine antibody molecules is the lowest, indicating that it has the ability to bind to TSLP. Better bonding ability.
  • CRLF2-Fc was coated with carbonate buffer solution of pH 9.6, 200 ng/well/100 ⁇ l, and coated overnight at 4° C. Wash five times with 300 ⁇ l/well PBST, then add 1% BSA-PBST to block at 37°C for 1 hour, first add 50 ⁇ l/well of TSLP-His diluted to 10 ⁇ g/ml with 1% BSA-PBST, and then add different Diluted concentrations of MA-I, MA-II, MA-III and MA-IV mouse antibodies and control antibodies, 50 ⁇ l/well, the initial maximum concentration of the five antibodies is 400 ⁇ g/ml, respectively, after 5-fold serial dilution, A total of 8 gradients were diluted for each antibody and incubated at 37°C for 2h.
  • the IC50 value of MA-I is the lowest , and significantly better than the control antibody, indicating that it can effectively inhibit the combination of TSLP and the receptor protein CRLF2.
  • Example 8 of the present invention further defines that the monoclonal antibody or its antigen-binding fragment is a chimeric antibody molecule, and the chimeric antibody molecule includes the heavy chain variable region of the murine antibody molecule in Example 2 and the light chain of the murine antibody molecule Variable regions and human antibody constant regions.
  • the human antibody constant region includes the human antibody heavy chain constant region and the human antibody light chain constant region, the human antibody heavy chain constant region is selected from the heavy chain constant region of human IgG1 type, IgG2 type or IgG4 type, IgG1 type
  • the amino acid sequence of the heavy chain constant region is shown in SEQ ID No:30
  • the amino acid sequence of the IgG2 type heavy chain constant region is shown in SEQ ID No:31
  • the IgG4 type heavy chain constant region amino acid sequence is shown in SEQ ID No:32
  • the light chain constant region of the human antibody is the light chain constant region of human C kappa type, and its amino acid sequence is shown in SEQ ID No: 33;
  • SEQ ID No: 30 amino acid sequence of heavy chain constant region of human IgG1 type:
  • SEQ ID No: 31 amino acid sequence of heavy chain constant region of human IgG2 type:
  • SEQ ID No: 32 amino acid sequence of heavy chain constant region of human IgG4 type:
  • SEQ ID No: 33 (light chain constant region amino acid sequence of human C kappa chain):
  • Example 9 of the present invention further defines the human antibody constant region on the basis of Example 8 to include the heavy chain constant region of human IgG1 type (its amino acid sequence is shown in SEQ ID No: 30) and the light chain region of human C kappa type. Chain constant region (its amino acid sequence is shown in SEQ ID No: 33).
  • the heavy chain variable region VH (SEQ ID No: 17) and the light chain variable region VL gene (SEQ ID No: 18) of the antibody molecule MA-I obtained by screening the immune phage antibody library in Example 2 maintained the mouse sequence Invariant, respectively cloned on the vector pTSE (as shown in Figure 3) containing the heavy chain constant region and light chain constant region genes, the heavy chain constant region is human IgG1 type (amino acid sequence is shown in SEQ ID NO:30) , the light chain constant region is human Ck type (amino acid sequence shown in SEQ ID NO: 33).
  • HEK293E cells purchasedd from: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, article number: GNHu43 were transiently transfected for antibody expression to obtain chimeric antibody CA-I.
  • Example 2 First use the sequence of the murine antibody molecule MA-1 in Example 2 to compare with the human antibody germline database (v-base) to find the human antibody light and heavy chain germlines with higher homology as candidate sequences, and then the mouse
  • the CDR sequence of the source antibody molecule MA-1 was grafted onto the human candidate sequence for homology modeling. Then, the key framework amino acid residues that may play an important role in maintaining the CDR loop structure are calculated by three-dimensional structure simulation, so as to design the back mutation of the humanized antibody.
  • the light and heavy chain variable regions of the designed humanized antibody containing back mutations were optimized and synthesized by Nanjing GenScript Biotechnology Co., Ltd., and then connected to the transient expression vector.
  • the following humanized antibody molecules were obtained: HA-I, HA-II, HA-III, HA-IV, and the sequences of the four monoclonal antibodies screened above were as follows:
  • SEQ ID No: 34 amino acid sequence of the heavy chain variable region of HA-I and HA-II:
  • SEQ ID No:35 amino acid sequence of the light chain variable region of HA-I:
  • SEQ ID No: 36 amino acid sequence of light chain variable region of HA-II and HA-IV:
  • SEQ ID No: 37 amino acid sequence of the heavy chain variable region of HA-III and HA-IV:
  • SEQ ID No: 38 amino acid sequence of the light chain variable region of HA-III:
  • Example 11 of the present invention further defines on the basis of Example 10 that the humanized antibody molecule also includes a human antibody constant region; the human antibody constant region includes a human antibody heavy chain constant region and a human antibody light chain constant region
  • the heavy chain constant region of the human antibody is selected from the heavy chain constant region of human IgG1 type, IgG2 type or IgG4 type, the amino acid sequence of the heavy chain constant region of IgG1 type is shown in SEQ ID No: 30, and the heavy chain constant region of IgG2 type
  • the region amino acid sequence is shown in SEQ ID No: 31, the heavy chain constant region amino acid sequence of IgG4 type is shown in SEQ ID No: 32, the human source antibody light chain constant region is the light chain constant region of human C kappa type, and its amino acid The sequence is shown in SEQ ID No:33.
  • Example 12 of the present invention further defines the constant region of human antibody on the basis of Example 11, including the heavy chain constant region of human IgG1 type (its amino acid sequence is shown in SEQ ID No: 30) and the human C kappa type. Light chain constant region (its amino acid sequence is shown in SEQ ID No: 33).
  • the heavy chain VH and light chain VL coding genes of the four humanized antibody molecules obtained by humanization in the above-mentioned Example 10 were respectively cloned into the vector pTSE containing the heavy chain constant region and light chain constant region genes (as shown in Figure 3 shown), the heavy chain constant region is human IgG1 type (amino acid sequence shown in SEQ ID NO: 30), and the light chain constant region is C k chain (amino acid sequence shown in SEQ ID NO: 33).
  • control antibody, and humanized antibody molecules HA-I, HA-II, HA-III, and HA-IV were transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Cat. No. GNHu43) for antibody expression.
  • Example 13 of the present invention further defines that the humanized antibody molecule is a full-length antibody or an antibody fragment on the basis of the above-mentioned examples, and the humanized antibody molecule includes one of Fab, F(ab)2, Fv or ScFv or several combinations.
  • Embodiment 14 of the present invention further defines the following scheme on the basis of the foregoing embodiments:
  • the present invention also provides a protein comprising the anti-TSLP monoclonal antibody defined in any one of the above embodiments or an antigen-binding fragment thereof.
  • the present invention also provides a polynucleotide molecule encoding the anti-TSLP monoclonal antibody or an antigen-binding fragment thereof as defined in any one of the above embodiments.
  • the present invention also provides a recombinant DNA expression vector, which comprises the polynucleotide molecule defined above.
  • the present invention also provides a host cell transfected with the recombinant DNA expression vector defined above, the host cell includes prokaryotic cells, yeast cells, insect cells or mammalian cells;
  • the host cell is a mammalian cell
  • the mammalian cell is HEK293 cell, CHO cell or NSO cell.
  • the present invention also provides a medicine, which comprises the anti-TSLP monoclonal antibody or antigen-binding fragment thereof defined in any one of the above embodiments.
  • the present invention also provides the use of the anti-TSLP monoclonal antibody or its antigen-binding fragment in the preparation of drugs for treating immune diseases or cancer;
  • the present invention provides a method for treating or preventing TSLP-mediated diseases, the method comprising administering to an individual in need thereof a therapeutically effective amount of an anti-TSLP monoclonal antibody, said diseases including immune diseases or cancer.
  • immune diseases include but are not limited to asthma, chronic obstructive pulmonary disease, chronic eosinophilic pneumonia, idiopathic pulmonary fibrosis, allergic dermatitis; said asthma includes severe asthma, eosinophilic or non-eosinophilic Eosinophilic asthma and hypoeosinophilic asthma.
  • the cancer includes, but is not limited to, pancreatic cancer, non-small cell lung cancer, melanoma, prostate cancer, kidney cancer, colorectal cancer or breast cancer.
  • TSLP has the best binding ability and the highest affinity; at the same time, the EC50 value of HA-I is similar to that of the chimeric antibody CA-I, indicating that the humanized HA-I retains the high affinity of the murine parental antibody MA-I for TSLP.
  • Coat TSLP-His with pH 9.6 carbonate buffer solution 100ng/well/100 ⁇ l, and coat overnight at 4°C. Wash five times with 300 ⁇ l/well PBST, then add 1% BSA-PBST to block at 37°C for 1 hour, first add HA-I, HA-II, HA-III diluted to 4 ⁇ g/ml with 1% BSA-PBST , HA-IV and chimeric antibody CA-I, 50 ⁇ l/well, and then add different dilutions of control antibodies, 50 ⁇ l/well and mix.
  • the initial maximum concentration of the control antibody was 400 ⁇ g/ml, which were serially diluted by 3 times, and 11 gradients were diluted in total, and incubated at 37°C for 2 hours. Wash five times with 300 ⁇ l/well PBST, then add Anti-Human IgG1-HRP (purchased from Sigma, product number: SAB 4200768) diluted 1:5000 with 1% BSA-PBST, and incubate at 37°C for 1 hour. TMB color development kit was used for color development, 100 ⁇ l/well, for 10 min at room temperature, and then 2M H 2 SO 4 was used to stop the color development.
  • the microplate reader reads at 450nm/630nm, and calculates the corresponding IC50 value, the specific data is as follows:
  • the screened 4 different humanized antibodies and chimeric antibodies can all inhibit the binding of TSLP to the control antibody, and the IC50 values of HA-I in these 4 humanized antibody molecules The lowest, the best inhibitory effect.
  • Carbonate buffer at pH 9.6 was used to coat human TSLP-His, mouse TSLP-His (purchased from Sino Biological Science and Technology Co., Ltd., article number: 51005-M08H), monkey TSLP-His (purchased from Inshore Protein Technology Co., Ltd., Cat. No.: CR62) 100ng/well/100 ⁇ l, coated overnight at 4°C.
  • TMB color development kit was used for color development, 100 ⁇ l/well, for 5 minutes at room temperature, and then 2M H 2 SO 4 was used to stop the color development.
