Anti-TSLP antibody and application thereof

Abstract

According to the present invention, the antibody specifically binding to TSLP or the antigen-binding fragment thereof is successfully obtained. The specific binding TSLP antibody or the antigen binding fragment thereof provided by the invention can be used as a medicine for treating or preventing diseases related to TSLP, especially a medicine for treating asthma and atopic dermatitis diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to anti-TSLP (Thymic stromal lymphopoietin) antibodies, methods of making the antibodies, pharmaceutical compositions comprising the antibodies, kits, and use of the antibodies in the manufacture of a medicament for the treatment and / or prevention of a thymic stromal lymphopoietin (TSLP) infection or a disease associated with TSLP. BACKGROUND

[0002] Asthma is one of the most common chronic allergic diseases worldwide, with more than 300 million patients globally. As of 2022, the prevalence of asthma among people aged 20 and above in China was 4.2%, with a total of 45.7 million patients. Asthma is characterized by paroxysmal cough, expectoration, chest tightness, and dyspnea, which can last for several weeks or longer. In severe cases, breathing is extremely difficult, and even life-threatening. There are three major categories of hot targets for treating asthma on the market, the first category is interleukins, such as interleukin 4, interleukin 5, interleukin 7, and interleukin 13, the second category is granulocyte macrophage colony-stimulating factor (GM-CSF), and the third category is TSLP. TSLP has been proven to play an important role in the development of asthma, therefore, the development of specific antibodies targeting TSLP and blocking its signaling pathway has a very important clinical value.

[0003] Thymic stromal lymphopoietin (TSLP) is a protein of the cytokine family. It plays an important role in the maturation of T cell populations by activating antigen-presenting cells. TSLP is mainly produced by non-hematopoietic cells, such as fibroblasts, epithelial cells, and different types of stromal or stromal-like cells. TSLP forms a ternary signaling complex with the thymic stromal lymphopoietin receptor CRLF2 (TSLPR) and the IL-7R alpha chain. Intracellular signals are then activated by activating STATs and JAK2 pathways. It mainly affects myeloid cells, inducing monocytes to release T cell-attracting chemokines and promoting the maturation of CD11c(+) dendritic cells. This protein promotes type 2 helper T cell (TH2) responses and is involved in immune responses in various inflammatory diseases, including asthma, allergic inflammation, and chronic obstructive pulmonary disease. Therefore, TSLP is considered a potential therapeutic target for the treatment of these diseases.

[0004] The TSLP receptor is a complex that includes two parts, one of which is TSLPR and the other of which is the IL-7 receptor alpha chain (IL-7Rα). TSLPR can only form a high-affinity receptor complex in cooperation with the IL-7 receptor alpha chain (IL-7Rα), and has very low affinity for TSLP itself. TSLP first binds to TSLPR with relatively low affinity, then recruits IL-7Rα for binding with high affinity, and finally activates signal pathways such as stat5, leading to the maturation of DCs and the differentiation of T cells.

[0005] Studies have found that glucocorticoids can reduce the expression of TSLP and TSLPR in asthmatic mice, and can change the function of DCs induced by TSLP, so that the antigen-induced Th2 response is shifted to Th1 response, and the production of IL-4, TL-5 and IL-13 is reduced, and the production of IL-10 and IFN-y is increased. This effect depends on the down-regulation of OX40L in DCs and the increase in IL-12 production. Research data from asthmatic patients show that the severity of airway inflammation in asthma is positively correlated with the expression level of TSLP.

[0006] TSLP-TSLPR and downstream signaling molecules play an important role in the occurrence and development of allergic inflammatory diseases such as allergic asthma.

[0007] At present, the drugs on the market that can treat asthma include Nucala (mepolizumab, targeting IL-5) of GSK, Cinqair (reslizumab, targeting IL-5) of Teva, and the biological therapies currently being developed to treat asthma, such as benralizumab (targeting IL-5 receptor alpha subunit [IL-5Rα]) of AstraZeneca and Dupixent (targeting IL-4 / IL-13) of Sanofi. All of these four therapies only target specific inflammatory molecules that drive asthma inflammation, and are only suitable for certain types of severe asthma patients, i.e. subgroups of patients, such as eosinophilic asthma. The Tezspire (targeting TSLP) developed by AstraZeneca and Amgen in conjunction has a much larger treatment population than the aforementioned biological therapies that have already been marketed.

[0008] Therefore, there is an urgent need to develop new anti-TSLP antibody drugs, especially drugs with good effects and low prices. SUMMARY

[0009] In order to solve the problems existing in the prior art, the purpose of the present application is to provide anti-TSLP antibodies and applications thereof.

[0010] The present application successfully obtains TSLP-specific antibodies with comparable or better properties than the previous TSLP-specific antibody Tezspire, including in vitro efficacy, drugability and animal efficacy results.

[0011] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to thymic stromal lymphopoietin (TSLP), comprising a heavy chain variable region, a light chain variable region, and a heavy chain constant region, wherein:

[0012] 1) The heavy chain variable region includes

[0013] (i) The three heavy chain complementarity-determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 contained within a peptide with the amino acid sequence SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86, or 88; or

[0014] (ii) The sequences of the three heavy chain complementarity-determining regions (HCDRs) described in (i) contain at least one and no more than five amino acid mutations (preferably amino acid substitutions, preferably conserved substitutions) in total.

[0015] 2) The light chain variable region includes,

[0016] (i) The three light chain complementarity-determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 contained within a peptide with the amino acid sequence SEQ ID NO: 8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87, or 89; or

[0017] (ii) A sequence comprising, relative to the three light chain complementarity-determining regions (LCDRs) described in (i), at least one and no more than five amino acid mutations (preferably amino acid substitutions, preferably conserved substitutions) in total on the three light chain complementarity-determining regions (LCDRs); and

[0018] 3) the heavy chain constant region comprises a heavy chain constant region of IgGl, IgG2 or IgG4 mutant, wherein the mutation is selected from at least one of S228, L234, L235, G236, G237, T250, M252, I253, S254, T256, D259, T307, V308, P329, A330, E380, M428 and N434 (numbering according to the Kabat EU index), preferably at least one of G237A, L234A / L235A, S228P, N434A, N434H, T307A / E380A / N434A, M428L / N434S (LS) and M252Y / S254T / T256E (numbering according to the Kabat EU index), more preferably M252Y / S254T / T256E (numbering according to the Kabat EU index); preferably the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 97.

[0019] The present application provides an antibody or antigen binding fragment thereof, which specifically binds to Thymic stromal lymphopoietin (TSLP), inhibits the binding of TSLP to its receptor, and has an IC50 of less than 100 nM in an in vitro assay. 50 The superior stability and in vitro drugability of the present antibody and the animal efficacy results are characterized by in vitro assay, PEG sedimentation method, animal experiment.

[0020] The present application provides an antibody or antigen binding fragment thereof, which specifically binds to Thymic stromal lymphopoietin (TSLP), inhibits the binding of TSLP to its receptor, and has an IC50 of less than 100 nM in an in vitro assay.

[0021] a) a heavy chain complementarity determining region HCDR3, which comprises or consists of any one of the group consisting of the following amino acid sequences: SEQ ID NO: 3, 15, 27, 39, 50, 60, 71 and 80, or which comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 3, 15, 27, 39, 50, 60, 71 or 80;

[0022] b) a light chain complementarity determining region LCDR3 comprising or consisting of any of the group of amino acid sequences of SEQ ID NO: 6, 18, 30, 42, 63 and 83, or comprising an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 6, 18, 30, 42, 63 or 83; and

[0023] c) a heavy chain constant region comprising a heavy chain constant region of an IgGl, IgG2 or IgG4 mutant, wherein the mutation is selected from at least one of S228, L234, L235, G236, G237, T250, M252, I253, S254, T256, D259, T307, V308, P329, A330, E380, M428 and N434 (numbering according to the Kabat EU index), preferably from at least one of G237A, L234A / L235A, S228P, N434A, N434H, T307A / E380A / N434A, M428L / N434S (LS) and M252Y / S254T / T256E (numbering according to the Kabat EU index), more preferably M252Y / S254T / T256E (numbering according to the Kabat EU index); preferably the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 97.

[0024] further comprising:

[0025] d) a heavy chain complementarity determining region HCDR1 comprising or consisting of any of the group of amino acid sequences of SEQ ID NO: 1, 13, 25, 37, 58 and 78, or comprising an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 1, 13, 25, 37, 58 or 78; and / or

[0026] e) a light chain complementarity determining region LCDR1 comprising or consisting of any of the group of amino acid sequences set forth in SEQ ID NOs: 4, 16, 28, 40, 61, and 81, or comprising an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 4, 16, 28, 40, 61, or 81;

[0027] Preferably, it further comprises:

[0028] f) a heavy chain complementarity determining region HCDR2 comprising or consisting of any of the group of amino acid sequences set forth in SEQ ID NOs: 2, 14, 26, 38, 49, 59, 70, and 79, or comprising an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 2, 14, 26, 38, 49, 59, 70, or 79; and / or

[0029] g) a light chain complementarity determining region LCDR2 comprising or consisting of any of the group of amino acid sequences set forth in SEQ ID NOs: 5, 17, 29, 41, 51, 62, and 82, or comprising an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 5, 17, 29, 41, 51, 62, and 82.

[0030] Optionally, the antibody or antigen binding fragment thereof, comprises three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, and a heavy chain constant region, wherein:

[0031] HCDR1 comprises or consists of SEQ ID NO: 1, 13, 25, 37, 58, or 78, or comprising an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 1, 13, 25, 37, 58, or 78;

[0032] HCDR2 comprises or consists of SEQ ID NO: 2, 14, 26, 38, 49, 59, 70, or 79, or the HCDR2 comprises an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 2, 14, 26, 38, 49, 59, 70, or 79;

[0033] HCDR3 comprises or consists of SEQ ID NO: 3, 15, 27, 39, 50, 60, 71, or 80, or the HCDR3 comprises an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 3, 15, 27, 39, 50, 60, 71, or 80;

[0034] LCDR1 comprises or consists of SEQ ID NO: 4, 16, 28, 40, 61, or 81, or the LCDR1 comprises an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 4, 16, 28, 40, 61, or 81 ;

[0035] LCDR2 comprises or consists of SEQ ID NO: 5, 17, 29, 41, 51, 62, or 82, or the LCDR2 comprises an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 5, 17, 29, 41, 51, 62, or 82;

[0036] LCDR3 comprises or consists of SEQ ID NO: 6, 18, 30, 42, 63, or 83, or the LCDR3 comprises an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 6, 18, 30, 42, 63, or 83; and

[0037] the heavy chain constant region comprises a heavy chain constant region of IgGl, IgG2 or IgG4 mutant, wherein the mutation is selected from at least one of S228, L234, L235, G236, G237, T250, M252, I253, S254, T256, D259, T307, V308, P329, A330, E380, M428 and N434 (numbering according to the Kabat EU index), preferably at least one of G237A, L234A / L235A, S228P, N434A, N434H, T307A / E380A / N434A, M428L / N434S (LS) and M252Y / S254T / T256E (numbering according to the Kabat EU index), more preferably M252Y / S254T / T256E (numbering according to the Kabat EU index); preferably the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 97.

[0038] Optionally, the antibody or antigen binding fragment thereof comprises:

[0039] (1) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having the amino acid sequence of SEQ ID NO: 7 or 11, and

[0040] three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having the amino acid sequence of SEQ ID NO: 8 or 12;

[0041] (2) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having the amino acid sequence of SEQ ID NO: 19, 23, 93 or 21, and

[0042] three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having the amino acid sequence of SEQ ID NO: 20, 24, 94 or 22;

[0043] (3) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having the amino acid sequence of SEQ ID NO: 31, 33 or 35, and

[0044] three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having the amino acid sequence of SEQ ID NO: 32, 34 or 36;

[0045] (4) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch of an amino acid sequence of SEQ ID NO: 43, 45 or 47, and

[0046] (4) three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch of an amino acid sequence of SEQ ID NO: 44, 46 or 48;

[0047] (5) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch of an amino acid sequence of SEQ ID NO: 52, 54 or 56, and

[0048] (5) three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch of an amino acid sequence of SEQ ID NO: 53, 55 or 57;

[0049] (6) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch of an amino acid sequence of SEQ ID NO: 64, 66 or 68, and

[0050] (6) three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch of an amino acid sequence of SEQ ID NO: 65, 67 or 69;

[0051] (7) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch of an amino acid sequence of SEQ ID NO: 72, 74 or 76, and

[0052] (7) three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch of an amino acid sequence of SEQ ID NO: 73, 75 or 77; or

[0053] (8) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch of an amino acid sequence of SEQ ID NO: 84, 86, 88 or 95, and

[0054] (8) three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch of an amino acid sequence of SEQ ID NO: 85, 87, 89 or 96.

