Anti-TSLP monoclonal antibodies, antigen-binding fragments thereof, and uses thereof

Abstract

The present invention provides an anti-TSLP monoclonal antibody, an antigen-binding fragment thereof, and uses thereof. Such an antibody has high affinity for the TSLP antigen and can effectively inhibit the binding of the TSLP antigen to its receptor complex, thereby preventing the release of pro-inflammatory cellular factors from immune cells targeted by TSLP, and can improve asthma control while preventing asthma attacks; the monoclonal antibody molecule screened in the present invention further has high thermal stability and good safety, and the present invention can be used to treat asthma, chronic obstructive pulmonary disease, chronic eosinophilic pneumonia, idiopathic pulmonary fibrosis, and allergic dermatitis; asthma includes severe asthma, eosinophilic or non-eosinophilic asthma, and hypoeosinophilic asthma.

Description

[Technical Field]

[0001] [CROSS-CIRCUMSTANCES OF RELATED APPLICATIONS] This application claims priority from Chinese application No. 202111461974.6 filed on December 2, 2021, the contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of biomedicine, and in particular to anti-TSLP monoclonal antibodies, antigen-binding fragments thereof, and uses thereof. [Background technology]

[0003] Asthma is currently one of the most common chronic diseases worldwide. The incidence rate of asthma is approximately 4.3%. There are approximately 300 million asthma patients worldwide. The prevalence rate varies from 1% to 18% across countries, with 10% to 20% of severe asthma patients experiencing difficulty controlling their condition with current treatments, accounting for 50% to 60% of asthma medical costs. Due to economic limitations, many patients in many developing countries, including China, lack access to effective diagnosis and treatment. On June 21, 2019, the internationally recognized medical journal Liuye Dao published another significant finding from the China Adult Lung Health Study (CPHS), a large-scale population study completed by Chinese scholars. The study revealed the prevalence of asthma in China, revealing that the prevalence rate among people aged 20 and over is 4.2%, with 45.7 million patients. In China, asthma has become a major public health and medical problem that must be seriously addressed. According to the Chinese Guidelines for the Prevention and Treatment of Bronchial Asthma, more than 70% of asthma patients in China currently have poorly controlled asthma, with the annual incidence of emergency visits and hospitalizations due to asthma attacks reaching 27% and 23%, respectively. Poor asthma control has a serious impact on patients' quality of life.

[0004] Asthma is a chronic inflammatory airway disease involving a variety of inflammatory cells and mediators. This chronic inflammation is associated with airway hyperresponsiveness and clinical manifestations include recurrent asthma attacks, shortness of breath, chest tightness, and cough. Attacks often worsen at night and / or early morning. Many patients are able to self-medicate or receive medical treatment. Pathologically, it manifests as chronic airway inflammatory changes and airway remodeling, including thickening of the airway wall, matrix and collagen deposition, subepithelial fibrosis, smooth muscle proliferation and hypertrophy, myofibroblast proliferation, and metaplasia and proliferation of mucous glands and goblet cells. This poses significant challenges for treatment. To date, there are many types of asthma medications available worldwide, most of which are chemicals. With the continuous development of biological drugs, the development of biological drugs for asthma has gradually gained attention.

[0005] T2-induced (T2-high) asthma is found in more than two-thirds of patients with severe asthma. Its typical feature is elevated levels of T2-type inflammatory biomarkers. The identification of diagnostic and predictive biomarkers (including blood eosinophils, serum IgE, exhaled nitric oxide (FeNO), and periostin) has revolutionized the field of targeted treatment for severe asthma. Monoclonal antibodies targeting Th2-induced inflammation are generally safe in adult patients with moderate to severe asthma. The remaining approximately one-third of severe asthmatics do not have the characteristics of an activated T2 inflammatory pathway, and these patients' non-T2-induced disease fails to be controlled by standard clinical guidelines. Research on non-T2 asthma remains unclear, and further research is needed to identify biomarkers to guide targeted treatment of different forms of non-T2 asthma.

[0006] Thymic stromal lymphopoietin (TSLP) is an epithelial cell factor produced in response to proinflammatory stimuli (e.g., intrapulmonary allergens, viruses, and other pathogens) and has the ability to enhance thymocyte proliferation. TSLP induces the release of downstream T2 cell factors, including IL-4, IL-5, and IL-13, leading to inflammation and asthma symptoms. TSLP can also activate multiple cell types involved in non-T2-induced inflammation. Therefore, the early upstream activity of TSLP in the inflammatory cascade has already identified it as a potential target in a broad range of asthma patients.

[0007] TSLP also regulates the immune system by activating immature DCs, lymphocytes, hypertrophic cells, basophils, and eosinophils. TSLP initiates intracellular signaling by forming a complex with its specific receptor, TSLPR, and the common receptor, IL-7Rα. TSLP first binds to TSLPR with high affinity and then forms a ternary complex with the extracellular domain of IL-7Rα. The two opposing faces of TSLP interact with TSLPR and IL-7Rα, respectively. STAT5 activation is a signal required for TSLP-mediated Th2 responses. Humanized anti-TSLP monoclonal antibodies can specifically bind to human TSLP and block its interaction with the receptor complex, thereby preventing the release of pro-inflammatory cellular factors from TSLP-targeted immune cells, thereby preventing asthma attacks and improving asthma control.

[0008] Currently, AstraZeneca and its partner Amgen's tezepelumab (also known as AMG157) is the first monoclonal antibody drug targeting TSLP, although it has not yet been launched. Clinical studies have shown that anti-TSLP humanized monoclonal antibodies act early upstream in the inflammatory cascade and are applicable to a wide range of severely uncontrolled asthma patients, including those with non-T2-induced asthma. A proof-of-concept challenge study of inhaled allergens in patients with mild atopic asthma demonstrated that they could suppress early and late asthma responses while reducing T2-type inflammatory biomarker levels. Given the importance of TSLP-targeted drugs in asthma treatment, the development of monoclonal antibody drugs that can be used as monotherapy or adjunctive therapy for asthma is necessary to meet the needs of asthma patients both domestically and internationally. Summary of the Invention [Problem to be solved by the invention]

[0009] To meet market demand, in the present invention, anti-TSLP monoclonal antibodies capable of specifically binding to TSLP and having high biological activity, antigen-binding fragments thereof, and uses thereof are obtained by screening immune libraries. [Means for solving the problem]

[0010] The specific technical solutions of the present invention are as follows:

[0011] The present invention provides an anti-TSLP monoclonal antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region containing three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and a light chain variable region containing three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), and the monoclonal antibody or antigen-binding fragment thereof is: AI: the heavy chain complementarity determining region HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 1, the heavy chain complementarity determining region HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 2, the heavy chain complementarity determining region HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 3, the light chain complementarity determining region LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO: 4, the light chain complementarity determining region LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO: 5, and the light chain complementarity determining region LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 6; A-II: 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 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 NO: 10, and the light chain complementarity determining region LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6; A-III: 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 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 NO: 13, and the light chain complementarity determining region LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; and A-IV: The present invention provides an anti-TSLP monoclonal antibody or an antigen-binding fragment thereof, the anti-TSLP monoclonal antibody being 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: 11; the heavy chain 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 NO: 15; and the light chain complementarity-determining region LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.

[0012] In the present invention, the above four types of monoclonal antibody molecules capable of binding with high affinity to the TSLP antigen were obtained by screening an immune library. The high binding activity of these antibody molecules prevents interaction with the receptor complex and further inhibits the release of pro-inflammatory cellular factors from immune cells targeted by TSLP, thereby preventing asthma attacks and improving asthma control. Furthermore, the monoclonal antibody molecules screened in the present invention also have relatively high thermal stability, meeting the requirements for drug development.

[0013] Furthermore, the monoclonal antibody or antigen-binding fragment thereof is a mouse-derived antibody molecule, and the mouse-derived antibody molecule is MA-I: the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 17, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 18; MA-II: the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 19, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 20; MA-III: the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:21 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:22; and MA-IV: the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 23, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 24; Preferably, the mouse-derived antibody molecule is MA-I.

[0014] In the present invention, mice were immunized with TSLP antigen, the immunization method was optimized, a phage display library was constructed, and the mouse-derived antibody molecules with high affinity, good activity, and stability were screened. Extensive cell-level experiments demonstrated that MA-I has higher biological activity than the other three mouse-derived antibody molecules. Therefore, MA-I is the preferred choice in the present invention.

