Anti-human thymic stromal lymphocyte neogenesis factor antibody, method for producing the same, and use

Abstract

The present invention discloses an antibody capable of binding to human thymic stromal lymphopoietin (hTSLP), as well as methods for preparing and using the same. The anti-human TSLP antibody of the present invention can specifically bind to the human TSLP receptor and has a potent inhibitory effect on TARC secretion by PBMCs, making it suitable for the treatment of TSLP-related diseases, such as immune-mediated inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to a human thymic stromal lymphopoietin (hTSLP) antibody, and particularly to an antibody that neutralizes hTSLP activity. Further, the present invention relates to a method for diagnosing or treating hTSLP-related diseases using an hTSLP antibody molecule, where the diseases include, for example, asthma, chronic obstructive pulmonary disease, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma.

Background Art

[0002] Bronchial asthma (also abbreviated as asthma) is one of the most common chronic diseases, and in recent years, the prevalence of asthma has been increasing worldwide. Currently, there are at least 300 million asthma patients in the world, and about 30 million in China. According to the data of the Asian Regional Asthma Epidemiology Survey, the prevalence of adult asthma in Asia is 0.7% - 11.9%, with an average of 5% or less, and in recent years, the average prevalence of asthma has been on the rise. The current standard treatment for asthma is inhaled glucocorticoids + / - long-acting β2 agonists, but it has strong side effects, leading to immune system disorders, and moreover, for 5 - 10% of patients, there is no effective treatment drug at present. According to the latest data of the Chinese Asthma Epidemiology Survey, only 40.5% of Chinese asthma patients can control their condition. There is a strong desire for drugs with high specificity, low toxicity and side effects, and that can effectively suppress the disease as a symptomatic treatment.

[0003] Human thymic stromal lymphopoietin (hTSLP) is a human interleukin 7 (hIL-7)-like cytokine that initiates allergic reactions by stimulating dendritic cells (DCs). hTSLP is a protein consisting of 159 amino acids, sharing 43% homology with mouse TSLP. It contains a signal sequence of 28 residues, six cysteine ​​molecules, and two putative N-glycosylation sites. Non-denatured sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a protein with a molecular weight of 23 kilodaltons (kDa) was expressed, while the calculated molecular weight of the mature protein was 14.9 kDa, indicating that hTSLP is glycosylated. hTSLP contains seven basic C-terminal amino acids and six cysteine ​​molecules thought to be involved in disulfide bond formation. The TSLP receptor complex is a heterodimer consisting of the TSLP receptor (TSLPR) and the IL-7 receptor α (IL-7Rα) chain. The receptor is mainly expressed in monocytes, bone marrow-derived dendritic cells (DCs), and B lymphocytes.

[0004] TSLPs are epithelial cell-derived cytokines produced in response to environmental and inflammatory stimuli, activating various inflammatory cells and downstream pathways. TSLPs are elevated in the airways of asthma patients and are associated with the expression of Th2 cytokines and chemokines, as well as disease severity. While TSLPs are important for regulating Th2 immunity, they may also play a significant role in other inflammatory pathways, thus contributing to a variety of asthma phenotypes.

[0005] Antibody drugs can efficiently block downstream signaling by specifically binding to target antigens with high affinity and inhibiting ligand-mediated receptor activation. Internationally, antibody drugs targeting the TSLP signaling pathway are not yet commercially available, but preliminary clinical studies suggest that anti-TSLP antibodies have the potential for widespread application in the treatment of severe persistent asthma, showing similarly efficient responses across patient populations with different blood eosinophil counts and other Th2 type biomarkers. This invention utilizes high-throughput hybridoma screening technology to develop and obtain high-affinity TSLP antibodies with higher biological activity than antibody drugs targeting the same organism, and further development is expected to provide new treatment options for asthma patients both domestically and internationally. [Brief explanation of the drawing]

[0006] [Figure 1] Figure 1 shows that TSLP-dependent STAT5 activation is inhibited by a humanized anti-human TSLP monoclonal antibody. [Figure 2] Figure 2 shows that humanized anti-human TSLP monoclonal antibodies inhibit TARC secretion by PBMCs. [Modes for carrying out the invention]

[0007] Through numerous experiments, the inventors of this invention were able to obtain a set of monoclonal antibodies that can block TSLP signaling by specifically blocking the binding of TSLP to cell surface TSLP receptors, thereby blocking TSLP-mediated biological activity.

[0008] In a first embodiment, the present application provides an antibody or antigen-binding moiety comprising a heavy chain variable region that specifically binds to human TSLP, wherein the heavy chain variable region comprises an HCDR3 sequence and optionally further comprises HCDR1 and / or HCDR2 sequences. In some embodiments, the HCDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs: 3, 6, 9, and 12. In some embodiments, the HCDR2 sequence comprises an amino acid sequence selected from SEQ ID NOs: 2, 5, 8, and 11. In some embodiments, the HCDR1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 1, 4, 7, and 10.

[0009] In some embodiments, the heavy chain variable region includes an amino acid sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NOs: 26, 32, 38, and 44, or the heavy chain variable region includes an amino acid sequence selected from SEQ ID NOs: 26, 32, 38, and 44.

[0010] In some embodiments, an antibody or its antigen-binding moiety that specifically binds to human TSLP further comprises a light chain variable region, the light chain variable region comprising LCDR1, LCDR2 and / or LCDR3 sequences. In some embodiments, the LCDR1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 13, 16, 19 and 22. In some embodiments, the LCDR2 sequence comprises an amino acid sequence selected from SEQ ID NOs: 14, 17, 20 and 23. In some embodiments, the LCDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs: 15, 18, 21 and 24.

[0011] In some embodiments, the light chain variable region includes an amino acid sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NOs: 29, 35, 41, and 47, or the light chain variable region includes an amino acid sequence selected from SEQ ID NOs: 29, 35, 41, and 47.

[0012] In some embodiments, the heavy chain of the antibody or its antigen-binding moiety that specifically binds to human TSLP includes an amino acid sequence selected from SEQ ID NOs. 27, 33, 39, and 45, or an amino acid sequence having at least 80% homology to the above sequences. Optionally, the light chain of the antibody or its antigen-binding moiety includes an amino acid sequence selected from SEQ ID NOs. 30, 36, 42, and 48, or an amino acid sequence having at least 80% homology to the above sequences.

[0013] In any embodiment, the antigen-binding moiety is selected from Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, Fd fragment, and single-domain antibody.

[0014] In some embodiments, the antibody that specifically binds to human TSLP as described in the first embodiment is a mouse-derived monoclonal antibody.

