Thymic stromal lymphopoietin-binding protein Z variants and their medical uses

Abstract

The present disclosure relates to a class of artificial polypeptides having binding affinity for thymic stromal lymphopoietin (TSLP), the polypeptides having the sequence EAVX4ALX7EIWX 11 LPNLX 16 X 17 X 18 QX 20 X 21 AFIX 25 X 26 The present disclosure also relates to the use of such TSLP-binding polypeptides as therapeutic, prognostic and / or diagnostic agents.

Description

[Technical field]

[0001] The present disclosure relates to a class of artificial polypeptides that have binding affinity for Thymic Stromal Lymphopoietin (hereinafter referred to as TSLP) protein. The present disclosure also relates to the use of such TSLP-binding polypeptides as therapeutic, prognostic and / or diagnostic agents. [Background technology]

[0002] Thymic stromal lymphopoietin (TSLP) is an epithelial and mast cell-derived cytokine that is involved in the pathogenesis of several inflammatory diseases, including the onset and persistence of airway inflammation. TSLP is a key regulator of many downstream inflammatory pathways. TSLP is a four-helix bundle cytokine closely related to IL-7, a member of the hematopoietin family of cytokines. TSLP was first isolated from a mouse thymic stromal cell line and found to be a growth factor for B cells. TSLP exerts its biological activity by binding to its receptor (TSLP-R). TSLP-R is a heterodimeric receptor consisting of the IL-7 receptor α chain (IL-7Rα) and the TSLP receptor α chain 1 (TSLP-Rα (also known as CRL2, TSLP-R, and CRLF2Y)), which is closely associated with the common receptor-γ chain (γc) found in the IL-2, IL-4, IL-9, and IL-15 receptor complexes. Functional TSLP-R is primarily expressed in hematopoietic cells, dendritic cells (DCs), T cells, B cells, natural killer (NK) cells, invariant natural killer T cells (iNKT), monocytes, basophils, mast cells, eosinophils, liver, brain, skeletal muscle, kidney, spleen, and thymus (Markovic and Savvides, 2020, Front Immunol 11:1557).

[0003] TSLP exists in two isoforms in humans. The short form of TSLP lacks the signal peptide and the first two α-helices. It is constitutively expressed in all epithelial cell layers and the skin epidermis, where it mediates homeostatic functions. The short form of TSLP has not been shown to exert proinflammatory activity, but rather is implicated as an antimicrobial peptide (Tsilingiri et al., 2017, Cell Mol Gastroenterol Hepatol 3(2):174-182). The long isoform is expressed at low / undetectable levels at steady state and increases in expression in some tissues during inflammation (hereafter, the long isoform is referred to as “TSLP” and the short isoform is referred to as “short form of TSLP” or “sfTSLP”). In addition, post-translational modifications such as glycosylation and furin cleavage may alter the functional activity of TSLP. Therefore, it appears important that drugs targeting TSLP block both the mature and active cleaved TSLP metabolites (Varricchi et al., 2018, Front Immunol 9:1595).

[0004] TSLP is produced in response to proinflammatory stimuli, such as viral infection, mechanical stress, allergens, and pollutants, inducing the inflammatory cascade. TSLP has been shown to promote inflammatory responses primarily through its activity on innate lymphoid cells (mainly ILC2), dendritic cells, and mast cells. TSLP plays a key role in the induction and maintenance of allergic inflammatory Th2, ILC2, and mast cell responses by producing allergen-induced cytokines, chemokines, and costimulatory molecules that induce naive T cells into Th2 cells producing IL-4, IL-5, and IL-13, all of which are important mediators of allergic inflammation (Gauvreau et al., 2020, Expert Opin Ther Targets 24(8):777-792).

[0005] Human TSLP expression has been reported to be increased in the airways of asthmatic patients and correlates with disease severity. Chronic allergic asthma is often characterized by Th2-type inflammation, whereas inflammation in nonallergic asthma is predominantly neutrophilic with a mixed Th1 and Th17 cytokine response. Because TSLP sits at the top of the signaling cascade, inhibition of TSLP could facilitate the treatment of both allergic and nonallergic asthma. In addition to its fundamental role in asthma development, TSLP is also known to be involved in the pathogenesis of other allergic conditions (Gauvreau et al., supra). Increased levels of TSLP protein have been found in skin lesions of patients with atopic dermatitis. Aberrant expression of TSLP has been observed in allergic diseases of the gastrointestinal tract, including Crohn's disease, eosinophilic esophagitis, and ulcerative colitis, as well as in cancer. Additionally, higher TSLP expression has been observed in patients with chronic obstructive pulmonary disease (COPD) compared to healthy individuals (Cianferoni and Spergel, 2014, Expert Rev Clin Immunol 10(11):1463-74).

[0006] Published data provide clinical evidence that inhibition of TSLP with the anti-TSLP monoclonal antibody tezepelumab reduces the annual incidence of asthma exacerbations compared with placebo (Corren and Ziegler, 2019, Nat Immunol 20(12):1603-1609). This result was independent of baseline eosinophil counts or other Th2 biomarkers. Among patients treated with long-acting beta-agonists and medium- to high-dose inhaled glucocorticoids, patients receiving tezepelumab had a lower rate of clinically significant asthma exacerbations than patients receiving placebo.

[0007] These findings highlight the potential benefit of targeting upstream cytokines such as TSLP, which may affect disease activity more broadly compared to inhibition of a single downstream pathway. Furthermore, in a phase III setting, tezepelumab reduced exacerbations independent of baseline blood eosinophil counts and improved lung function, asthma control, and health-related quality of life in a broad population of patients with severe, uncontrolled asthma (Menzies-Gow et al., 2021, N Engl J Med 384(19):1800-1809). Tezepelumab was recently approved for the treatment of severe asthma and is marketed under the trade name TezeSpire®. Tezepelumab is currently being tested in clinical trials for the treatment of COPD, rhinosinusitis, eosinophilic esophagitis, and chronic urticaria.

[0008] The prevalence of allergic diseases, such as asthma, allergic rhinitis, atopic dermatitis, and food allergies, appears to have increased in recent years, especially in developed countries, leading to an increasing proportion of the patient population (Rutowski et al., 2014, Postepy Dermatol Alergol 31(2):77-83).

[0009] In many inflammatory diseases, the means of diagnosis and monitoring of treatment are clinical evaluation and analysis of common biomarkers. Reliance on traditional clinical evaluation in the diagnosis and monitoring of treatment of inflammatory diseases may lead to suboptimal patient outcomes. Reliable methods are needed to diagnose inflammatory pathologies, evaluate disease status, and monitor response to treatment. Furthermore, the rational design and application of new therapies for inflammatory diseases requires the discovery, validation, and implementation of informative indicators of biological processes or pharmacological responses to therapeutic interventions. This may be facilitated by the development of appropriate companion diagnostic tools to enable a personalized medicine approach, tailoring medical decisions, practices, interventions, and / or products to individual patients based on predicted response or disease risk. Studies have shown that upregulation of TSLP in bronchoalveolar lavage fluid (BALF) is closely correlated with disease severity in asthma. TSLP levels in BALF have been shown to be inversely correlated with pulmonary function, suggesting that it may serve as a useful diagnostic marker (Li et al., 2018, J Immunol 200(7):2253-2262).

[0010] Since tissue penetration rate is negatively related to the size of the molecule, relatively large antibody molecules inherently have poor tissue distribution and penetration ability, and may not be suitable for local pulmonary delivery to treat respiratory diseases such as asthma and COPD. Although antibodies are widely used in various routine applications such as analytical, purification, diagnostic and therapeutic purposes due to their high affinity and specificity for many antigens, they also have some drawbacks. Such drawbacks include aggregation tendency, limited stability and limited solubility, which make antibodies less suitable for alternative administration routes such as inhalation.

[0011] Therefore, the use of monoclonal antibodies is not always optimal for treatment, and there is a continuing need to provide agents with high affinity for TSLP. It is also of great interest to provide for the use of such molecules in the treatment, diagnosis, and prognosis of disease. Summary of the Invention

[0012] It is an object of the present disclosure to provide new TSLP-binding agents that can be used, for example, for therapeutic, prognostic and diagnostic applications.

[0013] It is an object of the present disclosure to provide molecules that allow for the efficient treatment of, for example, various forms of inflammatory diseases, while mitigating the above and other drawbacks of current therapies.

[0014] It is further an object of the present disclosure to provide molecules suitable for prognostic and diagnostic applications, for example, for various forms of inflammatory diseases.

[0015] These and other objects that will become apparent to one skilled in the art from this disclosure are met by the various aspects of the present invention as claimed in the appended claims and disclosed herein as a whole.

[0016] Thus, in a first aspect of the disclosure, there is provided a TSLP-binding polypeptide comprising the TSLP-binding motif BM, which motif is i) EAVX4ALX7EIWX 11 LPNLX 16 X 17 X 18 QX 20 X 21 AFIX 25 X 26 LRD (SEQ ID NO: 1081), Independently of each other, X4 is selected from D, E, and H; X7 is selected from I, L, M, and V; X 11 is selected from A, D, E, K, N, Q, R, S, and T; X 16 is selected from N and T; X 17 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 18is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 20 is selected from H, N, Q, T, W and Y; X 21 is selected from D, E, G, H, K, M, N, Q, and R; X 25 is selected from A, H, I, K, L, Q, R, V and Y; and X 26 is selected from K and S and ii) an amino acid sequence having at least 93% identity to the sequence defined in i); The amino acid sequence is selected from TSLP-binding polypeptides are provided.

[0017] In one embodiment, in sequence i), X4 is selected from D, E and H; X7 is selected from I, L, M, and V; X 11 is selected from A, D, E, K, N, Q, R, S, and T; X 16 is selected from N and T; X 17 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 18 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 20 is selected from H, N, Q, T, W and Y; X 21 is selected from D, E, G, H, K, M, N, Q, and R; X 25 is selected from A, H, I, K, L, Q, R, V, and Y; and X 26 is selected from K and S TSLP-binding polypeptides are provided.

[0018] In another embodiment, in sequence i), X4 is selected from E and H; X7 is selected from I, L, and V; X 11 is selected from A, D, E, Q, R, S, and T; X 16 is selected from N and T; X 17 is selected from A, D, E, G, H, K, N, Q, R, S, T, V, W, and Y; X 18 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 20 is selected from H, N, Q, W and Y; X 21 is selected from D, E, H, K, M, N, Q, and R; X 25 is selected from H, I, K, L, Q, R, V, and Y; and X 26 is selected from K and S TSLP-binding polypeptides are provided.

[0019] In yet another embodiment, in sequence i), X4 is selected from E and H; X7 is selected from I, L, and V; X 11 is selected from A, D, E, Q, S, and T; X 16 is T; X 17 is selected from D, E, G, H, N, Q, R, S, W, and Y; X 18 is selected from A, D, E, F, G, H, I, L, N, Q, R, S, T, W, and Y; X 20 is selected from H, W and Y; X 21 is selected from D, E, H, N, and Q; X 25 is selected from I, L, R, V, and Y; and X 26 is K TSLP-binding polypeptides are provided.

[0020] In yet another embodiment, in sequence i), X4 is E; X7 is selected from I and V; X 11 is selected from A, D, E, Q, S, and T; X 16 is T; X 17 is selected from D, E, G, H, N, Q, R, and Y; X 18 is selected from A, D, E, F, G, I, L, N, Q, R, S, T, and Y; X 20 is selected from H, W and Y; X 21 is selected from D, E, H, N, and Q; X 25 is selected from I, L, V, and Y; and X 26 is K TSLP-binding polypeptides are provided.

[0021] In yet another embodiment, in sequence i), X4 is E; X7 is V; X 11 is selected from A and T; X 16 is T; X 17 is R; X 18 is selected from D and E; X 20 is W; X 21 is Q; X 25 is Y; and X 26 is K There is provided a TSLP-binding polypeptide according to any of the preceding claims.

[0022] As used herein, "X n " and "X m " is used to denote amino acids at positions n and m in sequence i) as defined above, n and m being integers indicating the position of the amino acid in said sequence counting from the N-terminus of said sequence. For example, X4 and X7 denote the amino acids at positions 4 and 7, respectively, from the N-terminus of sequence i).

[0023] In an embodiment according to the first aspect, X in sequence i) n are independently selected from the group of possible residues according to Table 1. n may be selected from any one of the enumerated groups of possible residues, and that the selection is m It will be appreciated that the present invention is independent of the choice of amino acid at position X in Table 1 (n ≠ m). n Any of the possible residues listed in can bind independently to any of the possible residues listed in any other variable position in Table 1.

[0024] The skilled artisan will understand that Table 1 should be read as follows: In one embodiment according to the first aspect, the amino acid residue "X" of sequence i) n " is selected from the "possible residues". Accordingly, Table 1 discloses some specific and individualized embodiments of the first aspect of the present disclosure. For example, in one embodiment according to the first aspect, a polypeptide is provided in which X4 of sequence i) is selected from D, E and H, and in another embodiment according to the first aspect, a polypeptide is provided in which X4 of sequence i) is selected from D and E. For the avoidance of doubt, the recited embodiments can be freely combined in further embodiments. For example, one such combination embodiment is a polypeptide in which X4 is selected from E and H, while X7 is selected from I, L and V, and X 25 is a polypeptide selected from H, I, K, L, Q, R, V and Y, etc.

[0025] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0026] In more specific embodiments defining a subclass of TSLP-binding polypeptides, sequence i) satisfies at least four of the eight conditions I to VIII: I. X4 is E or H; II.X7 is selected from I, L and V; III.X 11 is selected from A, D, E, Q, S and T; IV.X 16 is T; VX 20 is selected from H, W and Y; VI.X 21 is selected from D, E, H, N and Q; VII.X 25 is selected from I, L, R, V and Y; and VIII.X 26 is K.

[0027] In some examples of TSLP-binding polypeptides according to the first aspect, sequence i) satisfies at least five of the eight conditions I-VIII. More specifically, sequence i) can satisfy at least six of the eight conditions I-VIII, such as at least seven of the eight conditions I-VIII, such as all eight conditions I-VIII.

[0028] In some embodiments of the TSLP-binding polypeptide according to the first aspect, X4 is E, X7 is V and X 20 In some embodiments, X4 is E, X7 is V, and X 20 In some embodiments, X4 is E, X7 is V, and X 21 In some embodiments, X4 is E, X7 is V, and X 21 In some embodiments, X4 is E, X7 is V, and X 25 In some embodiments, X4 is E, X7 is V, and X 25 is L.

[0029] As described in detail in the experimental section below, the selection of TSLP-binding polypeptide variants led to the identification of a number of individual TSLP-binding motif (BM) sequences belonging to the class defined in the first aspect of the present disclosure. These sequences constitute individual embodiments of sequence i) or ii) according to this aspect. The individual TSLP-binding motif sequences correspond to amino acid positions 8-36 of SEQ ID NOs: 1-875 as presented in the sequence listing. In one embodiment of the TSLP-binding polypeptide according to this first aspect, sequence i) corresponds to the sequence at positions 8-36 in a sequence selected from the group consisting of SEQ ID NOs: 1-645. In one embodiment, sequence i) corresponds to the sequence at positions 8-36 in a sequence selected from the group consisting of SEQ ID NOs: 1-630 and 645. In one embodiment, sequence i) corresponds to the sequence at positions 8-36 in a sequence selected from the group consisting of SEQ ID NOs: 1-91 and 645. In one embodiment, sequence i) corresponds to the sequence at positions 8-36 in a sequence selected from the group consisting of SEQ ID NOs: 1-27. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 1-2.

[0030] As those skilled in the art will recognize, the function of any polypeptide, such as the TSLP-binding ability of the polypeptide of the present disclosure, depends on the tertiary structure of the polypeptide.Therefore, it is possible to make minor changes to the amino acid sequence of the polypeptide without affecting its function.Therefore, the present disclosure encompasses modified variants of TSLP-binding polypeptides that retain TSLP-binding properties.

[0031] Thus, TSLP-binding polypeptides comprising an amino acid sequence having 93% or greater identity, such as 96% or greater identity, to a polypeptide defined in i) are encompassed by the present disclosure. For example, an amino acid residue belonging to a group (e.g., hydrophobic, hydrophilic, polar, etc.) defined by a particular functionality of the amino acid residue can be replaced with another amino acid residue from the group with the same functionality.

[0032] In some embodiments, such changes can be made at any position in the sequence of a TSLP-binding polypeptide disclosed herein. In other embodiments, such changes can be made only at non-variable positions, also referred to as scaffold amino acid residues. In such cases, changes are not permitted at variable positions. In other embodiments, such changes can only be at variable positions.

[0033] According to one definition of such "variable positions", they are the positions designated with "X" in sequence i) defined above.

[0034] According to another definition, a "variable position" is a position that is randomized in a selection library of Z variants prior to selection, and thus may be, for example, positions 2, 3, 4, 6, 7, 10, 11, 17, 18, 20, 21, 25 and 28 of sequence i). This definition of "variable position" does not include positions 16 and 26, which may be either one of the two options at each position, but are scaffold positions in this context. See Nord et al. (1995), Prot Eng 8:601-608, and Lofblom et al. (2010), FEBS Letters, 584:2670-2680. Although directed to other targets, the polypeptides disclosed in Nord et al. and Lofblom et al., like the TSLP-binding Z variants of the present disclosure, are also based on a scaffold derived from a Z derivative of domain B of protein A from Staphylococcus aureus. As shown in Nord et al. (see, e.g., FIG. 4), the amino acids at position 23 (corresponding to position 16 of the TSLP-binding motif of the invention) and position 33 (corresponding to position 26 of the TSLP-binding motif of the invention) are N and S, respectively. Also, as shown in Lofblom et al., polypeptides having amino acid residues N and S at positions 23 and 33, respectively (corresponding to positions 16 and 26 of the TSLP-binding motif of the invention; see FIG. 2 of Lofblom et al.), and polypeptides having amino acid residues T and K at positions 23 and 33, respectively, all have a maintained basic structure and function. Thus, in the context of this definition of "variable position," it is contemplated that the amino acid residues at positions 16 and 26 form part of a common scaffold, with either N or T at scaffold position 16 and either S or K at scaffold position 26.

[0035] The term "% identity" used throughout the specification can be calculated, for example, as follows: The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al., 1994, Nucleic Acids Research, 22:4673-4680). Comparison is performed over a window that corresponds to the shortest of the aligned sequences. In some cases, the shortest of the aligned sequences may be the target sequence. In other cases, the shortest of the aligned sequences may be the query sequence. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have the same correspondence in the target sequence is reported as the % identity.

[0036] In another embodiment, a TSLP-binding polypeptide is provided that comprises a binding motif sequence corresponding to a sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 875; or a sequence having 93% or more identity thereto, for example 96% identity thereto.

