IL-17a binding polypeptides

Abstract

The present disclosure relates to a class of engineered polypeptides having binding affinity to interleukin-17A (IL-17A) and provides IL-17A binding polypeptides comprising the sequence EX2DX4AX6X7EIX 10 X 11 LPNL X 16 X 17 X 18 QX 20 X 21 AFIX 25 X 26 LX 28 X 29‑ The present disclosure relates to a class of engineered polypeptides having binding affinity to interleukin-17A (IL-17A) and provides IL-17A binding polypeptides comprising the sequence EX2DX4AX6X7EIX 10 X 11 LPNL X 16 X 17 X 18 QX 20 X 21 AFIX 25 X 26 LX 28 X 29‑ The present disclosure relates to a class of engineered polypeptides having binding affinity to interleukin-17A (IL-17A) and provides IL-17A binding polypeptides comprising the sequence EX2DX4AX6X7EIX 10 X 11 LPNL X 16 X 17 X

Description

[0001] This application is a divisional application of the application filed on January 12, 2016, with application number 201680011379.3, entitled "IL-17A-binding polypeptide". Invention Field

[0002] This disclosure relates to a class of engineered peptides having a binding affinity for interleukin-17A (hereinafter referred to as IL-17A). This disclosure also relates to the use of such interleukin-17A binding peptides as diagnostic agents, prognostic agents, and / or therapeutic agents. background

[0003] The interleukin-17 (IL-17) family is a family of pro-inflammatory cytokines that contribute to the pathogenesis of several inflammatory diseases. The primary source of IL-17 is the T cell lineage called helper T cells 17 (Th17 cells), which differ from the classic Th1 and Th2 cell subsets. Studies in mouse models and humans have identified the crucial roles of IL-17 and Th17 cells in the pathogenesis of inflammation and autoimmunity, as well as in host defense against certain pathogens. Based on these observations, IL-17 and Th17 cells are considered targets of interest for the treatment of several chronic inflammatory diseases such as psoriasis, rheumatoid arthritis (RA), ankylosing spondylitis (AS), systemic lupus erythematosus (SLE), and multiple sclerosis (MS) (Miossec and Kolls, 2012, Nat Rev Drug Discov 11:763-7).

[0004] The disulfide-linked homodimeric cytokine IL-17A is a member of the IL-17 family, which also includes IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. Within this family, IL-17A and IL-17F show the highest amino acid sequence homology (50%) with each other, and they bind to the same receptors: IL-17 receptor A (IL-17RA) and IL-17 receptor C (IL-17RC). Furthermore, IL-17A and IL-17F can be expressed together as a heterodimer. Despite their high amino acid sequence homology, IL-17A and IL-17F perform different functions. IL-17A is involved in the development of autoimmunity, inflammation, and tumors, and also plays an important role in host defense against bacterial and fungal infections. On the other hand, IL-17F is primarily involved in mucosal host defense mechanisms (Iwakura et al., 2011, Immunity 34:149-62).

[0005] When secreted, IL-17A promotes the production of various pro-inflammatory cytokines, chemokines, antimicrobial peptides, and metalloproteinases (MMPs) from fibroblasts, endothelial cells, and epithelial cells. A key role of IL-17A is the induction of granulocyte production and the recruitment of neutrophils to sites of inflammation. However, if left uncontrolled, this response can lead to chronic inflammation, accompanied by tissue destruction and angiogenesis (Iwakura et al. 2008, Immunol Rev 226:57-79; Reynolds et al. 2010, Cytokine Growth Factor Rev 21:413-23). ​​IL-17A is central to the pathogenesis of psoriasis, a common chronic inflammatory skin disease affecting approximately 2.5% of the world's population (reviewed in Chiricozzi and Krueger, 2013, Expert Opin. Investig. Drugs 22(8):993-1005). Studies in patients with rheumatoid arthritis (RA) have shown that IL-17A-positive cells are present in the inflamed synovium. In mouse models of RA, administration of IL-17A via intra-articular gene transfer resulted in severe worsening of clinical scores (Lubberts et al. 2002, Inflamm Res 51:102-4). Conversely, IL-17A is protected against the development and outcomes of arthritis by inhibition of monoclonal antibodies targeting this ligand or receptor (Lubberts et al. 2004, Arthritis Rheum 50:650-9). In MS patients, IL-17A gene overexpression has been reported (Lock et al. 2002, Nat Med 8:500-8), and IL-17A and Th17 cells have been clearly involved in mouse models of experimental autoimmune encephalitis (Cua et al. 2003, Nature 421:744-8; Uyttenhove and Van Snick 2006, Eur J Immunol 36:2868-74). Increased IL-17A levels have been shown to be clinically associated with various ocular inflammatory diseases, such as uveitis, scleritis, and dry eye disease (DED), in patients with arthritis (Kang et al. 2011, J Korean Med Sci 26:938-44). Recent studies have shown IL-17 and IFNγ-positive cells in clinical samples of coronary atherosclerosis, suggesting a local role in vascular dysfunction (Eid et al. 2009, J Cardiothorac Surg 4:58). IL-17A may also be significant for chronic obstructive pulmonary disease (COPD).The number of IL-17A-positive cells is increased in lung tissue from COPD patients (Chu et al. 2011, Int Immunopharmacol 11:1780-8; Di Stefano et al. 2009, Clin ExpImmunol 157:316-24). In COPD mouse models, IL-17RA-deficient mice are resistant to the development of emphysema, while overexpression of IL-17A accelerates the development of emphysema, indicating that IL-17A is sufficient to mediate this response (Chen et al. 2011, PLoS One 6:e20333; Shan et al. 2012, Sci Transl Med 4:117ra9). Therefore, the involvement of IL-17A in several different autoimmune and inflammatory diseases suggests the broad applicability of IL-17A-targeted therapies.

[0006] Targeting IL-17A or its receptor is the most direct way to block IL-17A-mediated function. Several biologics that neutralize IL-17A signaling, including the anti-IL-17A monoclonal antibodies secukinumab and ixekizumab, are currently in clinical development (Patel et al., 2013, Ann Rheum Dis 72 Supplement 2:ii116-23). ​​Secukinumab has been approved for the treatment of psoriasis and is currently being investigated for the treatment of psoriatic arthritis (PsA) and asthma. Ixekizumab is currently undergoing clinical trials for psoriasis, PsA, and RA. Blocking IL-17 receptor-mediated signaling is also under clinical investigation, including the human monoclonal anti-IL-17RA antibody brodalumab for the treatment of psoriasis, RA, and asthma (Hu et al., 2011, Ann NY Acad Sci 1217:60-76). Therefore, the clinical efficacy of IL-17A inhibition in various diseases, particularly psoriasis, has been confirmed, and the safety profile, including phase II and III data, shows good tolerability of IL-17A inhibitors (Genovese et al. 2010, Arthritis Rheum 62:929-39 and Hueber et al. 2010, Sci Transl Med 2:52ra72).

[0007] The unpredictable and chronic nature of psoriasis and other inflammatory diseases, along with the high degree of unmet medical needs, necessitates the development of new modes of treatment.

[0008] Because tissue permeability is negatively correlated with molecular size, relatively large antibody molecules inherently exhibit poor tissue distribution and penetration. Furthermore, although antibodies are widely used in a variety of routine applications (such as for analytical, purification, diagnostic, and therapeutic purposes) due to their high affinity and specificity for many possible antigens, they still suffer from several drawbacks. These drawbacks include the need for complex mammalian expression systems, a tendency to aggregate, limited solubility, the need to form and stably maintain disulfide bonds, and the risk of unwanted immune responses.

[0009] Therefore, the use of monoclonal antibodies is not always optimal for therapy, and there remains a persistent need for agents with high affinity for IL-17A. There is also great interest in providing the use of such molecules in the treatment, diagnosis, and prognosis of diseases. Invention Overview

[0010] The purpose of this disclosure is to provide novel IL-17A binders that can be used, for example, for diagnostic, prognostic, and therapeutic applications.

[0011] The purpose of this disclosure is to provide novel IL-17A binding agents that can be used as domains in fusion proteins containing one or more additional domains having similar or other functions.

[0012] The purpose of this disclosure is to provide a molecule that allows for effective therapies targeting multiple forms of inflammatory and autoimmune diseases while mitigating the aforementioned and other drawbacks of current therapies.

[0013] Another objective of this disclosure is to provide a molecule suitable for prognostic and diagnostic applications.

[0014] These and other objectives, which will be apparent to those skilled in the art from the content of this disclosure, are satisfied by the various aspects of the invention as claimed in the appended claims and as generally disclosed herein.

[0015] Therefore, in a first aspect of this disclosure, an IL-17A binding polypeptide is provided, the IL-17A binding polypeptide comprising an IL-17A binding motif BM, the motif being composed of an amino acid sequence selected from:

[0016] i) EX2DX4AX6X7EIX 10 X 11 LPNL X 16 X 17 X 18 QX 20 X 21 AFIX 25 X 26 LX28 X 29

[0017] Among them, independently of each other,

[0018] X2 is selected from A, H, M, and Y;

[0019] X4 is selected from A, D, E, F, K, L, M, N, Q, R, S, and Y;

[0020] X6 is selected from A, Q, and W;

[0021] X7 is selected from F, I, L, M, V, W, and Y;

[0022] X 10 Selected from A and W;

[0023] X 11 Selected from A, D, E, F, G, L, M, N, Q, S, T, and Y;

[0024] X 16 Selected from N and T;

[0025] X 17 Selected from H, W, and Y;

[0026] X 18 Selected from A, D, E, H, and V;

[0027] X 20 Selected from A, G, Q, S, and W;

[0028] X 21 Selected from A, D, E, F, H, K, N, R, T, V, W, and Y;

[0029] X 25 Selected from A, D, E, G, H, I, L, M, N, Q, R, S, T, and V;

[0030] X 26 Selected from K and S;

[0031] X 28 Selected from I, L, N, and R; and

[0032] X 29 Selected from D and R;

[0033] and

[0034] ii) An amino acid sequence that has at least 89% identity with the sequence defined in i).

[0035] The above definition of a class of sequence-related IL-17A-binding peptides is based on a statistical analysis of numerous random peptide variants of a parental scaffold selected for their interaction with IL-17A in several different selection experiments. The identified IL-17A-binding motif, or “BM,” corresponds to the target binding region of the parental scaffold, which constitutes two α-helices within a triple-helix bundle protein domain. In the parental scaffold, altered amino acid residues of the two BM helices constitute a binding surface for interaction with the constant Fc portion of the antibody. In this disclosure, random variations of the binding surface residues and subsequent variant selection have replaced Fc interaction capability with the ability to interact with IL-17A.

[0036] As those skilled in the art will recognize, the function of any polypeptide, such as the IL-17A binding capacity of the polypeptides of this disclosure, depends on the polypeptide's tertiary structure. Therefore, it is possible to make minor alterations to the amino acid sequence of a polypeptide without affecting its function. Consequently, this disclosure includes modified variants of the IL-17A-binding polypeptide, said variants in which the IL-17A binding characteristic is retained.

[0037] In this manner, this disclosure also includes an IL-17A-binding polypeptide comprising an amino acid sequence having 89% or greater identity with the polypeptide defined as in i). In some embodiments, the polypeptide may comprise at least 93%, such as at least 96%, of the same sequence as the polypeptide defined as in i). For example, it is possible to exchange amino acid residues belonging to a specific functional group of amino acid residues (e.g., hydrophobic, hydrophilic, polar, etc.) for another amino acid residue from the same functional group.

[0038] In some embodiments, such changes can be made at any position in the sequence of the IL-17A-binding peptide as disclosed herein. In other embodiments, such changes can be made only at non-variable positions, also known as scaffold amino acid residues. In such cases, changes at variable positions, i.e., positions indicated by "X" in sequence i), are not permitted.

[0039] The term "% identity," used throughout this specification, can be calculated, for example, as follows: The query sequence is aligned with the target sequence using the CLUSTAL W algorithm (Thompson et al., Nucleic Acids Research, 22: 4673-4680 (1994)). Comparisons are made on a window corresponding to the shortest aligned sequence. In some cases, the shortest aligned sequence may be the target sequence. In other cases, the query sequence may constitute the shortest aligned sequence. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have the same counterpart in the target sequence is reported as the identity %.

[0040] In a particular embodiment according to the first aspect, a polypeptide as defined above is provided, wherein, in sequence i),

[0041] X2 is selected from A, H, and M;

[0042] X4 is selected from A, D, E, F, L, M, N, Q, R, and Y;

[0043] X 11 Selected from A, D, E, F, G, L, M, N, S, T, and Y;

[0044] X 18 Selected from A, D, E, and V;

[0045] X 20 Selected from A, G, Q, and W;

[0046] X 21 Selected from E, F, H, N, R, T, V, W, and Y;

[0047] X 25 Selected from A, D, E, G, H, I, L, N, Q, R, S, T, and V; and

[0048] X 28 Selected from I, N, and R.

[0049] In another specific embodiment according to the first aspect, a polypeptide as defined in the preceding paragraph is provided, wherein, additionally, in sequence i),

[0050] X 16 Let T be the value of T.

[0051] X 17 For W;

[0052] X 21 Selected from E, F, H, W, T, and Y;

[0053] X 25 Selected from A, D, E, G, H, I, L, N, Q, R, S, and T;

[0054] X 26 Let K be K; and

[0055] X 29 The answer is D.

[0056] “X n "and "X m"In this document, the amino acids at positions n and m in sequence i) as defined above are used to indicate the positions of the amino acids in sequence i) when counted from the N-terminus. For example, X3 and X7 indicate the amino acids at positions 3 and 7 from the N-terminus of sequence i), respectively."

[0057] In an embodiment according to the first aspect, a polypeptide is provided, wherein X in sequence i) n Independently selected from a set of possible residues listed in Table 1. Those skilled in the art will understand that X n It can be selected from any of the listed possible residue groups and the selection is independent of X. m The selection of amino acids in the formula, where n ≠ m. Therefore, the positions X listed in Table 1... n Any of the possible residues in can be independently combined with any other possible residues in any of the other variable positions listed in Table 1.

[0058] Those skilled in the art will understand that Table 1 should be interpreted as follows: In one embodiment according to the first aspect, a polypeptide is provided, wherein the amino acid residue "X" in sequence i) n "Selected from "possible residues". Therefore, Table 1 discloses several specific and individualized embodiments of the first aspect of this disclosure. For example, in one embodiment according to the first aspect, a polypeptide is provided in which X4 in sequence i) is selected from A, D, E, F, L, N, Q, R, and Y, and in another embodiment according to the first aspect, a polypeptide is provided in which X4 in sequence i) is selected from D, E, N, and Y. For the avoidance of doubt, the listed embodiments can be freely combined in yet other embodiments. For example, one such combination embodiment is where X4 is selected from D, E, F, N, Q, R, and Y, and X7 is selected from F, V, and W, and X... 18 Polypeptides selected from A, D, E, and H, etc.

[0059] Table 1:

[0060]

[0061]

[0062] In a more specific embodiment defining the subclass of the IL-17A binding peptide, sequence i) satisfies at least 6 of the following 11 conditions I-XI:

[0063] I. X2 is A;

[0064] II. X4 is selected from D, E, and Q;

[0065] III. X6 is A;

[0066] IV. X7 is selected from F and V;

[0067] V. X 16 Let T be the value of T.

[0068] VI. X 17 For W;

[0069] VII. X 18 Choose from A and D;

[0070] VIII. X 20 For W;

[0071] IX. X 26 Let K be the value of K.

[0072] X. X 28 Let R be the value of R; and

[0073] XI. X 29 The answer is D.

[0074] In some examples of IL-17A binding peptides according to the first aspect, sequence i) satisfies at least 7 of the 11 conditions I-XI. More particularly, sequence i) may satisfy at least 8 of the 11 conditions I-XI, such as at least 9 of the 11 conditions I-XI, such as at least 10 of the 11 conditions I-XI, such as all of the 11 conditions I-XI.

[0075] In some embodiments of the IL-17A binding peptide according to the first aspect, X2X6, X2X 10 or X6X 10 For AA. In some implementations, X2X 17 X2X 20 X6X 17 X6X 20 X 10 X 17 or X 10 X 20 For AW. In some implementations, X2X 28 X6X 28 or X 10 X 28 For AR. In some implementations, X 17 X 28 or X 20 X 28 For WR. In some implementations, X 17 X 20 For WW.

[0076] As described in detail in the experimental section below, the selection of IL-17A-binding peptide variants has led to the identification of a large number of individual IL-17A-binding motif (BM) sequences. These sequences constitute individual embodiments according to sequence i). The sequences of the individual IL-17A-binding motifs correspond to... Figure 1 The amino acid positions 8-36 of SEQ ID NO:1-1216 are shown. Therefore, in one embodiment of the IL-17A binding polypeptide according to this aspect, sequence i) corresponds to the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO:1-1216. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO:1-66, 1200, 1206, and 1214. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO:1-66. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO:1-35. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO:1-27. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO:1-10. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO:1-7. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO:1-4. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1.

[0077] In some embodiments of this disclosure, BM, as defined above, "forms" a portion of a triple-helix bundle protein domain. This is understood to mean that the sequence of BM is "inserted" into or "transplanted" onto the sequence of the original triple-helix bundle domain, such that BM replaces a similar structural motif in the original domain. For example, not wanting to be bound by theory, BM is considered to constitute two of the three helices of the triple-helix bundle and thus can replace any such double-helix motif within the triple-helix bundle. As those skilled in the art will recognize, the replacement of two helices of the triple-helix bundle domain by two BM helices must be done in a manner that 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 consistent with the overall folding of the triple-helix bundle protein domain to which it forms a portion, for example, having the same secondary structural elements in the same order. Therefore, if the polypeptide according to this embodiment of this aspect has the same folding as the original domain, then the fact that BM "forms" a portion of the triple-helix bundle domain according to this disclosure implies shared basic structural characteristics, such as those that result in similar CD spectra. Other relevant parameters are known to those skilled in the art.

[0078] In certain embodiments, the IL-17A binding motif (BM) thus forms part of a triple-helix bundle protein domain. For example, the BM may substantially constitute two α-helices with interconnected loops within the triple-helix bundle protein domain. In certain embodiments, the triple-helix bundle protein domain is selected from domains of bacterial receptor proteins. Non-limiting examples of such domains include the five different triple-helix domains of protein A from Staphylococcus aureus, such as domain B, and its derivatives. In some embodiments, the triple-helix bundle protein domain is a variant of protein Z derived from domain B of Staphylococcus aureus protein A.

[0079] In embodiments where the IL-17A binding polypeptide forms part of a triple-helix bundle protein domain, as disclosed herein, the IL-17A binding polypeptide may comprise a binding module (BMod) selected from the following amino acid sequences:

[0080] iii) K-[BM]-DPSQS X a X b LLX c EAKKL X d X e X f Q;

[0081] in

[0082] [BM] is the IL-17A binding motif as defined in this paper, provided that X 29 The answer is D;

[0083] X a Selected from A and S;

[0084] X b Selected from N and E;

[0085] X c Selected from A, S, and C;

[0086] X d Selected from E, N, and S;

[0087] X e Selected from D, E, and S;

[0088] X f Selected from A and S; and

[0089] iv) An amino acid sequence that has at least 85% identity with the sequence defined by iii).

[0090] In some embodiments, it may be advantageous for the polypeptide to exhibit high structural stability during production or storage and in vivo, such as resistance to chemical modifications, changes in physical conditions, and protein hydrolysis. Therefore, in embodiments where the IL-17A-binding polypeptide, as disclosed herein, forms part of a triple-helix bundle protein domain, the IL-17A-binding polypeptide may comprise a binding module (BMod) selected from the amino acid sequence:

[0091] v) K-[BM]-QPEQS X a X b LLX c EAKKL X d X e X f Q,

[0092] in

[0093] [BM] is the IL-17A binding motif as defined in this paper, provided that X 29 R;

[0094] X a Selected from A and S;

[0095] X b Selected from N and E;

[0096] X c Selected from A, S, and C;

[0097] X d Selected from E, N, and S;

[0098] X e Selected from D, E, and S;

[0099] X f Selected from A and S; and

[0100] vi) An amino acid sequence that has at least 85% identity with the sequence defined by v).

[0101] As discussed above, polypeptides containing minor alterations compared to the above amino acid sequences are also within the scope of this disclosure, the alterations having little impact on the polypeptide's tertiary structure or function. Therefore, in some embodiments, sequences iv) and vi) respectively have at least 87% identity, such as at least 89% identity, such as at least 91% identity, such as at least 93% identity, such as at least 95% identity, such as at least 97% identity.

[0102] In one implementation, X in sequence iii) or v) a The answer is A.

[0103] In one implementation, X in sequence iii) or v) a Let it be S.

[0104] In one implementation, X in sequence iii) or v) b Let N be the number of elements in the array.

[0105] In one implementation, X in sequence iii) or v) b The value is E.

[0106] In one implementation, X in sequence iii) or v) c The answer is A.

[0107] In one implementation, X in sequence iii) or v) c Let it be S.

[0108] In one implementation, X in sequence iii) or v) c The answer is C.

[0109] In one implementation, X in sequence iii) or v) d The value is E.

[0110] In one implementation, X in sequence iii) or v) d Let N be the number of elements in the array.

[0111] In one implementation, X in sequence iii) or v) d Let it be S.

[0112] In one implementation, X in sequence iii) or v) e The answer is D.

[0113] In one implementation, X in sequence iii) or v) e The value is E.

[0114] In one implementation, X in sequence iii) or v) e Let it be S.

[0115] In one implementation, X in sequence iii) or v) d X e Selected from EE, ES, SD, SE, and SS.

[0116] In one implementation, X in sequence iii) or v) d X e For ES.

[0117] In one implementation, X in sequence iii) or v) d X e It is SE.

[0118] In one implementation, X in sequence iii) or v) d X e For SD.

[0119] In one implementation, X in sequence iii) or v) f The answer is A.

[0120] In one implementation, X in sequence iii) or v) f Let it be S.

[0121] In one implementation, in sequence iii) or v), X a For A; X b For N; X c For A and X f The answer is A.

[0122] In one implementation, in sequence iii) or v), X a S; X b For E; X c For A and X f The answer is A.

[0123] In one implementation, in sequence iii) or v), X a For A; X b For N; X c C and X f The answer is A.

[0124] In one implementation, in sequence iii) or v), X a S; X b For E; X c For S and X f Let it be S.

[0125] In one implementation, in sequence iii) or v), X a S; X b For E; X c For S and X f The answer is A.

[0126] In one implementation, in sequence iii) or v), X a S; X b For E; X c For A and X f Let it be S.

[0127] In one implementation, in sequence iii) or v), X a S; X b For E; X c C and X f Let it be S.

[0128] In one implementation, in sequence iii) or v), X a For A; X b For N; X c For A; X d X e For ND and X f The answer is A.

[0129] In one implementation, in sequence iii) or v), X a S; X b For E; X c For A; X d X e For ND and X f The answer is A.

[0130] In one implementation, in sequence iii) or v), X a For A; X b For N; X c For C; X d X e For ND and X f The answer is A.

[0131] In one implementation, in sequence iii) or v), X a S; X b For E; X c S; X d X eFor ND and X f Let it be S.

[0132] In one implementation, in sequence iii) or v), X a S; X b For E; X c S; X d X e For ND and X f The answer is A.

[0133] In one implementation, in sequence iii) or v), X a S; X b For E; X c For C; X d X e For ND and X f Let it be S.

[0134] In one implementation, in sequence iii) or v), X a For A; X b For N; X c For A; X d X e For SE and X f The answer is A.

[0135] In one implementation, in sequence iii) or v), X a S; X b For E; X c For A; X d X e For SE and X f The answer is A.

[0136] In one implementation, in sequence iii) or v), X a For A; X b For N; X c For C; X d X e For SE and X f The answer is A.

[0137] In one implementation, in sequence iii) or v), X a S; X b For E; X c S; X d X e For SE and X f Let it be S.

[0138] In one implementation, in sequence iii) or v), X a S; X b For E; X cFor A; X d X e For SE and X f Let it be S.

[0139] In one implementation, in sequence iii) or v), X a S; X b For E; X c For C; X d X e For SE and X f Let it be S.

[0140] In one implementation, in sequence iii) or v), X a For A; X b For N; X c For A; X d X e For ES and X f The answer is A.

[0141] In one implementation, in sequence iii) or v), X a S; X b For E; X c For A; X d X e For ES and X f The answer is A.

[0142] In one implementation, in sequence iii) or v), X a For A; X b For N; X c For C; X d X e For ES and X f The answer is A.

[0143] In one implementation, in sequence iii) or v), X a S; X b For E; X c S; X d X e For ES and X f Let it be S.

[0144] In one implementation, in sequence iii) or v), X a S; X b For E; X c For C; X d X e For ES and X f Let it be S.

[0145] In one implementation, in sequence iii) or v), X a For A; Xb For N; X c For A; X d X e For SD and X f The answer is A.

[0146] In one implementation, in sequence iii) or v), X a S; X b For E; X c For A; X d X e For SD and X f The answer is A.

[0147] In one implementation, in sequence iii) or v), X a For A; X b For N; X c For C; X d X e For SD and X f The answer is A.

[0148] In one implementation, in sequence iii) or v), X a S; X b For E; X c S; X d X e For SD and X f Let it be S.

[0149] In one implementation, in sequence iii) or v), X a S; X b For E; X c For A; X d X e For SD and X f Let it be S.

[0150] In one implementation, in sequence iii) or v), X a S; X b For E; X c For C; X d X e For SD and X f Let it be S.

[0151] In yet another embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO: 1-1216. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO: 1-66, 1200, 1206, and 1214. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO: 1-66. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO: 1-35. In another embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO: 1-27. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO: 1-10. In yet another embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO: 1-7. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in the sequence selected from the group consisting of SEQ ID NO:1-4, and in another embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1.

[0152] Furthermore, in another embodiment, an IL-17A binding polypeptide is provided, the IL-17A binding polypeptide comprising an amino acid sequence selected from the following:

[0153] vii) YA-[BMod]-AP;

[0154] Where [BMod] is the IL-17A binding module as defined above; and

[0155] viii) is an amino acid sequence that has at least 86% identity with the sequence defined by vii).

[0156] In an alternative embodiment, an IL-17A binding polypeptide is provided, the IL-17A binding polypeptide comprising an amino acid sequence selected from:

[0157] ix) FA-[BMod]-AP;

[0158] Where [BMod] is the IL-17A binding module as defined above; and

[0159] x) is an amino acid sequence that has at least 86% identity with the sequence defined by ix).

[0160] Optionally, an IL-17A binding polypeptide is provided, the IL-17A binding polypeptide comprising an amino acid sequence selected from the following:

[0161] xi) FN-[BMod]-AP;

[0162] Where [BMod] is the IL-17A binding module as defined above; and

[0163] xii) An amino acid sequence that has at least 86% identity with the sequence defined by xi).

[0164] As discussed above, polypeptides containing minor changes compared to the above amino acid sequences without significantly affecting their tertiary structure and function also fall within the scope of this disclosure. Therefore, in some embodiments, an IL-17A-binding polypeptide as defined above may, for example, have at least 88%, such as at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98%, identical to the sequence defined by vii), ix), or xi).

[0165] In some embodiments, the IL-17A binding motif can form part of a polypeptide comprising an amino acid sequence selected from:

[0166]

[0167]

[0168] Where [BM] is the IL-17A binding motif as defined above.

[0169] In one embodiment, the IL-17A binding polypeptide comprises an amino acid sequence selected from the following:

[0170] xiii) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0171] Where [BM] is the IL-17A binding motif as defined above; and

[0172] The amino acid sequence that has at least 86% identity with the sequences defined in xiv) and xiii).

[0173] Again, polypeptides containing minor alterations to the amino acid sequences described above that do not significantly affect their tertiary structure and function are also within the scope of this disclosure. Therefore, in some embodiments, an IL-17A-binding polypeptide as defined above may, for example, have at least 87%, such as at least 89%, such as at least 91%, such as at least 93%, such as at least 94%, such as at least 96%, such as at least 98% of the sequence defined by xiii).