[0015] Furthermore, the mouse-derived antibody molecule further comprises a mouse-derived antibody heavy chain constant region selected from mouse IgG1, IgG2a, IgG2b, or IgG3 heavy chain constant regions, and a mouse-derived antibody light chain constant region; provided that the amino acid sequence of the IgG1 heavy chain constant region is as shown in SEQ ID NO: 26, the amino acid sequence of the IgG2a heavy chain constant region is as shown in SEQ ID NO: 27, the amino acid sequence of the IgG2b heavy chain constant region is as shown in SEQ ID NO: 28, and the amino acid sequence of the IgG3 heavy chain constant region is as shown in SEQ ID NO: 29; and the mouse-derived antibody light chain constant region is selected from mouse C k a light chain constant region of the type IQIQ, and the amino acid sequence thereof is as set forth in SEQ ID NO: 25; Preferably, the mouse-derived antibody molecule comprises a mouse IgG1 heavy chain constant region and a mouse C k and a light chain constant region of the type

[0016] Furthermore, the monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody molecule, and the chimeric antibody molecule comprises a heavy chain variable region of the mouse-derived antibody molecule, a light chain variable region of the mouse-derived antibody molecule, and a human-derived antibody constant region.

[0017] The chimeric antibody molecule comprises the variable region sequence of a mouse-derived antibody molecule and the constant region of a human-derived antibody. The design of the chimeric antibody molecule is used to verify that the specific function of the CDR is not altered after humanization of the constant region in this invention, and provides a basis for further research and development in the study of humanized antibody molecules.

[0018] Furthermore, the monoclonal antibody or antigen-binding fragment thereof is a humanized antibody molecule, and the humanized antibody molecule is HA-I: the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 34, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 35; HA-II: the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 34, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 36; HA-III: the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 37 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 38; and HA-IV: the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 37, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 36; Preferably, the humanized antibody molecule is HA-I.

[0019] In the present invention, mouse-derived antibody molecules are humanized and then screened for humanized antibody molecules. Verification by in vitro and in vivo experiments shows that, among the four humanized antibody molecules proposed in the present invention, HA-I has the highest biological activity and the most pronounced pharmacological effect. Therefore, HA-I is preferred in the present invention.

[0020] Furthermore, the humanized antibody molecule further comprises a human-derived antibody constant region.

[0021] Furthermore, the humanized antibody molecule is a full-length antibody or an antibody fragment, and the humanized antibody molecule comprises one or a combination of several of Fab, F(ab)2, Fv, or ScFv.

[0022] Furthermore, the human-derived antibody constant region comprises a human-derived antibody heavy chain constant region selected from human IgG1, IgG2, or IgG4 heavy chain constant regions, and a human-derived antibody light chain constant region, wherein the amino acid sequence of the IgG1 heavy chain constant region is as shown in SEQ ID NO: 30, the amino acid sequence of the IgG2 heavy chain constant region is as shown in SEQ ID NO: 31, and the amino acid sequence of the IgG4 heavy chain constant region is as shown in SEQ ID NO: 32, and the human-derived antibody light chain constant region is selected from human C k a light chain constant region of the type IQI, and the amino acid sequence thereof is as set forth in SEQ ID NO: 33; Preferably, the human-derived antibody constant region is a combination of a human IgG1 heavy chain constant region and a human C k and a light chain constant region of the type

[0023] The present invention further provides a protein comprising the above-mentioned anti-TSLP monoclonal antibody or an antigen-binding fragment thereof.

[0024] The present invention further provides a polynucleotide molecule encoding the above-mentioned anti-TSLP monoclonal antibody or antigen-binding fragment thereof.

[0025] The present invention further provides a recombinant DNA expression vector comprising the above polynucleotide molecule.

[0026] The present invention further provides a host cell transfected with the recombinant DNA expression vector, the host cell including a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell; Preferably, the host cell is a mammalian cell, and it is proposed that the mammalian cell is a HEK293 cell, a CHO cell or an NS0 cell.

[0027] The present invention further proposes a drug comprising the above-mentioned anti-TSLP monoclonal antibody or an antigen-binding fragment thereof.

[0028] The present invention further relates to use of the anti-TSLP monoclonal antibody or its antigen-binding fragment in the manufacture of a therapeutic agent for immune diseases or cancer; Preferably, the immune disease includes asthma, chronic obstructive pulmonary disease, chronic eosinophilic pneumonia, idiopathic pulmonary fibrosis, and allergic dermatitis; the asthma includes severe asthma, eosinophilic or non-eosinophilic asthma, and hypoeosinophilic asthma; Preferably, the cancer comprises pancreatic cancer, non-small cell lung cancer, melanoma, prostate cancer, renal cancer, colorectal cancer or breast cancer.

[0029] The present invention further provides a method for treating or preventing a TSLP-mediated disease, comprising administering a therapeutically effective amount of the anti-TSLP monoclonal antibody described above to an individual in need thereof, wherein the disease includes an immune disease or cancer; The immune diseases include asthma, chronic obstructive pulmonary disease, chronic eosinophilic pneumonia, idiopathic pulmonary fibrosis, and allergic 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, renal cancer, colorectal cancer, or breast cancer. [Effects of the Invention]

[0030] The beneficial effects of the present invention include at least the following: the anti-TSLP monoclonal antibodies or antigen-binding fragments thereof provided by the present invention have high affinity for the TSLP antigen and can effectively inhibit the binding of the TSLP antigen to its receptor complex, thereby blocking the release of pro-inflammatory cellular factors from immune cells targeted by TSLP, and can prevent asthma attacks while improving asthma control. In addition, the monoclonal antibody molecules screened by the present invention also have high thermal stability and good safety. The anti-TSLP monoclonal antibodies or antigen-binding fragments thereof screened by the present invention can be used to treat immune diseases or cancer. Examples of immune diseases include, but are not limited to, asthma, chronic obstructive pulmonary disease, chronic eosinophilic pneumonia, idiopathic pulmonary fibrosis, and allergic dermatitis; examples of asthma include, but are not limited to, severe asthma, eosinophilic or non-eosinophilic asthma, and hypoeosinophilic asthma; and examples of cancer include, but are not limited to, pancreatic cancer, non-small cell lung cancer, melanoma, prostate cancer, renal cancer, colorectal cancer, and breast cancer. [Brief explanation of the drawings]

[0031] [Figure 1] 1 shows the plasmid profile of the vector pScFv-Disb-HS in Example 2 of the present invention. [Figure 2] FIG. 10 is a graph comparing the relative affinities of anti-TSLP phage monoclonal antibodies by serial dilution ELISA in Example 3 of the present invention. [Figure 3] 1 shows a profile of vector pTSE in Example 5 of the present invention. [Figure 4] FIG. 10 is a denaturing polyacrylamide gel electrophoresis diagram of mouse-derived antibody molecules in Example 5 of the present invention. [Figure 5] FIG. 10 is a diagram comparing the binding ability of mouse-derived antibodies and TSLP in Example 6 of the present invention. [Figure 6] FIG. 10 is a comparative diagram of a competitive inhibition experiment between a mouse-derived antibody and the TSLP receptor protein CRLF2 in Example 7 of the present invention. [Figure 7]FIG. 10 is a denaturing polyacrylamide gel electrophoresis diagram of the humanized antibody molecule in Example 12 of the present invention. [Figure 8] FIG. 10 is a diagram comparing the binding ability of the humanized antibody molecule and TSLP in Example 15 of the present invention. [Figure 9] FIG. 10 is a comparative diagram of a competitive inhibition experiment between a humanized antibody and a control antibody in Example 16 of the present invention. [Figure 10] FIG. 10 is a diagram showing a cross-linking experiment between a humanized antibody in Example 17 of the present invention and TSLP from different species. [Figure 11] FIG. 10 is a comparative diagram showing an experiment on the inhibition of the binding of TSLP to cell surface receptors by anti-TSLP monoclonal antibodies in Example 18 of the present invention. [Figure 12] FIG. 10 is a comparative diagram showing the detection (reporter gene method) of the biological activity of anti-TSLP monoclonal antibodies in Example 19 of the present invention. [Figure 13] FIG. 10 is a comparative diagram showing the inhibition of chemokine release from TSLP-induced mDC cells by anti-TSLP monoclonal antibodies in Example 20 of the present invention. [Figure 14] FIG. 10 is a diagram evaluating the thermal stability of anti-TSLP monoclonal antibody HA-1 in Example 21 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0032] In order to make the present invention more readily understandable, before describing the examples, some technical and scientific terms in the present invention will be explained below.