[0037] In some embodiments, the BM as defined above "forms part" of the three-helix bundle protein domain. This is understood to mean that the sequence of the BM is "inserted" or "grafted" into the sequence of the original three-helix bundle domain, such that the BM replaces a similar structural motif in the original domain. For example, without wishing to be bound by theory, the BM is believed to constitute two of the three helices of a three-helix bundle and can therefore replace such a two-helix motif in any three-helix bundle. As the skilled artisan will recognize, the replacement of two helices of a three-helix bundle domain with two BM helices must be done in such a way that it does not affect the basic structure of the polypeptide. That is, the overall folding of the Cα backbone of the polypeptide according to this embodiment of the invention is substantially the same as that of the three-helix bundle protein domain of which it forms a part, e.g., having the same elements of secondary structure in the same order. Thus, a BM according to the present disclosure "forms part of" a three-helix bundle domain if the polypeptide according to this embodiment has the same fold as the original domain, suggesting that basic structural properties are shared, which, for example, result in similar CD spectra. Those skilled in the art will recognize other parameters that are relevant.

[0038] In certain embodiments, the TSLP-binding motif (BM) thus forms part of a three-helix bundle protein domain. For example, the BM can essentially consist of two alpha helices with an interconnecting loop within said three-helix bundle protein domain. In certain embodiments, said three-helix bundle protein domain is selected from domains of bacterial receptor proteins. Non-limiting examples of such domains are the five different three-helix domains of protein A from Staphylococcus aureus, such as domain B and its derivatives. In some embodiments, the three-helix bundle protein domain is a variant of protein Z derived from domain B of Staphylococcus protein A (Wahlberg E et al. 2003, PNAS 100(6):3185-3190).

[0039] In some embodiments in which the TSLP-binding polypeptides disclosed herein form part of a three-helix bundle protein domain, the TSLP-binding polypeptide can comprise a binding module (BMod), the amino acid sequence of which is iii) K-[BM]-DPSQSX a X b LLX c EAKKLX d X e X f Q (SEQ ID NO: 1082), [BM] is a TSLP-binding motif as defined anywhere herein; X a is selected from A and S; X b is selected from E and N; X c is selected from A, S and C; X d is selected from E, N and S; X e is selected from D, E and S; X f is selected from A and S; Amino acid sequence and iv) an amino acid sequence having at least 91% identity to the sequence defined in iii); is selected from.

[0040] In some embodiments, the polypeptides can advantageously exhibit high structural stability, including resistance to chemical modification, changes in physical state, and proteolytic degradation during production and storage, as well as in vivo.

[0041] As mentioned above, polypeptides containing minor modifications compared to the above amino acid sequences that do not significantly affect the tertiary structure and function of the polypeptide are also within the scope of the present disclosure. Thus, in some embodiments, sequence iv) has at least 93% identity to the sequence defined in iii), such as at least 95%, such as at least 97%.

[0042] In one embodiment, the X a is A.

[0043] In one embodiment, the X a is S.

[0044] In one embodiment, the X b is N.

[0045] In one embodiment, the X b is E.

[0046] In one embodiment, the X c is A.

[0047] In one embodiment, the X c is S.

[0048] In one embodiment, the X c is C.

[0049] In one embodiment, the X d is E.

[0050] In one embodiment, the X d is N.

[0051] In one embodiment, the X d is S.

[0052] In one embodiment, the X e is D.

[0053] In one embodiment, the X e is E.

[0054] In one embodiment, the X e is S.

[0055] In one embodiment, the X d X e is selected from EE, ES, SD, SE and SS.

[0056] In one embodiment, the X d X e is ES.

[0057] In one embodiment, the X d X e is a SE.

[0058] In one embodiment, the X d X e is SD.

[0059] In one embodiment, the X f is A.

[0060] In one embodiment, the X f is S.

[0061] In one embodiment, in sequence iii), X a is A;Xb is N;X c is A and X f is A.

[0062] In one embodiment, in sequence iii), X a is S;X b is E;X c is A and X f is A.

[0063] In one embodiment, in sequence iii), X a is A;X b is N;X c is C and X f is A.

[0064] In one embodiment, in sequence iii), X a is S;X b is E;X c is S and X f is S.

[0065] In one embodiment, in sequence iii), X a is S;X b is E;X c is C and X f is S.

[0066] In one embodiment, in sequence iii), X a is A;X b is N;X c is A and X d X e is ND and X f is A.

[0067] In one embodiment, in sequence iii), X a is S;X b is E;X c is A and X d X e is ND and X f is A.

[0068] In one embodiment, in sequence iii), X a is A;X b is N;X c is C and X d X e is ND and X f is A.

[0069] In one embodiment, in sequence iii), X a is S;X b is E;X c is S and X d X e is ND and X f is S.

[0070] In one embodiment, in sequence iii), X a is S;X b is E;X c is C and X d X e is ND and X f is S.

[0071] In one embodiment, in sequence iii), X a is A;X b is N;X c is A and X d X e is SE, and X f is A.

[0072] In one embodiment, in sequence iii), X a is S;X b is E;X c is A and X d X e is SE, and X f is A.

[0073] In one embodiment, in sequence iii), X a is A;X b is N;X c is C and X d X eis SE, and X f is A.

[0074] In one embodiment, in sequence iii), X a is S;X b is E;X c is S and X d X e is SE, and X f is S.

[0075] In one embodiment, in sequence iii), X a is S;X b is E;X c is C and X d X e is SE, and X f is S.

[0076] In one embodiment, in sequence iii), X a is A;X b is N;X c is A and X d X e is SD and X f is A.

[0077] In one embodiment, in sequence iii), X a is S;X b is E;X c is A and X d X e is SD and X f is A.

[0078] In one embodiment, in sequence iii), X a is A;X b is N;X c is C and X d X e is SD and X f is A.

[0079] In one embodiment, in sequence iii), X a is S;X bis E;X c is S and X d X e is SD and X f is S.

[0080] In one embodiment, in sequence iii), X a is S;X b is E;X c is C and X d X e is SD and X f is S.

[0081] In a further embodiment of the first aspect of the present disclosure, sequence iii) corresponds to the amino acid sequence of positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 875. In one embodiment, sequence iii) is selected from the amino acid sequences corresponding to positions 7 to 55 in SEQ ID NOs: 1 to 645. In one embodiment, sequence iii) is selected from the amino acid sequences corresponding to positions 7 to 55 in SEQ ID NOs: 1 to 630 and 645. In another embodiment, sequence iii) is selected from the amino acid sequences corresponding to positions 7 to 55 in SEQ ID NOs: 1 to 91. In one embodiment, sequence iii) is selected from the amino acid sequences corresponding to positions 7 to 55 in SEQ ID NOs: 1 to 27. In yet another embodiment, sequence iii) is selected from the amino acid sequences corresponding to positions 7 to 55 in SEQ ID NOs: 1 to 2.

[0082] In yet a further embodiment, v) YA-[BMod]-AP (SEQ ID NO: 1083), an amino acid sequence in which [BMod] is a TSLP-binding module as defined above; and vi) an amino acid sequence having at least 86% identity to the sequence defined in v); The present invention provides a TSLP-binding polypeptide comprising an amino acid sequence selected from the group consisting of:

[0083] As mentioned above, polypeptides containing minor modifications compared to the above amino acid sequences that do not significantly affect the tertiary structure and function of the polypeptide are also within the scope of the present disclosure. Thus, in some embodiments, the TSLP-binding polypeptides defined above can have a sequence vi) that is at least 88% identical, such as at least 98%, such as at least 96%, such as at least 94%, such as at least 92%, such as at least 90%, to the sequence defined in v).

[0084] In some embodiments, the TSLP binding motif is ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK (SEQ ID NO: 1084); ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 1085); ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 1086); ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK (SEQ ID NO: 1087); AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK (SEQ ID NO: 1088); VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 1089); AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK (SEQ ID NO: 1090); VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK (SEQ ID NO: 1091); VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK (SEQ ID NO: 1092); AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1093); AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO: 1094); AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO: 1095); AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK (SEQ ID NO: 1096); VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1097); VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO: 1098); VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK (SEQ ID NO: 1099); VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK (SEQ ID NO: 1100); and AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK (SEQ ID NO: 1101) ([BM] is the IL-17A binding motif as defined above) The polypeptide may form part of a polypeptide comprising an amino acid sequence selected from:

[0085] In one embodiment, the TSLP-binding polypeptide is vii) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1097), [BM] is an amino acid sequence that is a TSLP-binding motif as defined above; and viii) an amino acid sequence having at least 86% identity to the sequence defined in vii); The amino acid sequence is selected from the group consisting of:

[0086] In a further embodiment, sequence vii) is selected from the group consisting of SEQ ID NOs: 1-636, 639-644 and 646-875, for example selected from the group consisting of SEQ ID NOs: 1-636 and 639-644.

[0087] In another further embodiment, the TSLP binding polypeptide is ix) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO: 1095), [BM] is an amino acid sequence that is a TSLP-binding motif as defined above; and x) an amino acid sequence having at least 86% identity to the sequence defined in ix); The amino acid sequence is selected from the group consisting of:

[0088] In a further embodiment, sequence ix) is selected from the group consisting of SEQ ID NO:637 and SEQ ID NO:638.

[0089] In another further embodiment, the TSLP binding polypeptide is xi) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1093), [BM] is an amino acid sequence that is a TSLP-binding motif as defined above; and xii) an amino acid sequence having at least 86% identity to the sequence defined in xi); The amino acid sequence is selected from the group consisting of:

[0090] In a further embodiment, sequence xi) is selected from the group consisting of SEQ ID NOs: 645 and 876-972.

[0091] In one embodiment, the TSLP-binding polypeptide of the first aspect has a K D The value is at most 1×10 -6 M, for example, at most 5 × 10 -7 M, for example, at most 1 × 10 -7 M, for example, at most 5 × 10 -8 M, for example, at most 1 × 10 -8 M binds to TSLP.

[0092] As used herein, the terms "TSLP binding" and "binding affinity for TSLP" refer to a property of a polypeptide that can be tested, for example, by ELISA, by surface plasmon resonance (SPR) technology, by quartz crystal microbalance (QCM) technology, and / or by Kinetic Exclusion Assay (KinExA®).

[0093] For example, as described in the Experimental Section below, TSLP binding affinity can be tested in an experiment in which a sample of a polypeptide is captured on an antibody-coated ELISA plate, biotinylated TSLP is added, followed by streptavidin-conjugated HRP. TMB substrate is added, and Victor 3 (PerkinElmer) or other multi-well plate reader is used to measure absorbance at 450 nm. Those skilled in the art can then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide to TSLP. If a quantitative measure is desired, ELISA can also be used, for example, to determine the EC50 value (half the maximum effective concentration) of the interaction. The response of the polypeptide to a dilution series of biotinylated TSLP is measured using ELISA as described above. Those skilled in the art can then interpret the results obtained by such experiments and calculate the EC50 value therefrom, for example, using GraphPad Prism 5 and nonlinear regression.

[0094] TSLP binding affinity can also be tested in experiments in which TSLP or a fragment thereof is immobilized on the sensor chip of a surface plasmon resonance (SPR) instrument and a sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on the sensor chip of the instrument and a sample containing TSLP or a fragment thereof is passed over the chip. One skilled in the art can then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide for TSLP. If a quantitative measure is desired, e.g., the K of the interaction can be determined. D Surface plasmon resonance can also be used to determine the K value. The binding value can be defined, for example, with a Biacore (Cytiva) or ProteOn XPR36 (Bio-Rad) instrument. TSLP is suitably immobilized on the sensor chip of the instrument, and samples of the polypeptide whose affinity is to be measured are prepared in serial dilutions and injected in random order. The K value is then calculated. DValues ​​can be calculated from the results using, for example, a 1:1 Langmuir binding model in the BIAevaluation4.1 software or other suitable software provided by the instrument manufacturer.

[0095] TSLP binding affinity can also be tested using a continuous flow system for automated analysis based on quartz crystal microbalance (QCM) technology. To monitor the binding interaction, one of the interacting molecules, or a fragment thereof, is immobilized on a sensor surface and a sample containing the other is injected over the sensor surface. The binding data is measured in real time. The signal output is given in frequency (Hz) and is directly related to the change in mass on the sensor surface. Quantitative measurements can be made, e.g., to determine the K of the interaction. D If desired, QCM methods can be used to determine the binding value. The binding value can be defined, for example, with an Attana A200® (Attana) instrument. For example, TSLP can be immobilized on the sensor chip of the instrument, and a sample of the polypeptide whose affinity is to be measured is prepared by serial dilution and injected. Data is collected with the Attester software, and the results are then used to determine the K D The values ​​can be calculated and subsequently processed in the evaluation software or other suitable software provided by the device manufacturer.

[0096] Another method for determining binding affinity to TSLP is the Kinetic Exclusion Assay (KinExA®; Sapidyne Instruments Inc; Darling and Brault, 2004, Assay and Drug Dev Tech 2(6):647-657) to measure equilibrium binding affinity and kinetics between unmodified molecules in solution. The affinity analysis involves the equilibrium dissociation constant, K D and the rate of binding, k a is experimentally measured, while the rate of dissociation, k d is the formula k d =K D *ka It can be calculated based on:

[0097] KinExA(R)K D The analysis requires the immobilization of one interaction partner (e.g., a titrated binding partner) on a solid phase, which is then used as a probe to capture the other interaction partner (e.g., a constant binding partner) that becomes free in solution when equilibrium is reached. In each experiment, a series of solutions is equilibrated with a constant concentration of one binding partner and a titration of the other binding partner. The solution is then briefly exposed to the solid phase, capturing a portion of the free constant binding partner and labeling it with a fluorescent secondary molecule. The short contact time with the solid phase is shorter than the time required for dissociation of the complex preformed in solution, meaning that competition between the solution and the titrated binding partner on the solid phase is "kinetically excluded". The solution equilibrium does not change during the measurement, since the solid phase is only used as a probe for the free constant binding partner in each sample. K D The value is calculated from the signal generated from the captured free constant binding partner, which is directly proportional to the concentration of free constant binding partner in the equilibrated sample. Data were analyzed using KinExA® Pro software and least squares analysis to obtain K D and the Active Binding site Concentration (ABC) optimum solution can be fitted to a stoichiometric related model, for example a curve representing a 1:1 reversible bimolecular interaction.

[0098] Binding kinetics determinations can be performed in a similar format to equilibrium analysis, except that measurements are taken "pre-equilibrium" and the binding signal is a function of time and total concentration of titrated binding partner. aThere are two methods that can be used to determine . The "direct method" holds the concentrations of the titrating and constant binding partners constant and probes the solution over time. The amount of free constant binding partner in solution will decrease as the sample approaches equilibrium. The "inject method" holds the incubation time constant, holds the concentration of one partner constant, and titrates the concentration of the other partner. As the concentration of the titrating binding partner increases, more complex is formed and so the amount of free constant binding partner will decrease.

[0099] As used in this disclosure, the terms "albumin binding" and "binding affinity for albumin" refer to a property of a polypeptide that can be tested in a manner similar to the examples described above for TSLP, for example, by SPR, by QCM, or by KinExA®.

[0100] Those of skill in the art will appreciate that various modifications and / or additions can be made to the TSLP-binding polypeptides in accordance with any of the embodiments disclosed herein to tailor the polypeptide for a particular use without departing from the scope of the disclosure.

[0101] For example, in one embodiment, a TSLP-binding polypeptide as described herein is provided, which is extended by and / or comprises additional amino acids at the C-terminus and / or N-terminus. Such a polypeptide should be understood as a polypeptide having one or more additional amino acid residues at the very beginning and / or very end of the polypeptide chain. Thus, a TSLP-binding polypeptide can include any suitable number of additional amino acid residues, e.g., at least one additional amino acid residue. Each additional amino acid residue can be added individually or collectively, e.g., to improve and / or simplify the production, purification, in vivo or in vitro stabilization, coupling, or detection of the polypeptide. Such additional amino acid residues can include one or more amino acid residues added for chemical coupling purposes. One example is the addition of a cysteine ​​residue. The additional amino acid residues may also provide a "tag" for purification or detection of the polypeptide, such as a His6 tag, a (HisGlu)3 tag ("HEHEHE" tag) or a "myc" (c-myc) tag or a "FLAG" tag, for interaction with a tag-specific antibody, or, in the case of a His6 tag, for interaction with immobilized metal affinity chromatography (IMAC).

[0102] In one embodiment, a TSLP-binding polypeptide as described herein is provided that comprises additional amino acids at the C-terminus and / or N-terminus. For example, in one embodiment of a TSLP-binding polypeptide disclosed herein, it consists of any one of the sequences disclosed herein with 0-15 additional C-terminus and / or N-terminus residues, such as 0-7 additional C-terminus and / or N-terminus residues. In one embodiment, the TSLP-binding polypeptide consists of any one of the sequences disclosed herein with 0-15 additional C-terminus residues, such as 0-4, such as 3.

[0103] The above-mentioned additional amino acids can be attached to the TSLP-binding polypeptide by chemical conjugation (using known organic chemistry methods) or by any other means, such as expression of the TSLP-binding polypeptide as a fusion protein, or linked in any other manner, either directly or via a linker, e.g., an amino acid linker.

[0104] Further polypeptide domains can further provide another TSLP-binding moiety. Thus, in a further embodiment, a multimeric form of TSLP-binding polypeptide is provided. It is understood that said multimer comprises at least two TSLP-binding polypeptides disclosed herein as monomer units, the amino acid sequences of which may be the same or different. The multimeric form of the polypeptide can comprise a suitable number of domains, each having a TSLP-binding motif, each forming a monomer within the multimer. These domains can have the same amino acid sequence, or they can have different amino acid sequences. In other words, the TSLP-binding polypeptide of the present invention can form homo- or heteromultimers, e.g. homo- or heterodimers. In one embodiment, a TSLP-binding polypeptide is provided in which the monomer units are covalently linked to each other. In another embodiment, the TSLP-binding polypeptide monomer units are expressed as a fusion protein. In one embodiment, a dimeric form of the TSLP-binding polypeptide is provided. In one particular embodiment, the dimeric form is a homodimeric form. In another embodiment, the dimeric form is a heterodimeric form.

[0105] For clarity, throughout this disclosure, the term "TSLP-binding polypeptide" is used to encompass TSLP-binding polypeptides in all forms, i.e., monomeric and multimeric forms.

[0106] The additional amino acids can include, for example, one or more additional polypeptide domain(s) that can provide the TSLP-binding polypeptide with additional functions, such as, for example, additional binding functions, or enzymatic functions, or toxic functions, or fluorescent signaling functions, or combinations thereof.

[0107] Furthermore, it may be advantageous for the TSLP-binding polypeptides defined herein to be part of a fusion protein or conjugate that includes a second or further moiety. The second or further moiety / moieties of such protein fusion polypeptides or conjugates may suitably possess a desired biological activity.

[0108] Thus, in a second aspect of the present disclosure, - a first part consisting of a TSLP-binding polypeptide according to the first aspect; and - a second portion comprising a polypeptide having a desired biological activity; A fusion protein or conjugate comprising:

[0109] In one embodiment, the fusion protein or conjugate may further comprise an additional moiety that comprises a desired biological activity that may be either the same or different from the biological activity of the second moiety.