[0174] Sequence xiii) of this type of polypeptide may be selected from the group consisting of SEQ ID NO:1-1216. In one embodiment, sequence xiii) is selected from the group consisting of SEQ ID NO:1-66, 1200, 1206, and 1214. In one embodiment, sequence xiii) is selected from the group consisting of SEQ ID NO:1-66. In one embodiment, sequence xiii) is selected from the group consisting of SEQ ID NO:1-35. In another embodiment, sequence xiii) is selected from the group consisting of SEQ ID NO:1-27. In one embodiment, sequence xiii) is selected from the group consisting of SEQ ID NO:1-10. In one embodiment, sequence xiii) is selected from SEQ ID NO:1-7. In one embodiment, sequence xiii) is selected from the group consisting of SEQ ID NO:1-4. In one embodiment, sequence xiii) is SEQ ID NO:1.

[0175] In one embodiment, the IL-17A binding polypeptide comprises an amino acid sequence selected from the following:

[0176] xv) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0177] Where [BM] is the IL-17A binding motif as defined above; and

[0178] The amino acid sequence that has at least 86% identity with the sequence defined in xvi) and xv).

[0179] Again, polypeptides containing minor alterations to the amino acid sequences described above that do not significantly affect their tertiary structure and function are also within the scope of this disclosure. Therefore, in some embodiments, an IL-17A-binding polypeptide as defined above may, for example, have at least 87%, such as at least 89%, such as at least 91%, such as at least 93%, such as at least 94%, such as at least 96%, such as at least 98% of the sequence defined by xv).

[0180] The sequence xv) in this type of polypeptide can be selected from the group consisting of SEQ ID NO:1217-1222. In one embodiment, the sequence xv) is selected from the group consisting of SEQ ID NO:1218-1222. In one embodiment, the sequence xv) is selected from the group consisting of SEQ ID NO:1219-1222. In another embodiment, the sequence xv) is selected from the group consisting of SEQ ID NO:1219 and SEQ ID NO:1222. In one embodiment, the sequence xv) is SEQ ID NO:1219.

[0181] The small size and robustness of the IL-17A binding domain disclosed in this invention offer several advantages over conventional monoclonal antibody-based therapies. These advantages include the possibility of subcutaneous (sc) administration at higher doses than antibodies, alternative routes of administration, flexibility in formulation for superior potency, and the absence of Fc-mediated side effects. The combination of small size with very high solubility (>100 mg / ml) and stability allows for extremely large molar amounts of drug within a small volume for sc injection. For systemic administration, this implies “home use” therapy for outpatients using convenient, small pre-filled syringes or auto-injectors to deliver small volumes and well-tolerated doses. Furthermore, the ability to achieve high molar concentrations in pharmaceutical products, combined with the ability to maintain functional stability in a variety of formulations, opens up local (skin, eye, lung) administration routes. Psoriasis, asthma, uveitis, and dry eye syndrome are examples of indications where alternative routes of administration may be particularly meaningful in IL-17A-mediated diseases.

[0182] As used herein, the terms "IL-17A binding" and "binding affinity for IL-17A" refer to properties that can be measured, for example, by ELISA, by surface plasmon resonance (SPR) technology, or by kinetic exclusion assay (KinExA). ® The properties of the peptide are tested. For example, as described in the following examples, the binding affinity of IL-17A can be tested in an experiment in which a peptide sample is captured on an antibody-coated ELISA plate and biotinylated IL-17A is added, followed by streptavidin-conjugated HRP. TMB substrate is added, and a multi-well plate reader, such as Victor, is used. 3The absorbance at 450 nm is measured using a Perkin Elmer. A technician can then interpret the results obtained through such experiments to establish at least a qualitative measurement of the binding affinity of the peptide to IL-17A. If a quantitative measurement is desired, for example, to determine the EC50 value (half-maximum effective concentration) of the interaction, an ELISA can also be used. The response of the peptide to a dilution series of biotinylated IL-17A is measured using an ELISA as described above. The technician can then interpret the results obtained through such experiments, and the EC50 value can be calculated from the results using, for example, GraphPad Prism 5 and nonlinear regression.

[0183] The binding affinity of IL-17A can also be tested in experiments in which IL-17A or a fragment thereof is immobilized on the sensor chip of a surface plasmon resonance (SPR) instrument, and a sample containing the peptide to be tested passes through the chip. Alternatively, the peptide to be tested is immobilized on the instrument's sensor chip, and a sample containing IL-17A or a fragment thereof passes through the chip. The results obtained by a technician can then be interpreted to at least establish a qualitative measurement of the binding affinity of the peptide to IL-17A. Quantitative measurements may also be desired, for example, to determine the Kx of the interaction. D The binding value can also be determined using surface plasmon resonance methods. For example, the binding value can be defined in a Biacore (GE Healthcare) or ProteOn XPR 36 (Bio-Rad) instrument. IL-17A is appropriately immobilized on the instrument's sensor chip, and samples of the peptides whose affinity to be determined are prepared by serial dilution and injected in a random order. Then, K... D The value can be calculated from the result using, for example, the 1:1 Langmui combined model of BIAevaluation 4.1 software provided by the instrument manufacturer, or other suitable software.

[0184] Another method for determining the binding affinity to IL-17A is the kinetic exclusion assay (KinExA). ® ;Sapidyne Instruments Inc, Boise, USA; Darling and Brault, 2004. Assay and Drug Dev Tech 2(6):647-657), which is used to measure the equilibrium binding affinity and kinetics between unmodified molecules in solution. For affinity analysis, the equilibrium dissociation constant K D and association rate k a Experimentally determined, the dissociation rate k d It can be based on equation k d = KD * k a calculate.

[0185] KinExA ® K D The analysis requires immobilizing an interacting partner (e.g., a titrated binding partner) on a solid phase, which is then used as a probe to capture other interacting partners (e.g., constant binding partners) free in solution after equilibrium is reached. For each experiment, a series of solutions at constant concentrations of one binding partner are titrated to equilibrium with another binding partner. The solutions are then briefly exposed to the solid phase, and a subset of the free constant binding partners is captured and labeled with fluorescent secondary molecules. The short contact time with the solid phase is less than the time required for the dissociation of the pre-formed complex in solution, meaning that competition between the solution and the titrated binding partners is "kinetically excluded." Since the solid phase is used only as a probe for the free constant binding partners in each sample, the solution equilibrium remains unchanged during the measurement. D The value is calculated from the signal generated by the captured free constant-bound pair, which is proportional to the concentration of the free constant-bound pair in the equilibrated sample. KinExA can be used. ® Pro software and least squares analysis are used to analyze the data to determine K D The optimal solution for the active binding site concentration (ABC) is fitted to a curve representing a stoichiometric model, such as a 1:1 reversible bimolecular interaction.

[0186] Besides the measurement results being collected in a “pre-equilibrium” state, the determination of binding kinetics can be performed in a format similar to equilibrium analysis, and the binding signal is a function of time and the total concentration of the titrated binding coupler. There are two methods that can be used to determine k. a The methods are as follows: The "direct method" keeps the concentrations of the titrated and constant-bound pair constants fixed and probes the solution over time. As the sample moves toward equilibrium, the amount of free constant-bound pair constants in the solution decreases. The "injection method" keeps the incubation time and the concentration of one pair constant while titrating the concentration of the other pair constant. As the concentration of the titrated pair constant increases, the amount of free constant-bound pair constants decreases as more complexes are formed.

[0187] In one embodiment, the IL-17A binding peptide is able to bind IL-17A, thereby enabling the interaction of K... D The value is at most 1 x 10 -6 M, such as at most 1 x 10 -7 M, such as at most 1 x 10 -8 M, such as at most 1 x 10 -9 M.

[0188] In one embodiment, the IL-17A binding peptide according to any aspect disclosed herein is capable of binding IL-17A molecules selected from the group consisting of human IL-17A and mouse IL-17A. In one embodiment, the IL-17A binding peptide is capable of binding human IL-17A. In one embodiment, the IL-17A binding peptide is capable of binding mouse IL-17A. In one embodiment, the IL-17A binding peptide is capable of binding both human IL-17A and mouse IL-17A. In this respect, human IL-17A may comprise the amino acid sequence SEQ ID NO:1226 or an antigenically effective fragment thereof. Similarly, mouse IL-17A may comprise the amino acid sequence SEQ ID NO:1227 or an antigenically effective fragment thereof.

[0189] Those skilled in the art will understand that various modifications and / or additions can be made to the IL-17A-binding peptide according to any aspect of the disclosure herein to tailor the peptide for a specific application without departing from the scope of this disclosure.

[0190] For example, in one embodiment, an IL-17A binding polypeptide as described herein is provided, said IL-17A binding polypeptide having been extended at the C-terminus and / or N-terminus and / or containing additional amino acids. Such polypeptides should be understood as polypeptides having one or more additional amino acid residues at the first and / or last position in the polypeptide chain (i.e., at the N- and / or C-terminus of sequence i) or ii). Therefore, the IL-17A binding polypeptide can contain any suitable number of additional amino acid residues, such as at least one additional amino acid residue. Each additional amino acid residue can be added individually or collectively to, for example, improve and / or simplify the production, purification, in vivo or in vitro stability, conjugation, or detection of the polypeptide. Such additional amino acid residues can comprise one or more amino acid residues added for the purpose of chemical conjugation. One such example is the addition of a cysteine ​​residue. Other amino acid residues can also provide "tags" for the purification or detection of peptides, such as His6 tags, (HisGlu)3 tags ("HEHEHE" tags), or "myc" (c-myc) tags or "FLAG" tags, for interaction with tag-specific antibodies or for immobilization metal affinity chromatography (IMAC) in the case of His6 tags.

[0191] Other amino acids, as discussed above, can be coupled to the IL-17A binding peptide by chemical conjugation (using known organic chemical methods) or by any other method, such as expressing the IL-17A binding peptide as a fusion protein or by any other means directly or via a linker, such as an amino acid linker.

[0192] Other amino acids, as discussed above, can, for example, constitute one or more polypeptide domains. These additional polypeptide domains can provide the IL-17A-binding polypeptide with another function, such as, for example, another binding function, an enzymatic function, a toxic function, a fluorescence signaling function, or a combination thereof.

[0193] Furthermore, additional polypeptide domains can provide another IL-17A binding moiety having the same IL-17A binding function. Therefore, in another embodiment, an IL-17A binding polypeptide in a multimeric form is provided. The multimer is understood to comprise at least two IL-17A binding polypeptides as disclosed herein as monomeric units, the amino acid sequences of which may be identical or different. The multimeric polypeptide may comprise a suitable number of domains, each domain having an IL-17A binding motif, and each domain forming a monomer within the multimer. These domains may have the same amino acid sequence, but optionally, they may have different amino acid sequences. In other words, the IL-17A binding polypeptide of the present invention can form homopolymers or heteropolymers, such as homodimers or heterodimers. In one embodiment, an IL-17A binding polypeptide is provided wherein the monomeric units are covalently coupled together. In another embodiment, the IL-17A binding polypeptide monomeric units are expressed as a fusion protein. In one embodiment, an IL-17A binding polypeptide in a dimer form is provided.

[0194] Furthermore, “heterogeneous” fusion peptides or proteins or conjugates in which the IL-17A-binding peptide or polymer thereof described herein constitutes a first domain or first portion, and where second and additional portions have functions other than binding IL-17A, are also anticipated and fall within the scope of this disclosure. The second and additional one or more portions of the fusion peptide or conjugate in such a protein suitably possess the desired biological activity.

[0195] Therefore, in a second aspect of this disclosure, a fusion protein or conjugate is provided comprising a first portion consisting of an IL-17A-binding polypeptide according to the first aspect, and a second portion consisting of a polypeptide having a desired biological activity. In another embodiment, the fusion protein or conjugate may additionally comprise a further portion having a desired biological activity, which may be the same as or different from the biological activity of the second portion.

[0196] Non-limiting examples of desired biological activities include therapeutic activity, binding activity, and enzymatic activity.

[0197] In one embodiment, the second part having the desired biological activity is a therapeutically active peptide.

[0198] Non-limiting examples of therapeutically active peptides are biomolecules, such as molecules selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines, and lymphokines. Non-limiting examples of anticipated cytokines include IL-2, IL-4, IL-7, IL-10, IL-11, IL-13, IL-21, IL-27, IL-35, IFNβ, and TGFβ. Non-limiting examples of anticipated chemokines include SDF-1 / CXCL12, BCL / BCA-1 / CXCL13, CXCL16, HCC1 / CCL14, TARC / CCL17, PARC / CCL18, MIP-3β / ELC / CCL19, SLC / CCL21, CCL25 / TECK, and CCL27 / CTACK.

[0199] Non-limiting examples of binding activity include binding activity that increases the in vivo half-life of the fusion protein or conjugate. In one particular embodiment, the binding activity is albumin-binding activity that 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 of a streptococcal G protein or a derivative thereof. In another particular embodiment, the binding activity is FcRn-binding activity that increases the in vivo half-life of the fusion protein or conjugate.

[0200] In one embodiment, the fusion protein or conjugate of the second aspect comprises two monomers of the IL-17A-binding polypeptide of the first aspect linked by an albumin-binding moiety, the two monomers having the same or different amino acid sequences. In a particular embodiment of the construct, the fusion protein or conjugate comprises two IL-17A-binding monomers having an albumin-binding moiety between them. The albumin-binding moiety may be, for example, the “GA” albumin-binding domain from a streptococcal G protein, such as “GA3” or a derivative thereof described in any of WO2009 / 016043, WO2012 / 004384, WO2014 / 048977, and WO2015 / 091957.

[0201] In one embodiment, such fusion proteins or conjugates are in the format "ZAZ", where each "Z" is individually an IL-17A-binding polypeptide as described herein, and "A" is an albumin-binding domain. In one embodiment, such IL-17A-binding polypeptides in the "ZAZ" format are capable of binding IL-17A, such that the interacting K... D The value is at most 1 x 10 -10 M, such as at most 1 x 10 -11 M, such as at most 1 x 10 -12M, such as at most 1 x 10 -13 M. In one embodiment, such a “ZAZ” polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1233-1247, such as the group consisting of SEQ ID NO:1236, 1237 and 1242-1247, such as the group consisting of SEQ ID NO:1236, 1244 and 1247 or the group consisting of SEQ ID NO:1237, 1244 and 1247. In even more specific embodiments, the “ZAZ” polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1244 and 1245. In one embodiment, the “ZAZ” polypeptide comprises SEQ ID NO:1244.

[0202] In one embodiment, the binding activity is the binding to angiogenesis-related factors. Non-limiting examples of angiogenesis-related factors include fibroblast growth factor (FGF), fibroblast growth factor 1 (FGF-1), basic FGF, angiogenesis protein 1 (Ang-1), angiogenesis protein 2 (Ang-2), angiopoietin 1 (Angpt-1), angiopoietin 2 (Angpt-2), angiopoietin 3 (Angpt-3), angiopoietin 4 (Angpt-4), tyrosine kinase with immunoglobulin-like domain 1 (TIE-1), tyrosine kinase with immunoglobulin-like domain 2 (TIE-2), vascular endothelial growth factor receptor 1 (VEGFR-1), vascular endothelial growth factor receptor 2 (VEGFR-2), vascular endothelial growth factor receptor 3 (VEGFR-3), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), and vascular endothelial growth factor E. (VEGF-E), placental growth factor (PlGF), transforming growth factor β1 (TGF-β1), transforming growth factor β2 (TGF-β2), transforming growth factor β receptor (type I, II, and III), matrix metalloproteinase (MMP), MET receptor tyrosine kinase (also known as cMET and hepatocyte growth factor receptor (HGFR)), members of the Notch receptor family, and beta-catenin.

[0203] In one embodiment, the binding activity is the binding to an immune response-related factor. Non-limiting examples of immune response-related factors include...

[0204] - T cell regulatory factors such as CD3, CD4, CD6, CD28, T-cell receptor α (TCRα), T-cell receptor β (TCRβ), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), herpes virus entry mediator (HVEM) / B- and T-lymphocyte attenuator (BTLA), killer inhibitory receptor (KIR), lymphocyte activation gene 3 (LAG3), galactagogue-9 (Gal9) / T-cell immunoglobulin mucin-3 (TIM3), and adenosine / α-2 adrenergic receptor (A2aR);

[0205] - NK cell recruitment factors such as CD16, natural killer cell lectin-like receptor gene 2D product (NKG2D), lymphocyte function-associated antigen 1 (LFA1), and natural cytotoxic receptors NKp30 and NKp40.

[0206] - Inflammation-related factors such as

[0207] • Cytokines and their receptors, including tumor necrosis factor (TNF); members of the tumor necrosis factor ligand superfamily (TNFSF) TNFSF11 / RANKL, TNFSF12 / TWEAK, TNFSF13, TNFSF13B / BAFF / BLys, TNFSF14, and TNFSF15; interleukins (ILs) IL-1α, IL-1β, IL-2, IL-5, IL-6, IL-10, IL-12, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-22, IL-23, IL-26, IL-32, IL-33, and IL-34; interferons INFα and INFγ; granulocyte colony-stimulating factor (GCSF) and granulocyte-macrophage colony-stimulating factor receptor (GM-CSF); and

[0208] • Inflammatory chemokines and their receptors, including IL-8 / CXCL8, ENA-78 / CXCL5, GROα / CXCL1, CTAP-III / CXCL7, IP-10 / CXCL10, Mig / CXCL9, PF4 / CXCL4, GCP-2 / CXCL6, MCP-1 / CCL2, MIP-1α / CCL3, MIP-3α / CCL20, RANTES / CCL5, lymphocyte chemokine / XCL1, and fractal chemokine / CX3CL1.

[0209] In a particularly selected embodiment, the binding activity of the second part is binding to a target selected from the group consisting of TNF, IL-1β, IL-6, IL-17F and IL-23.

[0210] In one embodiment of the first or second aspect of this disclosure, an IL-17A binding peptide, fusion protein, or conjugate is provided, said IL-17A binding peptide, fusion protein, or conjugate comprising an immunomodulatory agent. Non-limiting examples of such immunomodulators include immunosuppressants or immunomodulators or other anti-inflammatory agents. For example, the IL-17A binding peptide, fusion protein, or conjugate described herein may comprise agents selected from the group consisting of: disease-modifying antirheumatic drugs (DMARDs), such as gold salts, imidazoline, methotrexate, and leflunomide; calcineurin inhibitors, such as cyclosporine A or FK. 506; lymphocyte recirculation regulators; mTOR inhibitors, such as rapamycin; ascomycin with immunosuppressive properties; glucocorticoids; corticosteroids; cyclophosphamide; immunosuppressive monoclonal antibodies, such as monoclonal antibodies with affinity for leukocyte receptors such as MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or their ligands; adhesion molecule inhibitors, such as LFA-1 antagonists, ICAM-1 or... ICAM-3 antagonists, VCAM-4 antagonists, or VLA-4 antagonists; anti-TNF agents, such as etanercept, and monoclonal antibodies against TNF, such as infliximab and adalimumab; inhibitors of pro-inflammatory cytokines; IL-1 blockers such as anaerobicin or IL-1 traps; IL-6 blockers; chemokine inhibitors; nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin; and anti-infective agents and other immune response modulators; and any combination of two or more of the above.

[0211] In one embodiment of the first or second aspect of this disclosure, an IL-17A-binding polypeptide, fusion protein, or conjugate is provided, said IL-17A-binding polypeptide, fusion protein, or conjugate comprising a toxic compound. Non-limiting examples of such toxic compounds include calicheamicin, maytansinoid, neocarzinostatin, esperamicin, dynemicin, kedarcidin, madroopeptin, doxorubicin, daunorubicin, auristatin, ricin-A chain, modeccin, truncated Pseudomonas exotoxin A, diphtheria toxin, and recombinant gelonin.

[0212] Recent advancements have been made in the development of multispecific agents, such as antibodies capable of binding to more than one antigen. These advancements include engineering complementarity-determining regions (CDRs) to satisfy two antigens at a single antibody binding site (Bostrom et al., 2009, Science 323(5921):1610-1614; Schaefer et al., 2011, Cancer Cell20(4):472-486), constructing heterodimeric antibodies using engineered Fc units (Carter, 2001, JImmunol Methods 248(1-2):7-15; Schaefer et al., 2011, Proc Natl Acad Sci USA 108(27):11187-11192), and fusing helper recognition unit genes to the N-terminus or C-terminus of the light or heavy chain of a full-length antibody (Kanakaraj et al., 2012, MAbs). 4(5):600-613; LaFleur et al., 2013, MAbs 5(2):208-218).

[0213] As discussed above, the following may be beneficial, such as the peptides disclosed herein that have an affinity for IL-17A also exhibiting affinity for another factor, such as immune response-related factors, for example, inflammation-related factors.

[0214] Therefore, in a third aspect of this disclosure, a complex is provided comprising at least one IL-17A binding polypeptide as defined herein and at least one antibody or an antigen-binding fragment thereof.

[0215] When used herein to refer to a third aspect of this disclosure, the term "complex" is intended to refer to two or more associated polypeptide chains, one having an affinity for IL-17A through the action of its IL-17A-binding motif as defined above, and the other being an antibody or an antigen-binding fragment thereof. These polypeptide chains may each contain different protein domains as described above in detail for the IL-17A-binding polypeptides of the first and second aspects, and the resulting multi-protein complex may have multiple functions. "Complex" is also intended to refer to two or more polypeptide chains as defined above, covalently linked, for example, by expressing them as a recombinant fusion protein or by chemical conjugation.

[0216] The third aspect provides a complex comprising 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) through at least one antigen recognition site located in a variable region of an immunoglobulin molecule, such as a carbohydrate, polynucleotide, lipid, polypeptide, or others. As used herein, the term "antibody or antigen-binding fragment thereof" includes not only full-length or complete polyclonal or monoclonal antibodies, but also: antigen-binding fragments thereof, such as Fab, Fab′, F(ab′)2, Fab3, Fv, and variants thereof; fusion proteins comprising one or more antibody moieties; humanized antibodies; chimeric antibodies; minibodies; diabodies; triabodies; tetrabodies; linear antibodies; single-chain antibodies; multispecific antibodies (e.g., bispecific antibodies); and any other modifications to the immunoglobulin molecule containing an antigen recognition site of desired specificity, including glycosylated variants of the antibody, amino acid sequence variants of the antibody, and covalently modified antibodies. Other examples of modified antibodies and their antigen-binding fragments include nanobodies, AlbudAb, DART (dual-affinity retargeting), BiTE (bispecific T-cell engager), TandAb (tandem double-stranded antibody), DAF (dual-action Fab), two-in-one antibodies, SMIP (small modular immunopharmaceuticals), FynomAb (fynomer fused to an antibody), DVD-Ig (double variable domain immunoglobulin), CovX-body (peptide-modified antibody), duobody, and triomAb. This enumeration of variants of antibodies and their antigen-binding fragments is not intended to be limiting, and other suitable variants are known to those skilled in the art.

[0217] Full-length antibodies consist of two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (V0). H ) and the first constant region, the second constant region and the third constant region (C H 1. C H 2 and C H 3). Each light chain contains a light chain variable region (V). L ) and light chain constant region (C L Antibodies are classified into different classes based on the amino acid sequence of the constant structural domains of their heavy chains. There are six major classes of antibodies: IgA, IgD, IgE, IgG, IgM, and IgY, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. As used herein, the term "full-length antibody" refers to any class of antibody, such as IgD, IgE, IgG, IgA, IgM, or IgY (or any of its subclasses). The subunit structures and three-dimensional configurations of the different classes of antibodies are well known.

[0218] An "antigen-binding fragment" is a portion or region of an antibody molecule that retains all or important antigen-binding parts of the corresponding full-length antibody, or a derivative thereof. Antibody-binding fragments may contain heavy chain variable regions (V... H ), light chain variable region (V L ), or both. V H and V L Each typically contains three complementary determinant regions, CDR1, CDR2, and CDR3. H or V L The three CDRs in the image are flanked by the frame regions (FR1, FR2, FR3, and FR4). Examples of antigen-binding fragments, as briefly listed above, include, but are not limited to: (1) Fab fragments, which are V... L -C L Chain and V H -C H (1) a monovalent fragment of chain 1; (2) a Fab' fragment, wherein the Fab' fragment is a Fab fragment having a heavy chain hinge region; (3) an F(ab')2 fragment, wherein the F(ab')2 fragment is a dimer of Fab' fragments connected by heavy chain hinge regions, for example by disulfide bridges in the hinge regions; (4) an Fc fragment; (5) an Fv fragment, wherein the Fv fragment is a V-shaped fragment with an antibody-containing single arm. L and V H The smallest antibody fragment of the structural domain; (6) a single-chain Fv (scFv) fragment, wherein the single-chain Fv (scFv) fragment is the V of scFv therein. H and V LThe domains are linked by peptide linkers to single-chain polypeptides; (7) (scFv)2, wherein the (scFv)2 comprises two V H Structural domain and two V L The structural domain is connected to two V-shaped structures via disulfide bridges. H Domain association, and (8) domain antibody, said domain antibody may be an antibody monovariable domain (V) that specifically binds to the antigen. H or V L Polypeptide.

[0219] Antigen-binding fragments can be prepared using conventional methods. For example, the F(ab′)2 fragment can be produced by pepsin digestion of a full-length antibody molecule, and the Fab fragment can be produced by reducing the disulfide bridge of the F(ab′)2 fragment. Alternatively, fragments can be prepared via recombinant techniques by expressing heavy and light chain fragments in suitable host cells (e.g., E. coli cells, yeast cells, mammalian cells, plant cells, or insect cells) and assembling them to form the desired antigen-binding fragment in vivo or in vitro. Single-chain antibodies can be prepared via recombinant techniques by linking a nucleotide sequence encoding a variable region of the heavy chain and a nucleotide sequence encoding a variable region of the light chain. For example, a flexible linker can be incorporated between the two variable regions. Methods for preparing both full-length antibodies and their antigen-binding fragments are known to those skilled in the art.

[0220] Therefore, in one embodiment, this aspect of the present disclosure provides a complex as defined herein, wherein the at least one antibody or its antigen-binding fragment is selected from the group consisting of: full-length antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, Fc fragments, Fv fragments, single-chain Fv fragments, (scFv)2, and domain antibodies. In one embodiment, the at least one antibody or its antigen-binding fragment is selected from full-length antibodies, Fab fragments, and scFv fragments. In a particular embodiment, the at least one antibody or its antigen-binding fragment is a full-length antibody.

[0221] In one embodiment of the complex as defined herein, the antibody or its antigen-binding fragment is selected from the group consisting of monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies and their antigen-binding fragments.

[0222] As used herein, the term "monoclonal antibody" refers to an antibody with monovalent affinity, meaning that each antibody molecule in a sample binds to the same epitope on the antigen. The term "polyclonal antibody," as used herein, refers to a collection of antibodies that react to a specific antigen, but may contain different antibody molecules, for example, those identifying different epitopes on the antigen. Polyclonal antibodies are typically produced by inoculating suitable mammals and purified from mammalian serum. Monoclonal antibodies are produced from the same immune cells of a clone (e.g., a hybridoma cell line) that serve as the sole parent cell. The term "human antibody," as used herein, refers to an antibody having variable and constant regions that substantially correspond to or are derived from antibodies obtained from human subjects. The term "chimeric antibody," as used herein, refers to a recombinant antibody or genetically engineered antibody, such as, for example, a mouse monoclonal antibody containing polypeptides or domains from a different species, such as humans, which are introduced to reduce the immunogenicity of the antibody. The term "humanized antibody" refers to an antibody derived from a non-human species whose protein sequences have been modified to increase their similarity to naturally occurring antibody variants in humans, thereby reducing immunogenicity.

[0223] For a complex as defined herein, in addition to binding to IL-17A, targeting at least one other antigen, such as an antigen selected from the group consisting of antigens associated with angiogenesis-related disorders and antigens associated with immune responses, may be advantageous. In one embodiment, the other antigen is associated with angiogenesis. In another embodiment, the other antigen is associated with an immune response.