[0055] Optionally, the antibody or antigen-binding fragment thereof comprises:

[0056] 1) a HCDR1 comprising or consisting of SEQ ID NO: 1,

[0057] a HCDR2 comprising or consisting of SEQ ID NO: 2,

[0058] a HCDR3 comprising or consisting of SEQ ID NO: 3,

[0059] a LCDR1 comprising or consisting of SEQ ID NO: 4,

[0060] a LCDR2 comprising or consisting of SEQ ID NO: 5, and

[0061] a LCDR3 comprising or consisting of SEQ ID NO: 6; or

[0062] 2) a HCDR1 comprising or consisting of SEQ ID NO: 13,

[0063] a HCDR2 comprising or consisting of SEQ ID NO: 14,

[0064] a HCDR3 comprising or consisting of SEQ ID NO: 15,

[0065] a LCDR1 comprising or consisting of SEQ ID NO: 16,

[0066] a LCDR2 comprising or consisting of SEQ ID NO: 17, and

[0067] a LCDR3 comprising or consisting of SEQ ID NO: 18; or 3) a HCDR1 comprising or consisting of SEQ ID NO: 25,

[0068] a HCDR2 comprising or consisting of SEQ ID NO: 26,

[0069] a HCDR3 comprising or consisting of SEQ ID NO: 27,

[0070] a LCDR1 comprising or consisting of SEQ ID NO: 28,

[0071] a LCDR2 comprising or consisting of SEQ ID NO: 29, and

[0072] a LCDR3 comprising or consisting of SEQ ID NO: 30; or 4) a HCDR1 comprising or consisting of SEQ ID NO: 37,

[0073] a HCDR2 comprising or consisting of SEQ ID NO: 38,

[0074] HCDR3 comprising or consisting of SEQ ID NO:39,

[0075] LCDR1 comprising or consisting of SEQ ID NO:40,

[0076] LCDR2 comprising or consisting of SEQ ID NO:41, and

[0077] LCDR3 comprising or consisting of SEQ ID NO:42; or 5) HCDR1 comprising or consisting of SEQ ID NO:25,

[0078] HCDR2 comprising or consisting of SEQ ID NO:49,

[0079] HCDR3 comprising or consisting of SEQ ID NO:50,

[0080] LCDR1 comprising or consisting of SEQ ID NO:28,

[0081] LCDR2 comprising or consisting of SEQ ID NO:51, and

[0082] LCDR3 comprising or consisting of SEQ ID NO:30; or 6) HCDR1 comprising or consisting of SEQ ID NO:58,

[0083] HCDR2 comprising or consisting of SEQ ID NO:59,

[0084] HCDR3 comprising or consisting of SEQ ID NO:60,

[0085] LCDR1 comprising or consisting of SEQ ID NO:61,

[0086] LCDR2 comprising or consisting of SEQ ID NO:62, and

[0087] LCDR3 comprising or consisting of SEQ ID NO:63; or 7) HCDR1 comprising or consisting of SEQ ID NO:25,

[0088] HCDR2 comprising or consisting of SEQ ID NO:70,

[0089] HCDR3 comprising or consisting of SEQ ID NO:71,

[0090] LCDR1 comprising or consisting of SEQ ID NO:28,

[0091] LCDR2 comprising or consisting of SEQ ID NO: 51, and

[0092] LCDR3 comprising or consisting of SEQ ID NO: 30; or

[0093] 8) HCDR1 comprising or consisting of SEQ ID NO: 78,

[0094] HCDR2 comprising or consisting of SEQ ID NO: 79,

[0095] HCDR3 comprising or consisting of SEQ ID NO: 80,

[0096] LCDR1 comprising or consisting of SEQ ID NO: 81,

[0097] LCDR2 comprising or consisting of SEQ ID NO: 82, and

[0098] LCDR3 comprising or consisting of SEQ ID NO: 83.

[0099] The present application also discloses an antibody or antigen binding fragment thereof that specifically binds to TSLP, comprising:

[0100] a heavy chain variable region comprising or consisting of SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86, or 88, or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86, or 88; or

[0101] The light chain variable region comprises: SEQ ID NO:8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87 or 89; or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 identity with the sequence SEQ ID NO:8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87 or 89; or the light chain variable region is composed of SEQ ID NO:8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87 or 89. NO: 8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87, or 89, or composed of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence SEQ ID NO: 8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87, or 89; or the light chain variable region contains an amino acid sequence identical to .... Compared to IDNO: 8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87, or 89, an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid mutations (preferably amino acid substitutions, more preferably conserved amino acid substitutions), or consisting thereof, preferably, the amino acid mutations do not occur in the light chain complementarity-determining region; and

[0102] the heavy chain constant region comprises a heavy chain constant region of an IgGl, IgG2 or IgG4 mutant, wherein the mutation is selected from at least one of S228, L234, L235, G236, G237, T250, M252, I253, S254, T256, D259, T307, V308, P329, A330, E380, M428 and N434 (numbering according to Kabat EU index), preferably at least one of G237A, L234A / L235A, S228P, N434A, N434H, T307A / E380A / N434A, M428L / N434S (LS) and M252Y / S254T / T256E (numbering according to Kabat EU index), more preferably M252Y / S254T / T256E (numbering according to Kabat EU index); preferably the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 97.

[0103] Optionally, the antibody or antigen-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of which are paired with any of the groups consisting of: SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, SEQ ID NO: 72 and SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77, SEQ ID NO: 84 and SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89.

[0104] Optionally, the antibody or antigen-binding fragment thereof, wherein each CDR is according to the Kabat definition, the Chothia definition, the Abm definition, and / or the Contact definition; preferably, wherein each CDR is according to the Kabat definition or the Chothia definition. For example, the Kabat definition is used in the embodiments of the present application.

[0105] Optionally, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising: SEQ ID NO: 91, or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 91; or consisting of SEQ ID NO: 91, or consisting of an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 91.

[0106] Optionally, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the amino acid sequences of which are:

[0107] 1) a heavy chain as depicted in SEQ ID NO: 93 and a light chain as depicted in SEQ ID NO: 94; or

[0108] 2) a heavy chain as depicted in SEQ ID NO: 95 and a light chain as depicted in SEQ ID NO: 96.

[0109] Optionally, the antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (preferably scFv) or (Fab')2, single domain antibody, diabody (dAb) or linear antibody.

[0110] The second aspect of the application provides a nucleic acid for encoding the antibody or antigen-binding fragment thereof of any one of the first aspect. The nucleic acid can be synthetic, recombinant or isolated. Due to the degeneracy of the nucleic acid code, a variety of nucleic acids will encode the same amino acid and all are encompassed herein.

[0111] The third aspect of the application provides an expression vector comprising the nucleic acid of the second aspect.

[0112] The fourth aspect of the application provides a host cell comprising the expression vector of the third aspect.

[0113] The fifth aspect of the application provides a method of producing the antibody or antigen-binding fragment thereof, comprising culturing the host cell of the fourth aspect, and recovering the antibody or antigen-binding fragment expressed thereby from the culture.

[0114] The antibodies or antigen-binding fragments thereof described herein can be produced from a hybridoma that secretes the antibody, or from a recombinantly produced cell that has been transformed or transfected with one or more genes that encode the antibody or antigen-binding fragment thereof. The antibodies or antigen-binding portions thereof are produced by culturing the host cell under conditions such that the nucleic acid is expressed and the antibody is produced, followed by recovery of the antibody.

[0115] Recombinant expression utilizes construction of an expression vector that includes a polynucleotide that encodes the antibody or antigen-binding portion thereof. Once the polynucleotide has been obtained, a vector for production of the antibody can be produced by recombinant DNA technology well known in the art. The expression vector can include appropriate transcription and translation control signals. This can be accomplished using in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0116] Alternatively, the host cell is a prokaryotic or eukaryotic cell.

[0117] Alternatively, the host cell is an E. coli cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell.

[0118] Alternatively, the host cell is a Chinese hamster ovary cell (CHO), a CHO cell variant, a 293 cell, or an NSO cell. The cell lines include VERO, BHK, HeIa, COS, MDCK, 293F, 293T, 3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, CRL7030, and HsS78Bst cells. But the cell lines used in the present application include, but are not limited to, the above cell lines.

[0119] Once the antibody or antigen-binding portion of the antibody has been produced by recombinant expression, it can be purified by any method known in the art for purification of immunoglobulin molecules, for example, by chromatography, centrifugation, differential solubility, or by any other standard technique for protein purification.

[0120] The sixth aspect of the present application provides a pharmaceutical composition comprising at least one of any of the antibodies or antigen-binding fragments thereof of the first aspect, and a pharmaceutically acceptable excipient. For example, the antibody or antigen-binding fragment thereof is combined with water for injection, or with saline.

[0121] The seventh aspect of the present application provides a kit comprising at least one of any of the antibodies or antigen-binding fragments thereof or homologues, derivatives thereof of the first aspect.

[0122] The eighth aspect of the present application provides use of the antibody or antigen-binding fragment thereof of the first aspect or the pharmaceutical composition of the sixth aspect in the manufacture of a medicament for the treatment and / or prevention of a disease associated with TSLP.

[0123] The ninth aspect of the present application provides the antibody or antigen binding fragment thereof of the first aspect or the pharmaceutical composition of the sixth aspect for use in the treatment and / or prevention of a disease associated with TSLP.

[0124] The present application also provides a method for treating and / or preventing a disease associated with TSLP, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen binding fragment of any one of the first aspect or the pharmaceutical composition of the sixth aspect.

[0125] The disease associated with TSLP described above is asthma or dermatitis.

[0126] Advantages of the present application

[0127] Compared with the prior art, the technical scheme of the present application has the following advantages:

[0128] 1) The antibody of the present application can specifically and highly affinitively bind to TSLP, inhibit or block the binding of TSLP to TSLP / IL7R, and can inhibit or block the proliferation effect of TSLP on Ba / F3 cells in vitro, and block the activation and cytokine secretion ability of PBMC by TSLP. Therefore, the antibody of the present application has the potential for preventing and / or treating asthma, other allergic reactions or autoimmune diseases.

[0129] 2) Compared with the positive drug Tezepelumab, it has better thermal stability, pH stability, oxidation stability, solubility and affinity to TSLP.

[0130] 3) The TSLP antibody Q61-F of the present application has better anti-dermatitis and anti-asthma effects compared with the positive drug Tezepelumab.

[0131] 4) The TSLP antibody of the present application has a longer half-life. BRIEF DESCRIPTION OF DRAWINGS

[0132] Figure 1: The ability of different TSLP antibodies to block the binding of TSLP to TSLPR and TSLPR-IL7R complex, A is the experimental results of different TSLP antibodies blocking the binding of TSLP to TSLPR-Fc protein, B is the experimental results of different TSLP antibodies blocking the binding of TSLP to TSLPR-IL-7R-Fc protein. The horizontal axis is the concentration of TSLP antibody (unit: μg / mL), and the vertical axis is the inhibition rate.

[0133] Figure 2 : The results of TSLP antibody inhibiting the proliferation of Ba / F3 cells. The horizontal axis is the concentration of antibody (unit: μg / mL), and the vertical axis is the inhibition rate (%).

[0134] Figure 3 : Results of TSLP antibody inhibition of TARC (also known as CCL17) secretion from PBMC cells. The horizontal axis is the TSLP antibody concentration (unit: pg / mL), and the vertical axis is the amount of TARC secretion (unit: pg / mL).

[0135] Figure 4 : Solubility of anti-TSLP antibody was measured using PEG precipitation method.

[0136] Figure 5 : AD (atopic dermatitis) model graph.

[0137] Figure 6 : Comparison results of body weight of mice in each administration group. The horizontal axis is the number of days (unit: days), and the vertical axis is the body weight of mice (unit: g).

[0138] Figure 7 : Comparison results of ear thickness of mice in each administration group. The horizontal axis is the number of days (unit: days), and the vertical axis is the ear thickness of mice (unit: pm).

[0139] Figure 8 : Comparison results of IgE content in serum of mice in each administration group. The horizontal axis is the group, and the vertical axis is the IgE content in serum of mice (unit: ng / mL).