[0110] Non-limiting examples of desired biological activity include therapeutic activity, binding activity, and enzymatic activity. In one embodiment, the second moiety having the desired biological activity is a therapeutically active polypeptide. In one embodiment, the second moiety is an immune response modifier. In another embodiment, the second moiety is an anti-cancer drug.

[0111] In one embodiment of the first or second aspect of the disclosure, there is provided a TSLP-binding polypeptide, fusion protein, or conjugate comprising an immune response modifier, non-limiting examples of which include immunomodulatory agents or other anti-inflammatory agents.

[0112] Non-limiting examples of therapeutically active polypeptides are biomolecules such as molecules selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.

[0113] Non-limiting examples of binding activity are those that increase the in vivo half-life of the fusion protein or conjugate, and those that act to, for example, block, inhibit, activate, increase, antagonize, or agonize a biological activity. One example of such a binding activity is a binding activity that increases the in vivo half-life of the fusion protein or conjugate. In one embodiment of the fusion protein or conjugate, the in vivo half-life of the fusion protein or conjugate is longer than the in vivo half-life of the TSLP-binding polypeptide itself. In one embodiment, the in vivo half-life is increased by at least 10-fold, such as at least 25-fold, such as at least 50-fold, such as at least 75-fold, such as at least 100-fold, compared to the in vivo half-life of the TSLP-binding polypeptide itself.

[0114] As mentioned above, the fusion protein or conjugate may comprise at least one further moiety with binding activity to a target. In one particular embodiment, the target is albumin, the binding to which increases the in vivo half-life of the fusion protein or conjugate. In one embodiment, the albumin binding activity is provided by the albumin binding domain (ABD) of Streptococcus protein G or a derivative thereof. Thus, the fusion protein may comprise, for example, a TSLP-binding polypeptide in monomeric or multimeric form (e.g., homodimeric or heterodimeric form) as defined herein, and the albumin binding domain of Streptococcus protein G or a derivative thereof. Derivatives of the albumin binding domain of Streptococcus protein G are known to the skilled person, for example from WO 2009 / 016043, WO 2012 / 004384 and WO 2014 / 048977, all of which are incorporated herein by reference. It is understood that the albumin binding domain (ABD) can be located at the C-terminus of the TSLP binding polypeptide and / or at the N-terminus of the TSLP binding polypeptide. Advantageously, having an ABD at the N-terminus of the TSLP binding polypeptide reduces or prevents oligomerization.

[0115] In another embodiment, a fusion protein or conjugate is provided in which the second portion having the desired binding activity is a protein based on protein Z derived from the B domain of protein A from Staphylococcus aureus, which has binding affinity for a target other than TSLP.

[0116] In another embodiment, a fusion protein or conjugate is provided in which the second moiety having the desired binding activity is an antibody or an antigen-binding fragment thereof. As is well known, an antibody is an immunoglobulin molecule capable of specifically binding to a target (antigen), such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site of the immunoglobulin molecule. As used herein, the term "antibody or antigen-binding fragment thereof" encompasses full-length or intact polyclonal or monoclonal antibodies, as well as antigen-binding fragments of such antibodies, such as Fab, Fab', F(ab')2, Fab3, Fv, variants thereof, fusion proteins comprising one or more antibody moieties, humanized antibodies, chimeric antibodies, minibodies, diabodies, tribodies, tetrabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g. bispecific antibodies), and any other modified form of immunoglobulin molecule comprising an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Further examples of modified antibodies and antigen-binding fragments thereof include nanobodies, AlbudAbs, DART (dual affinity re-targeting), BiTE (bispecific T cell induction), TandAbs (tandem diabodies), DAF (dual acting Fab), 2-in-1 (two-in-one) antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulins), CovX-bodies (peptide modified antibodies), duobodies and triomAbs. This list of antibody variants and antigen-binding fragments thereof should not be considered as limiting and the skilled person will know other suitable variants.

[0117] In one embodiment, the at least one antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain Fab (scFab) fragment, an Fc fragment, an Fv fragment, a single-chain Fv (scFv) fragment, an (scFv)2, an scFv-Fc construct, and a domain antibody. In one embodiment, the at least one antibody or antigen-binding fragment thereof is selected from a full-length antibody, a Fab fragment, and an scFv fragment. In one particular embodiment, the at least one antibody or antigen-binding fragment thereof is a full-length antibody.

[0118] In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, and an antigen-binding fragment thereof.

[0119] Those skilled in the art recognize that construction of fusion proteins often involves the use of linkers between the functional moieties to be fused, and there are various types of linkers with different properties, such as flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers. Linkers have been used, for example, to increase the stability or improve folding of the fusion protein, increase expression, improve biological activity, enable targeting, and alter the pharmacokinetics of the fusion protein. Thus, in one embodiment, the polypeptide according to any aspect disclosed herein further comprises at least one linker, such as at least one linker selected from flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers. In one embodiment, the linker is disposed between the TSLP-binding polypeptide and a further polypeptide domain, such as between a TSLP-binding domain disclosed herein and its antibody or antigen-binding fragment thereof. Flexible linkers are often used in the art when the domains being linked require some degree of movement or interaction, and can be particularly useful in some embodiments. Such linkers are generally composed of small, non-polar (e.g., G) or polar (e.g., S or T) amino acids. Some flexible linkers consist primarily of a series of G and S residues, e.g. (GGGGS) pThe linker can be optimized by adjusting the copy number "p" to achieve appropriate separation between functional moieties or to maintain necessary moiety interactions. Apart from G and S linkers, other flexible linkers are known in the art, such as G and S linkers containing additional amino acid residues such as T and A to maintain flexibility, as well as polar amino acid residues to improve solubility. Further non-limiting examples of linkers include ASGS (SEQ ID NO: 1102), GAPGGGGS (SEQ ID NO: 1103), GGGGSLVPRGSGGGGS (SEQ ID NO: 1104), (GS)3 (SEQ ID NO: 1105), (GS)4 (SEQ ID NO: 1106), (GS)8 (SEQ ID NO: 1107), GGSGGHGMSGG (SEQ ID NO: 1108), GGSGGSGGSGG (SEQ ID NO: 1109), GGSGG (SEQ ID NO: 1110), GGSGGG These include GG (SEQ ID NO:1111), GGGSEGGGSEGGGSEGGG (SEQ ID NO:1112), AAGAATAA (SEQ ID NO:1113), GGGGG (SEQ ID NO:1114), GGSSG (SEQ ID NO:1115), GSGGGTGGGSG (SEQ ID NO:1116), GSGSGSGSGGSG (SEQ ID NO:1117), GSGGSGGSGGSGGS (SEQ ID NO:1118) and GSGGSGSGGSGGSG (SEQ ID NO:1119), as well as GT. Other suitable linkers are known to those skilled in the art.

[0120] In one embodiment, the linker is a flexible linker comprising glycine (G), serine (S) and / or threonine (T) residues. n S m ) p and (S n G m ) p(independently, n=1-7, m=0-7, n+m≦8, p=1-7). In one embodiment, n=1-5. In one embodiment, m=0-5. In one embodiment, p=1-5. In a more specific embodiment, n=4, m=1 and p=1-4. In one embodiment, the linker is selected from the group consisting of S4G (SEQ ID NO: 1120), (S4G)3 (SEQ ID NO: 1121) and (S4G)4 (SEQ ID NO: 1122). In one embodiment, the linker is selected from the group consisting of G4S (SEQ ID NO: 1123) and (G4S)3 (SEQ ID NO: 1124). In one particular embodiment, the linker is G4S, and in another embodiment, the linker is (G4S)3.

[0121] With regard to the above description of fusion proteins or conjugates incorporating TSLP-binding polypeptides according to the present disclosure, it should be noted that the designations of first, second and further moieties are made for reasons of clarity, in order to distinguish between the TSLP-binding polypeptide or polypeptides according to the present invention, on the one hand, and moieties exhibiting the same or other functions, on the other hand. These designations are not intended to refer to the actual order of the different domains in the polypeptide chain of the fusion protein or conjugate. Similarly, the designations of first and second monomeric units are made for the sake of clarity, in order to distinguish said units. Thus, for example, said first moiety (or monomeric unit) can appear at the N-terminus, middle or C-terminus of the fusion protein or conjugate, without being limited thereto.

[0122] The above aspects further include polypeptides in which the TSLP-binding polypeptide according to the first aspect or the TSLP-binding polypeptide contained in the fusion protein or conjugate according to the second aspect further comprises a label, such as a label selected from the group consisting of fluorescent dyes and metals, chromogenic dyes, chemiluminescent compounds, bioluminescent proteins, enzymes, radionuclides and radioactive particles. Such labels, which will be familiar to those skilled in the art, can be used, for example, for detection of the polypeptide.

[0123] In some embodiments, the labeled TSLP binding polypeptide is present as part of a fusion protein or conjugate that also includes a second or further moiety having the desired biological activity. The label can be attached only to the TSLP binding polypeptide in some cases and to both the TSLP binding polypeptide and the second moiety of the fusion protein or conjugate in other cases. It is further possible that the label can be attached to the second moiety and not to the TSLP binding moiety. Thus, in yet another embodiment, a TSLP binding polypeptide comprising a second moiety is provided, in which the label is attached only to the second moiety. Thus, when referring to a labeled polypeptide, it should be understood as a reference to all aspects of the polypeptides described herein, including TSLP binding polypeptides, fusion proteins and conjugates comprising the TSLP binding polypeptide.

[0124] Further aspects of the disclosure provide a polynucleotide encoding a TSLP-binding polypeptide, fusion protein, or conjugate described herein; an expression vector comprising said polynucleotide; and a host cell comprising said expression vector.

[0125] Also encompassed by the disclosure is a method of producing the above-described TSLP-binding polypeptide or fusion protein, comprising culturing the host cell under conditions permissive for expression of the polypeptide from its expression vector, and isolating the polypeptide.

[0126] Alternatively, the TSLP-binding polypeptides or fusion proteins of the disclosure can be made by non-biological peptide synthesis using amino acids and / or amino acid derivatives with protected reactive side chains, which non-biological peptide synthesis can include: - stepwise coupling of amino acids and / or amino acid derivatives to form a polypeptide or fusion protein as described herein having protected reactive side chains - removal of protecting groups from reactive side chains of the polypeptide or fusion protein, and - Folding of polypeptides in aqueous solution Includes.

[0127] The conjugates disclosed herein can be produced by conjugation of at least one of the TSLP-binding polypeptides or fusion proteins described herein to at least one additional moiety. Those skilled in the art are aware of conjugation methods, such as conventional chemical conjugation methods using, for example, charged succinimidyl esters or carbodiimides.

[0128] It should be understood that the TSLP-binding polypeptides according to the present disclosure are typically useful as therapeutic, diagnostic and / or prognostic agents themselves. A therapeutic effect can be achieved, for example, by antagonizing the action of TSLP.

[0129] Thus, in another aspect, there is provided a composition comprising a TSLP-binding polypeptide, fusion protein or conjugate described herein and at least one pharma- ceutically acceptable excipient or carrier, hi one embodiment, the composition further comprises at least one additional active agent, such as at least two additional active agents, such as at least three additional active agents.

[0130] The small size and robustness of the TSLP-binding polypeptides of the present disclosure provide several advantages over conventional monoclonal antibody-based therapies. Such advantages include formulation advantages, routes of administration, such as alternative routes of administration, administration at higher molar doses than antibodies, and the absence of Fc-mediated side effects. The short plasma half-life of the polypeptides described herein may be advantageous over monoclonal antibodies with their long residence times, particularly when treating acute inflammatory conditions. The agents of the present disclosure are contemplated for oral, respiratory (including inhalation and insufflation), topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration, such as, in particular, respiratory, intravenous, or subcutaneous administration.

[0131] In another aspect of the disclosure, there is provided a TSLP-binding polypeptide, fusion protein, conjugate or composition as described herein for use as a pharmaceutical, prognostic and / or diagnostic agent. In one embodiment, there is provided a TSLP-binding polypeptide, fusion protein, conjugate or composition for use in treating, diagnosing or prognosing a TSLP-associated disorder or disease, such as an inflammatory disease, an autoimmune disease or a cancer disease.

[0132] In one embodiment, said use in diagnosis is carried out in vivo. In another embodiment, said use in diagnosis is carried out in vitro. In one embodiment, said use in prognosis is carried out in vivo. In another embodiment, said use in prognosis is carried out in vitro.

[0133] In one embodiment, the TSLP-binding polypeptide, fusion protein, conjugate or composition is provided for use as a pharmaceutical. In a more specific embodiment, the TSLP-binding polypeptide, fusion protein, conjugate or composition described herein is provided for use as a pharmaceutical to modulate TSLP function, such as to modulate TSLP function in vivo. As used herein, the term "modulate" refers to altering activity, such as partially inhibiting or completely inhibiting TSLP function.

[0134] In one embodiment, a TSLP-binding polypeptide, fusion protein, conjugate or composition for use in treating a TSLP-associated disorder is provided.

[0135] In one embodiment, a TSLP-binding polypeptide, fusion protein, conjugate or composition is provided for use in the diagnosis of a TSLP-associated disorder. In a more specific embodiment, said use in diagnosis is performed in vivo. In another specific embodiment, said use in diagnosis is performed in vitro.

[0136] In one embodiment, a TSLP-binding polypeptide, fusion protein, conjugate or composition is provided for use in predicting the prognosis of a TSLP-associated disorder. In a more specific embodiment, said use in predicting the prognosis is performed in vivo. In another specific embodiment, said use in predicting the prognosis is performed in vitro.

[0137] As used herein, the term "TSLP-associated disorder or disease" refers to any disorder, disease, or condition in which TSLP action is implicated and / or in which modulation (e.g., inhibition) of TSLP may be beneficial. Such a disorder, disease, or condition may be selected from the group consisting of respiratory disorders, skin disorders, allergies, eye disorders, gastrointestinal disorders, and cancer.

[0138] Non-limiting examples include asthma, atopic dermatitis, atopic keratoconjunctivitis, urticaria, allergic rhinitis, chronic rhinosinusitis with nasal polyposis, eosinophilic esophagitis, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis (EGPA) / Churg-Strauss syndrome, breast cancer, prurigo nodularis, and bullous pemphigoid.

[0139] It should be understood that the TSLP-binding polypeptides, fusion proteins, conjugates or compositions can be used as the sole diagnostic or prognostic agent or as a companion diagnostic and / or prognostic agent.

[0140] In related embodiments, methods of treating a TSLP-associated disorder are provided, comprising administering to a subject in need thereof an effective amount of a TSLP-binding polypeptide, fusion protein, conjugate, or composition described herein. In more specific embodiments of the methods, the TSLP-binding polypeptide, fusion protein, conjugate, or composition described herein modulates TSLP function in vivo. Those skilled in the art will appreciate that any description of the use of a TSLP-binding polypeptide, fusion protein, conjugate, or composition described herein for the treatment of a disease or disorder is equally relevant to the associated method of treatment. For the sake of brevity, such description will not be repeated here.

[0141] In another aspect of the disclosure, an in vitro method for detecting TSLP is provided, comprising providing a sample suspected of containing TSLP, contacting the sample with a TSLP-binding polypeptide, fusion protein, conjugate, or composition described herein, and detecting binding of the TSLP-binding polypeptide, fusion protein, conjugate, or composition, indicating the presence of TSLP in the sample.

[0142] In one embodiment, the method further comprises an intermediate washing step to remove unbound polypeptide, fusion protein, conjugate or composition after contacting with the sample.

[0143] In another aspect of the disclosure, there is provided a diagnostic or prognostic method for determining the presence or absence of TSLP in a subject, comprising the steps of: a) contacting a subject, or a sample isolated from a subject, with a TSLP-binding polypeptide, fusion protein, conjugate, or composition described herein; and b) obtaining a value corresponding to the amount of TSLP-binding polypeptide, fusion protein, conjugate or composition in said subject or bound to said sample. A method is provided that includes:

[0144] In one embodiment, the method further comprises an intermediate washing step to remove unbound polypeptide, fusion protein, conjugate or composition after contacting with the subject or sample and prior to obtaining the value.

[0145] In one embodiment, the method further comprises comparing the value to a standard, which may be a numerical value, a threshold value, or a visual indicator, for example based on a color response. A person skilled in the art will appreciate that various ways of comparing to a standard are known in the art and may be suitable for use.

[0146] In one embodiment of such a method, the subject is a mammalian subject, such as a human subject. In one embodiment, the method is performed in vivo. In another embodiment, the method is performed in vitro.

[0147] In certain embodiments of the various therapeutic, diagnostic and prognostic medical uses and methods disclosed herein, the TSLP-related disorder is selected from the group consisting of inflammatory diseases, autoimmune diseases and cancer diseases. Thus, the TSLP-related disorder may be selected from respiratory diseases, skin diseases, allergies, eye diseases, gastrointestinal diseases and cancer.

[0148] Examples of specific indications within these categories, which are also embodiments of TSLP-related disorders in the context of the present disclosure for which polypeptides with affinity for TSLP are believed to be of therapeutic, diagnostic or prognostic relevance, are asthma, atopic dermatitis, atopic keratoconjunctivitis, urticaria, allergic rhinitis, chronic rhinosinusitis with nasal polyposis, eosinophilic esophagitis, chronic obstructive pulmonary disease (COPD), eosinophilic granulomatosis with polyangiitis (EGPA) / Churg-Strauss syndrome, breast cancer, prurigo nodularis, and bullous pemphigoid.

[0149] In another embodiment of any one of the foregoing therapeutic aspects of the disclosure, the TSLP-related disorder is selected from the group consisting of cryoglobulinemia, pulmonary fibrosis, idiopathic pulmonary fibrosis, Netherton syndrome, atherosclerosis, systemic sclerosis, lichen planus, pancreatic cancer, Behcet's disease, eczema herpetiformis, eosinophilic gastroenteritis, multiple sclerosis, Kawasaki disease-induced thrombosis, IgG4-related disease, and endometriosis.

[0150] More information about TSLP and its role as a target for therapeutic intervention or as a diagnostic and / or prognostic biomarker can be found in the reviews by Tsilingiri et al., Varricchi et al., Gauvreau et al., and Markovic and Savvides, all of which are mentioned above in the Background section.

