Antibodies that bind to TSLP and TSLPR, and methods of use.

Abstract

Thymic interstitial lymphocyte necrotizing factor (TSLP) is an epithelial cell-derived cytokine produced in response to pro-inflammatory stimuli. Variant TSLP or TSLPR-binding proteins and methods of use are provided herein. This disclosure provides antibodies against TSLP and TSLP receptor (TSLPR), as well as methods for treating diseases in which human TSLP and human TSLP receptor are involved in disease pathology. The antibodies of this disclosure may include Fc modifications and may exhibit a longer half-life than antibodies that are otherwise identical without Fc modifications (e.g., certain anti-TSLP and anti-TSLPR antibodies known in the art).

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority based on U.S. Provisional Patent Application No. 63 / 522,626, filed on June 22, 2023.

Background Art

[0002] Background Thymic stromal lymphopoietin (TSLP) is an epithelial - cell - derived cytokine produced in response to pro - inflammatory stimuli. TSLP has been found to promote allergic inflammatory responses mainly through its activity on dendritic cells and mast cells. Human TSLP expression has been reported to be increased in the airways of asthma in correlation with disease severity. Furthermore, TSLP protein levels are detectable in concentrated bronchoalveolar lavage (BAL) fluid from asthmatic patients and other patients suffering from allergic disorders. In addition, TSLP has also been found to promote fibrosis.

[0003] TSLP binds to a heterodimeric receptor consisting of the TSLP receptor (TSLPR) and the IL - 7 receptor α - chain (IL - 7Rα) in dendritic cells, thereby activating the dendritic cells. Upon activation, the dendritic cells express inflammatory cytokines such as thymus and activation - regulated chemokine (TARC(CCL17)), macrophage - derived chemokine (MDC(CCL22)), and the like.

[0004] Activation of dendritic cells by TSLP via the TSLP receptor is associated with disease pathologies including allergic inflammatory diseases (e.g., asthma) and autoimmune diseases (e.g., systemic scleroderma).

[0005] Z Thus, in the art, there is a need for antagonists against TSLP or the TSLP receptor (TSLPR) to prevent and treat diseases in which human TSLP and the human TSLP receptor are involved in disease pathologies (e.g., inflammatory and fibrotic disorders).

Brief Description of the Drawings

[0006] Brief explanation of the drawing [Figure 1] Figure 1 shows the ability of antibody 1 wild-type and antibody 1 YTE to inhibit STAT5-inducible expression of luciferase.

[0007] [Figure 2] Figure 2 shows the ability of antibody 1 wild-type and antibody 1 YTE to inhibit the proliferation of Baf3 cells.

[0008] [Figure 3-1] Figures 3A, 3B, and 3C show the ability of the antibodies shown to inhibit STAT5-inducible expression of luciferase in HEK293 cells. [Figure 3-2] Same as above.

[0009] [Figure 4] Figures 4A and 4B show the ability of the antibodies to inhibit STAT5-inducible expression of luciferase in Ba / F3 cells.

[0010] [Figure 5] Figures 5A and 5B demonstrate the ability of the antibodies shown to inhibit the proliferation of Baf3 cells.

[0011] [Figure 6-1] Figures 6A to 6C show the ability of the antibodies to inhibit TARC secretion from donor PMBC cells. [Figure 6-2] Same as above.

[0012] [Figure 7-1] Figures 7A to 7C show the ability of the antibodies shown to inhibit TARC secretion from isolated monocytes of the donor. [Figure 7-2] Same as above. [Overview of the project] [Means for solving the problem]

[0013] Summary of Disclosure This disclosure provides antibodies against TSLP and TSLP receptors (TSLPR), as well as methods for treating diseases in which human TSLP and human TSLP receptors are involved in disease pathology. The antibodies of this disclosure may include Fc modifications and may exhibit a longer half-life than antibodies that are otherwise identical without Fc modifications (e.g., certain anti-TSLP and anti-TSLPR antibodies known in the art).

[0014] In one embodiment, a TSLP or TSLPR-binding protein is provided herein, comprising: (a) a heavy chain variable region (VH) including (i) a CDR1 having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12, (ii) a CDR2 having the amino acid sequence shown in any one of SEQ ID NOs: 13 to 24, and (iii) a CDR3 having the amino acid sequence shown in any one of SEQ ID NOs: 25 to 36; (b) a light chain variable region (VL) including (i) a CDR1 having the amino acid sequence shown in any one of SEQ ID NOs: 37 to 48, (ii) a CDR2 having the amino acid sequence shown in any one of SEQ ID NOs: 49 to 56, and (iii) a CDR3 having the amino acid sequence shown in any one of SEQ ID NOs: 57 to 60; and (c) an Fc domain comprising at least one amino acid modification.

[0015] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 1, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 13, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 25; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 37, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 49, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 57.

[0016] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 2, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 14, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 26; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 38, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 50, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 58.

[0017] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 3, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 15, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 27; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 39, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 51, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 59.

[0018] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 4, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 16, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 28; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 40, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 52, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 60.

[0019] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 5, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 29; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 41, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 53, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 61.

[0020] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 6, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 30; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 42, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 54, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 62.

[0021] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 7, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 19, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 31; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 43, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 55, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 63.

[0022] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 8, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 20, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 32; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 44, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 56, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 60.

[0023] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 9, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 21, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 33; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 45, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 53, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 57.

[0024] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 10, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 22, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 34; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 46, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 54, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 58.

[0025] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 11, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 23, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 35; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 47, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 55, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 59.

[0026] In some embodiments, VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 12, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 24, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 36; VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 48, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 56, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 60.

[0027] In some embodiments, VH includes a sequence having at least 80% sequence identity with any one of the amino acid sequences from SEQ ID NOs. 65 to 69, and VL includes a sequence having at least 80% sequence identity with any one of the amino acid sequences from SEQ ID NOs. 70 to 74.

[0028] In some embodiments, VH includes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 65, and VL includes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 70.

[0029] In some embodiments, VH includes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 66, and VL includes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 71.

[0030] In some embodiments, VH includes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 67, and VL includes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 72.

[0031] In some embodiments, VH includes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 68, and VL includes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 73.

[0032] In some embodiments, VH includes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 69, and VL includes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 74.

[0033] In some embodiments, Fc is the immunoglobulin Fc domain of IgG1, IgG2, or IgG4.

[0034] In some embodiments, Fc is an IgG1 immunoglobulin domain, and at least one modification includes modified M252Y, S254T, and T256E(YTE).

[0035] In some embodiments, Fc is an IgG1 immunoglobulin domain, and at least one modification includes L234A / L235A(LALA).

[0036] In some embodiments, Fc is an IgG1 immunoglobulin domain, and at least one modification includes LALAGA and N434A.

[0037] In some embodiments, Fc is an IgG1 immunoglobulin domain, and at least one modification includes M252Y, S254T, and T256E(YTE), as well as / or M428L and N434S(LS).

[0038] In some embodiments, Fc is an IgG1 immunoglobulin domain, and at least one modification includes M252Y, S254T, and T256E(YTE), as well as L234A / L235A(LALA).

[0039] In some embodiments, Fc is the IgG2 immunoglobulin domain.

[0040] In some embodiments, Fc is the IgG4 immunoglobulin domain.

[0041] In one embodiment, a heavy chain variable region (VH) comprising (i) a CDR1 having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12, (ii) a CDR2 having the amino acid sequence shown in any one of SEQ ID NOs: 13 to 24, and (iii) a CDR3 having the amino acid sequence shown in any one of SEQ ID NOs: 25 to 36; a light chain variable region (VL) comprising (i) a CDR1 having the amino acid sequence shown in any one of SEQ ID NOs: 37 to 48, (ii) a CDR2 having the amino acid sequence shown in any one of SEQ ID NOs: 49 to 56, and (iii) a CDR3 having the amino acid sequence shown in any one of SEQ ID NOs: 57 to 60; and a modified Fc that extends the half-life of the TSLP or TSLPR binding protein compared to a TSLP or TSLPR binding protein that does not contain the modified Fc is provided herein.

[0042] In one embodiment, a TSLP or TSLPR-binding protein is provided herein, wherein the TSLP or TSLPR-binding protein specifically binds to an epitope of TSLP and comprises an Fc domain including amino acid modifications M252Y, S254T, and T256E(YTE), and / or M428L and N434S(LS).

[0043] In one embodiment, a method for treating an inflammatory disorder or disease in a patient requiring treatment of such disorder or disease is provided herein, the method comprising administering to the patient subcutaneously or intravenously an effective amount of one of the TSLP or TSLPR binding proteins described herein.

[0044] In some embodiments, the inflammatory disorder or disease is atopic dermatitis.

[0045] In some embodiments, the inflammatory disorder or disease is asthma.

[0046] In some embodiments, the administration of TSLP or TSLPR-binding protein is subcutaneous.

[0047] In some embodiments, the administration of TSLP or TSLPR-binding protein is intravenous. [Modes for carrying out the invention]

[0048] Detailed explanation To facilitate understanding of this disclosure, many terms and phrases are defined below.

