Il-1r-i binding polypeptide

Abstract

To provide a class of engineered polypeptides having a binding affinity for interleukin-1 receptor type-I (IL-1R-I) including a binding motif (BM)EX2X3X4X5X6X7EIX10X11 LPNLX16RX18QYX21AFIX25X26 LX28D, and to provide the use of such an IL-1R-I binding polypeptide as a therapeutic, prognostic and / or diagnostic agent.SOLUTION: The present disclosure provides a class of engineered polypeptides having a binding affinity for interleukin-1 receptor type-I (IL-1R-I) including a binding motif (BM)EX2X3X4X5X6X7EIX10X11 LPNLX16RX18QYX21AFIX25X26 LX28D. The present disclosure also provides the use of such an IL-1R-I binding polypeptide as a therapeutic, prognostic and / or diagnostic agent.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure relates to a class of engineered polypeptides having binding affinity for the interleukin-1 receptor type-I (hereinafter referred to as IL-1R-I). The present disclosure also relates to the use of such IL-1R-I binding polypeptides as therapeutic, prognostic, and / or diagnostic agents. [Background technology]

[0002] background The IL-1 system is an attractive target for pharmaceutical intervention because it plays a central role in the initiation and maintenance of inflammatory responses, as well as in the innate and adaptive immune systems (Dinarello, 2009, Annu Rev Immunol, 27:519-50; Sims and Smith, 2010, Nat Rev Immunol. 10(2):89-102). Because the IL-1 system often acts as an initiator and key cytokine in inflammatory responses, many aspects of the progression of pathological immune responses can be modulated by such intervention. The biological activity of IL-1β can be pharmacologically inhibited by antibodies that prevent or modulate the binding of IL-1β to the type IL-1 receptor IL-1R-I.

[0003] The biological activity of the IL-1 system can also be modulated by antagonizing IL-1R. A recombinant form of endogenous IL-1Ra, called anakinra (Kineret®), is described, for example, in US6599873, EP0343684, and EP0541920. Anakinra has shown clinical efficacy in rheumatoid arthritis, cryopyrin-associated periodic syndromes (CAPS; Kone-Paut and Galeotti, 2014, Expert Rev Clin Immunol, 10(1):7-18), and many other inflammatory and autoimmune conditions (Dinarello et al, 2011, Blood, 117(14):3720-32; Dinarello et al, 2012, Nat Rev Drug Discov, 11(8):633-52).

[0004] IL-1 receptor can also be antagonized by antibodies that bind to the IL-1R-I subunit and prevent ligand-mediated IL-1 receptor activation and signal transduction. Some such antibodies are described in WO2004 / 022718, which discloses IL-1R-I antagonists such as 15C4. Other examples of IL-1 receptor antagonist antibodies that bind to the IL-1R-I subunit are described in WO2010 / 052505, which discloses IL-1R-I antagonists such as the 9GL antibody. Yet another example of an IL-1 receptor antagonist antibody called 2D8 is described in WO2005 / 023872.

[0005] The unpredictable and chronic nature of inflammatory diseases, and the high unmet medical need, necessitate the development of new therapeutic modalities. Since tissue penetration rate is inversely proportional to molecular size, relatively large antibody molecules inherently have poor tissue distribution and penetration capabilities.

[0006] Thus, the use of antibodies or other large molecule drugs is not always optimal for treatment, and there is a continuing need to provide agents with high affinity for the IL-1 receptor. Summary of the Invention [Problem to be solved by the invention]

[0007] Summary of the Invention It is an object of the present disclosure to provide novel IL-1R-I binding agents that can be used, for example, for therapeutic, prognostic, and diagnostic applications.

[0008] It is an object of the present disclosure to provide molecules that enable efficient targeted treatment of various forms of inflammatory, autoinflammatory and autoimmune diseases.

[0009] It is a further object of the present disclosure to provide molecules suitable for prognostic and diagnostic applications.

[0010] These and other objects which will become apparent to those skilled in the art from this disclosure are met by the different aspects of the invention as set forth in the appended claims and generally disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Thus, in a first aspect of the disclosure there is provided an IL-1R-I binding polypeptide comprising an IL-1R-I binding motif BM, which motif is i)EX 2 X 3 X 4 X 5 X 6 X 7 EIX 10 X 11 LPNLX 16 RX 18 QYX 21 AFIX 25 X 26 LX 28 D (SEQ ID NO: 1686) (Independently of each other, X 2 is selected from A, D, E, F, H, I, L, Q, S, T, and V; X 3is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 4 is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 5 is selected from A, I, and V; X 6 is selected from F, H, I, Q, R, T, V, and Y; X 7 is selected from A, D, E, F, G, H, I, L, M, Q, S, T, V, W, and Y; X 10 is selected from F and Y; X 11 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 16 is selected from N and T; X 18 is selected from K, R, and S; X 21 is selected from Q, T, and V; X 25 is selected from I, M, R, V, and Y; X 26 is selected from K and S, and X 28 is selected from F, I, L, and M; and ii) X 5 an amino acid sequence having at least 96% identity to the sequence defined in i), provided that The present invention provides an IL-1R-I binding polypeptide, which comprises an amino acid sequence selected from:

[0012] The above definition of the related sequence class, IL-1R-I binding polypeptides, is based on a statistical analysis of several random polypeptide variants of a parent scaffold that were selected for interaction with IL-1R-I in several different selection experiments. The identified IL-1R-I binding motif (i.e., "BM") corresponds to the target binding region of the parent scaffold, which comprises two α-helices in a three-helical bundle protein domain. In the parent scaffold, the amino acid residues of the two BM helices are varied to provide a binding surface for interaction with the Fc constant part of an antibody. In the present disclosure, the interaction ability of Fc is replaced by the interaction ability with IL-1R-I by randomly varying the binding surface residues and then selecting the variants.

[0013] As will be appreciated by those skilled in the art, the function of any polypeptide, such as the IL-1R-I binding ability of the polypeptide of the present disclosure, depends on the tertiary structure of the polypeptide.Therefore, minor changes can be made to the amino acid sequence in the polypeptide without affecting the function of the polypeptide.Therefore, the present disclosure encompasses modified variants of IL-1R-I binding polypeptides that retain IL-1R-I binding properties.

[0014] Thus, IL-1R-I binding polypeptides comprising an amino acid sequence having 96% or greater identity to the polypeptide defined in i) are also encompassed by the present disclosure. Accordingly, certain sequence variants of the sequences defined in i) are encompassed by the above definition, with the proviso that within such sequence variants, X 5 is I or V. For example, an amino acid residue belonging to a certain functional group of amino acid residues (e.g., hydrophobic, hydrophilic, polar, etc.) can be replaced with another amino acid residue from the same functional group.

[0015] In some embodiments, such changes can be made at any position of the IL-1R-I binding polypeptide sequences disclosed herein. In other embodiments, such changes can be made only at the invariant positions, also referred to as scaffold amino acid residues. In such cases, changes are not allowed at the variable positions (i.e., positions indicated with an "X" in sequence i).

[0016] The term "% identity" as used throughout the 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, 1994, Nucleic Acids Research, 22:4673-4680). Comparison is made over a window corresponding to the shortest of the aligned sequences. In some cases, the shortest of the aligned sequences may be the target sequence. In other instances, the query sequence may constitute the shortest of the aligned sequences. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have identical corresponding sequences in the target sequence is reported as the % identity.

[0017] As used herein, "X n " and "X m " is used to denote amino acids at positions n and m in sequence i) as defined above, where n and m are integers indicating the position of the amino acid in said sequence counting from the N-terminus of said sequence. For example, X 3 and X 7 indicate the amino acids at positions 3 and 7, respectively, from the N-terminus of sequence i).

[0018] In an embodiment of the first aspect, X in sequence i) n is independently selected from the group of possible residues in Table 1. One of skill in the art can easily select X from any one of the groups of possible residues listed. n You can select, and this selection will be X m It will be appreciated that n ≠ m is independent of the choice of amino acid in nAny of the possible residues listed in can be combined independently with any of the possible residues listed in any other variable position in Table 1.

[0019] The skilled artisan will recognize that Table 1 should be read as follows: In one embodiment of the first aspect, the amino acid residue "X" in sequence i) is n " is selected from the "possible residues". Thus, Table 1 discloses several specific and individualized embodiments of the first aspect of the disclosure. For example, in one embodiment of the first aspect, X in sequence i) 4 is selected from A, D, E, I, K, Q, S, and V, and in another embodiment of the first aspect, X in sequence i) is 4 is selected from A, E, I, K, Q, and V. For the avoidance of doubt, the recited embodiments may be freely combined in further embodiments. For example, one such combined embodiment is 4 is selected from A, E, I, K, Q, and V, while X 6 is selected from H, Q, T, and Y, and X 11 is a polypeptide selected from H, R, and Y.