[0224] In one embodiment, the antigen is associated with angiogenesis and is selected from the group consisting of: fibroblast growth factor (FGF), fibroblast growth factor 1 (FGF-1), basic FGF, angiogenesis protein 1 (Ang-1), angiogenesis protein 2 (Ang-2), angiopoietin 1 (Angpt-1), angiopoietin 2 (Angpt-2), angiopoietin 3 (Angpt-3), angiopoietin 4 (Angpt-4), tyrosine kinase with immunoglobulin-like domain 1 (TIE-1), tyrosine kinase with immunoglobulin-like domain 2 (TIE-2), vascular endothelial growth factor receptor 1 (VEGFR-1), vascular endothelial growth factor receptor 2 (VEGFR-2), vascular endothelial growth factor receptor 3 (VEGFR-3), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), and vascular endothelial growth factor E. (VEGF-E), placental growth factor (PlGF), transforming growth factor β1 (TGF-β1), transforming growth factor β2 (TGF-β2), transforming growth factor β receptor (type I, II, and III), matrix metalloproteinase (MMP), MET receptor tyrosine kinase (also known as cMET and hepatocyte growth factor receptor (HGFR)), members of the Notch receptor family, and β-catenin. In one embodiment, the antibody or fragment thereof is selected from the group consisting of AMG 780, AMG 386, MEDI-3617, nesvacumab, CVX-241, bevacizumab, ranibizumab, VGX100, CVX-241, ABP 215, PF-06439535, fresolimumab, metelimumab, onartuzumab, emibetuzumab, and tarextumab.

[0225] In one implementation, the antigen is associated with an immune response or immune system disorder, and is selected from the group consisting of:

[0226] - T cell regulatory factors such as CD3, CD4, CD6, CD28, T-cell receptor α (TCRα), T-cell receptor β (TCRβ), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), herpesvirus invasion mediator (HVEM) / B- and T-lymphocyte attenuator (BTLA), killer cell inhibitory receptor (KIR), lymphocyte activation gene 3 (LAG3), galactagogue-9 (Gal9) / T-cell immunoglobulin mucin-3 (TIM3), and adenosine / α-2 adrenergic receptor (A2aR);

[0227] - NK cell recruitment factors such as CD16, natural killer cell lectin-like receptor gene 2D product (NKG2D), lymphocyte function-associated antigen 1 (LFA1), and natural cytotoxic receptors NKp30 and NKp40.

[0228] - Inflammation-related factors such as

[0229] • Cytokines and their receptors, including tumor necrosis factor (TNF); members of the tumor necrosis factor ligand superfamily (TNFSF) TNFSF11 / RANKL, TNFSF12 / TWEAK, TNFSF13, TNFSF13B / BAFF / BLys, TNFSF14, and TNFSF15; interleukins (ILs) IL-1α, IL-1β, IL-2, IL-5, IL-6, IL-10, IL-12, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-22, IL-23, IL-26, IL-32, IL-33, and IL-34; interferons INFα and INFγ; granulocyte colony-stimulating factor (GCSF) and granulocyte-macrophage colony-stimulating factor receptor (GM-CSF); and

[0230] • Inflammatory chemokines and their receptors, including IL-8 / CXCL8, ENA-78 / CXCL5, GROα / CXCL1, CTAP-III / CXCL7, IP-10 / CXCL10, Mig / CXCL9, PF4 / CXCL4, GCP-2 / CXCL6, MCP-1 / CCL2, MIP-1α / CCL3, MIP-3α / CCL20, RANTES / CCL5, lymphocyte chemokine / XCL1, and fractal chemokine / CX3CL1.

[0231] In one implementation, the antigen is selected from the group consisting of TNF, IL-1β, IL-6, IL-17F and IL-23.

[0232] In one embodiment, the antibody or fragment thereof is selected from the group consisting of visilizumab, otelixizumab, ipilimumab, tremelimumab, pembrolizumab, nivolumab, pidilizumab, MPDL3280A, MEDI-4736, MPDL3280A and lirilumab, and antigen-binding fragments thereof.

[0233] In one specific embodiment, the antigen is TNF. In one embodiment, the antibody or a fragment thereof is selected from the group consisting of adalimumab, infliximab, golimumab, certolimumabpegol, and their antigen-binding fragments. In another embodiment, the antibody or a fragment thereof is a full-length antibody selected from the group consisting of adalimumab, infliximab, golimumab, and certolimumabpegol. In one specific embodiment, the antibody or its antigen-binding fragment is adalimumab or its antigen-binding fragment, such as full-length adalimumab.

[0234] The complexes described herein can exist, for example, as fusion proteins or conjugates. Thus, the at least one IL-17A binding polypeptide and the at least one antibody or its antigen-binding fragment can be coupled by chemical conjugation (using known organic chemical methods) or by any other method such as expressing the complex as a fusion protein, or otherwise linked directly or via a linker such as an amino acid linker.

[0235] Therefore, in one embodiment, a complex as defined herein is provided, wherein the complex is a fusion protein or a conjugate. In one embodiment, the complex is a fusion protein. In another embodiment, the complex is a conjugate. In one embodiment of the complex, the IL-17A binding polypeptide is attached to the N-terminus or C-terminus of the heavy chain of the antibody or its antigen-binding fragment. In another embodiment, the IL-17A binding polypeptide is attached to the N-terminus or C-terminus of the light chain of the antibody or its antigen-binding fragment. In one embodiment, the IL-17A binding polypeptide is attached to the N-terminus and / or C-terminus of both the light and heavy chains of the antibody or its antigen-binding fragment. For example, the IL-17A binding peptide may be attached to the N-terminus of the heavy chain only, the N-terminus of the light chain only, the C-terminus of the heavy chain only, the C-terminus of the light chain only, both the N-terminus and C-terminus of the heavy chain, both the N-terminus and C-terminus of the light chain, only the C-terminus of the light chain and the N-terminus of the heavy chain, or only the C-terminus of the heavy chain and the N-terminus of the light chain.

[0236] As those skilled in the art will understand, the construction of fusion proteins typically involves the use of linkers between functional parts to be fused. Those skilled in the art are aware of different types of linkers with varying 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 of fusion proteins or improve their folding, thereby increasing fusion protein expression, improving their biological activity, affinity, and / or binding, enabling them to target, and altering their pharmacokinetics. Therefore, in one embodiment, an IL-17A-binding peptide, fusion protein, conjugate, or complex as defined herein further comprises at least one linker. The linker may be, for example, selected from the group consisting of flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers. Optionally, the linker may be a non-peptide linker. In one embodiment of a fusion protein or conjugate as disclosed herein, the linker is arranged between a first portion consisting of an IL-17A-binding peptide as defined herein and a second portion consisting of a peptide having the desired biological activity. In one embodiment of a complex as disclosed herein, the linker is arranged between the IL-17A-binding peptide and the antibody or its antigen-binding fragment. Those skilled in the art will appreciate that the presence of a joint arranged in any of the foregoing contexts does not preclude the presence of additional joints in the same or any other circumstances.

[0237] Flexible linkers are typically used when the linked domains require a certain degree of mobility or interaction, and they can be particularly useful in some embodiments of IL-17A-binding peptides, fusion proteins, conjugates, or complexes as defined herein. Such linkers typically comprise small, nonpolar amino acids (e.g., G) or polar amino acids (e.g., S or T). Some flexible linkers consist primarily of extensions of G and S residues, such as (GGGGS)p and (SSSSG). p Adjusting the copy number "p" allows for optimization of the linker to achieve proper separation between functional parts or to maintain necessary inter-part interactions. Besides the 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, A, K, and E, to maintain flexibility, and polar amino acid residues to improve solubility.

[0238] In one embodiment, the linker is a flexible linker comprising glycine (G), serine (S), and / or threonine (T) residues. In one embodiment, the linker comprises residues selected from glycine (G), serine (S), and / or threonine (T). n S m ) p and (S) n G m ) p A sequence of general forms, wherein, independently, n = 1-7, m = 0-7, n + m ≤ 8, and p = 1-10. 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.

[0239] In one embodiment, the connector comprises a sequence selected from the group consisting of G4S, (G4S)2, (G4S)3 and (G4S)4.

[0240] In one embodiment, the connector comprises a general-purpose GT(G) connector. n S m ) p The sequence, wherein independently, n = 1-7, m = 0-7, n + m ≤ 8, and p = 1-10. In one embodiment, n = 1-5. In one embodiment, m = 0-5. In one embodiment, p = 1-5. In a more particular embodiment, n = 4, m = 1, and p = 1-4. Therefore, in an embodiment where n = 4, the connector comprises GT(G4S). pIn one particular embodiment, n = 4 and p = 1, such that the connector comprises GTG4S. In one embodiment, the connector comprises GT(G4S) selected from... p TS, GT (G4S) p PR and GT (G4S) p The sequence of groups composed of PK.

[0241] In one embodiment, the connector comprises a general formula GAP (G n S m ) p The sequence, wherein independently, n = 1-7, m = 0-7, n + m ≤ 8, and p = 1-10. In one embodiment, n = 1-5. In another embodiment, m = 0-5. In yet another embodiment, p = 1-5. In a more specific embodiment, n = 4, m = 1, and p = 1-4. Therefore, in an embodiment where n = 4, the connector comprises GAP (G4S). p In one particular implementation, n = 4 and p = 1, such that the connector comprises GAPG4S. In one implementation, the connector comprises GAP(G4S) selected from GAP. p TS, GAP (G4S) p PR and GAP (G4S) p The sequence of groups composed of PK.

[0242] In one embodiment, the connector comprises a KL(G4S) selection. p LQ(G4S) p And YV (G4S) p The connector comprises a sequence of groups consisting of PK. In one embodiment, the connector comprises a sequence selected from the groups consisting of S4G, (S4G)3, (S4G)4, and (S4G)8. In one embodiment, the connector comprises a sequence selected from the groups consisting of VDGS, ASGS, and VEGS. In a particular embodiment, the connector comprises ASGS.

[0243] Regarding the above description of fusion proteins, conjugates, or complexes incorporating IL-17A-binding peptides according to this disclosure, it should be noted that the designations of first, second, and additional portions are made for clarity, to distinguish, on the one hand, from one or more IL-17A-binding peptides according to the invention, and on the other hand, from portions exhibiting other functions. These designations are not intended to indicate the actual order of the different domains in the polypeptide chain of the fusion protein, conjugate, or complex. Thus, for example, the first portion may appear without limitation at the N-terminus, middle, or C-terminus of the fusion protein, conjugate, or complex.

[0244] This disclosure also includes polypeptides in which the IL-17A-binding polypeptide according to the first aspect, as well as those contained in a fusion protein or conjugate according to the second aspect or in a complex according to the third aspect, further comprises a marker, such as a marker selected from the group consisting of fluorescent dyes and metals, chromophore dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles. For example, such markers can be used to detect polypeptides.

[0245] In embodiments of the IL-17A binding polypeptide labeled therein, comprising the IL-17A binding polypeptide and the label according to the first aspect of this disclosure, the labeled polypeptide can be used to indirectly label IL-17A-expressing cells, such as cells of inflammation-associated cancers. Non-limiting examples of inflammation-associated cancers include gastric cancer, colorectal cancer, non-small cell lung cancer, hepatocellular carcinoma, and adenocarcinoma (Wu et al., 2014 Tumour Biol. 35(6):5347-56; Wu et al., 2012 PLoS One 7(12); Zhang et al., 2012 Asian Pac J Cancer Prev 13(8):3955-60; Liu et al., 2011 BiochemBiophys Res Commun.407(2):348-54).

[0246] In other embodiments, the labeled IL-17A-binding polypeptide is present as a part of a fusion protein, conjugate, or complex that also comprises a second part having the desired biological activity and possibly additional parts. The label may be coupled to only the IL-17A-binding polypeptide in some cases, and to both the IL-17A-binding polypeptide and the second part of the fusion protein or conjugate and / or an antibody or antigen-binding fragment of the complex in other cases. Furthermore, it is possible for the label to be coupled to only the second part or to an antibody or its antigen-binding fragment without being coupled to the IL-17A-binding portion. Thus, in yet another embodiment, an IL-17A-binding polypeptide comprising a second part is provided, wherein the label is coupled to only the second part. In yet another embodiment, a complex as defined herein is provided, wherein the label is coupled to only an antibody or its antigen-binding fragment.

[0247] In one embodiment, the IL-17A binding peptide, fusion protein, conjugate, or complex described herein includes a chelating environment provided by a polyaminopolycarboxylic acid chelating agent conjugated to the IL-17A binding peptide via a thiol group of a cysteine ​​residue or an amino group of a lysine residue.

[0248] In embodiments where IL-17A binds to peptides, fusion proteins, conjugates, or complexes and is radiolabeled, such radiolabeled peptides may contain radionuclides. Most radionuclides are metallic, and metals typically cannot form stable covalent bonds with elements present in proteins and peptides. For this reason, labeling proteins and peptides with radioactive metals is carried out using chelating agents, i.e., polydentate ligands, which form non-covalent compounds with metal ions called chelates. In embodiments where IL-17A binds to peptides, fusion proteins, conjugates, or complexes, the incorporation of the radionuclide is achieved by providing a chelating environment through which the radionuclide can be coordinated, chelated, or complexed to the peptide.

[0249] One example of a chelating agent is a polyaminopolycarboxylic acid chelating agent. Such polyaminopolycarboxylic acid chelating agents can be divided into two categories: macrocyclic chelating agents and acyclic chelating agents.

[0250] In one embodiment, the IL-17A binding peptide, fusion protein, or conjugate includes a chelating environment provided by a polyamino-polycarboxylic acid chelating agent conjugated to the IL-17A binding peptide via a thio group of a cysteine ​​residue or an ε-amino group of a lysine residue.

[0251] The most commonly used macrocyclic chelating agent for radioisotopes of indium, gallium, yttrium, bismuth, radioactinides, and radiolanthanides is various derivatives of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). In one embodiment, the chelating environment for IL-17A-binding peptides, fusion proteins, conjugates, or complexes is provided by DOTA or its derivatives. More specifically, in one embodiment, the chelating peptides included in this disclosure are obtained by reacting the DOTA derivative 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidoethylacetamide (maleimidomonoamide-DOTA)) with said peptide.

[0252] Additionally, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and its derivatives can be used as chelating agents. Therefore, in one embodiment, an IL-17A-binding polypeptide, fusion protein, conjugate, or complex is provided, wherein the polyaminopolycarboxylic acid chelating agent is 1,4,7-triazacyclononane-1,4,7-triacetic acid or its derivative.

[0253] The most commonly used acyclic polyaminopolycarboxylic acid chelating agents are various derivatives of DTPA (diethylenetriaminepentaacetic acid). Therefore, peptides having a chelating environment provided by diethylenetriaminepentaacetic acid or its derivatives are also included in this disclosure.

[0254] In one embodiment, the IL-17A binding peptide, fusion protein, conjugate, or complex described herein further comprises one or more polyethylene glycol (PEG) moieties, for example, to improve the pharmacokinetic properties of the molecule.

[0255] In a fourth aspect of this disclosure, a polynucleotide is provided that encodes an IL-17A-binding polypeptide or fusion protein as described herein; an expression vector comprising the polynucleotide; and a host cell comprising the expression vector.

[0256] This disclosure also includes a method for producing a polypeptide or fusion protein as described above, the method comprising culturing the host cell under conditions that allow the polypeptide to be expressed by its expression vector, and isolating the polypeptide.

[0257] The IL-17A binding peptide of this disclosure can optionally be prepared via non-biological peptide synthesis using amino acids and / or amino acid derivatives with protected reactive side chains, the non-biological peptide synthesis including...

[0258] - Stepwise coupling of amino acids and / or amino acid derivatives to form a polypeptide with protected reactive side chains, according to the first aspect.

[0259] - Removing protecting groups from the reactive side chains of peptides, and

[0260] - Folding peptides in aqueous solutions.

[0261] It should be understood that the IL-17A binding peptides according to this disclosure can be used independently as therapeutic agents, diagnostic agents, or prognostic agents; or as means for targeting other therapeutic agents or diagnostic agents that have direct or indirect effects on IL-17A. Direct therapeutic effects can be achieved, for example, by inhibiting IL-17A signaling, such as by blocking the binding of IL-17A to one or more receptors.

[0262] On the other hand, compositions are provided comprising an IL-17A-binding peptide, fusion protein, conjugate, or complex as described herein, and at least one pharmaceutically acceptable excipient or carrier. In 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. Non-limiting examples of additional active agents that may prove useful in such compositions are the therapeutically active peptides, immune response modifiers, and toxic compounds described herein.

[0263] Those skilled in the art will appreciate that the IL-17 binding peptide, fusion protein, conjugate, or complex, or pharmaceutical composition comprising the IL-17A binding peptide, fusion protein, conjugate, or complex as described herein, can be administered to a subject using standard administration techniques, such as oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, percutaneous, intramuscular, intranasal, sublingual, sublingual, or suppository administration. Therefore, in one embodiment, an IL-17A binding peptide, fusion protein, conjugate, or complex, or pharmaceutical composition as described herein is provided for oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, percutaneous, intramuscular, intranasal, sublingual, sublingual, or suppository administration. In a particular embodiment, an IL-17A binding peptide, fusion protein, conjugate, or complex, or pharmaceutical composition as described herein is provided for oral administration. In another particular embodiment, an IL-17A binding peptide, fusion protein, conjugate, or complex, or pharmaceutical composition as described herein is provided for topical administration, such as topical application to the eye.

[0264] IL-17A can also be used as a valuable biomarker for the diagnosis and prognosis of certain cancers, such as inflammation-related cancers, including gastric cancer, colorectal cancer, non-small cell lung cancer, hepatocellular carcinoma, and adenocarcinoma. For example, IL-17 has been associated with poor prognosis and survival in patients with colorectal cancer and hepatocellular carcinoma.

[0265] Therefore, in another aspect of this disclosure, IL-17A-binding peptides, fusion proteins, conjugates, complexes or compositions as described herein are provided for use as pharmaceuticals, diagnostic agents or prognostic agents.

[0266] In one embodiment, an IL-17A binding peptide, fusion protein, conjugate, complex, or composition as described herein is provided for use as a pharmaceutical agent to regulate IL-17A function in vivo. As used herein, the term "regulation" refers to altering activity, such as partially or completely inhibiting IL-17A function by a sub-functional allele.

[0267] Non-limiting examples of IL-17A-binding peptides that can be used for the treatment, prognosis, and / or diagnosis of IL-17A-related conditions or diseases include: arthritis, such as rheumatoid arthritis, chronic arthritis, arthritis deformans, and rheumatic diseases involving bone loss; inflammatory pain; spondyloarthropathies, including ankylosing spondylitis, Reiter syndrome, reactive arthritis, psoriatic arthritis, enteritis arthritis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, and osteoarthritis; hypersensitivity conditions such as hypersensitivity reactions (including airway hypersensitivity reactions). Hypersensitivity (both hypersensitivity and skin hypersensitivity), allergies and responses to allergen exposure; gastrointestinal conditions or diseases such as autoimmune inflammatory bowel disease (including, for example, ulcerative colitis, Crohn's disease, and irritable bowel syndrome), celiac disease (idiopathic stomatitis), intraperitoneal abscesses and adhesions, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, viral hepatitis, chronic active hepatitis, and Helicobacter pylori-associated gastritis; ocular conditions and diseases such as endocrine ophthalmopathy, Graves' disease, uveitis (anterior and posterior), and keratoconjunctivitis sicca. Sicca (dry eye disease), vernal keratoconjunctivitis, herpetic stromal keratitis, and dry eye disease; nephrological conditions and diseases, such as glomerulonephritis (with or without nephrotic syndrome, including idiopathic nephrotic syndrome or minimal change disease);Acute conditions and diseases, such as acute and hyperacute inflammatory reactions, septic shock (e.g., endotoxic shock and adult respiratory distress syndrome), meningitis, pneumonia, severe burns, acute infections, sepsis, stroke, and ischemia; cachexia (wasting syndrome), such as cachexia associated with pathological TNF release, cachexia due to infection, cachexia associated with cancer, cachexia associated with organ dysfunction, and AIDS-related cachexia; bone-related conditions and diseases, such as bone metabolic diseases, including osteoarthritis, osteoporosis, inflammatory arthritis, and bone loss such as age-related bone loss and periodontal disease. Diseases, including bone implant loosening and bone erosion; juvenile conditions and diseases such as juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, and juvenile vasculitis; vasculitis, including vasculitis of large vessels such as polymyalgia rheumatica, Takayasu's sarteritis, and temporal arteritis, and vasculitis of medium vessels such as Buerger's disease, cutaneous vasculitis, and Kawasaki disease. Diseases and polyarteritis nodosa; small vessel vasculitis such as Behçet's syndrome, Chug-Strauss syndrome, cutaneous vasculitis, Henoch-Schönlein purpura, microscopic polyangiitis, Wegener's granulomatosis, and Golfer's vasculitis; vasculitis of variable vessels and arteritis; skin conditions or diseases such as psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, and atopic dermatitis;Lung conditions and diseases, such as obstructive or inflammatory airway diseases, asthma, bronchitis, COPD, pneumoconiosis, emphysema, acute and hyperacute inflammatory reactions, interstitial lung fibrosis, airway inflammation, and bronchial asthma; metabolic conditions and diseases, such as atherosclerosis, dyslipidemia, and type 1 diabetes; and other systemic autoimmune conditions and diseases, such as systemic lupus erythematosus (SLE), lupus nephritis, polychondritis, myasthenia gravis, Stevens-Johnson syndrome, tumors, myositis, dermatomyositis, adult-onset Still's disease, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, systemic sclerosis, Sjogren's syndrome, multiple sclerosis (MS), Guillain-Barré disease, and Addison's disease. (disease) and Raynaud's phenomenon.

[0268] The IL-17A binding peptide disclosed herein can also be used to treat recipients of heart, lung, combined heart-lung, liver, kidney, pancreas, skin, or corneal grafts, including allogeneic graft rejection or xenograft rejection, and to prevent graft-versus-host disease, such as after bone marrow transplantation and organ graft-related arteriosclerosis.

[0269] The IL-17A binding peptide disclosed herein can also be used for the treatment, diagnosis, or prognosis of inflammation-related cancers. As used herein, the term "cancer" refers to neoplastic diseases and / or cancers such as metastatic or invasive cancers, including lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, rectal cancer, anal cancer, stomach cancer, gastric cancer, colon cancer, colorectal cancer, small bowel cancer, esophageal cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, and renal pelvis carcinoma. Pelvis), mesothelioma, hepatocellular carcinoma, biliary tract cancer, vegetations of the central nervous system (CNS), spinal cord axis tumors, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, adenocarcinoma, lymphoma, lymphocytic leukemia or cancer of unknown origin, or other proliferative or neoplastic IL-17A-related conditions, including resistant forms of any of the above cancers or a combination of one or more cancers or hyperproliferative disorders.

[0270] Non-limiting examples of inflammation-related cancers include stomach cancer, colorectal cancer, non-small cell lung cancer, hepatocellular carcinoma, and adenocarcinoma.

[0271] In one embodiment, an IL-17A binding peptide, fusion protein, conjugate, complex, or composition is provided for use in the treatment, diagnosis, or prognosis of IL-17A-related conditions, such as those selected from inflammatory diseases, autoimmune diseases, and cancer, or conditions such as those comprising the group consisting of inflammatory diseases and autoimmune diseases. In one embodiment, the conditions are selected from the group consisting of inflammatory conditions, allergic conditions, hypersensitivity reactions, autoimmune diseases, severe infections, and graft rejection.

[0272] In one particular implementation, the IL-17A-related conditions are selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, uveitis, and dry eye disease.

[0273] In even more specific embodiments, the IL-17A-related condition is psoriasis.

[0274] In another implementation, the IL-17A-related condition is an inflammation-related cancer, such as cancer selected from the group consisting of gastric cancer, colorectal cancer, non-small cell lung cancer, hepatocellular carcinoma, and adenocarcinoma.

[0275] In a related aspect, a method for detecting IL-17A is provided, the method comprising: providing a sample suspected of containing IL-17A; contacting the sample with an IL-17A-binding peptide, fusion protein, conjugate, complex, or composition as described herein; and detecting the binding of the IL-17A-binding peptide, fusion protein, conjugate, complex, or composition to indicate the presence of IL-17A in the sample. In one embodiment, the method further comprises an intermediate washing step for removing unbound peptides, fusion proteins, conjugates, complexes, or compositions after contact with the sample.

[0276] In one embodiment, the method is a diagnostic or prognostic method for determining the presence of IL-17A in a subject, the method comprising the following steps:

[0277] - Contacting a subject or a sample isolated from a subject with an IL-17A-binding peptide, fusion protein, conjugate, complex, or composition as described herein, and

[0278] - Obtain a value corresponding to the amount of IL-17A-binding polypeptide, fusion protein, conjugate, complex, or composition that has been bound to the subject or the sample.

[0279] In one embodiment, the method further includes an intermediate washing step for removing unbound peptides, fusion proteins, conjugates, complexes, or compositions after contact with the subject or sample and before obtaining values.

[0280] In one embodiment, the method further includes the step of comparing the value with a reference. The reference can be scored by numerical value, threshold, or visual indicators, such as based on a colorimetric reaction. Those skilled in the art will appreciate that various methods of comparison with a reference known in the art may be suitable for use.

[0281] In one implementation of this method, the subject is a mammalian subject, such as a human subject.

[0282] In one implementation, the method is performed in vivo.

[0283] In one embodiment, the method is performed in vitro.

[0284] In a related aspect, a method for treating IL-17A-related conditions is provided, the method comprising administering an effective amount of an IL-17A-binding peptide, fusion protein, conjugate, complex, or composition as described herein to a subject in need of it. In a more specific embodiment of the method, the IL-17A-binding peptide, fusion protein, conjugate, complex, or composition as described herein modulates IL-17A function in vivo.

[0285] In one embodiment, the IL-17A-related condition is selected from the group consisting of inflammatory diseases, autoimmune diseases, and cancer, such as the group consisting of inflammatory diseases and autoimmune diseases. In a particular embodiment of the above aspect, the IL-17A-related condition is selected from the group consisting of inflammatory conditions, allergic conditions, hypersensitivity reactions, autoimmune diseases, severe infections, and graft rejection. In one embodiment, the IL-17A-related condition is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, uveitis, and dry eye disease. In a more specific embodiment, the IL-17A-related condition is psoriasis. In another embodiment, the IL-17A-related condition is cancer, such as cancer selected from the group consisting of gastric cancer, colorectal cancer, non-small cell lung cancer, hepatocellular carcinoma, and adenocarcinoma.

[0286] Administering a therapeutically effective amount of an IL-17A-binding polypeptide, fusion protein, conjugate, complex, or composition as described herein, together with at least one second pharmaceutical substance, such as an immune response modifier, toxic compound, or anticancer agent, may be beneficial.

[0287] As used herein, the term "co-administration" includes both concomitant administration and sequential administration. Therefore, in one embodiment, a method as defined above is provided, the method further comprising co-administering an immune response modulator. In another embodiment, a method as defined above is provided, the method further comprising co-administering an additional anti-inflammatory agent. In another embodiment, a method as defined above is provided, the method further comprising co-administering a toxic compound. In yet another embodiment, a method as defined above is provided, the method further comprising co-administering an anticancer agent.

[0288] The foregoing provides non-limiting examples of immune response modulators and toxic compounds. Non-limiting examples of anticancer agents include those selected from the group consisting of: oligristatin, anthracycline, cefotaxime, cobustatin, doxorubicin, duocarmycin, CC-1065 antitumor antibiotic, ecteinascidin, gerundmycin, maytansinoid, methotrexate, fungal toxins, paclitaxel, ricin, bouganin, white tree toxin, Pseudomonas exotoxin 38 (PE38), diphtheria toxin (DT), and their analogues, derivatives, and combinations thereof. Those skilled in the art will appreciate that non-limiting examples of cytotoxic agents include all possible variations of the aforementioned agents; for example, the agent oligristatin includes, for example, oligristatin E, oligristatin F, oligristatin PE, and their derivatives.