[0140] Figure 9 : Comparison results of body weight of mice in each administration group. The horizontal axis is the number of days (unit: days), and the vertical axis is the body weight change of mice.

[0141] Figure 10 : Comparison results of IgE content in serum of mice in each administration group. The horizontal axis is the group, and the vertical axis is the IgE content in serum of mice (unit: ng / mL).

[0142] Figure 11 : Results of TSLP antibody with YTE mutation in heavy chain inhibiting Ba / F3 cell proliferation. The horizontal axis is the antibody concentration (unit: pg / mL), and the vertical axis is the inhibition rate (%).

[0143] Figure 12 : Results of TSLP antibody with YTE mutation in heavy chain inhibiting TARC (also known as CCL17) secretion from PBMC cells. The horizontal axis is the TSLP antibody concentration (unit: pg / mL), and the vertical axis is the amount of TARC secretion (unit: pg / mL). DETAILED DESCRIPTION

[0144] The embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are intended to explain, but not limit, the scope of the present application. Unless otherwise specified, the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, but are conventional products that can be purchased on the market.

[0145] Abbreviations

[0146] PBS refers to Phosphate Buffer Solution.

[0147] PBST refers to PBS solution with Tween-20 added.

[0148] BSA refers to Bovine Serum Albumin. BSA blocking solution is prepared from clear albumin.

[0149] TMB refers to 3,3',5,5'-Tetramethylbenzidine

[0150] Solution.

[0151] IC 50 refers to the concentration of the antibody that produces 50% inhibition.

[0152] ELISA refers to Enzyme-Linked Immunosorbent Assay.

[0153] TARC refers to Thymus and Activation-Regulated Chemokine, also known as CCL17.

[0154] PEG refers to polyethylene glycol (PEG).

[0155] HIC refers to Hydrophobic interaction chromatography.

[0156] Tm refers to Melting temperature.

[0157] ND (Not Detected) refers to the experimental data is not detected under the experimental conditions.

[0158] NA (Not Apply) refers to the experimental data is beyond the detection limit of the instrument under the experimental conditions, and is not applicable.

[0159] Definitions

[0160] Unless otherwise indicated, any polypeptide chain is described herein as having an amino acid sequence starting at the N-terminus and ending at the C-terminus.

[0161] The term "antibodies and antigen-binding portions thereof" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0162] The term "antibody" as used herein refers to an immunoglobulin molecule comprised of four polypeptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain (CL1). The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each of VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, wherein the three CDRs of the VHare HCDR1, HCDR2 and HCDR3, and the three CDRs of the VLare LCDR1, LCDR2 and LCDR3. The assignment of amino acids to each domain is generally consistent with the definition at Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989).

[0163] There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM and some of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0164] The mutations to the constant region of the antibody of the present application refer to variants of the heavy chain constant region or the light chain constant region that do not change the structure and function of the variable region of the antibody, but can make the antibody have new properties, such as improving stability, prolonging half-life, etc. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants that are subjected to site-directed modification and amino acid substitution. Mutations are made to the heavy chain constant region of the antibody, including but not limited to the following positions: S228, L234, L235, G236, G237, T250, M252, I253, S254, T256, D259, T307, V308, P329, A330, E380, M428 and N434 (numbering according to the Kabat EU index), optionally, the heavy chain constant region of the antibody comprises at least one mutation selected from G237A, L234A / L235A, S228P, N434A, N434H, T307A / E380A / N434A, M428L / N434S (LS) and M252Y / S254T / T256E (YTE) (numbering according to the Kabat EU index).

[0165] Unless otherwise specified herein, the numbering of amino acid residues in the constant region is according to the EU numbering system, also known as the EU index of Kabat, as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0166] The term "antigen-binding portion" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a TSLP protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Binding fragments of an antibody are included within the term "antigen-binding portion" of an antibody and include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL1, and CH1 domains of a full-length antibody; (ii) a F(ab')2 fragment, bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains of a full-length antibody; (iv) a Fv fragment consisting of the VL and VH domains of a full-length antibody; (v) a dAb fragment, which consists of a VH domain; and (vi) a CDR. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single contiguous chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). Such single chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed.

[0167] Different analyses can be employed to determine or roughly estimate CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition. The Kabat definition is a standard for numbering residues in antibodies and is commonly used to determine CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the location of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses integrated computer programs that mimic antibody structure made by Oxford Molecular Group. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); "AbM™, A Computer Program for Modeling Variable Regions of Antibodies" Oxford, UK; Oxford Molecular, Ltd. The AbM definition models antibody tertiary structure from primary sequence using known databases and ab initio methods such as those described in Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0168] The antibody of the present application, which is not limited in its origin, can be a human antibody, a mouse antibody, a rat antibody, and the like from any animal. It can also be a chimeric antibody or a humanized antibody, and the like recombinant antibody. A humanized antibody is preferred.

[0169] The term "murine antibody" is a monoclonal antibody against human TSLP prepared according to the knowledge and skill in the art. When prepared, the subject is injected with a TSLP antigen, and then a hybridoma expressing an antibody having a desired sequence or functional property is isolated. The murine anti-TSLP antibody or antigen-binding fragment thereof can further comprise a light chain constant region of a murine kappa, lambda chain, or a variant thereof, or further comprise a heavy chain constant region of a murine IgGl, IgG2, IgG3, or a variant thereof.

[0170] The term "chimeric antibody" refers to an antibody in which the variable region of the heavy chain and the light chain of a mammal other than human, for example, a mouse antibody, and the constant region of the heavy chain and the light chain of a human antibody are fused to reduce the immune response reaction induced by the murine antibody. The chimeric antibody can be prepared using known methods. For example, the chimeric antibody can be prepared by inserting the genes of the hybridoma into an appropriate vector and introducing it into a host. Specifically, the cDNA of the variable region (V) of the antibody is synthesized from the mRNA of the hybridoma using a reverse transcriptase. When the DNA encoding the V region of the target antibody is obtained, it is ligated with the DNA encoding the constant region (C region) of the desired human antibody, and then inserted into an expression vector. Alternatively, the DNA encoding the V region of the antibody can be inserted into an expression vector containing the DNA of the C region of the human antibody. It is inserted into the expression vector so as to be expressed under the control of the expression regulatory region. Next, the host cell is transformed with the expression vector, and the chimeric antibody can be expressed.

[0171] The term "humanized antibody", also referred to as CDR-grafted antibody, refers to an antibody in which the CDR sequences of a mouse are grafted into the variable region framework of a human antibody, i.e., an antibody generated by grafting the CDR sequences of a mouse into a different type of human germline antibody framework sequence. The heterologous reaction induced by the chimeric antibody carrying a large amount of mouse protein component can be overcome. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk / vbase), and in Kabat, E. A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in immunogenicity while causing a decrease in activity, the human antibody variable region framework sequence can be subjected to a minimum of back or reverse mutations to maintain activity.

[0172] The term "amino acid mutation" refers to an amino acid substitution, insertion, deletion, or modification. Specifically, a mutation at one or more amino acid positions on a polypeptide fragment between a polypeptide and a variant thereof, wherein the variant can be obtained by substitution, insertion, deletion, or modification of amino acids at one or more positions on the polypeptide.

[0173] The term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence, a variant of an antibody obtained by conservative substitution of amino acids sufficiently retains the biological activity of the sequence from which it is derived. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue for another that has similar side chain properties, such as substitution of a residue with a side chain that is similar in size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a preferred substitution is to replace a given amino acid residue with another amino acid residue from the same side chain family.

[0174] The term "nucleic acid" refers to DNA or RNA. The nucleic acid can be single- or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is functionally related to another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0175] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof, means that, when optimally aligned and compared with another nucleic acid (or its complement), using any of the sequence comparison algorithms described above, such as FASTA, BLAST or Gap, the nucleotides are identical in at least about 80%, more preferably in at least about 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide bases.

[0176] The terms "substantially similar" or "substantially similar to" when applied to a polypeptide means that two peptide sequences have at least 80% sequence identity, more preferably at least 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity when optimally aligned, e.g., using programs such as Gap or BESTFIT using default gap weights. Differences at non-identical residue positions can be the result of amino acid substitutions, deletions or insertions, more preferably, differences at non-identical residue positions are the result of conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ, due to conservative substitutions, the percent sequence identity or a similar measure can be adjusted upwards to correct for the conservative nature of the variations. Means for making this adjustment are well known to those of ordinary skill in the art. Examples of groups of amino acids that have side chains of similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl group: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic- aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions can be based on the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is any substitution that has a non-negative value in the PAM250 log-likelihood matrix.

[0177] The term "Thymic Stromal Lymphopoietin (TSLP)" is a four-α-helix bundle type I cytokine and an epithelial cell-derived cytokine produced in response to proinflammatory stimuli, closely related to interleukin-7 (IL-7), which initiates allergic responses by stimulating dendritic cells (DCs) and is an important factor in regulating immune responses in humans. The term "TSLP" includes variants, isoforms, homologs, orthologs and paralogs of TSLP.

[0178] The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein coding information to a host cell.

[0179] The term "expression vector" or "expression construct" refers to a vector that is suitable for transforming a host cell and contains nucleic acid sequences that direct and / or modulate the expression of one or more heterologous coding regions operably linked thereto. Expression vectors can include, but are not limited to, sequences that affect or modulate transcription, translation, and, if introns are present, RNA splicing of a coding region operably linked thereto.

[0180] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of the original cell that has been transformed. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny of the transformed cell that have a mutation not present in the original cell, regardless of the number of passages. The nucleic acid content of the progeny can not be identical to that of the original transformed cell, but can contain mutations when the original transformed cell contains a mutation. Mutant progeny that have the same function or biological activity as the originally transformed cell are included herein.

[0181] The term "transfection" means the uptake of foreign or exogenous DNA by a cell, which is "transfected" when the exogenous DNA is introduced into the cell membrane. A variety of transfection techniques are well known in the art. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. The techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

[0182] The term "antibody framework" or "FR region" refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain. In essence, it is the variable domain without the CDRs.

[0183] The terms "specifically binds," "selectively binds," "selectively binds," and "specifically binds to" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody binds to an antigen with an affinity (KD) of about less than 10 -8 M, for example, about less than 10 -9 M, 10 - 10 M, 10 -11 M, 10 -12 M or less.

[0184] The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Generally, the antibodies of the present disclosure bind TSLP with a dissociation equilibrium constant (KD) of less than about 10 -7 M, for example less than about 10 -8 M or 10 -9 M. KD values are determined using FACS or Biacore methods for the affinity of the antibodies of the present disclosure to cell surface antigens.

[0185] The term "Tm" refers to the melting point of a protein, that is, the temperature at which the molar heat capacity reaches a maximum, and is an important parameter for characterizing the thermal stability of an antibody. Traditionally, thermal stability is measured by differential scanning calorimetry (DSC). A typical thermogram produced by DSC has three partially overlapping regions of unfolding. Each of these melting regions is associated with a particular melting temperature (or Tm value) that corresponds to the maximum rate of change of heat capacity with temperature in a given temperature region. In most cases, the first (lowest temperature) Tm1 corresponds to the CH2 domain of Fc, the second Tm2 corresponds to the Fab, and Tm3 corresponds to the CH3 domain of Fc. The Tm values for the CH2 and CH3 domains do not vary as much as the Fab Tm values. Thus, during developability assessments or formulation screening, the Tm2 (Fab) values are primarily referenced. The greater the Tm2 (Fab) value, the better the thermal stability of the protein.

[0186] The term "treatment" includes therapeutic treatment, prophylactic treatment, and use in the reduction of risk or other risk factors of developing a disease in a subject. Treatment does not require complete cure of the disease, but includes embodiments in which symptoms are alleviated or underlying risk factors are reduced.

[0187] The term "about" or "approximately" means an acceptable error for the particular value as determined by one of ordinary skill in the art to which the value pertains, which is dependent in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within one, two, or three standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term "about" or "approximately" precedes the first numerical value in a series of two or more numerical values, the term "about" or "approximately" is understood to apply to every numerical value in that series of numerical values.

[0188] The term "effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition, which is necessary to achieve a desired therapeutic result. For prophylactic use, the desired result includes eliminating or reducing the risk, lessening the severity, or delaying the onset of a disorder, including biochemical, histological, and / or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes presenting during the course of the disorder's development. For therapeutic applications, the desired result includes clinical results such as reducing the incidence of various target antigen-related disorders of the present disclosure or ameliorating one or more symptoms of the disorder, reducing the dosage of other medications required to treat the disorder, enhancing the effect of another medication, and / or delaying the progression of the target antigen-related disorder of the present disclosure in a patient.