[0020] [Table 1] TIFF2025078711000003.tif255170 TIFF2025078711000004.tif231154 TIFF2025078711000005.tif233154 TIFF2025078711000006.tif233154 TIFF2025078711000007.tif233154 TIFF2025078711000008.tif231154 TIFF2025078711000009.tif233154 TIFF2025078711000010.tif234154TIFF2025078711000011.tif45170

[0021] In one embodiment of the present disclosure, in i), X 2 is selected from A, E, F, H, I, L, Q, S, T, and V; X 3 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 4 is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 5 is selected from A, I, and V; X 6 is selected from F, H, Q, R, V, and Y; X 7 is selected from A, D, E, F, G, H, I, L, M, Q, S, T, V, W, and Y; X 10 is selected from F and Y; X 11 is selected from A, D, E, F, G, H, I, L, M, Q, S, T, V, W, and Y; X 16 is selected from N and T; X 18 is selected from K and R; X 21 is selected from T and V; X 25 is selected from I and R; X 26 is selected from K and S, and X 28 is selected from F and L; IL-1R-I binding polypeptides are provided.

[0022] In one embodiment of the present disclosure, in i), X 2 is selected from A, E, F, H, I, L, Q, S, T, and V; X 3 is selected from A, D, E, F, H, I, K, L, Q, R, S, T, V, W, and Y; X 4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 5 is selected from A, I, and V; X 6 is selected from H, Q, R, and Y; X 7 is selected from A, D, E, F, G, H, I, L, M, Q, S, V, W, and Y; X 10 is selected from F and Y; X 11 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 16 is selected from N and T; X 18 is selected from K and R; X 21 is selected from T and V; X 25 is selected from I and R; X 26 is selected from K and S, and X 28 is selected from F and L; IL-1R-I binding polypeptides are provided.

[0023] In one embodiment of the present disclosure, in i), X 2 is selected from A, I, L, T, and V; X 3 is selected from E, I, V, and Y; X 4 is selected from A, D, E, F, H, I, K, L, Q, R, S, T, V, W, and Y; X 5 is selected from A, I, and V; X 6 is selected from Q and Y; X 7 is selected from F, H, I, L, M, Q, V, W, and Y; X 10 is selected from F and Y; X 11 is selected from A, D, E, F, G, H, K, L, Q, R, S, T, V, W, and Y; X 16 is selected from N and T; X 18 is selected from K and R; X 21 is selected from T and V; X 25 is selected from I and R; X 26 is selected from K and S, and X 28 is selected from F and L; IL-1R-I binding polypeptides are provided.

[0024] In another embodiment of the disclosure, the IL-1R-I binding motif comprises in i) X 2 is selected from A, I, L, T, and V; X 3 is selected from E and Y; X 4 is selected from A, E, I, K, Q, R, T, V, and Y; X 5 is selected from I and V; X 6 is selected from Q and Y; X 7 is selected from F and M; X 10 is selected from F and Y; X 11 is selected from A, D, E, F, G, H, K, L, Q, R, S, T, V, and Y; X 16 is selected from N and T; X 18 is selected from K and R; X21 is selected from T and V; X 25 is selected from I and R; X 26 is selected from K and S, and X 28 is an amino acid sequence selected from the sequences: and an IL-1R-I binding polypeptide consisting of an amino acid sequence having at least 93% identity to the sequence defined above.

[0025] In another embodiment of the present disclosure, in i), X 2 is selected from A, I, L, T, and V; X 3 is selected from E and Y; X 4 is selected from A, D, E, F, H, K, L, Q, R, S, T, V, W, and Y; X 5 is selected from A, I, and V; X 6 is selected from Q and Y; X 7 is selected from F, H, I, L, M, Q, V, W, and Y (such as F, H, I, L, Q, V, W, and Y); X 10 is selected from F and Y; X 11 is selected from A, D, E, F, G, H, K, Q, R, S, V, W, and Y; X 16 is selected from N and T; X 18 is selected from K and R; X 21 is T; X 25 is selected from I and R; X 26 is selected from K and S, and X 28 is selected from F and L.

[0026] In one more particular embodiment of the subclass of IL-1R-I binding polypeptides, the sequence i) satisfies the following ten conditions I to X: IX 3 is E; II.X 5 is selected from I and V; III.X 6 is selected from Q and Y; IV.X 7 is selected from F and M; VX 10 is selected from F and Y; VI.X 18 is selected from K and R; VII.X 21 is T; VIII.X 25 is R; IX.X 26 is selected from K and S; and XX 28 is selected from F and L Meet at least five of the following:

[0027] In some examples of IL-1R-I binding polypeptides of the first aspect, sequence i) satisfies at least 6 of the ten conditions I-X. More specifically, sequence i) may satisfy at least 7 of the ten conditions I-X (such as at least 8 of the ten conditions I-X, such as at least 9 of the ten conditions I-X, such as all of the ten conditions I-X).

[0028] In some embodiments of the IL-1R-I binding polypeptide of the first aspect, X 2 X 3 X 6 In some embodiments, X is a IL-1R-I binding polypeptide. 2 X 3 X 6In some embodiments, X is an IL-1R-I binding polypeptide, 6 X 10 is selected from the group consisting of QF, QY, YF, and YY. 6 X 10 In some embodiments, X is QF. 10 X 18 is selected from the group consisting of FK, FR, YK, and YR. 10 X 18 is FK or FR. In some embodiments, X 18 X 25 X 28 is KRL or RRL. In some embodiments, X 5 X 7 is AM, IM, or VM. 5 X 7 is IM or VM.

[0029] In one embodiment, X 5 is selected from I and V. As demonstrated in Example 4, position X of the BM 5 It was found that an IL-1R-I binding polypeptide (Z18557, SEQ ID NO: 1205) exhibits high affinity for IL-1R-I (see Table 11). Replacement of alanine with valine or isoleucine at this position produces an IL-1R-I binding polypeptide with high affinity for IL-1R-I.

[0030] As detailed in the experimental section below, the selection of IL-1R-I binding polypeptide variants led to the identification of several individual IL-1R-I binding motif (BM) sequences. These sequences constitute individual embodiments of sequence i) of this aspect. The individual IL-1R-I binding motif sequences correspond to amino acid positions 8-36 of the amino acid sequences listed as SEQ ID NOs: 1-1632 and 1679 in FIG. 1. Thus, in one embodiment of the IL-1R-I binding polypeptide of this aspect, sequence i) corresponds to a sequence at positions 8-36 in a sequence selected from the group consisting of SEQ ID NOs: 1-1632 and 1679 (such as the group consisting of SEQ ID NOs: 20-1632 and 1679). In one embodiment, sequence i) corresponds to a sequence at positions 8-36 in a sequence selected from the group consisting of SEQ ID NOs: 1206-1632 and 1679 (such as the group consisting of SEQ ID NOs: 1210-1632 and 1679).

[0031] In one embodiment, the sequence i) corresponds to the sequence of positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1205 and 1250 to 1632.

[0032] In one embodiment, the sequence i) corresponds to the sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1206 to 1252 and 1679.

[0033] In another embodiment, sequence i) corresponds to the sequence at positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1253 to 1632. In another embodiment, sequence i) corresponds to the sequence at positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1253 to 1441 or the group consisting of SEQ ID NOs: 1442 to 1632.

[0034] In one embodiment, sequence i) is selected from the group consisting of SEQ ID NOs: 158, 227, 268, 294, 325, 1176, 1205, 1251, 1252, 1270, 1284, 1285, 1298, 1307, 1308, 1311, 1324, 1328, 1330, 1331, 1334, 1339, 1340, 1343, 1353, 1361, 1362, 1364, 1367, 1368, 1375, 1393, 1415, 1420, 1421, 1422, 1423, 1435, 1471, 1472, 1571, 1594, and 1662 (SEQ ID NOs: 268, 1205, 1206, 1207, 1208, 1209, 1310, 1311, 1324, 1328, 1330, 1331, 1334, 1339, 1340, 1343, 1353, 1361, 1362, 1364, 1367, 1368, 1375, 1393, 1415, 1420, 1421, 1422, 1423, 1435, 1471, 1472, 1571, 1594, and 1662 (SEQ ID NOs: 268, 1205, 120 252, 1270, 1284, 1285, 1298, 1307, 1308, 1324, 1328, 1330, 1331, 1334, 1339, 1340, 1343, 1361, 1364, 1367, 1368, 1375, 1393, 1415, 1420, 1421, 1422, 1423, and 571, and the like, SEQ ID NO: 1252, 1285, 1298, 1308, 1324, 1328, 1330, 1331, 1340, 1361, 1393, 1415, 1420, and 1421.

[0035] In one particular embodiment, sequence i) corresponds to the sequence at positions 8 to 36 in a sequence selected from SEQ ID NOs: 1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594, and 1679. In one embodiment, sequence i) corresponds to the sequence at positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1328, 1435, and 1679 (such as the group consisting of SEQ ID NOs: 1252, 1328, and 1679; the group consisting of SEQ ID NOs: 1252, 1435, 1679; the group consisting of SEQ ID NOs: 1252 and 1679; or the group consisting of SEQ ID NOs: 1328 and 1435). In one embodiment, sequence i) corresponds to the sequence at positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1328, and 1435.

[0036] Thus, Figure 1 illustrates polypeptide variants whose IL-1R-I binding ability has been experimentally established (at least by isolation in an affinity-based assay) that identify several distinct BM sequences that constitute distinct embodiments of the IL-1R-I binding motif (BM) that exhibit 93% identity or, where applicable, 96% identity with sequence i).