[0289] Although the invention has been described with reference to various exemplary aspects and embodiments, those skilled in the art will understand that many changes can be made and equivalents can be substituted for its elements without departing from the scope of the invention. Furthermore, many modifications can be made to adapt particular situations or molecules to the teachings of the invention without departing from its essential scope. Therefore, it is intended that the invention be limited to any particular embodiment contemplated, but rather that the invention encompass all embodiments falling within the scope of the appended claims. Brief description of the attached diagram

[0290] Figure 1 This document lists the amino acid sequences of examples of IL-17A binding peptides of this disclosure (SEQ ID NO: 1-1222), control peptides (SEQ ID NO: 1223), albumin-binding peptides PP013 (SEQ ID NO: 1224) and PEP07843 (SEQ ID NO: 1225), as well as the amino acid sequences of human IL-17A (SEQ ID NO: 1226), mouse IL-17A (SEQ ID NO: 1227), cynomolgus monkey IL-17A (SEQ ID NO: 1229), rhesus monkey IL-17A (SEQ ID NO: 1230), and human IL-17F (SEQ ID NO: 1228) used for selecting, screening, and / or characterizing examples of the invention. In the IL-17A binding peptides of this disclosure, the inferred IL-17A binding motif (BM) extends from residue 8 to residue 36 in each sequence. The amino acid sequence of the 49-amino acid residue long polypeptide (BMod) that constitutes the complete triple helix bundle in each of these Z variants is predicted to extend from residue 7 to residue 55.

[0291] Figure 2The inhibition of IL-17A-induced IL-6 production, evaluated by the NHDF assay described in Example 3, is shown. (A) His6-Z06260 (hollow rhombus), His6-Z06282 (hollow square), and His6-Z06455 (hollow circle) were titrated in a medium containing IL-17A. (B) Z06260-ABD (solid rhombus), His6-Z06282 (hollow square), Z06282-ABD (solid square), His6-Z06455 (hollow circle), and Z06455-ABD (solid circle) were titrated in a medium containing IL-17A and HSA.

[0292] Figure 3 The IL-17A binding capacity of a set of Z variants from the first and second mature libraries, as determined by ELISA, as described in Example 8, is shown. The results are presented as a percentage of binding capacity compared to IL-17A binding variant Z10241 (SEQ ID NO:11).

[0293] Figure 4 shows the results of the binding specificity analysis performed in the Biacore instrument as described in Example 8. Sensing maps were obtained by injecting 20 nM His6-tagged Z variants Z10508 (SEQ ID NO:2) (A), Z10532 (SEQ ID NO:1) (B), and Z15167 (SEQ ID NO:4) (C) onto the surface of the CM5 chip, respectively, with human IL-17A (black), human IL-17A / F (dark gray), and human IL-17F (light gray).

[0294] Figure 5 The inhibition of IL-17A-induced IL-6 production by a series of Z variants (solid curves) originating from the first mature selection, compared to a conjugate from the primary selection (dashed curve), is shown as described in Example 8. All conjugates inhibit IL-17A in a dose-dependent manner, and the mature conjugates exhibit increased blocking ability compared to the primary conjugate.

[0295] Figure 6 shows the binding of human IL-17A (gray) and cynomolgus IL-17A (black) to (A) ZAZ3363 (SEQ ID NO:1244), (B) ZAZ3364 (SEQ ID NO:1245), and (C) ZAZ3365 (SEQ ID NO:1246) as analyzed in a Biacore instrument as described in Example 11. The resulting curves, obtained by subtracting the responses from the blank surface, correspond to the respective IL-17A protein injected at concentrations of 2.5 nM, 10 nM, and 40 nM.

[0296] Figure 7 illustrates the inhibition of IL-17A-induced IL-6 production in the NHDF assay described in Example 14. Figure 7A shows the superior inhibitory efficacy of the dimer Z-ABD-Z peptide ZAZ3220 (SEQ ID NO:1236; solid black line) compared to the corresponding monomeric Z variant Z10241 (SEQ ID NO:11; dashed black line). Figure 7B shows the evaluation of different linker lengths between the Z and ABD moieties in the Z-ABD-Z peptide containing the Z variant Z06282 (SEQ ID NO:1206). Z-ABD-Z peptides ZAZ3174 (SEQ ID NO:1233), ZAZ3176 (SEQ ID NO:1235), ZAZ3234 (SEQ ID NO:1238), ZAZ3235 (SEQ ID NO:1239), ZAZ3236 (SEQ ID NO:1240), and ZAZ3237 (SEQ ID NO:1241) with different numbers of G4S repeats or minimal linkers on each side of the ABD were compared.

[0297] Figure 8 illustrates the dose-dependent inhibition of the Z-ABD-Z peptide in the hIL-17A-induced KC-model described in Example 15. (A) Complete inhibition of KC production by ZAZ3174 (SEQ ID NO:1233) was obtained at a dose of 2.5 mg / kg. (B) Complete inhibition of KC production by ZAZ3220 (SEQ ID NO:1236) was obtained at a dose of 0.4 mg / kg. The doses indicated on the x-axis are given in mg / kg and corresponding pmol doses. The numbers above the columns indicate the percentage of inhibition in each dose group; 72% inhibition was observed at 0.1 mg / kg ZAZ3220. LOQ = Limit of Quantification.

[0298] Figure 9 shows the pharmacokinetic profiles of Z-ABD-Z peptides (A) ZAZ3220 (SEQ ID NO: 1236) and (B) ZAZ3363 (SEQ ID NO: 1244) after a single intravenous (iv) (black line) or subcutaneous (sc) (gray dashed line) administration to SD rats, as described in Example 16. The mean serum concentration versus time is shown.

[0299] Figure 10 Results of topical application to rabbit eyes as described in Example 17 are shown. Uptake of the Z variant Z10199 (SEQ ID NO: 1217) and the Z-ABD-Z polypeptide ZAZ3174 (SEQ ID NO: 1233) in both the aqueous humor and vitreous humor was demonstrated, while the control IgG antibody did not penetrate the eye.

[0300] Figure 11 shows the activity of ZAZ3363 (SEQ ID NO: 1244) prepared and analyzed in OAF1 and OAF2, respectively, compared with that prepared in PBS, in the NHDF assay described in Example 18. A) OAF1 (solid circle), PBS (cross shape), and OAF1 without ZAZ3363 (hollow rhombus). B) OAF2 (hollow triangle), PBS (cross shape), and OAF2 without ZAZ3363 (hollow rhombus).

[0301] Figure 12 The pharmacokinetic profiles of ZAZ3363 formulated in OAF1 (solid circle), OAF2 (hollow triangle), and PBS (cross shape) for intraduodenal administration, as described in Example 18, are shown.

[0302] Figure 13 shows the complex HC in the NHDF assay described in Example 19. Ada -Z14253 and LC Ada Evaluation of Z14253. (A) Complex HC Ada -Z14253 (left) and LC Ada - Schematic diagram of Z14253 (right). (B) Inhibition of IL-6 production induced by IL-17A. (C) Inhibition of IL-8 production induced by TNF. (D) Inhibition of IL-8 production induced by TNF and IL-17A. Z04726-ABD is a negative control and was included in all three assays.

[0303] Figure 14 shows the pharmacokinetic profiles of the Z-ABD-Z peptide ZAZ3363 (SEQ ID NO: 1244) in cynomolgus monkeys after intravenous administration of 20 mg / kg (black) or 40 mg / kg (grey) on days 1, 4, 7, and 10, as described in Example 20. The mean plasma concentrations as a function of time are shown after the analysis of (A) the first injection on day 1 and (B) the fourth injection on day 10. Error bars represent standard deviations.

[0304] Figure 15 The pharmacokinetic profile of the Z-ABD-Z peptide ZAZ3363 (SEQ ID NO: 1244) in an individual beagle dog following oral administration is shown. 150 mg of ZAZ3363 was administered as an enteric-coated capsule on day 0. Example

[0305] Overview

[0306] The following examples disclose novel Z-variant molecules targeting interleukin-17A (IL-17A) developed based on phage display technology. The IL-17A binding peptides described herein were sequenced, and their amino acid sequences are enclosed in sequence identifier SEQ ID NO:1-1222. Figure 1 The examples are listed below. The examples also describe the characterization of the IL-17A-binding peptide and its in vitro and in vivo functions.

[0307] Example 1

[0308] IL-17A combined with Z variant selection and screening

[0309] Materials and methods

[0310] Biotinylation of target proteinsHuman IL-17A (hIL-17A Peprotech catalog 200-17; SEQ ID NO:1226) and mouse IL-17A (mIL-17A Peprotech catalog 210-17; SEQ ID NO:1227) were biotinylated at room temperature (RT) for 30 min using a 20 x mol excess, according to the manufacturer's recommendations. Subsequent buffer exchanges to phosphate-buffered saline (PBS, 10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) were performed using a dialysis cartridge (Slide-a-lyzer 3.5 K, 3500 MWCO, Thermo Scentific, catalog 66333) according to the manufacturer's instructions.

[0311] Phage display selection with IL-17A binding to the Z variant Essentially, a library of random variants of protein Z displayed on a phage, constructed in phage particle pAY02592 as described by Grönwall et al. (2007) J Biotechnol, 128:162-183, was used to select IL-17A-binding variants of Z. In this library, the albumin-binding domain (abbreviated ABD, corresponding to GA3 of the G protein from Streptococcus strain G148) was used as the fusion partner for the Z variant. This library was designated Zlib006Naive.II and had a size of 1.5 x 10⁻⁶. 10The size of each library member (Z variant). Escherichia coli RRIΔM15 cells (Rüther et al., (1982) Nucleic Acids Res 10:5765-5772) from a glycerol stock solution containing the phage particle library Zlib006Naive.II were inoculated in 20 μL of a defined proline-free medium supplemented with 100 µg / ml ampicillin [3 g / L KH2PO4, 2 g / L K2HPO4, 0.02 g / L uracil, 6.7 g / L YNB (Difco™ amino acid-free yeast nitrogen base, Becton Dickinson), 5.5 g / L glucose monohydrate, 0.3 g / L L-alanine, 0.24 g / L L-arginine monohydrochloride, 0.11 g / L L-asparagine monohydrate, 0.1 g / L L-cysteine, 0.3 g / L L-glutamate, 0.1 g / L L-glutamine, 0.2 g / L L-cysteine, 0.3 g / L L-glutamate, 0.1 g / L L-glutamine, 0.2 g / L L-cysteine, 0.2 ...cysteine, 0.2 g / L L-glutamine, 0.2 g / L L-cysteine, 0.2 g / L L-cysteine, 0.2 g / L L-cysteine, 0.2 g / L L-cysteine, 0.2 g / L L-cysteine, 0.2 g The culture was prepared with [g / l glycine, 0.05 g / l L-histidine, 0.1 g / l L-isoleucine, 0.1 g / l L-leucine, 0.25 g / l L-lysine monohydrochloride, 0.1 g / l L-methionine, 0.2 g / l L-phenylalanine, 0.3 g / l L-serine, 0.2 g / l L-threonine, 0.1 g / l L-tryptophan, 0.05 g / l L-tyrosine, 0.1 g / l L-valine]. The culture was grown in a fermenter (Belach Bioteknik, BR20) at 37°C. The optical density (OD) at 600 nm was measured to be 0.75. 600When using 10 x moles of M13K07 helper phage (New England Biolabs, catalog number N0315S), approximately 2.6 μL of culture was infected. Cells were incubated for 30 min, at which point they were switched to a culture medium supplemented with 100 µM isopropyl-β-D-1-thiogalactopyranoside (IPTG) (for induction of expression) and 50 µg / ml ampicillin, 12.5 µg / ml carbenicillin, 25 µg / ml kanamycin, 35 ml / l 1.217 M MgSO4, and 10 ml trace element solution [129 mM FeCl3; 36.7 mM ZnSO4; 10.6 mM CuSO4; 78.1 mM MnSO4; 94.1 mM CaCl2 dissolved in 1.2 M HCl] [2.5 g / l (NH4)2SO4; 5.0 g / l yeast extract (Merck 1.03753.0500); 25 g / l peptone (Scharlau 07-119); 2 g / l K2HPO4; 3 g / l [KH₂PO₄; 1.25 g / L Na₃C₆H₅O₇ ∙ 2 H₂O; 0.1 ml / L Breox FMT30 antifoamer] Fill the fermenter to 20 L. Begin glucose-restricted fed-batch culture, where a 600 g / L glucose solution is fed into the reactor (15 g / h initially, then 40 g / h at the end of fermentation after 17 h). Maintain the pH at 7 by automatically adding 25% NH₄OH. Add air (10 L / min) and set the stirrer to maintain dissolved oxygen levels above 30%. Remove cells from the culture by tangential flow filtration.

[0312] The phage particles were precipitated twice from the supernatant with PEG / NaCl (polyethylene glycol / sodium chloride), filtered, and dissolved in PBS and glycerol, such as Grönwall et al., as described above. The phage stock solution was stored at -80°C before use.

[0313] Selection for biotinylated hIL-17A (b-hIL-17A) and biotinylated mIL-17A (b-mIL-17A) was performed in four cycles divided into six different tracks (Table 2). Phage stock preparation, the selection procedure, and phage amplification between selection cycles were generally performed as described in WO2009 / 077175 for selection of another target, with the following exceptions. Exception 1: PBS supplemented with 3% bovine serum albumin (BSA, Sigma, catalog number A3059) and 0.1% Tween20 (Acros Organics, catalog number 233362500) was used as the selection buffer. Exception 2: Selection was performed using Dynabeads...® Incubate phage stock with M-280 streptavidin (SA beads, Invitrogen, catalog number 11206D) for pre-selection in cycles 1-3. Exception 3: Pre-block all tubes and beads used in selection with PBS supplemented with 5% BSA and 0.1% Tween 20. Exception 4: Perform selection in solution at RT, with selection occurring at 200 min in the first cycle and 120 min in subsequent cycles. Exception 5: After this, capture the target-phage complex on the SA beads with 1 mg beads / 6.4 µg b-hIL-17A or b-mIL-17A. Exception 6: Use *E. coli* strain XL1-Blue cells (Agilent Technologies, catalog number 200268) grown in medium supplemented with 10 µg / ml tetracycline (Exception 7) for infection. Exception 8: Use tryptone yeast extract plates (15 g / L agar, 10 g / L tryptone water (Merck), 5 g / L yeast extract, 3 g / L NaCl, 2% glucose) supplemented with 0.1 g / L ampicillin and 0.01 g / L tetracycline for bacterial dispersal. Exception 9: 10 x excess of M13K07 helper phage compared to bacteria is permitted to infect logarithmically growing bacteria.

[0314] Table 2 shows an overview of the selection strategy, describing the increased stringency obtained by using a reduced target concentration and an increased number of washes in subsequent cycles.

[0315] Table 2: Overview of selections from the primary library

[0316]

[0317] Track 1 was divided into the second through fourth cycles, resulting in a total of 4 tracks in cycle 2 (1-1 to 1-4), 5 tracks in cycle 3 (1-1-1 to 1-4-2), and 6 tracks in cycle 4 (1-1-1-1 to 1-4-2-1). The number of phage particles used for selection was approximately 2000 times the number of phage particles eluted in the previous cycle, but a higher amount was used in selection tracks 1-1, 1-1-1, and 1-1-1-1.

[0318] Wash with 0.1% PBST (PBS supplemented with 0.1% Tween-20) for 1 min and elute as described in WO2009 / 077175.

[0319] Generation of Z variants for ELISAZ variants were generated by seeding selected monoclonal antibodies into 1 ml TSB-YE medium supplemented with 100 µg / ml ampicillin and 0.1 mM IPTG in deep-well plates (Nunc, catalog number 278752). The plates were incubated at 37°C for 18–24 h. Cells were pelleted by centrifugation, resuspended in 400 µl of 0.05% PBST, and frozen at -80°C to release the periplasmic fraction. The frozen samples were thawed in a water bath, and the freeze-thaw procedure was repeated 6 times. 0.05% PBST was added to the thawed samples, and the cells were pelleted by centrifugation.

[0320] The final periplasmic extract supernatant contained the Z-variant as a fusion with ABD, denoted as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (Grönwall et al., ibid.). Z##### refers to the Z-variant of each of the 58 amino acid residues.

[0321] ELISA screening for Z variant The binding of the Z variant to human IL-17A was analyzed by ELISA. A 96-well ELISA plate (Costar, catalog 3690) was coated overnight at 4°C with anti-ABD goat antibody (internally generated) diluted in coating buffer (50 mM sodium carbonate, pH 9.6; Sigma, catalog C3041) to coat the half-region. The antibody solution was decanted, and the wells were washed in water and blocked with 100 μl of PBSC (PBS supplemented with 0.5% casein) at RT for 1 to 3 h. The blocking solution was discarded, and 50 µl of periplasmic solution was added to the wells and incubated at RT with gentle shaking for 1.5 h. As a blank control, 0.05% PBST was added instead of the periplasmic sample. The supernatant was decanted, and the wells were washed four times with 0.05% PBST. Then, 50 µl of b-hIL-17A at a concentration of 6.5 nM in PBSC was added to each well. The plate was incubated at RT for 1.5 h, followed by washing as described above. Streptoacid-conjugated HRP (Thermo Scientific, catalog number N100), diluted 1:30,000 in PBSC, was added to the wells and the plate was incubated for 1 h. After washing as described above, 50 μl of ImmunoPure TMB substrate (Thermo Scientific, catalog number 34021) was added to the wells, and the plate was treated according to the manufacturer's recommendations. Victor... 3 A perforated plate reader (Perkin Elmer) was used to measure absorbance at 450 nm.

[0322] sequencingAll clones were selected and sequenced in parallel with the ELISA screening. Essentially, as described in WO2009 / 077175, PCR fragments were amplified from individual colonies, sequenced, and analyzed.

[0323] result

[0324] Phage display selection with IL-17A binding to the Z variant After four cycles of phage display selection for b-hIL-17A and b-mIL-17A, individual clones were obtained.

[0325] ELISA screening for Z variant Clones obtained after four cycles of selection were amplified in 96-well plates and screened for hIL-17A binding activity in ELISA. Forty-two Z variants were found to respond to hIL-17A at a concentration of 6.5 nM, yielding 4x the response of the blank control or higher (0.43–3.2 AU). No response was obtained to the blank control.

[0326] sequencing After four cycles of selection, the resulting clones were sequenced. Each variant was assigned a unique identifier #####, and each variant was designated Z#####. The amino acid sequence of the 58-amino acid long Z variant is shown in the figure. Figure 1 The neutralizing sequences are listed as SEQ ID NO:1200-1216 in the sequence listing. In each sequence, the inferred IL-17A binding motif extends from residue 8 to residue 36. The amino acid sequence of the 49-amino acid residue long polypeptide predicted to constitute the complete triple helix bundle within each of these Z variants extends from residue 7 to residue 55.

[0327] Example 2

[0328] The generation of monomeric IL-17A binding to the Z variant

[0329] This embodiment describes the general procedure for subcloning and generating His-tagged Z variants and Z variants fused with ABD, which is used in the characterization experiments below.

[0330] Materials and methods

[0331] Subcloning of Z variants with His-tag DNA from the respective Z variants was amplified from the library vector pAY02592. A subcloning strategy for constructing monomeric Z variant molecules with an N-terminal His6 tag was applied using standard molecular biology techniques (essentially as described in WO2009 / 077175 for Z variants binding to another target). The Z gene fragment was subcloned into the expression vector pAY01448, resulting in the coding sequence MGSSHHHHHHLQ-[Z#####]-VD.

[0332] Subcloning of the Z variant fused with ABD DNA from the respective Z variants was amplified from the library vector pAY02592. PCR was performed using suitable primer pairs, and the resulting gene fragments were cleaved with restriction endonucleases PstI and AccI, and ligated into the expression vector pAY03362 digested with the same enzymes to generate the ABD fusion protein, wherein the ABD variant is PP013 (SEQ ID NO: 1224). The construct encoded by this expression vector is MGSSLQ-[Z#####]-VDSS-PP013.

[0333] nourish E. coli BL21(DE3) cells (Novagen) were transformed with plasmids containing gene fragments of each of the respective IL-17A-binding Z variants and cultured at 37°C in 800 ml or 1000 ml TSB-YE medium supplemented with 50 µg / ml kanamycin. To induce protein expression, [the following was observed] at OD [the following was observed]. 600 When the concentration was 2, IPTG was added to a final concentration of 0.2 mM and the culture was incubated at 37°C for another 5 h. Cells were harvested by centrifugation.

[0334] Purification of IL-17A-binding Z variant with His6-tag Resuspend approximately 2-5 g of each cell precipitate in 30 ml of a solution supplemented with Benzonase. ® (Merck) to a binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) at a concentration of 15 U / ml. After cell lysis, cell debris was removed by centrifugation, and each supernatant was applied 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 the IL-17A-binding Z variant was subsequently eluted with elution buffer 1 (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4).

[0335] Purification of IL-17A-binding Z variant fused with ABD Resuspend approximately 1-2 g of each cell precipitate in 30 ml of solution supplemented with Benzonase. ®TST-buffered (Merck) solution (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween 20, pH 8.0). After cell disruption by sonication and clarification by centrifugation, each supernatant was applied to a gravity flow column containing 1 ml of agarose immobilized with internally generated anti-ABD ligands. The ABD-fused Z variant was eluted with 0.1 M HAc after washing with TST-buffered and 5 mM NH4Ac pH 5.5 buffer. Acetonitrile (ACN) was added to the fraction eluted in the anti-ABD agarose affinity chromatography purification step at a final concentration of 10%, and the sample was loaded onto a 1 ml Resource 15 RPC column (GE Healthcare) pre-equilibrated with RPC solvent A (0.1% TFA, 10% ACN, 90% water). After washing the column with RPC solvent A, the bound protein was eluted with a linear gradient of 0%–50% RPC solvent B (0.1% TFA, 80% ACN, 20% water) in 20 ml. Fractions containing the purified ABD-fusion Z variant were identified and pooled by SDS-PAGE analysis. Following RPC purification, the pool buffer was exchanged for PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) using a PD-10 column (GE Healthcare). Finally, the ABD-fusion Z variant was elute in 1 ml EndoTrap. ® Purification on red (Hyglos) columns ensures low endotoxin levels.

[0336] Protein concentration was achieved using NanoDrop. ® The absorbance at 280 nm and the extinction coefficient of each protein were determined using an ND-1000 spectrophotometer. Purity was analyzed by SDS-PAGE stained with Coomassie Brilliant Blue, and the identity of each purified Z variant was confirmed by LC / MS analysis.

[0337] result

[0338] Cultivation and purificationThe IL-17A-binding Z variant with a His6-tag and the Z variant fused to ABD at its C-terminus were expressed as soluble gene products in *E. coli*. The amount of purified protein from approximately 1–5 g of bacterial precipitate was determined by spectrophotometric measurement of absorbance at 280 nm, ranging from approximately 5 mg to 20 mg for different IL-17A-binding Z variants. SDS-PAGE analysis of each final protein product showed that these products primarily contained the IL-17A-binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.

[0339] Example 3

[0340] Evaluation of the blocking ability of primary IL-17A binding to the Z variant

[0341] Normal human skin fibroblasts (NHDF) produce IL-6 upon stimulation with IL-17A (Chang and Dong 2007, Cell Research 17:435-440), and the amount of IL-6 released into the supernatant is correlated with the concentration of added IL-17A. Therefore, IL-17A blockade leads to a reduction in quantifiable IL-6 in the supernatant. In this embodiment, this assay was used to evaluate the blocking ability of IL-17A alone or in fusion with ABD (PP013; SEQ ID NO:1224) to bind to Z variants Z06260 (SEQ ID NO:1200), Z06282 (SEQ ID NO:1206), and Z06455 (SEQ ID NO:1214).

[0342] Materials and methods

[0343] NHDF determination of His6-tagged Z-variant NHDF cells (Lonza, catalog number CC-2511) were cultured in fibroblast basal medium (Lonza, catalog number CC-3132) supplied with growth promoting factors (Lonza, catalog number CC-5034). The day before the experiment, 10 cells were added to a 100 μl solution of 10... 5Cells / well were seeded into 96-well plates (Greiner, catalog number 655180). On the day of the experiment, dilutions of IL-17A-specific Z variants His6-Z06260, His6-Z06282, and His6-Z06455 were prepared in separate 96-well plates. The Z variants were titrated from 3130 nM to 0.2 nM in a three-step titration with medium containing 0.031 nM hIL-17A. Standard curves of hIL-17A (0.002–31 nM) and controls containing medium with 0.031 nM hIL-17A or medium alone were also prepared. The medium in plates containing overnight-cultured NHDF cells was discarded. Test samples, standard curve samples, and controls were transferred to plates containing cells. NHDF cells were stimulated at 37°C for 18–24 h, and the IL-6 content in the supernatant was subsequently quantified using an IL-6-specific ELISA as described below.

[0344] NHDF determination of His6-tagged and ABD-fused Z variants NHDF cells were prepared as described above. On the day of the experiment, dilutions of the IL-17A-specific Z variant constructs His6-Z06282, His6-Z06455, Z06260-ABD, Z06282-ABD, and Z06455-ABD were prepared in separate 96-well plates. The Z variant constructs were titrated from 780 nM to 0.2 nM in a four-fold increment in medium containing 0.031 nM IL-17A and 8 µM HSA. Standard IL-17A curves and controls were prepared and downstream analyses were performed as described above.

[0345] IL-6 ELISA96-well half-zone plates (Costar, catalog number 3690) were coated with 4 μg / ml anti-IL-6 monoclonal antibody MAB206 (R&D systems) in PBS (50 µl / well) and incubated overnight at 4°C. The next day, the plates were rinsed twice with tap water and blocked with PBS + 2% BSA (Sigma) for 2 h. IL-6 standard (R&D systems, catalog number 206-IL-50) titrated serially at 2-fold dilutions (0.2–50 ng / ml) and supernatant from the cell assay plate were added to the coated ELISA plates (50 µl / well) and incubated at RT for 1.5 h. The plates were washed four times in an automated ELISA washer, and 0.25 µg / ml (50 µl / well) biotinylated anti-IL-6 polyclonal antibody (R&D systems, BAF206) was added. After 1 hour of incubation, the plate was washed, and 50 μl of streptavidin-HRP (Thermo Fisher, catalog number N100), diluted 8000 times, was added to each well. After another hour of incubation and subsequent washing, the plate was developed with 50 µl of TMB (Thermo Fisher, catalog number 34021) per well, and the reaction was terminated with 50 µl of 2MH2SO4. The plate was then read using a 96-well plate reader (Victor). 3 The absorbance at 450 nm was measured, and the concentration of IL-6 in each sample was calculated. The results are presented as the percentage of maximum IL-6 production, calculated as follows:

[0346] 100-[(ABS IL-17A阻断剂 - ABS 背景 ) / (ABS 最大IL-6产生 - ABS 背景 )] x 100

[0347] result

[0348] Results from the first NHDF assay showed that all three Z variants, His6-Z06260, His6-Z06282, and His6-Z06455, blocked IL-17A-induced IL-6 production in a dose-dependent manner. Figure 2 A). The IC50 value is approximately 40-140 nM.

[0349] The NHDF assay was repeated with the same Z variant fused to ABD, and HSA was included in the assay to determine retention of antigen binding under simulated in vivo conditions. In vivo, ABD binds to albumin and delivers a longer circulating half-life. Results showed that all three conjugates fused to ABD blocked IL-17A-induced IL-6 production as well as or better than the His6-tagged Z variant. Figure 2 B). The IC50 values ​​from the second determination are summarized in Table 3.

[0350] Table 3: IC50 values ​​of primary Z-variant blocking IL-17A-induced IL-6 production in the presence of HSA.

[0351]

[0352] Example 4

[0353] Design and construction of the first mature library of IL-17A combined with the Z variant

[0354] In this embodiment, a mature library was designed and constructed. This library was used to select an additional IL-17A binding Z variant. Selection from a mature library is generally expected to produce binders with increased affinity (Orlova et al., (2006) Cancer Res 66(8):4339-48). In this study, split-pool DNA synthesis was used to generate randomized single-linked heads, allowing defined codons to be incorporated into the desired positions during synthesis.

[0355] Materials and methods

[0356] Document DesignThe library is based on the sequence of the IL-17A-binding Z variant identified in Example 1. In the new library, 13 variable positions in the Z molecular scaffold are biased toward certain amino acid residues according to a strategy based on the Z variant sequences defined in SEQ ID NO:1200-1216. DNA adapters containing the following 147 bp sequence ordered from DNA 2.0 (Menlo Park, CA, USA) were generated using split-cell synthesis: 5'-AA ATA AAT CTC GAG GTA GAT GCC AAA TACGCC AAA GAA NNN NNN NNN GCG NNN NNN GAG ATC NNN NNN TTA CCT AAC TTA ACC NNNNNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTATTT A-3' (SEQ ID NO:1248; randomized codons are represented as NNN), which contain partially randomized coding sequences for helices 1 and 2 in the corresponding amino acid sequences, flanked by restriction sites for XhoI and SacI. The theoretical distribution of amino acid residues in a new library comprising 13 variable Z positions (9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32, and 35) within the Z-molecule scaffold is given in Table 4. The resulting theoretical library has a size of 6.1 x 10⁻⁶. 9 Variations.