[0049] As used herein, unless otherwise indicated, the term “antibody” is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof (e.g., an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody)), or an antigen-binding fragment of an antibody containing an intact antibody (e.g., an antigen-binding fragment of a monoclonal antibody), an antigen-binding fragment, or an Fc fragment that has been modified, manipulated, or chemically conjugated. Generally, an antibody is a multimeric protein containing four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are linked by interchain disulfide bonds. The immunoglobulin heavy chains are linked by interchain disulfide bonds. The light chains consist of one variable region (VL) and one constant region (CL). The heavy chain consists of one variable region (VH) and at least three constant regions (CH1, CH2, and CH3). The variable region determines the antibody binding specificity. Each variable region contains three hypervariable regions known as complementarity-determining regions (CDRs), adjacent to four relatively conserved regions known as framework regions (FRs). The ranges of the FRs and CDRs are defined (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). The three CDRs (referred to as CDR1, CDR2, and CDR3) contribute to antibody binding specificity. Naturally occurring antibodies are used as starting materials for engineered antibodies (e.g., chimeric antibodies and humanized antibodies). Examples of antibody-based antigen-binding fragments include Fab, Fab', (Fab')2, Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies.Examples of modified or manipulated antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). An example of a chemically conjugated antibody is one in which the toxin portion is conjugated.

[0050] The terms "variable domain" and "variable region" refer to a portion of an antibody or immunoglobulin domain that is interchangeable, exhibits variability in its sequence, and is involved in determining the specificity and binding affinity of a particular antibody. Variability is not uniformly distributed throughout the antibody's variable domain; it is concentrated within the subdomains of the heavy and light chain variable regions. These subdomains are referred to as "hypervariable regions" or "complementarity-determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portion of the variable domain is called the "framework" region (FRM or FR), which provides a scaffold for six CDRs in three-dimensional space, forming the antigen-binding surface.

[0051] As used herein, the “Fc polypeptide” of dimer Fc refers to one of the two polypeptides that form the dimer Fc domain (i.e., the polypeptide containing the C-terminal constant region of the immunoglobulin heavy chain and possessing the ability to stably self-associate). For example, the Fc polypeptide of dimer IgG Fc contains the IgG CH2 and IgG CH3 constant domain sequences. Fc can be of classes IgA, IgD, IgE, IgG, and IgM. These classes are also denoted as α, δ, ε, γ, and μ, respectively. Some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0052] The terms “Fc receptor” and “FcR” are used to describe receptors that bind to the Fc region of an antibody. For example, an FcR can be a human FcR in its natural sequence. Generally, FcRs are those that bind to IgG antibodies (γ receptors) and include the FcγRI, FcγRII, and FcγRIII subclass receptors, which include allele variants and alternatively spliced ​​forms of these receptors. Examples of FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have different but similar amino acid sequences, primarily in their cytoplasmic domains. Other isotypes of immunoglobulins can also be bound by certain FcRs (see, e.g., Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) within its cytoplasmic domain (reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs (including those to be identified in the future) are encompassed by the term "FcR" as used herein. The above terms also include FcRn, the fetal receptor responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol. 24:249 (1994)).

[0053] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and, in some embodiments, refer to any mammalian subject, in particular human, for which diagnosis, treatment, or therapy is desired. “Mammal” for treatment purposes refers to any animal classified as a mammal (including humans, domesticated animals and livestock, as well as animals kept for laboratory, zoo, competition, or pet purposes (e.g., dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.)). In some embodiments, the mammal is human. None of these terms require the supervision of a medical professional.

[0054] As used herein, the term “effective dose” means an amount of a compound (e.g., a compound of this disclosure) sufficient to produce a beneficial or desired result. An effective dose may be administered in one or more doses, applications, or applications and is not intended to be limited to any particular formulation or route of administration. As used herein, the term “to treat” includes any effect that results in improvement of a condition, disease, disorder, etc., such as reducing, mitigating, regulating, improving or eliminating, or improving the symptoms thereof.

[0055] As used herein, the term “pharmaceutical composition” refers to a combination of an active agent and a carrier, inactive or active substance, which makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0056] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutically acceptable carriers (e.g., phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents). The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th edition, Mack Publ. Co., Easton, PA (1975).

[0057] The terms “a” and “an” as used herein mean “one or more,” and include the plural unless the context is appropriate.

[0058] As used herein, all numbers or numerical ranges include all integers within or encompassing such a range, and fractions of values ​​or integers within or encompassing such a range, unless the context otherwise clearly indicates otherwise. Thus, for example, a reference to the range 90–100% includes 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc. In another example, references to the range of 1 to 5,000 times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, as well as 1.1, 1.2, 1.3, 1.4, 1.5 times, 2.1, 2.2, 2.3, 2.4, 2.5 times, etc.

[0059] "Approximately" as used herein means a range that includes the number and extends from 10% below that number to 10% above that value. "Approximately" means a range that extends from 10% below the lower limit of that range to 10% above the upper limit of that range.

[0060] "Percent (%) identity" refers to the degree to which two sequences (nucleotides or amino acids) have the same residues at the same position in their alignment. For example, "an amino acid sequence is X% identical to sequence number Y" refers to the % identity of the amino acid sequence to sequence number Y, and is further detailed as X% of the residues in that amino acid sequence being identical to the residues in the sequence disclosed in sequence number Y. Generally, computer programs are employed for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0061] Throughout this description, where a composition is described as having, including, or comprising, certain components, or where a process and method is described as having, including, or comprising, certain steps, it is intended that the compositions of the Disclosure are essentially or consist of the components described, and that the processes and methods relating to the Disclosure are essentially or consist of the processing steps described.

[0062] As a general rule, percentages of compositions are expressed in units of weight unless otherwise specified. Furthermore, if a variable is not given a definition, the previous definition of that variable takes precedence.

[0063] TSLP-binding protein and TSLPR-binding protein TSLP or TSLPR-binding proteins that bind to TSLP or TSLPR, respectively, are provided herein. In certain embodiments, the TSLP or TSLPR-binding protein is an antibody. In certain embodiments, the TSLP or TSLPR-binding protein is an antibody comprising a modified Fc region. In certain embodiments, TSLP or TSLPR-binding proteins are described herein, comprising: a) a heavy chain variable region (VH) including (i) a CDR1 having the amino acid sequence of any one of SEQ ID NOs: 1 to 12, (ii) a CDR2 having the amino acid sequence of any one of SEQ ID NOs: 13 to 24, and (iii) a CDR3 having the amino acid sequence of any one of SEQ ID NOs: 25 to 36; b) a light chain variable region (VL) including (i) a CDR1 having the amino acid sequence of any one of SEQ ID NOs: 37 to 48, (ii) a CDR2 having the amino acid sequence of any one of SEQ ID NOs: 49 to 56, and (iii) a CDR3 having the amino acid sequence of any one of SEQ ID NOs: 57 to 60; and c) an Fc domain including amino acid modifications M252Y, S254T, and T256E(YTE), and / or M428L and N434S(LS).

[0064] In certain embodiments, TSLP or TSLPR-binding proteins are further described herein, including a) a heavy chain variable region (VH) comprising (i) a CDR1 having the amino acid sequence of any one of SEQ ID NOs: 1 to 12, (ii) a CDR2 having the amino acid sequence of any one of SEQ ID NOs: 13 to 24, and (iii) a CDR3 having the amino acid sequence of any one of SEQ ID NOs: 25 to 36; b) a light chain variable region (VL) comprising (i) a CDR1 having the amino acid sequence of any one of SEQ ID NOs: 37 to 48, (ii) a CDR2 having the amino acid sequence of any one of SEQ ID NOs: 49 to 56, and (iii) a CDR3 having the amino acid sequence of any one of SEQ ID NOs: 57 to 60; and c) a TSLP or TSLPR-binding protein comprising a modified Fc that extends the half-life of the TSLP or TSLPR-binding protein compared to a TSLP or TSLPR-binding protein without the modified Fc.

[0065] In certain embodiments, TSLP or TSLPR-binding proteins are further described herein, which specifically bind to an epitope of TSLP and include Fc domains comprising amino acid modifications M252Y, S254T, and T256E(YTE), and / or M428L and N434S(LS).

[0066] Exemplary CDR amino acid sequences of TSLP or TSLPR-binding proteins are provided in Table 1. [Table 1]

[0067] In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region comprising CDR1, CDR2, and CDR3 as described in Table 1. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of any one of SEQ ID NOs: 1 to 12, (b) CDR2 having the amino acid sequence of any one of SEQ ID NOs: 13 to 24, and (c) CDR3 having the amino acid sequence of any one of SEQ ID NOs: 25 to 36.

[0068] In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region comprising CDR1, CDR2, and CDR3 as described in Table 1. In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region comprising (a) CDR1 having the amino acid sequence of any one of SEQ ID NOs. 37-48, (b) CDR2 having the amino acid sequence of any one of SEQ ID NOs. 49-56, and (c) CDR3 having the amino acid sequence of any one of SEQ ID NOs. 57-60.