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

[0038] In certain embodiments, the IL-1R-I binding motif (BM) thus forms part of a three-helix bundle protein domain. For example, the BM may essentially consist of two α-helices with an interconnecting loop within said three-helix bundle protein domain. In certain embodiments, said three-helix bundle protein domain is selected from the domains of bacterial receptor proteins. Non-limiting examples of such domains are the five different three-helical domains (such as domain B) of protein A from Staphylococcus aureus and its derivatives. In some embodiments, the three-helical bundle protein domain is a variant of protein Z derived from domain B of staphylococcal protein A.

[0039] In some embodiments in which the IL-1R-I binding polypeptide disclosed herein forms part of a three-helix bundle protein domain, the IL-1R-I binding polypeptide may comprise a binding module (BMod), the amino acid sequence of which is iii) K-[BM]-DPSQSX a X b LLX c EAKKLX d X e X f Q; (SEQ ID NO: 1687) ([BM] is the IL-1R-I binding motif as defined herein; X a is selected from A and S; X b is selected from N and E; X c is selected from A, S, and C; X d is selected from E, N, and S; X e is selected from D, E, and S; X f is selected from A and S); and iv) an amino acid sequence having at least 91% identity to the sequence defined in iii); is selected from.

[0040] In some embodiments, the polypeptides may advantageously exhibit high structural stability (such as resistance to chemical modification, changes in physical conditions, and proteolytic activity) during production and storage, as well as in vivo.

[0041] As discussed above, polypeptides with minor changes compared to the above amino acid sequences and which do not significantly affect the tertiary structure and function of the polypeptide are also included within the scope of the present disclosure.Thus, in some embodiments, sequence iv) has at least 93% (such as at least 95% identity, such as at least 97% identity) with the sequence defined by iii).In certain embodiments, such minor changes as exemplified above may only exist in the flanking sequence (i.e., the part of the sequence adjacent to the BM in iii) above).In other embodiments, minor changes may exist in the BM and / or in the flanking sequence.

[0042] In one embodiment, in sequence iii), X a is A.

[0043] In one embodiment, in sequence iii), X a is S.

[0044] In one embodiment, in sequence iii), X b is N.

[0045] In one embodiment, in sequence iii), X b is E.

[0046] In one embodiment, in sequence iii), X c is A.

[0047] In one embodiment, in sequence iii), X c is S.

[0048] In one embodiment, in sequence iii), X c is C.

[0049] In one embodiment, in sequence iii), X d is E.

[0050] In one embodiment, in sequence iii), X d is N.

[0051] In one embodiment, in sequence iii), X d is S.

[0052] In one embodiment, in sequence iii), X e is D.

[0053] In one embodiment, in sequence iii), X e is E.

[0054] In one embodiment, in sequence iii), X e is S.

[0055] In one embodiment, in sequence iii), X d X e is selected from EE, ES, SE, and SS.

[0056] In one embodiment, in sequence iii), X d X e is ES.

[0057] In one embodiment, in sequence iii), X d X e is a SE.

[0058] In one embodiment, in sequence iii), X f is A.

[0059] In one embodiment, in sequence iii), X f is S.

[0060] In one embodiment, in iii), X a is A;X b is N;X c is A and X f is A.

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

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

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

[0064] In one embodiment, in iii), X a is A;X b is N;X c is A;X d X e is ND and X f is A.

[0065] In one embodiment, in iii), X a is A;X b is N;X c is C;X d X e is ND and X f is A.

[0066] In one embodiment, in iii), X a is S;X b is E;X c is S;X d X e is ND and Xf is S.

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

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

[0069] In one embodiment, in iii), X a is A;X b is N;X c is C;X d X e is SE, and X f is A.

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

[0071] In one embodiment, in iii), X a is S;X b is E;X c is C;X d X e is SE, and X f is S.

[0072] In one embodiment, in iii), X a is A;X b is N;X c is A;X dX e is ES and X f is A.

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

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

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

[0076] In one embodiment, sequence iii) or iv) corresponds to a sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NOs: 1-1638 and 1667-1670.

[0077] In yet a further embodiment, sequence iii) corresponds to a sequence at positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 1638, 1667 to 1668, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 20 to 1638 and 1670 to 1679). In one embodiment, sequence iii) corresponds to a sequence at positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1206 to 1638 and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 1206 to 1632 and 1670 to 1679, such as the group consisting of SEQ ID NOs: 1210 to 1638 and 1670 to 1679, such as the group consisting of SEQ ID NOs: 1210 to 1632 and 1670 to 1679).

[0078] In one embodiment, sequence iii) corresponds to a sequence from positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1205, 1250 to 1632, and 1670 to 1679.

[0079] In one embodiment, sequence iii) corresponds to the sequence at positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1206 to 1252, 1672, and 1679.

[0080] In another embodiment, sequence iii) corresponds to a sequence at positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1253 to 1632, 1670 to 1671, and 1673 to 1678. In another embodiment, sequence iii) corresponds to a sequence at positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1253 to 1441, 1670 to 1671, and 1674 to 1678 or the group consisting of SEQ ID NOs: 1442 to 1632, and 1673.

[0081] In one embodiment, sequence iii) is selected from the group consisting of SEQ ID NOs: 158, 227, 268, 294, 325, 1176, 1205, 1251, 1252, 1270, 1284, 1285, 1298, 1307, 1308, 1311, 1324, 1328, 1330, 1331, 1334, 1339, 1340, 1343, 1353, 1 The group consisting of 361, 1362, 1364, 1367, 1368, 1375, 1393, 1415, 1420, 1421, 1422, 1423, 1435, 1471, 1472, 1571, 1594, 1662, and 1670 to 1679 (SEQ ID NOs: 268, 1205, 1252, 1270, 1284, 1285, 129 8, 1307, 1308, 1324, 1328, 1330, 1331, 1334, 1339, 1340, 1343, 1361, 1364, 1367, 1368, 1375, 1393, 1415, 1420, 1421, 1422, 1423, 1571, 1670-1672, 1674, and 1676-1679 The group includes sequences corresponding to positions 7 to 55 of a sequence selected from the group consisting of SEQ ID NOs: 1252, 1285, 1298, 1308, 1324, 1328, 1330, 1331, 1340, 1361, 1393, 1415, 1420, 1421, 1670 to 1672, 1674, 1676 to 1677, and 1679.

[0082] In one particular embodiment, sequence iii) corresponds to a sequence at positions 7 to 55 in a sequence selected from SEQ ID NOs: 1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594, and 1670 to 1679. In one embodiment, sequence iii) corresponds to a sequence at positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1328, 1435, 1672, 1675 to 1676, and 1679 (such as the group consisting of SEQ ID NOs: 1252, 1328, and 1679; the group consisting of SEQ ID NOs: 1252, 1435, and 1679; or the group consisting of SEQ ID NOs: 1328 and 1435). In one embodiment, sequence i) corresponds to a sequence at positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1328, and 1435.

[0083] In one embodiment, sequence iii) corresponds to the sequence from positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1672, 1675 to 1676, and 1679 (such as the group consisting of SEQ ID NOs: 1672 and 1675 to 1676).

[0084] In yet a further embodiment, v) YA-[BMod]-AP; (SEQ ID NO: 1688) (wherein [BMod] is an IL-1R-I binding module as defined herein); and vi) an amino acid sequence having at least 90% identity to the sequence defined in v); The present invention provides an IL-1R-I binding polypeptide comprising an amino acid sequence selected from:

[0085] or, vii) FN-[BMod]-AP; (SEQ ID NO: 1689) (wherein [BMod] is an IL-1R-I binding module as defined herein); and viii) an amino acid sequence having at least 90% identity to the sequence defined in vii); The present invention provides an IL-1R-I binding polypeptide comprising an amino acid sequence selected from:

[0086] In one embodiment, ix) FNK-[BM]-DPSQS ANLLX c EAKKL NDAQA P; (SEQ ID NO: 1690) ([BM] is the IL-1R-I binding motif defined above, and X c is selected from A and C); and x) an amino acid sequence having at least 90% identity to the sequence defined in ix); The present invention provides an IL-1R-I binding polypeptide comprising an amino acid selected from the group consisting of:

[0087] In another embodiment, xi) FAK-[BM]-DPSQS SELLX cEAKKL SESQA P; (SEQ ID NO: 1691) ([BM] is the IL-1R-I binding motif defined above, and X c is selected from A, S, and C); and xii) an amino acid sequence having at least 90% identity to the sequence defined in xi); The present invention provides an IL-1R-I binding polypeptide comprising an amino acid selected from the group consisting of:

[0088] In another embodiment, xiii) FAK-[BM]-DPSQS SELLX c EAKKL NDSQA P; (SEQ ID NO: 1692) ([BM] is the IL-1R-I binding motif defined above, and X c is selected from A, S, and C); xiv) an amino acid sequence having at least 90% identity to the sequence defined in xiii); The present invention provides an IL-1R-I binding polypeptide comprising an amino acid selected from the group consisting of:

[0089] In yet another embodiment, xv) YAK-[BM]-DPSQS SELLX c EAKKL X d X e SQA P; (SEQ ID NO: 1693) ([BM] is the IL-1R-I binding motif defined above, and X c is selected from A, S, and C, and X d is selected from E, N, and S, and X e is selected from D, E, and S); and xvi) an amino acid sequence having at least 90% identity to the sequence defined in xv); The present invention provides an IL-1R-I binding polypeptide comprising an amino acid selected from the group consisting of:

[0090] In yet another embodiment, xvii) KYAK-[BM]-DPSQS SELLXc EAKKL X d X e SQA P; (SEQ ID NO: 1694) ([BM] is the IL-1R-I binding motif defined above, and X c is selected from A, S, and C, and X d is selected from E, N, and S, and X e is selected from D, E, and S); and xviii) an amino acid sequence having at least 90% identity to the sequence defined in xvii); The present invention provides an IL-1R-I binding polypeptide comprising an amino acid selected from the group consisting of:

[0091] As discussed above, polypeptides with minor changes compared to the above amino acid sequences and which do not significantly affect the tertiary structure and function of the polypeptide are also included within the scope of the present disclosure.Thus, in some embodiments, sequence vi), vii), x), xii), xiv), xvi), or xviii) may be 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 v), vii), ix), xi), xiii), xv), and xvii), respectively.In certain embodiments, such minor changes as exemplified above may only exist in the adjacent sequence (i.e., the part of the sequence adjacent to the BM in v), vii), ix), xi), xiii), xv), and xviii).In other embodiments, minor changes may only exist in the BM and / or in the adjacent sequence.