  • the microplate reader reads at 450nm/630nm, and calculates the corresponding EC50 value, the specific data is as follows:
  • HA-I has the lowest EC50 value with human TSLP and cynomolgus monkey TSLP, indicating that its binding ability is strong, and it can be used in experimental animal models of cynomolgus monkeys for pharmacological and toxicological research and safety. sex evaluation.
  • the diluted humanized antibodies HA-I, HA-II, HA-III, HA-IV and control antibodies were added to 96-well plates containing 100 ⁇ l of BaF/3-TSLPR cells, 50 ⁇ l/well.
  • Antigen TSLP was diluted to 8 ⁇ g/ml with sample diluent, and added to the above well plate containing BaF/3-TSLPR cells, humanized antibody and control antibody, 50 ⁇ l/well. After mixing gently, the 96-well plate was incubated at 4°C for 1 h. After the incubation, the supernatant was centrifuged at 3000 rpm, and the cell pellet was collected.
  • the screened 4 different humanized antibodies and control antibodies can all compete with TSLP for binding to the cell surface receptor TSLPR.
  • HA-I has the lowest IC50 value and is better than the control antibody, indicating that it has a better blocking effect on the binding of TSLP and its receptor at the cellular level.
  • BaF/3 cells expressing TSLPR, IL-7R ⁇ , and STAT5-Luc primary mouse B cells, purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, article number: 3111C0001CCC000095
  • sample diluent its composition Including 90% IMDM, 10% FBS, 300 ⁇ g/ml Hygromycin, 0.5 ⁇ g/ml Puromycin and 600 ⁇ g/ml Geneticin
  • TSLP-RAS-His antigen to make the concentration to 160 ng/ml ml, mix gently and add the cell solution to a 96-well plate, 50 ⁇ l/well.
  • the humanized antibodies HA-I, HA-II, HA-III, HA-IV and the control antibody were respectively diluted with the sample diluent to an initial concentration of 15 ⁇ g/ml, 3-fold serial dilution in total of 8 gradients, each sample concentration was two multiple holes.
  • the screened four different humanized antibodies and control antibodies can all bind to TSLP, and can compete to inhibit the binding of TSLP to the receptor complex and play a role, blocking intracellular signal path.
  • the construction of the engineered cell line BaF/3-TSLPR-IL7R ⁇ -STAT5-Luc can simulate the proliferation response of human mast cells under the action of TSLP.
  • TSLP stimulates and up-regulates the expression of intracellular proliferation signal (STAT5-Luc) by binding to cell surface TSLPR and IL7R ⁇ receptors.
  • STAT5-Luc intracellular proliferation signal
  • HA-I has the lowest IC50 value and is significantly better than the control antibody, indicating that it can block the binding of TSLP and its receptor at the cellular level, and has the best effect on inhibiting cell proliferation.
  • Example 20 Anti-TSLP monoclonal antibody blocks TSLP-induced release of chemokines from mDC cells
  • Resuscitate PBMC cells use the kit to sort mature DC cells, use the sample diluent (its components include 90% 1640 and 10% FBS) to adjust the cell density to 4 ⁇ 10 5 cells/mL, and add the cell suspension to the 96-well plate Medium, 50 ⁇ l/well.
  • the humanized antibodies HA-I, HA-II, HA-III, HA-IV and the control antibody were diluted to the initial concentration of 80ng/ml with the sample diluent, 3 times of serial dilution, a total of 8 gradients, and the concentration of each sample was two Multiple wells were added to a 96-well plate containing mature DC cells, 25 ⁇ l/well.
  • TSLP protein was diluted to 80ng/ml with sample diluent, and added to a 96-well plate containing mature DC cells, humanized antibody and control antibody, 25 ⁇ l/well. After gently mixing evenly, place the 96-well plate in a 37°C CO 2 incubator for overnight culture, and take the supernatant after about 24 hours, 50 ⁇ l/well.
  • TARC detection was performed according to the instructions of the human TARC ELISA kit (purchased from Dakowi Biotechnology Co., Ltd., catalog number: 1117542). First, use the diluent in the kit to dilute the supernatant 3 times and mix well.
  • the diluted supernatant and standard were added to the sample wells, 100 ⁇ l/well, and incubated at room temperature for 2 hours. After the incubation, wash the plate with washing solution 3 times. Add Biotinylated antibody diluent, 100 ⁇ l/well, and incubate at room temperature for 2h. After the incubation, wash the plate with washing solution 3 times. Add Streptavidin-HRP working solution, 100 ⁇ l/well, and incubate at room temperature for 20 minutes. After the incubation, wash the plate with washing solution 3 times. Add TMB color development solution, 100 ⁇ l/well, incubate at room temperature in the dark for about 15 minutes, and stop color development with 100 ⁇ l/well stop solution. The microplate reader reads the OD value at 450nm and calculates the corresponding IC50 value. The specific data are as follows:
  • the screened 4 different humanized antibodies and control antibodies can all inhibit TSLP from activating mDC cells to release the chemokine TARC.
  • HA-I has the lowest IC50 value and is significantly better than the control antibody, indicating that it can effectively inhibit the activation effect of TSLP on mDC at the cellular level, and the inhibitory effect is the best.
  • the temperature of the anti-TSLP monoclonal antibody HA-I is 71.7°C
  • the average Tagg is 83.0°C, showing good conformational stability and colloidal stability.
  • Example 21 Toxicity test of anti-TSLP monoclonal antibody subcutaneously administered to cynomolgus monkeys for 4 weeks
  • the purpose of the study is to observe the toxic reaction after subcutaneous injection of anti-TSLP monoclonal antibody in cynomolgus monkeys once a week for 4 consecutive weeks.
  • Test product information anti-TSLP monoclonal antibody HA-I, formulated into injection (excipients include histidine, histidine hydrochloride, sorbitol, polysorbate 80);
  • Reference substance information buffer solution of anti-TSLP monoclonal antibody HA-I (ingredients include histidine, histidine hydrochloride, sorbitol, polysorbate 80, except for anti-TSLP monoclonal antibody HA-I which does not contain pharmaceutical ingredients, Same as the other components of the injection) as a control;
  • Species and strains cynomolgus monkeys
  • Gender female, male
  • Subcutaneous injection administration the first administration is recorded as D1, and then administered once a week for 4 consecutive weeks, a total of 5 administrations;
  • Test results During the test period, the clinical observations, body weight, body temperature, electrocardiogram parameters, blood cell counts, blood biochemistry, urine examination, T lymphocyte subsets, and general observations of animals in groups 2-4 in each group showed no obvious indicators Abnormal changes or obvious abnormal changes without regularity.
  • the FIB of female animals in the 300mg/kg dose group increased before the second dose (D8) and the day after the third dose (D16), and returned to normal the day after the last dose (D30), and the rest of the animals showed no abnormalities.
  • the IL-6 of male animals in the 30mg/kg dose group increased, and the IL-10 of female animals in the 300mg/kg dose group increased, and they all recovered the next day.
  • Example 22 Toxicity test of anti-TSLP monoclonal antibody subcutaneously administered to cynomolgus monkeys for 13 weeks
  • Example 21 On the basis of Example 21, the purpose of this study is to evaluate the toxic reaction and toxicokinetics of the anti-TSLP monoclonal antibody given once a week and repeated subcutaneous injection in cynomolgus monkeys for 13 weeks by injecting anti-TSLP monoclonal antibody Medical conditions, and the recovery of toxicity after 6 weeks of drug withdrawal.
  • Test product information anti-TSLP monoclonal antibody HA-I, formulated into injection (excipients include histidine, histidine hydrochloride, sorbitol, polysorbate 80);
  • Reference substance information buffer solution of anti-TSLP monoclonal antibody HA-I (ingredients include histidine, histidine hydrochloride, sorbitol, polysorbate 80, except for anti-TSLP monoclonal antibody HA-I which does not contain pharmaceutical ingredients, Same as the other components of the injection) as a control;
  • Species and strains cynomolgus monkeys
  • Gender female, male
  • Subcutaneous injection administration the first administration was recorded as D1, and then administered once a week for 13 consecutive weeks, with a total of 14 administrations; the following parameters were evaluated in this experiment: clinical symptom observation, body weight, food intake, Body temperature, accompanying safety pharmacological index detection, electrocardiogram, blood pressure, ophthalmological examination, clinical pathological examination (blood count, coagulation function, blood biochemical analysis, urinalysis), immunological index (immune cell phenotype, cytokine, immunoglobulin , complement), anti-drug antibodies, toxicokinetics.