[0189] The term "pharmaceutical composition" refers to a preparation of a plurality of preparations. Containing an effective amount of an antibody, a pharmaceutically acceptable carrier, diluent or excipient, the preparation is a sterile liquid solution, a liquid suspension or a lyophilized form.

[0190] The antibodies of the present application can be used as a composition for separate administration, or can be used in combination with other active agents.

[0191] The term "YTE mutation" refers to M252Y / S254T / T256E, the amino acids at positions 252\254\256 are mutated from M, S, T to Y, T, E respectively, and the term "LS mutation" refers to M428L / N434S, the amino acids at positions 428\434 are mutated from M, N to L, S respectively, the numbering is according to Kabat EU index.

[0192] The term "kit" refers to a box for holding reagents such as antibodies, detection chemicals, drug residues, viral species, etc. For example, the kits described include antibodies, fragments thereof, homologues, derivatives thereof, etc. of the present application, for example, conjugates with labels or with cytotoxicity, as well as instructions for use of the antibodies, conjugates for killing specific types of cells, etc. The instructions can include guidance for use of the antibodies, conjugates, etc. in vitro, in vivo, or ex vivo. The antibodies can be in liquid form or solid, typically lyophilized. The kits can contain other reagents as appropriate, such as buffers, reconstitution solutions, and other necessary components for the intended use. It is contemplated that the combination of reagents packaged in predetermined amounts with instructions for their use, for example, for therapeutic use or for performing diagnostic assays. When the antibodies are labeled, for example, with an enzyme, then the kit can include substrates and the necessary cofactors for the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. can be included. The relative amounts of the various reagents can be varied to provide concentrates of reagent solutions, which provide user flexibility, space saving, reagent saving, etc. The reagents can also be provided in dry powder form, typically lyophilized, including excipients that, upon dissolution, provide reagent solutions with the appropriate concentrations.

[0193] The term "asthma" as used herein refers to allergic asthma, non-allergic asthma.

[0194] The term "allergic asthma" as used herein refers to asthma triggered by one or more inhaled allergens. Such patients have positive IgE fluorescence enzyme immunoassay (FEIA) levels to one or more allergens that trigger an asthmatic response.

[0195] Generally, most allergic asthma is associated with Th2-type inflammation.

[0196] The term "non-allergic asthma" refers to patients who have low eosinophils, low Th2, or low IgE at the time of diagnosis. Patients with "non-allergic asthma" have a negative response to a panel of allergens, including regional-specific allergens, in an IgE fluorescence enzyme immunoassay (FEIA). In addition to low IgE, those patients often have low or no eosinophil counts and low Th2 counts at the time of diagnosis.

[0197] The term "TSLP related disease" refers to, but is not limited to, asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic rhinosinusitis, urticaria, Netherton syndrome, eosinophilic esophagitis, food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungal rhinosinusitis, chronic pruritus, cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, rheumatoid arthritis, chronic obstructive pulmonary disease, systemic sclerosis, multiple sclerosis, keloid, ulcerative colitis, nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, Churg-Strauss syndrome, eosinophilic myalgic syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, scleroderma, interstitial lung disease, fibrosis induced by hepatitis B or C, radiation-induced fibrosis, and wound healing-induced fibrosis.

[0198] Example 1. Preparation of TSLP recombinant protein

[0199] According to the TSLP protein sequence (full length 159 aa, ACCESSION: AAH40592) in the NCBI database, base sequence optimization was performed, a pcDNA3.1-Myc-His vector was inserted with HindIII / SacII enzyme cutting sites, an expression plasmid of TSLP protein was constructed, after transfection of 293F cells, the supernatant expressed by the cells was cultured and collected, concentrated and purified by a nickel column (Cytiva) to obtain TSLP protein, and the amino acid sequence is shown in SEQ ID NO: 92 (SEQ ID NO: 92 MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLITYMSGTKST ENNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINAT QAMKKRTTNKCLEQVSQLQGLWRRFNRPLLKQQ).

[0200] Example 2. Construction and identification of recombinant TSLP receptor and IL7RA receptor cell lines

[0201] To screen the antibodies that can block the binding of TSLP to TSLP receptor, BaF3 cell strain expressing human TSLP receptor and human IL7Rα (TSLPR / IL7Rα) was constructed. The target gene TSLPR / IL7Rα was cloned into the target cell strain by electroporation to form a stable high expression cell strain. First, human TSLPR and human IL7Rα genes were cloned into pcDNA3.1-Myc-His plasmid, respectively, and then human TSLPR was cloned into BaF3 cell strain by electroporation, and selected for culture under 400 μg / ml G418 screening pressure for 1 week. On this basis, the second round of infection was carried out, and human IL7Rα gene was cloned into it, and selected for culture under 400 μg / ml G418 for two to three weeks. Finally, the monoclonal cell strain that simultaneously highly expressed TSLPR and IL7Rα was screened by flow sorting method.

[0202] Example 3. Preparation and screening of anti-human TSLP monoclonal antibody

[0203] Immunization of animals

[0204] The anti-human TSLP monoclonal antibody was produced by immunizing mice. Balb / c mice were used in the experiment, female, 6-8 weeks old (source: Vivotecnia). After the mice were purchased, they were raised in the laboratory environment for 1 week, and the light / dark cycle was adjusted for 12 / 12 hours. The mice that had adapted to the environment were immunized with recombinant protein, huTSLP-his (200 μg) and Alum adjuvant. After the 4th-5th immunization, the antibody titer in the mice was determined by ELISA method, and the mice with high antibody titer in the serum and the titer tending to plateau were selected, and after being executed by cervical dislocation, the spleen cells were taken and fused with myeloma cells. The spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells by using the optimized PEG-mediated fusion step to obtain hybridoma cells. The hybridoma cells were screened by traditional ELISA method and Biacore method.

[0205] Screening of hybridoma cells using ELISA method and Biacore method

[0206] ELISA method

[0207] (1) 100 μL of 1 μg / mL antigen protein solution was added to a 96-well plate, and the plate was coated at 4°C overnight;

[0208] (2) 100 μL of cell supernatant (or purified antibody) was added to the 96-well detection plate coated with the target protein TSLP;

[0209] (3) Incubate in a 37°C biochemical incubator for 1 h;

[0210] (4) Wash 3 times with PBST and tap off residual solution; (5) Add anti-mouse Fc or anti-human FC enzyme-labeled secondary antibody (diluted at 1:10000) with PBST+2% BSA, 100 μL per well;

[0211] (6) Incubate at 37°C for 1 h;

[0212] (7) Wash 4 times with PBST and tap off residual solution;

[0213] (8) Add TMB single-component color developing solution, 100 μL / well, develop color for about 8 min in the dark;

[0214] (9) Add 2M H2SO4 to stop the reaction, 50 μL / well;

[0215] (10) Read OD450 value on an enzyme-labeled instrument.

[0216] Biacore method

[0217] The Biacore 8K molecular interaction analyzer was used to capture the hybridoma supernatant with an Anti-mouse antibody CM5 chip, and then analyze the sample TSLP protein, so as to determine the affinity of the antibody and the protein, and to perform kinetic primary screening of the antibody. The specific operation steps are as follows: set up three cycles of Startup, and then set capture (capture antibody in supernatant) to 300 s, 30 μL / min; analysis (analysis sample) concentration is 200 nM, binding time is 120 s, 30 μL / min; dissociation time is 120 s, regeneration (10 mM glycine-HCl) is 30 μL / min, 30 s; then fill in the sample number and the corresponding protein concentration in the list, add the corresponding reagent according to the reagent position given by the program, cover the film, put the 96-well plate into the sample cabin, and start the program.

[0218] Sequencing of hybridoma subclone cell lines

[0219] The monoclonal hybridoma cell strains with good activity were screened, and the logarithmic growth period hybridoma cells were collected, RNA was extracted with TRNzol, and reverse transcription was performed All-in-One First-Strand cDNA Synthesis SuperMix for PCR, Transgene, AT321-01). The cDNA obtained by reverse transcription was sent to a sequencing company for sequencing after PCR amplification. Sequencing obtained the sequences of the mouse anti-TSLP antibodies: Q46, Q61, Q75, Q78, Q112, Q148, Q170, and Q187, and the variable region amino acid sequences are as follows:

[0220] Table 1 Sequencing results of mouse TSLP antibodies

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] To prepare a chimeric antibody, the variable region of the mouse antibody is fused with the constant region of a human antibody, and the chimeric antibody formed is screened. The sequence of the humanized constant region is as follows:

[0227] Heavy chain constant region (SEQ ID NO: 90):

[0228] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0229] Light chain constant region (SEQ ID NO: 91):

[0230] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0231] Example 4. Design of humanization of anti-human TSLP monoclonal antibody

[0232] In order to reduce the immunogenicity of the murine antibody, the Q61, Q187, Q78, Q75, Q46, Q148, Q112, Q170 antibodies, which have been screened for excellent in vivo and in vitro activity, were subjected to humanization. The humanization of the murine monoclonal antibody was performed according to the methods disclosed in many literatures in the art. Briefly, the murine CDR sequences were transplanted into the human antibody variable region framework to overcome the heterogeneity reaction induced by carrying a large amount of murine protein components in the chimeric antibody. Then, back-mutation was performed on the human antibody variable region framework sequence to obtain the final humanized molecule.

[0233] 4.1 Selection and back-mutation of human FR region of Q46

[0234] (1) Selection of human FR region

[0235] The humanized VH template of Q46 is IGHV1-69-2*01, and the humanized VL template is IGKV1-13*02. The CDR of Q46 was transplanted into the human template, and the variable region sequence obtained after transplantation is as follows:

[0236] huQ46VH-CDR:

[0237] EVQLVQSGAEVKKPGATVKISCKVSGYTFT DYEIH WVQQAPGKGLEWMG LIDPET GDTAYNQKFKG RVTITADTSTDTAYMELSSLRSEDTAVYYCAT EGQLGLSMDY WGQG TLVTVSS(SEQ ID NO:9)

[0238] huQ46VL-CDR:

[0239] DIQLTQSPSSLSASVGDRVTITC KASQDVGIVVA WYQQKPGKAPKLLIY WASTRHT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYSSYPLT FGGGTKVEIK(SEQ ID NO:10)

[0240] (2) Back-mutations of humanized antibody

[0241] The back-mutation sites of humanized antibody Q46 are as follows:

[0242] Table 2 Back-mutations of humanized antibody Q46

[0243]

[0244]

[0245] Note: V24A in the table means that the amino acid V at position 24 of the humanized antibody is back-mutated to A.

[0246] The VH and VL of humanized antibody huQ61 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and different back-mutated heavy and light chains were combined. The expressed proteins were subjected to affinity and cell activity determination, and finally the humanized antibody Q46-F with high degree of humanization and excellent activity was determined, and the back-mutation site thereof was heavy chain V24A, and the sequence was as follows:

[0247] Q46-F heavy chain variable region VH sequence:

[0248] EVQLVQSGAEVKKPGATVKISCKASGYTFT DYEIH WVQQAPGKGLEWMG LIDPET GDTAYNQKFKG RVTITADTSTDTAYMELSSLRSEDTAVYYCAT EGQLGLSMDY WGQG TLVTVSS(SEQ ID NO:11)

[0249] Q46-F light chain variable region VL sequence:

[0250] DIQLTQSPSSLSASVGDRVTITC KASQDVGIVVA WYQQKPGKAPKLLIY WASTRHT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYSSYPLT FGGGTKVEIK(SEQ ID NO:12)

[0251] 4.2 Selection of human FR region of Q61 and back-mutation

[0252] (1) Selection of human FR region

[0253] The humanized VH template of Q61 is IGHV1-18*01, and the humanized VL template is IGKV1-27*01. The CDR of Q61 is grafted onto the human template, and the variable region sequence obtained after grafting is as follows:

[0254] huQ61 VH-CDR:

[0255] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYDIN WVRQAPGQGLEWMG WMGWIFPG DGSTEYNEKFKV RVTMTTDTSTSTAYMELRSLRSDDTAVYFCAR TGDYVDFDY WGQGTLVTVSS (SEQ ID NO: 21)

[0256] huQ61 VL-CDR:

[0257] DIQMTQSPSSLSASVGDRVTITC SASQGISNFLN WYQQKPGKVPKLLIY YTSSLHS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QQHSQLPYT FGGGTKVEIK (SEQ ID NO: 22)

[0258] (2) The back-mutation of the humanized antibody

[0259] The back-mutation sites of the Q61 humanized antibody are as follows:

[0260] Table 3 The back-mutation of the Q61 humanized antibody

[0261]

[0262] Note: In the table, M48I means that the amino acid M at position 48 of the humanized antibody is back-mutated to I.