[0092] In some embodiments, the IL-1R-I binding motif may form part of a polypeptide comprising an amino acid sequence selected from the following: ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK SEQ ID NO:1695; ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK SEQ ID NO:1696; ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK SEQ ID NO:1697; ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK SEQ ID NO:1698; AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK SEQ ID NO:1699; VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK SEQ ID NO:1700; AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK SEQ ID NO:1701; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK SEQ ID NO:1702; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK SEQ ID NO:1703; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK SEQ ID NO:1704; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP SEQ ID NO:1705; AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK SEQ ID NO:1706; AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP SEQ ID NO:1707; AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK SEQ ID NO:1708; AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK SEQ ID NO:1709; AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP SEQ ID NO:1710; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK SEQ ID NO:1711; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP SEQ ID NO:1712; AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK SEQ ID NO:1713; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK SEQ ID NO:1714; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP SEQ ID NO:1715; AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK SEQ ID NO:1716; AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK SEQ ID NO:1717; AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP SEQ ID NO:1718; AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK SEQ ID NO:1719; AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK SEQ ID NO:1720; VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK SEQ ID NO:1721; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK SEQ ID NO:1722; VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK SEQ ID NO:1723; VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK SEQ ID NO:1724; VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK SEQ ID NO:1725; VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK SEQ ID NO:1726; VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK SEQ ID NO:1727; VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK SEQ ID NO:1728; VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK SEQ ID NO:1729; VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK SEQ ID NO:1730; AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK SEQ ID NO: 1731, and ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK SEQ ID NO: 1732 ([BM] is the IL-1R-I binding motif as defined above).

[0093] In one embodiment, said IL-1R-I binding polypeptide comprises an amino acid sequence having at least 89% identity to any one of the above defined sequences.

[0094] In one embodiment, the IL-1R-I binding polypeptide comprises an amino acid sequence selected from the following: xix) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1721) where [BM] is an IL-1R-I binding motif as defined herein; and xx) An amino acid sequence having at least 89% identity to a sequence defined in xix).

[0095] In one embodiment, the IL-1R-I binding polypeptide comprises an amino acid sequence selected from the following: xxi) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO: 1709) where [BM] is an IL-1R-I binding motif as defined herein; and xxii) An amino acid sequence having at least 89% identity to a sequence defined in xxi).

[0096] In one particular embodiment, the IL-1R-I binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1667-1668 and 1670-1679.

[0097] In one embodiment, the IL-1R-I binding polypeptide comprises an amino acid sequence selected from the following: xxiii) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO: 1711), where [BM] is an IL-1R-I binding motif as defined herein; and xxiv) An amino acid sequence having at least 89% identity to a sequence defined in xxiii).

[0098] Also included within the scope of the present disclosure are polypeptides that have minor changes compared to the above amino acid sequences, and these changes do not significantly affect the tertiary structure and function of the polypeptide.Thus, in some embodiments, sequence xx), xxii), or xxiv) may be 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%) identical to the sequence defined by xix), xxi), or xxiii), respectively.Furthermore, in certain embodiments, such minor changes as exemplified above may only be present in the sequence adjacent to the BM described in xix), xxi), or xxiii).In other embodiments, minor changes may be present in the BM and / or in the adjacent sequence.

[0099] The sequences xix) to xxiv) in such an IL-1R-I-binding polypeptide can be selected from the sequences at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 1638, 1667 to 1668, and 1670 to 1679 shown in FIG.

[0100] In one embodiment of the IL-1R-I-binding polypeptide of this aspect, sequence xix), xxi), or xxiii) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 1632, 1667 to 1668, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 20 to 1638, 1667 to 1668, and 1670 to 1679). In one embodiment, sequence xix), xxi), or xxiii) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1206 to 1632, 1667 to 1668, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 1210 to 1632, 1667 to 1668, and 1670 to 1679).

[0101] In one embodiment, sequence xvii), xix), or xxi) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1205, 1250 to 1632, and 1670 to 1679.

[0102] In one embodiment, the sequence xix), xxi), or xxiii) corresponds to the sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1206 to 1252, 1672, and 1679.

[0103] In another embodiment, sequence xix), xxi), or xxiii) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1253 to 1632, 1670 to 1671, and 1673 to 1678. In another embodiment, sequence xix), xxi), or xxiii) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1253 to 1441, 1670 to 1671, and 1674 to 1678 or the group consisting of 1442 to 1632 and 1673.

[0104] In one embodiment, the sequence xix), xxi), or xxiii) is selected from SEQ ID NOs: 158, 227, 268, 294, 325, 1176, 1205, 1251, 1252, 1270, 1284, 1285, 1298, 1307, 1308, 1311, 1324, 1328, 1330, 1331, 1334, 1339, 1340, The group consisting of 1343, 1353, 1361, 1362, 1364, 1367, 1368, 1375, 1393, 1415, 1420, 1421, 1422, 1423, 1435, 1471, 1472, 1571, 1594, 1662, and 1670 to 1679 (SEQ ID NOs: 268, 1205, 1252, 1270, 1284, 1 285, 1298, 1307, 1308, 1324, 1328, 1330, 1331, 1334, 1339, 1340, 1343, 1361, 1364, 1367, 1368, 1375, 1393, 1415, 1420, 1421, 1422, 1423, 1571, 1670-1672, 1674, and 1676-1679 The sequence corresponds to the sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1285, 1298, 1308, 1324, 1328, 1330, 1331, 1340, 1361, 1393, 1415, 1420, 1421, 1670 to 1672, 1674, 1676 to 1677, and 1679.

[0105] In one particular embodiment, the sequence xix), xxi), or xxiii) corresponds to the sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594, and 1670 to 1679. In one embodiment, the sequence xix), xxi), or xxiii) corresponds to the sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1328, 1435 1672, 1675 to 1676, and 1679 (such as the group consisting of SEQ ID NOs: 1252, 1328, and 1679; the group consisting of SEQ ID NOs: 1252, 1435, and 1679; or the group consisting of SEQ ID NOs: 1328 and 1435). In one embodiment, the sequence xix), xxi), or xxiii) corresponds to the sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1328, and 1435.

[0106] In one embodiment, sequence xix), xxi), or xxiii) corresponds to a sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NOs: 1672, 1675-1676, and 1679 (such as the group consisting of SEQ ID NOs: 1672, 1675, and 1676).

[0107] In one embodiment, the IL-1R-I binding polypeptide has a melting temperature above 45°C.

[0108] Binding of a polypeptide as defined herein to IL-1R-I can disrupt signal transduction via IL-1R-I either in vivo or in vitro. Thus, in one embodiment, there is provided an IL-1R-I binding polypeptide as defined herein that is capable of blocking IL-1R-I dependent signal transduction.

[0109] Half maximal inhibitory concentration (IC 50) is a measure of the inhibitory efficacy of a substance of a particular quantifiable biological or biochemical function. This quantitative measure indicates the amount of a particular substance required to inhibit a particular biological function by 50% and is commonly used in the art. In one particular embodiment, the median inhibitory concentration of blockade (IC 50 ) is up to 1×10 -7 M (maximum 5 × 10 -8 M, etc., up to 2 x 10 -8 M, etc., up to 1×10 -8 M, etc., up to 5 x 10 -9 M, etc., up to 2 x 10 -9 M, etc., up to 1×10 -9 M, etc., up to 5 x 10 -10 M, etc., up to 4 x 10 -10 M, etc., up to 3 x 10 -10 In one embodiment, the half maximal inhibitory concentration (IC) of blockade is provided. 50 ) is 1 × 10 -15 M and 1×10 -7 Between M (1×10 -15 M and 1×10 -8 Between M and 1×10 -15 M and 1×10 -9 Between M and 1×10 -15 M and 1×10 -10 Between M and 1×10 -15 M and 1×10 -11 Between M and 1×10 -15 M and 1×10 -12 (e.g. between M and

[0110] As used herein, the terms "IL-1R-I binding" and "binding affinity for IL-1R-I" refer to a property of a polypeptide that can be tested, for example, by using ELISA, biolayer interferometry (BLI), or surface plasmon resonance (SPR) technologies. For example, as described in the Examples below, IL-1R-I binding affinity can be tested in an experiment in which a polypeptide sample is captured on an antibody-coated ELISA plate and biotinylated IL-1R-I is added, followed by streptavidin-conjugated HRP. TMB substrate is added and absorbance at 450 nm is measured using a multiwell plate reader (Victor). 3 (Perkin Elmer, etc.). The results obtained by such experiments can then be interpreted by one of skill in the art to establish at least a qualitative measure of the binding affinity of the polypeptide for IL-1R-I. For example, the EC 50 ELISA can also be used if a quantitative measure is desired to determine the half maximal effective concentration (EC). The response of the polypeptide to a dilution series of biotinylated IL-1R-I is measured using the ELISA described above. The results obtained by such experiments can then be interpreted by one skilled in the art, for example, by deriving an EC value from the results using GraphPad Prism5 and nonlinear regression. 50 The value can be calculated.