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Abstract

抗TSLP单克隆抗体、其抗原结合片段及其用途。该抗体与TSLP抗原具有较高的亲和力，能够有效抑制TSLP抗原与其受体复合物的结合，进而阻止由TSLP靶向的免疫细胞释放促炎性细胞因子，防止哮喘发作并改善哮喘控制；本发明筛选得到的单克隆抗体分子还具有较高的热稳定性和较好的安全性，本发明能够用于治疗哮喘、慢性阻塞性肺病、慢性嗜酸性粒细胞性肺炎、特发性肺纤维化、过敏性皮炎；哮喘包括重度哮喘、嗜酸细胞性或非嗜酸细胞性哮喘和低嗜酸细胞哮喘。

Description

抗TSLP单克隆抗体、其抗原结合片段及其用途
相关申请的交叉引用
本申请要求2021年12月02日提交的中国专利申请202111461974.6的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及生物医药技术领域，特别涉及一种抗TSLP单克隆抗体、其抗原结合片段及其用途。
背景技术
哮喘是目前全球最常见的慢性疾病之一，哮喘的发病率约为4.3％，全球约有3亿哮喘病人，各国哮喘患病率1％-18％不等，其中10-20％的严重哮喘患者使用目前的治疗方法很难控制，这些患者消耗了哮喘医疗费用的50-60％，由于经济条件的制约，在广大发展中国家，很多患者未能得到有效的诊断和治疗，这其中也包括中国。2019年6月21日，国际权威医学期刊《柳叶刀》发表我国学者完成的大规模人群研究【中国成人肺部健康研究】(CPH Study)的另一项重要成果，揭示了我国哮喘的流行状况，明确我国20岁及以上人群哮喘患病率4.2％，患病人数达到4570万。在中国，哮喘已经成为主要的、需要认真面对和解决的公共卫生与医疗保健问题之一。中国支气管哮喘防治指南指出，目前仍有超过70％的国内哮喘患者控制不佳，患者一年中由于哮喘发作导致急诊及住院发生率达到27％及23％，哮喘控制不佳将严重影响患者的生活质量。
哮喘是一种由多种炎症细胞与介质参与的气道慢性炎症性疾病，这种慢性炎症与气道高反应性相关，临床表现为反复发作的喘息、气促、胸闷、咳嗽等症状，多在夜间和(或)清晨发作、加剧，多数患者可自行缓解或经治疗缓解；病理学上表现为气道的慢性炎症性改变，以及气道重塑，后者包括气道壁增厚、基质与胶原沉积上皮下纤维化、平滑肌增生和肥大，肌成纤维细胞增殖及黏液腺，杯状细胞化生及增生等，给治疗带来极大的困难。迄今全球上市的哮喘药物有很多种，其中大多为化学药物，随着生物药的不断发展，针对哮喘的生物药研发不断被人们关注。
T2炎症驱动(T2高)的哮喘存在于超过三分之二的重度哮喘患者中，其典型特征是T2炎性生物标志物水平升高，诊断和预测性生物标记物(包括血液嗜酸性粒细胞、血清IgE和呼出气一氧化氮(FeNO)、Periostin等)的鉴定，使重症哮喘的靶向治疗领域发生了革命性的变化。以Th2驱动的炎症为靶点的单克隆抗体在成人中重度哮喘患者中通常是安全的。其余大约三分之一的重度哮喘患者不具有激活的T2炎症通路特征，这类患者的非T2驱动疾病采用临床标准指南治疗症状仍不受控。对非2型哮喘研究尚不明确，需要进一步的研究来确定生物标记物来指导不同形式的非2型哮喘的靶向治疗。
胸腺基质淋巴细胞生成素TSLP(Thymic stromal lymphopoietin)是一种针对促炎性刺激(例如肺内过敏原、病毒及其他病原体)产生的上皮细胞因子，具有增强胸腺细胞增生的作用。TSLP驱动下游T2细胞因子的释放，包括IL-4、IL-5和IL-13，导致炎症和哮喘症状。TSLP也能激活参与非T2驱动炎症的多种类型细胞。因此，TSLP在炎症级联反应的早期上游活动已被确定为在广泛哮喘患者群体中的一个潜在靶点。
此外，TSLP通过激活未成熟DC、淋巴细胞、肥大细胞、嗜碱性粒细胞和嗜酸性粒细胞来调节免疫。TSLP通过与其特异性受体TSLPR和共同受体IL-7Rα形成复合物来启动细胞内信号传导。TSLP首先与TSLPR高亲和力结合，然后与IL-7Rα的胞外结构域形成三元复合物。TSLP的两个相对面分别与TSLPR和IL-7Rα相互作用。STAT5的激活是TSLP介导的Th2反应所必需的信号。抗TSLP的人源化单克隆抗体能够特异性地结合人TSLP并阻断其与受体复合物的相互作用，由此可能阻止由TSLP靶向的免疫细胞释放促炎性细胞因子，从而防止哮喘发作并改善哮喘控制。
目前，阿斯利康及其合作伙伴安进公司的Tezepelumab(又名AMG157)是首个靶向TSLP的单克隆抗体药物，但目前仍未上市，通过临床研究显示，抗TSLP的人源化单克隆抗体作用于炎症级联反应的早期上游，适用于广泛的重症不受控哮喘患者，包括非T2驱动的哮喘患者，在轻度、特应性哮喘患者中开展的一项概念验证吸入性过敏原挑战研究证实，证明其能够抑制早期和晚期的哮喘反应，并降低T2炎症生物标志物水平。鉴于TSLP靶点药物在哮喘治疗上的重要性，为了满足国内外哮喘患者的需求，急需开发针对哮喘具有单独治疗或者辅助治疗的单克隆抗体治疗药物。
发明内容
为了满足市场需求，本发明通过对免疫文库的筛选，得到了可以与TSLP特异性结合且具有较高生物学活性的抗TSLP单克隆抗体、其抗原结合片段及其用途。
本发明具体技术方案如下：
本发明提供了一种抗TSLP单克隆抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述重链可变区包括3个分别用HCDR1、HCDR2和HCDR3表示的重链互补决定区，所述轻链可变区包括3个分别用LCDR1、LCDR2和LCDR3表示的轻链互补决定区，所述单克隆抗体或其抗原结合片段选自以下任意一种：
A-Ⅰ：所述重链互补决定区HCDR1包含如SEQ ID No:1所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:2所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:3所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:4所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:5所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:6所示的氨基酸序列；
A-Ⅱ：所述重链互补决定区HCDR1包含如SEQ ID No:7所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:8所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:9所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:4所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:10所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:6所示的氨基酸序列；
A-Ⅲ：所述重链互补决定区HCDR1包含如SEQ ID No:1所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:11所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:3所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:12所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:13所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:14所示的氨基酸序列；
A-Ⅳ：所述重链互补决定区HCDR1包含如SEQ ID No:1所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:11所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:3所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:12所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:15所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:16所示的氨基酸序列。
本发明通过对免疫文库的筛选得到上述4种能够与TSLP抗原高亲和力结合的单克隆抗体分子，而且结合活性较好，从而阻断其与受体复合物的相互作用，进而阻止由TSLP靶向的免疫细胞释放促炎性细胞因子，防止哮喘发作并改善哮喘控制，此外，本发明筛选得到的单克隆抗体分子还具有较高的热稳定性，满足成药条件。
进一步的，所述单克隆抗体或其抗原结合片段为鼠源抗体分子，所述鼠源抗体分子选自以下任意一种：
MA-Ⅰ：所述重链可变区包含如SEQ ID No:17所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:18所示的氨基酸序列；
MA-Ⅱ：所述重链可变区包含如SEQ ID No:19所示的氨基酸序列，所述轻链可变区包含如SEQ  ID No:20所示的氨基酸序列；
MA-Ⅲ：所述重链可变区包含如SEQ ID No:21所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:22所示的氨基酸序列；
MA-Ⅳ：所述重链可变区包含如SEQ ID No:23所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:24所示的氨基酸序列；
优选的，所述鼠源抗体分子为MA-Ⅰ。
本发明通过用TSLP抗原免疫小鼠，优化免疫方法，创建噬菌体展示库，并筛选出上述亲和力较高，活性较好且较为稳定的鼠源抗体分子，通过大量的细胞水平实验验证，发现相对其他3个鼠源抗体分子，MA-Ⅰ具有更高的生物学活性，为此，本发明优选的选择MA-Ⅰ。
进一步的，所述鼠源抗体分子还包括鼠源抗体重链恒定区和鼠源抗体轻链恒定区，所述鼠源抗体重链恒定区选自鼠的IgG1型、IgG2a型、IgG2b型或IgG3型的重链恒定区；其中，所述IgG1型的重链恒定区氨基酸序列如SEQ ID No:26所示，所述IgG2a型的重链恒定区氨基酸序列如SEQ ID No:27所示，所述IgG2b型的重链恒定区氨基酸序列如SEQ ID No:28所示，所述IgG3型的重链恒定区氨基酸序列如SEQ ID No:29所示；所述鼠源抗体轻链恒定区为鼠C k型的轻链恒定区，且其氨基酸序列如SEQ ID No:25所示；
优选的，所述鼠源抗体分子包括鼠的IgG1型的重链恒定区和鼠C k型的轻链恒定区。
进一步的，所述单克隆抗体或其抗原结合片段为嵌合抗体分子，所述嵌合抗体分子包括所述鼠源抗体分子的重链可变区、所述鼠源抗体分子的轻链可变区和人源抗体恒定区。