[0069] In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region comprising (a) a CDR1 having the amino acid sequence of any one of SEQ ID NOs: 1 to 12, (b) a CDR2 having the amino acid sequence of any one of SEQ ID NOs: 13 to 24, and (c) a CDR3 having the amino acid sequence of any one of SEQ ID NOs: 35 to 36; and a light chain variable region comprising (a) a CDR1 having the amino acid sequence of any one of SEQ ID NOs: 37 to 48, (b) a CDR2 having the amino acid sequence of any one of SEQ ID NOs: 49 to 56, and (c) a CDR3 having the amino acid sequence of any one of SEQ ID NOs: 57 to 60.

[0070] In some embodiments, the TSLP-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 1, (b) CDR2 having the amino acid sequence of SEQ ID NO: 13, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 25; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 37, (b) CDR2 having the amino acid sequence of SEQ ID NO: 49, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 57.

[0071] In some embodiments, the TSLP-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 2, (b) CDR2 having the amino acid sequence of SEQ ID NO: 14, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 26; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 38, (b) CDR2 having the amino acid sequence of SEQ ID NO: 50, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 58.

[0072] In some embodiments, the TSLP-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 3, (b) CDR2 having the amino acid sequence of SEQ ID NO: 15, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 27; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 39, (b) CDR2 having the amino acid sequence of SEQ ID NO: 51, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 59.

[0073] In some embodiments, the TSLPR-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 4, (b) CDR2 having the amino acid sequence of SEQ ID NO: 16, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 28; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 40, (b) CDR2 having the amino acid sequence of SEQ ID NO: 52, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 60.

[0074] In some embodiments, the TSLP-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 5, (b) CDR2 having the amino acid sequence of SEQ ID NO: 17, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 41, (b) CDR2 having the amino acid sequence of SEQ ID NO: 53, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 61.

[0075] In some embodiments, the TSLP-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 6, (b) CDR2 having the amino acid sequence of SEQ ID NO: 18, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 30; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 42, (b) CDR2 having the amino acid sequence of SEQ ID NO: 54, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 62.

[0076] In some embodiments, the TSLP-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 7, (b) CDR2 having the amino acid sequence of SEQ ID NO: 19, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 31; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 43, (b) CDR2 having the amino acid sequence of SEQ ID NO: 55, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 63.

[0077] In some embodiments, the TSLPR-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 8, (b) CDR2 having the amino acid sequence of SEQ ID NO: 20, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 32; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 44, (b) CDR2 having the amino acid sequence of SEQ ID NO: 56, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 60.

[0078] In some embodiments, the TSLP-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 9, (b) CDR2 having the amino acid sequence of SEQ ID NO: 21, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 33; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 45, (b) CDR2 having the amino acid sequence of SEQ ID NO: 53, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 57.

[0079] In some embodiments, the TSLP-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 10, (b) CDR2 having the amino acid sequence of SEQ ID NO: 22, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 34; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 46, (b) CDR2 having the amino acid sequence of SEQ ID NO: 54, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 58.

[0080] In some embodiments, the TSLP-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 11, (b) CDR2 having the amino acid sequence of SEQ ID NO: 23, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 35; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 47, (b) CDR2 having the amino acid sequence of SEQ ID NO: 55, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 59.

[0081] In some embodiments, the TSLPR-binding protein includes a heavy chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 12, (b) CDR2 having the amino acid sequence of SEQ ID NO: 24, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 36; and a light chain variable region comprising (a) CDR1 having the amino acid sequence of SEQ ID NO: 48, (b) CDR2 having the amino acid sequence of SEQ ID NO: 56, and (c) CDR3 having the amino acid sequence of SEQ ID NO: 60.

[0082] Exemplary amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of TSLP or TSLPR-binding proteins are provided in Table 2. [Table 2-1] [Table 2-2]

[0083] In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region (VH) containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region (VH) containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region (VH) containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region (VH) containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region (VH) containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region (VH) containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region (VH) containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region (VH) containing an amino acid sequence having 100% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69.

[0084] In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region (VL) containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region (VL) containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region (VL) containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region (VL) containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region (VL) containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region (VL) containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region (VL) containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region (VL) containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a light chain variable region (VL) containing an amino acid sequence having 100% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74.

[0085] In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region (VH) containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the heavy chain variable region (VH) of the TSLP or TSLPR-binding protein disclosed in Table 2, and a light chain variable region (VL) containing at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) the same amino acid sequence as the light chain variable region (VL) of the same TSLP or TSLPR-binding protein disclosed in Table 2.

[0086] In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69; and a light chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69; and a light chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69; and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69; and a light chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69; and a light chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74.In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69; and a light chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69; and a light chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 65-69; and a light chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 70-74. In some embodiments, the TSLP or TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence according to any one of SEQ ID NOs. 65-69; and a light chain variable region containing an amino acid sequence according to any one of SEQ ID NOs. 70-74.

[0087] In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 65; and a light chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 70. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 65; and a light chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 70. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 65; and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 65; and a light chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 70. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 65; and a light chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 70. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 65; and a light chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 70. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 65; and a light chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 70.In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 65; and a light chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 70.

[0088] In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 66; and a light chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 66; and a light chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 66; and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 66; and a light chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 66; and a light chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 66; and a light chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 66; and a light chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 71.In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 66; and a light chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 71.

[0089] In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 67; and a light chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 72. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 67; and a light chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 72. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 67; and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 67; and a light chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 72. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 67; and a light chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 72. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 67; and a light chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 72. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 67; and a light chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 72.In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 67; and a light chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 72.

[0090] In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 68; and a light chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 73. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 68; and a light chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 73. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 68; and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 73. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 68; and a light chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 73. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 68; and a light chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 73. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 68; and a light chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 73. In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 68; and a light chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 73.In some embodiments, the TSLP-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 68; and a light chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 73.

[0091] In some embodiments, the TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 69; and a light chain variable region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 74. In some embodiments, the TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 69; and a light chain variable region containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 74. In some embodiments, the TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 69; and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 74. In some embodiments, the TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 69; and a light chain variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 74. In some embodiments, the TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 69; and a light chain variable region containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 74. In some embodiments, the TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 69; and a light chain variable region containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 74. In some embodiments, the TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 69; and a light chain variable region containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 74.In some embodiments, the TSLPR-binding protein includes a heavy chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 69; and a light chain variable region containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 74.

[0092] FC modification TSLP or TSLPR-binding proteins containing a modified Fc region are described herein. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0093] In some embodiments, the TSLP or TSLPR-binding protein comprises a modified Fc containing one or more modifications. In some embodiments, one or more modifications are located in an Fc derived from IgG1 (e.g., human IgG1 (hIgG1)). In some embodiments, one or more modifications are located in an Fc derived from IgG4 (e.g., human IgG4 (hIgG4)). In some embodiments, one or more modifications are located in an Fc derived from IgG2. In some embodiments, one or more modifications promote selective binding of the Fc-γ receptor.

[0094] An example amino acid sequence of an Fc sequence is provided in Table 3. [Table 3-1] Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19

[0095] In some embodiments, the TSLP or TSLPR-binding protein includes an Fc having one or more modifications in SEQ ID NO: 76. In some embodiments, the TSLP or TSLPR-binding protein includes an Fc having one or more modifications in SEQ ID NO: 77. In some embodiments, the TSLP or TSLPR-binding protein includes an Fc having one or more modifications in SEQ ID NO: 82. In some embodiments, the Fc includes an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 76, 77, and 82. In some embodiments, the Fc includes an amino acid sequence having at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 76, 77, and 82. In some embodiments, the Fc includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 76, 77, and 82. In some embodiments, the Fc includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 76, 77, and 82. In some embodiments, Fc includes an amino acid sequence having at least 96% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 76, 77, and 82. In some embodiments, Fc includes an amino acid sequence having at least 97% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 76, 77, and 82. In some embodiments, Fc includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 76, 77, and 82. In some embodiments, Fc includes an amino acid sequence having at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 76, 77, and 82. In some embodiments, Fc includes an amino acid sequence of any one of SEQ ID NOs: 76, 77, and 82.

[0096] In some embodiments, one or more modifications in the modified Fc are selected from the group consisting of S298A, E333A, K334A, K326A, F243L, R292P, Y300L, V305I, P396L, F243L, R292P, Y300L, L235V, P396L, F243L, S239D, I332E, A330L, S267E, L328F, D265S, S239E, K326A, A327H, G237F, K326E, G236A, D270L, H268D, S324T, L234F, N325L, V266L, and S267D. In some embodiments, one or more modifications in the modified Fc are selected from the group consisting of S228P, M252Y, S254T, T256E, T256D, T250Q, H285D, T307A, T307Q, T307R, T307W, L309D, Q411H, Q311V, A378V, E380A, M428L, N434A, N434S, N297A, D265A, L234A, L235A, and N434W.

[0097] In some embodiments, the modified Fc includes specific combinations of amino acid substitutions selected from the group consisting of L234A / L235A, V234A / G237A, L235A / G237A / E318A, S228P / L236E, H268Q / V309L / A330S / A331S, C220S / C226S / C229S / P238S, C226S / C229S / E3233P / L235V / L235A, L234F / L235E / P331S, C226S / P230S, L234A / G237A, L234A / L235A / G237A, and L234A / L235A / P329G.