[0151] Although the present invention has been described with reference to various exemplary aspects and embodiments, those skilled in the art will recognize that various modifications can be made and equivalents can be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope of the invention. Therefore, it is intended that the present invention not be limited to any of the specific embodiments contemplated, but rather to include all embodiments that fall within the scope of the appended claims. [Brief description of the drawings]

[0152] [Figure 1A] Figure 1 shows examples of circular dichroism (CD) spectra collected as described in Example 3 for N- or C-terminally His-tagged Z variants of two different scaffolds: (A) ZTP631 (SEQ ID NO: 631) and (C) ZTP637 (SEQ ID NO: 637) melting curves; (B) ZTP631 and (D) ZTP637 CD spectra collected before (black line) and after (gray line) thermal denaturation. [Figure 1B]CD spectra collected before (black line) and after (gray line) thermal denaturation of (B) ZTP631 and (D) ZTP637. [Figure 1C] (A) ZTP631 (sequence number 631) and (C) ZTP637 (sequence number 637) melting curves. [Figure 1D] CD spectra collected before (black line) and after (gray line) thermal denaturation of (B) ZTP631 and (D) ZTP637. [Diagram 2] Figure 2 shows an example of a sensorgram obtained for His6-tagged Z variant ZTP631 (SEQ ID NO: 631) when analyzed for interaction with immobilized human TSLP measured by Biacore as described in Example 3. ZTP631 was injected at concentrations of 11 nM (solid line), 33 nM (dashed line), and 100 nM (dotted line). [Diagram 3] FIG. 3 shows concentration-dependent inhibition of TARC production by TSLP in human PBMCs by the indicated Z variants, as described in Example 4. [Figure 4A] Figure 4 shows examples of circular dichroism (CD) spectra collected as described in Example 7 for mature C-terminal His-tagged Z variants of two different scaffolds: (A) ZTP876 (SEQ ID NO: 876) and (C) ZTP967 (SEQ ID NO: 967) melting curves; CD spectra collected before (black line) and after (gray line) thermal denaturation of (B) ZTP876 and (D) ZTP967. [Figure 4B] CD spectra collected before (black line) and after (gray line) thermal denaturation of (B) ZTP876 and (D) ZTP967. [Figure 4C] (A) ZTP876 (sequence number 876) and (C) ZTP967 (sequence number 967) melting curves. [Figure 4D] CD spectra collected before (black line) and after (gray line) thermal denaturation of (B) ZTP876 and (D) ZTP967. [Diagram 5]Figure 5 shows three example sensorgrams obtained for mature Z-ABD variants when analyzed for interaction with immobilized human TSLP measured by Biacore as described in Example 7. ZTPA028 (SEQ ID NO:1000) (solid line), ZTPA029 (SEQ ID NO:1001) (dashed line), and ZTPA030 (SEQ ID NO:1002) (dotted line) were injected at consecutive concentrations of 3.3, 10, 30, and 90 nM, respectively, in the SCK experiment. [Figure 6] Figure 6 shows example sensorgrams obtained from Biacore kinetic analysis of TSLP from different species, showing binding to cynomolgus monkey TSLP, but not rat or mouse TSLP. TSLP at concentrations of 4 nM (solid line), 16 nM (dashed line), and 64 nM (dotted line) was injected over HSA-captured ZTPA001 (SEQ ID NO: 973) fused to the ABD. [Figure 7]7 shows the results of a dot blot performed with TSLP-binding Z variants ZTPA104 (SEQ ID NO: 1076; panel A) and ZTPA001 (SEQ ID NO: 973; panel B) and an IL-17A-binding reference polypeptide (panel C). The Z variants were compared with highly abundant plasma proteins; HSA (1), IgG (2), alpha-2-macroglobulin (3), alpha 1-antitrypsin (4), complement component 3c (C3c) (5), haptoglobulin (6), alpha 1-antichymotrypsin (7), complement C4 (8), IgE (9), hemopexin (10), transthyretin (11), Fc fragment of IgG (12), holo-transferrin (13), fibrinogen (14), IgA (15), IgM (16); Structurally related IL-2 family cytokines, IL-2 (17), IL-4 (19), IL-7 (21), IL-9 (22), IL-15 (23), and IL-21 (25); unrelated cytokines, IL-3 (18), IL-5 (20), and IL-17A (24); and TSLP proteins, sfTSLP (26), full-length TSLP (uncleaved) (27), and full-length TSLP (cleaved) (28) (all from human), were incubated with the coated membrane. Development of the membrane with anti-Z polyclonal antibody yielded dots for positive binding events. Panel D shows the results of ZTPA104 preincubated with 10-fold excess of HSA prior to use in a dot blot with a subset of the proteins in panels A-C: HSA (1), IgG (2), complement component 3c (C3c) (5), α1-antichymotrypsin (7), complement C4 (8), IgE (9), Fc fragment of IgG (12), holo-transferrin (13), fibrinogen (14), IgA (15), IgM (16), sfTSLP (26), full-length TSLP (uncleaved) (27), and full-length TSLP (cleaved) (28). [Figure 8]8 shows a diagram of exemplary results from the PathHunter® eXpress IL7R / CRLF2 cell assay described in Example 8 from the analysis of ZTPA002 (SEQ ID NO:974; light grey circles), ZTPA001 (SEQ ID NO:973; dark grey triangles) and ZTPA093 (SEQ ID NO:1065; black diamonds). Results are presented as inhibition profile curves with relative light units (RLU) measured on an EnSpire multimode reader on the y-axis versus inhibitor concentration on the x-axis. [Figure 9A] FIG. 9 shows the serum concentration versus time profile in individual cynomolgus monkeys administered ZTPA001 (SEQ ID NO: 973) intravenously and analyzed as described in Examples 9(A) and 10(B). [Figure 9B] FIG. 9 shows the serum concentration versus time profile in individual cynomolgus monkeys administered ZTPA001 (SEQ ID NO: 973) intravenously and analyzed as described in Examples 9(A) and 10(B). [Figure 10A] Figure 10 shows cytokine and chemokine production in the TSLP-induced cynomolgus monkey model described in Example 10. Shown are serum IL-2 (A), IL-5 (B), TARC (C), and MDC (D) levels 4 and 8 hours after intravenous injection of 62.5 μg / kg TSLP in the absence (left side of graph) or presence (right side of graph) of ZTPA001 (SEQ ID NO: 973) injected 2 hours prior to injection of TSLP. Serum levels of cytokines / chemokines are expressed in pg / mL; LLOQ = lower limit of quantification. [Figure 10B] Figure 10 shows cytokine and chemokine production in the TSLP-induced cynomolgus monkey model described in Example 10. Shown are serum IL-2 (A), IL-5 (B), TARC (C), and MDC (D) levels 4 and 8 hours after intravenous injection of 62.5 μg / kg TSLP in the absence (left side of graph) or presence (right side of graph) of ZTPA001 (SEQ ID NO: 973) injected 2 hours prior to injection of TSLP. Serum levels of cytokines / chemokines are expressed in pg / mL; LLOQ = lower limit of quantification. [Figure 10C]Figure 10 shows cytokine and chemokine production in the TSLP-induced cynomolgus monkey model described in Example 10. Shown are serum IL-2 (A), IL-5 (B), TARC (C), and MDC (D) levels 4 and 8 hours after intravenous injection of 62.5 μg / kg TSLP in the absence (left side of graph) or presence (right side of graph) of ZTPA001 (SEQ ID NO: 973) injected 2 hours prior to injection of TSLP. Serum levels of cytokines / chemokines are expressed in pg / mL; LLOQ = lower limit of quantification. [Figure 10D] Figure 10 shows cytokine and chemokine production in the TSLP-induced cynomolgus monkey model described in Example 10. Shown are serum IL-2 (A), IL-5 (B), TARC (C), and MDC (D) levels 4 and 8 hours after intravenous injection of 62.5 μg / kg TSLP in the absence (left side of graph) or presence (right side of graph) of ZTPA001 (SEQ ID NO: 973) injected 2 hours prior to injection of TSLP. Serum levels of cytokines / chemokines are expressed in pg / mL; LLOQ = lower limit of quantification. [Figure 11] Figure 11 shows the mean serum concentration versus time profiles of ZTPA001 (SEQ ID NO: 973; filled circles) and ZTPA104 (SEQ ID NO: 1076; filled squares) in rats. Mean values ​​were included in the graph only if all values ​​(n=3) at a given time point were above the LLOQ. [Figure 12A] Figure 12: Concentrations of ZTPA001 (SEQ ID NO: 973) in (A) BAL fluid and (B) lung tissue homogenates of male (squares) and female (circles) cynomolgus monkeys assessed following administration by inhalation. No differences were observed between left (L) and right (R) lungs or between tissue sampling sites, i.e., prox and distal. [Figure 12B] Figure 12: Concentrations of ZTPA001 (SEQ ID NO: 973) in (A) BAL fluid and (B) lung tissue homogenates of male (squares) and female (circles) cynomolgus monkeys assessed following administration by inhalation. No differences were observed between left (L) and right (R) lungs or between tissue sampling sites, i.e., prox and distal. [Figure 13]FIG. 13 shows ZTPA001 (SEQ ID NO: 973) plasma concentration versus time profiles in individual cynomolgus monkeys evaluated following administration by inhalation. [Figure 14] Figure 14 shows the TSLP binding ability of ZTPA001 (SEQ ID NO: 973) in cynomolgus monkey plasma samples assessed after administration by inhalation. Data from two analyses are shown and are expressed as the mean ± standard deviation of duplicates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0153] overview The following examples disclose the development of novel Z variant molecules targeting human thymic stromal lymphopoietin (TSLP) based on phage display technology. Selected polypeptides were sequenced as described herein and their amino acid sequences are listed in the sequence listing under sequence identifiers SEQ ID NOs: 1 to 875. The examples further describe the characteristics of these selected TSLP-binding polypeptides, their variants and derivatives, as well as fusion proteins comprising them, and demonstrate their in vitro and in vivo functionality. EXAMPLES

[0154] Example 1 Selection and screening of TSLP-binding Z variants overview In this example, recombinantly produced human TSLP was used as a target in phage display selections using two different phage libraries of Z variants. Selected clones were DNA sequenced, produced in E. coli, and assayed against TSLP by ELISA (enzyme-linked immunosorbent assay).

[0155] Materials and Methods Biotinylation of target protein: Recombinantly produced human TSLP (R&D Systems, Catalog No. 1398-TS / CF; SEQ ID NO: 1079) was biotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific) at a 10-fold molar excess according to the manufacturer's recommendations. The reaction was carried out at 4° C. for 2.5 hours, followed by incubation at room temperature (RT) for 1 hour. Biotinylation was performed in a dialysis cassette, which was used for buffer exchange into phosphate buffered saline (PBS: Dulbecco Gibco DPBS; 9.6 mM phosphate, 138 mM NaCl, 2.67 mM KCl, pH 7.4) immediately following biotinylation.

[0156] Phage display selection of TSLP-binding Z variants: TSLP-binding polypeptides were selected using two different libraries of random variants of protein Z displayed on bacteriophage, constructed in phagemids pAY02592 or pAY04242, respectively, essentially as described in Gronwall et al., 2007 J Biotechnol 128:162-183. In these libraries, the albumin binding domain (ABD, domain GA3 of protein G from Streptococcus strain G148; SEQ ID NO: 1078) is used as a fusion partner for the Z variants. The libraries are designated Zlib006Naive.II and Zlib008Naive.II, respectively, and are 1.5×10 10 and 2.9 x 10 10 is a library member (Z variant).

[0157] Phage stocks of phagemid library Zlib006Naive.II were generated from glycerol stocks of the library using E. coli RRIΔM15 cells (Ruther et al., 1982 Nucleic Acids Res 10:5765-5772) essentially as previously described (see, e.g., PCT Publication WO 2017 / 072280).

[0158] Phage stocks were generated from the phagemid library "Naive.II" as follows: Glycerol stocks of E. coli XL-1 Blue cells (Agilent Technologies, Cat. No. 200268) containing the phagemid library Zlib008Naive.II were inoculated into 20 L of TSB+YE medium [30 g / L tryptic soy broth; 5.0 g / L yeast extract] supplemented with 11 g / L glucose monohydrate, 100 μg / mL carbenicillin, and 10 mg / mL tetracycline hydrochloride. Cultures were grown at 37°C in fermentors (Belach Bioteknik, BR20). When the cells reached an OD of 600 nm (OD 600When the culture reached an optical density of 0.62 at 37 °C, approximately 4 L of culture was infected with a 10-fold molar excess of M13K07 helper phage (New England Biolabs, catalog no. N0315S). The cells were incubated for 30 min and then immediately added to the culture medium (3.05 g / L (NH4)2SO4; 6.1 g / L yeast extract; 3.65 g / L K2HPO4; 5.45 g / L KH2PO4; 2.29 g / L Na3C6H5O7·2H2O; 0.1 mL / L Breox FMT30 antifoam); 16 mL of trace element solution [129 mM FeCl3; 36.7 mM ZnSO4; 10.6 mM CuSO4; 78.1 mM MnSO4; 94.1 mM CaCl2 in 1.2 M HCl]; 10.4 mL of vitamin solution [500 mg / L DL-pantothenic acid, calcium salt; 500 mg / L choline chloride; 500 mg / L folic acid; 1000 mg / L The fermentor was filled to 20 L with: myo-inositol; 500 mg / L niacinamide; 50 mg / L riboflavin; 500 mg / L thiamine hydrochloride; 36.8 mL of 30 g / L thiamine hydrochloride; 53 mL of 1.217 M MgSO4; 16 mL of 100 mg / mL carbenicillin; 14.4 mL of 25% NH4OH. The culture was incubated for 60 min. 20 mL of 50 mg / mL kanamycin and 2 mL of 1 M isopropyl-β-D-1-thiogalactopyranoside (IPTG) were added. The temperature was reduced to 30° C. A glucose-limited fed-batch culture was started, feeding 600 g / L glucose solution into the reactor (15 g / h at the start, linearly increasing to 75 g / h after 20 h, then constant feeding until harvest). The pH was adjusted to 7 through automatic addition of 25% NH4OH, air was supplemented (10 L / min), and the agitator was set to maintain the dissolved oxygen level above 30%. The culture was harvested 23 hours after induction with IPTG. The cells in the culture were removed by centrifugation.

[0159] For both libraries, phage particles were precipitated twice from the supernatant using polyethylene glycol / sodium chloride (PEG / NaCl), filtered, and dissolved in PBS and glycerol as described in Gronwall et al., supra. Phage stocks were stored at -80°C until use.

[0160] Selection against biotinylated TSLP was performed in solution in tracks 1, 3, 4, and 6, or using a solid phase of streptavidin beads (SA beads, Dynabeads M-280 Streptavidin, Thermo Fisher) in tracks 2, 5, and 7. Selections were performed at 37° C. in tracks 1, 2, 4, and 5, and at room temperature in tracks 3, 6, and 7. As selections progressed, tracks were further divided according to target concentration and number and / or time of washes.

[0161] To reduce the amount of background binders, preselection was performed using SA beads in cycles 1-3 and neutravidin beads (NA beads, Sera-Mag SpeedBeads neutravidin-coated magnetic particles, Cytiva) in cycle 4. During preselection, phage stocks were incubated with the coated beads for 30-60 min at room temperature in end-over-end rotation. Beads used for preselection or selection were preblocked with PBS supplemented with 3% bovine serum albumin (BSA, Sigma) and 0.1% Tween® 20 (PBSTB). All tubes used were of the nonstick type (AM12450 Nonstick RNase-free microcentrifuge tubes, Ambion).

[0162] Selections were performed in PBSTB solution supplemented with 1.5 μM human serum albumin (HSA, Recombumin, Novozyme) (PBSTBH) at room temperature or 37°C. The time for selection was between 60 and 120 min, followed by capture of the target-phage particle complexes on beads, alternating between SA and NA beads. For solid-phase selection, biotinylated TSLP was pre-immobilized on SA beads before selection. The various beads containing the target-phage particle complexes were then washed with PBS supplemented with 0.1% Tween®-20 (PBST0.1%).

[0163] In addition to the above, several tracks of cycle three were also performed in which selection was performed in solution followed by capture of target-phage particle complexes in wells of 96-well high-binding polystyrene plates (Corning 3690) coated with neutravidin protein (NA, Pierce, Cat. No. 31000) diluted to 5 μg / mL in carbonate buffer (Sigma). The plates were then washed extensively with PBST 0.1%.

[0164] In the final selection cycle, log-phase bacteria were infected with the eluate, diluted, and then spread onto TBAB plates (30 g / L tryptose blood agar base, Oxoid) supplemented with 0.2 g / L ampicillin to form single colonies for ELISA screening.

[0165] The selection strategy describing increasing stringency of successive cycles with decreasing target concentration and increasing number of washes is outlined in Table 2. Washes were performed with PBST for 1 min unless otherwise stated. Elution was performed as described in WO 2009 / 077175.

[0166] [Table 2-1] [Table 2-2]

[0167] Amplification and preparation of phage particles: Amplification of phage particles between different selection cycles was performed as follows. E. coli strain XL1-Blue (Agilent Technologies, Cat. No. 200268) was used for phage amplification, and M13K07 helper phage was used in 100-fold excess in the first cycle and 50-fold excess in the following cycles. XL-1 Blue was cultivated at 37°C until early logarithmic phase in TSB medium (tryptic soy broth, 30 g / L) supplemented with 1% glucose and 1 μg / ml tetracycline, and then infected with phage particles. A 10- to 100-fold excess of bacteria was used compared to the amount of phage particles. The infection was left for 30 min at 37°C, after which the medium volume was doubled by adding TSB medium supplemented with 1% glucose, 1 μg / mL tetracycline, and 200 μg / mL carbenicillin. After approximately 1 hour of incubation at 37°C, helper phage was added and incubated at 37°C for 1.5 hours. Superinfected bacteria were pelleted at 3500×g and resuspended in 50 mL (100 mL after the first cycle) of TSB+YE medium supplemented with 25 μg / mL kanamycin and 0.1 mM IPTG (isopropyl-β-D-1-thiogalactopyranoside) and incubated overnight at 30°C. The overnight culture was pelleted and the phage particles in the supernatant were precipitated twice with PEG / NaCl. The phage particles were then resuspended in selection buffer before entering the next selection cycle. In the final selection cycle, log phase bacteria were infected with the eluate, diluted, and then spread on TBAB plates (30 g / L tryptose blood agar base, Oxoid) supplemented with 0.2 g / L ampicillin to form single colonies for ELISA screening.

[0168] Preparation of Z variants for ELISA: Z variants were prepared by inoculating 1.2 mL of TSB-YE medium supplemented with 100 μg / mL ampicillin and 1 mM IPTG in deep-well plates (Nunc). Plates were incubated at 37 °C with rotation for 22-24 h. Cells were pelleted by centrifugation, resuspended in 150 μL of PBST 0.05% (PBS supplemented with 0.05% Tween® 20), sealed in aluminum foil and incubated at 82 °C in a water bath for 20 min. Plates were briefly spun down to remove droplets from the foil and the heat-treated suspension was then subjected to a filtration step using a 96-well filter plate (Merck Millipore). The final filtered supernatant containing the soluble portion of the extract (heat-treated (HT) lysate) contained the Z variant as a fusion to ABD and was expressed as AQHDEALE-[ZTP###]-VDYV-[ABD]-YVPG (SEQ ID NO: 1125; Gronwall et al. supra). ZTP### denotes the sequence of the individual 58 amino acid residue, TSLP-binding Z variant.

[0169] Sequencing: All individually selected clones were subjected to a sequencing procedure. PCR fragments were amplified from single colonies, sequenced and analyzed essentially as described in WO 2015 / 189430.