嵌合抗体分子包括鼠源抗体分子的可变区序列和人源抗体恒定区，嵌合抗体分子的设计用于验证本发明恒定区人源化后没有改变CDR的特异功能，为人源化抗体分子的研究提供了进一步的研发基础。
进一步的，所述单克隆抗体或其抗原结合片段为人源化抗体分子，所述人源化抗体分子选自以下任意一种：
HA-Ⅰ：所述重链可变区包含如SEQ ID No:34所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:35所示的氨基酸序列；
HA-Ⅱ：所述重链可变区包含如SEQ ID No:34所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:36所示的氨基酸序列；
HA-Ⅲ：所述重链可变区包含如SEQ ID No:37所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:38所示的氨基酸序列；
HA-Ⅳ：所述重链可变区包含如SEQ ID No:37所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:36所示的氨基酸序列；
优选的，所述人源化抗体分子为HA-Ⅰ。
本发明针对鼠源抗体分子进行人源化设计后筛选得到人源化抗体分子，通过体内外的实验验证发现本发明提供的4种人源化抗体分子中，HA-Ⅰ的生物学活性较高，药效最为显著，所以本发明优选HA-Ⅰ。
进一步的，所述人源化抗体分子还包括人源抗体恒定区。
进一步的，所述人源化抗体分子为全长抗体或抗体片段，所述人源化抗体分子包括Fab、F(ab)2、Fv或ScFv中的一种或几种组合。
进一步的，所述人源抗体恒定区包括人源抗体重链恒定区和人源抗体轻链恒定区，所述人源抗体重链恒定区选自人的IgG1型、IgG2型或IgG4型的重链恒定区，所述IgG1型的重链恒定区氨基酸序列如SEQ ID No:30所示，所述IgG2型的重链恒定区氨基酸序列如SEQ ID No:31所示，所述IgG4型的重链恒定区氨基酸序列如SEQ ID No:32所示，所述人源抗体轻链恒定区为人C k型的轻链恒定区，且其氨基酸序列如SEQ ID No:33所示；
优选的，所述人源抗体恒定区包括人的IgG1型的重链恒定区和人C k型的轻链恒定区。
本发明还提供了一种蛋白，其包含所述的抗TSLP单克隆抗体或其抗原结合片段。
本发明还提供了一种多核苷酸分子，所述多核苷酸分子编码所述的抗TSLP单克隆抗体或其抗原结合片段。
本发明还提供了一种重组DNA表达载体，所述重组DNA表达载体包含所述的多核苷酸分子。
本发明还提供了一种转染所述的重组DNA表达载体的宿主细胞，所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞；
优选的，所述宿主细胞为哺乳动物细胞，所述哺乳动物细胞为HEK293细胞、CHO细胞或NS0细胞。
本发明还提供了一种药物，所述药物包含所述的抗TSLP单克隆抗体或其抗原结合片段。
本发明还提供了所述的抗TSLP单克隆抗体或其抗原结合片段在制备治疗免疫性疾病或癌症药物中的用途；
优选的，所述免疫性疾病包括哮喘、慢性阻塞性肺病、慢性嗜酸性粒细胞性肺炎、特发性肺纤维化、过敏性皮炎；所述哮喘包括重度哮喘、嗜酸细胞性或非嗜酸细胞性哮喘和低嗜酸细胞性哮喘；
优选的，所述癌症包括胰腺癌、非小细胞肺癌、黑色素瘤、前列腺癌、肾癌、结直肠癌或乳腺癌。
本发明还提供了一种用于治疗或预防TSLP介导的疾病的方法，该方法包括向有需要的个体施用治疗有效量的所述的抗TSLP单克隆抗体，所述疾病包括免疫性疾病或癌症；
所述免疫性疾病包括哮喘、慢性阻塞性肺病、慢性嗜酸性粒细胞性肺炎、特发性肺纤维化、过敏性皮炎；所述哮喘包括重度哮喘、嗜酸细胞性或非嗜酸细胞性哮喘和低嗜酸细胞哮喘；
所述癌症包括胰腺癌、非小细胞肺癌、黑色素瘤、前列腺癌、肾癌、结直肠癌或乳腺癌。
本发明的有益效果至少包括：本发明提供的抗TSLP单克隆抗体或其抗原结合片段与TSLP抗原具有较高的亲和力，能够有效抑制TSLP抗原与其受体复合物的结合，进而阻止由TSLP靶向的免疫细胞释放促炎性细胞因子，防止哮喘发作并改善哮喘控制，此外，本发明筛选得到的单克隆抗体分子还具有较高的热稳定性和较好的安全性，本发明筛选得到的抗TSLP单克隆抗体或其抗原结合片段能够用于治疗免疫性疾病或癌症，免疫性疾病包括但不限于哮喘、慢性阻塞性肺病、慢性嗜酸性粒细胞性肺炎、特发性肺纤维化、过敏性皮炎；哮喘包括但不限于重度哮喘、嗜酸细胞性或非嗜酸细胞性哮喘和低嗜酸细胞哮喘；癌症包括但不限于胰腺癌、非小细胞肺癌、黑色素瘤、前列腺癌、肾癌、结直肠癌或乳腺癌。
附图说明
图1为本发明实施例2中pScFv-Disb-HS载体的质粒图谱；
图2为本发明实施例3中梯度稀释ELISA抗TSLP噬菌体单克隆抗体相对亲和力的比较图；
图3为本发明实施例5中载体pTSE的图谱；
图4为本发明实施例5中鼠源抗体分子的变性聚丙烯酰胺凝胶电泳图；
图5为本发明实施例6中鼠源抗体与TSLP的结合能力比较图；
图6为本发明实施例7中鼠源抗体与TSLP受体蛋白CRLF2的竞争抑制实验比较图；
图7为本发明实施例12中人源化抗体分子的变性聚丙烯酰胺凝胶电泳图；
图8为本发明实施例15中人源化抗体分子与TSLP的结合能力比较图；
图9为本发明实施例16中人源化抗体与对照抗体的竞争抑制实验比较图；
图10为本发明实施例17中人源化抗体与不同种属的TSLP交叉结合实验图；
图11为本发明实施例18中抗TSLP单克隆抗体抑制TSLP与细胞表面受体结合实验比较图；
图12为本发明实施例19中抗TSLP单克隆抗体生物学活性检测(报告基因法)比较图；
图13为本发明实施例20中抗TSLP单克隆抗体阻断TSLP诱导mDC细胞释放趋化因子比较图；
图14为本发明实施例21抗TSLP单克隆抗体HA-1热稳定性评价图。
具体实施方式
为了更加容易理解本发明，描述实施例之前，先对本发明某些技术和科学术语作以下说明：
本文所使用的术语“抗体”，包含全抗体及其任一抗原结合片段，抗体包括鼠源抗体、人源化抗体、双特异抗体或嵌合抗体，抗体也可以是Fab、F(ab)2、Fv或ScFv(单链抗体)，抗体可以是天然存在的抗体也可以是通过改变(例如突变、缺失、置换等)的抗体。
本文所使用的术语“可变区”和“恒定区”，即为抗体重链和轻链靠近N段的序列区为可变区(V区)，靠近C段的其余氨基酸序列相对稳定，为恒定区(C区)，可变区包括3个互补性决定区(CDR)和4个框架区(FR)，每条轻链可变区和重链可变区均有3个CDR区和4个FR区组成，重链的3个CDR区分别通过HCDR1、HCDR2和HCDR3表示，轻链的3个CDR区分别通过LCDR1、LCDR2和LCDR3表示。
本文所使用的术语“鼠源抗体分子”，其来源是用TSLP抗原免疫注射小鼠后得到的抗体。
本文所使用的术语“嵌合抗体分子”，是将鼠源抗体的可变区与人源抗体的恒定区融合而成的抗体，可以减轻鼠源抗体在人体内诱发的免疫应答反应。嵌合抗体是利用DNA重组技术，将鼠源单抗的轻、重链可变区基因插入含有人抗体恒定区的表达载体中，这样表达的抗体分子中轻重链的可变区是鼠源的，而恒定区是人源的，整个抗体分子的近2/3部分都是人源的。这样产生的抗体，减少了鼠源抗体的免疫原性，同时保留了亲本抗体特异性结合抗原的能力。
本文所使用的术语“人源化抗体分子”，其是将鼠源单抗的CDR移植至人源抗体可变区，替代人源抗体CDR，使人源抗体获得鼠源单抗的抗原结合特异性，同时减少其异源性。
术语“CHO细胞”为中国仓鼠卵巢细胞(chinese hamster ovary cell)；术语“HEK293细胞”为人胚肾293细胞(human embryonic kidney 293cell)，术语“NS0细胞”为小鼠NS0胸腺瘤细胞。
下面结合以下实施例对本发明作进一步详细说明。
实施例1
本发明实施例1提供了一种抗TSLP单克隆抗体或其抗原结合片段，具体包括重链可变区和轻链可变区，重链可变区包括3个分别用HCDR1、HCDR2和HCDR3表示的重链互补决定区，轻链可变区包括3个分别用LCDR1、LCDR2和LCDR3表示的轻链互补决定区，所述单克隆抗体或其抗原结合片段选自以下任意一种。以下CDR基于Kabat编号系统确定。
Figure PCTCN2022132848-appb-000001
实施例2鼠源抗体分子的筛选
本发明通过用TSLP抗原免疫小鼠，优化免疫方法，创建噬菌体展示库并建立抗原位点筛选方法，具体噬菌体展示库的构建与筛选鉴定如下：
步骤一：TSLP抗原免疫小鼠
1、实验动物：
种属品系：BALB/c，雌性，小鼠；
体重：18-20g；
实验动物提供商：北京华阜康生物科技股份有限公司。
2、免疫：对小鼠进行免疫，免疫抗原为人TSLP(南京金斯瑞生物科技有限公司合成基因，本公司构建载体并表达纯化)。
步骤二：噬菌体抗体库的构建
取效价较高的小鼠脾细胞，利用Trizol试剂(购买自Ambion，货号：15596026)，提取小鼠脾细胞中的总RNA，RT-PCR获得cDNA，以cDNA为模板，采用简并引物(所用简并引物参考文献：Journal of Immunological Methods 233(2000)167-177)进行PCR扩增，从而获得免疫小鼠抗体重链可变区基因库(VH)及轻链可变区基因库(VL)，轻重链分别双酶切，连接至同样分步骤酶切处理过的载体上，构建pScFv-Disb-HS-VH-VL基因库，PscFv-DisB-HS载体是采用一系列基因克隆的方法对载体pComb3载体(购自中国质粒载体菌株细胞株基因保藏中心)进行改造，使之用于噬菌体单链抗体库的构建和表达。改造后的载体命名pScFv-Disb-HS载体，获得其质粒图谱如图1所示，并以此载体为基础，构建小鼠免疫噬菌体抗体库。
步骤三：以TSLP为抗原包被免疫管，抗原包被量为5μg/500μl/管，4℃包被过夜，再用4％脱脂奶粉/PBST分别封闭免疫管和免疫噬菌体抗体库，室温封闭1h。封闭后的免疫噬菌体抗体库加入免疫管中进行抗原抗体结合，噬菌体投入量约为10 9～10 12个，室温反应1h后，使用PBST-PBS洗去未结合的噬菌体，通过0.1MpH2.2的Glycine-HCl洗脱，最后使用1.5M pH 8.8的Tris-HCl中和洗脱下来的噬菌体抗体溶液至pH7.0左右。
步骤四：将上述中和后的噬菌体感染10ml生长至对数期的TG1菌液，37℃培养箱中静置30min，取出部分菌液进行梯度稀释，涂布于2YTAG平板上，用于计算噬菌体产出量。剩余的菌液离心弃上清，将菌体沉淀重悬于少量培养基，吸出后涂布于2YTAG大平板，为下一轮筛选做准备。
步骤五：将上述感染后涂板的菌体从大平板上刮下，接菌至2YTAG液体培养基，摇至对数期后加入M13KO7辅助噬菌体超感染，在28℃条件下，220rpm培养过夜制备噬菌体，PEG/NaCl沉降纯化噬菌体用于下一轮筛选，共进行一轮噬菌体库富集筛选。
步骤六：TSLP噬菌体单链抗体阳性克隆的筛选：经过一轮筛选后，挑取分隔良好的单克隆菌落，接种于加有2YTAG液体培养基的96孔深孔板，在37℃条件下，220rpm的条件下培养至其对数生长期，每孔加入约10 10的辅助噬菌体M13KO7，在37℃的温度条件下静止感染30min。4000rpm，离心15min，弃去上清，菌体用2YTAK重悬沉淀，在28℃且220rpm的条件下培养过夜。4000rpm，4℃的条件下离心15min后，吸取扩增后的噬菌体上清进行ELISA鉴定，最终筛选得到四个亲和力较高的抗TSLP的鼠源抗体分子，分别命名为MA-Ⅰ，MA-Ⅱ，MA-Ⅲ和MA-Ⅳ，将上述得到的单克隆抗体进行基因测序确定为正确的抗体序列，经过测序，上述筛选到的4个单克隆抗体序列如下：