[0111] IL-1R-I binding affinity can also be tested in an experiment in which IL-1R-I or a fragment thereof is immobilized on the sensor chip of a surface plasmon resonance (SPR) device and a sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on the sensor chip of the device and a sample containing IL-1R-I or a fragment thereof is passed over the chip. The skilled person can then establish at least a qualitative measure of the binding affinity of the polypeptide to IL-1R-I by interpreting the results obtained by such an experiment. For example, the K DSurface plasmon resonance can also be used if a quantitative scale for determining the binding value is desired. Binding values ​​can be defined, for example, with a Biacore (GE Healthcare) or ProteOn XPR36 (Bio-Rad) instrument. IL-1R-I is appropriately immobilized on the sensor chip of the instrument, and samples of the polypeptide whose affinity is to be determined are prepared by serial dilution and injected in random order. Then, the K D Values ​​can be calculated from the results using, for example, a 1:1 Langmuir binding model in the BIAevaluation4.1 software or other suitable software provided by the instrument manufacturer.

[0112] Thus, in one embodiment, the EC 50 The maximum value is 1×10 -7 M (maximum 5 × 10 -8 M, etc., up to 1×10 -8 M, etc., up to 5 x 10 -9 M, etc., up to 1×10 -9 M, etc., up to 5 x 10 -10 M, etc., up to 4 x 10 -10 M, etc., up to 3 x 10 -10 M, etc., up to 2 x 10 -10 In one embodiment, the EC of the interaction is 50 The value is 1 x 10 -15 M and 1×10 -7 Between M (1×10 -15 M and 1×10 -8 Between M and 1×10 -15 M and 1×10 -9 Between M and 1×10 -15 M and 5×10 -10 Between M and 1×10 -15 M and 1×10 -10 Between M and 1×10 -15 M and 5×10 -11 Between M and 1×10 -15 M and 1×10 -11 Between M and 1×10 -15 M and 5×10-12 Between M and 1×10 -15 M and 1×10 -12 M, etc.).

[0113] In one particular embodiment, the K D The maximum value is 1×10 -6 M (maximum 1 × 10 -7 M, etc., up to 5 x 10 -8 M, etc., up to 4 x 10 -8 M, etc., up to 3 x 10 -8 M, etc., up to 2 x 10 -8 M, etc., up to 1×10 -8 M, etc., up to 7 x 10 -9 M, etc., up to 5 x 10 -9 M, etc., up to 4 x 10 -9 M, etc., up to 3 x 10 -9 M, etc., up to 2 x 10 -9 M, etc., up to 1×10 -9 M, etc., up to 9 x 10 -10 M, etc., up to 7 x 10 -10 M, etc., up to 5 x 10 -10 In one embodiment, the K of interaction with IL-1R-I is preferably 0.01 to 0.1 M. D The value is 1 x 10 -15 M and 1×10 -6 Between M and 1×10 -15 M and 1×10 -7 Between M and 1×10 -15 M and 1×10 -8 Between M and 1×10 -15 M and 1×10 -9 Between M and 1×10 -15 M and 1×10 -10 Between M and 1×10 -15 M and 1×10 -11 Between M and 1×10 -15 M and 1×10 -12 For example, between M.

[0114] In one embodiment, there is provided an IL-1R-I binding polypeptide as defined herein, wherein said IL-1R-I is human IL-1R-I or cynomolgus IL-1R-I (such as human IL-1R-I).

[0115] An IL-1R-I binding polypeptide as defined herein may be capable of blocking the interaction of IL-1R-I with IL-1 cytokines, such as the interaction of IL-1R-I with IL-1α and / or IL-1β. Thus, in one embodiment, said IL-1R-I binding polypeptide is capable of blocking the interaction of IL-1R-I with IL-1 cytokines, such as the interaction of IL-1R-I with IL-1α and / or IL-1β.

[0116] Those skilled in the art will appreciate that various modifications and / or additions can be made to the IL-1R-I binding polypeptide of any embodiment disclosed herein to tailor the polypeptide to a particular application without departing from the scope of the disclosure.

[0117] For example, in one embodiment, an IL-1R-I binding polypeptide as described herein is provided, which is extended by and / or comprises additional amino acids at the C-terminus and / or N-terminus. Such a polypeptide should be understood as a polypeptide having one or more amino acid residues at the initial and / or final position in the polypeptide chain. Thus, an IL-1R-I binding polypeptide may comprise any suitable number of additional amino acid residues (e.g., at least one additional amino acid residue). Each additional amino acid residue may be added individually or collectively, for example, to improve the production, purification, in vivo or in vitro stabilization, coupling or detection of the polypeptide. Such additional amino acid residues may include one or more amino acid residues added for chemical coupling. An example of this is the addition of a cysteine ​​residue. The additional amino acid residues may also provide a "tag" for purification or detection of the polypeptide (such as a His residue for interaction with a tag-specific antibody). 6 Tag, (HisGlu) 3 tag ("HEHEHE" tag), "myc" (c-myc) tag, or "FLAG" tag, or His for immobilized metal affinity chromatography (IMAC) 6 -Tags, etc.).

[0118] The additional amino acids discussed above may be coupled to the IL-1R-I binding polypeptide by chemical conjugation (using known organic chemistry methods) or any other means (such as expression of the IL-1R-I binding polypeptide as a fusion protein) or linked in any other manner, either directly or via a linker (e.g., an amino acid linker).

[0119] The additional amino acids discussed above can include, for example, one or more polypeptide domains that can provide the IL-1R-I binding polypeptide with additional functions, such as additional binding functions, enzymatic functions, toxic functions, fluorescent signaling functions, or combinations thereof.

[0120] The further polypeptide domain may further provide another IL-1R-I binding moiety having the same IL-1R-I binding function. Thus, in a further embodiment, an IL-1R-I binding polypeptide in the form of a multimer is provided. The multimer comprises at least two IL-1R-I binding polypeptides disclosed herein as monomer units, where it is understood that the amino acid sequences may be the same or different. The polypeptide in the form of a multimer may comprise a suitable number of domains, each having an IL-1R-I binding motif, each forming a monomer within the multimer. These domains may have the same amino acid sequence, but alternatively may have different amino acid sequences. In other words, the IL-1R-I binding polypeptide of the present invention may form a homomultimer or a heteromultimer (e.g., a homodimer or a heterodimer). In one embodiment, an IL-1R-I binding polypeptide is provided in which the monomer units are covalently linked to each other. In one embodiment, an IL-1R-I binding polypeptide is provided in which the monomer units are non-covalently linked to each other. In another embodiment, the IL-1R-I binding polypeptide monomer unit is expressed as a fusion protein. In one embodiment, a dimeric form of the IL-1R-I binding polypeptide is provided. An example of an IL-1R-I binding polypeptide that forms a disulfide-bonded dimer after production is the polypeptide having the sequence set forth in SEQ ID NO: 1658. The hinge region of IgG1 Fc (disclosed in FIG. 1 as positions 1-16 of SEQ ID NO: 1662) can be used to obtain the dimeric form of the IL-1R-I binding polypeptide.

[0121] Furthermore, "heterologous" fusion polypeptides or proteins, or conjugates, in which an IL-1R-I binding polypeptide or multimer thereof as described herein constitutes a first domain (i.e., a first portion), and second and further portions have a function other than binding to IL-1R-I, are also contemplated and are included within the scope of this disclosure. The second and further portions of the fusion polypeptide or conjugate in such proteins suitably also possess the desired biological activity.

[0122] Thus, in a second aspect of the disclosure there is provided a fusion protein or conjugate comprising a first portion consisting of an IL-1R-I binding polypeptide of 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 further comprise a further portion comprising a desired biological activity which may be the same or different from the biological activity of the second portion.

[0123] Thus, in one embodiment, said IL-1R-I binding polypeptide comprises an IL-1R-I binding motif BM according to sequence i) or ii); a binding module BMod according to sequence iii) or iv) or an amino acid sequence according to any one of sequences v) to xxiv). In particular, said IL-1R-I binding motif may correspond to the amino acid sequence of positions 8 to 36; said binding module may correspond to the amino acid sequence of positions 7 to 55; said sequence xix), xxi) or xxiii) may be selected from the group consisting of the sequences selected from the SEQ ID NOs shown in Figure 1 and the SEQ ID NOs listed above. Preferred embodiments of IL-1R-I binding polypeptides are disclosed in the first aspect.