[0033] The term "antibody" as used herein includes whole antibodies and any one of their antigen-binding fragments. Antibodies include murine antibodies, humanized antibodies, bispecific antibodies, or chimeric antibodies. Antibodies may be Fab, F(ab)2, Fv, or ScFv (single-chain antibodies). Antibodies may be naturally occurring antibodies or antibodies that have been altered (e.g., by mutation, deletion, substitution, etc.).

[0034] As used herein, the terms "variable region" and "constant region" refer to the sequence regions adjacent to the N-terminus of the heavy and light chains of an antibody, which are variable regions (V regions). The remaining amino acid sequences adjacent to the C-terminus are relatively stable and are constant regions (C regions). The variable regions contain three complementarity-determining regions (CDRs) and four framework regions (FRs). Each light chain variable region and heavy chain variable region consists of three CDR regions and four FR regions. The three CDR regions of the heavy chain are designated HCDR1, HCDR2, and HCDR3, respectively. The three CDR regions of the light chain are designated LCDR1, LCDR2, and LCDR3, respectively.

[0035] As used herein, the term "murine-derived antibody molecule" is derived from an antibody obtained after immunizing a mouse with a TSLP antigen.

[0036] As used herein, the term "chimeric antibody molecule" refers to an antibody in which the variable region of a mouse-derived antibody is fused with the constant region of a human-derived antibody, and can reduce the immune response elicited by mouse-derived antibodies in the human body. Chimeric antibodies are produced by using DNA recombinant technology to insert the light and heavy chain variable region genes of a mouse-derived single antibody into an expression vector containing a human antibody constant region. In the antibody molecule expressed in this manner, the light and heavy chain variable regions are mouse-derived, while the constant regions are human-derived, meaning that nearly two-thirds of the entire antibody molecule is human-derived. Antibodies produced in this manner have reduced immunogenicity compared to mouse-derived antibodies, while retaining the parent antibody's ability to specifically bind to antigens.

[0037] As used herein, the term "humanized antibody molecule" refers to an antibody molecule in which the CDRs of a mouse-derived antibody are grafted into the variable region of a human antibody in place of the CDRs of a human-derived antibody, thereby allowing the human antibody to acquire the antigen-binding specificity of the mouse-derived antibody and simultaneously reducing its heterogeneity.

[0038] The term "CHO ​​cells" refers to Chinese hamster ovary cells. The term "HEK293 cells" refers to human embryonic kidney 293 cells. The term "NS0 cells" refers to mouse NS0 thymoma cells.

[0039] The present invention will now be described in more detail in conjunction with the following examples.

[0040] Example 1 Example 1 of the present invention provides an anti-TSLP monoclonal antibody or an antigen-binding fragment thereof. Specifically, the antibody comprises a heavy chain variable region containing three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and a light chain variable region containing three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3). The monoclonal antibody or antigen-binding fragment thereof is any one selected from the following. The following CDRs are determined according to the Kabat numbering system:

[0041] [Table 1]

[0042] Example 2: Screening of mouse-derived antibody molecules In the present invention, mice were immunized with TSLP antigen, the immunization method was optimized, a phage display library was constructed, and an antigen site screening method was established. The specific construction and screening of the phage display library are as follows:

[0043] Step 1: Immunize mice with TSLP antigen. 1. Experimental Animals Type and breed: BALB / c, female, mouse; Weight: 18-20g; Experimental animals were provided by Beijing Huafukang Biotechnology Co., Ltd. 2. Immunization: Mice were immunized with human TSLP (a synthetic gene from Nanjing Jinsirui Biotechnology Co., Ltd., which was expressed and purified using a vector constructed by our company) as an immunizing antigen.

[0044] Step 2: Construct a phage antibody library. High-titer mouse spleen cells were isolated, and total RNA was extracted using Trizol reagent (purchased from Ambion, product number 15596026). RT-PCR was then performed to obtain cDNA. Using the cDNA as a template, PCR amplification was performed using degenerate primers (reference for the degenerate primers used: Journal of Immunological Methods 233 (2000) 167-177). This resulted in the generation of a heavy chain variable region gene library (VH) and a light chain variable region gene library (VL) of immunized mouse antibodies. Double enzymatic digestion was performed on each of the light and heavy chains, and the resulting libraries were then ligated into similarly digested vectors to construct the pScFv-Disb-HS-VH-VL gene library. The pScFv-Disb-HS vector was modified from the pComb3 vector (purchased from the China Plasmid and Vector Bacterial and Cell Lines Gene Collection Center) using a series of gene cloning procedures, and then used to construct and express the phage single-chain antibody library. The modified vector was named PscFv-Disb-HS vector, and its plasmid profile is shown in Figure 1. Furthermore, a mouse immune phage antibody library based on this vector was constructed.

[0045] Step 3: TSLP is used as an antigen to coat the immunotube. The antigen coating amount is 5 μg / 500 μl / tube, and the coating is left overnight at 4°C. Next, the immunotube and the immunophage-antibody library are blocked with 4% nonfat dry milk / PBST and then blocked at room temperature for 1 hour. The blocked immunophage-antibody library is then placed into the immunotube to allow antigen-antibody binding, and the phage input amount is approximately 10 9 ~10 12After reacting at room temperature for 1 hour, unbound phage are removed with PBST-PBS and eluted with 0.1 M Glycine-HCl, pH 2.2. Finally, the eluted phage-antibody solution is neutralized to approximately pH 7.0 with 1.5 M Tris-HCl, pH 8.8.

[0046] Step 4: 10 ml of TG1 bacterial solution grown to logarithmic phase is infected with the neutralized phage and left to stand in a 37°C incubator for 30 minutes. A portion of the bacterial solution is serially diluted and coated onto 2YTAG plates, and the amount of phage produced is calculated. The remaining bacterial solution is centrifuged and the supernatant is discarded. The bacterial pellet is resuspended in a small amount of medium, aspirated, and coated onto a large 2YTAG plate for the next screening.

[0047] Step 5: After the infection, the bacterial cells coated on the plate are scraped off the large plate and inoculated into 2YTAG liquid medium. After rocking until the plate reaches the logarithmic phase, the auxiliary phage M13KO7 is added for superinfection. The plate is then cultured overnight at 28°C and 220 rpm to produce phages. The phages are precipitated and purified with PEG-NaCl before being prepared for the next round of screening. A total of one phage library enrichment screening is performed.

[0048] Step 6: Screening for TSLP phage single-chain antibody positive clones: After one round of screening, well-isolated monoclonal colonies are screened and inoculated into 96-well deep well plates containing 2YTAG liquid medium. Culture is performed at 37°C and 220 rpm until the colonies reach the logarithmic growth phase. Approximately 10 per well are used. 10The auxiliary phage M13KO7 was added and allowed to infect for 30 minutes at 37°C. After 15 minutes of centrifugation at 4000 rpm, the supernatant was discarded. The bacterial pellet was resuspended in 2YTAK and incubated overnight at 28°C and 220 rpm. After 15 minutes of centrifugation at 4000 rpm and 4°C, the amplified phage supernatant was aspirated and subjected to ELISA assay. Finally, four high-affinity anti-TSLP mouse antibody molecules, designated MA-I, MA-II, MA-III, and MA-IV, were screened. The resulting monoclonal antibodies were sequenced to determine their exact antibody sequences. The sequences of the four monoclonal antibodies screened as described above were as follows:

[0049] [Table 2]

[0050] in particular, SEQ ID NO: 17 (amino acid sequence of the heavy chain variable region of MA-I): [ka] SEQ ID NO: 18 (amino acid sequence of the light chain variable region of MA-I): [ka] SEQ ID NO: 19 (amino acid sequence of the heavy chain variable region of MA-II): [ka] SEQ ID NO: 20 (amino acid sequence of the light chain variable region of MA-II): [ka] SEQ ID NO: 21 (amino acid sequence of the heavy chain variable region of MA-III): [ka] SEQ ID NO: 22 (amino acid sequence of the light chain variable region of MA-III): [ka] SEQ ID NO: 23 (amino acid sequence of the heavy chain variable region of MA-IV): [ka] SEQ ID NO: 24 (amino acid sequence of the light chain variable region of MA-IV): [ka]

[0051] In another aspect, the present invention further relates to an anti-TSLP monoclonal antibody or an antigen-binding fragment thereof, as follows: Specifically, the antibody comprises a heavy-chain variable region including three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), and a light-chain variable region including three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), respectively, where HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region represented by SEQ ID NO: 17, 19, 21, or 23, respectively; and LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light-chain variable region represented by SEQ ID NO: 18, 20, 22, or 24, respectively. Those skilled in the art can determine the CDR sequences of heavy-chain or light-chain variable regions whose amino acid sequences are already known using commonly used numbering systems (e.g., Kabat, AbM, Chothia, Contact, or IMGT). When the Kabat numbering system is used to determine the CDRs, the CDR sequences of the heavy and light chain variable regions are as shown in Example 1.