鼠源抗体分子 重链可变区序列 轻链可变区序列
MA-Ⅰ SEQ ID No:17 SEQ ID No:18
MA-Ⅱ SEQ ID No:19 SEQ ID No:20
MA-Ⅲ SEQ ID No:21 SEQ ID No:22
MA-Ⅳ SEQ ID No:23 SEQ ID No:24
具体的，SEQ ID No:17(MA-Ⅰ的重链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000002
SEQ ID No:18(MA-Ⅰ的轻链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000003
SEQ ID No:19(MA-Ⅱ的重链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000004
SEQ ID No:20(MA-Ⅱ的轻链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000005
SEQ ID No:21(MA-Ⅲ的重链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000006
SEQ ID No:22(MA-Ⅲ的轻链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000007
SEQ ID No:23(MA-Ⅳ的重链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000008
SEQ ID No:24(MA-Ⅳ的轻链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000009
从另一个角度而言，本发明还涉及一种抗TSLP单克隆抗体或其抗原结合片段，具体包括重链可变区和轻链可变区，重链可变区包括3个分别用HCDR1、HCDR2和HCDR3表示的重链互补决定区，轻链可变区包括3个分别用LCDR1、LCDR2和LCDR3，其中，HCDR1、HCDR2和HCDR3分别为SEQ ID No:17、19、21或23所示的重链可变区的HCDR1、HCDR2和HCDR3；LCDR1、LCDR2和LCDR3分别为SEQ ID No:18、20、22或24所示的轻链可变区的LCDR1、LCDR2和LCDR3。本领域技术人员可以基于常见的编号系统(如Kabat、AbM、Chothia、Contact或IMGT等)确定氨基酸序列已知的重链可变区或轻链可变区的CDR序列。当使用Kabat编号系统确定CDR时，重链可变区和轻链可变区的CDR序列如实施例1所示。
实施例3梯度稀释ELISA比较抗TSLP噬菌体单克隆抗体的亲和力
将实施例2中获得的4个鼠源抗体分子(MA-Ⅰ，MA-Ⅱ，MA-Ⅲ和MA-Ⅳ)进行单克隆噬菌体的展示和纯化，然后进行噬菌体梯度稀释ELISA实验鉴定亲和力，对照抗体选择安进公司的抗TSLP单克隆抗体Tezepelumab(又名AMG157，专利申请号为CN201880026131.3，专利名称为用抗TSLP抗体治疗哮喘)，具体方法如下：
用pH9.6的碳酸盐缓冲液包被TSLP，100ng/孔/100μl，在4℃温度条件下包被过夜，使用PBST洗涤三次，将实施例2中筛选得到的4个噬菌体单克隆抗体分别用PBST四倍梯度稀释，每孔加入100μl稀释后的样品，在室温下静置1小时。用PBST洗涤ELISA板，将PBST稀释后的HRP-anti-M13(购买自Bio-viewshine，货号：GE27-9421-01)单克隆抗体加入ELISA板中，在室温放置1h。TMB显色试剂盒显色，室温显色10分钟，用2M H 2SO 4终止后，酶标仪在450nm/630nm下读数，并计算对应的EC50值，具体数据如下：
克隆 MA-Ⅰ MA-Ⅱ MA-Ⅲ MA-Ⅳ 对照抗体
EC50 2.28 5.515 8.038 41.38 23.14
通过上述数据及如图2所示，实施例2筛选出的4个不同的鼠源抗体分子均能够与TSLP结合，但是与其他3个鼠源抗体分子及对照抗体相比，本发明提供的单克隆抗体MA-Ⅰ与TSLP具有更高的亲和力。
实施例4
本发明实施例4在实施例2的基础上进一步限定了鼠源抗体分子还包括鼠源抗体重链恒定区和鼠源抗体轻链恒定区，所述鼠源抗体重链恒定区选自鼠的IgG1型、IgG2a型、IgG2b型或IgG3型的重链恒定区；其中，IgG1型的重链恒定区氨基酸序列如SEQ ID No:26所示，IgG2a型的重链恒定区氨基酸序列如SEQ ID No:27所示，IgG2b型的重链恒定区氨基酸序列如SEQ ID No:28所示，IgG3型的重链恒定区氨基酸序列如SEQ ID No:29所示；鼠源抗体轻链恒定区为鼠C k型的轻链恒定区，且其氨基酸序列如SEQ ID No:25所示；具体序列如下：
SEQ ID No:25(鼠C k型的轻链恒定区氨基酸序列)：
Figure PCTCN2022132848-appb-000010
SEQ ID No:26(鼠的IgG1型的重链恒定区氨基酸序列)：
Figure PCTCN2022132848-appb-000011
SEQ ID No:27(鼠的IgG2a型的重链恒定区氨基酸序列)：
Figure PCTCN2022132848-appb-000012
SEQ ID No:28(鼠的IgG2b型的重链恒定区氨基酸序列)：
Figure PCTCN2022132848-appb-000013
SEQ ID No:29(鼠的IgG3型的重链恒定区氨基酸序列)：
Figure PCTCN2022132848-appb-000014
实施例5抗TSLP鼠源抗体分子制备
本发明实施例5在实施例4的基础上优选的限定了鼠源抗体分子包括鼠的IgG1型的重链恒定区(其氨基酸序列如SEQ ID No:26所示)和鼠C k型的轻链恒定区(其氨基酸序列如SEQ ID No:25所示)。抗体制备方法具体如下：
1、在将实施例2筛选出来的4个单克隆抗体的重链VH和轻链VL的编码基因分别克隆至装有重链和轻链恒定区基因的载体pTSE(如图3所示)，优选的重链恒定区为鼠的IgG1型恒定区(氨基酸序列如SEQ ID No:26所示)，轻链恒定区为鼠源C k链(氨基酸序列如SEQ ID No:25所示)，pTSE载体结构如图3所示(pTSE载体制备过程参见CN103525868A说明书第3页第[0019]段)。
2、瞬时转染HEK293E细胞(购自中国医学科学院基础医学研究所，货号为GNHu43)，进行抗体表达，使用AKTA仪器通过protein A亲和柱纯化获得4个单克隆抗体，同时使用BCA试剂盒(购买自：北京汇天东方科技有限公司，货号：BCA0020)进行蛋白浓度测定，之后通过SDS-PAGE鉴定蛋白大小，结果如图4所示，从左侧到右侧依次为非还原MA-Ⅰ，MA-Ⅱ，MA-Ⅲ和MA-Ⅳ和蛋白质分子量Marker及还原MA-Ⅰ，MA-Ⅱ，MA-Ⅲ和MA-Ⅳ鼠源抗TSLP单克隆抗体，每条带的分子量大小与理论一致。
实施例6鼠源抗体与TSLP的结合实验
用pH9.6的碳酸盐缓冲液包被TSLP，100ng/孔/100μl，在4℃的温度条件下过夜包被。用300μl/孔PBST洗涤五次，再加入1％BSA-PBST在37℃温度条件下封闭1h，加入不同稀释浓度的MA-Ⅰ，MA-Ⅱ，MA-Ⅲ和MA-Ⅳ鼠源抗体，4种抗体的起始最高浓度均是1μg/ml，分别经过5倍梯度稀释，每个抗体共稀释8个梯度，在37℃温度条件下孵育1h。用300μl/孔PBST洗涤五次，再加入用1％BSA-PBST 1:2000稀释的Goat Anti-Mouse IgG-HRP(购买自solarbio，货号：SE131)，在37℃温度条件下孵育1h。TMB显色试剂盒显色，100μl/孔，室温显色8min，然后用2MH 2SO 4终止显色。酶标仪在450nm/630nm下读数，并计算对应的EC50值，具体数据如下：
克隆 MA-Ⅰ MA-Ⅱ MA-Ⅲ MA-Ⅳ
EC50(ng/ml) 1.099 2.041 1.983 5.572
通过上述数据及如图5所示，筛选出的4个不同的鼠源抗体均能与TSLP进行结合，此外，这4个鼠源抗体分子中MA-Ⅰ的EC50值最低，说明其与TSLP具有更好的结合能力。
实施例7鼠源抗体与TSLP受体蛋白CRLF2的竞争抑制实验
用pH9.6的碳酸盐缓冲液包被CRLF2-Fc，200ng/孔/100μl，在4℃的温度条件下过夜包被。用300μl/孔PBST洗涤五次，再加入1％BSA-PBST在37℃温度条件下封闭1h，先加入经过1％BSA-PBST稀释至10μg/ml的TSLP-His，50μl/孔，再加入不同稀释浓度的MA-Ⅰ，MA-Ⅱ，MA-Ⅲ和MA-Ⅳ鼠源抗体及对照抗体，50μl/孔，5种抗体的起始最高浓度均是400μg/ml，分别经过5倍梯度稀释，每个抗体共稀释8个梯度，在37℃温度条件下孵育2h。用300μl/孔PBST洗涤五次，再加入用2％BSA-PBST 1:5000稀释的Anti-His-Tag Mouse-HRP(购买自北京康为世纪生物科技有限公司，货号：CW0285)，在37℃温度条件下孵育1h。TMB显色试剂盒显色，100μl/孔，室温显色10min，然后用2MH 2SO 4终止显色。酶标仪在450nm/630nm下读数，并计算对应的IC50值，具体数据如下：
克隆 MA-Ⅰ MA-Ⅱ MA-Ⅲ MA-Ⅳ 对照抗体
IC50(ng/ml) 1523 15626 2402 11816 3460
通过上述数据及如图6所示，筛选出的4个不同的鼠源抗体均能与受体蛋白CRLF2产生竞争，此外，本发明提供的4个鼠源抗体分子中MA-Ⅰ的IC50值最低，且明显优于对照抗体，说明其能够有效的抑制了TSLP与受体蛋白CRLF2的结合。
实施例8
本发明实施例8进一步的限定了单克隆抗体或其抗原结合片段为嵌合抗体分子，嵌合抗体分子包括实施例2中鼠源抗体分子的重链可变区、鼠源抗体分子的轻链可变区和人源抗体恒定区。人源抗体恒定区包括人源抗体重链恒定区和人源抗体轻链恒定区，人源抗体重链恒定区选自人的IgG1型、IgG2型或IgG4型的重链恒定区，IgG1型的重链恒定区氨基酸序列如SEQ ID No:30所示，IgG2型的重链恒定区氨基酸序列如SEQ ID No:31所示，IgG4型的重链恒定区氨基酸序列如SEQ ID No:32所示，人源抗体轻链恒定区为人C k型的轻链恒定区，且其氨基酸序列如SEQ ID No:33所示；
SEQ ID No:30(人的IgG1型的重链恒定区氨基酸序列)：
Figure PCTCN2022132848-appb-000015
SEQ ID No:31(人的IgG2型的重链恒定区氨基酸序列)：
Figure PCTCN2022132848-appb-000016