[0098] M428L / N434S(LS)、M252Y / S254T / T256E(YTE) (、T250Q / M428L、T307A / E380A / N434A、T256D / T307Q(DQ)、T256D / T307W) DW)、M252Y / T256D(YD)、T307Q / Q311V / A378V(QVV)、T256D / H285D / T307R / Q311V / A378V(DDRVV)、L309D / Q311H / N434S(DHS)、S228P / L235E(SPLE) L234A / L235A(LALA), M428L / N434A(LA), L234A / G237A(LAGA), L234A / L235A / G237A(LALAGA), L234A / L235A / P329G(LALAPG), N297A / YTE, D265A / YTE, LALA / YTE, LAGA / YTE, LALAGA / YTE, LALAPG / YTE, N297A / LS, D265A / LS, LALA / LS, LAGA / LS, LALAGA / LS, LALAPG / LS, N297A / DHS, D265A / DHS, LA LA / DHS、LAGA / DHS、LALAGA / DHS、LALAPG / DHS、SP / YTE、SPLE / YTE、SP / LS、SPLE / LS、SP / DHS、SPLE / DHS、N297A / LA、D265A / LA、LALA / LA、LAGA / LA、LA LAGA / LA、LALAPG / LA、N297A / N434A、D265A / N434A、LALA / N434A、LAGA / N4 34A、LALAGA / N434A、LALAPG / N434A、N297A / N434W、D265A / N434W、LALA / N4 34W、LAGA / N434W、LALAGA / N434W、LALAPG / N434W、N297A / DQ、D265A / DQ、L ALA / DQ、LAGA / DQ、LALAGA / DQ、LALAPG / DQ、N297A / DW、D265A / DW、LALA / DW 、LAGA / DW、LALAGA / DW、LALAPG / DW、N297A / YD、D265A / YD、LALA / YD、LAGA / YD、LALAGA / YD、LALAPG / YD、N297A / QVV、D265A / QVV、LALA / QVV、LAGA / QVV,The modified Fc includes specific combinations of amino acid substitutions selected from the group consisting of LALAGA / QVV, LALAPG / QVV, N297A / DDRVV, D265A / DDRVV, LALA / DDRVV, LAGA / DDRVV, LALAGA / DDRVV, and LALAPG / DDRVV. In some embodiments, the modified Fc includes specific combinations of amino acid substitutions selected from the group consisting of M428L / N434S(LS) and M252Y / S254T / T256E(YTE). In some embodiments, the modified Fc includes modifications of M428L / N434S(LS) (e.g., SEQ ID NO: 93, SEQ ID NO: 110, SEQ ID NO: 117). In some embodiments, the modified Fc includes modifications of M252Y / S254T / T256E(YTE) (e.g., SEQ ID NO: 86, SEQ ID NO: 107, SEQ ID NO: 116).

[0099] In some embodiments, the TSLP or TSLPR binding proteins described herein include modifications to improve their ability to mediate effector function. Such modifications are known in the art and include defucosylation or manipulation of the affinity of Fc to activating receptors (primarily FCGR3a for antibody-dependent cell-mediated cytotoxicity (ADCC)) and to C1q for complement-dependent cell-mediated cytotoxicity (CDC).

[0100] In some embodiments, the TSLP or TSLPR-binding proteins provided herein contain an Fc domain (e.g., IgG1) having a reduced fucose content at position Asn 297 (EU numbering) compared to naturally occurring Fc domains. Such Fc domains are known to have improved ADCCs. In some embodiments, such antibodies contain no fucose at position Asn 297.

[0101] In some embodiments, the TSLP or TSLPR-binding proteins described herein include an Fc region having one or more amino acid substitutions that improve ADCC (e.g., substitutions at one or more positions among positions 298, 333, and 334 of the Fc region). In some embodiments, the TSLP or TSLPR-binding proteins described herein include an Fc region having one or more amino acid substitutions at positions 239, 332, and 330.

[0102] In some embodiments, Fc includes an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 75-175 and 178-188. In some embodiments, Fc includes an amino acid sequence having at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 75-175 and 178-188. In some embodiments, Fc includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 75-175 and 178-188. In some embodiments, Fc includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 75-175 and 178-188. In some embodiments, Fc includes an amino acid sequence having at least 96% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 75-175 and 178-188. In some embodiments, Fc includes an amino acid sequence having at least 97% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 75-175 and 178-188. In some embodiments, Fc includes an amino acid sequence having at least 98% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 75-175 and 178-188. In some embodiments, Fc includes an amino acid sequence having at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs. 75-175 and 178-188. In some embodiments, Fc includes an amino acid sequence of any one of SEQ ID NOs. 75-175 and 178-188.

[0103] In some embodiments, the TSLP or TSLPR-binding proteins described herein include an Fc region having at least one galactose residue within an oligosaccharide bound to the Fc region. Such variants may have improved CDC function.

[0104] In some embodiments, the TSLP or TSLPR binding proteins described herein include one or more modifications that improve or reduce C1q binding and / or CDC.

[0105] In certain embodiments, the Fc region comprises one or more amino acid substitutions, where one or more substitutions result in an increase in one or more of the following compared to an Fc region without one or more substitutions: antibody half-life, ADCC activity, ADCP activity, or CDC activity. In certain embodiments, one or more amino acid substitutions result in an increased antibody half-life at pH 6.0 compared to an antibody containing a wild-type Fc region. In a particular embodiment, the antibody has an increased half-life that is longer by approximately 10,000 times, 1,000 times, 500 times, 100 times, 50 times, 20 times, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.95 times, 1.9 times, 1.85 times, 1.8 times, 1.75 times, 1.7 times, 1.65 times, 1.6 times, 1.55 times, 1.50 times, 1.45 times, 1.4 times, 1.35 times, 1.3 times, 1.25 times, 1.2 times, 1.15 times, 1.1 times, or 1.05 times compared to an antibody containing a wild-type Fc region.

[0106] In a particular embodiment, the Fc region comprises one or more amino acid substitutions, where one or more substitutions result in a decrease in one or more of the ADCC activity, ADCP activity, or CDC activity compared to an Fc without one or more substitutions.

[0107] In a particular embodiment, the Fc region binds to an Fcγ receptor selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In a particular embodiment, the Fc region binds to the Fcγ receptor with higher affinity at pH 6.0 compared to an antibody containing a wild-type Fc region.

[0108] In some embodiments, the TSLP or TSLPR binding proteins described herein include a long half-life (i.e., serum half-life). In some embodiments, the TSLP or TSLPR binding proteins described herein include a half-life of at least about 14, 28, 42, 56, 70, 84, 96, or more than 96 weeks. In some embodiments, the TSLP or TSLPR-binding proteins described herein include half-lives in the range of about 14 to about 96 days, about 14 to about 84 days, about 14 to about 70 days, about 14 to about 56 days, about 14 to about 42 days, about 14 to about 28 days, about 28 to about 96 days, about 28 to about 84 days, about 28 to about 70 days, about 28 to about 56 days, about 28 to about 42 days, about 42 to about 96 days, about 42 to about 84 days, about 42 to about 70 days, or about 42 to about 56 days. In some embodiments, the TSLP or TSLPR-binding proteins described herein include half-lives in the range of about 42 to about 56 days. In some embodiments, the TSLP or TSLPR-binding proteins described herein include half-lives in the range of about 42 to about 56 days. In some embodiments, the TSLP or TSLPR-binding proteins described herein have a half-life of about 50 days. Methods for measuring half-life are known in the art. In some embodiments, the half-life is measured in non-human primates. In some embodiments, the half-life is measured in humans. In some embodiments, the half-life is measured after intravenous administration. In some embodiments, the half-life is measured after subcutaneous administration.

[0109] In some embodiments, the TSLP or TSLPR-binding protein described herein has a half-life at least 20% longer than that of the comparative antibody. In some embodiments, the comparative antibody contains the same complementarity-determining region and variable region, but contains a different Fc region. In some embodiments, the half-life of the TSLP or TSLPR-binding protein described herein is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% longer than that of the comparative antibody. In some embodiments, the half-life of the TSLP or TSLPR-binding protein described herein is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times longer than that of the comparative antibody.

[0110] light chain constant region TSLP or TSLPR-binding proteins containing a light chain constant (LC) region are described herein. Unless otherwise specified herein, the numbering of amino acid residues in the light chain constant region follows the EU numbering system (also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0111] In some embodiments, the TSLP or TSLPR-binding protein includes a light chain constant (LC) region as disclosed herein. In some embodiments, the TSLP or TSLPR-binding protein includes an LC region comprising SEQ ID NO: 176. In some embodiments, the TSLP or TSLPR-binding protein includes an LC comprising SEQ ID NO: 177.