[0357] Table 4: Library Design, First Mature

[0358]

[0359] Library ConstructionThe library was amplified using AmpliTaq Gold polymerase (Applied Biosystems, catalog 4311816) for 12 cycles as recommended by the supplier, and the pooled products were purified using the QIAquick PCR Purification Kit (QIAGEN, catalog 28106) as recommended by the supplier. The purified pools of randomized library fragments were digested with restriction endonucleases XhoI and SacI-HF (New England Biolabs, catalog 0146L and catalog 3156M, respectively) and concentrated using the QIAquick PCR Purification Kit. Subsequently, the products were subjected to preparative 2.5% agarose gel electrophoresis (Nuisieve GTC agarose, Cambrex, Invitrogen) as recommended by the supplier, and purified from the gel using the QIAGEN Gel Extraction Kit (QIAGEN, catalog 28706).

[0360] The phage vector pAY02592 (essentially the same as pAffi1 described above by Grönwall et al.) was restriction endonucleated with the same enzyme and purified using phenol / chloroform extraction and ethanol precipitation. The restriction endonuclease fragment and the restriction endonuclease vector were ligated at a 5:1 molar ratio using T4 DNA ligase (Fermentas, catalog number EL0011) at RT for 2 h, followed by overnight incubation at 4°C. The ligated DNA was recovered by phenol / chloroform extraction and ethanol precipitation, and then dissolved in 10 mM Tris-HCl, pH 8.5. Thus, the resulting library in the vector pAY02592 encodes Z variants, each fused to an albumin-binding domain (ABD) derived from a streptococcal G protein.

[0361] The ligation reaction (approximately 160 ng DNA / transformation) was electroporated into electrocompetent *E. coli* ER2738 cells (Lucigen, Middleton, WI, USA, 50 µl). Immediately following electroporation, approximately 1 ml of recovery medium (supplying *E. coli* ER2738 cells) was added. The transformed cells were incubated at 37°C for 60 min. Samples were collected for titration and to determine the number of transformants. Cells were then pooled and cultured overnight at 37°C in 1 L of TSB-YE medium supplemented with 2% glucose, 10 μg / ml tetracycline, and 100 µg / ml ampicillin. Cells were pelleted at 4,000 g for 15 min and resuspended in PBS / glycerol solution (approximately 40% glycerol). Cells were aliquoted and stored at -80°C. Library clones from the Z variant were sequenced to validate inclusions and to evaluate the results compared to the designed library. Sequencing was performed as described in Example 1, and the amino acid distribution was confirmed.

[0362] Preparation of bacteriophage stock solution A phage stock containing a phage particle library was prepared in a 20 L fermenter (Belach Bioteknik). Cells from the glycerol stock containing this phage particle library were seeded into 10 L of TSB-YE supplemented with 1 g / L glucose, 100 mg / L ampicillin, and 10 mg / L tetracycline. The cells were cultured until they reached an optical density (OD) of 0.52 at 600 nm. 600 At this time, approximately 1.7 μL of culture was infected with 5 x mol excess M13K07 helper phage. The cells were incubated for 30 min, at which point the fermenter was filled to 10 μL with a compound fermentation medium [2.5 g / L (NH4)2SO4; 5.0 g / L yeast extract; 30 g / L tryptone, 2 g / L K2HPO4; 3 g / L KH2PO4, 1.25 g / L; Na3C6H5O7 • 2 H2O; Breox FMT30 antifoamer 0.1 ml / L]. The following components were added: 10 ml carbenicillin 25 mg / ml; 5 ml kanamycin 50 mg / ml; 1 ml 1 M IPTG; 17.5 ml / L 1.217 M MgSO4; and 5 ml of trace element solution [129 mM FeCl3, 36.7 mM ZnSO4, 10.6 mM CuSO4, 78.1 mM MnSO4, and 94.1 mM CaCl2 dissolved in 1.2 M HCl]. A glucose-restricted fed-batch culture was initiated, with a 600 g / L glucose solution fed into the reactor (3.5 g / h initially, increasing to 37.5 g / h after 20 h until the end of the culture). The pH was maintained at pH 7 by automatic addition of 25% NH4OH. Air was added (5 L / min), and the stirrer was set to 500 rpm. After 24 h of fed-batch culture, OD... 600 The value was 19.4. Cells in the culture were precipitated by centrifugation at 15,900 g. Phage particles were precipitated twice from the supernatant with PEG / NaCl as described in Example 1, filtered, and dissolved in PBS and glycerol. The phage stock solution was stored at -80°C until selected for use.

[0363] result

[0364] Library Construction A new library was designed based on a set of IL-17A binding Z variants (Examples 1 and 3) with proven binding properties. The theoretical size of the designed library is 6.1 x 10⁻⁶. 9 The Z variant was used. The actual size of the library, determined by titration after transformation into *E. coli* ER2738 cells, was 4.5 x 10⁻⁶.9 Transformed strains.

[0365] Library quality was tested by sequencing 96 transformants and comparing their actual sequences with the theoretical design. The contents of the actual library showed satisfactory results compared to the designed library. Therefore, a mature library of potential IL-17A binders was successfully constructed.

[0366] Example 5

[0367] Selection, screening, and characterization of Z variants from first-mature libraries

[0368] Materials and methods

[0369] Phage display selection with IL-17A binding to the Z variant As described in Example 1, the target proteins hIL-17A and mIL-17A were biotinylated. Phage display selection was performed for hIL-17A and mIL-17A in four cycles, essentially as described in Example 1, using a new library of Z variant molecules constructed as described in Example 4, with the following exceptions: Exception 1: 0.1% PBST was used as the selection buffer. During selection, fetal bovine serum (FCS, Gibco, catalog 10108-165) and human serum albumin (HSA, Albucult, Novozymes, catalog 230-005) were added to the selection buffer to final concentrations of 10% and 1.5 µM, respectively. Exception 2: A pre-selection step was performed by incubating the phage stock solution with SA beads. Exception 3: All tubes and beads used in the selection were pre-blocked with PBS supplemented with 3% BSA and 0.1% Tween 20. Exception 4: The selected times are the 140th minute, 70th minute, 60th minute, and 50th minute in cycles 1, 2, 3, and 4, respectively.

[0370] Table 5 shows an overview of the selected strategies, describing the increased stringency achieved in subsequent cycles by using reduced target concentrations and increased wash counts.

[0371] Table 5: Overview of selections from the first mature library

[0372]

[0373]

[0374] Tracks 1-3 in cycle 1 were divided into the second through fourth cycles, resulting in a total of 6 tracks in cycle 2 (1-1 to 3-3), 6 tracks in cycle 3 (1-1-1 to 3-3-1), and 24 tracks in cycle 4 (1-1-1-1a to 3-3-1-2b). Washing was performed for 1 min using 0.1% PBST. However, for tracks 1-3 in cycle 3, one wash lasted 10 min.

[0375] After the final wash in cycle 4, the target phage complexes from all orbitals on the SA beads were divided into two aliquots. The bound phage particles from the first aliquot were eluted immediately, while the second aliquot underwent an overnight wash before elution. The bound phage particles were eluted using two different procedures: 1) glycine-HCl, pH 2.2 as in Example 1, or 2) 500 μl of 100 mM sodium phosphate, 150 mM sodium chloride, pH 5.5, neutralized with 500 µl of PBS.

[0376] Amplification of phage particles The amplification of phage particles selected between cycles 1 and 2 was generally performed as described in Example 1, with the following exceptions: Exception 1: *E. coli* ER2738 was used for phage amplification. Exception 2: M13K07 helper phage was used in excess at 5x. Exception 3: The amplification of phage particles selected between cycles 2 and 4 was performed as follows: After infecting logarithmically growing *E. coli* ER2738 with phage particles, TSB supplemented with 2% glucose, 10 μg / ml tetracycline, and 100 μg / ml ampicillin was added, followed by incubation at 37°C with rotation for 30 min. The bacteria were then infected with M13K07 helper phage. The infected bacteria were centrifuged, resuspended in TSB-YE medium supplemented with 100 µM IPTG, 25 µg / ml kanamycin, and 100 µg / ml ampicillin, and grown overnight at 30°C. Centrifuge the overnight culture and precipitate the phage particles in the supernatant twice with PEG / NaCl buffer. Finally, resuspend the phage in selection buffer and proceed to the next selection cycle.

[0377] In the final selection cycle, logarithmic growth phase bacteria are infected with eluent, diluted, and then plated on TBAB plates (30 g / l tryptone agar, Oxoid catalog number CMO233B) supplemented with 0.2 g / L ampicillin to form single colonies to be used in ELISA screening.

[0378] Sequencing of potential binders Individual clones from different selection orbitals were selected for sequencing. Gene fragment amplification and sequence analysis were performed, essentially as described in Example 1.

[0379] ELISA screening for Z variant Single colonies containing the Z variant (expressed as the Z variant ABD fusion protein as described in Example 1) were randomly picked from selected clones of a mature library and grown in culture medium substantially as described in Example 1. Periplasmic supernatant preparation and ELISA screening were also performed substantially as described in Example 1, with the following two exceptions: Exception 1: Biotinylated hIL-17A was used at a concentration of 0.4 nM. Exception 2: Periplasmic fractions from the primary selected Z variant Z06282 (SEQ ID NO: 1206) were used as a positive control.

[0380] EC50 analysis of the Z variant This study analyzed the response of a series of IL-17A-binding Z variants to serial dilutions against b-hIL-17A using ELISA, as described above. The biotinylated target protein was added at a concentration of 6 nM and step-diluted 1:3 up to 8 pM. As a background control, the Z variant was also measured without the addition of the target protein. Periplasmic samples containing the initial IL-17A conjugate Z06282 (SEQ ID NO: 1206) were included as positive controls. Data were analyzed using GraphPad Prism 5 and nonlinear regression, and EC50 values ​​(half-maximum effective concentration) were calculated.

[0381] result

[0382] Phage display selection of mature IL-17A combined with Z variant Selection was made from a total of 24 parallel orbitals, each consisting of 4 cycles. Different selection orbitals varied in target concentration, target species origin (human IL-17A or mouse IL-17A), selection time, washing conditions, and the pH of the elution buffer. Clones originating from the selection orbitals, eluted with only human IL-17 at pH 2.2, showed the best performance in ELISA screening.

[0383] sequencing Sequencing was performed on randomly selected clones. Each individual Z variant was provided with an identification number Z##### as described in Example 1. A total of 932 new and unique Z variant molecules were identified. For the 494 best-performing variants in the ELISA screening below, the amino acid sequence of the 58-amino acid residue-long Z variant was... Figure 1The sequences are listed as SEQ ID NO:1-3, SEQ ID NO:11-16, SEQ ID NO:28-31, SEQ ID NO:36-63, and SEQ ID NO:67-519. In each sequence, the inferred IL-17A binding motif extends from residue 8 to residue 36. The amino acid sequence of the 49-amino acid residue long polypeptide predicted to constitute the complete triple helix bundle within each of these Z variants extends from residue 7 to residue 55.

[0384] ELISA screening for Z variant Clones obtained after four selection cycles were generated in 96-well plates and screened for human IL-17A binding activity using ELISA. All randomly selected clones were analyzed. 494 of the 932 unique Z variants were found to give a response of 2 x blank control or higher (0.15–2.0 AU) to 0.4 nM hIL-17A. Clones originating from all selected orbitals showed positive signals. The blank control had absorbance of 0.055–0.075 AU.

[0385] EC50 analysis of the Z variant Subgroups of the Z variant were selected based on the results of the ELISA experiments described above (absorbance above 1.25 AU) or based on changes in the amino acid sequence, and underwent target titration in ELISA format. Periplasmic samples were incubated with a series of dilutions of b-hIL-17A ranging from 6 nM to 8 pM. Periplasmic samples containing Z06282 (SEQ ID NO: 1206) identified in the primary selection were included as positive controls. The obtained values ​​were analyzed and their respective EC50 values ​​were calculated (Table 6).

[0386] Table 6: EC50 values ​​calculated from ELISA titration analysis

[0387]

[0388] Example 6

[0389] Design and construction of a second mature library of IL-17A combined with the Z variant

[0390] In this embodiment, a second mature library is constructed essentially as described in Embodiment 4. This library is then used to select an additional IL-17A-binding Z variant.

[0391] Materials and methods

[0392] Document DesignThe library was based on the sequence of the IL-17A-binding Z variant selected and characterized in Example 5. In the new library, 13 positions in the modified Z molecular scaffold described in Examples 4 and 5 were biased towards certain amino acid residues, according to a strategy based on the Z variant sequences of the 37 best-performing variants in EC50 analysis (Table 6). DNA adapters were generated using cleavage synthesis and ordered from DNA 2.0. It contains the following 147 bp of partially randomized helices 1 and 2 encoding amino acid sequences: 5'-AA ATA AAT CTC GAG GTA GAT GCC / GCA AAA TAC GCC AAA GAA / GAG NNN NNNNNN GCG NNN NNN GAG ATC / ATT NNN NNN TTA / CTG CCT / CCC AAC TTA / CTC ACC NNN NNNCAA / CAG NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTATTT A-3' (SEQ ID NO:1249; randomized codons are represented as NNN), flanked by restriction sites XhoI and SacI. Table 7 presents the theoretical distribution of amino acid residues in the new library, including six variable amino acid positions (11, 14, 18, 25, 28, and 32) and seven constant amino acid positions (9, 10, 13, 17, 24, 27, and 35) within the Z-molecule scaffold. The resulting theoretical library size is 3.9 x 10⁻⁶. 6 Variations.

[0393] Library Construction The library was constructed essentially as described in Example 4, with the following exception: the cells were cultured overnight in 0.5 l of TSB-YE medium supplemented with 2% glucose, 10 µg / ml tetracycline and 100 µg / ml ampicillin.

[0394] Preparation of bacteriophage stock solution A phage stock solution containing a phage particle library was prepared in a shake flask. Cells from the glycerol stock solution containing this phage particle library were seeded in 0.5 μL of TSB-YE medium supplemented with 2% glucose, 10 µg / mL tetracycline, and 100 µg / mL ampicillin. The cultures were grown at 37°C until OD (dose expiratory time). 600The concentration was reached to 0.6, and approximately 83 ml of culture was then infected with a 5 x mol excess of M13K07 helper phage and incubated at 37°C for 40 min. Cells in the culture were pelleted by centrifugation, dissolved in TSB-YE medium supplemented with 100 µg / ml ampicillin, 25 µg / ml kanamycin, and 0.1 mM IPTG, and grown at 30°C for 18 h. After incubation, cells were pelleted by centrifugation at 4,000 g, and the phage particles remaining in the medium were subsequently precipitated twice with PEG / NaCl as described in Example 1, filtered, and dissolved in PBS and glycerol. The phage stock solution was stored at -80°C until selected for use.

[0395] Table 7: Library Design, Second Maturity

[0396]

[0397] result

[0398] Library Construction A new library was designed based on a set of IL-17A binding Z variants (Example 5) with proven binding properties. The theoretical size of the designed library is 3.9 x 10⁻⁶. 6 The Z variant was used. The actual size of the library, determined by titration after transformation into *E. coli* ER2738 cells, was 1.4 x 10⁻⁶. 9 Transformed strains.

[0399] Library quality was tested by sequencing 96 transformants and comparing their actual sequences with the theoretical design. The contents of the actual library showed satisfactory results compared to the designed library. Therefore, a mature library of potential IL-17A binders was successfully constructed.

[0400] Example 7

[0401] Selection, screening, and characterization of Z variants from second-mature libraries

[0402] Materials and methods

[0403] Phage display selection with IL-17A binding to the Z variantThe target protein hIL-17A was biotinylated as described in Example 1. Using a second mature library of the Z variant molecule constructed as described in Example 6, phage display selection for hIL-17A was performed essentially as described in Example 5, with the following exceptions: Exception 1: Selection was performed in solution or on the solid phase at RT. Exception 2: Selection was performed at 60 min, 10 min, or 1 min in cycle 1 and at 30 min, 4 min, or 10 sec in cycles 2, 3, and 4. Following selection in solution, the target phage complex was captured on SA beads. Exception 3: During solid-phase selection, the target was captured on SA beads prior to selection.

[0404] Table 8 shows an overview of the selection strategies, outlining the differences between selection in solution and selection on the solid phase, as well as the increased stringency achieved in subsequent cycles by using reduced target concentrations and increased washing cycles.

[0405] Tracks 1-6 in cycle 1 were distributed into cycles 2 through 4, resulting in a total of 7 tracks in cycle 2 (1-1 to 6-1), 8 tracks in cycle 3 (1-1-1 to 6-1-1), and 16 tracks in cycle 4 (1-1-1-1 to 6-1-1-1). Washing was performed with 0.1% PBST over a 1-minute period. However, one wash in cycles 3 (tracks 2-1-1, 3-1-1, and 5-1-1) lasted for 15 minutes.

[0406] After the final wash in cycle 4, the target phage complexes from the SA beads of the five orbitals (1-1-1-1, 1-2-1-2, 3-1-1-1, 4-1-1-1, and 5-1-1-2) were divided into two aliquots. The bound phage particles from the first aliquot were immediately eluted, while the second aliquot underwent washing for approximately 65 hours before elution of the phage particles. The bound phages were eluted using glycine-HCl at pH 2.2, as described in Example 1.

[0407] Basically as described in Example 1, phage particle amplification is performed between selection cycles.

[0408] Table 8: Overview of selections from the second-mature library

[0409]

[0410]

[0411] Sequencing of potential binders Individual clones from different selection orbitals were selected for sequencing. Gene fragment amplification and sequence analysis were performed, essentially as described in Example 1.

[0412] ELISA screening for Z variant Single colonies containing the Z variant (expressed as the Z variant ABD fusion protein as described in Example 1) were randomly picked from selected clones of the second mature IL-17A library and grown in culture as described in Example 1. Periplasmic supernatant preparation and ELISA screening were performed substantially as described in Example 1, with the following two exceptions: Exception 1: Biotinylated hIL-17A was used at a concentration of 0.2 nM or 0.33 nM. Exception 2: The periplasmic fraction of the Z variant Z10241 (SEQ ID NO:11) identified from the selection of the first mature library was used as a positive control, and a blank control was generated by adding 0.05% PBST for a periplasmic exchange step.

[0413] EC50 analysis of the Z variant A series of IL-17A-binding Z variants were subjected to analysis of responses to serial dilutions against b-hIL-17A using ELISA as described in Example 5. Biotinylated protein was added at a concentration of 10 nM and step-diluted eight times at a 1:2 ratio, followed by a single 1:5 dilution up to 8 pM. As a background control, the Z variant was also measured without the target protein. Periplasmic samples containing the previously matured Z variant Z10241 (SEQ ID NO:11) were included as positive controls. Data were analyzed using GraphPad Prism 5 and nonlinear regression, and EC50 values ​​were calculated.

[0414] result

[0415] Phage display selection of IL-17A-binding Z variant from the second mature library The selection is made from a total of 16 parallel orbitals, each containing 4 cycles. The orbital selection varies in terms of target concentration, selection time, washing conditions, and whether the selection is performed in solution or on the solid phase.

[0416] sequencing Sequencing was performed on randomly selected clones. Each individual Z variant was provided with an identification number Z##### as described in Example 1. A total of 759 new and unique Z variant molecules were identified.

[0417] Of the 704 best-performing variants screened in the ELISA test below, the amino acid sequences of the 58-amino acid residue long Z variant were... Figure 1The sequences are listed as SEQ ID NO:5-10, SEQ ID NO:17-27, SEQ ID NO:32-35, SEQ ID NO:64-66, and SEQ ID NO:520-1199 in the sequence listing. In each sequence, the inferred IL-17A binding motif extends from residue 8 to residue 36. The amino acid sequence of the 49-amino acid residue long polypeptide predicted to constitute the complete triple helix bundle within each of these Z variants extends from residue 7 to residue 55.

[0418] ELISA screening for Z variant Clones obtained after four selection cycles were generated in 96-well plates and screened for hIL-17A binding activity using ELISA. All randomly selected clones were analyzed. 704 of the 759 unique Z variants were found to give a response of 2 x blank control or higher (0.22–2.2 AU) to hIL-17A at concentrations of 0.2 nM or 0.33 nM. Clones originating from all selected orbitals were collected with positive signals. Based on the representative group of the plate, the mean response of the blank control was 0.11 AU.

[0419] EC50 analysis of the Z variant The subgroups of IL-17A binding to the Z variant were selected based on the results of the ELISA experiment described above (the Z variant had a higher absorbance than the positive control Z10241, SEQ ID NO:11) and underwent target titration in ELISA format as described in Example 5. The obtained values ​​were analyzed and their respective EC50 values ​​were calculated (Table 9).

[0420] Table 9: EC50 values ​​calculated from ELISA titration analysis

[0421]

[0422] Example 8

[0423] In vitro characterization of a subset of mature IL-17A binding to the Z variant

[0424] Materials and methods

[0425] Subcloning and generation of a Z variant with an N-terminal His6-tag were performed as described in Example 2. Site-directed mutagenesis of His6-Z10532 (SEQ ID NO:1) resulted in the substitution of D with A at position 25, generating another variant, His6-Z15167 (SEQ ID NO:4). The generation of His6-Z15167 was carried out as described above for the other Z variants.

[0426] Circular dichroism (CD) spectral analysisThe purified His6-tagged Z variant was diluted to 0.5 mg / ml in PBS. For each diluted Z variant, CD spectra in the 250–195 nm range were obtained at 20 °C. Variable temperature measurement (VTM) was also performed to determine the melting temperature (Tm). In VTM, absorbance was measured at 221 nm while the temperature was increased from 20 °C to 90 °C at a rate of 5 °C / min. New CD spectra were obtained at 20 °C after the heating procedure to investigate the refolding ability of the Z variant. CD measurements were performed using cuvettes with a 1 mm optical path length on a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB).

[0427] IL-17 combined with ELISA screening A 96-well half-zone plate (Costar, 3690) was coated overnight at 4°C with 1 µg / ml hIL-17A in PBS at a volume of 50 µl / well. On the day of analysis, the plate was rinsed twice with tap water and then blocked with PBS + 2% BSA (Sigma) for 2 h. Z10241 (SEQ ID NO:11) was used as a standard and titrated serially at 3-fold dilutions (300-0.005 ng / ml), and other Z variants were added to the coated ELISA plates at 4 different dilutions (50 µl / well) and incubated at RT for 1.5 h. The plates were washed 4 times in an automated ELISA washer and 2 µg / ml (50 µl / well) of goat anti-Z antibody was added. After 1 h of incubation, the plates were washed and 50 μl of anti-goat IgG-HRP (DAKO) diluted 5000-fold was added to each well. After another hour of incubation and washing, the plate was developed with 50 µl TMB (ThermoFisher, 34021) per well, and the reaction was terminated with 50 µl 2 M H2SO4. The reaction was then performed using a multi-label reader (Victor). 3 Read the board in Perkin Elmer.

[0428] Biacore dynamics analysis Determine the kinetic constants (k) of human IL-17A on 27 His6-tagged Z variants. on and k off ) and affinity (K DhIL-17A was immobilized on a carboxylated dextran layer on the surface of a CM5 chip (GE Healthcare, catalog BR100012) in the flow cell. Immobilization was performed using an amine coupling chemistry and HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v surfactant P20, GE Healthcare, catalog BR100188) as the run buffer, according to the manufacturer's protocol. One flow cell surface on the chip was activated and inactivated as a blank during analyte injection. In kinetic experiments, HBS-EP was used as the run buffer at a flow rate of 50 µl / min. The analyte, the Z variant, was diluted to final concentrations of 100 nM, 20 nM, and 4 nM in HBS-EP buffer and injected for 4 min, followed by dissociation in the run buffer for 8 min. After 8 min of dissociation, the surface was regenerated with two injections of 10 mM HCl. The kinetic constants were calculated from the sensor plots using the Langmuir 1:1 model in BiaEvaluation software 4.1 (GE Healthcare).

[0429] Biacore combines specificity analysis The interactions of three His6-tagged IL-17A binding Z variants (Z10508 (SEQ ID NO:2), Z10532 (SEQ ID NO:1), and Z15167 (SEQ ID NO:4)) with hIL-17A (SEQ ID NO:1226), hIL-17F (SEQ ID NO:1228; R&D Systems, catalog 1335-IL / CF), and human IL-17A / F heterodimer (R&D Systems, catalog 5194-IL-025 / CF) were analyzed in a Biacore 2000 instrument. The three different IL-17 variants were immobilized in flow cells on a carboxylated dextran layer on the surface of a CM5 chip. Immobilization was performed using amine coupling chemistry and HBS-EP as a run buffer, according to the manufacturer's protocol. One flow cell surface on the chip was activated and deactivated to serve as a blank during analyte injection. In the binding experiments, HBS-EP was used as the run buffer at a flow rate of 50 µl / min. The analyte, the Z variant, was diluted individually in HBS-EP run buffer to final concentrations of 4 nM, 20 nM, 100 nM, and 500 nM, and injected for 4 min. After dissociation for 15 min, the surface was regenerated with four injections of 10 mM HCl. The results were analyzed using BiaEvaluation software.

[0430] Blocking IL-17A-induced IL-6 production in NHDF assay NHDF was determined and quantified essentially as described in Example 3 using an IL-6-specific ELISA. Briefly, 5000 cells / well were seeded at 100 μl into a half-zone 96-well plate the day before the experiment. On the day of the experiment, dilutions of 36 IL-17A-specific Z variants were prepared in separate 96-well plates. The Z variants were titrated from 4690 nM to 0.08 nM in a three-fold increment in medium containing 0.9 nM hIL-17A. A standard curve for hIL-17A (6.2–0.0001 nM) and controls containing medium with 0.9 nM hIL-17A or medium alone were prepared.

[0431] result

[0432] CD Analysis CD spectra of IL-17A-binding Z variants with the His6 tag show that each variant has an α-helical structure at 20 °C. The results of variable temperature measurements that determined the melting temperature (Tm) are shown in Table 10. For all IL-17A-binding Z variants, reversible folding was observed when the spectra measured before and after heating to 90 °C were overlapped.

[0433] IL-17A binding ability was analyzed by ELISA. The ability of purified His6-tagged Z variant molecules from the first and second maturation rings to bind IL-17A was screened in an ELISA assay. Results were... Figure 3 The value is shown as a percentage of binding ability compared to variant Z10241.

[0434] Biacore dynamics analysis The interaction between 28 His6-tagged IL-17A-binding Z variants and hIL-17A was analyzed using a Biacore instrument by injecting different concentrations of the Z variant onto surfaces containing a fixed amount of IL-17A. The kinetic parameters (KL) of the binding between the Z variant and hIL-17A were determined using a 1:1 interaction model. D k a (k on ) and k d (k off The summary of )) is given in Table 11.

[0435] Table 10: Depolymerization Temperature (Tm)

[0436]

[0437] Biacore combines specificity analysisThe binding of three His6-tagged IL-17A-binding Z-variants (Z10508, Z10532, and Z15167) to hIL-17A, hIL-17F, and hIL-17A / F was tested in a Biacore instrument by injecting Z-variants onto surfaces containing IL-17 variants. Ligand immobilization levels on the surfaces were 657 RU, 977 RU, and 770 RU for IL-17A, IL-17F, and IL-17A / F, respectively. All tested Z-variants showed binding to human IL-17A, with weaker binding to IL-17A / F, while binding to IL-17F was undetectable. Figure 4 shows the curves obtained by subtracting the responses from the blank surface. Z15167 showed the fastest association curve with human IL-17A.

[0438] Blockade of IL-17A-induced IL-6 production in NHDF assay The ability of purified His6-tagged Z variants from the first and second mature libraries to block IL-17A-induced IL-6 production was screened using NHDF assays. Results from the NHDF assays showed that all tested mature conjugates exhibited increased IL-17A-specific blocking ability compared to the primary conjugate His6-Z06282. A schematic diagram showing the typical inhibition spectra of a series of conjugates from the first mature library is provided. Figure 5 The calculated IC50 values ​​for all analyzed conjugates are shown in Table 12.