Figure PCTCN2022132848-appb-000017
SEQ ID No:32(人的IgG4型的重链恒定区氨基酸序列)：
Figure PCTCN2022132848-appb-000018
SEQ ID No:33(人的C k链的轻链恒定区氨基酸序列)：
Figure PCTCN2022132848-appb-000019
实施例9嵌合抗体分子的制备
本发明实施例9在实施例8的基础上进一步限定了人源抗体恒定区包括人的IgG1型的重链恒定区(其氨基酸序列如SEQ ID No:30所示)和人C k型的轻链恒定区(其氨基酸序列如SEQ ID No:33所示)。
具体的制备方法：
将实施例2中免疫噬菌体抗体库筛选得到的抗体分子MA-Ⅰ的重链可变区VH(SEQ ID No:17)和轻链可变区VL基因(SEQ ID No:18)保持鼠源序列不变，分别克隆至装有重链恒定区和轻链恒定区基因的载体pTSE(如图3所示)上，重链恒定区为人的IgG1型(氨基酸序列如SEQ ID NO:30所示)，轻链恒定区为人的C k型(氨基酸序列如SEQ ID NO:33所示)。瞬时转染HEK293E细胞(购买自：中国医学科学院基础医学研究所，货号为：GNHu43)，进行抗体表达，得到嵌合抗体CA-Ⅰ。
实施例10鼠源抗体分子MA-Ⅰ进行人源化
首先使用实施例2中鼠源抗体分子MA-1的序列和人抗体种系数据库(v-base)比较，寻找同源性较高的人抗体轻、重链种系作为候选序列，然后将鼠源抗体分子MA-1的CDR的序列移植到人源候选序列上进行同源建模。然后通过三维结构模拟计算可能对于维持CDR环状结构起重要作用的关键框架氨基酸残基，从而设计人源化抗体的回复突变。将设计好的包含回复突变的人源化抗体的轻、重链可变区分别由南京金斯瑞生物科技有限公司优化合成，然后再连接到瞬时表达载体上，对人源化得到的轻重链组合分析，得到如下人源化抗体分子：HA-Ⅰ，HA-Ⅱ，HA-Ⅲ，HA-Ⅳ，上述筛选到的4个单克隆抗体序列如下：
单克隆抗体 重链可变区 轻链可变区
HA-Ⅰ SEQ ID No:34 SEQ ID No:35
HA-Ⅱ SEQ ID No:34 SEQ ID No:36
HA-Ⅲ SEQ ID No:37 SEQ ID No:38
HA-Ⅳ SEQ ID No:37 SEQ ID No:36
具体的，SEQ ID No:34(HA-Ⅰ和HA-Ⅱ的重链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000020
SEQ ID No:35(HA-Ⅰ的轻链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000021
SEQ ID No:36(HA-Ⅱ与HA-Ⅳ的轻链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000022
SEQ ID No:37(HA-Ⅲ和HA-Ⅳ的重链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000023
SEQ ID No:38(HA-Ⅲ的轻链可变区的氨基酸序列)：
Figure PCTCN2022132848-appb-000024
实施例11
本发明实施例11在实施例10的基础上进一步的限定了人源化抗体分子还包括人源抗体恒定区；人源抗体恒定区包括人源抗体重链恒定区和人源抗体轻链恒定区，人源抗体重链恒定区选自人的IgG1型、IgG2型或IgG4型的重链恒定区，IgG1型的重链恒定区氨基酸序列如SEQ ID No:30所示，IgG2型的重链恒定区氨基酸序列如SEQ ID No:31所示，IgG4型的重链恒定区氨基酸序列如SEQ ID No:32所示，人源抗体轻链恒定区为人C k型的轻链恒定区，且其氨基酸序列如SEQ ID No:33所示。
上述人源抗体恒定区具体序列与实施例8相同。
实施例12人源化抗体分子的制备
本发明实施例12在实施例11的基础上进一步的限定了人源抗体恒定区包括人的IgG1型的重链恒定区(其氨基酸序列如SEQ ID No:30所示)和人C k型的轻链恒定区(其氨基酸序列如SEQ ID No:33所示)。
将上述实施例10人源化得到的4个人源化抗体分子的重链VH和轻链VL的编码基因分别克隆至装有重链恒定区和轻链恒定区基因的载体pTSE(如图3所示)，重链恒定区为人的IgG1型(氨基酸序列如SEQ ID NO:30所示)，轻链恒定区为C k链(氨基酸序列如SEQ ID NO:33所示)。
将对照抗体、和人源化抗体分子HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ分别瞬时转染HEK293细胞(购自中国医学科学院基础医学研究所，货号为GNHu43)，进行抗体表达，使用AKTA仪器通过protein A亲和柱纯化获得单克隆抗体，同时使用BCA试剂盒(购买自：北京汇天东方科技有限公司，货号：BCA0020)进行蛋白浓度测定，之后通过SDS-PAGE鉴定蛋白大小，结果如图7所示，从左侧到右侧依次为非还原蛋白质分子量HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ、实施例9中制备的嵌合抗体CA-Ⅰ、对照抗体、非还原蛋白质分子量Marker1和还原蛋白质分子量Marker2、HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ、嵌合抗体CA-Ⅰ、对照抗体，每条带的分子量大小与理论一致。
实施例13
本发明实施例13在上述实施例的基础上进一步的限定了人源化抗体分子为全长抗体或抗体片段，人源化抗体分子包括Fab、F(ab)2、Fv或ScFv中的一种或几种组合。
实施例14
本发明实施例14在上述实施例的基础上进一步的限定了如下方案：
进一步的，本发明还提供了一种蛋白，其包含上述任意一个实施例限定的抗TSLP单克隆抗体或其抗原结合片段。
本发明还提供了一种多核苷酸分子，多核苷酸分子编码上述任意一个实施例限定的抗TSLP单克隆抗体或其抗原结合片段。
本发明还提供了一种重组DNA表达载体，重组DNA表达载体包含上述限定的多核苷酸分子。
本发明还提供了一种转染上述限定的重组DNA表达载体的宿主细胞，宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞；
优选的，宿主细胞为哺乳动物细胞，哺乳动物细胞为HEK293细胞、CHO细胞或NS0细胞。
本发明还提供了一种药物，药物包含上述任意一个实施例限定的抗TSLP单克隆抗体或其抗原结合片段。
本发明还提供了所述的抗TSLP单克隆抗体或其抗原结合片段在制备治疗免疫性疾病或癌症药物中的用途；
本发明提供了一种用于治疗或预防TSLP介导的疾病的方法，该方法包括向有需要的个体施用治疗有效量的抗TSLP单克隆抗体，所述疾病包括免疫性疾病或癌症。优选的，免疫性疾病包括但不限于哮喘、慢性阻塞性肺病、慢性嗜酸性粒细胞性肺炎、特发性肺纤维化、过敏性皮炎；所述哮喘包括重度哮喘、嗜酸细胞性或非嗜酸细胞性哮喘和低嗜酸细胞哮喘。
优选的，癌症包括但不限于胰腺癌、非小细胞肺癌、黑色素瘤、前列腺癌、肾癌、结直肠癌或乳腺癌。
实施例15人源化抗体分子与TSLP结合实验
用pH9.6的碳酸盐缓冲液包被TSLP-His，200ng/孔/100μl，在4℃的温度条件下过夜包被。用300μl/孔PBST洗涤五次，再加入1％BSA-PBST在37℃温度条件下封闭1h，加入不同稀释浓度的人源化抗体HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ和实施例9中制备的嵌合抗体CA-Ⅰ及对照抗体，6个抗体的起始最高浓度均是5μg/ml，分别经过5倍稀释后每个抗体均做8个梯度，在37℃温度条件下孵育1h。用300μl/孔PBST洗涤五次，再加入用1％BSA-PBST 1:5000稀释的Goat Anti Human IgG-HRP(购买自北京中杉金桥生物技术有限公司，货号：ZB-2304)，在37℃温度条件下孵育1h。TMB显色试剂盒显色，100μl/孔，室温显色5min，然后用2M H 2SO 4终止显色。酶标仪在450nm/630nm下读数，并计算对应的EC50值，具体数据如下：
克隆 HA-Ⅰ HA-Ⅱ HA-Ⅲ HA-Ⅳ 嵌合抗体CA-Ⅰ 对照抗体
EC50(ng/ml) 10.16 20.12 32.9 25.57 13.06 54.99
通过上述数据及实验结果如图8所示，4个不同的人源化抗体分子均能与TSLP进行结合，本发明提供的4个不同的单克隆抗体的EC50值均明显低于对照抗体，说明本发明提供的单克隆抗体与TSLP的结合能力强，亲和力高，此外，从图8及上述数据中还可以得出，4个不同的单克隆抗体中HA-Ⅰ的EC50值最低，说明其与TSLP结合能力最好，亲和力最高；同时HA-Ⅰ的EC50值与嵌合抗体CA-Ⅰ相似，说明人源化后的HA-Ⅰ保留了鼠源亲本抗体MA-Ⅰ与TSLP的高亲和力。
实施例16人源化抗体与对照抗体的竞争抑制实验
用pH9.6的碳酸盐缓冲液包被TSLP-His，100ng/孔/100μl，在4℃的温度条件下过夜包被。用300μl/孔PBST洗涤五次，再加入1％BSA-PBST在37℃温度条件下封闭1h，先加入经过1％BSA-PBST稀释至4μg/ml的HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ及嵌合抗体CA-Ⅰ，50μl/孔，再加入不同稀释浓度的对照抗体，50μl/孔混合。对照抗体的起始最高浓度是400μg/ml，分别经过3倍梯度稀释，共稀释11个梯度，在37℃温度条件下孵育2h。用300μl/孔PBST洗涤五次，再加入用1％BSA-PBST 1:5000稀释的Anti-Human IgG1-HRP(购买自Sigma，货号：SAB 4200768)，在37℃温度条件下孵育1h。TMB显色试剂盒显色，100μl/孔，室温显色10min，然后用2M H 2SO 4终止显色。酶标仪在450nm/630nm下读数，并计算对应的IC50值，具体数据如下：
克隆 HA-Ⅰ HA-Ⅱ HA-Ⅲ HA-Ⅳ 嵌合抗体CA-Ⅰ
IC50(ng/ml) 475.4 626.4 1633 977.7 627.3
通过上述数据及如图9所示，筛选出的4个不同的人源化抗体及嵌合抗体均能够抑制TSLP与对照抗体的结合，同时这4个人源化抗体分子中HA-Ⅰ的IC50值最低，其抑制效果最佳。
实施例17人源化抗体与不同种属的TSLP交叉结合实验
用pH9.6的碳酸盐缓冲液分别包被人TSLP-His、鼠TSLP-His(购买自义翘神州科技股份有限公司，货号：51005-M08H)、猴TSLP-His(购买自近岸蛋白质科技有限公司，货号：CR62)100ng/孔/100μl，在4℃的温度条件下过夜包被。用300μl/孔PBST洗涤五次，再加入1％BSA-PBST在37℃温度条件下封闭1h，加入不同稀释浓度的人源化抗体HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ，4个人源化抗体的起始最高浓度均是16μg/ml，分别经过4倍稀释后每个抗体均做8个梯度，在37℃温度条件下孵育1h。用300μl/孔PBST洗涤五次，再加入用1％BSA-PBST 1:5000稀释的Goat Anti Human  IgG-HRP，在37℃温度条件下孵育1h。TMB显色试剂盒显色，100μl/孔，室温显色5min，然后用2M H 2SO 4终止显色。酶标仪在450nm/630nm下读数，并计算对应的EC50值，具体数据如下：