[0112] An example amino acid sequence of the Fc constant region is provided in Table 4. [Table 4]

[0113] Treatment method As described herein, in certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of inflammatory disorders or diseases. In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of atopic dermatitis. In certain embodiments, the treatment reduces disease severity in the subject, where disease severity is assessed by the Atopic Dermatitis Disease Severity Outcome Scale. In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of asthma. In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of idiopathic pulmonary fibrosis. In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of alopecia areata. In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of chronic rhinosinusitis with nasal polyps. In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of chronic rhinosinusitis without nasal polyps (CRSsNP). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of eosinophilic esophagitis (EoE). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of eosinophilic gastrointestinal disorders or diseases (ENID) selected from the group consisting of eosinophilic gastritis (EoG), eosinophilic enteritis (EoN), eosinophilic colitis (EoC), and eosinophilic gastroenteritis (EGE). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of Churg-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of prurigo nodularis (PN). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of chronic idiopathic urticaria (CSU). In a particular embodiment, TSLP or TSLPR-binding proteins are used in the treatment of chronic pruritus of unknown cause (CPUO).In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of bullous pemphigoid (BP). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of cold-induced urticaria (ColdU). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of allergic fungal rhinosinusitis (AFRS). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of allergic bronchopulmonary aspergillosis (ABPA). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of chronic obstructive pulmonary disease (COPD). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis). In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of psoriasis. In certain embodiments, TSLP or TSLPR-binding proteins are used in the treatment of lupus. In certain embodiments, the isolated antibody is used in the treatment of rheumatoid arthritis.

[0114] In certain embodiments, a method for treating an inflammatory disorder or disease in a mammalian subject in need, the method comprising administering a therapeutically effective amount of a TSLP or TSLPR-binding protein or a pharmaceutical composition described herein to the mammalian subject, is described herein. In certain embodiments of the method described herein, the inflammatory disorder or disease is atopic dermatitis. In certain embodiments, the inflammatory disorder or disease is asthma. In certain embodiments, the inflammatory disorder or disease is idiopathic pulmonary fibrosis. In certain embodiments, the inflammatory disorder or disease is alopecia areata. In certain embodiments, the inflammatory disorder or disease is chronic rhinosinusitis with nasal polyps. In certain embodiments, the inflammatory disorder or disease is chronic rhinosinusitis without nasal polyps (CRSsNP). In certain embodiments, the inflammatory disorder or disease is eosinophilic esophagitis (EoE). In certain embodiments, the inflammatory disorder or disease is an eosinophilic gastrointestinal disorder or disease (ENID) selected from the group consisting of eosinophilic gastritis (EoG), eosinophilic enteritis (EoN), eosinophilic colitis (EoC), and eosinophilic gastroenteritis (EGE). In certain embodiments, the inflammatory disorder or disease is Churg-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA). In certain embodiments, the inflammatory disorder or disease is prurigo nodosa (PN). In certain embodiments, the inflammatory disorder or disease is chronic idiopathic urticaria (CSU). In certain embodiments, the inflammatory disorder or disease is chronic pruritus of unknown etiology (CPUO). In certain embodiments, the inflammatory disorder or disease is bullous pemphigoid (BP). In certain embodiments, the inflammatory disorder or disease is cold-induced urticaria (ColdU). In certain embodiments, the inflammatory disorder or disease is allergic fungal rhinosinusitis (AFRS). In certain embodiments, the inflammatory disorder or disease is allergic bronchopulmonary aspergillosis (ABPA). In certain embodiments, the inflammatory disorder or disease is chronic obstructive pulmonary disease (COPD).In certain embodiments, the inflammatory disorder or disease is inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis). In certain embodiments, the inflammatory disorder or disease is psoriasis. In certain embodiments, the inflammatory disorder or disease is lupus. In certain embodiments, the inflammatory disorder or disease is rheumatoid arthritis.

[0115] In a particular embodiment, a method for treating an inflammatory disorder or disease in a patient in need, the method comprising subcutaneously or intravenously administering an effective amount of a TSLP or TSLPR-binding protein containing a modified Fc region to the patient, is described herein. In a particular embodiment, a method for treating an inflammatory disorder or disease in a patient in need, the method comprising a) a heavy chain variable region (VH) comprising (i) a CDR1 having the amino acid sequence of any one of SEQ ID NOs: 1 to 12, (ii) a CDR2 having the amino acid sequence of any one of SEQ ID NOs: 13 to 24, and (iii) a CDR3 having the amino acid sequence of any one of SEQ ID NOs: 25 to 36; b) a light chain variable region (VL) comprising (i) a CDR1 having the amino acid sequence of any one of SEQ ID NOs: 37 to 48, (ii) a CDR2 having the amino acid sequence of any one of SEQ ID NOs: 49 to 56, and (iii) a CDR3 having the amino acid sequence of any one of SEQ ID NOs: 57 to 60; and c) an effective amount of TSLP or TSLPR-binding protein comprising an Fc domain comprising amino acid modifications M252Y, S254T, and T256E(YTE), and / or M428L and N434S(LS), is described herein, comprising subcutaneous or intravenous administration to the patient.

[0116] In a particular embodiment, a method for treating an inflammatory disorder or disease in a patient in need, the method further described herein, comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having the amino acid sequence of any one of SEQ ID NOs: 1 to 12, (ii) a CDR2 having the amino acid sequence of any one of SEQ ID NOs: 13 to 24, and (iii) a CDR3 having the amino acid sequence of any one of SEQ ID NOs: 25 to 36; b) a light chain variable region (VL) comprising (i) a CDR1 having the amino acid sequence of any one of SEQ ID NOs: 37 to 48, (ii) a CDR2 having the amino acid sequence of any one of SEQ ID NOs: 49 to 56, and (iii) a CDR3 having the amino acid sequence of any one of SEQ ID NOs: 57 to 60; and c) subcutaneous or intravenous administration to the patient an effective amount of TSLP or TSLPR binding protein comprising a modified Fc that extends the half-life of the TSLP or TSLPR binding protein compared to a TSLP or TSLPR binding protein without the modified Fc.

[0117] In certain embodiments, a method for treating an inflammatory disorder or disease in a patient in need, the method comprising subcutaneously or intravenously administering an effective amount of TSLP or TSLPR-binding protein to the patient, wherein the TSLP or TSLPR-binding protein specifically binds to an epitope of TSLP and comprises an Fc domain including amino acid modifications M252Y, S254T, and T256E(YTE), and / or M428L and N434S(LS), is further described herein.

[0118] In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 75 mg to approximately 150 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 250 mg to approximately 750 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg to approximately 700 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg to approximately 600 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg to approximately 500 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg to approximately 400 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 400 mg to approximately 700 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 400 mg to approximately 600 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg to approximately 500 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 500 mg to approximately 700 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 500 mg to approximately 600 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 600 mg to approximately 700 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, or 700 mg.

[0119] In some embodiments, administration of TSLP or TSLPR-binding protein is intravenous, intratumoral, intramuscular, subcutaneous, intralesional, intraintestinal, intracolonic, intrarectal, in a pouch, or intraperitoneal. In some embodiments, administration of TSLP or TSLPR-binding protein is via parenteral routes (e.g., intravenous, intramuscular, subcutaneous, intra-arterial, or intraperitoneal administration). In some embodiments, administration of TSLP or TSLPR-binding protein is intravenous or subcutaneous. In some embodiments, administration of TSLP or TSLPR-binding protein is intravenous. In some embodiments, administration of TSLP or TSLPR-binding protein is subcutaneous.

[0120] Administration of TSLP or TSLPR-binding protein may occur at various intervals. In some embodiments, TSLP or TSLPR-binding protein is administered to the patient at least once at intervals longer than 8 weeks. In some embodiments, the interval is about 12 weeks to about 26 weeks. In some embodiments, the interval is about 12 weeks to about 22 weeks. In some embodiments, the interval is about 12 weeks to about 18 weeks. In some embodiments, the interval is about 12 weeks to about 14 weeks. In some embodiments, the interval is about 16 weeks to about 26 weeks. In some embodiments, the interval is about 16 weeks to about 22 weeks. In some embodiments, the interval is about 16 weeks to about 18 weeks. In some embodiments, the interval is about 20 weeks to about 26 weeks. In some embodiments, the interval is about 20 weeks to about 22 weeks. In some embodiments, the interval is about 12 weeks. In some embodiments, the interval is about 16 weeks. In some embodiments, the interval is about 26 weeks.

[0121] Pharmaceutical composition This disclosure also features a pharmaceutical composition comprising a therapeutically effective amount of a TSLP or TSLPR-binding protein as described herein. The composition may be formulated for use in various drug delivery systems. 1 or more physiologically acceptable excipients or carriers may also be included in the composition for appropriate formulation. Formulations suitable for use in this disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th edition, 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0122] In some embodiments, the pharmaceutical composition may include, for example, formulation materials for modifying, maintaining, or preserving the pH, volume osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or osmosis of the composition.In such embodiments, suitable formulation materials include, but are not limited to, the following: amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulin); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); and low molecular weight polypeptides. Hydrogenated ions; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., Pluronic®, PEG, sorbitan esters, polysorbate (e.g., polysorbate 20), polysorbate, triton®, tromethamine, lecithin, cholesterol, tyroxapole); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (Remington's See Pharmaceutical Sciences, 18th edition (Mack Publishing Company, 1990).

[0123] In some embodiments, the pharmaceutical composition is citric acid-free.

[0124] In some embodiments, the pharmaceutical composition may include nanoparticles, such as polymer nanoparticles, liposomes, or micelles.