[0170] ELISA screening of Z variants to TSLP: Binding of Z variants to TSLP was analyzed by ELISA assay. Half-area 96-well ELISA plates (Corning) were coated overnight at 4°C with 50 μL of 2 μg / mL anti-ABD goat antibody (homemade) diluted in PBS. The antibody solution was discarded and the wells were blocked with 100 μL of Blocker® casein in PBS (Thermo Scientific) for 1 h at room temperature. The blocking solution was discarded and 50 μL of HT lysate diluted 1:8 in PBST 0.05% was added to the wells and incubated for 1 h at room temperature. As a negative control, HT lysate prepared using only the ABD part was added. The supernatant was discarded and the wells were washed 4 times with PBST 0.05%. Then, 50 μL of biotinylated TSLP at a concentration of 10 nM diluted in Blocker® casein in PBS was added to each well, respectively. Plates were incubated at room temperature for 2 hours, followed by washing as above. Streptavidin-conjugated HRP (Thermo Scientific) diluted 1:30,000 in Blocker® casein in PBS was added to the wells, and the plates were incubated for 45 minutes. After washing as above, 50 μL of TMB substrate (1-Step Ultra TMB-ELISA Pierce Thermo Fisher Scientific) was added to the wells, and the plates were processed according to the manufacturer's recommendations. Absorbance at 450 nm was measured using a multi-well plate reader (EnSpire, PerkinElmer). Additionally, binders were subjected to ELISA assays using the steps and reagents described above, all steps performed at 37°C. A subset of binders was also subjected to ELISA assays using the protocol described above, but with biotinylated TSLP at the following concentrations: 3.3 nM, 1.7 nM, or 0.37 nM, respectively.

[0171] result Phage display selection of TSLP-binding Z variants: After 3-4 cycles of phage display selection against biotinylated TSLP, individual clones were obtained.

[0172] Sequencing: Clones obtained after 3 or 4 cycles of selection were sequenced. Each variant was given a unique identification number ### and the individual variants are referred to as ZTP###. The amino acid sequences of the Z variants 58 amino acid residues in length are listed in the sequence listing as SEQ ID NOs: 631-644, 809-828, and 846-875. A putative TSLP-binding motif extends from residue 8 to residue 36 in each sequence. The amino acid sequence of the 49 amino acid residue long polypeptide predicted to constitute a complete three-helix bundle in each of these Z variants extends from residue 7 to residue 55.

[0173] ELISA screening of Z variants against TSLP: Clones obtained after 3 and 4 cycles of selection were prepared as soluble unpurified samples in 96-well plates and screened for TSLP binding activity by ELISA. Binders were shown to give responses of 0.5-3.2 AU at a target concentration of 10 nM TSLP, which is at least 3.5 times higher than the blank control. ELISAs performed at 37°C gave similar results to ELISAs performed at room temperature. Multiple binders assayed against biotinylated TSLP at concentrations of 3.3 nM, 1.7 nM, or 0.37 nM were shown to give responses of 1.9-3.2 AU with 0.08 AU for the blank control, 0.3-3.0 AU with 0.095 AU for the blank control, and 0.06-1.5 AU with 0.06 AU for the blank control, respectively.

[0174] Example 2 Generation of TSLP-binding Z variants overview This example describes the general procedures for subcloning and generation of His-tagged Z variants and Z variants fused to an albumin binding domain, which were used throughout the following characterization experiments.

[0175] Materials and Methods Subcloning of His6-tagged Z variants: DNA encoding Z variants ZTP632 (SEQ ID NO: 632), ZTP634 (SEQ ID NO: 634), ZTP633 (SEQ ID NO: 633), ZTP635 (SEQ ID NO: 635), ZTP636 (SEQ ID NO: 636), and ZTP631 (SEQ ID NO: 631) was amplified from the Zlib006Naive.II library vector pAY02592. DNA encoding Z variants ZTP637 (SEQ ID NO: 637) and ZTP638 (SEQ ID NO: 638) was amplified from the Zlib008Naive.II library vector pAY04242. The subcloning strategy for construction of monomeric Z variant molecules with an N-terminal His6 tag was applied to the Zlib006Naive.II library member, and the subcloning strategy for construction of variant molecules with a C-terminal His6 tag was applied to the Zlib008Naive.II library member, both using standard molecular biology techniques and essentially as described in PCT Publication WO 2009 / 077175. Z gene fragments were subcloned into T7 promoter-driven expression vectors, resulting in the coding sequences MGSSHHHHHHLQ-[ZTP###]-VD (SEQ ID NO:1126) and [ZTP###]-LEHHHHHH (SEQ ID NO:1127), respectively.

[0176] Subcloning of Z variants fused to ABD: The gene encoding Z variant ZTP631 (SEQ ID NO: 631) cloned into a custom plasmid was ordered from ATUM (Newark, CA). The Z variant was cloned as a fusion protein with ABD variant PP013 (SEQ ID NO: 1077). The construct encoded by the expression vector was in the format [ZTP631]-ASGS-[PP013] (ZTPA092; SEQ ID NO: 1064).

[0177] Expression of TSLP-binding Z variants: Generation of Z variants was achieved essentially as follows: E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing sequence-verified gene fragments of each respective TSLP-binding Z variant and grown at 37°C in a 50 mL scale using the EnPresso protocol (Enpresso GmbH). To induce protein expression, IPTG was added after 8 hours of growth to a final concentration of 0.5 mM and the temperature was set to 30°C. After induction, cultures were incubated for 16 hours before cells were harvested by centrifugation.

[0178] Purification of His6-tagged TSLP-binding Z variants: Approximately 1–2 g of each cell pellet was resuspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) supplemented with Benzonase® (Merck). After cell disruption, cell debris was removed by centrifugation and each supernatant was added to a 1 mL His GraviTrap IMAC column (GE Healthcare). Contaminants were removed by washing with wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4) and Z variants were subsequently eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4). The Z variants were subjected to a second purification step by reversed-phase chromatography (RPC), where each Z variant was loaded onto a 1 mL Resource 15 RPC column (GE Healthcare) pre-equilibrated with RPC solvent A (0.1% trifluoroacetic acid (TFA), 10% acetonitrile (ACN), 90% water). After column washing with RPC solvent A, bound proteins were eluted with an 18 mL linear gradient of 0–60% RPC solvent B (0.1% TFA, 80% ACN, 20% water). The buffer was then exchanged into DPBS (Corning) using a PD-10 desalting column.

[0179] Protein concentration was measured by absorbance measurement at 280 nm. Samples with concentrations less than approximately 1 mg / ml were concentrated using Amicon Ultra-4, Ultracel-3K filters (Merck Millipore). Purity was analyzed by SDS-PAGE stained with Coomassie blue, and the identity of each purified Z variant was confirmed using LC / MS analysis.

[0180] Purification of TSLP-binding Z variants fused with ABD: Approximately 5 g of cell pellet was resuspended in TST-buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween® 20, pH 8.0) supplemented with Benzonase® (Merck). After cell disruption and clarification by centrifugation and filtration (0.45 μm filter), the supernatant was added to a gravity-flow column together with 5 mL agarose immobilized with anti-ABD ligand (homemade). After washing with TST-buffer and 5 mM NH4Ac pH 5.5 buffer, the ABD-fused Z variants were eluted with 0.1 M HAc. The eluate was then subjected to a second purification step by RPC. 10% ACN was added to each elution, followed by loading onto a 3 mL Resource RPC column (GE Healthcare) equilibrated with solvent A (10% ACN, 0.1% TFA, 90% Milli-Q water) and elution using a 54 mL gradient of 0-60% solvent B (80% ACN, 0.1% TFA, 20% Milli-Q water). Elution fractions were analyzed by SDS-PAGE and HPLC-MS and pooled. Eluates were buffer exchanged into DPBS using a PD-10 desalting column. Protein concentration measurements and purity and identity analyses were performed as described above for the His6-tagged Z variants.

[0181] result Generation of TSLP-binding Z variants: TSLP-binding Z variants with His6 tags or fused to the ABD were successfully cloned and expressed as soluble gene products in E. coli. Each DNA construct was confirmed by DNA sequencing. SDS-PAGE analysis of each final protein preparation showed that they contained predominantly the TSLP-binding Z variant. The exact identity and molecular weight of each Z variant was confirmed by HPLC-MS analysis.

[0182] Example 3 Characterization of primary TSLP-binding Z variants overview In this example, N-terminal His6-tagged Z variants ZTP632 (SEQ ID NO: 632), ZTP634 (SEQ ID NO: 634), ZTP633 (SEQ ID NO: 633), ZTP635 (SEQ ID NO: 635), ZTP636 (SEQ ID NO: 636) and ZTP631 (SEQ ID NO: 631); and C-terminal His6-tagged Z variants ZTP637 (SEQ ID NO: 637) and ZTP638 (SEQ ID NO: 638) were characterized in terms of secondary structure, stability and binding profile. Melting temperature and secondary structure content were analyzed by circular dichroism (CD) spectroscopy. Biacore was used to characterize the interaction of Z variants with TSLP.

[0183] Materials and Methods Circular dichroism (CD) spectroscopy: His6-Z and Z-His6 variants prepared as described in Example 2 were diluted to 0.5 mg / mL in PBS. CD spectra from 250 to 195 nm were obtained at 20°C. Additionally, variable temperature measurements (VTM) were performed to measure melting temperatures (Tm). In VTM, absorbance was measured at 221 nm while the temperature was increased from 20°C to 90°C with a temperature gradient of 5°C / min. To study the refolding ability of the Z variants, new CD spectra were obtained at 20°C after the heating run. CD measurements were performed on a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) using a cell with a path length of 1 mm.

[0184] Biacore affinity analysis: The affinity (K) of TSLP for TSLP-binding His6-Z and Z-His6 variants was determined using a Biacore 8K instrument (Cytiva). D ) was measured. In this experiment, human TSLP was immobilized on the carboxylated dextran layer of one CM5 chip surface (Cytiva) to an immobilization level of approximately 210-280 RU. Immobilization was performed using HBS-EP+ (Cytiva) as the running buffer with amine coupling chemistry according to the manufacturer's protocol. One flow cell surface was activated and deactivated and used as a blank during analyte injection. In the kinetics experiment, HBS-EP+ was used as the running buffer and the flow rate was 30 μL / min. The analytes, i.e., His6-Z or Z-His6 variants, were diluted in HBS-EP+ buffer to concentrations of 100, 33 and 11 nM, respectively, and injected into the flow cell. The concentrated analytes were injected for 2 min, followed by a 10 min dissociation time. The surface was regenerated by one injection of regeneration buffer (10 mM glycine pH 1.5, Cytiva) and a 10 min wait time was set before the next injection round. A blank cycle with HBS-EP+ was performed before or near each analyte cycle to subtract background. Target binding analysis was performed using Biacore 8K Evaluation software (Cytiva) and a Langmuir 1:1 model. All data were double-referenced prior to kinetic fitting, i.e., responses from a blank surface and from a blank cycle in which buffer was injected instead of analyte were subtracted.

[0185] result CD analysis: CD spectra of eight TSLP-bound Z variants with His6 tags showed that all variants had α-helical structure at 20°C, as judged by typical minima at 208 and 222 nm. Reversible folding was observed for all Z variants when spectra taken before and after heating to 90°C were overlaid. Melting temperatures (Tm) are summarized in Table 3. Exemplary CD spectra of His6-ZTP631 and ZTP637-His6 are shown in Figure 1.

[0186] [Table 3]

[0187] Biacore affinity analysis: The interaction of the His6-Z and Z-His6 variants with TSLP was analyzed on a Biacore 8K instrument by injecting various concentrations of purified Z variants over the TSLP-immobilized surface. The dissociation equilibrium constants (K ) were calculated based on data for each TSLP binder at three concentrations (11, 33, and 100 nM, respectively) injected over the TSLP surface. D ) are summarized in Table 4. An example of a sensorgram obtained for one TSLP-binding polypeptide (ZTP631, SEQ ID NO: 631) is shown in FIG.

[0188] [Table 4]

[0189] Example 4 Characterization of TSLP-binding polypeptides in an in vitro blocking assay overview This example describes the evaluation of select Z variants for their ability to block the effects of TSLP-induced thymus- and activation-regulated chemokine (TARC) production in human peripheral blood mononuclear cells (PBMCs).

[0190] Materials and Methods One hundred thousand PBMCs per well were plated in assay medium [RPMI1640 (Gibco), 5% human serum (BIOIVT) and 1% pen-strep (Lonza)] in 96-well culture plates (Greiner Bio One). Serially titrated His-tagged Z variants (diluted in assay medium in seven steps from 10 to 0.0002 μM final concentration in the cell assay plate) and TSLP (final concentration 1 ng / mL in the cell assay plate) were pre-incubated for 1 h at room temperature before being added to the cells. Cells with added reagents were incubated for 24 h at 37 °C in a CO2 incubator. Supernatants were collected by transferring 120 μL from each well to a V-bottom dilution plate (Greiner Bio One) and centrifuged at 400 × g for 5 min. Then, 2 × 60 μL were transferred to two U-bottom plates (Greiner Bio One), the plates were sealed and stored at -80 °C until further analysis. TARC levels were measured using the Human CCL17 / TARC-DuoSet® ELISA (R&D Systems, Cat. No. DY364). The ELISA was performed essentially as described by the manufacturer, except that a) half-area ELISA plates were used, b) half the volumes listed were used, and c) the plates were washed four times with 175 μL of wash buffer. Absorbance was measured at 450 nm using a plate reader.

[0191] result Z variants ZTP631 (sequence number 631), ZTP632 (sequence number 632), ZTP633 (sequence number 633), ZTP634 (sequence number 634), ZTP635 (sequence number 635), ZTP636 (sequence number 636), ZTP673 (sequence number 637) and ZTP638 (sequence number 638) were shown to concentration-dependently block TARC production by TSLP in human PBMCs (Figure 3).

[0192] Example 5 Design and construction of a maturation library of TSLP-binding Z variants overview In this example, a new library was designed based on the TSLP binding variants described in Examples 2, 3, and 4. The mature library contained approximately 5.9×10 9 Each clone contained 10 individual clones.

[0193] Materials and Methods Design of affinity matured TSLP library: A new library was designed based on the sequences of TSLP-binding Z variants selected, generated, and characterized as described in Examples 1-4. Thirteen surface-exposed positions of the Z molecular scaffold were biased towards specific amino acid residues following a strategy based primarily on the binding motifs of the Z variants defined in SEQ ID NOs: 631-644, 809-828, and 846-875. The design of the new library is shown in Table 5, showing the percentage proportion of amino acids used at each of the 13 randomized positions.

[0194] [Table 5]

[0195] Two oligonucleotides with complementary 3′ ends (one forward complementary and one reverse complementary) were generated using the TRIM technique. These oligos were ordered from Ella Biotech GmbH (Martinsried, Germany).

[0196] Library construction was performed essentially as previously described (e.g., PCT Publication WO 2017 / 072280) in a vector designated pAY02592, with the following exceptions: Approximately 230 ng was used per electroporation to transform electrocompetent XL-1 Blue cells. After electroporation, cells were pooled and incubated in recovery medium (Lucigen) at 37° C. for 60 minutes, then cultured in 3 L of TSB-YE medium supplemented with 10 μg / mL tetracycline and 100 μg / mL carbenicillin. Library quality and amino acid distribution were confirmed by sequencing essentially as described in WO 2009 / 077175.

[0197] Preparation of phage stocks: Cells from a glycerol stock containing the phagemid library were inoculated into 2 L of TSB supplemented with 100 μg / mL carbenicillin, 10 μg / mL tetracycline, and 1% glucose and grown at 80 rpm and 37° C. The cultures reached an OD of 600 nm. 600 When the culture reached an optical density of 0.7 at 37° C., a 50-fold molar excess of M13K07 helper phage was used to infect the culture, and the cells were incubated at 37° C. for 1.5 hours. The cells were pelleted and resuspended in 2 L of TSB+YE supplemented with 100 μg / mL carbenicillin, 25 μg / mL kanamycin, and 0.1 mM IPTG. The culture was incubated at 30° C. and 70 rpm, and the culture was harvested after 21 hours. The cells in the culture were removed by centrifugation (10,700×g, 30 min at 4° C.). Phage particles were precipitated twice from the supernatant using PEG / NaCl, filtered as described in Example 1, and dissolved in PBSB and glycerol. Phage stocks were stored at −80° C. until used for selection.

[0198] result Library construction: A new library was designed based on the set of TSLP binding variants described in Examples 1-4. The theoretical size of the designed library was 4.7 × 10 8 The actual size of the library, determined by titration after transformation into E. coli XL-1 Blue cells, was 5.9 × 10 9 Transformants were identified. The quality of the library was checked by sequencing 96 transformants and comparing their actual sequences to the theoretical design. Sequence analysis of individual library members confirmed that the codon distribution was in accordance with the theoretical design. The library was designated Zlib006TSLP.I.

[0199] Example 6 Selection and screening of affinity matured TSLP-binding Z variants overview In this example, TSLP was used as a target in phage display selections using a TSLP maturation phage library of Z variants. Selected clones were DNA sequenced, prepared in E. coli periplasmic fractions, and assayed against various target proteins by ELISA and Biacore.

[0200] Materials and Methods Protein biotinylation: Human TSLP was biotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin as described in Example 1.

[0201] Phage display selection of TSLP-binding Z variants: Phage display selections were performed using phage stocks of the newly generated maturation library. Selection against biotinylated TSLP was performed in solution in all tracks essentially as described in Example 1, but with the specific conditions described in Table 6.

[0202] Cycles 1, 3, and 4 were performed using streptavidin-coated beads (Sera-Mag SpeedBeads streptavidin-coated magnetic particles, Cytiva) as solid phase, whereas cycle 2 used neutravidin beads. In some tracks, a wash step was performed by capture of target-phage particle complexes in wells of a 96-well high-binding polystyrene plate (Corning) coated with SA protein (Millipore) diluted to 5 mg / mL in carbonate buffer. For overnight washes, non-biotinylated TSLP was added to the wash buffer. Selections were performed at 37°C or room temperature using preblocked tubes (Protein LowBind, Eppendorf). To reduce the degree of nonspecific binding, a preselection was performed in cycles 1-3 using the corresponding solid phase used in that specific cycle and track. Additionally, in some tracks, phage stocks were preheated at 70°C for 15 min and subsequently spun down in a centrifuge at 13,000 rpm before being used for selection.

[0203] As the selection proceeded, the tracks were further divided according to target concentration and number and / or time of washes.

[0204] [Table 6]

[0205] Amplification and preparation of phage particles: Preparation of phage stocks and amplification of phage between selection cycles were performed essentially as described in Example 1. For the last selection cycle, log-phase bacteria were infected with the eluate and spread onto TBAB plates supplemented with 0.2 g / L ampicillin to form single colonies for use in ELISA screening.

[0206] Generation of Z variants for ELISA: Z variants were generated and HT lysates of each individual variant were prepared as described in Example 1. The final filtered supernatant, containing the soluble portion of the extract, contained the Z variant as a fusion to ABD represented as AQHDEALE-[ZTP###]-VDYV-[ABD]-YVPG (SEQ ID NO: 1125; Gronwall et al. supra). ZTP### refers to the individual 58 amino acid residue Z variant.