[0439] Table 11: Kinetic parameters of Z variant binding to hIL-17A

[0440]

[0441] Table 12: IC50 values ​​of mature Z-variants

[0442]

[0443] Example 9

[0444] Production of IL-17 binding to Z-ABD-Z peptide

[0445] IL-17A is a homodimeric cytokine with two receptor-binding sites. It is presumed that a polypeptide containing both parts of the IL-17A-binding Z variant of the present disclosure will block IL-17A more effectively than a polypeptide containing only one such part. This embodiment describes a general procedure for subcloning and generating polypeptides comprising two Z variants fused in the generic format Z-[L1]-ABD-[L2]-Z with the albumin-binding domain variant PP013 (SEQ ID NO: 1224), wherein [L1] and [L2] are linkers separating the Z and ABD parts.

[0446] Materials and methods

[0447] Subcloning of Z-[L1]-ABD-[L2]-Z peptides with different [L1] and [L2] linker lengths DNA from the library vector pAY02592 was amplified by PCR using PfuTurbo DNA polymerase (Agilent Technologies, catalog number 600254) along with suitable primer pairs for Z06282 (SEQ ID NO:1206), Z10241 (SEQ ID NO:11), and Z10532 (SEQ ID NO:1). The DNA encoding each motif was ligated into the expression vector pET26b(+) (Novagen, Madison, WI) in three subsequent cloning steps using T4 DNA ligase (Fermentas, catalog number EL0011), resulting in the Z-[L1]-ABD-[L2]-Z construct. The DNA encoding the three motifs was encoded by different numbers of (GGGGS) sequences. n DNA segregation from the repeating hybridization adapter is flanked by restriction endonuclease sites. The construct encoded by the expression vector is Z#####-[GAP-(G4S)]. n -TS]-PP013-[GT-(G4S) n -PR]-Z#####, where each n is individually 1-4, and as further specified in Table 13.

[0448] Table 13: Dimeric Z variants fused with ABD via different [L1] and [L2] lengths

[0449]

[0450] The restriction sites in the DNA encoding Z#####-[(I)-(G4S)n-(II)]-PP013-[(III)-(G4S)n-(IV)]-Z##### can be cleaved by restriction endonucleases AscI (I), SpeI (II), KpnI (III), and SacII (IV), respectively.

[0451] Subcloning of the Z-[L1]-ABD-[L2]-Z polypeptide with the smallest [L1] linker: DNA encoding an additional dimeric Z variant containing Z06282 (SEQ ID NO: 1206) but with a minimum linker [L1] was generated using standard molecular biology techniques. The construct encoded by the expression vector was Z06282-VDGS-PP013-GT-(G4S). n -PR-Z06282, and as further specified in Table 14.

[0452] Genes encoding Z12876 (SEQ ID NO: 1218), Z14253 (SEQ ID NO: 1219), Z14254 (SEQ ID NO: 1220), and Z14255 (SEQ ID NO: 1221) (corresponding to Z10241 (SEQ ID NO: 11), Z10532 (SEQ ID NO: 1), Z10508 (SEQ ID NO: 2), and Z10863 (SEQ ID NO: 3), respectively, but starting with the amino acid residue AE instead of VD) were amplified by PCR using Pfu Turbo DNA polymerase with appropriate primer pairs. Each fragment was ligated into the expression vector pET26b(+) in three subsequent cloning steps using T4 DNA ligase to generate the Z-[L1]-ABD-[L2]-Z construct. The DNA encoding the three segments was further isolated using site-directed mutagenesis-modified adapters using standard molecular biology techniques. The constructs encoded by the expression vector are Z####-[VDGS]-PP013-[GT-G4S-PK]-Z#### and Z#####-[ASGS]-PP013-[GT-G4S]–Z#####, and as further specified in Table 14.

[0453] Table 14: Dimeric Z variants fused with ABD via the smallest [L1] joint

[0454]

[0455] nourish E. coli BL21(DE3) cells (Novagen) were transformed with plasmids containing gene fragments of each of the respective Z-ABD-Z polypeptides and cultured at 37°C in 800 ml or 1000 ml of TSB-YE medium supplemented with 50 µg / ml kanamycin. To induce protein expression, [the following was observed] at OD [the following was observed]. 600 When the concentration was 2, IPTG was added to a final concentration of 0.2 mM and the culture was incubated at 37°C for another 5 h. Cells were harvested by centrifugation.

[0456] Purification of IL-17A-binding Z-ABD-Z peptide Resuspend the cell pellet in a solution supplemented with Benzonase. ®TST buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween 20, pH 8.0) was used. After cell disruption by sonication and clarification by centrifugation, each supernatant was applied to a column containing agarose and immobilized anti-ABD ligands (internal generation). After washing with TST buffer and 5 mM NH4Ac pH 5.5 buffer, the Z-ABD-Z peptide was eluted with 0.1 M HAc. Acetonitrile (ACN) was added to the eluted fraction at a final concentration of 10%, and the sample was loaded onto a Source 15 RPC column (GE Healthcare) pre-equilibrated with RPC solvent A (0.1% TFA, 10% ACN, 90% water). After washing the column with RPC solvent A, the bound protein was eluted with a linear gradient from RPC solvent A to RPC solvent B (0.1% TFA, 80% ACN, 20% water). Fractions containing purified Z-ABD-Z peptides were identified and pooled by SDS-PAGE analysis. Following RPC purification, the pool buffer was exchanged for PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) using a Sephadex G-25 column (GE Healthcare). Finally, the Z-ABD-Z variant was purified using an EndoTrap column. ® Purification on red (Hyglos) columns ensures low endotoxin levels.

[0457] Protein concentration was achieved using NanoDrop. ® The absorbance at 280 nm and the extinction coefficient of each protein were determined using an ND-1000 spectrophotometer. Purity was analyzed by SDS-PAGE stained with Coomassie Brilliant Blue, and the identity of each purified Z-ABD-Z variant was confirmed by LC / MS analysis.

[0458] result

[0459] Cultivation and purification The IL-17A-binding Z variant fused with ABD was expressed as a soluble gene product in *E. coli*. SDS-PAGE analysis of each final protein product showed that they primarily contained the desired IL-17A-binding Z-ABD-Z peptide. HPLC-MS analysis confirmed the correct identity and molecular weight of each Z-ABD-Z peptide.

[0460] Example 10

[0461] Solubility of Z-ABD-Z peptide

[0462] The solubility of three Z-ABD-Z peptides in physiological buffer was investigated by continuously concentrating the samples using ultrafiltration, followed by concentration measurements by absorbance readings at 280 nm and visual inspection of the samples.

[0463] Materials and methods

[0464] Z-ABD-Z peptides ZAZ3363 (SEQ ID NO:1244), ZAZ3364 (SEQ ID NO:1245), and ZAZ3422 (SEQ ID NO:1247) were diluted to 2.5 mg / ml in PBS, pH 7.4. Twelve Amicon Ultra centrifuge filter units (Millipore, catalog number UFC800324) with a 3 kDa cut-off were pre-washed with PBS by centrifugation at 4000 g for 10 min in a float centrifuge. The concentrators were emptied, and 4 ml of each Z-ABD-Z peptide was added to the first set of three different centrifuge filter units. Centrifugation at 4000 g for 13–16 min at 20 °C yielded approximately 1 ml of concentrate. 20 μl of sample (UF sample 1) was taken from each concentrate for further analysis, and the remaining sample volume was transferred to the second set of three centrifuge filter units. Centrifugation and sample removal were repeated three times, with rotation times of 8–10 min, 7 min, and 13 min, respectively (UF samples 2, 3, and 4). Absorbance readings were taken using a NanoDrop® ND-1000 spectrophotometer and by diluting UF samples 1–4 in PBS by 3, 6, 12, and 24 times, respectively. Concentrations were calculated using the theoretical extinction coefficient 1 Abs280 = 0.612 mg / ml (the same for all three Z-ABD-Z peptides). Absorbance readings were also taken for the undiluted filtrate.

[0465] result

[0466] The concentrations determined by absorbance readings at 280 nm after each centrifugation step are shown in Table 15. No aggregates were detected by visual inspection of the concentrate. Therefore, the solubility of ZAZ3363, ZAZ3364, and ZAZ3422 in PBS, pH 7.4, was determined to be at least 60 mg / ml. Absorbance readings of the undiluted filtrate showed concentrations very close to 0 mg / ml.

[0467] Table 15: Concentrations after continuous concentration of Z-ABD-Z samples

[0468]

[0469] Example 11

[0470] Biacore's cross-species analysis

[0471] Materials and methods

[0472] The interactions of Z-ABD-Z peptides ZAZ3363 (SEQ ID NO:1244), ZAZ3364 (SEQ ID NO:1245), and ZAZ3365 (SEQ ID NO:1246) with hIL-17A and cynomolgus monkey IL-17A (cIL-17A, SEQ ID NO:1229; Evitria, custom order), and the interactions of ZAZ3220 (SEQ ID NO:1236) with hIL-17A and rhesus monkey IL-17A (rmIL-17-A, SEQ ID NO:1230; Cusabio, catalog number CSB-EP011597MOV), were analyzed in a Biacore 2000. HSA was immobilized in flow cells on a carboxylated dextran layer on the surface of a CM5 chip. Immobilization was performed using amine coupling chemistry and HBS-EP as a run buffer, according to the manufacturer's protocol. The HSA fixation levels on the surface were 953 RU (for ZAZ3363, ZAZ3364, and ZAZ3365) and 493 RU (for ZAZ3220), respectively. A flow cell surface on the chip was activated and inactivated as a blank during analyte injection. In binding experiments, HBS-EP was used as the run buffer at a flow rate of 30 µl / min. ZAZ3363, ZAZ3364, and ZAZ3365 were diluted to a final concentration of 200 nM in HBS-EP run buffer and injected for 5 min, followed by injection of the IL-17A variant. ZAZ3220 was diluted to a final concentration of 500 nM in HBS-EP run buffer and injected for 4 min, followed by injection of the IL-17A variant. hIL-17A and cIL-17A were diluted to final concentrations of 2.5 nM, 10 nM, and 40 nM, respectively, in HBS-EP running buffer and injected for 5 min onto surfaces containing ZAZ3363, ZAZ3364, and ZAZ3365 captured on HSA. After dissociation for 10 min, the surfaces were regenerated with two injections of 10 mM HCl. hIL-17A and cIL-17A were also diluted to final concentrations of 0.1 nM, 0.3 nM, 0.9 nM, 2.7 nM, and 8.1 nM, and 2.7 nM, 8.1 nM, 24.3 nM, 72 nM, and 216 nM, respectively, in HBS-EP running buffer and injected for 6 min onto surfaces containing ZAZ3220 captured on HSA. After dissociation for 5 min, the surfaces were regenerated with two injections of 10 mM HCl. The results were analyzed using BiaEvaluation software.

[0473] result

[0474] The binding of hIL-17A and cIL-17A to ZAZ3363, ZAZ3364, and ZAZ3365, and the binding of hIL-17A and rmIL-17A to ZAZ3220, were tested in a Biacore instrument by injecting the Z-ABD-Z peptide followed by an IL-17A variant into a surface containing HSA. All tested Z variants showed binding to the tested IL-17A variants, namely ZAZ3363, ZAZ3364, and ZAZ3365, which showed binding to human and cynomolgus monkey IL-17A (Figure 6), and ZAZ3220, which showed binding to human and rhesus monkey IL-17A.

[0475] Example 12

[0476] Biacore combines specificity analysis

[0477] In this embodiment, the specificity of the two Z-ABD-Z peptides was tested by analyzing their potential interactions with a group of proteins in the IL-17 family, as well as with other interleukins and other abundant plasma proteins. The potential interactions of the same group of analyzed proteins with the ABD variant PEP07843 were also analyzed.

[0478] Materials and methods

[0479] The interactions of ZAZ3363 (SEQ ID NO: 1244) or ZAZ3422 (SEQ ID NO: 1247) with groups of 24 or 12 different proteins (specified in Table 16) were analyzed using a Biacore 2000 instrument. HSA and the ABD variant PEP07843 (SEQ ID NO: 1225, corresponding to PP013 (SEQ ID NO: 1224) with an N-terminal extension of GSS) were immobilized on the surface of a CM5 chip, and blank surfaces were prepared as described in Example 11. The ligand immobilization levels on the surfaces were 1315 RU and 99 RU for HSA and PEP07843, respectively, for the chip used in the ZAZ3363 run, and 791 RU and 100 RU, respectively, for the chip used in the ZAZ3422 run. In the binding experiments, HBS-EP was used as the running buffer, and the flow rate was 30 µl / min. ZAZ3363 and ZAZ3422 were diluted to a final concentration of 200 nM in HBS-EP or HBS-EP supplemented with 500 mM NaCl and injected for 7 min, followed by the injection of analytes. The analytes, i.e., groups of 24 or 12 different proteins, were each diluted to a final concentration of 0.4 nM to 250 nM in HBS-EP or HBS-EP supplemented with 500 mM NaCl and injected for 5 min. After dissociation for 7 min (ZAZ3363) or 10 min (ZAZ3422), the surface was regenerated by four injections of 10 mM NaOH and two injections of 10 mM HCl, respectively, for ZAZ3363 or ZAZ3422. The results were analyzed using BiaEvaluation software.

[0480] Table 16: Protein analyte testing using ZAZ3363 and ZAZ3422

[0481]

[0482]

[0483] supplier: 1 Peprotech; 2 R&D Systems; 3 Roche / Apoteket AB; 4 Bethyl; 5 ProSpec; 6 Sino Biological Inc. 7 Sigma

[0484] result

[0485] The specificity of ZAZ3363 and ZAZ3422 was tested in a Biacore instrument by studying their interactions with 24 and 12 analyte proteins, respectively. The Z-ABD-Z peptide was injected onto an HSA-containing surface, followed by the analyte protein. The only interactions detected for ZAZ3363 and ZAZ3422 were strong binding to hIL-17A and weaker binding to hIL-17A / F, as expected and consistent with the results presented in Example 8. Potential interactions between the analyte protein and the ABD variant PEP07843 were also evaluated, but no interaction was detected. Therefore, the Z-ABD-Z peptide exhibits high specificity.

[0486] Example 13

[0487] Kinetic measurements of Z-ABD-Z, IL-17A, and HSA complexes using KinExA®

[0488] The technical limitations of SPR in accurately determining the kinetic parameters of high-affinity interactions, and the increased complexity of determining the affinity between two dimer targets via SPR, necessitate the use of kinetic exclusion assays (KinExA). ® Further analysis of the binding of the Z-ABD-Z peptide complexed with HSA to IL-17A, as well as the binding of the Z-ABD-Z peptide alone or in complex with IL-17A to HSA, was conducted using KinExA technology. ® Measuring equilibrium binding affinity and kinetics between unmodified molecules in solution (Darling and Brault, 2004. Assay and Drug Dev Tech 2(6):647-657).

[0489] Materials and methods

[0490] Z-ABD-Z peptides ZAZ3220 (SEQ ID NO: 1236), ZAZ3363 (SEQ ID NO: 1244), and ZAZ3422 (SEQ ID NO: 1247), as well as HSA (Novozymes, catalog number 230-005), human IL-17A (Peprotech, catalog number 200-17), biotinylated (as described in Example 1) human IL-17A, mouse monoclonal anti-HSA antibody (Abcam, catalog number 10241), and internally generated goat polyclonal anti-Z antibody were shipped to Sapidyne Instruments Inc (Boise, Idaho, USA) for KinExA® measurement and analysis.

[0491] Combination of Z-ABD-Z / HSA and IL-17A To determine affinity, i.e., K DThe respective Z-ABD-Z peptides complexed with HSA were used as constant binding partners (CBPs), and IL-17A was used as a titrant. KinExA was used. ® Pro software and applications use least squares analysis for data analysis to determine K D The optimal solution for the active binding site concentration (ABC) is fitted to a curve representing the 1:1 reversible bimolecular interaction.

[0492] For k a The determination was made using the same fixed IL-17A used in the kinetic and equilibrium experiments, applied by direct binding curve analysis. The amount of free Z-ABD-Z / HSA in the sample was measured before equilibrium, generating data points that monitored the decrease in free Z-ABD-Z / HSA as the sample moved toward equilibrium.

[0493] Combination of Z-ABD-Z and Z-ABD-Z / IL-17A with HSA Further analysis was conducted on the binding of Z-ABD-Z peptide ZAZ3363 to HSA, with or without IL-17A. To determine K... D Free or IL-17A-conjugated ZAZ3363 is used as a constant binding partner (CBP), and HSA is used as a titrant. KinExA as described above is used. ® Use Pro software for data analysis.

[0494] For k a The determination was made using the same fixed HSA used in the kinetic experiments and the trapping reagent used in the equilibrium experiments, and direct binding curve analysis was applied. The amount of ZAZ3363 / IL-17A in the sample was measured before equilibrium, generating data points that monitored the decrease of free ZAZ3363 / IL-17A as the sample moved toward equilibrium.

[0495] result

[0496] In the first group KinExA ® In the measurements, the Z-ADB-Z peptides ZAZ3220, ZAZ3363, and ZAZ3422, respectively complexed with HSA, showed very high affinity for IL-17A, and their K... D The values ​​range from subpicomolar to femtomolar. The kinetic parameters calculated when assuming monovalent binding between these Z-ABD-Z peptides and the dimer IL-17A are shown in Table 17.

[0497] In the second group KinExA ®In the measurements, the interaction between ZAZ3363 in its free state or in combination with IL-17A and HSA was measured. The calculated kinetic parameters from these analyses are shown in Table 18. The affinity of ZAZ3363 for HSA was not significantly affected by the presence of IL-17A.

[0498] In summary, using KinExA ® Technical measurements indicate that the Z-ABD-Z peptide has a very high affinity for IL-17A. The measured K... D <0.33 pM is superior to two clinically advanced comparable drugs, secukinumab (K). D = 122 pM; WO2006 / 013107 and WO2012 / 125680) and ixekizumab (K D = 2 pM; WO2007 / 070750) reported affinity. Furthermore, it was demonstrated that both IL-17A and albumin are bound simultaneously, meaning that both binding functions are intact in the Z-ABD-Z fusion protein.

[0499] Table 17: Kinetic parameters of Z-ABD-Z peptide binding to IL-17A

[0500]

[0501] * 95% confidence interval

[0502] ** For K D 95% confidence interval not distinguished

[0503] Table 18: Kinetic parameters of Z-ABD-Z peptide binding to HSA

[0504]

[0505] * 95% confidence interval

[0506] Example 14

[0507] Characterization of Z-ABD-Z peptide in NHDF assay

[0508] Materials and methods

[0509] Blockade of IL-17A-induced IL-6 production in NHDF assayNHDF cells (Lonza, catalog number CC-2511) were cultured in fibroblast basal medium (Lonza, catalog number CC-3132) supplied with growth factor (Lonza, catalog number CC-5034). The day before the experiment, 5000 cells / well were seeded at 100 µl into a half-zone 96-well plate (Greiner, catalog number 675180). On the day of the experiment, dilutions of IL-17A-specific Z-ABD-Z peptides (ZAZ3174 (SEQ ID NO:1233), ZAZ3175 (SEQ ID NO:1234), ZAZ3176 (SEQ ID NO:1235), ZAZ3220 (SEQ ID NO:1236), ZAZ3221 (SEQ ID NO:1237), ZAZ3234 (SEQ ID NO:1238), ZAZ3235 (SEQ ID NO:1239), ZAZ3236 (SEQ ID NO:1240), ZAZ3237 (SEQ ID NO:1241), ZAZ3269 (SEQ ID NO:1242), and ZAZ3270 (SEQ ID NO:1243)) with different linker lengths between the Z and ABD portions were prepared in separate 96-well plates. The Z-ABD-Z peptide was titrated from 190 nM to 0.003 nM in a three-step titration in a medium containing 0.9 nM hIL-17A and 8 µM HSA. Standard IL-17A curves (6.2–0.0001 nM) and controls containing medium with 0.9 nM IL-17A or medium alone were also prepared. The medium in plates containing overnight cultured NHDF cells was discarded, and 100 µl / well of sample was transferred to the cell plate and incubated at 37°C for 18–24 h. The next day, the IL-6 content in the supernatant was quantified using an IL-6-specific ELISA as described in Example 3. In subsequent NHDF assays, essentially as described above, but using NHDF cell lines from ATCC (catalog number PSC-201-012), additional Z-ABD-Z peptides (ZAZ3363 (SEQ ID NO: 1244), ZAZ3364 (SEQ ID NO: 1245), ZAZ3365 (SEQ ID NO: 1246), and ZAZ3422 (SEQ ID NO: 1247)) were analyzed. The blocking ability of these Z-ABD-Z peptides was also analyzed after incubation at 40°C for 2 and 4 weeks.

[0510] result

[0511] The ability of the Z-ABD-Z peptide to block IL-17A-induced IL-6 production was investigated in an NHDF assay. First, the Z-ABD-Z formulation was observed to significantly increase the blocking ability compared to the monomeric His6-Z formulation, as shown in Figure 7A, which displays the inhibition curves for His6-Z10241 and ZAZ3220 (including Z10241). The shape of the ZAZ3220 curve indicates that the detection limit of this cellular assay was reached, demonstrating a one-to-one inhibitory effect of ZAZ3220, and it is possible that the Z-ABD-Z peptide exhibits even better inhibitory activity than expected from this experiment. It was anticipated that the potency of ZAZ3220 would increase to the detection limit due to the strong affinity effect obtained by binding to the homodimeric IL-17A. Secondly, a comparison of Z-ABD-Z peptides with different linker lengths and containing Z variants Z06282 (SEQ ID NO: 1206) and Z12876 (SEQ ID NO: 1218) showed that linker length has a relatively small effect on blocking ability. A group of Z-ABD-Z peptides containing Z06282 with different linker lengths is shown in Figure 7B. The Z-ABD-Z peptides ZAZ3363, ZAZ3364, ZAZ3365, and ZAZ3422 were also analyzed in subsequent NHDF assays after incubation at 40°C for 2 and 4 weeks. Even after four weeks of incubation at 40°C, IL-17A inhibitory ability was retained. The calculated IC50 values ​​reflecting the ability of different Z-ABD-Z peptides to inhibit IL-17A are summarized in Table 19. These IC50 values ​​are more than three times lower than the reported hIL-6 neutralizing activity (IC50 = 2.1 ± 0.1 nM) of the IL-17A inhibitory monoclonal antibodies secukinumab AIN457 (WO2006 / 013107 and WO2012 / 125680) currently in clinical development.

[0512] Table 19: Calculated IC50 values ​​from NHDF measurements

[0513]

[0514]

[0515] Example 15

[0516] In vivo neutralization of hIL-17A

[0517] Human IL-17A can bind to and stimulate the mouse IL-17 receptor, leading to an increase in mouse KC (CXCL1) chemokine and subsequent secretion.

[0518] Materials and methods

[0519] In vivo neutralization of hIL-17A in a KC mouse model Dosage range experiments were conducted to identify the optimal dose of human IL-17A for inducing KC in mice. These experiments revealed that a dose of 150 μg / kg of human IL-17A induced appropriate KC levels in mouse serum collected 2 hours after IL-17A administration. ZAZ3174 (SEQ ID NO: 1233) and ZAZ3220 (SEQ ID NO: 1236) were analyzed in this model under two different scenarios. In the first experiment, ZAZ3174 was subcutaneously administered to mice at three different doses: 0.25 mg / kg, 2.5 mg / kg, and 25 mg / kg 9 hours prior to human IL-17A administration. Mice were sacrificed 2 hours after human IL-17A administration, and KC levels were determined by ELISA using a commercially available kit (KC Quantikine; R&D Systems, catalog number D1700) according to the manufacturer's instructions. In the second experiment, ZAZ3220 was subcutaneously administered to mice at three different doses: 0.05 mg / kg, 0.1 mg / kg, and 0.4 mg / kg, nine hours prior to the administration of human IL-17A. Mice were sacrificed two hours after administration of human IL-17A, and KC levels were determined by ELISA as described above.

[0520] result

[0521] In vivo neutralization of hIL-17A in a KC mouse model The Z-ABD-Z peptide was determined to block the ability of human IL-17A to stimulate mouse IL-17 receptors, resulting in a dose-dependent inhibition of KC elevation in mice. ZAZ3174 completely blocked KC induction at a dose of 2.5 mg / kg under the described conditions. Figure 8A As shown in the diagram. The second experiment revealed that the Z-ABD-Z peptide ZAZ3220, containing the affinity-matured Z variant, completely blocked the IL-17A-induced KC response at a dose of 0.4 mg / kg. Figure 8B This corresponds to a 6-fold increase in in vivo blocking effect compared to ZAZ3174. 72% inhibition was achieved with 0.1 mg / kg ZAZ3220.

[0522] Example 16

[0523] Pharmacokinetics of Z-ABD-Z peptide in rats

[0524] This example describes two separate experiments in which pharmacokinetic parameters of two different Z-ABD-Z peptides were determined in rats following subcutaneous (sc) and / or intravenous (iv) injection.

[0525] Materials and methods

[0526] In the first experiment, ZAZ3220 (SEQ ID NO: 1236) in PBS was administered intravenously (n=3) to male Sprague Dawley (SD) rats (Charles River, Germany) at a dose of 1.2 mg / kg corresponding to 57 nmol / kg. Blood samples were collected from the tail vein of each rat at different time points after administration: 0.08 h, 8 h, 24 h, 48 h, 72 h, 120 h, 168 h, 240 h, 336 h, 408 h, and 504 h.

[0527] In the second experiment, ZAZ3363 (SEQ ID NO:1244) in PBS was administered to male SD rats via intravenous (iv) (n=5) or subcutaneous (sc) at a dose of 1.2 mg / kg corresponding to 64 nmol / kg. Blood samples were collected from the tail vein of each rat at time points of 0 h, 0.08 h, 0.5 h, 1 h, 3 h, 8 h, 24 h, 72 h, 120 h, 168 h, 216 h, 264 h, 336 h, 408 h, 456 h, and 504 h after iv administration, or at time points of 0 h, 0.08 h, 0.5 h, 1 h, 3 h, 8 h, 24 h, 72 h, 120 h, and 168 h after sc administration.

[0528] Serum was prepared from blood samples and stored at -20°C until analysis. ZAZ3220 and ZAZ3363 in rat serum were quantified using PK-ELISA.

[0529] PK-ELISA96-well, half-zone plates (50 µl / well) were coated overnight at 4°C with 4 µg / well of goat anti-Z Ig (in-house manufactured) in coating buffer (Sigma, catalog number C3041). The next day, the plates were blocked with PBS + 0.5% casein for 1.5 h. Individual rat serum, at a minimum dilution of 10x in a PBS-casein + 10% rat serum pool (assay matrix), was added to the plates and titrated serially in 2-fold dilutions. One plate contained a standard for each Z-ABD-Z peptide (titrated between 300 ng / ml and 3 pg / ml), and each plate contained four controls for each Z-ABD-Z peptide diluted to concentrations within the linear range of the assay. The standards and controls were diluted in the assay matrix. The diluted standards, controls, and samples were prepared in separate plates and transferred to the coated ELISA plates. The plate was incubated at RT for 1.5 h, followed by incubation for 1.5 h with a custom-made detection rabbit anti-ABD Ig (4 µg / ml, CUV002) and then for 1 h with donkey anti-rabbit IgG-HRP (Jackson Immunoresearch, catalog number 711-035-152). The reaction was developed with TMB (Thermo Fisher), and the reaction was terminated with 2 M H2SO4 after 15 min. The plate was then read from an ELISA reader (Victor). 3 Absorbance was measured at 450 nm using a Perkin Elmer. The concentrations of the respective Z-ABD-Z variants in the samples were calculated from the standard curve using a GraphPad Prism5 and four-parameter logarithmic (4-PL) curve fitting.

[0530] Pharmacokinetic analysis Pharmacokinetic analysis was based on the relationship between individual rat serum concentrations and time. The terminal half-life (T½) and bioavailability were estimated using Microsoft Excel and GraphPad Prism, and a two-compartment model was applied.