[0125] In some embodiments, the pharmaceutical composition may include sustained-release or controlled-release formulations. Techniques for formulating sustained-release or controlled-release means (e.g., liposome carriers, biodegradable microparticles or porous beads and depot injections) are also known to those skilled in the art. Sustained-release preparations may include, for example, porous polymer microparticles or semipermeable polymer matrices in the form of molded articles (e.g., films) or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, poly(2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. The sustained-release composition may also include liposomes, which can be prepared by any of several methods known in the art.

[0126] The pharmaceutical compositions comprising TSLP or TSLPR-binding proteins disclosed herein may be presented in dosage unit form and may be prepared by any suitable method. The pharmaceutical compositions should be formulated to be compatible with their intended route of administration. Examples of routes of administration include intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. In some embodiments, the TSLP or TSLPR-binding proteins disclosed herein are administered intravenously or subcutaneously. In some embodiments, the TSLP or TSLPR-binding proteins disclosed herein are administered intravenously. In some embodiments, the TSLP or TSLPR-binding proteins disclosed herein are administered subcutaneously.

[0127] Useful formulations can be prepared by methods known in the pharmaceutical field. See, for example, Remington's Pharmaceutical Sciences, 18th edition (Mack Publishing Company, 1990). Suitable formulation components for parenteral administration include sterile diluents (e.g., water for injection, physiological saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents); antimicrobial agents (e.g., benzyl alcohol or methylparaben); antioxidants (e.g., ascorbic acid or sodium sulfite); chelating agents (e.g., EDTA); buffers (e.g., acetates, citrates, or phosphates); and agents for tonic adjustment (e.g., sodium chloride or dextrose). In some embodiments, formulations for parenteral administration are citrate-free.

[0128] For intravenous or subcutaneous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered physiological saline (PBS). The carrier should be stable under manufacturing and storage conditions and should be protected from microorganisms. The carrier may be a solvent or dispersion medium (e.g., water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof).

[0129] Intravenous or subcutaneous drug delivery formulations may be contained in syringes, pens, or bags. In some embodiments, the bag may be connected to a channel including a tube and / or needle. In some embodiments, the formulation may be a lyophilized formulation or a liquid formulation.

[0130] These compositions may be sterilized by conventional sterilization techniques or by sterile filtration. The resulting aqueous solutions may be packaged for immediate use or lyophilized, and the lyophilized preparations may be combined with a sterile aqueous carrier before administration.

[0131] Polyols (which can act as isotonic agents and stabilize TSLP or TSLPR-binding proteins) may also be included in the formulation. Polyols are added to the formulation in amounts that may vary with respect to the desired isotonicity of the formulation. In some embodiments, aqueous formulations are isotonic. The amount of polyol added may also be varied in relation to the molecular weight of the polyol. For example, small amounts of monosaccharides (e.g., mannitol) are added compared to disaccharides (e.g., trehalose). In some embodiments, the polyol used in the formulation as a tonicity agent is mannitol.

[0132] Detergents or surfactants may also be added to the formulation. Exemplary detergents include nonionic detergents (e.g., polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188)). The amount of detergent added is such that it reduces aggregation of the formulated TSLP or TSLPR-binding protein and / or minimizes the formation of granular material in the formulation and / or reduces adsorption. In some embodiments, the formulation may contain a surfactant, which is a polysorbate. In some embodiments, the formulation may contain a detergent, polysorbate 80, or Tween® 80. Tween® 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edition, 1996).

[0133] In some embodiments, the protein products of the present disclosure are formulated as liquid formulations. In some embodiments, the liquid formulations are prepared in combination with a sugar at a stabilizing level. In some embodiments, the liquid formulations are prepared in an aqueous carrier. In some embodiments, the stabilizer is added in an amount less than or equal to the amount that would result in a viscosity undesirable or unsuitable for intravenous administration. In some embodiments, the sugar is a disaccharide, such as sucrose. In some embodiments, the liquid formulations may also contain one or more of buffers, surfactants, and preservatives.

[0134] In some embodiments, the pH of the liquid formulation is set by the addition of a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid is hydrochloric acid. In some embodiments, the base is sodium hydroxide.

[0135] In this specification, the aqueous carrier of interest is pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for the preparation of liquid formulations. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH-buffered solutions (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0136] Preservatives may be added to the formulations herein as needed to reduce bacterial activity. The addition of preservatives may facilitate, for example, the production of multi-dose formulations.

[0137] TSLP or TSLPR-binding proteins can be lyophilized to produce a lyophilized formulation containing the protein and a lyophilized protective agent. The lyophilized protective agent may be a sugar, for example, a disaccharide. In some embodiments, the lyophilized protective agent is sucrose or maltose. The lyophilized formulation may also contain one or more of the following: buffering agents, surfactants, fillers, and / or preservatives.

[0138] The amount of sucrose or maltose useful for stabilizing lyophilized drug products can be at least 1:2 protein to sucrose or maltose by weight ratio. In some embodiments, the protein to sucrose or maltose weight ratio is 1:2 to 1:5. In some embodiments, the pH of the formulation is set by adding a pharmaceutically acceptable acid and / or base before lyophilization. In some embodiments, the pharmaceutically acceptable acid is hydrochloric acid. In some embodiments, the pharmaceutically acceptable base is sodium hydroxide.

[0139] In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 75 mg to approximately 150 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 250 mg to approximately 750 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg to approximately 700 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg to approximately 600 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg to approximately 500 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg to approximately 400 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 400 mg to approximately 700 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 400 mg to approximately 600 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg to approximately 500 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 500 mg to approximately 700 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 500 mg to approximately 600 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 600 mg to approximately 700 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, or approximately 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, or 700 mg. In some embodiments, TSLP or TSLPR-binding protein is administered in doses of approximately 300 mg.The actual dose levels of the active ingredient in the pharmaceutical compositions of this disclosure may be about 250 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, or 700 mg, to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0140] The specific dose may be a uniform dose of approximately 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg of protein per patient. Alternatively, the patient's dose may be adjusted to the patient's approximate body weight or surface area. Other factors in determining the appropriate dose may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and medical condition. Further refinements of the calculations required to determine the appropriate dose for treatment are customarily made by those skilled in the art, particularly in light of the dose information and assays disclosed herein. The dose may also be determined through the use of known assays for dose determination, used in conjunction with appropriate dose-response data. The dose for individual patients may be adjusted while monitoring the progression of the disease. Blood levels of the targetable construct or complex in the patient may be measured to determine whether the dose needs to be adjusted to achieve or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes, as well as their dosages, are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer et al., Clinica Chimica Acta 308: 33-41, 2001).

[0141] Preparation method The TSLP or TSLPR binding proteins described above can be prepared using recombinant DNA techniques well known to those skilled in the art. For example, one or more isolated polynucleotides encoding a TSLP or TSLPR binding protein can be ligated to other suitable nucleotide sequences (including, for example, constant region coding sequences and expression regulatory sequences) to produce a conventional gene expression construct (i.e., an expression vector) encoding the desired TSLP or TSLPR binding protein. The production of the defined gene construct is within the scope of the conventional art in this field.

[0142] Nucleic acids encoding the desired TSLP or TSLPR binding protein can be incorporated (ligated) into an expression vector (which can be introduced into host cells via conventional transfection or transformation techniques). Exemplary host cells include Escherichia coli (E. coli) cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and melanoma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that allow the host cells to express the gene encoding the TSLP or TSLPR binding protein.

[0143] Specific expression and purification conditions vary depending on the expression system employed. For example, if the gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter (e.g., Trp or Tac) and a prokaryotic signaling sequence. The expressed protein may be secreted. The expressed protein may accumulate in a refractive body or inclusion body, which can be collected after the cells are disrupted by French press or sonication. The refractive body is then solubilized, and its protein can be refolded and / or cleaved by methods known in the art.

[0144] If the manipulated gene is to be expressed in a eukaryotic host cell (e.g., a CHO cell), it is first inserted into an expression vector containing an appropriate eukaryotic promoter, secretory signal, poly(A) sequence, and stop codon. Optionally, the vector or gene construct may include enhancers and introns. In embodiments involving a fusion protein containing a TSLP or TSLPR binding protein or a portion thereof, the expression vector may optionally include a sequence encoding all or part of the constant region, allowing for the expression of the entire heavy chain or light chain or a portion thereof. The gene construct can be introduced into a eukaryotic host cell using conventional techniques.

[0145] In some embodiments, an N-terminal signal sequence is included in the protein construct to express a TSLP or TSLPR-binding protein. Exemplary N-terminal signal sequences include those derived from interleukin-2, CD-5, IgG κ light chain, trypsinogen, serum albumin, and prolactin.

[0146] Following transfection, a single clone can be isolated for cell banking using methods known in the art (e.g., limiting dilution, ELISA, FACS, microscopy, or Clonepix). The clone can be cultured under conditions suitable for bioreactor scale-up, and the expression of TSLP or TSLPR-binding protein can be maintained.