Figure PCTCN2022132848-appb-000025
通过上述数据及如图10所示，筛选出的4个不同的人源化抗体均能与人TSLP、食蟹猴TSLP进行结合，与鼠TSLP均无结合。此外，这4个人源化抗体分子中，HA-Ⅰ与人TSLP和食蟹猴TSLP的EC50值最低，说明其结合能力强，可在食蟹猴的实验动物模型中进行药理毒理性研究及安全性评价。
实施例18抗TSLP单克隆抗体抑制TSLP与细胞表面受体结合实验
将BaF/3-TSLPR工程细胞株消化计数，利用样品稀释液(其成分包括90％IMDM、10％FBS、300μg/ml Hygromycin)将细胞稀释至1×10 6cells/ml，加入96孔板中，100μl/孔。人源化抗体HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ和对照抗体分别利用样品稀释液稀释至初始浓度为200μg/ml，3倍梯度稀释共10个梯度。将稀释好的人源化抗体HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ和对照抗体分别加入含有100μl BaF/3-TSLPR细胞的96孔板中，50μl/孔。抗原TSLP利用样品稀释液稀释至8μg/ml，加入上述含有BaF/3-TSLPR细胞、人源化抗体及对照抗体的孔板中，50μl/孔。轻轻混匀后，将96孔板置于4℃条件下孵育1h。孵育结束后3000rpm离心弃上清，收集细胞沉淀。向板中加入提前稀释好的Goat Anti Human IgG-Fc抗体(购买自SouthernBiotech，货号：2048-30)，100μl/孔，与每孔细胞沉淀混合均匀，4℃条件下孵育1h。孵育结束后每孔加入100μl PBS缓冲液清洗细胞，3000rpm离心弃上清，每孔加入100μl PBS缓冲液重悬细胞沉淀，流式细胞仪上机检测。收集数据并计算对应的IC50值，具体数据如下：
克隆 HA-Ⅰ HA-Ⅱ HA-Ⅲ HA-Ⅳ 对照抗体
IC50(μg/ml) 0.724 0.921 1.241 1.133 1.477
通过上述数据及如图11所示，筛选出的4个不同的人源化抗体及对照抗体均能与TSLP竞争结合细胞表面受体TSLPR。此外，这4个人源化抗体分子中HA-Ⅰ的IC50值最低且优于对照抗体，说明其在细胞水平上对TSLP与其受体的结合有较好的阻断效果。
实施例19抗TSLP单克隆抗体生物学活性检测(报告基因法)
将表达TSLPR、IL-7Rα、STAT5-Luc的BaF/3(小鼠原B细胞，购买自中国医学科学院基础医学研究所细胞资源中心，货号：3111C0001CCC000095)细胞消化计数，利用样品稀释液(其成分包括90％IMDM、10％FBS、300μg/ml Hygromycin、0.5μg/ml Puromycin和600μg/ml Geneticin)将细胞稀释至1×10 6cells/ml，加入TSLP-RAS-His抗原使其浓度至160ng/ml，轻轻混匀后将细胞液加入96孔板，50μl/孔。人源化抗体HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ和对照抗体分别利用样品稀释液稀释至初始浓度为15μg/ml，3倍梯度稀释共8个梯度，每个样品浓度两个复孔。将稀释好的HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ和对照抗体分别加入含有50μl细胞混合液的96孔板中，轻轻混匀后，将96孔板放入细胞培养箱孵育5h，培养条件37℃、5％CO 2。5h后将96孔板取出，3000rpm/min离心5min，甩弃溶液，加入Glo Lysis Buffer(购买自Promega，货号：E2661)50μl/孔，室温裂解5分钟，轻拍细胞板将细胞裂解液混匀，转移细胞裂解液至384孔板，10μl/孔，加入等量的Bright-GolTMLuciferase Assay System，室温反应2～15min，在酶标仪下读荧光数值，并计算对应的 IC50值，具体数据如下：
克隆 HA-Ⅰ HA-Ⅱ HA-Ⅲ HA-Ⅳ 对照抗体
IC50(ng/ml) 289.2 378.3 469.4 444.9 514.8
通过上述数据及如图12所示，筛选出的4个不同的人源化抗体及对照抗体均能与TSLP进行结合，且能够竞争抑制TSLP与受体复合物结合并发挥作用，阻断胞内信号通路。工程细胞株BaF/3-TSLPR-IL7Rα-STAT5-Luc的构建，可以模拟人肥大细胞在TSLP作用下的增殖反应。TSLP通过与细胞表面TSLPR和IL7Rα受体的结合，激发并上调细胞内增殖信号(STAT5-Luc)的表达。这4个人源化抗体分子能够有效阻断TSLP与细胞表面受体的结合作用，进而抑制细胞内增殖信号的发生发展。此外，这4个人源化抗体分子中HA-Ⅰ的IC50值最低且明显优于对照抗体，说明其在细胞水平上能够阻断TSLP与其受体的结合，抑制细胞增殖效果最优。
实施例20抗TSLP单克隆抗体阻断TSLP诱导mDC细胞释放趋化因子
复苏PBMC细胞，利用试剂盒分选获得mature DC细胞，使用样品稀释液(其成分包括90％1640和10％FBS)调整细胞密度至4×10 5cells/mL，将细胞悬液加入96孔板中，50μl/孔。人源化抗体HA-Ⅰ、HA-Ⅱ、HA-Ⅲ、HA-Ⅳ及对照抗体分别利用样品稀释液稀释至初始浓度80ng/ml，3倍梯度稀释，共8个梯度，每个样品浓度两个复孔，加入到含有mature DC细胞的96孔板中，25μl/孔。TSLP蛋白用样品稀释液稀释至80ng/ml，加入到含有mature DC细胞、人源化抗体及对照抗体的96孔板中，25μl/孔。轻轻混合均匀后，将96孔板置于37℃CO 2培养箱中过夜培养，约24h后取上清，50μl/孔。依据人TARC ELISA试剂盒(购买自达科为生物技术有限公司，货号：1117542)说明书，进行TARC检测。首先利用试剂盒中的稀释液对上清进行3倍稀释，混合均匀。将稀释上清及标准品加入样本孔中，100μl/孔，室温孵育2h。孵育结束后洗液清洗孔板3次。加入Biotinylated antibody稀释液，100μl/孔，室温孵育2h。孵育结束后洗液清洗孔板3次。加入Streptavidin-HRP工作液，100μl/孔，室温孵育20min。孵育结束后洗液清洗孔板3次。加入TMB显色液，100μl/孔，避光室温孵育约15min，100μl/孔终止液终止显色。酶标仪读取450nm处OD值，并计算对应的IC50值，具体数据如下：
克隆 HA-Ⅰ HA-Ⅱ HA-Ⅲ HA-Ⅳ 对照抗体
IC50(ng/ml) 100.1 241.1 313.4 261.3 584.0
通过上述数据及图13所示，筛选出的4个不同的人源化抗体及对照抗体均可以抑制TSLP激活mDC细胞释放趋化因子TARC。此外，这4个人源化抗体分子中HA-Ⅰ的IC50值最低且明显优于对照抗体，说明其在细胞水平上能够有效抑制TSLP对mDC的激活效应，且抑制效果最好。
实施例21抗TSLP单克隆抗体HA-Ⅰ热稳定性评估
将抗TSLP单克隆抗体HA-Ⅰ超滤换液到PBS缓冲体系中，12000rpm，在4℃条件下，离心5min，使用多功能蛋白热稳定性分析系统(购买自Unchained Labs)对抗TSLP单克隆抗体HA-Ⅰ的热稳定性进行评估。通过监测蛋白内源性荧光随温度变化(从25℃开始，以0.3℃/min的升温速度升温至95℃)检测蛋白构象的变化，从而确定蛋白熔解温度Tm，评估蛋白构象稳定性。样品发生聚集时，会导致散射光波发生干涉，散射光信号增加，通过静态光散射测定蛋白的胶体稳定性(以Tagg进行表征)，结果参考如下表和附图14所示。
样品 Tm(℃) Tagg 266(℃)
2mg/ml抗TSLP单克隆抗体HA-Ⅰ 71.7 83.0
如上表和图14显示，抗TSLP单克隆抗体HA-Ⅰ的温度为71.7℃，平均Tagg为83.0℃，显示出较好的构象稳定性和胶体稳定性。
实施例21抗TSLP单克隆抗体皮下注射给予食蟹猴4周毒性试验
研究目的是为观察食蟹猴皮下注射抗TSLP单克隆抗体，每周给药1次，连续给药4周后的毒性反应情况。
(1)供试品信息：抗TSLP单克隆抗体HA-Ⅰ，配制成注射液(辅料包括组氨酸、盐酸组氨酸、山梨醇、聚山梨酯80)；
对照品信息：抗TSLP单克隆抗体HA-Ⅰ的缓冲液(成分包括组氨酸、盐酸组氨酸、山梨醇、聚山梨酯80，除不含药物成分抗TSLP单克隆抗体HA-Ⅰ外，与注射液其他成分相同)作为对照；
(2)提供单位：北京东方百泰生物科技股份有限公司；
(3)实验动物：
种属和品系：食蟹猴；
年龄：2.5-5岁；
体重：2-5kg；
性别：雌、雄；
动物数量：8只，雌、雄各4只；
(4)分组与给药
Figure PCTCN2022132848-appb-000026
皮下注射给药，第1次给药记为D1，之后每周给药1次，连续给药4周，共给药5次；
在此实验中评估以下参数：临床症状观察、体重、食量、体温、伴随的安全药理指标检测、心电图、血压、眼科检查、临床病理检测(血细胞计数、凝血功能、血液生化分析、尿液分析)、免疫学指标(免疫细胞表型、细胞因子、免疫球蛋白、补体)、抗药抗体、毒代动力学。
试验结果：试验期间，各组动物临床观察、体重、体温、心电图参数、血细胞计数、血液生化，尿液检查、T淋巴细胞亚群以及2-4组动物解剖大体观察各项指标均未见明显异常变化或未见规律性的明显异常变化。
此外，300mg/kg剂量组雌性动物第2次药前(D8)以及第3次药后次日(D16)FIB可见升高，末次药后次日(D30)恢复正常，其余动物未见异常。首次药后1-2小时(D1),30mg/kg剂量组雄性动物IL-6升高，300mg/kg剂量组雌性动物IL-10升高，次日均可恢复。首次药后次日(D2)，300mg/kg剂量组雌雄性动物IL-6均有升高，D8药前恢复正常，第2次药前(D8)，300mg/kg剂量组雌性动物还可见IL-10升高，D29恢复正常，其余未见明显异常变化。
试验期间，各组动物均未检测到ADA。
毒代动力学结果显示，抗TSLP单克隆抗体HA-Ⅰ分别以30、100、300mg/kg的剂量皮下注射给予食蟹猴，首次和第4次给药后，血药浓度及暴露量随剂量增加而增加，主要毒代动力学参数未见明显性别差异。每周给药1次，连续给药4次后，EB070在食蟹猴体内有轻微蓄积(蓄积因子为1.49-2.71)。
实施例22抗TSLP单克隆抗体皮下注射给予食蟹猴13周毒性试验
在实施例21的基础上，本试验研究目的是通过注射抗TSLP单克隆抗体，评价抗TSLP单克隆抗体每周给予1次，重复皮下注射给予食蟹猴13周后的毒性反应和毒代动力学情况，以及停药恢复6周后毒性的恢复情况。
(1)供试品信息：抗TSLP单克隆抗体HA-Ⅰ，配制成注射液(辅料包括组氨酸、盐酸组氨酸、山梨醇、聚山梨酯80)；