[0015] In some embodiments, the antibody that specifically binds to human TSLP as described in the first embodiment is a humanized antibody.

[0016] In some embodiments, the antibodies or antigen-binding moieties disclosed herein can inhibit TSLP-dependent STAT5 activation. In some other embodiments, the antibodies or antigen-binding moieties can inhibit TARC secretion by PBMCs.

[0017] In a second aspect, the present invention provides an antibody that specifically binds to the human TSLP described above, or a nucleotide molecule encoding an antigen-binding portion thereof.

[0018] In a third aspect, the present invention provides an expression vector comprising the nucleotide molecules described above.

[0019] In some embodiments, the expression vector is pTT5, pUC57, pDR1, pcDNA3.1(+), pDHFF, or pCHO 1.0, etc.

[0020] In a fourth aspect, the present application provides a host cell comprising the expression vector described above. In some embodiments, the host cell is HEK293, COS, CHO, NS0, sf9, sf21, DH5α, BL21(DE3), or TG1, etc.

[0021] In a fifth aspect, the present application provides a method for preparing an antibody or an antigen-binding portion thereof that specifically binds to human TSLP according to the first aspect, the method comprising the following steps: a) Culturing the host cell under expression conditions capable of producing the antibody or an antigen-binding portion thereof in the host cell according to the fourth aspect to express the antibody or an antigen-binding portion thereof; and b) Separating and purifying the antibody or an antigen-binding portion thereof expressed in step a).

[0022] In a sixth aspect, the present application provides a pharmaceutical composition comprising an anti-human TSLP antibody or an antigen-binding portion thereof according to the first aspect and a pharmaceutically acceptable carrier.

[0023] In some embodiments, the composition is applied to the treatment of human TSLP-related diseases.

[0024] In some embodiments, the human TSLP-related diseases include asthma, chronic obstructive pulmonary disease, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin's lymphoma.

[0025] In other aspects, the present application provides a method for preventing or treating human TSLP-related diseases, comprising administering to an individual in need thereof the antibody or an antigen-binding portion thereof according to the first aspect, or the pharmaceutical composition according to the sixth aspect.

[0026] The anti-human TSLP antibody or antigen-binding portion thereof according to the present invention can specifically bind to human TSLP and has one or more of the following effects: blocking the binding of human TSLP to hTSLPR; inhibiting TSLP-dependent STAT5 activation; inhibiting TARC secretion by PBMCs. The anti-human TSLP antibody or antigen-binding portion thereof according to the present invention can be applied to the prevention or treatment of human TSLP-related diseases, such as immune-mediated inflammatory diseases.

[0027] This application provides a novel anti-hTSLP antibody or antigen-binding portion thereof that specifically binds to human TSLP. In a preferred embodiment, the antibody or antigen-binding portion thereof according to this application binds to human TSLP with high affinity and inhibits the binding activity of TSLP to TSLPR. This application further relates to a polynucleotide encoding the antibody or antigen-binding fragment thereof, a vector containing the polynucleotide, a host cell containing the polynucleotide or vector, a method for preparing and purifying the antibody, and the medical and biological uses of the antibody or antigen-binding portion thereof, such as its use in the prevention or treatment of diseases or conditions related to TSLP. This application further relates to an hTSLP detection method and an hTSLP activity regulation method using the antibody or antigen-binding fragment thereof.

[0028] To facilitate a brief understanding of this application, first, some terms used in the text are defined.

[0029] As used in this text, the term "antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains, i.e., two heavy chains (H) and two light chains (L) interconnected via disulfide bonds, and its polymer (e.g., IgM). Each heavy chain contains a variable heavy chain region (abbreviated as VH) and a constant heavy chain region (abbreviated as CH). The constant heavy chain region contains three domains: CH1, CH2, and CH3. Each light chain contains a variable light chain region (abbreviated as VL) and a constant light chain region (abbreviated as CL). The constant light chain region contains one domain (CL1). The VH and VL regions can be further divided into highly variable regions called complementarity-determining regions (CDRs), and conserved regions called framing regions (FRs) are interposed between them.

[0030] As used in this text, the term “antigen-binding region” of an antibody refers to a part or segment of a complete antibody molecule that is responsible for binding to an antigen. The antigen-binding domain may include the heavy chain variable region (VH), the light chain variable region (VL), or both. Antigen-binding fragments of antibodies can be prepared from complete antibody molecules using any suitable standard technique, such as protein hydrolysis digestion or recombinant genetic engineering. Non-restrictive examples of antigen-binding regions include Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv(scFv) molecules, single-domain antibodies, dAb fragments, and minimal recognition units (e.g., isolated CDRs) consisting of amino acid residues that mimic the highly variable region of an antibody. The term “antigen-binding region” further includes engineered molecules such as biantibodies, tripoantibodies, quadriantibodies, and microantibodies.

[0031] As used in this text, the terms “heavy chain variable region (VH)” and “light chain variable region (VL)” refer to the variable heavy chain and light chain regions of a single antibody, respectively, including FR1, 2, 3, and 4, and CDR1, 2, and 3.

[0032] It is well known to those skilled in the art that the complementarity-determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions within the variable region that have the greatest influence on the affinity and specificity of an antibody. There are two general definitions of VH or VL CDR sequences: the Kabat definition and the Chothia definition, see, for example, Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); A1-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci.USA86:9268-9272 (1989). For a given antibody's variable region sequence, the CDR region sequences in the VH and VL sequences can be determined according to the Kabat or Chothia definition. In the embodiments of this application, the CDR sequence is defined using the Kabat definition. In this text, the heavy chain variable regions CDR1, CDR2, and CDR3 are abbreviated as HCDR1, HCDR2, and HCDR3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 are abbreviated as LCDR1, LCDR2, and LCDR3, respectively.

[0033] For the variable region sequence of a given antibody, the sequence of the CDR region within the variable region sequence can be analyzed in many ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0034] In this text, "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope. For example, it refers to the ability of an antibody to bind to a specific antigen with an affinity at least twice as high as its affinity to the nonspecific antigen. However, it should be understood that an antibody can specifically bind to two or more antigens related to its sequence. For example, the antibody according to the present invention can specifically bind to TSLPs of humans and non-humans (e.g., mice and non-human primates).