[0147] TSLP or TSLPR-binding proteins can be isolated and purified using methods known in the art, including centrifugation, deep filtration, cell lysis, homogenization, freeze-thaw cycles, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography. [Examples]

[0148] Examples Examples of specific embodiments for carrying out the present invention are provided herein. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any sense. While efforts have been made to ensure accuracy with respect to the figures used (e.g., quantities, temperatures, etc.), some experimental errors and deviations should naturally be accepted.

[0149] The implementation of the present invention will, unless otherwise indicated, utilize conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the scope of the art of the present art. Such techniques are well described in the literature. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., latest edition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan, Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); and Carey and Sundberg, Advanced Organic Chemistry, 3rd edition (Plenum Press) Vols A and B (1992).

[0150] method Gene synthesis and plasmid construction The coding sequences for the HC and LC of the antibody were generated by DNA synthesis and PCR, and subsequently subcloned into plasmids for mammalian cell expression for protein expression in a mammalian cell system. The gene sequences in the expression vectors were confirmed by DNA sequencing.

[0151] Expression of antibody constructs Transient antibody expression was achieved by co-transfection of paired HC and LC constructs into CHO cells using the PEI method. Briefly, approximately 5.5 × 10⁶ cells in a shaking flask. 6 CHO cells at a concentration of / mL were used as the host. Transfection was initiated by adding a mixture of 1 mg / L DNA and 7 mg / L PEI in OptiMEM® medium (Invitrogen) to the cells, followed by gentle mixing. The cells were then cultured in an incubator shaker at 120 rpm, 37°C, and 8% CO2 for 9 days. Peptone and glucose were supplied after 24 hours, and thereafter every 2-3 days depending on cell density and viability. Cell culture was terminated on day 9 when cell viability decreased to <80%. The conditioned medium was collected for protein purification.

[0152] Purification of antibody constructs Protein purification by affinity chromatography and ion exchange chromatography were performed using an AKTA pure instrument (GE Lifesciences). The conditioned medium expressing the target antibody was collected by centrifugation at 4000 rpm for 50 minutes and filtered through a 0.22 μm filter. The supernatant was loaded onto a Mabselect® SuRe® (GE Healthcare) column. After washing the column with buffer A (PBS, pH 7.4), the protein was eluted with buffer B (1 M glycine, pH 2.7) and immediately neutralized with 1 / 10 volume of buffer D (1 M sodium citrate, pH 6.0). The affinity-purified antibody was then buffer-exchanged to 20 mM sodium acetate, pH 5.5.

[0153] SEC-HPLC analysis of antibody constructs Analytical SEC-HPLC was performed using a Shimadzu LC-10 HPLC instrument (Shimadzu Corp.). 20 μl of sample per 1 mg / mL was loaded onto a Superdex® 200 Increase 5 / 150GL column (GE Lifesciences). The mobile phase was 2×PBS, flow rate 0.3 mL / min, for 15 minutes.

[0154] Measurement of TSLP or TSLPR binding kinetics of antibodies using surface plasmon resonance The binding kinetics and affinity constants were determined at 25°C and in HBS-EP+ electrophoresis buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) using a Biacore 8K SPR system (GE HealthCare) equipped with a Series S Sensor Chip Protein G (Cytiva, Cat. 29179315). After a stabilization period in the electrophoresis buffer, the anti-TSLP mAb construct, diluted to 1 μg / mL, was captured onto flow cell 2 (active) for 60 seconds at a flow rate of 10 μL / min. Recombinant human TSLP or TSLPR protein with His tag was prepared at concentrations of 0 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, and 0 nM, and injected into flow cell 1 (reference) and flow cell 2 (active) for 180 seconds at a flow rate of 50 μL / min. Recombinant cynomolgus monkey TSLP protein with His tag was prepared at concentrations of 0 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, 25 nM, and 0 nM, and injected into flow cell 1 (reference) and flow cell 2 (active) for 180 seconds at a flow rate of 50 μL / min. The sample was injected onto newly captured mAbs in a multi-cycle manner by regenerating the capture surface by injecting glycine pH 1.5 at a flow rate of 30 μL / min for 30 seconds. The data was processed and analyzed as follows using Biacore Insight Evaluation Software Version 2.0.15.12933 (GE Healthcare): The response from flow cell 1 (reference) was subtracted from the response from flow cell 2 (active). Then, the responses from the two buffer blank injections were subtracted from the reference subtracted data (2-1) to obtain dual reference data, which was fitted to a 1:1 binding model to determine the apparent association rate constant (ka) and dissociation rate constant (kd). Their ratio provides the apparent equilibrium dissociation constant or affinity constant (KD = kd / ka).

[0155] Evaluation of blockade by ELISA A 96-well plate (Costar #9018) is coated overnight at 4°C with 2 μg / ml TSLP R (Acro, TSR-H525a) in PBS pH 7.4, and then blocked at 37°C for 2 hours with PBST + 1% BSA. Sequentially diluted test samples (double strips, 10 nM to 1 / 5 dilution, 7 doses + blank) are mixed with 5 ng / ml biotin-TSLP (Acro, TSP-H82Eb) at RT for 30 minutes. The TA-TSLP mixture is then added to the blocked plate and incubated at 37°C for 1 hour. After washing, the plate is incubated with streptavidin-HRP (SIGMA, 21140) at 37°C for 1 hour. After washing, TMB is added to each well and incubated at room temperature until color development occurs (approximately 10 minutes). The reaction is stopped by adding 1N HCl, and the optical density (OD) is read at 450 nm. The inhibition % is calculated as 1 - (OD450 of sample / OD450 of "ligand only"). The IC50 of TA is calculated through nonlinear regression.

[0156] Example 1: Evaluation of the ability of modified TSLP and TSLPR antibodies to improve the affinity and efficacy of blocking TSLP or TSLPR. result Determination of antibody affinity for TSLP or TSLPR Using the method described above, the affinity of anti-TSLP antibodies to TSLP and their binding kinetics were evaluated using surface plasmon resonance (SPR) in comparison to a control antibody, antibody 1 (which contains an IgG2 Fc domain). Antibodies based on antibody 1 but containing modified M252Y, S254T, and T256E (YTE), as well as IgG1 Fc domains (SEQ ID NO: 89) including L234A / L235A (LALA), are referred to herein as "antibody 1 YTE".

[0157] When measured by SPR, the antibody showed strong affinity for TSLP. The antibody was shown to bind to human TSLP at sub-nanomolar concentrations and cross-reactive to cynomolgus monkey TSLP at sub-nanomolar concentrations, as summarized in Table 5. [Table 5]

[0158] Example 2: Inhibition of TSLP binding to TSLPR and / or IL-7Rα The TSLP-responsive luciferase reporter Ba / F3 cell line is a mouse Ba / F3 cell line engineered to express both TSLPR (CRLF2, also known as cytokine receptor-like factor 2) and IL-7Ra isolated by the self-cleaving P2A peptide. The construct was delivered by lentiviral transduction of STAT5 luciferase reporter Ba / F3 cells (which express a firefly luciferase reporter driven by a STAT5 response element located upstream of the promoter). Following activation by TSLP, the endogenous transcription factor STAT5 binds to the response element and induces transcription of the luciferase reporter gene.

[0159] Inhibition of luciferase expression in Baf3 cells was used to block TSLP-induced biological activity, and the functional activity of the antibody was evaluated. Briefly, Baf3 cells were collected and seeded at 50,000 cells per well in 50 μL. Cells were incubated overnight at 37°C and 5% CO2. Meanwhile, serial dilutions of the antibody and TSLP were incubated in assay medium at 4°C for 30 minutes. A 50 μL mixture of TSLP and purified antibody (1:1 by volume) was added to each well. Cells were incubated at 37°C for 6 hours. 100 μL of ONE-Glo luciferase reagent was added to each well, and cell lysis was allowed by shaking at room temperature for approximately 2 minutes. The RLU of cells in each well was recorded using a luminescent plate reader, and the subsequent data were analyzed using GraphPad Prism. The IC50 value was determined as the antibody concentration required to inhibit 50% of the maximum RLU of luciferase detected in TSLP-only incubation. The relative IC50 value was determined relative to antibody 1, which served as the reference antibody. The results are summarized in Table 6 and Figure 1. [Table 6]

[0160] Antibodies were generated based on the sequences of antibody 2 and antibody 3a, as well as antibody 2 and antibody 3 having IgG1 Fc domains (SEQ ID NO: 86) containing modified M252Y, S254T, and T256E (YTE). These antibodies, along with antibody 1 and antibody 1 YTE (which also has the LALA modification shown in Example 1), were tested for their ability to inhibit luciferase expression in Baf3 and HEK293 cells as described in this example. The results are summarized in Table 7, Figures 3A-3C (HEK293 cells), and Figures 4A-4B (Ba / F3 cells). Antibody 4, the anti-TSLPR antibody, did not show significant inhibition of luciferase expression in HEK293 cells (Figure 3C) and Ba / F3 cells (Figure 4B). [Table 7]

[0161] Example 3: Inhibition of cell proliferation in Ba / F3 cells Ba / F3 cells were collected, plated, and washed twice with PBS. 5,000 cells / well were seeded into a 96-well culture plate in 50 μL of assay medium (1640 containing 10% FBS). Serial dilutions of antibody (500 nM, 1:5 dilution) were prepared with human TSLP protein (EC50) in assay medium. 50 μL of the antibody and TSLP protein mixture in assay medium was added to the cells in the wells. Meanwhile, 50 μL of assay medium was added to an unstimulated control well. Cells were incubated at 37°C with 5% CO2 for 72 hours. 100 μL of CellTiter-Glo reagent was added per well, and the mixture was shaken at room temperature for approximately 2 minutes to allow cell lysis. The RLU of the plates was read using a luminescent plate reader, and the subsequent data was analyzed using GraphPad Prism. The IC50 value was determined as the antibody concentration required to inhibit 50% of the maximum RLU of luciferase detected by incubation with TSLP alone. The results are summarized in Tables 6 and 7, Figure 2, and Figures 5A-5B.