[0263] The VH and VL of the humanized antibody huQ61 are subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and different back-mutated heavy and light chains are combined, and the expressed proteins are subjected to affinity and cell activity determination, and finally the humanized antibody Q61-F with high humanization degree and excellent activity is determined, and the back-mutation sites are heavy chain Y95F and light chain F72Y, and the sequences are as follows:

[0264] The Q61-F heavy chain variable region VH sequence is:

[0265] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYDIN WVRQAPGQGLEWMG WIFPG DGSTEYNEKFKV RVTMTTDTSTSTAYMELRSLRSDDTAVYFCAR TGDYVDFDYWGQGTLVTVSS (SEQ ID NO: 23)

[0266] Q61-F light chain variable region VL sequence:

[0267] DIQMTQSPSSLSASVGDRVTITC SASQGISNFLN WYQQKPGKVPKLLIY YTSSLHS GVPSRFSGSGSGTDYTLTISSLQPEDVATYYC QQHSQLPYT FGGGTKVEIK (SEQ ID NO: 24)

[0268] 4.3 Selection and back mutation of humanized FR regions of Q75

[0269] (1) Selection of humanized FR regions

[0270] The humanized VH template of Q75 is IGHV1-46*01, and the template of humanized VL is IGKV3-15*01. The CDR of Q75 is grafted onto the humanized template, and the variable region sequence obtained after grafting is as follows:

[0271] huQ75VH-CDR:

[0272] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMN WVRQAPGQGLEWMG QIYP GDGDTDYNGKFEG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GSYGSGYHYAMD F WGQGTTVTVSS (SEQ ID NO: 33)

[0273] huQ75VL-CDR:

[0274] EIVMTQSPATLSVSPGERATLSC SVSSSISSSNLH WYQQKPGQAPRLLIY GTSYLAS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQWSSYPLT FGGGTKVEIK (SEQ ID NO: 34)

[0275] (2) Back mutation of humanized antibody

[0276] The back mutation sites of Q75 humanized antibody are as follows:

[0277] Table 4 Back mutation of Q75 humanized antibody

[0278]

[0279] Note: In the table, M48I means that the amino acid M at position 48 of the humanized antibody is reverted to I.

[0280] The VH and VL of the humanized antibody huQ75 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and different revertant heavy and light chains constructed were combined, and the expressed proteins were subjected to affinity and cell activity determination, and finally the humanized antibody Q75-F with high degree of humanization and excellent activity was determined, and the revertant mutation sites thereof were heavy chain R72A and light chain F72Y, and the sequences were as follows:

[0281] Q75-F heavy chain variable region VH sequence:

[0282] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMN WVRQAPGQGLEWMG QIYP GDGDTDYNGKFEG RVTMTADTSTSTVYMELSSLRSEDTAVYYCAR GSYGSGYHYAM DF WGQGTTVTVSS (SEQ ID NO: 35)

[0283] Q75-F light chain variable region VL sequence:

[0284] EIVMTQSPATLSVSPGERATLSC SVSSSISSSNLH WYQQKPGQAPRLLIY GTSYLAS GIPARFSGSGSGTEYTLTISSLQSEDFAVYYC QQWSSYPLT FGGGTKVEIK (SEQ ID NO: 36)

[0285] 4.4 Selection and revertant mutation of human FR region of Q78

[0286] (1) Selection of human FR region

[0287] The humanized VH template of Q78 is IGHV1-18*01, and the humanized VL template is IGKV1-33*01. The CDR of Q78 is grafted onto the human template, and the variable region sequence obtained after grafting is as follows:

[0288] huQ78 VH-CDR:

[0289] QVQLQESGPGLVKPSQTLSLTCAVYGGSFS SYGVH WIRQPPGKGLEWIG LIWPGGS TNYNSALMSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR RRDGFGAMDY WGQGTL VTVSS (SEQ ID NO: 45)

[0290] huQ78 VL-CDR:

[0291] DIQMTQSPSSLSASVGDRVTITC TASQDINKYIA WYQQKPGKAPKLLIY YTSTLQP G VPSRFSGSGSGTDFTFTISSLQPEDIATYYC LQYDNLYT FGGGTKVEIK (SEQ ID NO: 46)

[0292] (2) Back-mutations of the humanized antibody

[0293] The back-mutation sites of the humanized antibody Q78 are as follows:

[0294] Table 5 Back-mutations of the humanized antibody Q78

[0295]

[0296] Note: I37V in the table means that the amino acid I at position 37 of the humanized antibody is back-mutated to V.

[0297] The VH and VL of the humanized antibody huQ78 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and different back-mutated heavy and light chains were combined, and the expressed proteins were subjected to affinity and cell activity determination, and finally the humanized antibody Q78-F with high degree of humanization and excellent activity was determined, and the back-mutation sites thereof were heavy chain V71K and light chain F71Y, and the sequences were as follows:

[0298] Q78-F heavy chain variable region VH sequence:

[0299] QVQLQESGPGLVKPSQTLSLTCAVYGGSFS SYGVH WIRQPPGKGLEWIG LIWPGGS TNYNSALMS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR RRDGFGAMDY WGQGTL VTVSS (SEQ ID NO: 47)

[0300] Q78-F light chain variable region VL sequence:

[0301] DIQMTQSPSSLSASVGDRVTITC TASQDINKYIA WYQQKPGKAPKLLIY YTSTLQPG VPSRFSGSGSGTDYTFTISSLQPEDIATYYC LQYDNLYT F GGGTKVEIK (SEQ ID NO: 48)

[0302] 4. Selection and back-mutation of humanized FR regions of Q112

[0303] (1) Selection of humanized FR regions

[0304] The humanized VH template of Q112 is IGHVl-69*10, and the humanized VL template is IGKV3-7*02. The CDRs of Q112 are grafted onto the humanized templates, and the variable region sequences obtained after grafting are as follows:

[0305] huQ112 VH-CDRs:

[0306] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYWMN WVRQAPGQGLEWMG QIFP GDGDTNYNGKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR SAYSGNYYYAMD Y WGQGTTVTVSS (SEQ ID NO: 54)

[0307] huQ112 VL-CDRs:

[0308] EIVMTQSPPTLSLSPGERVTLSC SVSSSISSSNLH WYQQKPGQAPRLLIY GTSNLAS G IPARFSGSGSGTDFTLTISSLQPEDFAVYYC QQWSSYPLT F GGGTKVEIK (SEQ ID NO: 55)

[0309] (2) Back-mutation of humanized antibodies

[0310] The back-mutation sites of the Q112 humanized antibodies are as follows:

[0311] Table 6 Back-mutation of Q112 humanized antibodies

[0312]

[0313] Note: In the table, M48I means that the amino acid M at position 48 of the humanized antibody is back-mutated to I.

[0314] The VH and VL of the humanized antibody huQ112 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and different combinations of the constructed reverse mutation heavy and light chains were made, the expressed proteins were subjected to affinity and cell activity determination, and finally the humanized antibody Q112-F with high humanization degree and excellent activity was determined, and the reverse mutation site thereof was light chain F72Y, and the sequence was as follows:

[0315] Q112-F heavy chain variable region VH sequence:

[0316] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYWMN WVRQAPGQGLEWMG QIFP GDGDTNYNGKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR SAYSGNYYYAMD Y WGQGTTVTVSS (SEQ ID NO: 56)

[0317] Q112-F light chain variable region VL sequence:

[0318] EIVMTQSPPTLSLSPGERVTLSC SVSSSISSSNLH WYQQKPGQAPRLLIY GTSNLAS G IPARFSGSGSGTDYTLTISSLQPEDFAVYYC QQWSSYPLT FGGGTKVEIK (SEQ ID NO: 57)

[0319] 4.6 Selection of human FR region of Q148 and reverse mutation

[0320] (1) Selection of human FR region

[0321] The humanized VH template of Q148 is IGHV2-70*04, and the humanized VL template is IGKV3-11*01. The CDR of Q148 is grafted onto the human template, and the variable region sequence obtained after grafting is as follows:

[0322] huQ148 VH-CDR:

[0323] QVTLKESGPALVKPTQTLTLTCTFSGFSLS PSGMGVG WIRQPPGKALEWLA HIWW DDDKRYNPALKS RLTISKDTSKNQVVLTMTNMDPVDTATYYCAR IGYYGEGFTY WGQ GTLVTVSS (SEQ ID NO: 66)

[0324] huQ148 VL-CDRs:

[0325] EIVLTQSPATLSLSPGERATLSC KASQSVDFDGDSYMN WYQQKPGQAPRLLIY AAS NRES GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQSDEDPYT FGGGTKVEIK (SEQ ID NO: 67)

[0326] (2) Back-mutations of the humanized antibody

[0327] The back-mutation sites of the humanized antibody Q148 are as follows:

[0328] Table 7 Back-mutations of the humanized antibody Q148

[0329]

[0330] Note: L4M in the table means that the 4th amino acid L of the humanized antibody is back-mutated to M.

[0331] The VH and VL of the humanized antibody Q148 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and the expressed proteins were subjected to affinity and cell activity determination after combination of the constructed different back-mutated heavy and light chains, and finally the humanized antibody Q148-F with high degree of humanization and excellent activity was determined, and the back-mutation site thereof was light chain L4M, and the sequence was as follows:

[0332] Q148-F heavy chain variable region VH sequence:

[0333] QVTLKESGPALVKPTQTLTLTCTFSGFSLS PSGMGVG WIRQPPGKALEWLA HIWW DDDKRYNPALKS RLTISKDTSKNQVVLTMTNMDPVDTATYYCAR IGYYGEGFTY WGQ GTLVTVSS (SEQ ID NO: 68)

[0334] Q148-F light chain variable region VL sequence:

[0335] EIVMTQSPATLSLSPGERATLSC KASQSVDFDGDSYMN WYQQKPGQAPRLLIY AA SNRES GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQSDEDPYT FGGGTKVEIK (SEQ ID NO: 69)

[0336] 4. Selection and back-mutation of human FR regions of Q170

[0337] (1) Selection of human FR regions

[0338] The humanized VH template of Q170 is IGHV1-46*01, and the VL template is IGKV1-37*01. The CDRs of Q170 are grafted onto the humanized templates, and the variable region sequences obtained after grafting are as follows:

[0339] huQ170 VH-CDR:

[0340] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMN WVRQAPGQGLEWMG QIYP GDGDTDYNGKFKG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGYVNYYYAMD Y WGQGTTVTVSS (SEQ ID NO: 74)

[0341] huQ170 VL-CDR:

[0342] DIQLTQSPSSLSASVGDRVTITC SVSSSISSSNLH WYRQKPGKVPKLLIY GTSNLAS G VPSRFSGSGSGTDFTLTISSLQPEDVATYYG QQWSSYPLT FGGGTKVEIK (SEQ ID NO: 75)

[0343] (2) Back-mutation of humanized antibodies

[0344] The back-mutation sites of the Q170 humanized antibody are as follows:

[0345] Table 8 Back-mutation of Q170 humanized antibody

[0346]

[0347] Note: V68A in the table indicates that the amino acid V at position 68 of the humanized antibody is back-mutated to A.