[0035] As used in this text, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, an antibody in which, apart from the possibility of spontaneous mutations occurring in a small number of individuals, the individual antibodies constituting the population are identical. The monoclonal antibodies described in this text include, in particular, “chimeric” antibodies in which part of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody belonging to a particular antibody class or subclass, and the remainder of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody belonging to a different antibody class or subclass, provided that such antibodies exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0036] As used in this text, the term "homology" is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after sequence alignment and the introduction of null positions, and, where necessary, the highest percentage achieved is considered homology. The methods and computer programs used for comparison are well known in this field. "At least 80% homology" as used in this text means that the homology is any value between 80% and 100%, for example, 85%, 90%, 95%, 99%, etc.

[0037] As used in this text, the term “TSLP-related disease” includes diseases and / or conditions associated with the activation of the TSLP signaling pathway, such as asthma, chronic obstructive pulmonary disease, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma.

[0038] On the other hand, the present application provides an antibody or its antigen-binding moiety that includes a heavy chain variable region and / or a light chain variable region and specifically binds to human TSLP. Tables 3-6 below exemplify the CDR, VH, VL, heavy chain and light chain amino acid sequences and corresponding nucleotide sequences applicable to the antibodies disclosed in the present application.

[0039] In a specific embodiment, HCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 3, 6, 9, and 12. In another specific embodiment, HCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 6, 9, and 12. In a preferred embodiment, HCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 9 and 12.

[0040] In a specific embodiment, HCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 2, 5, 8, and 11. In another specific embodiment, HCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 5, 8, and 11. In a preferred embodiment, HCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 8 and 11.

[0041] In a specific embodiment, HCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 1, 4, 7, and 10. In another specific embodiment, HCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 4, 7, and 10. In a preferred embodiment, HCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 7 and 10.

[0042] In some embodiments, the heavy chain variable region of the antibody disclosed herein includes an amino acid sequence selected from SEQ ID NOs: 26, 32, 38, and 44. In specific embodiments, the heavy chain variable region consists of an amino acid sequence selected from SEQ ID NOs: 26, 32, 38, and 44.

[0043] The antibodies or antigen-binding regions disclosed herein may include a heavy chain variable region and further include a light chain variable region.

[0044] In some embodiments, the CDR3 (LCDR3) of the light chain variable region is selected from the amino acid sequences shown in SEQ ID NOs: 15, 18, 21, and 24, or from the amino acid sequences shown in SEQ ID NOs: 18, 21, and 24. In preferred embodiments, LCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 21 and 24.

[0045] In some embodiments, LCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 14, 17, 20, and 23, or from the amino acid sequences shown in SEQ ID NOs: 17, 20, and 23. In preferred embodiments, LCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 20 and 23.

[0046] In some embodiments, LCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 13, 16, 19, and 22, or from the amino acid sequences shown in SEQ ID NOs: 16, 19, and 22. In preferred embodiments, LCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 19 and 22.

[0047] In some embodiments, the light chain variable region of the antibody disclosed herein includes an amino acid sequence selected from SEQ ID NOs: 29, 35, 41, and 47. In specific embodiments, the light chain variable region consists of an amino acid sequence selected from SEQ ID NOs: 29, 35, 41, and 47.

[0048] In some embodiments, the heavy chain or heavy chain variable region, light chain or light chain variable region of the antibody disclosed herein may have at least one amino acid substituted, deleted, or added based on the respective corresponding specific amino acid sequences listed above, and the resulting variants still retain the activity to bind to human TSLP.

[0049] In one embodiment, the number of such amino acid substitutions, deletions, or additions is 1 to 30, preferably 1 to 20, and more preferably 1 to 10. In a preferred embodiment, the difference between the sequence variant and the original amino acid sequence is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In a more preferred embodiment, the difference between the sequence variant and the original amino acid sequence is about 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions. In a specific embodiment, the amino acid substitutions are conservative substitutions.

[0050] In a preferred embodiment, the antibody disclosed herein is antibody 111H or 163H, wherein antibody 111H has a heavy chain sequence shown in SEQ ID NO: 39 and a light chain sequence shown in SEQ ID NO: 42, in which the LCDR sequence is the same as that of antibody 111, HCDR1 and HCDR3 are the same as those of antibody 111, and the HCDR2 sequence is preferably SEQ ID NO: 49; antibody 163H has a heavy chain sequence shown in SEQ ID NO: 45 and a light chain sequence shown in SEQ ID NO: 48, in which the CDR sequence is the same as that of antibody 163.

[0051] In some embodiments, the antibodies disclosed herein are monoclonal antibodies. In specific embodiments, the antibodies disclosed herein are humanized antibodies.

[0052] The antibodies or antigen-binding moieties disclosed herein can specifically bind to human TSLP. In specific embodiments, the antibody or antigen-binding moiety specifically binds to human TSLP or cynomolgus monkey TSLP. In preferred embodiments, the antibody or antigen-binding moiety specifically binds to human TSLP.

[0053] In some embodiments, the antibodies or antigen-binding moieties disclosed herein can block the binding of human TSLP to hTSLPR; inhibit TSLP-dependent STAT5 activation; and inhibit TARC secretion by PBMCs.

[0054] The present invention further provides a nucleotide molecule encoding an antibody or an antigen-binding portion thereof as disclosed herein, a vector comprising the polynucleotide, a host cell comprising the polynucleotide or the vector, and a method for preparing and purifying the antibody.

[0055] In some embodiments, the antibody or the nucleotide molecule encoding its antigen-binding portion is operably linked to a control sequence recognizable by the host cell transformed with the vector.

[0056] In some embodiments, any suitable expression vector can be applied to the present invention. For example, the expression vector may be one of pTT5, pUC57, pDR1, pcDNA3.1(+), pDHFF, and pCHO 1.0. The expression vector may also include a fusion DNA sequence linked to a suitable transcriptional and translational regulatory sequence.

[0057] In some embodiments, the available host cells are cells containing the expression vector, and may be eukaryotic cells. For example, mammalian or insect host cell culture systems can be used to express the antibody or its antigen-binding moiety according to this invention. For example, HEK293 cells, COS, CHO, NS0, sf9, and sf21 are all applicable to the present invention. The host cells may also be prokaryotic cells containing the expression vector, such as DH5α, BL21(DE3), and TG1.

[0058] In some embodiments, the method for preparing the anti-human TSLP monoclonal antibody disclosed herein includes: expressing the anti-human TSLP monoclonal antibody by culturing host cells under expression conditions; and separating and purifying the expressed anti-human TSLP monoclonal antibody. Using the above method, recombinant proteins can be purified into a substantially homogeneous substance, for example, as a single band in SDS-PAGE electrophoresis.