[0531] result

[0532] The mean serum concentration-time spectra of Z-ABD-Z peptides ZAZ3220 and ZAZ3363 after a single administration of 1.2 mg / kg to SD rats are shown in Figures 9A and 9B, respectively, and the pharmacokinetic parameters calculated using two-compartment analysis are summarized in Table 20. The estimated half-life (t½) of both ZAZ3220 and ZAZ3363 after intravenous administration is approximately 49 h. The t½ of ZAZ3363 administered subcutaneously is 45 h, and the bioavailability is calculated to be 45%.

[0533] Table 20: Mean pharmacokinetic parameters of Z-ABD-Z peptide after a single intravenous or subcutaneous administration to SD rats

[0534]

[0535] Cmax: Peak serum concentration; Tmax: Time to reach peak serum concentration; AUC 0-t : - Area under the time curve from time zero to the final quantifiable concentration: T 1 / 2 Half-life; MRT: Mean Retention Time; CL: Clearance; F: Absolute Bioavailability Percentage, calculated as F = [(mean AUC)] s.c. × Dosage i.v. ) / (Average AUC) i.v. × Dosage s.c. )]×100

[0536] Example 17

[0537] Topical application of the Z variant to rabbit eyes

[0538] Topical application to the eye is beneficial for ocular disorders such as uveitis or dry eye. Topical application allows for extremely high local drug concentrations with minimal risk of systemic side effects. In this example, the feasibility of topical application of the Z variant molecule to the eye was investigated in rabbits. After four repeated topical administrations, the concentrations of the Z variant molecule and control IgG were measured in the anterior chamber (aqueous humor), vitreous fluid, and serum.

[0539] Materials and methods

[0540] The generation of Z variants The Z variant Z10199 (SEQ ID NO: 1217) was derived from Z06282 (SEQ ID NO: 1206), but the starting AE was subcloned without any tag, except that the C-terminus of which was supplemented with the amino acid residue VD. It was expressed essentially as described in Example 2 and purified by anion exchange and reversed-phase chromatography. The sample buffer was exchanged for PBS pH 7.2 and EndoTrap was used. ® Hyglos (Red 10 Pillars) removes endotoxins.

[0541] Animal researchAt time points 0, 1, 2, and 3 hours, 48 ​​nmol (1 drop, 50 µl) of Z10199 and ZAZ3174 (SEQ ID NO: 1233) were topically applied to each eye of rabbits (n=2 / molecule). A control human IgG antibody (Xolair; Novartis) was administered to two different rabbits in the same manner. As a negative control, one rabbit was administered PBS only. After each application, the eyelids were kept closed for approximately 30 seconds to retain the sample on the cornea. After 4 hours, vitreous fluid, aqueous humor, and serum were collected. The samples were centrifuged to remove tissue debris and aggregates, and the levels of Z10199, ZAZ3174, and the antibody were quantified by ELISA.

[0542] Quantitative analysis using ELISA A sandwich ELISA was performed using internally produced polyclonal goat anti-protein Z immunoglobulin for capture, internally produced polyclonal rabbit anti-protein Z immunoglobulin as the primary antibody, and goat anti-rabbit IgG-HRP (Dako catalog number P0448) as the secondary antibody to analyze the concentrations of Z10199 and ZAZ3174 in the collected samples. Detection was performed by incubation at RT with ImmunoPure TMB for 15 min, and the reaction was terminated by adding 2 M H2SO4. The results were analyzed using a Victor microplate reader. 3 Absorption was measured at 450 nm. The concentration of Z10199 was calculated using a standard curve prepared with the same molecule and a nonlinear regression formula using GraphPad Prism5.

[0543] The IgG concentration in the collected samples was analyzed using an IgG ELISA kit (Abcam 100547) and analyzed using the provided standard curve as described by the manufacturer and by nonlinear regression as described above.

[0544] result

[0545] Figure 10 It has been shown that both Z10199 and ZAZ3174 permeate the eye after topical application and are present in the aqueous humor and vitreous humor at low nM concentrations. In contrast, control IgG antibodies were not detected in the aqueous humor or vitreous humor. Serum samples can be considered negative for both the Z variant and IgG. Therefore, the Z variant molecules of this disclosure can be delivered via this alternative route of administration, which cannot be used for antibody administration, and can achieve a local effect that avoids systemic exposure.

[0546] Example 18

[0547] Pharmacokinetic analysis of ZAZ3363 product administered duodenally in rats

[0548] Materials and methods

[0549] Test Project ZAZ3363 (SEQ ID NO:1244) was prepared at a concentration of 50 mg / ml (OAF1 and OAF2) or 100 mg / ml (PBS) in 1) OAF1: 0.12 M sodium chenodeoxycholate (Sigma, catalog number C8261) and 0.12 M propyl gallate (Sigma, catalog number P3130), pH 7.4; 2) OAF2: 50 mg / m sodium decanoate (Sigma, catalog number C4151); or 3) 50 mM sodium phosphate (PBS), pH 7.0.

[0550] NHDF cell assay The activity of ZAZ3363 (SEQ ID NO:1244) formulated in OAF1, OAF2 or PBS was confirmed in the IL-17-dependent NHDF cell assay described in Example 14.

[0551] Duodenal administration Male Sprague Dawley rats (Charles River) were anesthetized with isoflurane, and a small incision was made to locate the duodenum. An indwelling catheter (R-DUOD, AgnTho's, Sweden) was surgically inserted into the duodenum, 20 mm from its origin in the region of least vascular system. The catheter was percutaneously inserted into the animal's back. The abdominal muscle tissue was closed with sutures, and the abdominal skin incision and the subscapular external site were closed with stainless steel wound clips. These animals were left to recover for 5–6 days before the study. Postoperative analgesia (carprofen 5 mg / kg and buprenorphine 0.05 mg / kg) was administered subcutaneously before surgery. Carprofen was also administered once daily for two days postoperatively. Additional doses of buprenorphine were administered as needed. Antibiotics (enrofloxacin 0.3 mg / ml) were administered in drinking water during the recovery period (3–5 days) and throughout the experiment. To mask the bitterness of enflufloxacin, add a piece of sugar to 500 ml of drinking water. To ensure patency, flush the duodenal catheter daily with sterile water (0.2–0.5 ml).

[0552] At time point zero, ZAZ3363 was administered directly into the duodenum in 0.5 ml volumes at a dose of 5.4 µmol / kg body weight in OAF1 or OAF2 (n=2) or at a dose of 10.8 µmol / kg in PBS (n=2). Blood samples were collected under isoflurane anesthesia at 1 h, 3 h, 8 h, 24 h, 72 h, 120 h, and 168 h post-administration, and serum was prepared using standard procedures. The concentration of ZAZ3363 in serum was measured using the quantitative PK-ELISA described in Example 16, but ZAZ3363 titrated between 70 ng / ml and 1 ng / ml was used as a standard.

[0553] Pharmacokinetic analysis Pharmacokinetic analysis was based on the relationship between individual rat serum concentrations and time. Terminal half-life (T½) and bioavailability were estimated using Microsoft Excel and GraphPad Prism.

[0554] result

[0555] The activity of the prepared ZAZ3363 In the NHDF cell assay, the activities of ZAZ3363 formulated in OAF1 and OAF2 were compared with those in PBS. Figure 11 shows the titration curves of OAF1 (Figure 11A) and OAF2 (Figure 11B) compared with ZAZ3363 formulated in PBS. The experiment showed that the activity of ZAZ3363 in both formulations was the same as that in the PBS formulation, that is, the biological activity of ZAZ3363 is not affected by different excipients.

[0556] Duodenal uptake of ZAZ3363 In a rat model of intraduodenal administration (id), the intestinal uptake of OAF1, OAF2, and PBS-prepared ZAZ3363 was examined. The experiment showed that, compared with PBS, the uptake of OAF1 and OAF2 formulations was increased (…). Figure 12 As shown in Table 21, the bioavailability of OAF1, OAF2, and PBS were 0.2, 0.8, and 0.0007, respectively. Despite observing significant individual variability, the OAF2 formulation of ZAZ3363 showed optimal uptake, averaging 1160 times better than the PBS formulation. The OAF1 formulation of ZAZ3363 was averaging 260 times better than the PBS formulation.

[0557] Table 21: Bioavailability and T½ of ZAZ3363 after intraduodenal administration in three different formulations

[0558]

[0559] Example 19

[0560] Characterization of the anti-IL-17A / anti-TNF complex in NHDF assay

[0561] Materials and methods

[0562] Production of complex and control antibody This study aimed to construct two different complexes targeting IL-17A and TNF, as well as an antibody with affinity for TNF. Heavy chain (HC) and light chain (LC) sequences were used. Ada (SEQ ID NO:1231) and LC Ada (SEQ ID NO:1232) An antibody designated "Ada" was constructed with the same CDR sequence and specificity as the commercially available monoclonal antibody adalimumab. The IL-17A-targeting Z variant Z14253 (SEQ ID NO:1219) was partially genetically fused to HC via a flexible 15-residue (GGGGS)3 linker. Ada or LC Ada The C-terminus of each compound generates the HC complex. Ada -Z14253 and LC Ada -Z14253. A schematic diagram of the constructed complex is shown in Figure 13A. Gene synthesis, cloning, production via transient gene expression in CHO cells, and purification using protein A affinity chromatography were performed using Evitria AG (Switzerland).

[0563] Blockade of IL-17A-induced IL-6 production in NHDF assay The NHDF determination was basically performed as described in Example 14, and the HC content of the study sample was... Ada -Z14253 and LC Ada Z14253 and its comparatives were titrated from 63 nM to 0.003 nM in a three-fold step in a medium containing 0.9 nM rhIL-17A. Comparatives included ZAZ3363 (SEQ ID NO: 1244), the anti-TNF antibody Ada, and the negative control variant Z04726 (SEQ ID NO: 1223; targeting Taq polymerase) (denoted as Z04726-PP013) which was recombinantly fused to the ABD variant PP013 (SEQ ID NO: 1224) via a VDSS linker as described in Example 2. Standard IL-17A curves (6.2–0.0001 nM) and controls containing medium or a separate medium containing 0.9 nM IL-17A were also prepared. The IL-6 content in the supernatant was quantified using an IL-6-specific ELISA as described in Example 3.

[0564] Blockade of TNF or TNF / IL-17A-induced IL-8 production in NHDF assayNHDF (Lonza, catalog number CC-2511) cells were cultured in fibroblast basal medium (Lonza, catalog number CC-3132) supplied with growth factor (Lonza, catalog number CC-5034). The day before the experiment, 5000 cells / well were seeded at 100 µl into a half-zone 96-well plate (Greiner, catalog number 675180). On the day of the experiment, HC cells were prepared in separate 96-well plates. Ada -Z14253 and LC Ada -Z14253 and dilutions of the comparatives described above. The complex and comparatives were titrated from 5 nM to 0.0007 nM in three-step increments in a medium containing 0.1 nM human TNF (R&D Systems, catalog number 2210-TA / CF) or a mixture of 0.05 nM and 0.1 nM rhIL-17A. Controls were also prepared containing either 0.1 nM TNF or a mixture of 0.05 nM and 0.1 nM rhIL-17A, or a medium alone. The medium in the plates containing overnight-cultured NHDF cells was discarded, and 100 µl / well of sample was transferred to the cell plate. The plates were incubated at 37°C for 18–24 h. The next day, the IL-8 content in the supernatant was quantified using an IL-8-specific ELISA.

[0565] IL-8 ELISA IL-8 was quantified using the DuoSet ELISA kit (R&D Systems, catalog number DY208). Half-zone plates (Costar, catalog number 3690) were coated overnight at 4°C with 50 µl / well of 4 µg / ml anti-IL-8 capture antibody in PBS. On the day of analysis, the plates were rinsed twice with tap water and blocked with PBS + 1% BSA for 2 h. IL-8 standards (R&D Systems, catalog number 890806) titrated serially at 2-fold dilutions (20–0.01 ng / ml) and supernatant from cell assay plates were added to the coated ELISA plates (50 µl / well) and incubated at RT for 1.5 h. The plates were washed four times in an automated ELISA washer, and 20 ng / ml (50 µl / well) of biotinylated anti-IL-8 detection antibody was added. After an additional 1 hour of incubation, the plate was washed, and 50 μl of streptavidin-HRP (ThermoFisher, catalog number N100), diluted 8000 x, was added to each well. After another hour of incubation and washing, the plate was developed with 50 µl of TMB (ThermoFisher, catalog number 34021) per well, and the reaction was terminated with 50 µl of 2 M H2SO4. The reaction was then performed using a multi-label reader (Victor).3 The absorbance was read from the Perkin Elmer.

[0566] result

[0567] Blockade of IL-17A-induced IL-6 production in NHDF assay Study of two complex HC in NHDF determination Ada -Z14253 and LC Ada -Z14253 regarding their ability to block IL-17A-induced IL-6 production. Results from NHDF measurements are shown in Figure 13B. HC Ada -Z14253 and LC Ada Both Z14253 and ZAZ3363 possess similar IL-17A blocking capabilities to ZAZ3363. As expected, no inhibition was observed with Ada or the negative control Z04726-PP013.

[0568] Blockade of TNF or TNF / IL-17A-induced IL-8 production in NHDF assay Study of two complex HC in NHDF determination Ada -Z14253 and LC Ada -Z14253 Regarding their ability to block TNF or TNF / IL-17A mixture-induced IL-8 production. Results from NHDF assays show that, regarding specific TNF blocking ability, HC Ada -Z14253 and LC Ada -Z14253 and the anti-TNF antibody Ada have similar inhibitory properties (Figure 13C). However, in a combined assay investigating the blocking effects on both TNF and IL-17, HC showed significantly better inhibition compared to Ada and ZAZ3363. Ada -Z14253 and LC Ada -Z14253 exhibits excellent suppression properties (Figure 13D).

[0569] Example 20

[0570] Pharmacokinetics of Z-ABD-Z peptide in monkeys

[0571] This example describes a 10-day repeated-dose study in cynomolgus monkeys administered the Z-ABD-Z peptide ZAZ3363. The results were used to estimate the half-life of ZAZ3363 in cynomolgus monkeys.

[0572] Materials and methods

[0573] ZAZ3363 (SEQ ID NO:1244) was administered as a short intravenous infusion on days 1, 4, 7, and 10 at 20 mg / kg (n=2; 1 male and 1 female) and 40 mg / kg (n=4; 2 males and 2 females). Plasma samples were collected to determine ZAZ3363 concentrations in combination with the first dose at time point 0 (before administration), 5 min, 0.5 h, 1 h, 2 h, 6 h, 24 h, and 48 h after administration, and the last dose at day 10 (before administration), 5 min, 0.5 h, 1 h, 2 h, 6 h, 24 h, 48 h, 5 days, 7 days, 10 days, 12 days, 14 days, and 21 days after administration. ZAZ3363 in plasma samples was quantified by LC-MS / MS based on peptides obtained after trypsin digestion of ZAZ3363. Concentration-time spectra were evaluated using both a non-partitioned approach (where day 1 and day 10 were analyzed separately) and a partitioned approach (where day 1 and day 10 data were evaluated for each animal combination).

[0574] result

[0575] The mean plasma concentration-time profiles of ZAZ3363 after administration on day 1 and day 10 are shown in Figures 14A and 14B, respectively. Although ZAZ3363 was not administered as a single dose evaluated in full PK, the available results after multiple dosings allowed for prediction of the concentration-time profile after a single dose. Based on bipartite behavior, plasma concentrations decreased in both phases. The t½ for the second phase, estimated from this repeated-dose study and a second repeated-dose study (data not shown), was approximately 4–7.5 days, consistent with the reported t½ for monkey albumin (Deo et al., 1974, J Nutr 104:858–64). No significant sex differences were observed.

[0576] Example 21

[0577] Oral administration of Z-ABD-Z peptide in dogs

[0578] Materials and methods

[0579] Capsule preparation ZAZ3363 (SEQ ID NO: 1244) was formulated in OAF1 (see Example 18) at a concentration of 100 mg / ml. The formulation was lyophilized and filled into hard-shell capsules, followed by enteric coating (performed by Catalent PharmaSolutions, Beinheim, France). Each capsule contained approximately 25 mg of ZAZ3363.

[0580] Animal researchAnimal studies were conducted at Huntingdon Life Science (Cambridgeshire, UK). Fasted beagles (n = 3; females) each received six capsules (approximately 150 mg) containing ZAZ3363. Serum samples were collected at 0.5, 1, 2, 4, 6, 8, 12, 24 and 96 hours after administration.

[0581] Quantitative The concentration of ZAZ3363 in serum samples was quantified using a PK sandwich ELISA, which is essentially as described in Example 16, but with the use of internally generated monoclonal antibody-Z IgG in the first coating step.

[0582] result

[0583] ZAZ3363 canine serum concentrations and time profiles are shown in... Figure 15 The results showed that ZAZ3363 was absorbed in the intestine in all three animal groups, but with some individual variation. Serum concentrations of ZAZ3363 reached a maximum of 2–30 nM. After circulation, serum levels of ZAZ3363 remained stable for at least 96 h, attributed to the interaction between the ABD portion PP013 (SEQ ID NO: 1223) within ZAZ3363 and canine albumin. The ability of PP013 to bind to canine albumin has been previously demonstrated (WO2012 / 004384).

[0584] Item-by-item listing of the implementation plan

[0585] 1. An IL-17A binding polypeptide comprising an IL-17A binding motif BM, the motif being composed of an amino acid sequence selected from:

[0586] i) EX2DX4AX6X7EIX 10 X 11 LPNL X 16 X 17 X 18 QX 20 X 21 AFIX 25 X 26 LX 28 X 29

[0587] Among them, independently of each other,

[0588] X2 is selected from A, H, M, and Y;

[0589] X4 is selected from A, D, E, F, K, L, M, N, Q, R, S, and Y;

[0590] X6 is selected from A, Q, and W;

[0591] X7 is selected from F, I, L, M, V, W, and Y;

[0592] X 10 Selected from A and W;

[0593] X 11 Selected from A, D, E, F, G, L, M, N, Q, S, T, and Y;

[0594] X 16 Selected from N and T;

[0595] X 17 Selected from H, W, and Y;

[0596] X 18 Selected from A, D, E, H, and V;

[0597] X 20 Selected from A, G, Q, S, and W;

[0598] X 21 Selected from A, D, E, F, H, K, N, R, T, V, W, and Y;

[0599] X 25 Selected from A, D, E, G, H, I, L, M, N, Q, R, S, T, and V;

[0600] X 26 Selected from K and S;

[0601] X 28 Selected from I, L, N, and R; and

[0602] X 29 Selected from D and R;

[0603] and

[0604] ii) An amino acid sequence that has at least 89% identity with the sequence defined in i).

[0605] 2. The IL-17A binding peptide according to Project 1, wherein, in sequence i):

[0606] X2 is selected from A, H, and M;

[0607] X4 is selected from A, D, E, F, L, M, N, Q, R, and Y;

[0608] X 11 Selected from A, D, E, F, G, L, M, N, S, T, and Y;

[0609] X 18 Selected from A, D, E, and V;

[0610] X 20 Selected from A, G, Q, and W;

[0611] X 21 Selected from E, F, H, N, R, T, V, W, and Y;

[0612] X 25 Selected from A, D, E, G, H, I, L, N, Q, R, S, T, and V; and

[0613] X 28 Selected from I, N, and R.

[0614] 3. The IL-17A binding peptide according to Project 2, wherein, in sequence i):

[0615] X 16 Let T be the value of T.

[0616] X 17 For W;

[0617] X 21 Selected from E, F, H, W, T, and Y;

[0618] X 25 Selected from A, D, E, G, H, I, L, N, Q, R, S, and T;

[0619] X 26 Let K be K; and

[0620] X 29 The answer is D.

[0621] 4. The IL-17A binding polypeptide according to any of the preceding items, wherein sequence i) satisfies at least 6 of the following 11 conditions I-XI:

[0622] I. X2 is A;

[0623] II. X4 is selected from D, E, and Q;

[0624] III. X6 is A;

[0625] IV. X7 is selected from F and V;

[0626] V. X 16 Let T be the value of T.

[0627] VI. X 17 For W;

[0628] VII. X 18 Choose from A and D;

[0629] VIII. X 20 For W;

[0630] IX. X 26 Let K be the value of K.

[0631] X. X 28 Let R be the value of R; and

[0632] XI. X 29 The answer is D.

[0633] 5. The IL-17A binding polypeptide according to Project 4, wherein sequence i) satisfies at least 7 of the 11 conditions I-XI.

[0634] 6. The IL-17A binding polypeptide according to Project 5, wherein sequence i) satisfies at least 8 of the 11 conditions I-XI.

[0635] 7. The IL-17A binding polypeptide according to Project 6, wherein sequence i) satisfies at least 9 of the 11 conditions I-XI.

[0636] 8. The IL-17A binding polypeptide according to Project 7, wherein sequence i) satisfies at least 10 of the 11 conditions I-XI.

[0637] 9. The IL-17A binding polypeptide according to Project 8, wherein sequence i) satisfies all of the 11 conditions I-XI.

[0638] 10. The IL-17A binding polypeptide according to any of the foregoing items, wherein X2X6, X2X 10 or X6X 10 It is AA.

[0639] 11. The IL-17A binding polypeptide according to any of the foregoing items, wherein X2X 17 X2X 20 X6X 17 X6X 20 X 10 X 17 or X 10 X 20 It is AW.

[0640] 12. The IL-17A binding polypeptide according to any of the foregoing items, wherein X2X 28 X6X 28 or X 10 X 28 For AR.

[0641] 13. The IL-17A binding polypeptide according to any of the foregoing items, wherein X 17 X 28 or X20 X 28 For WR.

[0642] 14. The IL-17A binding polypeptide according to any of the preceding items, wherein X 17 X 20 For WW.

[0643] 15. The IL-17A binding polypeptide according to any of the preceding items, wherein sequence i) is a sequence selected from position 8 to position 36 of the group consisting of SEQ ID NO: 1-1216.

[0644] 16. The IL-17A binding polypeptide according to item 15, wherein sequence i) is the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO: 1-66, 1200, 1206 and 1214.

[0645] 17. The IL-17A binding polypeptide according to item 16, wherein sequence i) is the sequence from position 8 to position 36 of the sequence selected from the group consisting of SEQ ID NO:1-66.

[0646] 18. The IL-17A binding polypeptide according to item 17, wherein sequence i) is the sequence from position 8 to position 36 of the sequence selected from the group consisting of SEQ ID NO:1-35.

[0647] 19. The IL-17A binding polypeptide according to item 18, wherein sequence i) is the sequence from position 8 to position 36 of the sequence selected from the group consisting of SEQ ID NO:1-27.

[0648] 20. The IL-17A binding polypeptide according to item 19, wherein sequence i) is the sequence selected from position 8 to position 36 of the group consisting of SEQ ID NO: 1-10.

[0649] 21. The IL-17A binding polypeptide according to Item 20, wherein sequence i) is the sequence selected from position 8 to position 36 of the group consisting of SEQ ID NO: 1-7.

[0650] 22. The IL-17A binding polypeptide according to item 21, wherein sequence i) is the sequence selected from position 8 to position 36 of the group consisting of SEQ ID NO: 1-4.

[0651] 23. The IL-17A binding polypeptide according to item 22, wherein sequence i) is the sequence from position 8 to position 36 in SEQ ID NO:1.

[0652] 24. The IL-17A binding polypeptide according to any of the preceding items, wherein the IL-17A binding motif forms part of a triple-helix bundle protein domain.

[0653] 25. The IL-17A binding polypeptide according to Item 24, wherein the IL-17A binding motif substantially forms part of a double helix with interconnected loops within the triple-helix bundle protein domain.

[0654] 26. The IL-17A binding polypeptide according to Item 25, wherein the triple-helix bundle protein domain is selected from the bacterial receptor domain.

[0655] 27. The IL-17A binding polypeptide according to Item 26, wherein the triple-helix bundle protein domain is selected from the domain of protein A from Staphylococcus aureus or a derivative thereof.

[0656] 28. The IL-17A binding polypeptide according to any of the foregoing items, wherein the IL-17A binding polypeptide comprises an amino acid sequence binding module BMod selected from:

[0657] iii) K-[BM]-DPSQS X a X b LLX c EAKKL X d X e X f Q;

[0658] in

[0659] [BM] is the IL-17A binding motif as defined in any of items 1-23, provided that X 29 The answer is D;

[0660] X a Selected from A and S;

[0661] X b Selected from N and E;

[0662] X c Selected from A, S, and C;

[0663] X d Selected from E, N, and S;

[0664] X e Selected from D, E, and S;

[0665] X f Selected from A and S; and

[0666] iv) An amino acid sequence that has at least 85% identity with the sequence defined by iii).

[0667] 29. The IL-17A binding polypeptide according to any one of items 1-27, wherein the IL-17A binding polypeptide comprises an amino acid sequence binding module BMod selected from the following:

[0668] v) K-[BM]-QPEQS X a X b LLX c EAKKL X d X e X f Q;

[0669] in

[0670] [BM] is the IL-17A binding motif as defined in any of items 1-23, provided that X 29 R;

[0671] X a Selected from A and S;

[0672] X b Selected from N and E;

[0673] X c Selected from A, S, and C;

[0674] X d Selected from E, N, and S;

[0675] X e Selected from D, E, and S;

[0676] X f Selected from A and S; and

[0677] vi) An amino acid sequence that has at least 85% identity with the sequence defined by v).

[0678] 30. The IL-17A binding polypeptide according to item 28 or 29, wherein X in sequence iii) or v) a The answer is A.

[0679] 31. The IL-17A binding polypeptide according to item 28 or 29, wherein X in sequence iii) or v) a Let it be S.

[0680] 32. The IL-17A binding polypeptide according to any one of items 28-31, wherein X in sequence iii) or v) b Let N be the number of elements in the array.

[0681] 33. The IL-17A binding polypeptide according to any one of items 28-31, wherein X in sequence iii) or v) b The value is E.

[0682] 34. The IL-17A binding polypeptide according to any one of items 28-33, wherein X in sequence iii) or v) c The answer is A.

[0683] 35. The IL-17A binding polypeptide according to any one of items 28-33, wherein X in sequence iii) or v) c Let it be S.

[0684] 36. The IL-17A binding polypeptide according to any one of items 28-33, wherein X in sequence iii) or v) c The answer is C.

[0685] 37. The IL-17A binding polypeptide according to any one of items 28-36, wherein X in sequence iii) or v) d The value is E.

[0686] 38. The IL-17A binding polypeptide according to any one of items 28-36, wherein X in sequence iii) or v) d Let N be the number of elements in the array.

[0687] 39. The IL-17A binding polypeptide according to any one of items 28-36, wherein X in sequence iii) or v) d Let it be S.

[0688] 40. The IL-17A binding polypeptide according to any one of items 28-39, wherein X in sequence iii) or v) e The answer is D.

[0689] 41. The IL-17A binding polypeptide according to any one of items 28-39, wherein X in sequence iii) or v) e The value is E.

[0690] 42. The IL-17A binding polypeptide according to any one of items 28-39, wherein X in sequence iii) or v) e Let it be S.

[0691] 43. The IL-17A binding polypeptide according to any one of items 37 and 39-42, wherein X in sequence iii) or v) d X e Selected from EE, ES, SD, SE, and SS.

[0692] 44. The IL-17A binding polypeptide according to item 43, wherein X in sequence iii) or v) d X e For ES.

[0693] 45. The IL-17A binding polypeptide according to item 43, wherein X in sequence iii) or v) d X e It is SE.

[0694] 46. ​​The IL-17A binding polypeptide according to item 43, wherein X in sequence iii) or v) d X e For SD.

[0695] 47. The IL-17A binding polypeptide according to any one of items 28-46, wherein X in sequence iii) or v) f The answer is A.

[0696] 48. The IL-17A binding polypeptide according to any one of items 28-46, wherein X in sequence iii) or v) f Let it be S.

[0697] 49. The IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X a For A; X b For N; X c For A and X f The answer is A.

[0698] 50. The IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X a S; X b For E; X c For A and X f The answer is A.

[0699] 51. The IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X a For A; X b For N; X c C and X f The answer is A.

[0700] 52. The IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X a S; X b For E; X c For S and X f Let it be S.

[0701] 53. The IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X a S; X b For E; X c For S and X f The answer is A.

[0702] 54. The IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X a S; X b For E; X c For A and X f Let it be S.

[0703] 55. The IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X a S; X b For E; X c C and X f Let it be S.