[0052] Example 3: Comparison of the affinity of anti-TSLP phage monoclonal antibodies by serial dilution ELISA Monoclonal phage were displayed and purified against the four mouse-derived antibody molecules (MA-I, MA-II, MA-III, and MA-IV) obtained in Example 2, and then serial dilution ELISA experiments were performed to assess the affinity of the phage. The control antibody was the anti-TSLP monoclonal antibody Tezepelumab (also known as AMG157, patent application number CN201880026131.3, patent title: Treatment of Asthma with Anti-TSLP Antibodies) from Amgen. The specific method is as follows:

[0053] TSLP was coated with a pH 9.6 carbonate buffer solution at 100 ng / well / 100 μl, coated overnight at 4°C, and washed three times with PBST. Each of the four phage monoclonal antibodies screened in Example 2 was serially diluted 4-fold with PBST, and 100 μl of the diluted sample was added per well and allowed to stand at room temperature for 1 hour. The ELISA plate was washed with PBST, and HRP-anti-M13 monoclonal antibody (purchased from Bio-viewshine, product number: GE27-9421-01) diluted in PBST was added to the ELISA plate and allowed to stand at room temperature for 1 hour. Color development was performed using a TMB color development kit for 10 minutes at room temperature. After the color development was terminated with 2M H2SO4, the values ​​were read at 450 nm / 630 nm on a microplate reader, and the corresponding EC 50 The specific data is as follows:

[0054] [Table 3]

[0055] As shown in the data above and Figure 2, all four different mouse-derived antibody molecules screened in Example 2 can bind to TSLP. However, compared with the other three mouse-derived antibody molecules and the control antibody, the monoclonal antibody MA-I provided by the present invention has a higher affinity for TSLP.

[0056] Example 4 Example 4 of the present invention further limits the following in addition to Example 2: the mouse-derived antibody molecule further comprises a mouse-derived antibody heavy chain constant region selected from mouse IgG1, IgG2a, IgG2b, or IgG3 heavy chain constant regions, and a mouse-derived antibody light chain constant region; provided that the amino acid sequence of the IgG1 heavy chain constant region is as set forth in SEQ ID NO: 26, the amino acid sequence of the IgG2a heavy chain constant region is as set forth in SEQ ID NO: 27, the amino acid sequence of the IgG2b heavy chain constant region is as set forth in SEQ ID NO: 28, and the amino acid sequence of the IgG3 heavy chain constant region is as set forth in SEQ ID NO: 29; and the mouse-derived antibody light chain constant region is selected from mouse C k The light chain constant region of this type has the amino acid sequence shown in SEQ ID NO: 25. The specific sequence is as follows:

[0057] SEQ ID NO: 25 (mouse C k light chain constant region amino acid sequence of type ( [ka] SEQ ID NO: 26 (amino acid sequence of the heavy chain constant region of mouse IgG1 type): [ka] SEQ ID NO: 27 (amino acid sequence of the heavy chain constant region of mouse IgG2a type): [ka] SEQ ID NO: 28 (amino acid sequence of the heavy chain constant region of mouse IgG2b type): [ka] SEQ ID NO: 29 (amino acid sequence of the heavy chain constant region of mouse IgG3 type): [ka]

[0058] Example 5: Production of anti-TSLP mouse-derived antibody molecules In addition to Example 4, Example 5 of the present invention preferably limits the following: the mouse-derived antibody molecule is a heavy chain constant region of mouse IgG1 type (the amino acid sequence of which is shown in SEQ ID NO: 26) and a mouse C k and a light chain constant region of the same type (the amino acid sequence of which is shown in SEQ ID NO: 25). Specifically, the antibody can be produced as follows.

[0059] 1. The heavy chain VH and light chain VL coding genes of the four monoclonal antibodies obtained by screening in Example 2 were each cloned into the vector pTSE incorporating heavy and light chain constant region genes (as shown in Figure 3), with the preferred heavy chain constant region being a mouse IgG1 type constant region (the amino acid sequence of which is shown in SEQ ID NO: 26), and the light chain constant region being a mouse-derived C k The structure of the vector pTSE is shown in Figure 3 (for the construction process of the vector pTSE, see paragraph

[0019] on page 3 of the specification of CN103525868A).

[0060] 2. HEK293E cells (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, product code: GNHu43) were transfected and transfected to express the antibodies. Four monoclonal antibodies were obtained by purifying them on a protein A affinity column using an AKTA instrument. Protein concentration was measured using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., product code: BCA0020). Protein size was then determined using SDS-PAGE. As shown in Figure 4, from left to right, the results are non-reduced MA-I, MA-II, MA-III, MA-IV, protein molecular weight markers, and reduced MA-I, MA-II, MA-III, and MA-IV mouse anti-TSLP monoclonal antibodies. The molecular weights of each band are consistent with theory.

[0061] Example 6: Binding experiment between mouse-derived antibodies and TSLP TSLP was coated in a pH 9.6 carbonate buffer solution at 100 ng / well / 100 μl overnight at 4°C. After washing five times with 300 μl / well of PBST, the plate was blocked with 1% BSA-PBST at 37°C for 1 hour. Mouse antibodies MA-I, MA-II, MA-III, and MA-IV were added at different dilutions. Starting at a maximum concentration of 1 μg / ml, each antibody was serially diluted 5-fold for a total of eight dilutions per antibody. The plate was then incubated at 37°C for 1 hour. After washing five times with 300 μl / well of PBST, Goat Anti-Mouse IgG-HRP (purchased from Solarbio, product number SE131) diluted 1:2000 in 1% BSA-PBST was added and the plate was incubated at 37°C for 1 hour. The color was developed with TMB color development kit, 100 μl / well, at room temperature for 8 min, and then the color development was terminated with 2 M H2SO4. The values ​​were read at 450 nm / 630 nm on a microplate reader, and the corresponding EC 50 The specific data is as follows:

[0062] [Table 4]

[0063] As shown in the data above and in Figure 5, all four different mouse-derived antibodies screened can bind to TSLP. Among these four mouse-derived antibody molecules, only MA-I had the highest EC 50 This indicates that MA-I has a better binding ability to TSLP.

[0064] Example 7: Competitive inhibition experiment between mouse-derived antibodies and the TSLP receptor protein CRLF2 CRLF2-Fc was coated in a pH 9.6 carbonate buffer solution at 200 ng / well in 100 μl, and incubated overnight at 4°C. After washing five times with 300 μl / well of PBST, the plate was blocked with 1% BSA-PBST at 37°C for 1 hour. TSLP-His diluted to 10 μg / ml in 1% BSA-PBST was added at 50 μl / well, followed by mouse antibodies MA-I, MA-II, MA-III, MA-IV, and a control antibody at various dilutions at 50 μl / well. The starting concentration of each antibody was 400 μg / ml, and each antibody was serially diluted five-fold for a total of eight dilutions. The plate was then incubated at 37°C for 2 hours. After washing five times with 300 μl / well of PBST, Anti-His-Tag Mouse-HRP (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., product number: CW0285) diluted 1:5000 in 2% BSA-PBST was added and incubated at 37°C for 1 hour. Color development was performed using a TMB color development kit, with 100 μl / well added at room temperature for 10 minutes, and then 2M H2SO4 was added to terminate the color development. The values ​​were read at 450 nm / 630 nm on a microplate reader, and the corresponding IC 50 The specific data is as follows:

[0065] [Table 5]

[0066] As shown in the above data and Figure 6, all four different mouse-derived antibodies screened can compete with the receptor protein CRLF2. Among the four mouse-derived antibody molecules provided by the present invention, only one antibody had an IC value of MA-I. 50 This value was the lowest and clearly superior to the control antibody, demonstrating that MA-I can effectively inhibit the binding of TSLP to the receptor protein CRLF2.