[0059] In some embodiments, the anti-TSLP antibodies disclosed herein can be separated and purified using affinity chromatography, and depending on the characteristics of the affinity column used, the anti-TSLP antibodies bound to the affinity column can be eluted using conventional methods such as high-salt buffering or pH changes.

[0060] In some embodiments, the humanized anti-human TSLP monoclonal antibodies disclosed herein are obtained by immunizing Balb / c mice with laboratory-prepared human TSLP antigens, repeating the immunization process until higher titers are achieved, then collecting mouse spleen cells and fusing them with hybridoma cells, followed by screening for hybridoma cell lines exhibiting TSLP inhibitory activity. More specifically, the inventors of the present invention, through numerous experiments, first individually expressed human TSLP antigens, then immunized mice with human TSLP antigens mixed with different adjuvants, fused the mouse spleen cells with the hybridoma cell line sp2 / 0, and screened the fused hybridomas for positive cell lines using human TSLP antigens, demonstrating definite inhibition of human TSLPR binding and TSLP function, thereby obtaining target cell lines. After humanizing the target molecule, both the light chain and heavy chain genes were cloned into the eukaryotic expression vector pCHO1.0. The expression vector was transfected into CHO cells using liposome technology, and then positive cell clones were screened with puromycin and methotrexate. The high-expression clones obtained from the screening were amplified and cultured in serum-free medium, and the humanized anti-human TSLP antigen monoclonal antibody was separated or purified using a protein A affinity column.

[0061] In several other embodiments, chimeric or humanized forms of the antibody, or other variant forms, can be produced by further modifying a mouse-derived parent antibody using, for example, conventional techniques of the art such as PCR mutagenesis. The parent antibody of this application can produce mutant antibodies by inducing mutations, for example, within the complementarity-determining region (CDR) domain of the antigen, which can then be screened for the presence of target properties such as binding affinity (lower KD), IC50, specificity, and preferential binding. Preferably, the target properties in the mutant antibody are improved compared to those in the parent antibody. Amino acid substitution mutant antibodies are preferred, in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues of the parent antibody molecule are removed and replaced by different residues. The most notable sites used for substitution mutagenesis are one or more CDR regions, but modifications in the framework region (FR) are also considered. Conservative amino acid substitutions are preferred, but non-conservative amino acid modifications can also be introduced to screen for target properties in the resulting mutant antibody.

[0062] In some embodiments, the serum half-life of an antibody is extended by modifying the Fc region of the antibody. The mutation sites identified as being able to improve the antibody-binding ability of human FcRn are mainly T250Q, M252Y, S254T, T256E, V308P, M428L, N434A, and N434S, and in this embodiment, the serum half-life of the antibody can be extended by mutations in the amino acids at these positions. The Fc region of the antibody is modified to reduce the ADCC effect, and the reported mutation sites in the Fc region are mainly L234A, L235A, L234F, and L235E, and in this embodiment, the reduction of the ADCC effect can be achieved by mutations in the amino acids at these positions.

[0063] This application provides a pharmaceutical composition comprising an antibody or its antigen-binding moiety as disclosed herein and a pharmaceutically acceptable carrier. The anti-TSLP antibody disclosed herein, for example, an anti-human TSLP monoclonal antibody, can exert a more stable therapeutic effect when incorporated into a pharmaceutical formulation together with a pharmaceutically acceptable carrier. In some embodiments, these formulations can ensure the integrity of the amino acid core sequence conformation of the anti-TSLP antibody disclosed herein, for example, an anti-human TSLP monoclonal antibody, and protect the polyfunctional groups of the protein from degradation (including, but not limited to, aggregation, deamidation, or oxidation). In some embodiments, liquid formulations can be stored stably for at least one year at 2°C to 8°C. In some embodiments, lyophilized formulations can be stored stably for at least six months at 30°C.

[0064] In some embodiments, the anti-human TSLP antibody monoclonal antibody preparation may be a formulation commonly used in the pharmaceutical field, such as a suspension, aqueous injection, or lyophilized preparation, preferably an aqueous injection or lyophilized preparation. For the aqueous injection or lyophilized preparation of the anti-human TSLP monoclonal antibody disclosed herein, pharmaceutically acceptable adjuvants include, but are not limited to, surfactants, solution stabilizers, isotonic modifiers, buffers, or combinations thereof. In some embodiments, the surfactant is polyoxyethylene sorbitol fatty acid ester (Tween 20 or 80), poloxamer (e.g., poloxamer 188), Triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, tetradecyl sarcosine, oleyl sarcosine, or octadecyl sarcosine, Pluronics, MONAQUAT TMThe solution stabilizer includes, but is not limited to, nonionic surfactants such as, and should be added in an amount that minimizes the tendency of the anti-human TSLP monoclonal antibody to granulate. In some embodiments, the solution stabilizer includes, but is not limited to, one of the following or a combination thereof: sugars such as reducing sugars and non-reducing sugars; amino acids such as sodium glutamate or histidine; alcohols such as tertiary alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol, etc. The solution stabilizer should be added in an amount that allows the final formulation to remain stable for the period that a person skilled in the art would consider stable. The isotonic modifier includes, but is not limited to, sodium chloride, mannitol, or a combination thereof. The buffer includes, but is not limited to, Tris, histidine buffer, phosphate buffer, or a combination thereof.

[0065] This application further provides a method for preventing or treating TSLP-related diseases, comprising administering to an individual an anti-human TSLP antibody or a composition comprising an anti-human TSLP monoclonal antibody. In some embodiments, the effect of anti-immune-mediated inflammatory responses is significant after administration to animals, including humans. Specifically, the anti-human TSLP antibodies disclosed herein can effectively prevent and / or treat immune-mediated inflammatory responses and can be used as anti-inflammatory agents.

[0066] The present invention further provides the use of anti-human TSLP antibodies or compositions comprising anti-human TSLP antibodies in the preparation of pharmaceuticals for the prevention or treatment of human TSLP-related diseases or symptoms. In some embodiments, the human TSLP-related diseases or symptoms are immune-mediated inflammatory reactions or immune-mediated inflammatory diseases.

[0067] In some embodiments, the immune-mediated inflammatory response or immune-mediated inflammatory disease includes, but is not limited to, asthma, chronic obstructive pulmonary disease, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma.