[0348] The VH and VL of the humanized antibody Q170 are subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and different back-mutated heavy and light chains are combined. The expressed proteins are subjected to affinity and cell activity determination, and finally the humanized antibody Q170-F with high degree of humanization and excellent activity is determined, and the back-mutation site is heavy chain R72A and light chain F72Y, and the sequence is as follows:

[0349] Q170-F heavy chain variable region VH sequence:

[0350] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMN WVRQAPGQGLEWMG QIYP GDGDTDYNGKFKG RVTMTADTSTSTVYMELSSLRSEDTAVYYCAR GGYVNYYYAMD Y WGQGTTVTVSS (SEQ ID NO: 76)

[0351] Q170-F light chain variable region VL sequence:

[0352] DIQLTQSPSSLSASVGDRVTITC SVSSSISSSNLH WYRQKPGKVPKLLIY GTSNLAS G VPSRFSGSGSGTDYTLTISSLQPEDVATYYG QQWSSYPLT FGGGTKVEIK (SEQ ID NO: 77)

[0353] 4.8 Selection and back-mutation of humanized FR regions of Q187

[0354] (1) Selection of humanized FR regions

[0355] The humanized VH template of Q187 is 1T3F, and the humanized VL template is 1T3F. The CDRs of Q187 are grafted onto the humanized templates, and the variable region sequences obtained after grafting are as follows:

[0356] huQ187 VH-CDR grafted:

[0357] EVQLVQSGAELKKPGSSVKVSCKASGYIFT TYWMH WVKQAPGQGLEWIG VIDPS DSDTTYNQKFKG KATLTVDKSTNTAYMELSSLRSEDTAVYYCTR SLDGYLDY WGQGT LVTVSS (SEQ ID NO: 86)

[0358] huQ187 VL-CDR grafted:

[0359] DIQMTQSPSTLSASVGDRVTITC RASENIYSYFA WYQQKPGKAPKLLIY NAKTLPAGVPSRFSGSGSGTDFTLTISSLQPDDFATYYC QHHYGTPYT FGQGTKVEVK (SEQ ID NO: 87)

[0360] (2) Back-mutations of the humanized antibody

[0361] The back-mutation sites of the humanized antibody Q187 are as follows:

[0362] Table 9 Back-mutations of the humanized antibody Q187

[0363]

[0364] Note: In the table, I28T means that the amino acid I at position 28 of the humanized antibody is back-mutated to T.

[0365] The VH and VL of the humanized antibody Q187 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and different back-mutated heavy and light chains were combined. The expressed proteins were subjected to affinity and cell activity assays, and finally the humanized antibody Q187-F with high degree of humanization and excellent activity was determined, which had the back-mutation sites of heavy chain I28T, T30I and K74T, and light chain I48V, and the sequence was as follows:

[0366] The sequence of the heavy chain variable region VH of Q187-F is as follows:

[0367] EVQLVQSGAELKKPGSSVKVSCKASGYTFI TYWMH WVKQAPGQGLEWIG VIDPS DSDTTYNQKFKG KATLTVDTSTNTAYMELSSLRSEDTAVYYCTR SLDGYLDY WGQGT LVTVSS (SEQ ID NO: 88)

[0368] The sequence of the light chain variable region VL of Q187-F is as follows:

[0369] DIQMTQSPSTLSASVGDRVTITC RASENIYSYFA WYQQKPGKAPKLLVY NAKTLPA GVPSRFSGSGSGTDFTLTISSLQPDDFATYYC QHHYGTPYT FGQGTKVEVK (SEQ ID NO: 89)

[0370] Example 5. Affinity determination of the anti-TSLP humanized antibody

[0371] The affinity of the anti-TSLP humanized antibody binding to TSLP was determined by the Biacore method. The affinity of the antibody to the protein was determined by the molecular interaction analyzer Biacore 8K, and the antibody was subjected to kinetic primary screening. The specific operation steps were as follows: three cycles of Startup were set, and then capture (capture of the antibody in the supernatant) was set to 300 s, 30 μL / min; analysis (analysis sample) concentration was 200 nM, binding time was 120 s, 30 μL / min; dissociation time was 120 s, regeneration (10 mM glycine-HCl) was 30 μL / min, 30 s; then the sample number and the corresponding protein concentration of the protein were filled in the list, and the corresponding reagents were added according to the reagent position given by the program, then the film was covered, the 96-well plate was placed in the sample chamber, and the program was started. The affinity of the anti-TSLP humanized antibody binding to TSLP was determined, and the results are shown in Table 10.

[0372] Table 10 Affinity results of anti-TSLP humanized antibody binding to TSLP

[0373]

[0374] The affinity of the humanized antibodies Q61-F, Q187-F, Q75-F and Q112-F obtained by the present application is higher than nM (10 -10 ) level, which can effectively bind to TSLP, and the binding capacity is better than the positive control drug Teze.

[0375] Example 6. Determination of the thermal stability of the anti-TSLP humanized antibody

[0376] The Tm value of the anti-TSLP antibody was determined by the DSF (differential fluorescence scanning technology) method:

[0377] (1) The Tm value of the antibody to be tested was determined by MicroCal TM VP Cap DSC system (Malvern Panalytical, Northampton, USA)

[0378] (2) Preparation of the antibody: The antibody to be tested was replaced with D-PBS buffer, and the antibody was concentrated to 0.5 mg / mL, filtered by a 0.22 μm filter, and then placed at 4°C for testing.

[0379] (3) Preparation of the instrument: clean the instrument with 40% Decon 90, set 9 groups of cleaning procedures, the scanning temperature from 10°C to 60°C, the heating rate is set to 180°C / h. After cleaning, set 3 groups of water, the scanning temperature from 10°C to 130°C, the heating rate is set to 180°C / h. If the DSC curves of the three groups of water are consistent, the instrument cleaning procedure is completed, and the sample is ready for determination.

[0380] (4) Determination of the procedure: set three groups of buffer, the scanning temperature from 10°C to 130°C, the heating rate is set to 180°C / h, as a control for instrument calibration. Buffer, sample, 14% decon and 14% decon together constitute a group of samples to be tested. Buffer and sample scanning temperature from 10°C to 130°C, the heating rate is set to 180°C / h; 14% decon cleaning scanning temperature from 10°C to 60°C, the heating rate is set to 180°C / h, and set clean before load before each sample.

[0381] (5) Sample addition and determination: set the program and data storage path according to the above method. According to the program, add 400 μL of the corresponding solution to be tested in the 96 deep well plate. After the sample addition is completed, place the 96 well plate into the sample storage rack. Click Start to start the determination of Tm value.

[0382] The results of the determination of the Tm value of the humanized antibody are shown in Table 11.

[0383] Table 11. Determination of the Tm value of the antibody

[0384]

[0385] It was determined that the Tm values of the humanized antibodies Q61-F and Q187-F were higher than that of the positive control antibody Teze under the same concentration and determination conditions, and were both greater than 70°C, indicating good thermal stability.

[0386] Example 7. Determination of the hydrophobicity of the anti-TSLP humanized antibody

[0387] The hydrophobicity of the antibody to be tested was determined by HPLC. The hydrophobicity can indirectly reflect the strength of the interaction between antibody molecules, thereby providing a reference for the tendency of aggregation and non-specific binding of the antibody molecules. Stronger hydrophobicity can indicate that the molecules are more likely to aggregate and exhibit non-specific binding. The experimental steps of hydrophobic interaction chromatography (HIC) are as follows:

[0388] (1) Equilibrium liquid phase system to system stability: according to the standard operation procedures of Waters e2695 / LC-20AT type high performance liquid chromatograph, the instrument was started and the preparation of the solvent management system was completed. First, connect double universal ultrapure water to flush the pipeline at a flow rate of 1.0 mL / min for 30 minutes, then use A liquid (1.5M ammonium sulfate + 25mM sodium phosphate, pH = 6.8) to flush the pipeline at a flow rate of 1.0 mL / min for 30 min, reduce the flow rate to 0 mL / min, connect the required chromatographic column, and then use A liquid to balance the chromatographic column to the baseline stability at a flow rate of 0.5 mL / min, about 30 min.

[0389] (2) Sample treatment: dilute the sample to a concentration of 2.0 mg / mL using PBS.

[0390] (3) After injecting 3 PBS, inject 2.0 mg / mL of the sample to be tested, and the injection volume is 10.0 μL

[0391] (4) Linear gradient elution of the sample with B liquid (75% 25mM sodium phosphate + 25% isopropanol, pH = 6.8).

[0392] (4) Data processing: hydrophobicity analysis according to sample retention time.

[0393] (5) Instrument and chromatographic column treatment: after the experiment, flush the chromatographic column with ultrapure water at a flow rate of 0.5 mL / min for 60 minutes, save the chromatographic column, and fill in the "HPLC use record"; then flush the pipeline with 20% methanol solution at a flow rate of 0.2 mL / min for 30 minutes, and turn off the liquid chromatograph.

[0394] The HIC results are shown in Table 12. The shorter the retention time, the weaker the hydrophobicity of the molecule. From the results, it can be seen that the retention time of Q61-F is lower than that of the positive control antibody Teze, and the hydrophobicity is better than that of the positive drug.

[0395] Table 12. Antibody hydrophobicity

[0396]

[0397] Example 8. Determination of the stability of anti-TSLP humanized antibodies

[0398] The stability of the antibodies to be tested was determined by HPLC, and the stability of different anti-TSLP antibodies under pH accelerated conditions and oxidation accelerated conditions was determined.

[0399] 8.1 Determination of the stability of anti-TSLP humanized antibodies under pH accelerated conditions

[0400] Accelerated assay to determine the effects of acidic, extremely acidic, and alkaline pH on the stability of humanized antibodies against TSLP: The solvents of 2.0 mg of humanized antibody and positive control antibody were replaced with 50 mM sodium acetate (adjusted to pH 5.5 with acetic acid), 20 mM citrate buffer (pH 3.0), 20 mM Tris, and 10 mM EDTA (adjusted to pH 9.0 with NaOH), respectively, with the total volume adjusted to 1.0 mL. The antibody sample at pH 5.5 was incubated in a 40°C oven for 14 days, the antibody sample at pH 3.0 was placed at room temperature in the dark for 24 hours, and the antibody sample at pH 9.0 was incubated in a 40°C oven for 7 days. The solvents were then replaced with PBS, and changes in aggregate and fragmentation were measured to investigate the effect of different pH values ​​on antibody stability. The results are shown in Table 13.

[0401] Table 13. Antibody stability under pH-accelerated conditions

[0402]

[0403]

[0404] As shown in Table 13, under different pH treatment conditions, the degree of aggregation and fragmentation of humanized antibodies Q61-F and Q187-F was not significant and was slightly better than that of positive control drugs, indicating that humanized antibodies Q61-F and Q187-F have good stability under different pH accelerated treatment conditions.

[0405] 8.2 Determination of the stability of anti-TSLP antibodies under accelerated oxidation conditions

[0406] 3.3 μL of 30% H2O2 was added to 1.0 mL of humanized antibody (2.0 mg / mL) and positive control antibody, and the mixture was incubated at room temperature in the dark for 24 h. After acceleration, the buffer solution was replaced with PBS and stored at -20 °C. Subsequently, changes in aggregate and fragmentation, as well as cell viability, were measured to investigate the effect of oxidation treatment on antibody stability. The results are shown in Table 14.

[0407] Table 14. Antibody stability under accelerated oxidation conditions

[0408]

[0409]

[0410] As shown in Table 14, under oxidative treatment conditions, the degree of aggregation and fragmentation of humanized antibodies Q61-F and Q187-F was not significant, which was better than that of the positive control drug, indicating that humanized antibodies Q61-F and Q187-F have good stability under oxidative treatment conditions.

[0411] Example 9. Determination of solubility of anti-TSLP humanized antibodies

[0412] The solubility of anti-TSLP antibodies was determined by PEG precipitation method, and the experimental steps were as follows:

[0413] (1) Preparation of PEG stock solution: Dissolve the weighed PEG solid in PBS to prepare a 40% mass fraction (w / v) stock solution, and adjust the pH to 7.2.

[0414] (2) High-throughput assay of microplate: Dilute the antibody to be tested to 10 mg / mL with PBS, and add 150 μL to a 96-well plate; dilute PEG with PBS to a mass fraction of 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, and 24%, and add 150 μL to the corresponding antibody wells in turn, mix thoroughly by blowing, so that the final concentration of the antibody is 5 mg / mL and the final concentration of PEG is 3%-12%. Different PEG concentrations will result in different degrees of precipitation, and the mixture will be placed at room temperature for 15 min. The turbidity of the mixture reflects the amount of precipitation. The OD value was determined by observation or spectrophotometer. 500 The solubility was evaluated. From Figure 4 It can be seen that, under the same antibody concentration, buffer system and temperature conditions, the positive control antibody TEZE first precipitated, while the Q61-F humanized antibody precipitated at 8% PEG mass fraction; Q187-F precipitated from 7% PEG mass fraction, but was significantly better than the positive control antibody TEZE. Therefore, the solubility of Q61-F and Q187-F is greater than that of TEZE.