对照品信息：抗TSLP单克隆抗体HA-Ⅰ的缓冲液(成分包括组氨酸、盐酸组氨酸、山梨醇、聚山梨酯80，除不含药物成分抗TSLP单克隆抗体HA-Ⅰ外，与注射液其他成分相同)作为对照；
(2)提供单位：北京东方百泰生物科技股份有限公司；
(3)实验动物：
种属和品系：食蟹猴；
年龄：3-5岁；
体重：2-5kg；
性别：雌、雄；
动物数量：40只，雌、雄各20只；
(4)分组与给药
Figure PCTCN2022132848-appb-000027
皮下注射给药，第1次给药记为D1，之后每周给药1次，连续给药13周，共给药14次；在此实验中评估以下参数：临床症状观察、体重、食量、体温、伴随的安全药理指标检测、心电图、血压、眼科检查、临床病理检测(血细胞计数、凝血功能、血液生化分析、尿液分析)、免疫学指标(免疫细胞表型、细胞因子、免疫球蛋白、补体)、抗药抗体、毒代动力学。
初步试验结果显示：食蟹猴重复皮下注射给予抗TSLP单克隆抗体HA-Ⅰ后，动物临床观察、体重、体温、眼科检查及大体尸检均未见明显异常。
本发明不局限于上述最佳实施方式，任何人在本发明的启示下都可得出其他各种形式的产品，但不论在其形状或结构上作任何变化，凡是具有与本申请相同或相近似的技术方案，均落在本发明的保护范围之内。

Claims (15)

  1. 一种抗TSLP单克隆抗体或其抗原结合片段，其特征在于，包括重链可变区和轻链可变区，所述重链可变区包括3个分别用HCDR1、HCDR2和HCDR3表示的重链互补决定区，所述轻链可变区包括3个分别用LCDR1、LCDR2和LCDR3表示的轻链互补决定区，所述单克隆抗体或其抗原结合片段选自以下任意一种：
    A-Ⅰ：所述重链互补决定区HCDR1包含如SEQ ID No:1所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:2所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:3所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:4所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:5所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:6所示的氨基酸序列；
    A-Ⅱ：所述重链互补决定区HCDR1包含如SEQ ID No:7所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:8所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:9所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:4所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:10所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:6所示的氨基酸序列；
    A-Ⅲ：所述重链互补决定区HCDR1包含如SEQ ID No:1所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:11所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:3所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:12所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:13所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:14所示的氨基酸序列；
    A-Ⅳ：所述重链互补决定区HCDR1包含如SEQ ID No:1所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:11所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:3所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:12所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:15所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:16所示的氨基酸序列。
  2. 如权利要求1所述的抗TSLP单克隆抗体或其抗原结合片段，其特征在于，所述单克隆抗体或其抗原结合片段为鼠源抗体分子，所述鼠源抗体分子选自以下任意一种：
    MA-Ⅰ：所述重链可变区包含如SEQ ID No:17所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:18所示的氨基酸序列；
    MA-Ⅱ：所述重链可变区包含如SEQ ID No:19所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:20所示的氨基酸序列；
    MA-Ⅲ：所述重链可变区包含如SEQ ID No:21所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:22所示的氨基酸序列；
    MA-Ⅳ：所述重链可变区包含如SEQ ID No:23所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:24所示的氨基酸序列；
    优选的，所述鼠源抗体分子为MA-Ⅰ。
  3. 如权利要求2所述的抗TSLP单克隆抗体或其抗原结合片段，其特征在于，所述鼠源抗体分子还包括鼠源抗体重链恒定区和鼠源抗体轻链恒定区，所述鼠源抗体重链恒定区选自鼠的IgG1型、IgG2a型、IgG2b型或IgG3型的重链恒定区；其中，所述IgG1型的重链恒定区氨基酸序列如SEQ ID No:26所示，所述IgG2a型的重链恒定区氨基酸序列如SEQ ID No:27所示，所述IgG2b型的重链恒定区氨基酸序列如SEQ ID No:28所示，所述IgG3型的重链恒定区氨基酸序列如SEQ ID No:29所示；所述鼠源抗体轻链恒定区为鼠C k型的轻链恒定区，且其氨基酸序列如SEQ ID No:25所示；
    优选的，所述鼠源抗体分子包括鼠的IgG1型的重链恒定区和鼠C k型的轻链恒定区。
  4. 如权利要求2所述的抗TSLP单克隆抗体或其抗原结合片段，其特征在于，所述单克隆抗体或其抗原结合片段为嵌合抗体分子，所述嵌合抗体分子包括所述鼠源抗体分子的重链可变区、所述鼠源抗体分子的轻链可变区和人源抗体恒定区。
  5. 如权利要求1所述的抗TSLP单克隆抗体或其抗原结合片段，其特征在于，所述单克隆抗体或其抗原结合片段为人源化抗体分子，所述人源化抗体分子选自以下任意一种：
    HA-Ⅰ：所述重链可变区包含如SEQ ID No:34所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:35所示的氨基酸序列；
    HA-Ⅱ：所述重链可变区包含如SEQ ID No:34所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:36所示的氨基酸序列；
    HA-Ⅲ：所述重链可变区包含如SEQ ID No:37所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:38所示的氨基酸序列；
    HA-Ⅳ：所述重链可变区包含如SEQ ID No:37所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:36所示的氨基酸序列；
    优选的，所述人源化抗体分子为HA-Ⅰ。
  6. 如权利要求5所述的抗TSLP单克隆抗体或其抗原结合片段，其特征在于，所述人源化抗体分子还包括人源抗体恒定区。
  7. 如权利要求5或6所述的抗TSLP单克隆抗体或其抗原结合片段，其特征在于，所述人源化抗体分子为全长抗体或抗体片段，所述人源化抗体分子包括Fab、F(ab)2、Fv或ScFv中的一种或几种组合。
  8. 如权利要求4或6所述的抗TSLP单克隆抗体或其抗原结合片段，其特征在于，所述人源抗体恒定区包括人源抗体重链恒定区和人源抗体轻链恒定区，所述人源抗体重链恒定区选自人的IgG1型、IgG2型或IgG4型的重链恒定区，所述IgG1型的重链恒定区氨基酸序列如SEQ ID No:30所示，所述IgG2型的重链恒定区氨基酸序列如SEQ ID No:31所示，所述IgG4型的重链恒定区氨基酸序列如SEQ ID No:32所示，所述人源抗体轻链恒定区为人C k型的轻链恒定区，且其氨基酸序列如SEQ ID No:33所示；
    优选的，所述人源抗体恒定区包括人的IgG1型的重链恒定区和人C k型的轻链恒定区。
  9. 一种蛋白，其特征在于，其包含权利要求1-8中任一项所述的抗TSLP单克隆抗体或其抗原结合片段。
  10. 一种多核苷酸分子，其特征在于，所述多核苷酸分子编码权利要求1-8中任一项所述的抗TSLP单克隆抗体或其抗原结合片段。
  11. 一种重组DNA表达载体，其特征在于，所述重组DNA表达载体包含权利要求10所述的多核苷酸分子。
  12. 一种转染如权利要求11所述的重组DNA表达载体的宿主细胞，其特征在于，所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞；
    优选的，所述宿主细胞为哺乳动物细胞，所述哺乳动物细胞为HEK293细胞、CHO细胞或NS0细胞。
  13. 一种药物，其特征在于，所述药物包含权利要求1-8中任一项所述的抗TSLP单克隆抗体或其抗原结合片段。
  14. 权利要求1-8中任一项所述的抗TSLP单克隆抗体或其抗原结合片段在制备治疗免疫性疾病或癌症药物中的用途；
    优选的，所述免疫性疾病包括哮喘、慢性阻塞性肺病、慢性嗜酸性粒细胞性肺炎、特发性肺纤维化、过敏性皮炎；所述哮喘包括重度哮喘、嗜酸细胞性或非嗜酸细胞性哮喘和低嗜酸细胞哮喘；
    优选的，所述癌症包括胰腺癌、非小细胞肺癌、黑色素瘤、前列腺癌、肾癌、结直肠癌或乳腺癌。
  15. 一种用于治疗或预防TSLP介导的疾病的方法，该方法包括向有需要的个体施用治疗有效量的权利要求1-8中任一项所述的抗TSLP单克隆抗体，所述疾病包括免疫性疾病或癌症；
    所述免疫性疾病包括哮喘、慢性阻塞性肺病、慢性嗜酸性粒细胞性肺炎、特发性肺纤维化、过敏性皮炎；所述哮喘包括重度哮喘、嗜酸细胞性或非嗜酸细胞性哮喘和低嗜酸细胞哮喘；
    所述癌症包括胰腺癌、非小细胞肺癌、黑色素瘤、前列腺癌、肾癌、结直肠癌或乳腺癌。
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