[0068] In some embodiments, the anti-human TSLP antibodies disclosed herein can be used as anti-immune-mediated inflammatory agents. An anti-immune-mediated inflammatory agent as described herein means a drug having the ability to suppress and / or treat immune-mediated inflammatory responses, for example, a drug that can delay the progression of symptoms associated with immune-mediated inflammatory responses and / or reduce the severity of those symptoms. In some embodiments, the drug can alleviate symptoms associated with existing inflammatory responses and prevent the appearance of other symptoms. In some embodiments, the drug can further reduce or prevent the progression of inflammatory responses.

[0069] The dosage of the anti-human TSLP monoclonal antibody and its composition disclosed herein will vary depending on the patient's age and weight, the characteristics and severity of the disease, and the route of administration when administered to animals, including humans. While the results of animal experiments and the overall situation may be referenced, the total dosage must not exceed the specified range.

[0070] The dosage and frequency of administration of antibodies or compositions thereof may vary depending on whether the disease is being prevented or treated. In prophylactic use, a composition containing the antibodies or mixtures thereof, in an amount defined as the “effective prophylactic dose,” is administered to a patient who is not yet diseased to enhance the patient’s resistance. In this use, the specific dosage also depends on the patient’s health condition and systemic immunity. Typically, relatively low doses are administered over a long period at relatively infrequent intervals. In therapeutic use, it may be necessary to administer relatively high doses at relatively short intervals until the progression of the disease slows or ends, preferably until the patient shows partial or complete improvement of disease symptoms. Then, a prophylactic program can be administered to the patient. The specific dosage and frequency will be readily apparent to those skilled in the art as required by the actual needs.

[0071] In this specification and in the claims, the words “includes,” “have,” and “contain” are intended to mean “include, but not limited to,” and are not intended to exclude other parts, additives, ingredients, or processes.

[0072] It should be understood that any features, characteristics, components, or processes described in any particular aspect, embodiment, or example of this Application may be applied to any other aspect, embodiment, or example described herein, insofar as they do not conflict with the descriptions therein.

[0073] The above disclosure is a general description of the present invention. The following specific examples further illustrate the present invention and should not be understood as limiting it. The examples do not include a detailed description of conventional methods, which are well known to those skilled in the art and are described in numerous publications, such as the Handbook of Molecular Cloning and the Cold Spring Harbor Manual of Antibody Technology. Reagents whose origin is not specified are conventional. [Examples]

[0074] <Example 1: Preparation of human TSLP hFc-tagged and Flag-tagged antigens, reference antibody Tezepelumab, and hFc-tagged protein of the extracellular segment of the human TSLP receptor> The human TSLP antigen sequence was obtained from UniProt (UniProtKB-Q969D9), and codon optimization was performed according to Homo sapiens codon preference. The amino acid fragment from positions 29 to 159 at the N-terminus was synthesized, and its sequence was subcloned into the pUC57 vector to obtain pUC57-hTSLP. The hFc fragment and Flag tag (DYKDDDDK) were inserted into the C-terminus of the hTSLP fragment by PCR and constructed in a pTT5 expression vector (stored in the laboratory). After obtaining pTT5(hTSLP-hFc) and pTT5(hTSLP-Flag), their sequences were determined, clones with completely correct sequences were selected, plasmids were extracted, and transfected.

[0075] The amino acid sequence of Tezepelumab was obtained from IMGT, and after codon optimization, the variable region of the heavy chain and the nucleotide sequence of the light chain were synthesized as a whole gene. The variable region of the heavy chain was ligated to the constant region of IgG2 by PCR, then cloned into a pTT5 expression vector, and the light chain sequence was also cloned into a pTT5 expression vector. After verification and confirmation by sequencing, the plasmid was extracted and prepared for transfection.

[0076] The human TSLPR sequence was obtained from UniProt (UniProtKB-Q9HC73), and codon optimization was performed according to Homo sapiens codon preference to synthesize an amino acid fragment from positions 23 to 231 at the N-terminus. This sequence was then subcloned into the pUC57 vector. The hFc fragment and Flag tag (DYKDDDDK) were inserted into the C-terminus of the hTSLPR-ECD fragment by PCR to construct a pTT5 expression vector. After sequencing, clones with the correct sequence were selected and transfected.

[0077] Plasmids were transfected into HEK293E cell lines (stored in the laboratory) using the PEI method. After culturing for 5 days in Freestyle 293 medium (purchased from Gibco) containing 3 mM valproic acid, the target protein was purified from the cell culture supernatant by protein A affinity chromatography (purchased from Pharmacia) or Flag affinity chromatography (purchased from Genscript). Protein quantification was performed by the bicinchoninic acid (BCA) method, and the purified protein was used for further analysis and research described below. The purified protein was used for mouse immunization and further analysis and research described below.

[0078] <Example 2: Immunization with hTSLP-hFc> The hTSLP-hFc antigen at 100 μg / mouse was diluted to 75 μl with physiological saline, then mixed with an equal volume of Freund's complete adjuvant and completely emulsified by ultrasonic waves. After that, it was subcutaneously injected at multiple points into 4- to 5-week-old Balb / c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd., animal production license number: SCXK (Shanghai) 2013-0018). Three weeks later, the protein at 50 μg / mouse was similarly diluted to 75 μl, then mixed with an equal volume of Freund's incomplete adjuvant and completely emulsified by ultrasonic waves, and then subcutaneously injected at multiple points into the mice. Two weeks later, this immunization was repeated again. All mice had their tails cut to collect blood and separate serum 1 week after the third immunization, and the serum titer was measured by ELISA coated with the hTSLP-hFc antigen. For mice with a serum antibody titer > 10,000, an impact immunization (ictus immunisatorius) was performed 1 week after blood collection: 10 μg antigen / 100 μl physiological saline / mouse was injected via the tail vein.

[0079] Titer measurement was performed by ELISA: ELISA plates were coated overnight at 4°C with 100 μl of hTSLP-hFc antigen per well at a coating concentration of 1 μg / ml. The plates were washed twice with PBST (PBS containing 0.5% Tween-20) and then beaten to dry. 200 μl of 1% BSA-containing coating solution was added to each well, blocked at room temperature for 4 hours, then beaten to dry and stored in a refrigerator at -20°C until use. For measurement, 100 μl of mouse serum of different concentrations was added to each well of the ELISA plate, creating two duplicate wells, and incubated at room temperature for 1.5 hours. The plates were washed three times with PBST and then beaten to dry. 100 μl of HRP-labeled rabbit anti-mouse Ig antibody (purchased from Sigma), diluted 1:10,000 times with PBST, was added and incubated at room temperature for 1 hour. The plates were washed three times with PBST and then beaten to dry. 100 μl of chromogenic solution (ELISA chromogenic solution A and chromogenic solution B, uniformly mixed in a 1:1 volume ratio immediately before use) was added to each well, and then 100 μl of 2M H2SO4 termination solution was added to each well to terminate the reaction. Immediately afterward, the OD value of each well was measured at a wavelength of 450 nm using a microplate reader (molecular device).