[0124] Non-limiting examples of desirable biological activity include therapeutic activity, binding activity, and in vivo half-life enhancing activity.

[0125] In one embodiment, the desired biological activity is an in vivo half-life enhancing activity, such that the second moiety increases the in vivo half-life of the fusion protein or conjugate.

[0126] In vivo half-life enhancing activity should be understood in this context as an activity that increases the in vivo half-life of a fusion protein or conjugate. The half-life of the first moiety (and possibly further moieties) is increased by fusion or conjugation to a second moiety having in vivo half-life enhancing activity. It will be recognized that the corresponding fusion protein or conjugate may comprise the above-mentioned first IL-1R-I binding moiety, the second moiety having in vivo half-life enhancing activity, and optionally one or more further moieties (such as further IL-1R-I binding moieties). For example, the above-mentioned fusion protein or conjugate may comprise two IL-1R-I binding polypeptides and a second moiety, which has an increased in vivo half-life compared to a fusion or conjugate that only comprises two IL-1R-I binding polypeptides. Examples of fusion proteins comprising IL-1R-I binding polypeptides and half-life extending moieties have been produced and experimentally tested (Example 10). In one embodiment, the fusion protein or conjugate comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1639-1657.

[0127] The half-life of the resulting fusion protein or conjugate can be extended by a second moiety that has an inherently long in vivo half-life. A polypeptide that increases the hydrodynamic size of the fusion protein or conjugate can, for example, increase the half-life of the fusion protein or conjugate. Alternatively, the second moiety can consist of a natural protein with a long in vivo half-life, such as serum albumin or the Fc portion of an antibody.

[0128] In one embodiment, the second portion comprises one of the albumin binding domain of streptococcal protein G or a derivative thereof; albumin; the Fc portion of an antibody, and transferrin. In one embodiment, the fusion protein or conjugate comprises a sequence selected from the group consisting of SEQ ID NOs: 1639-1658.

[0129] In one embodiment, the second moiety is an Fc portion of an antibody, such as an IgG1 Fc (SEQ ID NO: 1662) or an IgG4 Fc. An IgG4 Fc that can be used as a half-life extending moiety is the Fc portion set forth in Uniprot Accession Number P01861, amino acid residues 99-327. In one embodiment, the fusion protein or conjugate comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1646-1654.

[0130] In one embodiment, the Fc portion of the antibody is a mutated Fc having improved affinity for FcRn and / or reduced effector response compared to the unmutated form of Fc. Thus, the Fc portion is a variant of a naturally occurring Fc into which one or more mutations have been introduced. For example, the fusion protein or conjugate comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1734-1737.

[0131] In another non-limiting example, the second moiety has in vivo half-life enhancing activity, the second moiety is transferrin. In certain embodiments of the fusion proteins or conjugates disclosed herein, a polypeptide selected from the group consisting of SEQ ID NO: 1657 is provided.

[0132] In one particular embodiment, the in vivo half-life increasing activity is albumin binding activity. A non-limiting example of a moiety that provides such albumin binding activity is albumin-binding camelid Ig V HH Domain and albumin binding engineered single Ig V H It is a domain.

[0133] In one embodiment, said albumin binding activity is obtained by the albumin binding domain of the Peptostreptococcal Albumin Binding (PAB) protein from Finegoldia magna or a derivative thereof.

[0134] In one embodiment, the albumin binding activity is obtained by an albumin binding domain of streptococcal protein G or a derivative thereof. An example of such an albumin binding domain is a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1659-1661. Preferably, the albumin binding domain is set forth in SEQ ID NO: 1661. In one embodiment, the fusion protein or conjugate comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1639-1645.

[0135] In another non-limiting example, the in vivo half-life enhancing activity is provided by polyethylene glycol (PEG) or hydroxyethyl starch (HES).

[0136] For example, the fusion protein or conjugate comprising at least one additional moiety may comprise: [IL-1R-I binding polypeptide]-[half-life increasing moiety]-[moiety having affinity for a selected target]. It should be understood that the three moieties in this example can be arranged in any order from the N-terminus to the C-terminus of the polypeptide.

[0137] Another non-limiting example is a binding activity that can modify the tissue distribution of the fusion protein compared to the non-fused IL-1R-I binding polypeptide. In one embodiment, the binding activity is binding to the transferrin receptor. Such a binding activity may be expected to cause the conjugate or fusion protein to cross the blood-brain barrier and enter brain tissue (Yu et al, 2011, Sci Transl Med 3(84):84ra44). In a preferred embodiment, such a binding activity is obtained by an antibody fragment.

[0138] Another non-limiting example of a binding activity is an activity that acts to block a biological activity.

[0139] In one embodiment, an IL-1R-I binding polypeptide, fusion protein, or conjugate is provided whose binding activity acts to block biological activity.

[0140] With respect to the above description of the fusion protein or conjugate incorporating the IL-1R-I binding polypeptide of the present disclosure, it should be noted that the designation of the first part, the second part, and the further part is for clearly distinguishing the IL-1R-I binding polypeptide or polypeptide of the present invention on the one hand, and the part exhibiting other functions on the other hand. These designations are not intended to refer to the actual order of the different domains in the polypeptide chain of the fusion protein or conjugate. Similarly, the designation of the first and second unit is for clearly distinguishing the units. Thus, for example, the first part (or monomer unit) may appear at, but is not limited to, the N-terminus, the middle, or the C-terminus of the fusion protein or conjugate.

[0141] Those skilled in the art recognize that in the construction of fusion proteins, linkers are often used between the functional moieties to be fused, and there are different types of linkers with different properties, such as flexible amino acid linkers, fixed amino acid linkers, and cleavable amino acid linkers. Linkers are used, for example, to increase the stability or improve the folding of fusion proteins, to increase the expression of fusion proteins, to improve biological activity, to enable targeting, and to change pharmacokinetics. Thus, in one embodiment, the polypeptide of any aspect disclosed herein further comprises at least one linker, such as at least one linker selected from flexible amino acid linkers, fixed amino acid linkers, and cleavable amino acid linkers.

[0142] In one embodiment, the linker is disposed between a first portion consisting of an IL-1R-I binding polypeptide as defined herein and a second portion consisting of a polypeptide having a desired biological activity. In another embodiment, the linker is disposed within the first portion. For example, one or more linkers can be disposed between the monomer units of the polypeptide as defined herein. In yet another embodiment, a linker can be disposed within the first portion and between the first portion and the second portion.

[0143] Flexible linkers are often used in the art when a certain degree of movement or interaction is required for the linked domains, and can be particularly useful in some embodiments. Such linkers are generally composed of small non-polar (e.g., G) or polar (e.g., S or T) amino acids. Some flexible linkers consist primarily of stretches of G and S residues (e.g., (GGGGS) p ). The copy number "p" can be adjusted to optimize the linker to provide a suitable separation between the functional moieties or to maintain the necessary interactions between the moieties. Apart from G and S linkers, other flexible linkers are known in the art, such as G and S linkers that contain additional amino acid residues (such as T and A) to maintain flexibility and polar amino acid residues to improve solubility. In one embodiment, the linker is a flexible linker that contains at least one amino acid residue selected from the group consisting of glycine, serine, and alanine. The skilled artisan will be aware of other suitable linkers.

[0144] In one embodiment, the linker is GS, VDSS, VDGS, VEGS, ASGS, (GGGGS) 2 (SEQ ID NO: 1683), AS(GGGGS) 2 (SEQ ID NO: 1684), and ((KEAAA) 3 KELAA) 2 (SEQ ID NO: 1685). In one particular embodiment, the linker is selected from AS(GGGGS) 2(SEQ ID NO:1684) or ((KEAAA) 3 KELAA) 2 (SEQ ID NO: 1685). In one embodiment, the above-mentioned fusion protein or conjugate containing a linker comprises a polypeptide having an amino acid sequence set forth in SEQ ID NOs: 1639 to 1658. In particular, the linker AS(GGGGS) 2 Exemplary fusion proteins or conjugates comprising the amino acid sequences set forth in SEQ ID NOs: 1646-1658 (such as SEQ ID NOs: 1648 and 1657).

[0145] Further aspects of the disclosure provide a polynucleotide encoding an IL-1R-I binding polypeptide or fusion protein described herein; an expression vector comprising said polynucleotide; and a host cell comprising said expression vector.

[0146] Also included in the disclosure is a method for producing the above-mentioned polypeptide or fusion protein, comprising culturing the host cell under conditions that allow expression of the above-mentioned polypeptide from its expression vector, and isolating the polypeptide.

[0147] Alternatively, the IL-1R-I binding polypeptides of the present disclosure can be produced by non-biological peptide synthesis using amino acids and / or amino acid derivatives having protected reactive side chains, which non-biological peptide synthesis can include: - stepwise coupling of amino acids and / or amino acid derivatives to form a polypeptide of the first aspect having protected reactive side chains, - removing protecting groups from reactive side chains of the polypeptide; and - folding of polypeptides in aqueous solution Includes.

[0148] It should be understood that the IL-1R-I binding polypeptides of the disclosure may be useful independently as therapeutic, diagnostic, or prognostic agents, or may be useful as a means to target other therapeutic or diagnostic agents (e.g., to affect IL-1R-I directly or indirectly). For example, inhibition of IL-1R-I signaling can have a direct therapeutic effect.