[0067] Example 8 Example 8 of the present invention further defines the following: the monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody molecule. The chimeric antibody molecule comprises a heavy chain variable region of the mouse-derived antibody molecule in Example 2, a light chain variable region of the mouse-derived antibody molecule, and a human-derived antibody constant region. The human-derived antibody constant region comprises a human-derived antibody heavy chain constant region selected from human IgG1, IgG2, or IgG4 heavy chain constant regions, and a human-derived antibody light chain constant region. The amino acid sequence of the IgG1 heavy chain constant region is as shown in SEQ ID NO: 30, the amino acid sequence of the IgG2 heavy chain constant region is as shown in SEQ ID NO: 31, and the amino acid sequence of the IgG4 heavy chain constant region is as shown in SEQ ID NO: 32. The human-derived antibody light chain constant region comprises a human C k The light chain constant region of this type is the same as that of the IgG1A-type ...

[0068] SEQ ID NO: 30 (amino acid sequence of the heavy chain constant region of human IgG1 type): [ka] SEQ ID NO: 31 (amino acid sequence of the heavy chain constant region of human IgG2 type): [ka] SEQ ID NO: 32 (amino acid sequence of the heavy chain constant region of human IgG4 type): [ka] SEQ ID NO: 33 (human C k light chain constant region amino acid sequence of type ( [ka]

[0069] Example 9: Production of chimeric antibody molecules Example 9 of the present invention further defines the following in addition to Example 8: the human-derived antibody constant region is a heavy chain constant region of human IgG1 type (the amino acid sequence of which is shown in SEQ ID NO: 30) and a human C kand a light chain constant region of the type (the amino acid sequence of which is set forth in SEQ ID NO:33).

[0070] The specific manufacturing method is as follows. The heavy chain variable region VH (SEQ ID NO: 17) and light chain variable region VL (SEQ ID NO: 18) genes of the antibody molecule MA-I screened from the immune phage antibody library in Example 2 were cloned into the vector pTSE incorporating the heavy chain constant region and light chain constant region genes, with the mouse-derived sequences remaining unchanged (as shown in Figure 3). The heavy chain constant region is of human IgG1 type (amino acid sequence shown in SEQ ID NO: 30). The light chain constant region is of human C type. k The amino acid sequence is shown in SEQ ID NO: 33. HEK293E cells (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, product code: GNHu43) were transiently transfected to express the antibody, and the chimeric antibody CA-I was obtained.

[0071] Example 10: Humanization of mouse-derived antibody molecule MA-I First, the sequence of the mouse-derived antibody molecule MA-I in Example 2 was compared with the human antibody species database (v-base) to identify candidate sequences for highly homologous human antibody light and heavy chain species. The CDR sequence of the mouse-derived antibody molecule MA-I was then grafted onto the human-derived candidate sequence for homology modeling. Subsequently, 3D structure simulation was performed to calculate key frame amino acid residues that may play an important role in maintaining the CDR cyclic structure, and further restoration mutations for the humanized antibody were designed. The light and heavy chain variable regions of the humanized antibody containing the designed restoration mutations were optimized and synthesized by Nanjing Jinsirui Biotechnology Co., Ltd., and then inserted into a rapid expression vector. The light and heavy chain combinations obtained by humanization were analyzed to obtain the humanized antibody molecules HA-I, HA-II, HA-III, and HA-IV. The four monoclonal antibody sequences screened as described above are as follows:

[0072] [Table 6]

[0073] in particular, SEQ ID NO: 34 (amino acid sequence of the heavy chain variable region of HA-I and HA-II): [ka] SEQ ID NO: 35 (amino acid sequence of the light chain variable region of HA-I): [ka] SEQ ID NO: 36 (amino acid sequence of the light chain variable region of HA-II and HA-IV): [ka] SEQ ID NO: 37 (amino acid sequence of the heavy chain variable region of HA-III and HA-IV): [ka] SEQ ID NO: 38 (amino acid sequence of the light chain variable region of HA-III): [ka]

[0074] Example 11 Example 11 of the present invention further limits the following in addition to Example 10: the humanized antibody molecule further comprises a human-derived antibody constant region; the human-derived antibody constant region comprises a human-derived antibody heavy chain constant region selected from human IgG1, IgG2, or IgG4 heavy chain constant regions, and a human-derived antibody light chain constant region. The amino acid sequence of the IgG1 heavy chain constant region is shown in SEQ ID NO: 30, the amino acid sequence of the IgG2 heavy chain constant region is shown in SEQ ID NO: 31, and the amino acid sequence of the IgG4 heavy chain constant region is shown in SEQ ID NO: 32. The human-derived antibody light chain constant region comprises a human C k The light chain constant region of this type is the same as that of the IgG1A-type ... The specific sequences of the constant regions of the above human-derived antibodies are the same as those in Example 8.

[0075] Example 12: Production of humanized antibody molecules Example 12 of the present invention further defines the following in addition to Example 11: the human-derived antibody constant region is a heavy chain constant region of human IgG1 type (the amino acid sequence of which is shown in SEQ ID NO: 30) and a human C k and a light chain constant region of the type (the amino acid sequence of which is set forth in SEQ ID NO:33).

[0076] The heavy chain VH and light chain VL coding genes of the four humanized antibody molecules obtained by humanization in Example 10 above were each cloned into the vector pTSE incorporating the heavy chain constant region and light chain constant region genes (as shown in Figure 3). The heavy chain constant region was human IgG1 type (amino acid sequence shown in SEQ ID NO: 30), and the light chain constant region was C k The amino acid sequence is as shown in SEQ ID NO: 33.

[0077] For each of the control antibody and humanized antibody molecules HA-I, HA-II, HA-III, and HA-IV, HEK293 cells (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, product code: GNHu43) were transfected and expressed. Monoclonal antibodies were obtained by purification on a protein A affinity column using an AKTA instrument. Protein concentration was measured using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., product code: BCA0020), followed by protein size determination using SDS-PAGE. As shown in Figure 7, from left to right, the non-reduced protein molecular weights were HA-I, HA-II, HA-III, HA-IV, the chimeric antibody CA-I prepared in Example 9, the control antibody, non-reduced protein molecular weight marker 1, reduced protein molecular weight marker 2, HA-I, HA-II, HA-III, HA-IV, the chimeric antibody CA-I, and the control antibody. The molecular weights of each band were consistent with theory.

[0078] Example 13 In addition to the above examples, Example 13 of the present invention further limits that the humanized antibody molecule is a full-length antibody or an antibody fragment, and includes one or a combination of several of Fab, F(ab)2, Fv, or ScFv.

[0079] Example 14 In addition to the above embodiments, the fourteenth embodiment of the present invention further defines the following technical solutions.

[0080] Furthermore, the present invention provides a protein comprising an anti-TSLP monoclonal antibody or an antigen-binding fragment thereof defined in any one of the above examples.

[0081] The present invention further provides a polynucleotide molecule encoding an anti-TSLP monoclonal antibody or antigen-binding fragment thereof defined by any one of the above embodiments.

[0082] The present invention further provides a recombinant DNA expression vector comprising a polynucleotide molecule defined as above.

[0083] The present invention further provides a host cell transfected with a recombinant DNA expression vector as defined above, the host cell including a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell; Preferably, the host cell is a mammalian cell, and the mammalian cell is provided as a host cell that is a HEK293 cell, a CHO cell, or an NS0 cell.

[0084] The present invention further provides a drug comprising an anti-TSLP monoclonal antibody or an antigen-binding fragment thereof defined in any one of the above examples.

[0085] The present invention further provides use of the above-mentioned anti-TSLP monoclonal antibody or antigen-binding fragment thereof in the manufacture of a therapeutic agent for immune diseases or cancer.

[0086] The present invention provides a method for treating or preventing a TSLP-mediated disease, comprising administering a therapeutically effective amount of an anti-TSLP monoclonal antibody to an individual in need thereof, wherein the disease is an immune disease or cancer. Preferably, the immune disease includes, but is not limited to, asthma, chronic obstructive pulmonary disease, chronic eosinophilic pneumonia, idiopathic pulmonary fibrosis, and allergic dermatitis; the asthma includes severe asthma, eosinophilic or non-eosinophilic asthma, and hypoeosinophilic asthma.

[0087] Preferably, the cancer includes, but is not limited to, pancreatic cancer, non-small cell lung cancer, melanoma, prostate cancer, renal cancer, colorectal cancer or breast cancer.