[0080] <Example 3: Hybridoma Fusion and Screening> Hybridoma sp2 / 0 cells (Cell Bank of the Committee for the Preservation of Typical Cultures, Chinese Academy of Sciences, preservation number TCM-18) were cultured in an incubator at 37°C and 5% CO2, with the fusion medium changed the day before fusion. Three days after shock immunization of mice, spleen cells were collected from the mice and fused. The fusion and screening methods were as follows: Mouse spleens were collected, polished and washed, and the spleen cells were counted. Spleen cells and sp2 / 0 cells were mixed in a 10:1 ratio and centrifuged at 1500 rpm for 7 minutes. The supernatant was washed away. Within 1 minute, 1 ml of PEG (1450) was added, and the mixture was gently shaken for 90 seconds. Within 2.5 minutes, 5 ml of serum-free DMEM culture medium (purchased from Gibco) was added all at once, and another 5 ml of serum-free medium was added to terminate the reaction. The mixture was left for 5 minutes and centrifuged at 1280 rpm for 8 minutes. Each 96-well plate contained 2 million sp2 / 0 cells, with 200 μl of cells uniformly inoculated into each well. The cells were first screened in HAT medium containing hypoxanthine (H), aminopterin (A), and thymidine (T). Half of the medium was replaced every 3-4 days, and on day 10, the medium was switched to HT medium. After 10 days, when more than 10% of the bottom of the 96-well plate was filled with hybridoma cells, the supernatant was collected and measured by ELISA on a microplate coated with hTSLP-hFc antigen. The ELISA measurement method was the same as described in Example 2. Positive hybridoma clones were selected, amplified and cultured in 24-well plates, and subcloned using the limiting dilution method to obtain hybridoma strains that stably expressed the target antibody. These strains were then stored and a bank was established.

[0081] <Example 4: Conjugation of mouse-derived anti-human hTSLP monoclonal antibody with human TSLP> Preferred mouse-derived anti-human hTSLP monoclonal antibodies were affinity-purified using a protein G affinity chromatography column, and then quantified by BCA. The EC50 of the anti-human TSLP monoclonal antibodies bound to hTSLP was measured by ELISA. The measurement method involved diluting recombinant human TSLP flag to 0.2 μg / ml with coating solution, adding 100 μl / well to a 96-well microplate using a multichannel pipette, and incubating at room temperature for 1.5 hours. The coating solution was discarded, the plate was washed once with PBST, and the plate was blocked with 200 μl / well of 1% BSA-containing coating solution and incubated at room temperature for 2 hours. The blocking solution was discarded, the plate was tapped to dry, and prepared for use. The anti-human TSLP monoclonal antibody was diluted 3-fold from a starting concentration of 100 μg / ml with 1% BSA-containing PBST over 12 gradients. 100 μl of the gradient-diluted antibody was aspirated using a multichannel pipette and added to an ELISA plate, with one duplicate well set up, and incubated at room temperature for 1.5 hours. The ELISA plates were washed four times with PBST and dried by tapping them with absorbent paper.

[0082] [Table 1]

[0083] 100 μl of sheep anti-human IgG Fc-HRP antibody (diluted 1000-fold with PBST containing 1% BSA immediately before use) was added to each well and incubated at room temperature for 1 hour. The plates were washed four times with PBST and dried by tapping them with absorbent paper. 100 μl of chromogenic solution (ELISA chromogenic solution A and chromogenic solution B were uniformly mixed in a 1:1 volume ratio immediately before use) was added to each well, and 80 μl of the final solution was added to each well 5 minutes after color development to terminate the reaction. Immediately afterward, the OD value of each well was measured at a wavelength of 450 nm using a microplate reader. The data was analyzed using GraphPad Prism 6 software. The experimental results are shown in Table 1.

[0084] <Example 5: Inhibition of TSLP and TSLPR binding by mouse-derived anti-human hTSLP monoclonal antibody> Recombinant human TSLPR-ECD-hFc was coated with coating buffer at a concentration of 2 μg / ml, and 100 μl / well was added to a 96-well microplate using a multichannel pipette. The plates were incubated at room temperature for 1.5 hours. The coating solution was discarded, and the plates were washed once with PBST.

[0085] [Table 2]

[0086] Blocked with 200 μl / well of 1% BSA-containing coating solution, incubated at room temperature for 2 hours, then tapped to dry and prepared for use. Mouse-derived TSLP antibody was diluted 3-fold in 11 gradients with 1% BSA-containing PBST from a starting concentration of 100 μg / ml, and an equal volume of TSLP-flag-biotin was added to the gradient-diluted antibody at a concentration of 2 ng / ml. Incubated at room temperature for 1 hour. 100 μl of the antibody and TSLP-flag-biotin mixture was aspirated using a multichannel pipette and added to an ELISA plate, with one duplicate well set up. Incubated at room temperature for 1.5 hours. Washed twice with PBST, and the ELISA plate was tapped to dry with absorbent paper. 100 μl of streptavidin-HRP antibody (diluted 1000-fold in 1% BSA-containing PBST immediately before use) was added to each well and incubated at room temperature for 1 hour. Washed four times with PBST, and the ELISA plate was tapped to dry with absorbent paper. 100 μl of chromogenic solution (ELISA chromogenic solution A and chromogenic solution B, uniformly mixed in a 1:1 volume ratio immediately before use) was added to each well. After color development, 80 μl of the final solution was added to each well 5 minutes later to terminate the reaction. Immediately afterward, the OD value of each well was measured at a wavelength of 450 nm using a microplate reader, and the data was analyzed using GraphPad Prism 6 software. The experimental results are shown in Table 2.

[0087] <Example 6: Measurement of mouse-derived anti-human TSLP monoclonal antibody sequence> Based on the experimental results of Examples 4 and 5, 30 hybridoma cell lines were selected and antibody sequences were extracted. Total RNA was extracted from each hybridoma cell line using Trizol (purchased from Shanghai Biotechnology), and the mRNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from ABI). Using primers reported in the literature, the light chain variable region and heavy chain variable region genes of mouse-derived anti-human hTSLP monoclonal antibodies were amplified by PCR. The PCR products were then cloned into pGEM-T vectors, sequenced, and the variable region gene sequences were analyzed. When the obtained sequences were aligned and analyzed using GenBank, all sequences matched the characteristics of the mouse IgG variable region gene. Preferred amino acid sequences of the CDR region of the antibodies are listed in Tables 3 and 4.