[0149] Thus, in another aspect, a composition is provided that comprises an IL-1R-I binding polypeptide, fusion protein, or conjugate as described herein, and at least one pharma- ceutically 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 combinations are immune response modifiers and anti-cancer agents as described herein. In one embodiment, the composition comprises a fusion protein or conjugate in which the desired biological activity is an in vivo half-life enhancing activity, such that the second moiety increases the in vivo half-life of the fusion protein or conjugate. Examples of moieties that provide such half-life enhancing activity are disclosed elsewhere herein.

[0150] Thus, in another aspect of the disclosure, there is provided an IL-1R-I binding polypeptide, fusion protein, conjugate, or composition as described herein for use as a medicament, prognostic agent, or diagnostic agent. In one embodiment, there is provided an IL-1R-I binding polypeptide as described above for use as a medicament.

[0151] In one embodiment, there is provided an IL-1R-I binding polypeptide, fusion protein, or conjugate, or composition as described herein for use as a medicament for modulating IL-1R-I function in vivo. As used herein, the term "modulate" refers to a change in activity, such as rendering IL-1R-I function hypomorphic, partially inhibiting or completely inhibiting IL-1R-I function.

[0152] In one embodiment, there is provided an IL-1R-I binding polypeptide, fusion protein, or conjugate, or composition described herein for use in the treatment, prognosis, or diagnosis of an IL-1R-I associated disorder.

[0153] As used herein, the term "IL-1R-I-associated disorder" refers to any disorder, disease, or condition in which IL-1R-I plays a regulatory role in a signal transduction pathway. Thus, in one embodiment, there is provided an IL-1R-I-binding polypeptide, fusion protein, or conjugate, or composition described herein for use in treating an IL-1R-I-associated disorder, such as a disorder selected from the group consisting of inflammatory disease, autoinflammatory syndrome, autoimmune disease, infectious disease, cardiovascular disease, ischemic disease, cancer, and diabetes. In one embodiment, the IL-1R-I-associated disorder is selected from the group consisting of inflammatory disease, autoinflammatory syndrome, and autoimmune disease.

[0154] In one embodiment, the IL-1R-I associated disorder is Familial Mediterranean Fever (FMF); Cryopyrin-Associated Periodic Syndrome (CAPS); TNF Receptor-Associated Periodic Syndrome (TRAPS); Hyper IgD Syndrome (HIDS); Periodic Fever; Aphthous Stomatitis; Pharyngitis; Adenitis (PFAPA); Rheumatoid Arthritis (RA), Juvenile RA, Juvenile Idiopathic Arthritis, Systemic Juvenile Idiopathic Arthritis, Adult-Onset Still's Disease; Schnitzler's Syndrome; Muckle-Wells Syndrome; Macrophage Activation Syndrome; Behcet's Disease; Uveitis; Acne Vulgaris; Pyoderma Gangrenosum; Gout; type 2 diabetes, incipient diabetes; dry eye syndrome; sweat gland abscess; neutrophilic dermatoses, especially histiocytic panniculitis, Weber-Christian disease, and neutrophilic panniculitis; cardiovascular disease, myocardial infarction, stroke; liver failure, renal failure; acute lung injury; pseudogout, calcium pyrophosphate deposition disease, chondrocalcinosis, IL-1 receptor antagonist deficiency (DIRA), IL-36 receptor antagonist deficiency (DITRA), ADAM2 deficiency (DADA2), septic arthritis, pyoderma gangrenosum-acne (PAPA) syndrome, pyoderma gangrenosum-acne-suppurative Psychiatric hidradenitis (PASH) syndrome, PAPA-hidradenitis suppurativa (PAPASH) syndrome, autoinflammatory syndrome with lymphedema (AISLE), phospholipase C-gamma-2 mutation autoinflammatory syndrome, NALP12-associated periodic syndrome (NAPS12), mevalonate kinase deficiency (MKD), psoriatic arthritis, reactive arthritis, ankylosing spondylitis, hemochromatosis-related arthritis, periarticular calcinosis, osteoarthritis, inflammatory osteoarthritis, osteoarthritis of the wrist, pustular psoriasis (generalized pustular psoriasis (GPP), pustular pustulosis of the hands and feet (PPP), acrodermatitis continua of Alobeau ( ACH, etc.); Blau syndrome; Sweet syndrome; various vasculitis (such as giant cell arteritis (GCA), polymyalgia rheumatica (PMR), Takayasu's arteritis, Kawasaki disease, urticarial vasculitis, and Henoch-Schönlein purpura (HSP)); neutrophilic urticaria and idiopathic cold urticaria; lichen planus; polymyositis, dermatomyositis, juvenile dermatomyositis, and inclusion body myositis; various stages of myeloma (such as smoldering myeloma (SMM), asymptomatic myeloma, Waldenström's macroglobulinemia, and multiple myeloma); neoplastic cachexia; solid tumor growth;Neonatal disorders (such as bronchopulmonary dysplasia (prophylaxis), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP), cerebral palsy due to perinatal cerebral ischemia, and infantile respiratory distress syndrome (IRDS)); Whipple's disease; traumatic brain injury; refractory epilepsy; systemic inflammatory response syndrome (SIRS); cutaneous lupus; Jessner-Kanoff disease; amyotrophic lateral sclerosis; systemic sclerosis (scleroderma); septic shock; acute pancreatitis; chronic recurrent multifocal osteomyelitis, nonbacterial osteitis (NBO), synovitis-acne-pustulosis-osteitis-osteitis (SAPHO) syndrome, and Majeed syndrome; recurrent multifocal osteomyelitis (MASH); Chondritis; idiopathic relapsing pericarditis (IRP); myocarditis; Erdheim-Chester disease; juvenile xanthogranuloma; islet cell transplantation; hemodialysis-induced systemic inflammation; graft-versus-host disease; ANCA-associated and recurrent glomerulonephritis; Cogan's syndrome; autoimmune inner ear disease; chronic granulomatous disease (CGD); Castleman's disease; heart failure; diastolic heart failure; antisynthase syndrome; acute ACL injury; acute hemorrhagic leukoencephalitis; AA amyloidosis; DiGeorge syndrome; generalized fatigue; chronic fatigue syndrome (CFS); Gulf War illness (GWI); and narcolepsy.

[0155] In another embodiment, the IL-1R-I associated disorder is cancer, such as a cancer selected from the group consisting of colon cancer, breast cancer, lung cancer, head and neck cancer, melanoma, and prostate cancer.

[0156] Those skilled in the art will recognize that the IL-1R-I binding polypeptide, fusion protein, or conjugate, or a composition comprising an anti-IL-1R-I binding polypeptide, fusion protein, or conjugate described herein, can be administered to a subject using standard administration techniques, including oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, duodenal, or suppository administration. Thus, in one embodiment, the IL-1R-I binding polypeptide, fusion protein, or conjugate, or composition described herein is provided for oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, duodenal, or suppository administration. In one embodiment, the administration is oral, intravenous, subcutaneous, or duodenal, and in one particular embodiment, the administration is oral.

[0157] As used herein, the term "subject" refers to a mammalian subject, such as a human subject.

[0158] Non-limiting examples of indications in which a topical administration route may be particularly relevant in IL-1R-I mediated diseases include ophthalmic diseases (such as dry eye, uveitis, scleritis, and ulcerative keratitis); dermatological diseases (such as ichthyosis and generalized pustular psoriasis); pulmonary diseases (such as adult respiratory distress syndrome, bronchopulmonary dysplasia, chronic obstructive pulmonary disease, asbestosis, and silicosis).

[0159] In some embodiments, it may be advantageous to administer the polypeptide, fusion protein, conjugate, or composition for the above use repeatedly within a certain period of time (e.g., within 24 hours from the onset of the disease). In one particular embodiment, the IL-1R-I binding polypeptide, fusion protein, conjugate, or composition is administered repeatedly to a subject in need within 24 hours from the onset of the disease (such as at least twice within 24 hours from the onset of the disease, such as at least three times within 24 hours from the onset of the disease). In a further particular embodiment, the IL-1R-I binding polypeptide, fusion protein, conjugate, or composition is administered continuously for a limited time from the onset (such as 24 hours). Repeated or continuous administration may be preferred when distribution to the brain is required (such as acute treatment of stroke).

[0160] In some embodiments, where the fusion protein or conjugate comprises a second moiety having in vivo half-life enhancing activity, the fusion protein or conjugate or a composition comprising such a fusion protein or conjugate is administered once a week to a subject in need thereof.

[0161] In a related aspect, there is provided a method for treating an IL-1R-I associated disorder comprising administering to a subject in need thereof an effective amount of an IL-1R-I binding polypeptide, fusion protein, conjugate, or composition described herein. In a more particular embodiment of the method, the IL-1R-I binding polypeptide, fusion protein, conjugate, or composition described herein modulates IL-1R-I function in vivo.

[0162] In one embodiment, said IL-1R-I associated disorder is selected from the group defined above in relation to the previous aspect.