[0088] Example 15: Binding experiment of humanized antibody molecules with TSLP TSLP-His was coated in a pH 9.6 carbonate buffer solution at 200 ng / well / 100 μl and incubated overnight at 4°C. After washing five times with 300 μl / well of PBST, the plate was blocked with 1% BSA-PBST at 37°C for 1 hour. Various dilutions of humanized antibodies HA-I, HA-II, HA-III, and HA-IV, the chimeric antibody CA-I prepared in Example 9, and a control antibody were added. The starting maximum concentration of each of the six antibodies was 5 μg / ml, and each was serially diluted 5-fold for a total of eight dilutions per antibody. The plate was then incubated at 37°C for 1 hour. After washing five times with 300 μl / well of PBST, Goat Anti-Human IgG-HRP (purchased from Beijing Zhongsugi Jinqiao Biotechnology Co., Ltd., product number ZB-2304) diluted 1:5000 in 1% BSA-PBST was added and incubated at 37°C for 1 hour. The color was developed with TMB color development kit, 100 μl / well, at room temperature for 5 min, and then the color development was terminated with 2 M H2SO4. The values ​​were read at 450 nm / 630 nm on a microplate reader, and the corresponding EC 50 The specific data is as follows:

[0089] [Table 7]

[0090] According to the above data, experimental results, and as shown in Figure 8, all four different humanized antibody molecules can bind to TSLP. 50 All of the values ​​were clearly lower than those of the control antibody. This demonstrates that the monoclonal antibodies provided by the present invention have strong binding ability and high affinity to TSLP. Furthermore, as is clear from Figure 8 and the above data, among the four different monoclonal antibodies, the EC of HA-I was 50 This proves that HA-I has the best binding ability and highest affinity to TSLP. 50 The values ​​were similar to those of the chimeric antibody CA-I, demonstrating that the humanized HA-I maintains the high affinity for TSLP of the mouse-derived parent antibody MA-I.

[0091] Example 16: Competitive inhibition experiment between humanized antibody and control antibody TSLP-His was coated in a pH 9.6 carbonate buffer solution at 100 ng / well / 100 μl overnight at 4°C. After washing five times with 300 μl / well of PBST, the plate was blocked with 1% BSA-PBST at 37°C for 1 hour. HA-I, HA-II, HA-III, HA-IV, and chimeric antibody CA-I diluted to 4 μg / ml in 1% BSA-PBST were added at 50 μl / well, followed by the addition of control antibodies at different dilutions at 50 μl / well. The control antibodies, all starting at a maximum concentration of 400 μg / ml, were serially diluted three-fold for a total of 11 dilutions, and the plate was incubated at 37°C for 2 hours. After washing five times with 300 μl / well of PBST, Anti-Human IgG1-HRP (purchased from Sigma, product number: SAB4200768) diluted 1:5000 with 1% BSA-PBST was added and incubated at 37°C for 1 hour. Color development was performed using a TMB color development kit, with 100 μl / well added at room temperature for 10 minutes, and the color development was terminated with 2M H2SO4. The values ​​were read at 450 nm / 630 nm on a microplate reader, and the corresponding IC50 The specific data is as follows:

[0092] [Table 8]

[0093] As shown in the data above and Figure 9, all four different humanized and chimeric antibodies screened were able to inhibit the binding of TSLP to the control antibody. At the same time, among these four humanized antibody molecules, the IC of HA-I was significantly higher than that of the control antibody. 50 This is the lowest value, and therefore the best inhibitory effect on HA-I.

[0094] Example 17: Cross-binding experiments of humanized antibodies with TSLP from different species Human TSLP-His, mouse TSLP-His (purchased from Yiqiao Shenzhou Technology Co., Ltd., product number: 51005-M08H), and monkey TSLP-His (purchased from Kin'an Protein Technology Co., Ltd., product number: CR62) were coated at 100 ng / well / 100 μl in a pH 9.6 carbonate buffer solution and incubated overnight at 4°C. After washing five times with 300 μl / well of PBST, the plates were blocked with 1% BSA-PBST at 37°C for 1 hour. Then, humanized antibodies HA-I, HA-II, HA-III, and HA-IV were added at different dilutions. The starting maximum concentration of each of the four humanized antibodies was 16 μg / ml, and each was diluted 4-fold for a total of eight dilutions per antibody. The plates were then incubated at 37°C for 1 hour. After washing five times with 300 μl / well of PBST, Goat Anti-Human IgG-HRP diluted 1:5000 in 1% BSA-PBST was added and incubated at 37°C for 1 hour. Color development was performed using a TMB color development kit, with 100 μl / well of the kit added at room temperature for 5 minutes, and then 2M H2SO4 was added to terminate the color development. The values ​​were read at 450 nm / 630 nm on a microplate reader, and the corresponding EC 50 The specific data is as follows:

[0095] [Table 9]

[0096] As shown in the data above and in Figure 10, all four different humanized antibodies screened were able to bind to human TSLP and cynomolgus TSLP, but not to mouse TSLP. Furthermore, among these four humanized antibody molecules, the EC of HA-I with human TSLP and cynomolgus TSLP was the highest. 50 The lowest value proves the strong binding ability of HA-I, allowing for pharmacological toxicity studies and safety evaluations in the cynomolgus monkey experimental animal model.

[0097] Example 18: Experiment to inhibit binding of TSLP to cell surface receptors by anti-TSLP monoclonal antibodies The BaF / 3-TSLPR engineered cell line was digested and counted, and the cells were diluted to 1 × 10 in sample diluent (containing 90% IMDM, 10% FBS, and 300 μg / ml hygromycin). 6The humanized antibodies HA-I, HA-II, HA-III, and HA-IV, and the control antibody, were each diluted to an initial concentration of 200 μg / ml and serially diluted three-fold for a total of 10 dilutions. The diluted humanized antibodies HA-I, HA-II, HA-III, and HA-IV, and the control antibody, were each added at 50 μl / well to a 96-well plate containing 100 μl of BaF / 3-TSLPR cells. The antigen TSLP was diluted to 8 μg / ml in sample diluent and added at 50 μl / well to the same well plate containing BaF / 3-TSLPR cells, humanized antibodies, and control antibodies. After gentle mixing, the 96-well plate was incubated at 4°C for 1 hour. After incubation, the plate was centrifuged at 3000 rpm, the supernatant discarded, and the cell pellet collected. Pre-diluted Goat Anti-Human IgG-Fc antibody (purchased from SouthernBiotech, product number: 2048-30) was added to the plate at 100 μl / well, uniformly mixed with the cell pellet in each well, and incubated at 4°C for 1 hour. After incubation, the cells were washed with 100 μl of PBS buffer per well, centrifuged at 3000 rpm, the supernatant was discarded, and 100 μl of PBS buffer per well was added to resuspend the cell pellet, followed by measurement using a flow cytometer. Data were collected and the corresponding IC 50 The specific data is as follows:

[0098] [Table 10]

[0099] As shown in the data above and in Figure 11, all four different humanized antibodies screened, as well as the control antibody, can compete with TSLP for binding to the cell surface receptor TSLPR. Furthermore, among these four humanized antibodies, only one antibody showed the highest IC value for HA-I. 50 This is the lowest value and is superior to the control antibody, demonstrating that HA-I has a good inhibitory effect on the binding of TSLP to its receptor at the cellular level.

[0100] Example 19: Detection of biological activity of anti-TSLP monoclonal antibodies (reporter gene method) BaF / 3 cells (mouse-derived B cells, purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, product number: 3111C0001CCC000095) expressing TSLPR, IL-7Rα, and STAT5-Luc were digested and counted, and then diluted to 1 × 10 cells in sample diluent (containing 90% IMDM, 10% FBS, 300 μg / ml hygromycin, 0.5 μg / ml puromycin, and 600 μg / ml geneticin). 6 The cells were diluted to 160 ng / ml with TSLP-RAS-His antigen and mixed gently. The cell mixture was then added to a 96-well plate at 50 μl per well. The humanized antibodies HA-I, HA-II, HA-III, HA-IV, and control antibodies were each diluted with sample diluent to an initial concentration of 15 μg / ml. A three-fold serial dilution was performed for a total of eight dilutions, with two replicate wells for each sample concentration. The diluted HA-I, HA-II, HA-III, HA-IV, and control antibodies were each added to the 96-well plate containing 50 μl of the cell mixture. After mixing gently, the 96-well plate was placed in a cell incubator and incubated for 5 hours at 37°C and 5% CO2. After 5 hours, the 96-well plate was removed and centrifuged at 3000 rpm / min for 5 minutes. The solution was then shaken off and discarded. 50 μl of Glo Lysis Buffer (purchased from Promega, product number: E2661) was added to each well and lysed at room temperature for 5 minutes. The cell plate was gently tapped to mix the cell lysate evenly. The cell lysate was then transferred to a 384-well plate at 10 μl / well and mixed with an equal volume of Bright-Gol TM Add the Luciferase Assay System, react at room temperature for 2 to 15 minutes, read the fluorescence value with a microplate reader, and calculate the corresponding IC 50 The specific data is as follows:

[0101] [Table 11]

[0102] As shown in the data above and Figure 12, all four different humanized antibodies and control antibodies screened were able to bind to TSLP and exert their effects by competitively inhibiting the binding of TSLP to the receptor complex, thereby blocking the intracellular signaling pathway. The construction of the engineered cell line BaF / 3-TSLPR-IL7Rα-STAT5-Luc simulated the proliferation response of human hypertrophic cells under the action of TSLP. TSLP stimulates and enhances the expression of the intracellular proliferation signal (STAT5-Luc) by binding to the cell surface TSLPR and IL7Rα receptor. These four humanized antibody molecules effectively inhibit the binding of TSLP to the cell surface receptor and further suppress the development of the intracellular proliferation signal. Furthermore, among these four humanized antibody molecules, only one antibody showed the IC of HA-I. 50 This was the lowest value and clearly superior to the control antibody, demonstrating that HA-I can inhibit the binding of TSLP to its receptor at the cellular level and has the most effective cell proliferation inhibitory effect.

[0103] Example 20: Inhibition of chemokine release from TSLP-induced mDC cells by anti-TSLP monoclonal antibodies PBMC cells were revived and selected using a kit to obtain mature DC cells, which were then diluted to a cell density of 4 × 10 in sample diluent (containing 90% 1640 and 10% FBS). 5The cell suspension was adjusted to 80 cells / mL and added at 50 μL per well to a 96-well plate. The humanized antibodies HA-I, HA-II, HA-III, and HA-IV, as well as the control antibody, were each diluted in sample diluent to an initial concentration of 80 ng / mL. 3-fold serial dilutions were performed for a total of eight dilutions, with two replicate wells for each sample concentration, and added at 25 μL per well to the 96-well plate containing mature DC cells. TSLP protein was diluted to 80 ng / mL in sample diluent and added at 25 μL per well to the 96-well plate containing mature DC cells, humanized antibodies, and control antibodies. After gentle mixing, the 96-well plate was placed in a CO2 incubator at 37°C and incubated overnight. After approximately 24 hours, the supernatant was removed and added at 50 μL per well. The TARC test was performed according to the instructions for the human TARC ELISA kit (purchased from Dakowei Biotechnology Co., Ltd., product number: 1117542). First, dilute the supernatant 3-fold with the diluent provided in the kit and mix evenly. Add the diluted supernatant and standard to the sample wells at 100 μl / well and incubate at room temperature for 2 hours. After incubation, wash the well plate three times with washing solution. Add 100 μl / well of biotinylated antibody dilution and incubate at room temperature for 2 hours. After incubation, wash the well plate three times with washing solution. Add 100 μl / well of Streptavidin-HRP working solution and incubate at room temperature for 20 minutes. After incubation, wash the well plate three times with washing solution. Add 100 μl / well of TMB color development solution and incubate at room temperature in the dark for approximately 15 minutes. Stop the color development with 100 μl / well of stop solution. Read the OD value at 450 nm on a microplate reader and determine the corresponding IC 50 The specific data is as follows:

[0104] [Table 12]

[0105] As shown in the data above and in Figure 13, all four different humanized antibodies and the control antibody screened were able to inhibit the release of the chemokine TARC from TSLP-activated mDC cells. Furthermore, among these four humanized antibody molecules, only one antibody showed the highest IC of HA-I. 50 This was the lowest value and clearly superior to the control antibody, demonstrating that HA-I can effectively inhibit the activating effect of TSLP on mDCs at the cellular level and has the most potent inhibitory effect.

[0106] Example 21: Evaluation of the thermal stability of anti-TSLP monoclonal antibody HA-I The anti-TSLP monoclonal antibody HA-I was ultrafiltered into a PBS buffer solution and centrifuged at 12,000 rpm and 4°C for 5 minutes. The thermal stability of the anti-TSLP monoclonal antibody HA-I was evaluated using a multifunctional protein thermal stability analysis system (purchased from Unchained Labs). The temperature-dependent change in the protein's intrinsic fluorescence (heated from 25°C to 95°C at a rate of 0.3°C / min) was monitored to detect protein conformational changes, thereby determining the protein melting temperature (Tm) and assessing the stability of the protein structure. Sample aggregation causes interference of scattered light waves, increasing the scattered light signal. The colloidal stability of the protein (characterized by Tag) was measured by static light scattering. The results are shown in the table below and Figure 14.

[0107] [Table 13]

[0108] As shown in the above table and Figure 14, the anti-TSLP monoclonal antibody HA-I exhibited good structural and colloidal stability, with a temperature of 71.7°C and an average Tagg of 83.0°C.

[0109] Example 22: Toxicity study of anti-TSLP monoclonal antibody administered subcutaneously to cynomolgus monkeys for 4 weeks The purpose of the study was to administer anti-TSLP monoclonal antibody subcutaneously to cynomolgus monkeys once a week for four consecutive weeks, and to observe the toxicity after administration.

[0110] (1) Experimental product information: Anti-TSLP monoclonal antibody HA-I prepared as an injection solution (auxiliary materials include histidine, histidine hydrochloride, sorbitol, and polysorbate 80); Control product information: The buffer solution for anti-TSLP monoclonal antibody HA-I (containing histidine, histidine hydrochloride, sorbitol, and polysorbate 80, but not containing the drug component anti-TSLP monoclonal antibody HA-I, and the other components are the same as those of the injection solution) was used as the control; (2) Provided by: Beijing Oriental Baitai Biotechnology Co., Ltd. (3) Experimental animals: Type and breed: Cynomolgus monkey; Age: 2.5-5 years; Weight: 2-5 kg; Gender: female, male; Number of animals: 8 males and 4 females;

[0111] [Table 14]

[0112] It will be administered by subcutaneous injection, with the first dose designated as D1, followed by weekly administration for 4 consecutive weeks, for a total of 5 doses; The parameters evaluated in this experiment are clinical observation, body weight, food intake, body temperature, accompanying safety pharmacological indicator tests, electrocardiogram, blood pressure, ophthalmological examination, clinical pathology tests (blood cell count, coagulation function, blood biochemistry analysis, urine analysis), immunological indicators (immune cell phenotype, cellular factors, immunoglobulins, complement), antidrug antibodies, and toxicokinetics.

[0113] Experimental results: During the experiment, clinical observations, body weight, body temperature, electrocardiogram parameters, blood cell counts, blood biochemistry, urinalysis, and T lymphocyte subpopulation measurements were performed on the animals in each group, and animals in groups 2 to 4 were dissected for general observation. As a result, no obvious abnormal changes or irregularly obvious abnormal changes were found in each index.

[0114] In addition, in female animals in the 300 mg / kg dose group, FIB increased before the second dose (D8) and the day after the third dose (D16), returning to normal the day after the final dose (D30), while no abnormalities were observed in the remaining animals. IL-6 increased in male animals in the 30 mg / kg dose group 1 to 2 hours after the first dose (D1), while IL-10 increased in female animals in the 300 mg / kg dose group, with both levels returning to normal the following day. IL-6 increased in both male and female animals in the 300 mg / kg dose group the day after the first dose (D2), returning to normal before the D8 dose. IL-10 also increased in female animals in the 300 mg / kg dose group before the second dose (D8), returning to normal on D29, with no other significant abnormal changes observed.

[0115] No ADA was detected in any of the animals in either group during the experiment.

[0116] Toxicokinetic results showed that anti-TSLP monoclonal antibody HA-I was administered subcutaneously to cynomolgus monkeys at doses of 30, 100, and 300 mg / kg. After the first and fourth doses, blood drug concentrations and exposure increased with increasing dose, and no significant gender differences were observed in major toxicokinetic parameters. After four consecutive weeks of weekly administration, slight accumulation of EB070 in the cynomolgus monkeys was observed (accumulation factor: 1.49-2.71).

[0117] Example 23: Toxicity study of anti-TSLP monoclonal antibody administered subcutaneously to cynomolgus monkeys for 13 weeks In addition to the objectives of Example 21, the objective of this experimental study was to evaluate the toxicity and toxicokinetics after 13 weeks of repeated subcutaneous administration of anti-TSLP monoclonal antibody once weekly to cynomolgus monkeys, as well as the recovery from toxicity 6 weeks after discontinuation of the drug.