[0088] [Table 3]

[0089] [Table 4]

[0090] <Example 7: Humanization of anti-human TSLP monoclonal antibody> Based on sequence analysis results, antibodies 27, 101, 111, and 163 were selected for the construction of chimeric antibodies and humanized antibodies. Chimeric antibodies were constructed by excising the heavy chain variable region and light chain variable region of mouse-derived antibodies and connecting them to the constant regions of the light and heavy chains of human IgG1, respectively, using overlap PCR.

[0091] Using the Kabat scheme, the amino acid sequences of the light chain variable region and heavy chain variable region of mouse-derived anti-human TSLP monoclonal antibody were analyzed, and three CDRs and four FRs were determined. Using antibody 163 as an example, IGHV3-23*04 was selected as the heavy chain CDR transplantation template by comparing its homology with human IgG germline sequences (Germline) using NCBI IgBlast. The heavy chain CDR region of mouse-derived anti-human TSLP monoclonal antibody 163 was transplanted into the framework region of IGHV3-23*04 to construct a heavy chain CDR transplantation antibody. Similarly, IGKV3-11*01 was selected as the light chain CDR transplantation template by comparing its homology with human IgG germline sequences. The light chain CDR region of mouse-derived anti-human TSLP monoclonal antibody 163 was transplanted into the framework region of IGKV3-11*01 to construct a light chain CDR transplantation antibody. Simultaneously, based on this, some amino acid sites in the framework region were reverse-mutated. During the reverse mutation, the amino acid sequence was numbered using Kabat numbering, and the location of the site was indicated by the Kabat number. Preferably, for the light chain variable region sequence, the A at position 43 of Kabat numbering was reverse-mutated to S derived from mouse, and the I at position 58 was reverse-mutated to V. For the heavy chain variable region, the A at position 49 was reverse-mutated to S. The gene sequence of the above variable region was synthesized by codon optimization at Bio-Biological Co., Ltd. according to the codon usage preference of Cricetulus griseus. The antibody to which the synthesized humanized variable region sequence was linked to the human IgG1 constant region was defined as the humanized antibody of antibody number 16 (163-Humanization, 163H).

[0092] Using the same principle as described above, the remaining three antibodies were similarly humanized. The sequences of the humanized antibodies are shown in Tables 5 and 6. Transient expression vectors for the humanized heavy chain and light chain were constructed using the pTT5 vector, and the above light chain and heavy chain combinations were transiently transfected using the HEK293E system to express the antibodies. HEK293E cells were cultured in Free Style 293 expression medium (purchased from Gibco), and the plasmid was transfected into the cells using the PEI transfection method. Five days later, the cell supernatant was harvested and purified with protein A to obtain the individual humanized monoclonal antibodies.

[0093] Ultimately, the nucleotide sequence of the humanized heavy chain variable region gene of antibody 27 is shown in SEQ ID NO: 25, and its amino acid sequence is shown in SEQ ID NO: 26; the nucleotide sequence of the humanized light chain variable region is shown in SEQ ID NO: 28, and its amino acid sequence is shown in SEQ ID NO: 29. By ligating to the human IgG1 constant region, the 27H humanized heavy chain (sequence shown in SEQ ID NO: 27) and the 27H humanized light chain (sequence shown in SEQ ID NO: 30) were finally obtained.

[0094] The nucleotide sequence of the humanized heavy chain variable region gene of antibody 101 is shown in SEQ ID NO: 31, and the amino acid sequence is shown in SEQ ID NO: 32; the nucleotide sequence of the humanized light chain variable region is shown in SEQ ID NO: 34, and the amino acid sequence is shown in SEQ ID NO: 35. By ligating to the human IgG1 constant region, the 101H humanized heavy chain (sequence shown in SEQ ID NO: 33) and the 101H humanized light chain (sequence shown in SEQ ID NO: 36) were ultimately obtained.

[0095] The nucleotide sequence of the humanized heavy chain variable region gene of antibody 111 is shown in SEQ ID NO: 37, and the amino acid sequence is shown in SEQ ID NO: 38; the nucleotide sequence of the humanized light chain variable region is shown in SEQ ID NO: 40, and the amino acid sequence is shown in SEQ ID NO: 41. By ligating to the human IgG1 constant region, the 111H humanized heavy chain (sequence shown in SEQ ID NO: 39) and the 101H humanized light chain (sequence shown in SEQ ID NO: 42) were ultimately obtained.

[0096] The nucleotide sequence of the humanized heavy chain variable region gene of antibody 163 is shown in SEQ ID NO: 43, and the amino acid sequence is shown in SEQ ID NO: 44; the nucleotide sequence of the humanized light chain variable region is shown in SEQ ID NO: 46, and the amino acid sequence is shown in SEQ ID NO: 47. By ligating to the human IgG1 constant region, the 163H humanized heavy chain (sequence shown in SEQ ID NO: 45) and the 101H humanized light chain (sequence shown in SEQ ID NO: 48) were ultimately obtained.

[0097] [Table 5]

[0098] [Table 6]

[0099] <Example 8: Inhibition of TSLP and TSLPR binding by a humanized anti-human TSLP antibody> The inhibitory effect of the humanized TSLP antibody on the binding of TSLP to TSLPR was measured using the method of Example 5. Details of the experimental results are shown in Table 7.

[0100] [Table 7]

[0101] <Example 9: Inhibition of TSLP-dependent STAT5 activation by a humanized anti-human TSLP antibody> First, we constructed a stable BaF3 cell line co-expressing two receptors, hIL-7R and hTSLPR. Based on this, we obtained a stable IL-7R / TSLPR-BaF3-Luc(STAT5) cell line by stably transfecting it with pGL4.52 (purchased from Promega).