[0415] Example 10. Blocking of TSLP binding to TSLPR and TSLPR-IL7R complex by anti-TSLP humanized antibodies

[0416] Different TSLP antibodies have different abilities to block TSLP binding to TSLPR and TSLPR-IL7R complex. TSLP binds to TSLPR and further recruits IL-7R to form a complex to function. In vitro competitive binding experiments were used to verify the ability of antibodies to block TSLP binding to TSLPR and TSLPR-IL7R complex. After co-incubation of biotin-labeled TSLP protein with different concentrations of antibodies for 30 min, they were added to a 96-well plate coated with TSLPR-IL7R complex, incubated at 37°C for 1 h, and then horseradish peroxidase-labeled Streptavidin was added for color development. After termination, the OD value was measured at 450 nm. As shown in Figure 1 and Table 15, the ability of Q61-F to block TSLP binding to TSLPR and TSLPR-IL7R complex was better than that of the control antibody Teze.

[0417] Table 15. IC of anti-TSLP humanized antibodies blocking TSLP binding to TSLPR and TSLPR-IL7R complex 50 Concentration

[0418]

[0419] Example 11. Anti-TSLP humanized antibodies inhibit Ba / F3 cell proliferation

[0420] Ba / F3 cells were electrotransformed to express both human TSLPR and IL-7R proteins and to be dependent on TSLP for proliferation. The activity of anti-TSLP antibodies was measured by adding different concentrations of antibodies to inhibit the proliferation of BaF3 cells dependent on TSLP by binding to TSLP factor added in the culture medium. TSLP factor and different concentrations of antibodies were added to 96-well cell culture plates, and 1.5 x 10 4 cells were added to each well, and after 2 days of incubation at 37°C in a 5% CO2 incubator, CCK8 was added to detect the proliferation of cells, and OD values were measured at 450 nm. As shown in Table 15 and Table 16, Q61-F had better ability to inhibit the proliferation of Ba / F3 cells than the control antibody Teze. Figure 2

[0421] Table 16. IC of anti-TSLP humanized antibodies inhibiting Ba / F3 cell proliferation 50 Concentration

[0422]

[0423] Example 12. Anti-TSLP humanized antibodies reduce TARC content

[0424] TSLP antibodies inhibit the secretion of TARC (thymus and activation-regulated chemokine, also known as CCL17) by PBMC cells, and TARC can bind to CCR4 receptors to mediate the occurrence of inflammatory, cancer and autoimmune-related diseases. TSLP can induce PBMC cells to produce chemokines such as TARC, but the binding of anti-TSLP antibodies to TSLP can inhibit the production of such chemokines, so the detection of TARC concentration can be used for in vitro pharmacodynamic evaluation experiments. TSLP factor and different concentrations of antibodies were added to 96-well cell culture plates, and 8 x 10 5 PBMC cells were added to each well, and after 2 days of incubation at 37°C in a 5% CO2 incubator, the concentration of TARC in the cell supernatant was detected using a Human CCL17 / TARC ELISA Kit kit (Shanghai Keaibo Biological). As shown in Table 16, Q61-F and Q87-F had comparable ability to inhibit TARC secretion compared to TEZE. Figure 3 ​​

[0425] Example 13. Anti-dermatitis efficacy experiment of anti-TSLP humanized antibody

[0426] Animal description

[0427] Species: Mouse

[0428] Breed: B-hIL4hIL4RA mice

[0429] Age: 6-8 weeks

[0430] Weight: 18-20g

[0431] Source: Biocytogen

[0432] breeding

[0433] Mice were housed in IVCs (independently ventilated cages) with access to sterile water. They were fed twice daily (AM and PM) except on study days. The overall health of the animals was assessed and their weight recorded before the start of the study. The study proceeded once the mice were deemed healthy.

[0434] AD (Atopic dermatitis) model construction: according to Figure 5 On day 0, mice were sensitized by uniformly applying 25 μL of 0.8% Oxazolone solution to their right ear and back. Subsequently, on days 7, 9, 11, 14, 16, 18, 21, 23, and 25, 25 μL of 0.4% Oxazolone solution was applied to the same site for challenge. Mice were weighed twice a week after the start of the experiment, and ear thickness was measured. Subcutaneous administration began on day 6. The dosing regimen, frequency, dosage, and volume for each treatment group are shown in Table 9. On day 26, blood was collected from the orbital venous plexus, and serum IgE levels were detected using the MOUSE IgE ELISA KIT kit.

[0435] Table 17 Dosing regimens, dosing frequency, dosage, and volume for each experimental group

[0436]

[0437] like Figure 6 As shown in the graph of mouse body weight changes, throughout the entire anti-dermatitis efficacy experiment, the body weight of mice in each treatment group continuously increased, with a generally consistent rate of increase, indicating that the mice in each treatment group were in normal health and that the drug administration had no toxic side effects on the mice; Figure 7The results of the comparison of the ear thickness of the mice in each administration group, the ear thickness of the mice in the model control group (MC) was continuously increasing compared with the blank control group (NC), and the phenomenon of ear fissure thickening was also observed by naked eye. However, the degree of ear thickening of the mice in each administration group, especially the Q61-F group, was significantly alleviated, and the drug efficacy was comparable to and slightly better than that of Teze; for example Figure 8 The results of the comparison of the total IgE concentration in the serum of the mice in each administration group, before modeling, the background serum IgE level of the mice in each group was similar and maintained at a low level. After modeling, the serum IgE of the MC group was significantly increased compared with the NC group. After treatment with Teze, Q61-F and Q187-F antibodies, the IgE level was significantly reduced, and the drug efficacy was comparable. The above indicates that in the animal body, the Q61-F antibody has an anti-dermatitis effect better than the positive drug TEZE.

[0438] Example 14. Anti-asthma efficacy experiment of anti-TSLP humanized antibody

[0439] Animal description

[0440] Species: mouse

[0441] Breed: B-hTSLP / hTSLPR mice

[0442] Age: 8 weeks

[0443] Weight: 16-22 g

[0444] Source: Biosearch Technologies

[0445] The mice were raised in IVC (independent ventilation cage box) and provided with sterilized water for drinking. The mice were fed twice a day (AM and PM) except on the study day. Before starting the study, the general health status of the animals was evaluated and the body weight was recorded. After determining the health of the mice, the study was conducted.

[0446] 25 animals were randomly divided into 5 groups according to body weight on Day-3, and were labeled as G1-G5, 5 animals in each group. Among them, G1 is the unmodeled group, G2-G5 is the modeled group, G2 is the negative control group, G3 is the test product 1 group, G4 is the test product 2 group, and G5 is the test product 3 group.

[0447] On days 0, 7 and 14, each animal in the G1 group was intraperitoneally injected with 200 μL of PBS for sensitization, and each animal in the G2-G5 group was intraperitoneally injected with 200 μL of 200 μg / mL OVA for sensitization. On days 21-25, the animals in the G2-G5 group inhaled 2% OVA by atomization for 30 minutes each day for 5 consecutive days. On day 26, the in vivo efficacy experiment was completed, and the lung alveolar lavage fluid, serum and lung tissue were taken for subsequent in vitro detection.

[0448] Table 18: Dosing regimen, dosing frequency, dosing amount and volume of each experimental group

[0449]

[0450] Figure 9 Figure 6 is a comparison of the body weight of mice in each administration group, and the results show that the body weight of mice increased steadily during the entire duration of the efficacy experiment (26 days), and the increase was the same as that of the control group, indicating that the basic health status of the mice was normal (consistent with the observation of good mental state and normal appetite of mice), and was not affected by modeling and administration.

[0451] Figure 10 Figure 7 is a comparison of the IgE content in the serum of mice in each administration group, and the results show that the IgE in the serum of the MC group increased significantly compared with the NC group, indicating that the modeling was successful. After treatment with Teze, Q61-F and Q187-F antibodies, the IgE level decreased, indicating that the antibodies can exert efficacy in the asthma model, but the efficacy of Teze is not obvious, while Q61-F shows certain efficacy, indicating that the efficacy of Q61-F is better than that of the positive drug Teze.

[0452] Example 15. Design of constant region of anti-TSLP humanized antibody

[0453] The heavy chain constant region of the above-mentioned antibodies Q61-F and Q187-F was designed to contain M252Y / S254T / T256E (YTE) mutations, respectively Q61-YA and Q187-YA, and the specific sequences are as follows:

[0454] Heavy chain sequence of Q61-YA

[0455] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWIFPGDGSTEYNEKFKVRVTMTTDTSTSTAYMELRSLRSDDTAVYFCARTGDYVDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 93)

[0456] Light chain sequence of Q61-YA

[0457] DIQMTQSPSSLSASVGDRVTITCSASQGISNFLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQHSQLPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 94)

[0458] Heavy chain sequence of Q187-YA

[0459] EVQLVQSGAELKKPGSSVKVSCKASGYTFITYWMHWVKQAPGQGLEWIGVIDPSDSDTTYNQKFKGKATLTVDTSTNTAYMELSSLRSEDTAVYYCTRSLDGYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 95)

[0460] Light chain sequence of Q187-YA

[0461] DIQMTQSPSTLSASVGDRVTITCRASENIYSYFAWYQQKPGKAPKLLVYNAKTLPAGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQHHYGTPYTFGQGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 96)

[0462] Heavy chain constant region sequence

[0463] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 97)

[0464] Example 16. Anti-TSLP humanized antibodies with heavy chain comprising YTE mutation inhibit Ba / F3 cell proliferation

[0465] Ba / F3 cells were electrotransferred to express both human TSLPR and IL-7R proteins and to proliferate in dependence on TSLP. The activity of anti-TSLP antibodies was measured by adding different concentrations of antibodies to bind to TSLP factor added in the culture medium, thereby inhibiting the proliferation of BaF3 cells that need to depend on TSLP for growth. TSLP factor and different concentrations of antibodies were added in 96-well cell culture plates, and 1.5 x 10 4 cells were added per well, and after incubation at 37°C in a 5% CO2 incubator for 2 days, CCK8 was added to detect the proliferation ability of cells, and OD value was measured at 450 nm. As shown in Table 18 and Table 19, Q61-YA had better ability to inhibit the proliferation of Ba / F3 cells than control antibody Tezepelumab. Figure 11

[0466] Table 19. IC50 of anti-TSLP humanized antibodies with heavy chain comprising YTE mutation to inhibit Ba / F3 cell proliferation 50 concentration

[0467]

[0468] Example 17. Anti-TSLP humanized antibodies with heavy chain comprising YTE mutation reduce TARC content

[0469] ​TSLP antibody inhibition of TARC (thymus and activation-regulated chemokine, also known as CCL17) secretion from PBMC cells, which can bind to CCR4 receptor and mediate inflammation, cancer and autoimmune related diseases. TSLP can induce PBMC cells to produce chemokines such as TARC, but the binding of anti-TSLP antibody to TSLP can inhibit the production of this chemokine, so the concentration detection of TARC can be used for in vitro efficacy evaluation experiments. Add TSLP factor and different concentrations of antibody in 96-well cell culture plate, add 8x10 5 PBMC cells per well, incubate at 37°C in a 5% CO2 incubator for 2 days, and detect the concentration of TARC in the cell supernatant using a Human CCL17 / TARC ELISA Kit kit. As Figure 12 and Table 20, the TARC secretion inhibition ability of Q61-YA and Q87-YA is better than Tezepelumab.

[0470] Table 20. Anti-TSLP humanized antibody with heavy chain containing YTE mutation reduces TARC content results

[0471]

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to thymic stromal lymphopoietin (TSLP), comprising a heavy chain variable region, a light chain variable region, and a heavy chain constant region, wherein: 1) the heavy chain variable region comprises, (i) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 contained within a peptide segment having the amino acid sequence of SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86, or 88; or (ii) a sequence comprising collectively at least one and not more than 5 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) on the three heavy chain complementarity determining regions (HCDRs) relative to the sequence of the three heavy chain complementarity determining regions (HCDRs) recited in (i); 2) the light chain variable region comprises, (i) three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 contained within a peptide segment having the amino acid sequence of SEQ ID NO: 8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87, or 89; or (ii) a sequence comprising collectively at least one and not more than 5 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) on the three light chain complementarity determining regions (LCDRs) relative to the sequence of the three light chain complementarity determining regions (LCDRs) recited in (i); and 3) the heavy chain constant region comprises a heavy chain constant region of an IgGl, IgG2, or IgG4 mutant, wherein the mutation is selected from at least one of S228, L234, L235, G236, G237, T250, M252, I253, S254, T256, D259, T307, V308, P329, A330, E380, M428, and N434 (numbering according to the Kabat EU index), preferably at least one of G237A, L234A / L235A, S228P, N434A, N434H, T307A / E380A / N434A, M428L / N434S, and M252Y / S254T / T256E (numbering according to the Kabat EU index), more preferably M252Y / S254T / T256E (numbering according to the Kabat EU index); preferably the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

97.