[0704] 56. The IL-17A binding polypeptide according to item 49, wherein, in sequence iii) or v), X a For A; X b For N; X c For A; X d X e For ND and X f The answer is A.

[0705] 57. The IL-17A binding polypeptide according to item 50, wherein, in sequence iii) or v), X a S; X b For E; X c For A; X d X e For ND and X f The answer is A.

[0706] 58. The IL-17A binding polypeptide according to item 51, wherein, in sequence iii) or v), X a For A; X b For N; X c For C; X d X e For ND and X f The answer is A.

[0707] 59. The IL-17A binding polypeptide according to item 52, wherein, in sequence iii) or v), X a S; X b For E; X c S; Xd X e It is ND; and X f Let it be S.

[0708] 60. The IL-17A binding polypeptide according to item 53, wherein, in sequence iii) or v), X a S; X b For E; X c S; X d X e For ND and X f The answer is A.

[0709] 61. The IL-17A binding polypeptide according to item 55, wherein, in sequence iii) or v), X a S; X b For E; X c For C; X d X e For ND and X f Let it be S.

[0710] 62. The IL-17A binding polypeptide according to item 49, wherein, in sequence iii) or v), X a For A; X b For N; X c For A; X d X e For SE and X f The answer is A.

[0711] 63. The IL-17A binding polypeptide according to item 50, wherein, in sequence iii) or v), X a S; X b For E; X c For A; X d X e For SE and X f The answer is A.

[0712] 64. The IL-17A binding polypeptide according to item 51, wherein, in sequence iii) or v), X a For A; X b For N; X c For C; X d X e For SE and X f The answer is A.

[0713] 65. The IL-17A binding polypeptide according to item 52, wherein, in sequence iii) or v), X a S; X b For E; X c S; X dX e For SE and X f Let it be S.

[0714] 66. The IL-17A binding polypeptide according to item 54, wherein, in sequence iii) or v), X a S; X b For E; X c For A; X d X e For SE and X f Let it be S.

[0715] 67. The IL-17A binding polypeptide according to item 55, wherein, in sequence iii) or v), X a S; X b For E; X c For C; X d X e For SE and X f Let it be S.

[0716] 68. The IL-17A binding polypeptide according to item 49, wherein, in sequence iii) or v), X a For A; X b For N; X c For A; X d X e For ES and X f The answer is A.

[0717] 69. The IL-17A binding polypeptide according to item 50, wherein, in sequence iii) or v), X a S; X b For E; X c For A; X d X e For ES and X f The answer is A.

[0718] 70. The IL-17A binding polypeptide according to item 51, wherein, in sequence iii) or v), X a For A; X b For N; X c For C; X d X e For ES and X f The answer is A.

[0719] 71. The IL-17A binding polypeptide according to item 52, wherein, in sequence iii) or v), X a S; X b For E; X c S; X d Xe For ES and X f Let it be S.

[0720] 72. The IL-17A binding polypeptide according to item 55, wherein, in sequence iii) or v), X a S; X b For E; X c For C; X d X e For ES and X f Let it be S.

[0721] 73. The IL-17A binding polypeptide according to item 49, wherein, in sequence iii) or v), X a For A; X b For N; X c For A; X d X e For SD and X f The answer is A.

[0722] 74. The IL-17A binding polypeptide according to Item 50, wherein, in sequence iii) or v), X a S; X b For E; X c For A; X d X e For SD and X f The answer is A.

[0723] 75. The IL-17A binding polypeptide according to item 51, wherein, in sequence iii) or v), X a For A; X b For N; X c For C; X d X e For SD and X f The answer is A.

[0724] 76. The IL-17A binding polypeptide according to Item 52, wherein, in sequence iii) or v), X a S; X b For E; X c S; X d X e For SD and X f Let it be S.

[0725] 77. The IL-17A binding polypeptide according to Item 54, wherein, in sequence iii) or v), X a S; X b For E; X c For A; X d X eFor SD and X f Let it be S.

[0726] 78. The IL-17A binding polypeptide according to item 55, wherein, in sequence iii) or v), X a S; X b For E; X c For C; X d X e For SD and X f Let it be S.

[0727] 79. The IL-17A binding polypeptide according to item 28, wherein the sequence iii) is a sequence selected from position 7 to position 55 of the group consisting of SEQ ID NO:1-1216.

[0728] 80. The IL-17A binding polypeptide according to item 79, wherein sequence iii) is the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO: 1-66, 1200, 1206 and 1214.

[0729] 81. The IL-17A binding polypeptide according to item 80, wherein sequence iii) is the sequence selected from position 7 to position 55 of the group consisting of SEQ ID NO: 1-66.

[0730] 82. The IL-17A binding polypeptide according to item 81, wherein sequence iii) is the sequence selected from position 7 to position 55 of the group consisting of SEQ ID NO: 1-35.

[0731] 83. The IL-17A binding polypeptide according to item 82, wherein sequence iii) is the sequence selected from position 7 to position 55 of the group consisting of SEQ ID NO:1-27.

[0732] 84. The IL-17A binding polypeptide according to item 83, wherein sequence iii) is the sequence selected from position 7 to position 55 of the group consisting of SEQ ID NO: 1-10.

[0733] 85. The IL-17A binding polypeptide according to item 84, wherein sequence iii) is the sequence selected from position 7 to position 55 of the group consisting of SEQ ID NO: 1-7.

[0734] 86. The IL-17A binding polypeptide according to item 85, wherein sequence iii) is the sequence selected from position 7 to position 55 of the group consisting of SEQ ID NO: 1-4.

[0735] 87. The IL-17A binding polypeptide according to item 86, wherein sequence iii) is the sequence from position 7 to position 55 in SEQ ID NO:1.

[0736] 88. The IL-17A binding polypeptide according to any of the preceding items, wherein the IL-17A binding polypeptide comprises an amino acid sequence selected from:

[0737] vii) YA-[BMod]-AP;

[0738] Where [BMod] is the IL-17A combination module as defined in any of items 28-87; and

[0739] viii) is an amino acid sequence that has at least 86% identity with the sequence defined by vii).

[0740] 89. The IL-17A binding polypeptide according to any one of items 1-87, wherein the IL-17A binding polypeptide comprises an amino acid sequence selected from:

[0741] ix) FA-[BMod]-AP;

[0742] Where [BMod] is the IL-17A combination module as defined in any of items 28-87; and

[0743] x) is an amino acid sequence that has at least 86% identity with the sequence defined by ix).

[0744] 90. The IL-17A binding polypeptide according to any one of items 1-87, wherein the IL-17A binding polypeptide comprises an amino acid sequence selected from:

[0745] xi) FN-[BMod]-AP;

[0746] Where [BMod] is the IL-17A combination module as defined in any of items 28-87; and

[0747] xii) An amino acid sequence that has at least 86% identity with the sequence defined by xi).

[0748] 91. The IL-17A binding polypeptide according to any of the preceding items, wherein the IL-17A binding polypeptide comprises an amino acid sequence selected from:

[0749] ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK;

[0750] ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;

[0751] DNAKFNK-[BM]-DPSVSCALEAKLNQAPK;

[0752] ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK!

[0753] AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK!

[0754] VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK!

[0755] AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK;

[0756] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK!

[0757] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK!

[0758] AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0759] AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP;

[0760] AEAKYAK-[BM]-DPSQSSELLSEAKKLNDAQAPK!

[0761] AEAKYAK-[BM]-DPSQSSELLSEAKKLNDAQAP;

[0762] AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0763] AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP;

[0764] AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK!

[0765] AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK!

[0766] AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP;

[0767] AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

[0768] AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP;

[0769] AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK;

[0770] AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK;

[0771] AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK;

[0772] AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP;

[0773] AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK;

[0774] AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK;

[0775] AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK;

[0776] AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP;

[0777] AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK;

[0778] AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK;

[0779] AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK;

[0780] VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0781] VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

[0782] VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK;

[0783] VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK;

[0784] VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK;

[0785] VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK;

[0786] VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK;

[0787] VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK;

[0788] VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK;

[0789] VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK;

[0790] VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK;

[0791] VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK;

[0792] AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and

[0793] ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0794] Wherein [BM] is the IL-17A binding motif as defined in any of items 1-23.

[0795] 92. The IL-17A binding polypeptide according to any of the preceding items, wherein the IL-17A binding polypeptide comprises an amino acid sequence selected from:

[0796] xiii) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0797] Where [BM] is the IL-17A binding motif as defined in any of items 1-23; and

[0798] The amino acid sequence that has at least 86% identity with the sequences defined in xiv) and xiii).

[0799] 93. The IL-17A binding polypeptide according to item 92, wherein sequence xiii) is selected from SEQ ID NO:1-1216.

[0800] 94. The IL-17A binding polypeptide according to Item 93, wherein sequence xiii) is selected from 1-66, 1200, 1206 and 1214.

[0801] 95. The IL-17A binding polypeptide according to item 94, wherein sequence xiii) is selected from SEQ ID NO:1-66.

[0802] 96. The IL-17A binding polypeptide according to item 95, wherein sequence xiii) is selected from SEQ ID NO:1-35.

[0803] 97. The IL-17A binding polypeptide according to item 96, wherein sequence xiii) is selected from SEQ ID NO:1-27.

[0804] 98. The IL-17A binding polypeptide according to item 97, wherein sequence xiii) is selected from SEQ ID NO:1-10.

[0805] 99. The IL-17A binding polypeptide according to item 98, wherein sequence xiii) is selected from SEQ ID NO:1-7.

[0806] 100. The IL-17A binding polypeptide according to item 99, wherein sequence xiii) is selected from SEQ ID NO:1-4.

[0807] 101. The IL-17A binding polypeptide according to item 100, wherein sequence xiii) is SEQ ID NO:1.

[0808] 102. The IL-17A binding polypeptide according to any one of items 1-91, wherein the IL-17A binding polypeptide comprises an amino acid sequence selected from:

[0809] xv) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0810] Where [BM] is the IL-17A binding motif as defined in any of items 1-23; and

[0811] The amino acid sequence that has at least 86% identity with the sequence defined in xvi) and xv).

[0812] 103. The IL-17A binding polypeptide according to item 102, wherein the sequence (xv) is selected from SEQ ID NO:1217-1222.

[0813] 104. The IL-17A binding polypeptide according to item 103, wherein the sequence (xv) is selected from SEQ ID NO:1218-1222.

[0814] 105. The IL-17A binding polypeptide according to item 104, wherein the sequence (xv) is selected from SEQ ID NO:1219-1222.

[0815] 106. The IL-17A binding polypeptide according to item 105, wherein the sequence (xv) is selected from SEQ ID NO:1219 and SEQ ID NO:1222.

[0816] 107. The IL-17A binding polypeptide according to item 106, wherein the sequence (xv) is SEQ ID NO:1219.

[0817] 108. The IL-17A binding polypeptide according to any of the preceding items, wherein the IL-17A binding polypeptide is capable of binding IL-17A such that the interacting K D The value is at most 1 x 10 -6 M, such as at most 1 x 10 -7 M, such as at most 1 x 10 -8 M, such as at most 1 x 10 -9 M.

[0818] 109. The IL-17A binding polypeptide according to any of the preceding items, wherein the IL-17A binding polypeptide is capable of binding IL-17A molecules selected from the group consisting of human IL-17A and murine IL-17A.

[0819] 110. The IL-17A binding polypeptide according to item 109, wherein the IL-17A binding polypeptide is capable of binding human IL-17A.

[0820] 111. The IL-17A binding polypeptide according to item 109, wherein the IL-17A binding polypeptide is capable of binding mouse IL-17A.

[0821] 112. The IL-17A binding polypeptide according to any one of items 109-111, wherein the IL-17A binding polypeptide is capable of binding human IL-17A and mouse IL-17A.

[0822] 113. The IL-17A binding polypeptide according to any one of items 109, 110 and 112, wherein the human IL-17A comprises the amino acid sequence SEQ ID NO:1226 or an antigenically effective fragment thereof.

[0823] 114. The IL-17A binding polypeptide according to any one of items 109, 111 and 112, wherein the mouse IL-17A comprises the amino acid sequence SEQ ID NO:1227 or an antigenically effective fragment thereof.

[0824] 115. The IL-17A binding polypeptide according to any of the preceding items, wherein the IL-17A binding polypeptide contains additional amino acids at the C-terminus and / or N-terminus.

[0825] 116. The IL-17A binding peptide according to item 115, wherein the additional amino acids improve and / or simplify the generation, purification, in vivo or in vitro stabilization, conjugation or detection of the peptide.

[0826] 117. The IL-17A binding polypeptide according to any of the preceding items, wherein the IL-17A binding polypeptide is in a polymeric form, the polymeric form comprising at least two IL-17A binding polypeptide monomer units, the amino acid sequences of the monomer units being identical or different.

[0827] 118. The IL-17A binding polypeptide according to item 117, wherein the IL-17A binding polypeptide monomer units are covalently coupled together.

[0828] 119. The IL-17A binding polypeptide according to item 118, wherein the IL-17A binding polypeptide monomer unit is expressed as a fusion protein.

[0829] 120. The IL-17A binding polypeptide according to any one of items 117-119, wherein the IL-17A binding polypeptide is in dimer form.

[0830] 121. A fusion protein or conjugate, said fusion protein or conjugate comprising:

[0831] - A first portion, the first portion comprising the IL-17A binding polypeptide according to any of the preceding items; and

[0832] - The second part consists of a polypeptide having the desired biological activity.

[0833] 122. The fusion protein or conjugate according to item 121, wherein the desired biological activity is a therapeutic activity.

[0834] 123. The fusion protein or conjugate according to item 121, wherein the desired biological activity is binding activity.

[0835] 124. The fusion protein or conjugate according to item 123, wherein the binding activity is albumin-binding activity, and the albumin-binding activity increases the in vivo half-life of the fusion protein or conjugate.

[0836] 125. The fusion protein or conjugate according to item 124, wherein the fusion protein or conjugate comprises two IL-17A binding polypeptides, each as defined in any one of items 1-116, having an albumin-binding moiety between the two IL-17A binding polypeptides.

[0837] 126. The fusion protein or conjugate according to item 125, wherein the fusion protein or conjugate is capable of binding IL-17A to enable the interacting K D The value is at most 1 x 10 -10 M, such as at most 1 x 10 -11 M, such as at most 1 x 10 -12 M, such as at most 1 x 10 -13 M. -

[0838] 127. The fusion protein or conjugate according to any one of items 124-126, wherein the second portion comprises an albumin-binding domain of a streptococcal G protein or a derivative thereof.

[0839] 128. The fusion protein or conjugate according to item 123, wherein the binding activity exerts an inhibitory effect on biological activity.

[0840] 129. The fusion protein or conjugate according to item 123, wherein the binding activity exerts a stimulating effect on biological activity.

[0841] 130. The fusion protein or conjugate according to item 121, wherein the desired biological activity is enzymatic activity.

[0842] 131. The fusion protein or conjugate according to item 122, wherein the second part is a therapeutically active polypeptide.

[0843] 132. The fusion protein or conjugate according to item 131, wherein the second part is an immune response modifier, such as an anti-inflammatory agent.

[0844] 133. The fusion protein or conjugate according to any one of items 121-122 and 130-132, wherein the second part is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines, and their agonists, antagonists and inhibitors.

[0845] 134. The fusion protein or conjugate according to item 131, wherein the second part is a toxic compound.

[0846] 135. The fusion protein or conjugate according to item 123, wherein the binding activity is binding to immune response-related factors, such as inflammation-related factors.

[0847] 136. A complex comprising at least one IL-17A binding polypeptide according to any one of items 1-117 or at least one fusion protein or conjugate according to any one of items 121-135, and at least one antibody or an antigen-binding fragment thereof.

[0848] 137. The complex according to item 136, wherein the at least one antibody or its antigen-binding fragment is selected from the group consisting of full-length antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, Fc fragments, Fv fragments, single-chain Fv fragments, (scFv)2 and domain antibodies.

[0849] 138. The complex according to item 137, wherein the at least one antibody or its antigen-binding fragment is selected from the group consisting of a full-length antibody, a Fab fragment, and an scFv fragment.

[0850] 139. The complex according to item 138, wherein the at least one antibody or its antigen-binding fragment is a full-length antibody.

[0851] 140. The complex according to any one of items 136-139, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0852] 141. The complex according to any one of items 136-140, wherein the antibody or its antigen-binding fragment is selected from the group consisting of human antibodies, humanized antibodies and chimeric antibodies, and their antigen-binding fragments.

[0853] 142. The complex according to item 141, wherein the antibody or its antigen-binding fragment is a human antibody or a humanized antibody, or its antigen-binding fragment.

[0854] 143. The complex according to any one of items 136-142, wherein the antibody or its antigen-binding fragment has an affinity for an antigen, said antigen being selected from the group consisting of antigens associated with angiogenesis-related disorders and antigens associated with immune responses or immune system disorders.

[0855] 144. The complex according to item 143, wherein the antigen is associated with angiogenesis-related disorders.

[0856] 145. The complex according to item 143, wherein the antigen is associated with an immune response or immune system disorder, such as inflammation.

[0857] 146. The complex according to any one of items 136-145, wherein the complex is a fusion protein or a conjugate.

[0858] 147. The complex according to any one of items 136-146, wherein the IL-17A binding polypeptide is attached to the N-terminus or C-terminus of the heavy chain of the antibody or its antigen-binding fragment.

[0859] 148. The complex according to any one of items 136-146, wherein the IL-17A binding polypeptide is attached to the N-terminus or C-terminus of the light chain of the antibody or its antigen-binding fragment.

[0860] 149. The complex according to any one of items 136-146, wherein the IL-17A binding polypeptide is attached to the N-terminus and / or C-terminus of the light and heavy chains of the antibody or its antigen-binding fragment.

[0861] 150. The complex according to any one of items 146-149, wherein the complex is a fusion protein.

[0862] 151. The IL-17A binding peptide, fusion protein, conjugate, or complex according to any of the preceding items, wherein the IL-17A binding peptide, fusion protein, conjugate, or complex further comprises at least one adapter, for example, selected from the group consisting of non-peptide adapters, flexible amino acid adapters, rigid amino acid adapters, and cleavable amino acid adapters.

[0863] 152. The fusion protein or conjugate according to item 151, wherein the connector is disposed between the first portion and the second portion.

[0864] 153. The complex according to item 151, wherein the linker is disposed between the IL-17A binding peptide and the antibody or its antigen-binding fragment.

[0865] 154. The IL-17A binding polypeptide, fusion protein, conjugate, or complex according to any one of items 151-153, wherein the linker is a flexible linker comprising amino acid residues selected from the group consisting of glycine, serine, and threonine.

[0866] 155. The IL-17A binding polypeptide, fusion protein, conjugate, or complex according to item 154, wherein the linker comprises a sequence having a sequence selected from the following general formulas:

[0867] (G n S m ) p and (S) n G m ) p ,

[0868] Among them, independently,

[0869] n = 1-7,

[0870] m = 0-7,

[0871] n + m ≤ 8 and

[0872] p = 1-10.

[0873] 156. An IL-17A-binding polypeptide, fusion protein, conjugate, or complex as described in item 155, wherein n = 1-5.

[0874] 157. An IL-17A-binding polypeptide, fusion protein, conjugate or complex according to any one of items 155-156, wherein m = 0-5.

[0875] 158. An IL-17A-binding polypeptide, fusion protein, conjugate or complex according to any one of items 155-157, wherein p = 1-5.

[0876] 159. An IL-17A-binding polypeptide, fusion protein, conjugate or complex according to any one of items 156-158, wherein n = 4, m = 1 and p = 1-4.

[0877] 160. The IL-17A binding polypeptide, fusion protein, conjugate, or complex according to item 159, wherein the linker comprises a sequence selected from the group consisting of G4S, (G4S)2, (G4S)3, and (G4S)4.

[0878] 161. The IL-17A binding polypeptide, fusion protein, conjugate, or complex according to item 160, wherein the linker comprises the sequence G4S.

[0879] 162. The IL-17A binding polypeptide, fusion protein, conjugate, or complex according to any one of items 155-159, wherein the general formula is GT(G n S m ) p .

[0880] 163. The IL-17A binding polypeptide, fusion protein, conjugate, or complex according to item 162, wherein the linker comprises the sequence GTG4S.

[0881] 164. The IL-17A binding polypeptide, fusion protein, conjugate, or complex according to any of the preceding items, wherein the IL-17A binding polypeptide, fusion protein, conjugate, or complex further comprises a marker.

[0882] 165. The IL-17A-binding polypeptide, fusion protein, conjugate, or complex according to item 164, wherein the marker is selected from the group consisting of fluorescent dyes and metals, chromophore dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides, and particles.

[0883] 166. The IL-17A binding polypeptide, fusion protein, conjugate, or complex according to any of the preceding items, wherein the IL-17A binding polypeptide, fusion protein, conjugate, or complex comprises a chelating environment provided by a polyamino polycarboxylate chelating agent conjugated to the IL-17A binding polypeptide via a thiol group of a cysteine ​​residue or an amino group of a lysine residue.

[0884] 167. The IL-17A binding polypeptide, fusion protein, conjugate, or complex according to any of the preceding items, wherein the IL-17A binding polypeptide, fusion protein, conjugate, or complex comprises one or more polyethylene glycol moieties.

[0885] 168. A polynucleotide, said polynucleotide encoding a polypeptide according to any one of items 1-163.

[0886] 169. Including expression vectors of polynucleotides as described in item 168.

[0887] 170. Host cells including the expression vectors described in item 169.

[0888] 171. A method for preparing a polypeptide according to any one of items 1-163, said method comprising:

[0889] -Culturing host cells according to item 170 under conditions that allow the polypeptide to be expressed by the expression vector, and

[0890] - Isolate the polypeptide.

[0891] 172. A composition comprising an IL-17A binding polypeptide, a fusion protein, a conjugate or complex according to any one of items 1-167, and at least one pharmaceutically acceptable excipient or carrier.

[0892] 173. The composition according to item 172, wherein the composition further comprises at least one additional active agent, such as an agent selected from the group consisting of therapeutically active peptides, immune response modifiers, anti-inflammatory agents, and toxic compounds.

[0893] 174. An IL-17A-binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-167, or a composition according to any one of items 172-173, for oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, sublingual, sublingual or suppository administration, such as for oral administration or such for topical administration.

[0894] 175. An IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-167, or a composition according to any one of items 172-173, for use as a pharmaceutical, diagnostic or prognostic agent.

[0895] 176. The IL-17A binding peptide, fusion protein, conjugate, complex, or composition for use according to item 175, wherein the peptide, fusion protein, conjugate, complex, or composition regulates IL-17A function in vivo.

[0896] 177. An IL-17A binding polypeptide, fusion protein, conjugate, complex or composition according to any one of items 175-176 for use, wherein the polypeptide, fusion protein, conjugate, complex or composition inhibits IL-17A signaling.

[0897] 178. An IL-17A binding polypeptide, fusion protein, conjugate, complex or composition according to any one of items 175-177 for use, wherein the polypeptide, fusion protein, conjugate, complex or composition blocks the binding of IL-17A to at least one of its homologous receptors.

[0898] 179. The IL-17A binding polypeptide, fusion protein, conjugate, complex or composition according to any one of items 175-178 for use in the treatment, diagnosis or prognosis of IL-17A-related conditions.

[0899] 180. An IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use as described in item 179, wherein the IL-17A-related condition is selected from the group consisting of inflammatory diseases, autoimmune diseases and cancer.

[0900] 181. An IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use as described in item 180, wherein the IL-17A-related condition is selected from the group consisting of inflammatory diseases and autoimmune diseases.

[0901] 182. An IL-17A-binding polypeptide, fusion protein, conjugate, complex, or composition for use as described in item 181, wherein the IL-17A-related condition is selected from the group consisting of inflammatory conditions, allergic conditions, hypersensitivity reactions, autoimmune diseases, severe infections, and graft rejection.

[0902] 183. The IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 182, wherein the IL-17A-related condition is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, uveitis and dry eye disease.

[0903] 184. The IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use as described in item 183, wherein the IL-17A-related condition is psoriasis.

[0904] 185. An IL-17A binding polypeptide, fusion protein, conjugate, or complex as described in item 179 for use in prognosis, wherein the IL-17A-associated condition is cancer, such as cancer selected from the group consisting of gastric cancer, colorectal cancer, non-small cell lung cancer, hepatocellular carcinoma, and adenocarcinoma.

[0905] 186. A method for detecting IL-17A, the method comprising: providing a sample suspected of containing IL-17A; contacting the sample with an IL-17A-binding polypeptide, fusion protein, conjugate, or complex according to any one of items 1-167 or a composition according to any one of items 172-173; and detecting the binding of the IL-17A-binding polypeptide, fusion protein, conjugate, complex, or composition to indicate the presence of IL-17A in the sample.

[0906] 187. A method for determining the presence of IL-17A in a subject, the method comprising the steps of:

[0907] - Contact the subject, or a sample isolated from the subject, with the IL-17A-binding polypeptide, fusion protein, conjugate, or complex according to any one of items 1-167, or the composition according to any one of items 172-173, and

[0908] - Obtain a value corresponding to the amount of IL-17A-binding peptide, fusion protein, conjugate, complex, or composition that has been bound in the subject or bound to the sample.

[0909] 188. The method according to item 187, the method further comprising the step of comparing the value with a reference.

[0910] 189. The method according to item 187 or 188, wherein the subject is a mammalian subject, such as a human subject.

[0911] 190. The method according to any one of items 187-189, wherein the method is performed in vivo.

[0912] 191. The method according to any one of items 187-189, wherein the method is performed in vitro.

[0913] 192. A method for treating IL-17A-related conditions, the method comprising administering an effective amount of an IL-17A-binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-167, or a composition according to any one of items 172-173, to a subject in need of it.

[0914] 193. The method according to Project 192, wherein the IL-17A-related condition is selected from the group consisting of inflammatory diseases, autoimmune diseases, and cancer.

[0915] 194. The method according to item 193, wherein the IL-17A-related condition is selected from the group consisting of inflammatory diseases and autoimmune diseases.

[0916] 195. The method according to item 194, wherein the IL-17A-related condition is selected from the group consisting of inflammatory conditions, allergic conditions, hypersensitivity reactions, autoimmune diseases, severe infections, and graft rejection.

[0917] 196. The method according to item 195, wherein the IL-17A-related condition is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, uveitis, and dry eye disease.

[0918] 197. The method according to item 196, wherein the IL-17A-related condition is psoriasis.

Claims

1. An IL-17A binding polypeptide, wherein the IL-17A binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1233-1247.

2. The IL-17A binding polypeptide according to claim 1, wherein the IL-17A binding polypeptide is composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 1236, 1237 and 1242-1247.

3. The IL-17A binding polypeptide according to claim 1, wherein the IL-17A binding polypeptide is composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 1236, 1244 and 1247.

4. The IL-17A binding polypeptide according to claim 1, wherein the IL-17A binding polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1237, 1244 and 1247.

5. The IL-17A binding polypeptide according to claim 1, wherein the IL-17A binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1244 and 1245.

6. The IL-17A binding polypeptide according to claim 1, wherein the IL-17A binding polypeptide is composed of the amino acid sequence SEQ ID NO: 1244.

7. A polynucleotide, wherein the polynucleotide encodes a polypeptide according to any one of claims 1-6.

8. A composition comprising an IL-17A binding polypeptide according to any one of claims 1-6 and at least one pharmaceutically acceptable excipient or carrier.

9. Use of the IL-17A binding polypeptide according to any one of claims 1-6 or the composition according to claim 8 in the preparation of a medicament for the treatment of IL-17A-related conditions selected from the group consisting of psoriasis, psoriatic arthritis, ankylosing spondylitis, uveitis, and aortitis.

10. Use of the IL-17A binding polypeptide according to any one of claims 1-6 or the composition according to claim 8 in the preparation of a diagnostic agent for the diagnosis of IL-17A-related conditions selected from the group consisting of psoriasis, psoriatic arthritis, ankylosing spondylitis, uveitis, and aortitis.

11. Use of the IL-17A binding polypeptide according to any one of claims 1-6 or the composition according to claim 8 in the preparation of a prognostic agent for the prognosis of IL-17A-related conditions selected from the group consisting of psoriasis, psoriatic arthritis, ankylosing spondylitis, uveitis, and aortitis.

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