[0102] IL-7R / TSLPR-BaF3-Luc(STAT5) cells in the logarithmic growth phase were replaced with serum-free IMDM medium and cultured overnight in a 37°C incubator. The following day, the antibody was diluted from an initial concentration of 100 μg / ml to 100 μl per well, 3-fold over 11 gradients. Then, 50 μl of 4 ng / ml TSLP-flag was added to each well and incubated for 30 minutes. The serum-free IL-7R / TSLPR-BaF3-Luc(STAT5) cells cultured overnight were centrifuged in serum-free IMDM medium for a cell concentration of 1 × 10⁶. 6 After adjusting to the final concentration and thoroughly mixing, 50 μl of the cell suspension per well was added to the antibody solution and incubated at 37°C for 4 hours. After homogeneous mixing by flicking the pipette tip three times, 75 μl of the mixture was taken per well on an assay white plate, creating one duplicate well. 75 μl of the firefly fluorescein luciferase substrate (purchased from Promega) was added to each well, left for 20 minutes, and then read at 560 nm. The data was analyzed using GraphPad Prism 6 software. The experimental results are shown in Figure 1, and the IC50s of TEZ-1, 111H, 163H, and TEZ-2 were 957.9 ng / ml, 184.4 ng / ml, 377.9 ng / ml, and 853.8 ng / ml, respectively. As can be seen from the experimental results, the effect of the preferred antibody in inhibiting TSLP-dependent STAT5 activation was significantly better than that of the reference antibody.

[0103] <Example 10: Inhibition of TARC secretion by PBMCs by a humanized anti-human TSLP antibody> Humanized anti-human TSLP antibody was diluted to concentrations of 80 μg / ml and 16 μg / ml in RPMI 1640 medium containing 5% fetal bovine serum, and added to 96-well cell culture plates at a rate of 50 μl / well. TSLP-flag protein was diluted to a concentration of 10 ng / ml in RPMI 1640 medium containing 5% fetal bovine serum, and added to the wells of the 96-well cell culture plates containing the TSLP antibody at a rate of 50 μl / well. The culture plates were incubated in a 37°C incubator for 1 hour. Fresh PBMCs (purchased from TPCS) were incubated in RPMI 1640 medium containing 5% fetal bovine serum at a rate of 2 × 10⁶ 6 The antibody was adjusted to cells / ml and inoculated at a rate of 100 μl per well into a 96-well cell culture plate containing the antibody, with three duplicate wells. After incubating the 96-well plate in a 37°C incubator for 2 days, the culture supernatant was harvested, and the TARC content in the supernatant was measured using the Human CCL17 / TARC Quantikine ELISA Kit (R&D Systems). The experimental results are shown in Figure 2. As can be seen from the experimental results, the activity of the preferred antibody in inhibiting TARC secretion by PBMCs was significantly superior to that of the reference antibody.

[0104] <Example 11: Affinity of humanized anti-human TSLP monoclonal antibody for TSLP> The affinity of the expressed and purified humanized antibody was measured using Biacore T200 (GE Healthcare), with Tezepelumab used as the reference antibody. The specific experimental method involved using a Protein A CM5 sensing chip (GE Healthcare), with FC1 (Flow cell 1) as the reference channel and FC2 (Flow cell 2) as the sample channel. Human antibodies or control antibodies were captured in the FC2 channel, and then different concentrations of hTSLP-Flag were injected. The cycling conditions involved injecting the analyte at 50 μl / min for 4 minutes into all FC channels, followed by a dissociation time of 20 minutes. Surface regeneration was then performed by injecting 6M guanidine hydrochloride (Sinopharm Group Chemical Reagents Co., Ltd.) at 10 μl / min for 30 seconds. Subsequently, the difference between the signals of the captured antibody and the uncaptured antibody, as well as the affinity of the interaction, were calculated using Biacore T200 evaluation software Ver1.0. As shown in Table 8, the affinity of the humanized antibody 163H for hTSLP was equivalent to that of the reference antibody Tezepelumab.

[0105] [Table 8]

[0106] Conclusion: The antibody of the present invention consistently exhibits stronger biological activity than the control antibody Tezepelumab in affinity and multiple in vitro functional activity assays.

[0107] While this application has been described in some way, it should be understood that it is not limited to what is shown and described herein. It will be obvious to those skilled in the art that various modifications can be made to the embodiments and / or certain features or parameters without departing from the scope of this application. All such modifications fall within the scope of the protection claimed herein.

Claims

1. An antibody or antigen-binding moiety thereof that specifically binds to human thymic stromal lymphocyte necrosis factor (hTSLP), comprising a heavy chain variable region and a light chain variable region, The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, and, The light chain variable region includes LCDR1, LCDR2, and LCDR3. An antibody or its antigen-binding portion, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

24.

2. The antibody or antigen-binding moiety according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 44, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

47.

3. The antibody or antigen-binding moiety according to claim 1, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 45, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

48.

4. The antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein the antigen-binding portion is selected from the group consisting of Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, and scFv fragment.

5. An antibody or its antigen-binding portion according to any one of claims 1 to 3, which can specifically bind to human thymic interstitial lymphocyte necrotizing factor or cynomolgus monkey thymic interstitial lymphocyte necrotizing factor, wherein the antigen-binding portion binds to human thymic interstitial lymphocyte necrotizing factor with a KD of less than 60 pM.

6. An antibody or antigen-binding moiety according to any one of claims 1 to 3, which can inhibit TSLP-dependent STAT5 activation and effectively inhibit TARC secretion by PBMCs.

7. A pharmaceutical composition comprising an antibody or an antigen-binding moiety thereof according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

8. An antibody according to any one of claims 1 to 6, or a nucleotide molecule encoding the antigen-binding portion thereof.

9. An expression vector comprising the nucleotide molecule described in claim 8.

10. A host cell comprising the nucleotide molecule described in claim 8 or the expression vector described in claim 9.

11. a) A step of culturing the host cells described in claim 10 under expression conditions that enable the host cells to produce the antibody or the antigen-binding portion thereof, thereby expressing the antibody or the antigen-binding portion thereof; and b) A step of separating and purifying the antibody or its antigen-binding portion expressed in step a), A method for preparing an antibody or its antigen-binding portion according to any one of claims 1 to 6, including the method described in any one of claims 1 to 6.

12. An antibody or antigen-binding portion thereof according to any one of claims 1 to 6, for use in the prevention or treatment of TSLP-related diseases.

13. The antibody or antigen-binding portion thereof according to claim 12, wherein the TSLP-related disease is an immune-mediated inflammatory disease.

14. The antibody or antigen-binding portion thereof according to claim 13, wherein the immune-mediated inflammatory disease is selected from asthma, chronic obstructive pulmonary disease, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma.

15. The pharmaceutical composition according to claim 7, for use in the prevention or treatment of TSLP-related diseases.

16. The pharmaceutical composition according to claim 15, wherein the TSLP-related disease is an immune-mediated inflammatory disease.

17. The pharmaceutical composition according to claim 16, wherein the immune-mediated inflammatory disease is selected from asthma, chronic obstructive pulmonary disease, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma.

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