2. An antibody or antigen-binding fragment thereof that specifically binds to thymic stromal lymphopoietin (TSLP), comprising: a) a heavy chain complementarity determining region HCDR3 comprising or consisting of any of the group of amino acid sequences set forth in SEQ ID NOs: 3, 15, 27, 39, 50, 60, 71, and 80, or comprising an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 3, 15, 27, 39, 50, 60, 71, or 80; b) a light chain complementarity determining region LCDR3 comprising or consisting of any of the group of amino acid sequences set forth in SEQ ID NOs: 6, 18, 30, 42, 63, and 83, or comprising an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 6, 18, 30, 42, 63, or 83; and c) the heavy chain constant region comprises a heavy chain constant region of an IgGl, IgG2, or IgG4 mutant, wherein, the mutations are selected from at least one of S228, L234, L235, G236, G237, T250, M252, I253, S254, T256, D259, T307, V308, P329, A330, E380, M428, and N434 (numbering according to the Kabat EU index), preferably at least one of G237A, L234A / L235A, S228P, N434A, N434H, T307A / E380A / N434A, M428L / N434S, and M252Y / S254T / T256E (numbering according to the Kabat EU index), more preferably M252Y / S254T / T256E (numbering according to the Kabat EU index); preferably the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

97.

3. The antibody or antigen binding fragment thereof of claim 2, further comprising: d) a heavy chain complementarity determining region HCDR1 comprising or consisting of any of the group of amino acid sequences set forth in SEQ ID NOs: 1, 13, 25, 37, 58, and 78, or comprising an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 1, 13, 25, 37, 58, or 78; and / or d) a heavy chain complementarity determining region HCDR1 comprising or consisting of any of the group of amino acid sequences set forth in SEQ ID NOs: 1, 13, 25, 37, 58, and 78, or comprising an amino acid sequence having 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 1, 13, 25, 37, 58, or 78; and / or e) a light chain complementarity determining region LCDR1 comprising or consisting of any of the group of amino acid sequences of SEQ ID NOs: 4, 16, 28, 40, 61 and 81, or the HCDR2 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 4, 16, 28, 40, 61 or 81; Preferably, it further comprises: f) a heavy chain complementarity determining region HCDR2 comprising or consisting of any of the group of amino acid sequences of SEQ ID NOs: 2, 14, 26, 38, 49, 59, 70 and 79, or the LCDR1 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 2, 14, 26, 38, 49, 59, 70 or 79; and / or g) a light chain complementarity determining region LCDR2 comprising or consisting of any of the group of amino acid sequences of SEQ ID NOs: 5, 17, 29, 41, 51, 62 and 82, or the LCDR2 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 5, 17, 29, 41, 51, 62 and 82.

4. An antibody or antigen binding fragment thereof comprising three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, and a heavy chain constant region, wherein: HCDR1 comprises or consists of SEQ ID NO: 1, 13, 25, 37, 58 or 78, or the HCDR1 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 1, 13, 25, 37, 58 or 78; HCDR2 comprises or consists of SEQ ID NO: 2, 14, 26, 38, 49, 59, 70 or 79, or the HCDR2 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 2, 14, 26, 38, 49, 59, 70 or 79; HCDR3 comprises or consists of SEQ ID NO: 3, 15, 27, 39, 50, 60, 71 or 80, or the HCDR3 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 3, 15, 27, 39, 50, 60, 71 or 80; LCDR1 comprises or consists of SEQ ID NO: 4, 16, 28, 40, 61 or 81, or the LCDR1 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 4, 16, 28, 40, 61 or 81; LCDR2 comprises or consists of SEQ ID NO: 5, 17, 29, 41, 51, 62 or 82, or the LCDR2 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 5, 17, 29, 41, 51, 62 or 82; LCDR3 comprises or consists of SEQ ID NO: 6, 18, 30, 42, 63 or 83, or the LCDR3 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 6, 18, 30, 42, 63 or 83; the heavy chain constant region comprises a heavy chain constant region of an IgGl, IgG2 or IgG4 mutant, wherein the mutation is selected from at least one of S228, L234, L235, G236, G237, T250, M252, I253, S254, T256, D259, T307, V308, P329, A330, E380, M428 and N434 (numbering according to the Kabat EU index), preferably at least one of G237A, L234A / L235A, S228P, N434A, N434H, T307A / E380A / N434A, M428L / N434S and M252Y / S254T / T256E (numbering according to the Kabat EU index), more preferably M252Y / S254T / T256E (numbering according to the Kabat EU index); preferably the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

97.

5. The antibody or antigen-binding fragment thereof of claim 1, comprising: (1) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 7 or 11, and (2) three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 8 or 12. three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 8 or 12; (2) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 19, 23, 93 or 21, and three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 20, 24, 94 or 22; (3) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 31, 33 or 35, and three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 32, 34 or 36; (4) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 43, 45 or 47, and three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 44, 46 or 48; (5) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 52, 54 or 56, and three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 53, 55 or 57; (6) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 64, 66 or 68, and three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 65, 67 or 69; (7) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 72, 74 or 76, and three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 73, 75 or 77; or (8) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 84, 86, 88 or 95, and three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch having an amino acid sequence as set forth in SEQ ID NO: 85, 87 or 96. three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within a peptide stretch of amino acid sequence as set forth in SEQ ID NO: 85, 87, 89 or 96.

6. The antibody or antigen binding fragment thereof of any one of claims 2-4, comprising: 1) HCDR1 comprising or consisting of SEQ ID NO: 1, HCDR2 comprising or consisting of SEQ ID NO: 2, HCDR3 comprising or consisting of SEQ ID NO: 3, LCDR1 comprising or consisting of SEQ ID NO: 4, LCDR2 comprising or consisting of SEQ ID NO: 5, and LCDR3 comprising or consisting of SEQ ID NO: 6; or 2) HCDR1 comprising or consisting of SEQ ID NO: 13, HCDR2 comprising or consisting of SEQ ID NO: 14, HCDR3 comprising or consisting of SEQ ID NO: 15, LCDR1 comprising or consisting of SEQ ID NO: 16, LCDR2 comprising or consisting of SEQ ID NO: 17, and LCDR3 comprising or consisting of SEQ ID NO: 18; or 3) HCDR1 comprising or consisting of SEQ ID NO: 25, HCDR2 comprising or consisting of SEQ ID NO: 26, HCDR3 comprising or consisting of SEQ ID NO: 27, LCDR1 comprising or consisting of SEQ ID NO: 28, LCDR2 comprising or consisting of SEQ ID NO: 29, and LCDR3 comprising or consisting of SEQ ID NO: 30; or 4) HCDR1 comprising or consisting of SEQ ID NO: 37, HCDR2 comprising or consisting of SEQ ID NO: 38, HCDR3 comprising or consisting of SEQ ID NO: 39, LCDR1 comprising or consisting of SEQ ID NO: 40, LCDR2 comprising or consisting of SEQ ID NO: 41, and LCDR3 comprising or consisting of SEQ ID NO: 42; or 5) HCDR1 comprising or consisting of SEQ ID NO: 25, HCDR2 comprising or consisting of SEQ ID NO: 49, HCDR3 comprising or consisting of SEQ ID NO: 50, LCDR1 comprising or consisting of SEQ ID NO: 28, LCDR2 comprising or consisting of SEQ ID NO: 51, and LCDR3 comprising or consisting of SEQ ID NO: 30; or 6) HCDR1 comprising or consisting of SEQ ID NO: 58, HCDR2 comprising or consisting of SEQ ID NO: 59, HCDR3 comprising or consisting of SEQ ID NO: 60, HCDR3 comprising or consisting of SEQ ID NO: 3, HCDR3 comprising or consisting of SEQ ID NO: 15, HCDR3 comprising or consisting of SEQ ID NO: 27, HCDR3 comprising or consisting of SEQ ID NO: 39, HCDR3 comprising or consisting of SEQ ID NO: 50, HCDR3 comprising or consisting of SEQ ID NO: 60, LCDR1 comprising or consisting of SEQ ID NO: 61, LCDR2 comprising or consisting of SEQ ID NO: 62, and LCDR3 comprising or consisting of SEQ ID NO: 63; or 7) HCDR1 comprising or consisting of SEQ ID NO: 25, HCDR2 comprising or consisting of SEQ ID NO: 70, HCDR3 comprising or consisting of SEQ ID NO: 71, LCDR1 comprising or consisting of SEQ ID NO: 28, LCDR2 comprising or consisting of SEQ ID NO: 51, and LCDR3 comprising or consisting of SEQ ID NO: 30; or 8) HCDR1 comprising or consisting of SEQ ID NO: 78, HCDR2 comprising or consisting of SEQ ID NO: 79, HCDR3 comprising or consisting of SEQ ID NO: 80, LCDR1 comprising or consisting of SEQ ID NO: 81, LCDR2 comprising or consisting of SEQ ID NO: 82, and LCDR3 comprising or consisting of SEQ ID NO:

83.

7. An antibody or antigen-binding fragment thereof that specifically binds to thymic stromal lymphopoietin (TSLP), comprising: a heavy chain variable region comprising or consisting of SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86, or 88, or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86, or 88; or comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid mutations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) as compared to SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86, or 88, preferably the amino acid mutations do not occur in the heavy chain complementarity determining regions; a heavy chain variable region comprising or consisting of SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86, or 88, or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86, or 88; or comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid mutations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) as compared to SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86, or 88, preferably the amino acid mutations do not occur in the heavy chain complementarity determining regions; a heavy chain variable region comprising: SEQ ID NO: 1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39, 41, 49, 51, 59, 61, 63, 71, 79, 81, 83, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 130 the heavy chain constant region comprises a heavy chain constant region of an IgGl, IgG2, or IgG4 mutant, wherein, ​ 8. The antibody or antigen-binding fragment thereof of claim 7, which comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of which are selected from any one of the groups consisting of the following pairs of amino acid sequences: SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, SEQ ID NO: 72 and SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77, SEQ ID NO: 84 and SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, and SEQ ID NO: 88 and SEQ ID NO:

89.

9. The antibody or antigen-binding fragment thereof of claim 1 or 5, wherein each CDR is defined according to the Kabat definition scheme, the Chothia definition scheme, the Abm definition scheme, the IMGT definition scheme, and / or the Contact definition scheme; preferably, wherein each CDR is defined according to the Kabat definition scheme or the Chothia definition scheme.

10. The antibody or antigen-binding fragment thereof of any one of claims 1-9, comprising a light chain constant region comprising: SEQ ID NO: 91, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 91; or consisting of SEQ ID NO: 91, or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:

91.

11. The antibody or antigen-binding fragment thereof of claim 7 or 8, comprising a heavy chain and a light chain, the amino acid sequences of which are: 1) a heavy chain as set forth in SEQ ID NO: 93 and a light chain as set forth in SEQ ID NO: 94; or 2) a heavy chain as set forth in SEQ ID NO: 95 and a light chain as set forth in SEQ ID NO:

96.

12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (preferably scFv) or (Fab')2, single domain antibody, diabody (dAb), or linear antibody.

13. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-12.

14. An expression vector comprising the nucleic acid of claim 13.

15. A host cell comprising the expression vector of claim 14.

16. A method of producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 15, and recovering the antibody or antigen-binding fragment thereof expressed by the host cell from the culture.

17. The method of claim 16, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

18. The method of claim 16 or 17, wherein the host cell is an E. coli cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell.

19. The method of claim 18, wherein the host cell is a Chinese hamster ovary cell (CHO), a CHO cell variant, a 293 cell, or an NSO cell.

20. A pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof of any one of claims 1-12, and a pharmaceutically acceptable excipient.

21. A kit comprising at least one antibody or antigen-binding fragment thereof of any one of claims 1-12, homologues, and derivatives thereof.

22. Use of the antibody or antigen-binding fragment of any one of claims 1-12 or the pharmaceutical composition of claim 20 for the manufacture of a medicament for the treatment and / or prevention of a disease associated with TSLP; preferably, the disease associated with TSLP is asthma or dermatitis. ​ ​ 23. The antibody or antigen binding fragment of any one of claims 1-12 or the pharmaceutical composition of claim 20 for use in the treatment and / or prevention of a TSLP- associated disease, wherein the TSLP-associated disease is asthma or dermatitis.

24. A method of treating and / or preventing a TSLP-associated disease, the method comprising administering to a subject in need thereof the antibody or antigen binding fragment of any one of claims 1-12 or the pharmaceutical composition of claim 20, wherein the TSLP-associated disease is asthma or dermatitis.

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