[0162] Example 4: Inhibition of TSLP-induced release of TARC in PBMC cells or monocytes Inhibition of TARC secretion by peripheral blood mononuclear (PBMC) cells was used to evaluate the functional activity of antibodies and block TSLP-induced biological activity. Briefly, PBMC cells were seeded at 20,000 cells in 100 μL of DMEM + 10% FBS and cultured overnight at 37°C. The following day, the cell culture medium was discarded and the cells were gently washed with fresh medium. A 150 μL mixture (1:1:1 by volume) of TSLP, purified antibody, and hTNFa was added to the cells, resulting in final concentrations of 20 ng / mL TSLP, 0–100 nM purified antibody, and 200 ng / mL hTNFa or 1.5 ng / mL hIL-4, 0–100 nM purified antibody, and 50 ng / mL TNFa. The cells were incubated in this mixture at 37°C for 2 days. After incubation, the culture supernatant was collected, and the amount of TARC present was analyzed using a commercially available TARC ELISA kit according to the manufacturer's instructions. The determined TARC concentration in each well was analyzed using GraphPad Prism. The IC50 value was determined as the antibody concentration required to inhibit 50% of the maximum TARC concentration detected in incubation with 20 ng / mL TSLP and 200 ng / mL hTNFa alone. The results are summarized in Tables 6 and 8, and Figures 6A-6C.

[0163] A similar assay was performed using monocyte cells. Monocyte cells were isolated from recovered PBMCs (3 donors) using the EasySep® Human Monocyte Enrichment Kit. The isolated monocytes were resuspended in assay medium at 800,000 cells / ml and seeded at 150 μl / well into assay plates (120,000 cells / well). 50 μl / well of 4×TSLP (1 ng / ml) was added to each well in assay medium. 50 μl / well of serially diluted 4× antibody (1,000 nM diluted to 1 / 3, with 11 doses (one dose being a blank)) was also added to each well in assay medium and incubated at 37°C and 5% CO2. After 2 days of incubation, the assay plates were centrifuged and 170 μl of the supernatant was collected for TARC detection. The amount of TARC in the culture supernatant was determined by ELISA according to the manufacturer's instructions. The results are summarized in Table 8 and Figures 7A-7C. [Table 8]

[0164] Equal parts This disclosure can be embodied in other specific forms without departing from its intent or essential features. Accordingly, the embodiments described herein should be considered illustrative in all respects, and not limiting the disclosure described herein. The scope of this disclosure is therefore indicated by the appended claims rather than by the foregoing description, and all modifications that fall within the meaning and scope of the claims are intended to be encompassed therein.

Claims

1. a) A heavy chain variable region (VH) comprising (i) CDR1 having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12, (ii) CDR2 having the amino acid sequence shown in any one of SEQ ID NOs: 13 to 24, and (iii) CDR3 having the amino acid sequence shown in any one of SEQ ID NOs: 25 to 36; b) A light chain variable region (VL) comprising (i) CDR1 having the amino acid sequence shown in any one of SEQ ID NOs: 37 to 48, (ii) CDR2 having the amino acid sequence shown in any one of SEQ ID NOs: 49 to 56, and (iii) CDR3 having the amino acid sequence shown in any one of SEQ ID NOs: 57 to 60; and c) Fc domain containing at least one amino acid modification, TSLP or TSLPR-binding proteins containing these proteins.

2. The TSLP-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 1, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 13, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 25; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 37, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 49, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

57.

3. The TSLP-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 2, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 14, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 26; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 38, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 50, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

58.

4. The TSLP-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 3, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 15, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 27; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 39, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 51, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

59.

5. The TSLPR-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 4, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 16, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 28; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 40, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 52, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

60.

6. The TSLP-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 5, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 29; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 41, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 53, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

61.

7. The TSLP-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 6, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 30; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 42, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 54, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

62.

8. The TSLP-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 7, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 19, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 31; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 43, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 55, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

63.

9. The TSLPR-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 8, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 20, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 32; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 44, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 56, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

60.

10. The TSLP-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 9, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 21, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 33; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 45, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 53, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

57.

11. The TSLP-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 10, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 22, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 34; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 46, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 54, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

58.

12. The TSLP-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 11, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 23, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 35; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 47, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 55, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

59.

13. The TSLPR-binding protein according to claim 1, wherein the VH comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 12, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 24, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO: 36; and the VL comprises (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 48, (ii) CDR2 having the amino acid sequence shown in SEQ ID NO: 56, and (iii) CDR3 having the amino acid sequence shown in SEQ ID NO:

60.

14. The TSLP or TSLPR binding protein according to any one of claims 1 to 13, wherein the VH comprises a sequence having at least 80% sequence identity with any one amino acid sequence from sequence numbers 65 to 69, and the VL comprises a sequence having at least 80% sequence identity with any one amino acid sequence from sequence numbers 70 to 74.

15. The TSLP-binding protein according to claim 14, wherein VH comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 65, and VL comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

70.

16. The TSLP-binding protein according to claim 14, wherein VH comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 66, and VL comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

71.

17. The TSLP-binding protein according to claim 14, wherein VH comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 67, and VL comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

72.

18. The TSLP-binding protein according to claim 14, wherein VH comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 68, and VL comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

73.

19. The TSLPR-binding protein according to claim 14, wherein VH comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 69, and VL comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

74.

20. The TSLP or TSLPR-binding protein according to any one of claims 1 to 18, wherein the Fc is the immunoglobulin Fc domain of IgG1, IgG2, or IgG4.

21. The TSLP or TSLPR-binding protein according to claim 20, wherein the Fc is an IgG1 immunoglobulin domain, and the at least one modification comprises modified M252Y, S254T, and T256E (YTE).

22. The TSLP or TSLPR-binding protein according to claim 20, wherein Fc is an IgG1 immunoglobulin domain, and the at least one modification comprises L234A / L235A (LALA).

23. The TSLP or TSLPR-binding protein according to claim 20, wherein Fc is an IgG1 immunoglobulin domain, and the at least one modification comprises LALAGA and N434A.

24. The TSLP or TSLPR-binding protein according to claim 20, wherein the Fc is an IgG1 immunoglobulin domain, and the at least one modification comprises M252Y, S254T, and T256E (YTE), and / or M428L and N434S (LS).

25. The TSLP or TSLPR-binding protein according to claim 20, wherein the Fc is an IgG1 immunoglobulin domain, and the at least one modification comprises M252Y, S254T, and T256E (YTE), and L234A / L235A (LALA).

26. The TSLP or TSLPR-binding protein according to claim 20, wherein Fc is an IgG2 immunoglobulin domain.

27. The TSLP or TSLPR binding protein according to claim 20, wherein Fc is an IgG4 immunoglobulin domain.

28. a) A heavy chain variable region (VH) comprising (i) CDR1 having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12, (ii) CDR2 having the amino acid sequence shown in any one of SEQ ID NOs: 13 to 24, and (iii) CDR3 having the amino acid sequence shown in any one of SEQ ID NOs: 25 to 36; b) A light chain variable region (VL) comprising (i) CDR1 having the amino acid sequence shown in any one of SEQ ID NOs: 37 to 48, (ii) CDR2 having the amino acid sequence shown in any one of SEQ ID NOs: 49 to 56, and (iii) CDR3 having the amino acid sequence shown in any one of SEQ ID NOs: 57 to 60; and c) A modified Fc that extends the half-life of the TSLP or TSLPR binding protein compared to a TSLP or TSLPR binding protein that does not contain the modified Fc, TSLP or TSLPR-binding proteins, including those mentioned above.

29. A TSLP or TSLPR-binding protein, wherein the TSLP or TSLPR-binding protein specifically binds to an epitope of TSLP and comprises an Fc domain including amino acid modifications M252Y, S254T, and T256E (YTE), and / or M428L and N434S (LS).

30. A method for treating an inflammatory disorder or disease in a patient requiring treatment for such disorder or disease, the method comprising administering to the patient subcutaneously or intravenously an effective amount of a TSLP or TSLPR-binding protein according to any one of claims 1 to 29.

31. The method according to claim 30, wherein the inflammatory disorder or disease is atopic dermatitis.

32. The method according to claim 31, wherein the inflammatory disorder or disease is asthma.

33. The method according to any one of claims 30 to 32, wherein the administration of TSLP or TSLPR-binding protein is subcutaneous.

34. The method according to any one of claims 30 to 32, wherein the administration of the TSLP or TSLPR-binding protein is intravenous.

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