[0207] ELISA screening of Z variants: The binding of Z variants to TSLP was analyzed in a series of ELISA assays. Each Z variant was analyzed against biotinylated TSLP at concentrations of 1, 0.2, 0.04 and 0 nM, respectively. The ELISA was performed essentially as described in Example 1, except that a high-binding 384-well plate (Greiner) was used. In addition, a selected subset of binders was subjected to ELISA using a target concentration of 0.2 nM. All samples were run in triplicate. In addition, a further selected subset of binders was subjected to ELISA using a target concentration of 0.2 nM, and all samples were run in triplicate. As a negative control, HT lysate containing the fusion protein ABD without the Z fusion partner, i.e., AQHDEALEVDYV-[ABD]-YVPG (SEQ ID NO: 1128), was used on all plates. The absorbance at 450 nm was measured using a multi-well plate reader, EnSpire (PerkinElmer).

[0208] Sequencing: All clones were sequenced in parallel with the ELISA screening. PCR fragments were amplified from single colonies, sequenced and analyzed as described in Example 1.

[0209] result Phage display selection of TSLP-binding Z variants: Individual clones were obtained after 1 to 4 cycles of phage display selection against biotinylated TSLP.

[0210] ELISA screening of Z variants: Clones obtained after 1-4 cycles of selection were prepared in 96-well plates and screened for binding activity to TSLP. In the first ELISA, all clones were analyzed against TSLP at concentrations of 1, 0.2, 0.04, and 0 nM, respectively. In two subsequent ELISA assays, a subset of binders was assayed in triplicate against 0.2 nM TSLP. Binders were considered positive if their response was 3-fold above background at 0.2 nM TSLP (>0.26 AU) in the first set and reasonable target-dependent responses at the other concentrations, or if their mean response was 3-fold above background in either of the latter two ELISAs (>0.195 AU or >0.293 AU, respectively).

[0211] Sequencing: Clones obtained after 1-4 cycles of selection were sequenced. Each variant was given a unique identification number ### and the individual variants are referred to as ZTP###. The amino acid sequences of the Z variants 58 amino acid residues in length are listed in the sequence listing as SEQ ID NOs: 1-630, 646-808 and 829-845. A putative TSLP-binding motif extends from residue 8 to residue 36 of each sequence. The amino acid sequence of a polypeptide 49 amino acid residues in length predicted to constitute a complete three-helix bundle in each of these Z variants extends from residue 7 to residue 55.

[0212] Example 7 Generation and characterization of mature TSLP-binding Z variants overview This example describes the cloning, production, and characterization of mature Z variants. CD spectroscopy was used to determine melting temperatures and evaluate secondary structure content. SPR was used to measure target binding affinity and kinetic values ​​and to analyze binding to TSLP from different species. Specificity was analyzed in a dot blot assay to evaluate possible off-target binding to selected abundant plasma proteins or other related or unrelated proteins. TSLP is a member of the interleukin 2 (IL-2) family of cytokines that also includes IL-4, IL-7, IL-9, IL-15, and IL-21. Each member of the IL-2 cytokine family consists of a four-alpha helix bundle and shares a common cytokine receptor gamma chain. However, there is low amino acid sequence similarity, and to confirm the specificity of the Z variants selected here for TSLP, all cytokines of the IL-2 cytokine family were included in the dot blot assay. In addition, the unrelated IL-17A cytokine was included in the specificity assay.

[0213] Materials and Methods Subcloning of Z variants fused to ABD: A subset of clones selected based on their scores in the ELISA assay were ordered from Twist Biosciences (San Francisco, CA) as cloned genes in custom vectors. In the genes ordered from Twist, the N-terminal amino acids at positions 1 and 2 of the Z variants (originally V and D, respectively) were mutated to amino acid residues A and E, respectively (SEQ ID NOs: 876-966). One set of these Z variants was then further mutated at the scaffold positions as follows: Y5F, N52S, and D53E (SEQ ID NOs: 967-972). The synthesized Z variants were subcloned into an expression vector containing the ABD variant PP013 (SEQ ID NO: 1077). The construct encoded by the expression vector was in the format [ZTP###]-ASGS-[PP013] and was designated ZTPA### (SEQ ID NOs: 973-1070). In addition, two Z variants, ZTP970 (SEQ ID NO: 970) and ZTP876 (SEQ ID NO: 876), were subcloned as fusions to the ABD with different sets of linkers. The constructs were made according to the methods described in Example 2 and were in the formats: [ZTP970]-A(EAAAK)2-AGS-[PP013] (ZTPA099; SEQ ID NO:1071), [ZTP970]-A(EAAAK)4-AGS-[PP013] (ZTPA100; SEQ ID NO:1072), [ZTP970]-(KEAAA)2-KAK-[PP013] (ZTPA101; SEQ ID NO:1073), [ZTP970]-(KEAAA)2-[PP013] (ZTPA102; SEQ ID NO:1074), [ZTP970]-(GGGGS)3-GS-[PP013] (ZTPA103; SEQ ID NO:1075), and GS-[PP013]-GGGGS-[ZTP876] (ZTPA104; SEQ ID NO:1076). One Z variant (SEQ ID NO: 645) was created by fusing with PP013 in the format [ZTP645]-ASGS-[PP013] (ZTPA105; SEQ ID NO: 1129).

[0214] Subcloning of Z variants fused to His6: A subset of Z variants were subcloned with a His6 tag as described in Example 2. The cloned variants were in the format [ZTP###]-LEHHHHHH (SEQ ID NO: 1127).

[0215] Expression of TSLP-binding Z variants: Expression of TSLP-binding Z variants was performed as described in Example 2 or on a smaller scale in 24-well format. Production in 24-well format followed the same procedure as described in Example 2, except that a culture volume of 3 mL was used and the temperature was maintained at 37° C. after addition of IPTG.

[0216] Purification of TSLP-binding Z variants fused with ABD in 96-well plate format: Approximately 0.1 mg of cell pellet was resuspended in TST buffer containing 3 mM EDTA, and cells were disrupted by heat treatment at 90° C. and subsequent DENARASE® (c-LEcta) (in 5 mM MgSO4) treatment, followed by centrifugation and filtration through a 3.0 / 0.2 μm filter (Pall). The clarified lysate was stored frozen and, after thawing, passed through a 0.45 μm filter plate (AcroPrep) before being added to a 1 mL filter plate (1.2 μm, Acroprep) prepacked with 100 μl of anti-ABD ligand (homemade). After washing with TST buffer and 5 mM NH4Ac pH 5.5 buffer, the ABD-fused Z variants were eluted with 0.1 M HAc. All washing and elution steps were performed using centrifugal force. Buffer exchange into DPBS was performed using a PD MultiTrap G-25 desalting plate (Cytiva).

[0217] Protein concentrations were determined by absorbance measurements at 280 nm. LC / MS analysis was used to confirm the identity of each purified Z variant.

[0218] Purification of TSLP-binding Z variants fused with ABD: Approximately 4 g of cell pellet was resuspended in TST buffer supplemented with DENARASE® (c-Lecta). After cell disruption and clarification by centrifugation and filtration (0.45 μm filter), the supernatant was applied to a gravity-flow column containing 2 × 1 mL agarose immobilized with anti-ABD ligand (homemade). After washing with TST-buffer and 5 mM NH4Ac pH 5.5 buffer, the ABD-fused Z variants were eluted with 0.1 M HAc. The eluates were then subjected to a second purification step by RPC. 10% ACN was added to each eluate, followed by loading onto a 1 mL Resource RPC column (Cytiva) equilibrated with solvent A (10% ACN, 0.1% TFA, 90% Milli-Q water) and elution using a gradient of 18 mL 0–60% solvent B (80% ACN, 0.1% TFA, 20% Milli-Q water). The eluted fractions were analyzed by SDS-PAGE and HPLC-MS and then pooled. The eluate buffer was exchanged into DPBS using a PD-10 desalting column (Cytiva).

[0219] Protein concentration was measured by absorbance measurement at 280 nm. Samples with concentrations less than 1 mg / mL were concentrated using Amicon Ultra-4, Ultracel-3K (Merck Millipore). Purity was analyzed by SDS-PAGE stained with Coomassie blue, and the identity of each purified Z variant was confirmed using LC / MS analysis.

[0220] Purification of TSLP-binding Z variants fused to His6: Z variants cloned as fusions to C-terminal His6 were purified using the IMAC strategy described in Example 2.

[0221] CD Analysis: A subset of Z variants cloned as Z-His6 were subjected to CD analysis essentially as described in Example 3.

[0222] Biacore Kinetic Screening: Affinity and kinetic values ​​of binding to TSLP were measured for the Z-ABD variants generated in this example using a 96-well strategy, as well as for the Z-ABD variant ZTPA092 (SEQ ID NO: 1064) generated in Example 2 using a Biacore 8K instrument. TSLP was immobilized on the surface of a CM5 chip as described in Example 3, resulting in immobilization levels of approximately 510-550 RU. A single cycle kinetic (SCK) method (injection of a series of analytes in one cycle, with no regeneration in between) was used for injection of each Z-ABD variant. A blank cycle with HBS-EP+ was performed before each analyte cycle to subtract background. The Z variants were injected at 3.3, 10, 30 and 90 nM, respectively, over the TSLP-immobilized sensor chip surface. Each of the four analyte concentrations was injected for 240 seconds, followed by a dissociation time of 15 minutes. The flow rate was 30 μL / min. HBS-EP+ was used as the running buffer. The surface was regenerated by three injections of regeneration buffer (10 mM glycine pH 1.5) followed by a waiting period before the next round of injections. The resulting sensorgrams were subjected to reference subtraction and analyzed with Biacore Insight evaluation software using a Langmuir 1:1 binding model to obtain the kinetic constant: binding rate constant (k on ), dissociation rate constant (k off ) and dissociation equilibrium constant (K D ) was sought.

[0223] Biacore kinetic analysis for TSLP from different species: Affinity and kinetic values ​​for Z-ABD variants ZTPA002 (SEQ ID NO: 974), ZTPA001 (SEQ ID NO: 973), and ZTPA093 (SEQ ID NO: 1065) were measured for a set of TSLP from different species: cynomolgus monkey (cynoTSLP, Sino Biological Cat. No. 90911-C08H), rat (rTSLP, MyBiosource, Cat. No. MBS2010776), and mouse (mTSLP, R&D Systems, Cat. No. 555-TS-010 / CF) using a Biacore 8K instrument. HSA was diluted to 10 μg / mL in immobilization buffer and immobilized on a CM5 sensor chip by amine chemistry using an amine coupling kit type 2 (Cytiva), 10 mM sodium acetate at pH 4.5 as immobilization buffer, and HBS-EP+ as running buffer, all according to the manufacturer's instructions. Both flow cells of each channel were immobilized (in sequence) at approximately 6500 RU.

[0224] Binding properties were evaluated using multi-cycle kinetics (MCK) with ligand capture. Z variants were captured by injecting 20 nM onto immobilized HSA in flow cell 2 at 5 μL / min for 150 s (capture reaction was approximately 50–100 RU), followed by injection of analyte in a concentration series of 4 nM, 16 nM, and 64 nM onto both flow cells 1 and 2. Binding interactions were evaluated in HBS-EP+ running buffer. Association time was 180 s (30 μL / min) and dissociation time was 460 s (30 μL / min). Reference cell (flow cell 1) and blank cycle injections (HBS-EP+) were subtracted from the sensorgrams prior to analysis. Regeneration buffer was 10 mM HCl (two pulses of 30 s / 30 μL / min).

[0225] Dot blot analysis: Binding of Z-ABD variants ZTPA104 (sequence number 1076) and ZTPA001 (sequence number 973) to two different forms of full-length human TSLP: hTSLP (uncleaved) (Acro Biosystems, catalog number TSP-H52Ha) and hTSLP (cleaved) (Acro Biosystems, catalog number TSP-H52Hb), as well as the short form of TSLP (sfTSLP; sequence number 1080; synthesized by Almac, Craigavon, UK) was assessed. Furthermore, the Z variants were compared with a series of different highly abundant plasma proteins: HSA (Recombumin Elite, Albumedix, Cat. No. 205-005); human IgG (Sigma, Cat. No. G4386); α-2-macroglobulin (human A2M, Sino Biological, Cat. No. 10952-H08B); α1-antitrypsin (SERPINA1 human, ProSpec, Cat. No. PRO-529); complement component 3c (C3c, Lee Biosolutions, Cat. No. 194-32); haptoglobulin, (ProSpec, Cat. No. PRO-567); α1-antichymotrypsin (SERPINA3, Fitzgerald / Kem-En-Tech, Cat. No. 30-AC47); C4 complement (Complement Technology, Cat. No. A105); IgE (Fitzgerald / Kem-En-Tech, Cat. No. 31-Al01); hemopexin (human HPX, Sino Biological, Catalog No. 10870-H08H); transthyretin (TTR, Sino Biological, Catalog No. 12091-H08H); IgG Fc fragment, (Jackson ImmunoResearch / Nordic BioSite, Catalog No. 009-060-008); holo-transferrin human (Sigma, Catalog No. T4132); fibrinogen (Abcamab, Catalog No. 81752); IgA (Bethyl Laboratories / Nordic Biosite, Catalog No. P80-102); IgM (Sigma, Catalog No. I8260); structurally related cytokines of the IL-2 family: (IL-2, Peprotech, Catalog No. 200-02);IL-4 (R&D Systems, Catalog No. 6507-IL / CF); IL-7 (kindly provided through a collaborative research effort); IL-9 (R&D Systems, Catalog No. 209-ILB-010 / CF); IL-15 (Peprotech, Catalog No. 200-15);and IL-21 (R&D Systems, Catalog No. 8879-IL-010 / CF), as well as the irrelevant IL-3 (R&D Systems, Catalog No. 203-IL-010 / CF), IL-5 (R&D Systems, Catalog No. 205-IL-025 / CF), and IL-17A (Peprotech, Catalog No. 200-17). A nitrocellulose membrane was divided into three areas and the proteins were applied to create identical sections on the membrane. 1 μL (0.1 mg / mL) of each protein was immobilized at a predefined location on each section. The membrane was blocked with Blocker® Casein for 2 hours at room temperature. The blocking solution was removed and the membrane was cut into three identical strips. Each strip was incubated with 2 μg / mL of two different TSLP-binding Z variants ZTPA104 (SEQ ID NO: 1076) and ZTPA001 (SEQ ID NO: 973) or IL-17A-binding Z variants included as control polypeptides (all diluted in Blocker® Casein), or with Blocker® Casein alone (negative control) for 1 h at room temperature and then washed 4×2 min with PBST. Bound Z variants were detected by first adding 5 μg / mL of goat anti-Z variant Ig antibody (homemade) for 1 h at room temperature and then washing 4×2 min with PBST. Next, anti-goat antibody-HRP conjugate (Dako, Cat. No. P0449) diluted 1:10000 was added, incubated for 1.5 h at room temperature and washed 5×5 min with PBST. All dilutions were made in Blocker® Casein and all incubation and washing steps were performed with gentle rocking. Signals were developed by adding approximately 5 mL SuperSignal™ West Pico PLUS (Thermo Scientific) followed by incubation for 5 min. The resulting chemiluminescence was detected using a Luminescent Image Analyzer (Fujifilm, Fuji Photo Film) and images were taken after 20 s exposure.

[0226] Additionally, other assays performed as described above used the same protocol, but in the presence of HSA, against a subset of proteins from human plasma or serum: IgG, complement component 3c (C3c), α1-antichymotrypsin, C4 complement, IgE, Fc fragment of IgG, holo-transferrin, fibrinogen, IgA, and IgM; as well as against HSA, sfTSLP, full-length TSLP (uncleaved), and full-length TSLP (cleaved). Z variant ZTPA104 (SEQ ID NO: 1076) was diluted in Blocker® casein supplemented with 10× molar excess of HSA and incubated at room temperature for 30 minutes before assaying against proteins on nitrocellulose membranes.

[0227] result Generation of TSLP-binding Z variants: TSLP-binding Z variants fused to the ABD were expressed as soluble gene products in E. coli. SDS-PAGE analysis of each final protein preparation showed that they contained predominantly TSLP-binding Z variants. The exact identity and molecular weight of each Z variant was confirmed by HPLC-MS analysis.

[0228] CD analysis: CD spectra measured for TSLP-bound Z variants with a C-terminal His6 tag showed that all variants had an α-helical structure at 20° C., as judged by typical minima at 208 and 222 nm. Reversible folding was observed for all Z variants when spectra measured before and after heating to 90° C. were overlaid. Melting temperatures (Tm) are summarized in Table 7. Exemplary CD spectra for Z variants ZTP876 and ZTP967 are shown in FIG. 4.

[0229] [Table 7]

[0230] Biacore kinetic screening: Z variants fused to each ABD were injected at various concentrations over a surface containing immobilized TSLP and their binding was analyzed. A summary of the calculated kinetic parameters is shown in Table 8. Exemplary sensorgrams for Z-ABD variants ZTPA028, ZTPA029 and ZTPA030 are shown in Figure 5.

[0231] [Table 8-1] [Table 8-2] [Table 8-3]

[0232] Biacore kinetic analysis for TSLP from different species: Using a capture strategy on a Biacore 8K instrument, the affinity and kinetic values ​​of binding to TSLP from cynomolgus monkey, rat, and mouse were measured for Z-ABD variants ZTPA002 (SEQ ID NO: 974), ZTPA001 (SEQ ID NO: 973), and ZTPA093 (SEQ ID NO: 1065), respectively. HSA was immobilized and then injected with each Z-ABD, followed by TSLP from different species. A summary of the kinetic parameters calculated based on data from three concentrations of cynomolgus monkey TSLP protein (4, 16, and 64 nM) injected onto the Z-ABD surface is shown in Table 9. No binding to TSLP from rat or mouse was detectable. An example of a sensorgram obtained for one TSLP-binding polypeptide (ZTPA001, SEQ ID NO: 973) is shown in FIG. 6.

[0233] [Table 9]

[0234] Dot blot analysis: A dot blot assay was used to assess the specificity of ZTPA104 and ZTPA001. These Z-ABD variants were bound to a nitrocellulose membrane containing dots of 28 different proteins, including two forms of full-length hTSLP (uncleaved or cleaved) and sfTSLP, 16 abundant plasma proteins, and 6 structurally related and 3 unrelated cytokines. Results for Z-ABD variants ZTPA104 and ZTPA001 are shown in Figure 7A-C, in comparison with an IL-17A binding Z variant included as a negative control. To examine whether the weak dots seen in Figure 7A-C for some proteins purified from plasma or serum (dots 5, 7, 14, and 16) could be due to binding of residual traces of HSA, binding in the presence of a 10-fold molar excess of HSA was also performed for those proteins, and the results of that analysis are shown in Figure 7D. Of the proteins examined in this dot blot, binding was detectable only to full-length hTSLP and HSA.