[0118] (1) Experimental product information: Anti-TSLP monoclonal antibody HA-I prepared as an injection solution (auxiliary materials include histidine, histidine hydrochloride, sorbitol, and polysorbate 80); Control product information: The buffer solution for anti-TSLP monoclonal antibody HA-I (containing histidine, histidine hydrochloride, sorbitol, and polysorbate 80, but not containing the drug component anti-TSLP monoclonal antibody HA-I, and the other components are the same as those of the injection solution) was used as the control; (2) Provided by: Beijing Oriental Baitai Biotechnology Co., Ltd. (3) Experimental animals: Type and breed: Cynomolgus monkey; Age: 3-5 years; Weight: 2-5 kg; Gender: female, male; Number of animals: 20 males and 20 females, total 40;

[0119] [Table 15]

[0120] The drug will be administered by subcutaneous injection, with the first dose designated as D1, followed by weekly doses for 13 consecutive weeks, for a total of 14 doses; the parameters evaluated in this study are clinical symptom observation, body weight, food intake, body temperature, accompanying safety pharmacological index tests, electrocardiogram, blood pressure, ophthalmological examination, clinical pathology tests (blood cell count, coagulation function, blood biochemistry analysis, urine analysis), immunological indexes (immune cell phenotype, cellular factors, immunoglobulins, complement), antidrug antibodies, and toxicokinetics.

[0121] As is clear from the results of preliminary experiments, after repeated subcutaneous administration of anti-TSLP monoclonal antibody HA-I to cynomolgus monkeys, no obvious abnormalities were found in animal clinical observations, body weight, body temperature, ophthalmological examinations, or gross necropsies.

[0122] The present invention is not limited to the above-mentioned preferred embodiment. Anyone can obtain various other forms of products under the teaching of the present invention. However, any changes in shape or structure that are the same or similar technical solutions as those in the present application shall be included within the scope of protection of the present invention.

Claims

1. An anti-TSLP monoclonal antibody or antigen-binding fragment thereof, a heavy chain variable region comprising three heavy chain complementarity determining regions designated HCDR1, HCDR2, and HCDR3, respectively; and a light chain variable region comprising three light chain complementarity determining regions designated LCDR1, LCDR2, and LCDR3, respectively; The monoclonal antibody or antigen-binding fragment thereof may be: A-I: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as set forth in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as set forth in SEQ ID NO: 2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as set forth in SEQ ID NO: 3, the amino acid sequence of the light chain complementarity determining region LCDR1 is as set forth in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determining region LCDR2 is as set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as set forth in SEQ ID NO: 6; A-II: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as set forth in SEQ ID NO:7, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as set forth in SEQ ID NO:8, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as set forth in SEQ ID NO:9, the amino acid sequence of the light chain complementarity determining region LCDR1 is as set forth in SEQ ID NO:4, the amino acid sequence of the light chain complementarity determining region LCDR2 is as set forth in SEQ ID NO:10, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as set forth in SEQ ID NO:6; A-III: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is as set forth in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is as set forth in SEQ ID NO: 11, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is as set forth in SEQ ID NO: 3, the amino acid sequence of the light chain complementarity determining region LCDR1 is as set forth in SEQ ID NO: 12, the amino acid sequence of the light chain complementarity determining region LCDR2 is as set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain complementarity determining region LCDR3 is as set forth in SEQ ID NO: 14; and A-IV: An anti-TSLP monoclonal antibody or an antigen-binding fragment thereof, characterized in that the anti-TSLP monoclonal antibody is any one selected from the group consisting of the heavy chain complementarity determining region HCDR1, which has the amino acid sequence shown in SEQ ID NO: 1; the heavy chain complementarity determining region HCDR2, which has the amino acid sequence shown in SEQ ID NO: 11; the heavy chain complementarity determining region HCDR3, which has the amino acid sequence shown in SEQ ID NO: 3; the light chain complementarity determining region LCDR1, which has the amino acid sequence shown in SEQ ID NO: 12; the light chain complementarity determining region LCDR2, which has the amino acid sequence shown in SEQ ID NO: 15; and the light chain complementarity determining region LCDR3, which has the amino acid sequence shown in SEQ ID NO:

16.

2. The monoclonal antibody or antigen-binding fragment thereof is a mouse-derived antibody molecule, and the mouse-derived antibody molecule is MA-I: the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 17 and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 18; MA-II: the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 19 and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 20; MA-III: the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 21 and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 22; and MA-IV: the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 23 and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 24; The anti-TSLP monoclonal antibody or antigen-binding fragment thereof according to claim 1.

3. The mouse-derived antibody molecule further comprises a mouse-derived antibody heavy chain constant region selected from mouse IgG1, IgG2a, IgG2b, or IgG3 heavy chain constant regions, and a mouse-derived antibody light chain constant region; wherein the amino acid sequence of the IgG1 heavy chain constant region is as shown in SEQ ID NO: 26, the amino acid sequence of the IgG2a heavy chain constant region is as shown in SEQ ID NO: 27, the amino acid sequence of the IgG2b heavy chain constant region is as shown in SEQ ID NO: 28, and the amino acid sequence of the IgG3 heavy chain constant region is as shown in SEQ ID NO: 29; and the mouse-derived antibody light chain constant region is selected from mouse C ｋ a light chain constant region of the type IHVIII, and the amino acid sequence thereof is as set forth in SEQ ID NO: 25; The anti-TSLP monoclonal antibody or antigen-binding fragment thereof according to claim 2.

4. The anti-TSLP monoclonal antibody or antigen-binding fragment thereof according to claim 2, characterized in that the monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody molecule, and the chimeric antibody molecule comprises a heavy chain variable region of the mouse-derived antibody molecule, a light chain variable region of the mouse-derived antibody molecule, and a human-derived antibody constant region.

5. The monoclonal antibody or antigen-binding fragment thereof is a humanized antibody molecule, and the humanized antibody molecule includes: HA-I: the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 35; HA-II: the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 36; HA-III: the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 37 and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 38; and HA-IV: the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 37 and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 36; The anti-TSLP monoclonal antibody or antigen-binding fragment thereof according to claim 1.

6. The anti-TSLP monoclonal antibody or antigen-binding fragment thereof of claim 5, wherein the humanized antibody molecule further comprises a human-derived antibody constant region.

7. The anti-TSLP monoclonal antibody or antigen-binding fragment thereof may be Fab, F(ab) ２ 2. The anti-TSLP monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that it comprises one or a combination of several of Fv, ScFv or ScFv.

8. The human-derived antibody constant region comprises a human-derived antibody heavy chain constant region selected from human IgG1, IgG2, or IgG4 heavy chain constant regions, and a human-derived antibody light chain constant region, wherein the amino acid sequence of the IgG1 heavy chain constant region is as shown in SEQ ID NO: 30, the amino acid sequence of the IgG2 heavy chain constant region is as shown in SEQ ID NO: 31, and the amino acid sequence of the IgG4 heavy chain constant region is as shown in SEQ ID NO: 32, and the human-derived antibody light chain constant region is selected from human C ｋ a light chain constant region of the type A, and the amino acid sequence thereof is as set forth in SEQ ID NO: 33; The anti-TSLP monoclonal antibody or antigen-binding fragment thereof according to claim 4 or 6.

9. A protein comprising an anti-TSLP monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7.

10. A polynucleotide molecule encoding the anti-TSLP monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

11. A recombinant DNA expression vector comprising the polynucleotide molecule of claim 10.

12. A host cell transfected with the recombinant DNA expression vector of claim 11, The host cell comprises a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell; The host cell is characterized in that the mammalian cell is a HEK293 cell, a CHO cell, or an NS0 cell.

13. A drug comprising the anti-TSLP monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

14. Use of the anti-TSLP monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in the manufacture of a therapeutic agent for immune diseases or cancer; The immune diseases include asthma, chronic obstructive pulmonary disease, chronic eosinophilic pneumonia, idiopathic pulmonary fibrosis, and allergic dermatitis; the asthma includes severe asthma, eosinophilic or non-eosinophilic asthma, and hypoeosinophilic asthma; The cancer comprises pancreatic cancer, non-small cell lung cancer, melanoma, prostate cancer, renal cancer, colorectal cancer or breast cancer.

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