[0235] Example 8 Characterization of TSLP-binding polypeptides in in vitro potency assays overview The potency of polypeptides targeting TSLP was evaluated using the PathHunter® eXpress IL7R / CRLF2 dimerization assay (Eurofins / DiscoverX). The PathHunter® dimerization assay detects TSLP-induced dimerization of the IL-7 receptor alpha chain (IL-7Rα) and the TSLP receptor alpha chain 1 (TSLP-R; encoded by CRLF). The assay uses Enzyme Complementation Technology (EFC), which is based on two recombinant β-galactosidase enzyme fragments, the enzyme acceptor (EA) and the enzyme donor (ED), each fused to one of the two TSLP receptor subunits. Binding of TSLP to one receptor subunit induces receptor dimerization and subsequent complementation of the enzyme fragment, resulting in an active enzyme capable of generating a chemiluminescent signal upon substrate hydrolysis.

[0236] Materials and Methods On the day of the assay, 10,000 cells per well were plated in Cell Plating 25 reagent (Eurofins / DiscoverX) in white 96-well TC plates (Eurofins / DiscoverX) and incubated for 4 hours at 37°C in a CO2 incubator. During this time, Z variants fused with ABD variant PP013 (SEQ ID NO: 1077) were serially titrated in Cell Plating 25 reagent (Albumedix) containing Recombumin Elite. TSLP (R&D Systems, Cat. No. 1398-TS / CF, SEQ ID NO: 1079) was diluted to a final concentration of 1 ng / mL. Serially titrated polypeptides and TSLP target protein were pre-incubated together for 30 minutes before being added to the cells. 10 μL of mixed polypeptide and TSLP protein were added to the cells and incubated for 16 hours at 37°C. After incubation, 110 μL of Flash detection reagent (a 1:4 mixture of Flash cell assay buffer and Flash substrate reagent, both from Eurofins / DiscoverX) was added to each well and the plate was incubated for 1 h at room temperature in the dark. Luminescence signals were measured using an EnSpire multimode reader (PerkinElmer).

[0237] result All TSLP-binding Z variants fused to ABD tested demonstrated potency in inhibiting TSLP-induced dimerization. Calculated IC50 values ​​ranging from 0.03 to 23 nM are shown in Table 10. As an illustration, a representative graph is shown in Figure 8.

[0238] [Table 10]

[0239] Example 9 Pharmacokinetic studies of ZTPA001 in non-human primates overview This example describes a single-dose pharmacokinetic (PK) study of ZTPA001 (SEQ ID NO: 973) administered intravenously (iv) to cynomolgus monkeys. Blood samples were collected up to 3 weeks after dosing and analyzed using an antibody-based sandwich PK-ELISA.

[0240] Materials and Methods PK study in NHP: On day 1, non-naive, non-fasted cynomolgus monkeys (n=3; 5.0-5.3 kg) were administered ZTPA001 at 1.33 mg / kg by bolus intravenous injection. A dose volume of 0.5 mL / kg was used for each injection. Blood samples (0.5 mL) were collected at the following time points: pre-dose, 10 and 30 minutes after dosing, and 1, 6, 10, 26, 50, 72, and 96 hours, as well as on days 6, 7, 8, 11, 13, 15, 18, and 22. Blood samples were allowed to clot for at least 30 minutes at room temperature before centrifugation. After centrifugation, serum was extracted, aliquoted, and stored at -70°C until further analysis.

[0241] Quantification of ZTPA001 by ELISA: 96-well half-area plates were coated with mouse anti-Z monoclonal antibody (2 μg / mL) in PBS (50 μL / well) and incubated overnight at 4°C. Plates were washed with PBST and blocked with Blocker® Casein (Thermo Scientific) for 1.5 h at 22°C. ZTPA001 standards were titrated in a 1.5-fold dilution series (50-1.3 pM) in 1% Cynomolgus serum pool in Blocker® Casein. Serum samples were diluted 1:100 in Blocker® Casein, followed by 1:50 in 1% Cynomolgus serum pool and serial dilution 1:4 in 1% Cynomolgus serum pool. Calibration standards and diluted serum samples were added to the coated ELISA plates (50 μL / well) and incubated at 22°C for 1.5 h. After washing with PBST, rabbit anti-ABD polyclonal antibody (2 μg / mL) was added. After 1.5 h incubation at 22°C, the plates were washed with PBST and 100 ng / mL of HRP-conjugated donkey anti-rabbit IgG (Jackson Immuno Research Cat. No. 711-035-152) was added to each well. After an additional 1 h incubation and subsequent washing with PBST, the plates were developed with TMB (50 μL / well) for 15 min at room temperature and the reaction was stopped by adding 0.2 M H2SO4 (50 μL / well). Absorbance at 450 nm was measured on a microplate reader (PerkinElmer Enspire LF).

[0242] Evaluation of PK profile: The serum concentration versus time profile of ZTPA001 was evaluated by noncompartmental analysis using linear up-log down extrapolation in Phoenix WinNonlin version 8.3.

[0243] result The PK profile of ZTPA001 after a single intravenous dose in cynomolgus monkeys is shown in Figure 9 A. In cynomolgus monkeys 1 and 2, the terminal half-life was determined to be 3-4 days, and the area under the concentration vs. time curve up to the last observation time point was 11 h*mg / mL.

[0244] Example 10 Demonstration of the mechanism of action of ZTPA001 in non-human primates overview This example describes a validation study of the single-dose mode of action (POM) of ZTPA001 administered intravenously to cynomolgus monkeys. Blood samples were collected up to 3 weeks after ZTPA001 administration and analyzed using an antibody-based sandwich PK-ELISA. Cytokine and chemokine levels were measured on a Meso Scale Discovery (MSD) platform (MSD).

[0245] Materials and Methods POM study in NHP: TSLP was administered by intravenous bolus at 6.25, 31.25, and 62.5 μg / kg to non-fasted female cynomolgus monkeys (n=3; 3.5-4.1 kg; 1 naive and 2 non-naive) on days 1, 8, and 16, respectively. A dose volume of 0.5 mL / kg was used for each injection. Serum samples were obtained pre-dose and 4, 8, 24, and 48 hours after TSLP administration. On day 30, ZTPA001 was administered on day 1 as a single 1.33 mg / kg intravenous (bolus) dose. Two hours after intravenous ZTPA001 administration, 62.5 μg of TSLP was administered by intravenous injection. A dose volume of 0.5 mL / kg was used for each injection. Blood samples (0.5 mL) for ZTPA001 serum concentrations were collected at the following time points: pre-dose, 10, and 130 min (10 min post-TSLP administration), and 3, 6, 10, 26, 50, 72, and 96 hours and days 8, 11, 15, 18, and 22 post-ZTPA001 administration. Blood samples for cytokine and chemokine levels were obtained pre-dose and 4, 8, 24, and 48 hours post-TSLP administration. Blood samples were allowed to clot for at least 30 minutes at room temperature before centrifugation. After centrifugation, serum was extracted, aliquoted, and stored at -70°C until further analysis.

[0246] Quantification of ZTPA001 by ELISA: The assay was performed as described in Example 9.

[0247] Evaluation of PK Profile: The serum concentration versus time profile of ZTPA001 was evaluated by noncompartmental analysis using linear up-log-down extrapolation in Phoenix WinNonlin version 8.3.

[0248] Cytokine and chemokine quantification by MSD: Cytokine and chemokine analyses were performed on serum obtained pre-treatment, 4, 8, 24 and 48 hours, and 1, 8, 16 and 30 days after TSLP treatment. Cytokines (IL-1β, IL-2, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 / IL-23p40, IL-15, IL-16, IL-17A, TNF-β, VEGF-A, IFN-γ, GM-CSF) and chemokines (MIP-1β, eotaxin-3, TARC, IP-10, MIP-1α, IL-8 HA, MCP-1, MDC, MCP-4) were measured using a V-PLEX Plus NHP Cytokine 24-Plex Kit (Meso Scale Discovery, Rockville, MD, Cat. No. K15058G) and analyzed on a MESO QuickPlex SQ120 reader (Meso Scale Discovery, Rockville, MD). Serum samples were diluted 1:4 and analyzed according to the manufacturer's instructions for the Cytokine Panel 1 (NHP) Kit, Proinflammatory Panel 1 (NHP) Kit, and Chemokine Panel 1 (NHP) Kit, respectively. Serum concentrations of cytokines and chemokines were assessed with MSD Discovery Workbench version 4.0.

[0249] result The ZTPA001 serum concentration versus time profile in cynomolgus monkeys after a single intravenous dose is shown in Figure 9B. The mean terminal half-life was determined to be 4 days, with an area under the concentration versus time curve to the time of the last quantifiable concentration of 5.7 h*mg / mL. Of the 24 cytokines and chemokines measured in the NHP PoM study, 16 were above both the LOD (limit of detection) and LLOQ (lower limit of quantification), thereby meeting the acceptance criteria (IL-2, IL-5, IL-6, IL-7, IL-8, IL-12 / IL-23p40, IL-15, IL-16, VEGF-A, MIP-1α, MIP-1β, TARC, IP-10, MCP-1, MDC, and MCP-4). Eight cytokines and chemokines did not meet the acceptance criteria because they were below the LOD or LLOQ (IL-1β, IL-10, IL-17A, TNF-β, IFN-γ, GM-CSF, eotaxin-3, and IL-8 HA). Administration of TSLP at a dose of 6.25 μg / kg did not lead to upregulation of cytokine / chemokine levels. Administration of TSLP at a dose of 31.25 μg / kg led to increased production of several cytokines / chemokines, most notably IL-2, TARC, MDC, MCP-1, and MCP-4. TSLP at the highest dose of 62.5 μg / kg induced upregulation of several pro-inflammatory and asthma-related cytokines / chemokines, most notably IL-2, IL-5, IL-6, IL-8, IL-16, TARC, MDC, MCP-1, MCP-4 and IP-10, with maximum concentrations reached 4-8 hours after administration. A single intravenous administration of ZTPA001 led to a reduction in some of those pro-inflammatory cytokines and chemokines. The serum concentration versus time profiles of asthma-related cytokines and chemokines after TSLP administration in the absence or presence of ZTPA001 are shown in Figure 10. The figure shows that a single intravenous administration of ZTPA001 leads to a reduction in asthma-related cytokines.

[0250] Example 11 Pharmacokinetic study of ZTPA001 and ZTPA104 in rats overview This example describes a single-dose pharmacokinetic (PK) study of ZTPA001 (SEQ ID NO: 973) and ZTPA104 (SEQ ID NO: 1076) administered intravenously (iv) to Sprague-Dawley rats. Blood samples were collected up to 3 weeks after dosing and serum concentrations were analyzed using an antibody-based sandwich PK-ELISA.

[0251] Materials and Methods PK study in rats: On day 1, ZTPA001 and ZTPA104 were each administered at 1.2 mg / kg by intravenous injection to male Sprague-Dawley rats (Charles River, Germany; n=6 / test article). A dose volume of 0.5 mL / kg was used for each injection. Due to the blood collection limit of 8 mL / kg, blood collection time points were divided into two cohorts as follows: cohort A (n=3 per test article) collected blood before dosing, 5 min, 1, 8, 72, 168, 264, 408, and 504 h after dosing; and cohort B (n=3 per test article) collected blood before dosing, 30 min, and 3, 24, 120, 216, 336, and 456 h after dosing. Blood samples were allowed to clot for at least 30 min at room temperature before centrifugation. After centrifugation, serum was extracted, aliquoted, and stored at −80°C until further analysis.

[0252] Quantification by ELISA: ELISA assays were performed essentially as described in Example 9 with the following exceptions: Exception 1: ZTPA001 standards were titrated in a 1.5-fold dilution series (50-0.87 pM) into 1% rat serum pool (Sprague Dawley; Bio IVT) in Blocker® casein. Serum samples were diluted 1:100 into Blocker® casein followed by 1:5 serial dilutions into 1% rat serum pool. Exception 2: ZTPA104 standards were titrated in a 1.5-fold dilution series (25-0.43 pM) into 1% rat serum pool in Blocker® casein. Serum samples were diluted 1:100 into Blocker® casein followed by 1:100 dilutions into 1% rat serum pool (except for samples taken pre-dose) followed by 1:5 serial dilutions into 1% rat serum pool. The lower limit of quantification (LLOQ) was 1.03 ng / mL for ZTPA001 and 0.52 ng / mL for ZTPA104.

[0253] Evaluation of PK profiles: Mean ZTPA001 and ZTPA104 serum concentration versus time profiles were evaluated by noncompartmental analysis using linear up-log-down extrapolation in Phoenix WinNonlin version 8.3. Concentration values ​​below the LLOQ were excluded from the analysis.

[0254] result The PK profiles of ZTPA001 and ZTPA104 in rats after a single intravenous administration are shown in Figure 11. The two polypeptides showed similar PK profiles, with maximum serum concentrations (C max ) were 41 and 35 μg / mL, the dose-normalized areas under the concentration-time curve for the last quantifiable serum concentration or time extrapolated to infinity (AUC / dose) were 1.2 and 1.1 h*kg / mL, and the terminal half-life (t 1 / 2 ) were 29 and 36 hours.

[0255] Example 12 Inhalation study of ZTPA001 in non-human primates overview This example describes a study in which cynomolgus monkeys were repeatedly dosed with ZTPA001 (SEQ ID NO: 973) via inhalation. Blood, bronchoalveolar lavage (BAL) and lung tissue samples were collected approximately 24 hours after the last dose of ZTPA001 and compound concentrations were analyzed by LC-MS / MS. Retention of TSLP-binding functionality of systemically distributed ZTPA001 was confirmed by ELISA.

[0256] Materials and Methods Inhalation study in NHP: ZTPA001 was administered nominally at 1.2, 6.7 or 12 mg / kg to cynomolgus monkeys (3.2-4.7 kg; 1 male and 1 female per dose level) seven times daily via 1-h inhalation. Test aerosols were generated using a jet nebulizer to facilitate inhalation through a face mask. On days 1 (first dose) and 7 (last dose), blood samples (0.5 ml) were collected in K2EDTA blood tubes at the following time points: pre-dose, immediately after dosing, and 1, 3, 7 and 23 h after the end of dosing. Plasma was obtained within 2 h of blood collection by centrifugation at 2000×g at 4°C for 10 min. After centrifugation, plasma was extracted, aliquoted and stored at -70°C until further analysis. At termination, bronchoalveolar lavage (BAL) and proximal and distal lung tissue samples were obtained and stored frozen at -70°C until bioanalysis.

[0257] Quantification of ZTPA001: Plasma, lung tissue homogenate and BAL fluid concentrations were measured by liquid chromatography tandem mass spectrometry (LC-MS / MS). A SMART Digest Trypsin Kit (Thermo Fisher Scientific, Cat. No. 60113-01) was used for sample digestion. Unique characteristic tryptic digest peptides were used for quantification, and corresponding stable isotope-labeled peptides were used as internal standards.

[0258] Assessment of PK Profile: The profile of mean ZTPA001 serum concentrations versus time was assessed by noncompartmental analysis using linear up-log-down extrapolation in Phoenix WinNonlin version 8.3.

[0259] TSLP-binding ELISA: Four samples with high plasma levels were selected for testing in the human TSLP-binding ELISA. The samples were from one male and one female monkey receiving a nominal dose of 6.7 mg / kg and one male and one female monkey receiving a nominal dose of 12 mg / kg, taken 23 hours after the end of dosing on day 7. TSLP-binding capacity of plasma samples was compared to cynomolgus plasma spiked with ZTPA001 over the same range as the samples (spike low) or at a high concentration (spike high). A negative control of unspiked blank plasma was also included.

[0260] Two half-area assay plates were coated with recombinant human TSLP (RnD Systems, Cat. No. 1398-TC / CF) at 1 μg / mL in PBS overnight at 4° C. The next day, the plates were washed with PBST 0.05% and blocked with Blocker® Casein in PBS for 1 h at 22° C. Meanwhile, samples and controls were diluted in matrix (Blocker® Casein in PBS supplemented with 1% Cynomolgus Plasma). Samples and spike low controls were each diluted to 100 nM, and spike high controls were diluted to 5000 nM. Duplicate dilutions were performed for all samples and controls, and a 3-fold dilution series was made in matrix. Samples were added to the wells of the blocked plate, incubated for 1 h at 22° C., and washed with PBST 0.05%. Monoclonal mouse anti-ABD Ig was used for primary detection, and anti-mouse Ig-HRP for secondary detection. Plates were developed with TMB substrate and the reaction was then stopped by the addition of 0.2M H2SO4. Absorbance was measured at 450 nm using an EnSpire plate reader.

[0261] result Repeated administration of ZTPA001 by inhalation resulted in high BAL fluid and lung tissue levels, with serum / plasma distribution already observed after the first dose. Higher doses resulted in increased ZTPA001 concentrations in BAL 24 hours after the start of the last of seven daily inhalation doses (Figure 12A). In lung tissue homogenates, ZTPA001 concentrations were more variable, but generally concentrations were lowest in the 1.2 mg / kg / day group (Figure 12B). No differences were observed between left and right lungs or between tissue sampling sites (proximal or distal). The ZTPA001 plasma concentration versus time relationship in cynomolgus monkeys after the first (day 1) and last (day 7) daily dose of ZTPA001 is shown in Figure 13. On day 1, plasma ZTPA001 concentrations gradually increased over time, reaching a maximum concentration 23 hours after the end of inhalation, i.e., before the second dose. Repeated dosing further increased ZTPA001 plasma concentrations on day 7.

[0262] The TSLP-binding ability of ZTPA001 in four selected plasma samples (showing high concentrations of ZTPA001 measured by LC-MS / MS) from four different animals was evaluated by human TSLP-binding ELISA. The titration curves of the test samples, spiked low control and spiked high control were well overlapped (Figure 14), i.e., all four test samples showed similar binding to human TSLP as the spiked control. In the case of the negative control, no binding to TSLP was detected. This indicates that ZTPA001 retains human TSLP-binding function after nebulization, inhalation and systemic distribution. Furthermore, since anti-ABD antibody is used to detect ZTPA001 in the TSLP ELISA, the results also indicate that the ABD portion of the ZTPA001 polypeptide is intact.

[0263] ITEMIZED LISTING OF EMBODIMENTS 1. A TSLP-binding polypeptide comprising a TSLP-binding motif BM, said motif comprising: i) EAVX4ALX7EIWX 11 LPNLX 16 X 17 X18 QX 20 X 21 AFIX 25 X 26 LRD (SEQ ID NO: 1081), Independently of each other, X4 is selected from D, E, and H; X7 is selected from I, L, M, and V; X 11 is selected from A, D, E, K, N, Q, R, S, and T; X 16 is selected from N and T; X 17 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 18 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 20 is selected from H, N, Q, T, W and Y; X 21 is selected from D, E, G, H, K, M, N, Q, and R; X 25 is selected from A, H, I, K, L, Q, R, V and Y; and X 26 is selected from K and S and ii) an amino acid sequence having at least 93% identity to the sequence defined in i); A TSLP-binding polypeptide consisting of an amino acid sequence selected from the group consisting of: 2. A TSLP-binding polypeptide according to item 1, comprising in sequence i) X4 is selected from D, E and H; X7 is selected from I, L, M, and V; X 11 is selected from A, D, E, K, N, Q, R, S, and T; X 16 is selected from N and T; X 17 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 18 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 20 is selected from H, N, Q, T, W and Y; X 21 is selected from D, E, G, H, K, M, N, Q, and R; X 25 is selected from A, H, I, K, L, Q, R, V, and Y; and X 26 is selected from K and S TSLP-binding polypeptide. 3. In sequence i), X4 is selected from E and H; X7 is selected from I, L, and V; X 11 is selected from A, D, E, Q, R, S, and T; X 16 is selected from N and T; X 17 is selected from A, D, E, G, H, K, N, Q, R, S, T, V, W, and Y; X 18 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 20 is selected from H, N, Q, W and Y; X 21 is selected from D, E, H, K, M, N, Q, and R; X 25 is selected from H, I, K, L, Q, R, V, and Y; and X 26 is selected from K and S A TSLP-binding polypeptide according to any of the preceding items. 4. In sequence i), X4 is selected from E and H; X7 is selected from I, L, and V; X 11 is selected from A, D, E, Q, S, and T; X 16 is T; X 17 is selected from D, E, G, H, N, Q, R, S, W, and Y; X 18 is selected from A, D, E, F, G, H, I, L, N, Q, R, S, T, W, and Y; X 20 is selected from H, W and Y; X 21 is selected from D, E, H, N, and Q; X 25 is selected from I, L, R, V, and Y; and X 26 is K A TSLP-binding polypeptide according to any of the preceding items. 5. In sequence i), X4 is E; X7 is selected from I and V; X 11 is selected from A, D, E, Q, S, and T; X 16 is T; X 17 is selected from D, E, G, H, N, Q, R, and Y; X 18 is selected from A, D, E, F, G, I, L, N, Q, R, S, T, and Y; X 20 is selected from H, W and Y; X 21 is selected from D, E, H, N, and Q; X 25 is selected from I, L, V, and Y; and X 26 is K A TSLP-binding polypeptide according to any of the preceding items. 6. In sequence i), X4 is E; X7 is V; X 11 is selected from A and T; X 16 is T; X 17 is R; X 18is selected from D and E; X 20 is W; X 21 is Q; X 25 is Y; and X 26 is K A TSLP-binding polypeptide according to any of the preceding items. 7. A TSLP-binding polypeptide according to any of the preceding paragraphs, wherein sequence i) satisfies at least four of the following eight conditions I to VIII: I. X4 is E or H; II.X7 is selected from I, L and V; III.X 11 is selected from A, D, E, Q, S and T; IV.X 16 is T; VX 20 is selected from H, W and Y; VI.X 21 is selected from D, E, H, N, and Q; VII.X 25 is selected from I, L, R, V and Y; and VIII.X 26 is K. 8. The TSLP-binding polypeptide according to item 7, wherein sequence i) satisfies at least five of the eight conditions I to VIII. 9. The TSLP-binding polypeptide according to item 8, wherein sequence i) satisfies at least six of the eight conditions I to VIII. 10. The TSLP-binding polypeptide according to item 9, wherein sequence i) satisfies at least seven of the eight conditions I to VIII. 11. The TSLP-binding polypeptide according to item 10, wherein sequence i) satisfies all eight conditions I to VIII. 12. X4 is E, X7 is V, and X 20 is W. 13. X4 is E, X7 is V, and X 21is Q. 14. X4 is E, X7 is V, and X 25 is Y. 15. The TSLP-binding polypeptide according to item 1, wherein the binding motif sequence corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 875. 16. The TSLP-binding polypeptide according to item 15, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 645. 17. The TSLP-binding polypeptide according to item 16, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 91. 18. The TSLP-binding polypeptide according to item 17, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 27. 19. The TSLP-binding polypeptide according to item 18, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 2. 20. A TSLP-binding polypeptide according to any preceding item, wherein the binding motif forms part of a three-helix bundle protein domain. 21. The TSLP-binding polypeptide according to item 20, wherein the binding motif essentially forms part of two α-helices with an interconnecting loop within the three-helix bundle protein domain. 22. The TSLP-binding polypeptide according to item 20 or 21, wherein the three-helix bundle protein domain is selected from bacterial receptor domains. 23. The TSLP-binding polypeptide according to any one of items 20 to 22, wherein the three-helix bundle protein domain is selected from the domain of protein A from Staphylococcus aureus or a derivative thereof. 24. A binding module (BMod), the amino acid sequence of which is iii) K-[BM]-DPSQSXa X b LLX c EAKKLX d X e X f Q (SEQ ID NO: 1082), [BM] is a TSLP-binding motif defined in any one of items 1 to 19; X a is selected from A and S; X b is selected from E and N; X c is selected from A, S and C; X d is selected from E, N and S; X e is selected from D, E and S; X f is selected from A and S; Amino acid sequence and iv) an amino acid sequence having at least 91% identity to the sequence defined in iii); TSLP-binding polypeptide according to any of the preceding claims, selected from the group consisting of: 25. The TSLP-binding polypeptide according to item 24, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 875. 26. A TSLP-binding polypeptide according to any of the preceding items, v) YA-[BMod]-AP (SEQ ID NO: 1083), [BMod] is an amino acid sequence as defined in item 24 or 25; and vi) an amino acid sequence having at least 86% identity to the sequence defined in v); A TSLP-binding polypeptide comprising an amino acid sequence selected from: 27. A TSLP-binding polypeptide according to any of the preceding items, vii) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1097), [BM] is an amino acid sequence as defined in any one of items 1 to 19; and viii) an amino acid sequence having at least 86% identity to the sequence defined in vii); A TSLP-binding polypeptide comprising an amino acid sequence selected from 28. The TSLP-binding polypeptide according to item 27, wherein sequence vii) is selected from the group consisting of SEQ ID NOs: 1 to 636, 639 to 644 and 646 to 875. 29. A TSLP-binding polypeptide according to any one of items 1 to 25, ix) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO: 1095), [BM] is an amino acid sequence as defined in any one of items 1 to 19; and x) an amino acid sequence having at least 86% identity to the sequence defined in ix); A TSLP-binding polypeptide comprising an amino acid sequence selected from: 30. The TSLP-binding polypeptide according to item 29, wherein sequence ix) is selected from the group consisting of SEQ ID NO: 637 and SEQ ID NO: 638. 31. A TSLP-binding polypeptide according to any one of items 1 to 26, xi) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1093), [BM] is an amino acid sequence as defined in any one of items 1 to 19; and xii) an amino acid sequence having at least 86% identity to the sequence defined in xi); A TSLP-binding polypeptide comprising an amino acid sequence selected from: 32. The TSLP-binding polypeptide according to item 31, wherein sequence xi) is selected from the group consisting of SEQ ID NOs: 645 and 876 to 972. 33.K of interaction with TSLP D The value is at most 1×10 -6 M, for example, at most 5 × 10 -7 M, for example, at most 1 × 10-7 M, for example, at most 5 × 10 -8 M, for example, at most 1 × 10 -8 A TSLP-binding polypeptide according to any preceding item, which is capable of binding to TSLP such that M. 34. A TSLP-binding polypeptide according to any preceding item, comprising additional amino acids at the C-terminus and / or N-terminus. 35. The TSLP-binding polypeptide of item 34, wherein the additional amino acid(s) improve production, purification, in vitro or in vivo stabilization, binding, or detection of the polypeptide. 36. A TSLP-binding polypeptide according to any of the preceding items in a multimeric form comprising at least two TSLP-binding polypeptide monomer units, the amino acids of which may be the same or different. 37. The TSLP-binding polypeptide of item 36, wherein the TSLP-binding polypeptide monomer units are covalently linked together. 38. The TSLP-binding polypeptide of claim 36, wherein the TSLP-binding polypeptide monomer unit is expressed as a fusion protein. 39. A TSLP-binding polypeptide according to any one of items 36 to 38 in dimeric form. 40. A fusion protein or conjugate comprising: - a first portion consisting of a TSLP-binding polypeptide according to any of the preceding items; and - a second portion comprising a polypeptide having a desired biological activity; A fusion protein or conjugate comprising: 41. A fusion protein or conjugate according to item 40, wherein the desired biological activity is a therapeutic activity. 42. A fusion protein or conjugate according to item 40, wherein the desired biological activity is binding activity. 43. A fusion protein or conjugate according to item 40, wherein the desired biological activity is an enzymatic activity. 44. A fusion protein or conjugate according to item 42, wherein the desired binding activity is albumin binding activity that increases the in vivo half-life of the fusion protein or conjugate. 45. A fusion protein or conjugate according to item 44, wherein the second part comprises the albumin binding domain of Streptococcus protein G or a derivative thereof. 46. ​​The fusion protein or conjugate according to item 45, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 973 to 1076 and 1129. 47. The fusion protein or conjugate according to item 46, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 973 to 1076. 48. The fusion protein or conjugate according to item 47, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 973-999 and 1064-1076. 49. The fusion protein or conjugate according to item 48, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 973, 974, 1065 and 1076. 50. The fusion protein or conjugate according to item 49, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 973 and 1076. 51. The fusion protein or conjugate according to item 50, comprising the amino acid sequence of SEQ ID NO: 973. 52. The fusion protein or conjugate according to item 50, comprising the amino acid sequence of SEQ ID NO: 1076. 53. A fusion protein or conjugate according to item 42, wherein the binding activity acts to block a biological activity. 54. The fusion protein or conjugate according to item 41, wherein the second part is a therapeutically active polypeptide. 55. The fusion protein or conjugate according to any one of items 40 to 43 and 53 to 54, wherein the second moiety is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines. 56. The fusion protein or conjugate according to any one of items 40 to 43, wherein the second part is selected from the group consisting of antibodies and antigen-binding fragments thereof. 57. The fusion protein or conjugate according to item 56, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain Fab (scFab) fragment, an Fc fragment, an Fv fragment, a single-chain Fv (scFv) fragment, an (scFv)2, an scFv-Fc construct and a domain antibody. 58. The fusion protein or conjugate according to item 57, wherein the at least one antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab fragment and an scFv fragment. 59. The fusion protein or conjugate according to item 58, wherein the at least one antibody or antigen-binding fragment thereof is a full-length antibody. 60. The fusion protein according to any one of items 40 to 59, wherein the second portion further comprises a linker. 61. A TSLP-binding polypeptide, fusion protein or conjugate according to any of the preceding items, further comprising a label selected from the group consisting of, for example, fluorescent dyes and metals, chromogenic dyes, chemiluminescent compounds, bioluminescent proteins, enzymes, radionuclides and radioactive particles. 62. A polynucleotide encoding a TSLP-binding polypeptide, fusion protein, or conjugate according to any of the preceding items. 63. An expression vector encoding the polynucleotide according to item 62. 64. A host cell comprising the expression vector according to item 63. 65. A method for producing a polypeptide, a fusion protein or a conjugate according to any one of items 1 to 60, comprising: - culturing the host cell according to item 64 under conditions allowing expression of said polypeptide from said expression vector; and - isolating said polypeptide. The method includes: 66. A composition comprising a TSLP-binding polypeptide, fusion protein or conjugate according to any one of items 1 to 61 and at least one pharma- ceutically acceptable excipient or carrier. 67. A TSLP-binding polypeptide, fusion protein or conjugate according to any one of items 1 to 60 or a composition according to item 66, for oral, respiratory, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration, such as for respiratory, intravenous or subcutaneous administration. 68. A TSLP-binding polypeptide, fusion protein or conjugate according to any one of items 1 to 60 or a composition according to item 66 for use as a pharmaceutical, diagnostic or prognostic agent. 69. A TSLP-binding polypeptide, fusion protein, conjugate or composition according to item 68 for use as a medicament. 70. A TSLP-binding polypeptide, fusion protein, conjugate or composition for use according to item 68 as a diagnostic and / or prognostic agent. 71. A TSLP-binding polypeptide, fusion protein, conjugate or composition for use as a pharmaceutical according to item 69, which regulates TSLP function in vivo. 72. A TSLP-binding polypeptide, fusion protein, conjugate or composition according to any one of items 68 to 71 for use in the treatment, prognosis or diagnosis of a TSLP-related disorder. 73. The TSLP-binding polypeptide, fusion protein, conjugate or composition for use according to item 72, wherein the TSLP-related disorder is selected from inflammatory diseases, autoimmune diseases and cancer diseases. 74. The TSLP-binding polypeptide, fusion protein, conjugate or composition for use according to item 72 or 73, wherein the TSLP-related disorder is selected from the group consisting of respiratory diseases, skin diseases, allergies, eye diseases, gastrointestinal diseases and cancer. 75. The TSLP-binding polypeptide, fusion protein, conjugate or composition for use according to any one of items 72 to 74, wherein the TSLP-related disorder or disease is selected from the group consisting of asthma, atopic dermatitis, atopic keratoconjunctivitis, urticaria, allergic rhinitis, chronic rhinosinusitis with nasal polyposis, eosinophilic esophagitis, chronic obstructive pulmonary disease (COPD), eosinophilic granulomatosis with polyangiitis (EGPA) / Churg-Strauss syndrome, breast cancer, prurigo nodularis, and bullous pemphigoid. 76. A method for treating a TSLP-associated disorder, comprising administering to a subject in need thereof an effective amount of a TSLP-binding polypeptide, fusion protein or conjugate according to any one of items 1 to 60, or a composition according to item 66. 77. The method of claim 76, wherein the TSLP-related disorder is selected from inflammatory diseases, autoimmune diseases, and cancer diseases. 78. The method of claim 77, wherein the TSLP-related disorder is selected from the group consisting of respiratory diseases, skin diseases, allergies, eye diseases, digestive diseases, and cancer. 79. The method according to item 78, wherein the TSLP-related disorder or disease is selected from the group consisting of asthma, atopic dermatitis, atopic keratoconjunctivitis, urticaria, allergic rhinitis, chronic rhinosinusitis with nasal polyposis, eosinophilic esophagitis, chronic obstructive pulmonary disease (COPD), eosinophilic granulomatosis with polyangiitis (EGPA) / Churg-Strauss syndrome, breast cancer, prurigo nodularis, and bullous pemphigoid. 80. A method for detecting TSLP in vitro, comprising providing a sample suspected of containing TSLP, contacting the sample with a TSLP-binding polypeptide, fusion protein or conjugate described in any one of items 1 to 61 or a composition described in item 66, and detecting binding of the TSLP-binding polypeptide, fusion protein, conjugate or composition, indicating the presence of TSLP in the sample. 81. A method for determining the presence or absence of TSLP in a subject, comprising the steps of: a) contacting a subject or a sample isolated from a subject with a TSLP-binding polypeptide, fusion protein or conjugate according to any one of items 1 to 61 or a composition according to item 66, and b) obtaining a value corresponding to the amount of TSLP-binding polypeptide, fusion protein, conjugate or composition in said subject or bound to said sample. The method includes: 82. The method of claim 81, further comprising the step of comparing the value with a reference. 83. The method according to item 81 or 82, wherein the subject is a mammalian subject, such as a human subject. 84. The method according to any one of items 81 to 83, which is carried out in vivo.

Claims

1. TSLP binding polypeptides comprising a TSLP binding motif BM, wherein the motif is i) EAVX ４ ALX ７ EIWX １１ LPNLX １６ X １７ X １８ QX ２０ X ２１ AFIX ２５ X ２６ LRD (SEQ ID NO: 1081), Independently of each other, X ４ is selected from D, E, and H; X ７ is selected from I, L, M, and V; X １１ is selected from A, D, E, K, N, Q, R, S, and T; X １６ is selected from N and T; X １７ is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W and Y; X １８ is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y; X ２０ is selected from H, N, Q, T, W and Y; X ２１ is selected from D, E, G, H, K, M, N, Q, and R; X ２５ is selected from A, H, I, K, L, Q, R, V and Y; and X ２６ is selected from K and S; Amino acid sequence and ii) an amino acid sequence having at least 93% identity to the sequence defined in i); A TSLP-binding polypeptide consisting of an amino acid sequence selected from:

2. In sequence i), X ４ is E; X ７ is V; X １１ is selected from A and T; X １６ is T; X １７ is R; X １８ is selected from D and E; X ２０ is W; X ２１ is Q; X ２５ is Y; and X ２６ is K; 2. The TSLP binding polypeptide of claim 1.

3. 2. The TSLP-binding polypeptide of claim 1, wherein the binding motif sequence corresponds to a sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 875, for example a sequence selected from the group consisting of SEQ ID NOs: 1 to 645.

4. 4. A TSLP-binding polypeptide according to any one of claims 1 to 3, wherein the binding motif forms part of a three-helix bundle protein domain.

5. a link module (BMod) having the amino acid sequence iii) K-[BM]-DPSQSX ａ X ｂ LLX ｃ EAKKLX ｄ X ｅ X ｆ Q (SEQ ID NO: 1082), [BM] is a TSLP binding motif as defined in claim 1; X ａ is selected from A and S; X ｂ is selected from E and N; X ｃ is selected from A, S, and C; X ｄ is selected from E, N and S; X ｅ is selected from D, E, and S; X ｆ is selected from A and S; Amino acid sequence and iv) an amino acid sequence having at least 91% identity to the sequence defined in iii); 2. The TSLP-binding polypeptide of claim 1, selected from:

6. 6. The TSLP-binding polypeptide of claim 5, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 875.

7. xi) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1093), [BM] is an amino acid sequence as defined in any one of claims 1 to 3; and xii) an amino acid sequence having at least 86% identity to the sequence defined in xi); 2. The TSLP-binding polypeptide of claim 1, comprising an amino acid sequence selected from:

8. 8. The TSLP-binding polypeptide of claim 7, wherein sequence xi) is SEQ ID NO: 645 and 876-972.

9. K of interaction with TSLP Ｄ The value is at most 1 x 10 －６ 2. The TSLP binding polypeptide of claim 1, which is capable of binding to TSLP such that M.

10. A fusion protein or conjugate comprising: - a first part consisting of a TSLP-binding polypeptide according to claim 1; and a second portion consisting of a polypeptide having a desired biological activity; A fusion protein or conjugate comprising:

11. A polynucleotide encoding the TSLP-binding polypeptide of claim 1 or the fusion protein of claim 10.

12. 11. A composition comprising a TSLP binding polypeptide according to claim 1, a fusion protein or conjugate according to claim 10, and at least one pharmaceutically acceptable excipient or carrier.

13. 13. A TSLP binding polypeptide according to claim 1, a fusion protein or conjugate according to claim 10, or a composition according to claim 12 for use as a pharmaceutical, diagnostic or prognostic agent.

14. 14. A TSLP binding polypeptide, fusion protein, conjugate or composition for use as claimed in claim 13 in the treatment, prognosis or diagnosis of a TSLP-associated disorder or disease, such as an inflammatory disease, an autoimmune disease or a cancer disease.

15. 15. The TSLP binding polypeptide, fusion protein, conjugate or composition for use according to claim 14, wherein the disorder or disease is selected from the group consisting of asthma, atopic dermatitis, atopic keratoconjunctivitis, urticaria, allergic rhinitis, chronic rhinosinusitis with nasal polyposis, eosinophilic esophagitis, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis (EGPA) / Churg-Strauss syndrome, breast cancer, prurigo nodularis, and bullous pemphigoid.

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