[0163] In some embodiments of the above-mentioned treatment method, it may be advantageous to repeatedly administer the above-mentioned polypeptide, fusion protein, conjugate, or composition within a certain period of time (e.g., within 24 hours from the onset of the disease). In one particular embodiment, the above-mentioned IL-1R-I binding polypeptide, fusion protein, conjugate, or composition is administered repeatedly to a subject in need within 24 hours from the onset of the disease (such as at least twice within 24 hours from the onset of the disease, such as at least three times within 24 hours from the onset of the disease). In a further particular embodiment, the above-mentioned IL-1R-I binding polypeptide, fusion protein, conjugate, or composition is administered continuously for a limited time from the onset (such as within 24 hours). Repeated or continuous administration may be preferred when distribution to the central nervous system is required (such as acute treatment of stroke).

[0164] In other embodiments of the above methods of treatment, the fusion protein or conjugate includes a second moiety having in vivo half-life enhancing activity or the composition includes a fusion protein or conjugate that includes a second moiety having in vivo half-life enhancing activity. In such methods, the fusion protein, conjugate, or composition can be administered once a week to a subject in need thereof.

[0165] It may be advantageous to administer a therapeutically effective amount of an IL-1R-I binding polypeptide, fusion protein, or conjugate, or composition described herein, and at least one second drug, such as an immune response modifier or an anti-cancer drug as described above.

[0166] As used herein, the term "co-administration" includes both combined administration and sequential administration.Therefore, in one embodiment, the method as defined above is provided further comprising co-administration of the immune response modifier as described above.In another embodiment, the method as defined above is provided further comprising co-administration of the anti-cancer agent as described above.

[0167] Another aspect of the disclosure provides a method for detecting IL-1R-I, comprising providing a sample suspected of containing IL-1R-I, contacting the sample with an IL-1R-I-binding polypeptide, fusion protein, conjugate, or composition described herein, and detecting binding of the IL-1R-I-binding polypeptide, fusion protein, conjugate, or composition, indicating the presence of IL-1R-I in the sample. In one embodiment, the method further comprises an intermediate wash step to remove unbound polypeptide, fusion protein, conjugate, or composition after contacting with the sample.

[0168] While the present invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents substituted for elements thereof without departing from the scope of the present invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the present invention without departing from its essential scope. Thus, the present invention should not be limited to any particular embodiment contemplated, but is intended to include all embodiments falling within the scope of the appended claims. [Brief description of the drawings]

[0169] [Figure 1]1 is a listing of the amino acid sequences of exemplary IL-1R-I binding polypeptides (SEQ ID NOs: 1-1638 and 1667-1679), albumin binding polypeptides (SEQ ID NOs: 1659-1661); human IgG1 Fc (SEQ ID NO: 1662), human albumin (SEQ ID NO: 1663), and human transferrin (SEQ ID NO: 1664) of the present disclosure, exemplary IL-1R-1 binding fusion polypeptides comprising the above polypeptides (SEQ ID NOs: 1639-1658 and 1734-1737), optionally including a linker, a control polypeptide (GS-hIL-1β, SEQ ID NO: 1733), and the amino acid sequences of cynomolgus IL-1R-I-His6 (SEQ ID NO: 1665) and cynomolgus IL-1R-I-Fc (SEQ ID NO: 1666) used for selection, screening, and / or characterization to illustrate the present invention. The predicted IL-1R-I binding motif (BM) of the IL-1R-I binding polypeptides disclosed herein spans from residue 8 to residue 36 in the sequences having SEQ ID NOs: 1-1638, 1667-1668 and 1670-1679. In SEQ ID NO: 1669, said BM spans from residue 5 to residue 33. The amino acid sequence of the 49 amino acid residue long polypeptide (BMod) predicted to constitute a complete three-helix bundle within each of these Z variants spans from residue 7 to residue 55, with the exception of the N-terminally truncated Z variant with SEQ ID NO: 1669, whose amino acid sequence spans from residue 4 to residue 52. [Diagram 2] FIG. 1 shows concentration-response curves of inhibition of IL-1β-induced IL-6 release in NHDF cells for six anti-IL-1R-I binding fusion proteins (PSI0536, SEQ ID NO: 1648; PSI0537, SEQ ID NO: 1649; PSI0534, SEQ ID NO: 1646; PSI0535, SEQ ID NO: 1647; PSI0538, SEQ ID NO: 1650; PSI0539, SEQ ID NO: 1651). Anakinra (dotted line) is included as a reference. [Diagram 3]1 is a diagram showing the concentration of IL-1R-I binding Z variants at 50% inhibition (IC50) of IL-1β-induced IL-6 release in human whole blood from 5-10 individual donors. The median IC50 values ​​for PSI0536 (SEQ ID NO: 1648), PSI537 (SEQ ID NO: 1649), and anakinra were 0.31, 1.25, and 0.30 nM, respectively.

[0170] Working Example overview The following examples disclose the development of novel Z variant molecules targeting interleukin 1 receptor I (IL-1R-I) based on phage display technology. The IL-1R-I binding polypeptides described herein have been sequenced and their amino acid sequences are listed in FIG. 1 with sequence identifiers SEQ ID NOs: 1-1632. Further IL-1R-I binding polypeptides disclosed herein are listed in FIG. 1 with sequence identifiers SEQ ID NOs: 1633-1638 and 1667-1679. The examples further describe the characterization of the IL-1R-I binding polypeptides and the in vitro functionality of said polypeptides. As used herein, the term "IL-1R-I binding Z variant" refers to a 58 amino acid long IL-1R-I binding polypeptide comprising the IL-1R-I binding motif disclosed herein.

[0171] Example 1 Selection and screening of IL-1R-I binding Z variants In this example, human and cynomolgus IL-1R-I (hIL-1R-I and cIL-1R-I, respectively) were used as target proteins in phage display selections using a phage library of Z variants. DNA of selected clones was sequenced, and Z variants were produced in E. coli as periplasmic fractions and assayed against IL-1R-I in an ELISA (enzyme-linked immunosorbent assay).

[0172] Materials and Methods Biotinylation of target proteins: Human IL-1R-I-Fc (hIL-1RI-Fc Creative BioMart catalog number IL1RI-771H) and human IL-1R-I (hIL-1RI, RnD Systems catalog number 269-1R / CF) were biotinylated for 30 minutes at room temperature (RT) using a 10-fold molar excess of No-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, catalog number 21327) according to the manufacturer's recommendations. Prior to biotinylation, buffer was exchanged into phosphate buffered saline (PBS, 10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) for hIL-1R-I-Fc and hIL-1R-I was dissolved in 100 mM phosphate buffer (pH 6.5) using a dialysis cassette (Slide-a-lyzer 3.5 K, 3500 MWCO, Thermo Scientific, Cat. No. 66333) according to the manufacturer's instructions. After biotinylation, buffer was exchanged into PBS for both proteins as described above.

[0173] Expression of his-tagged target protein: Cynomolgus IL-1R-I-His 6 (cIL-1R-I-His 6 cIL-1R-I-His was expressed using the FreeStyle293-F Expression System (Thermo Fisher Scientific) essentially according to the manufacturer's protocol. Supernatants were harvested by centrifugation 5 days after transfection of the expression vector and stored at -70°C. Frozen supernatants from FreeStyle293-F cultures were thawed and filtered (0.22 μm). 6 The supernatant containing the IgG was purified using affinity chromatography with an IMAC column. The purified protein was buffer exchanged into PBS. Protein purity was analyzed by SDS-PAGE stained with Coomassie Blue and molecular weight was analyzed using mass spectrometry (HPLC / MS or MALDI-TOF / MS).

[0174] Phage display selection of IL-1R-I binding Z variants: IL-1R-I binding Z variants were selected using a library of random variants of protein Z displayed on bacteriophage in fusion to the wild type albumin binding domain (abbreviated as ABD001, SEQ ID NO: 1660). The size of the library, designated Zlib006Naive.II, was 1.5×10 10 Library members (Z variants). Construction of the phage library vector pAY02592 and generation of phage stocks were previously described in WO2016 / 113246.

[0175] hIL-1R-I-Fc, biotinylated hIL-1R-I-Fc (b-hIL-1R-I-Fc), biotinylated hIL-1R-I (b-hIL-1R-I), and cIL-1R-I-His 6Selection against (SEQ ID NO: 1665) was performed in four selection cycles. In the first cycle, four selection tracks were performed in parallel (1-1 to 1-4). Selection tracks 1-1 and 1-4 were split into two parts in cycle 2, thereby obtaining a total of six parallel tracks during rounds 2, 3, and 4, respectively; 2-1 to 2-9, 3-1 to 3-9, and 4-1 to 4-9, as shown in Table 2. The number of phage particles used for selection was 2000 times the number of eluted phage particles in the previous cycle, but a smaller amount was used in selection tracks 4-1 to 4-5. Preparation of phage stocks, selection procedure, and amplification of phage between selection cycles were performed essentially as described in WO2009 / 077175, with the following exceptions 1 to 8. Exception 1: PBS supplemented with 10% fetal calf serum (FCS, Gibco, Cat. No. 10108-165), 1.5 μM human serum albumin (HSA, Novozymes, Cat. No. 230-005), and 0.1% Tween 20 (Acros Organics, Cat. No. 233362500) was used as the selection buffer. Exception 2: Dynabeads® M-280 Streptavidin (SA beads, Life technologies / Invitrogen, Cat. No. 11206D), SA beads precoated with biotinylated human IgG1-Fc (b-hFc, Jackson Immuno Research Cat. No. 009-060-008) (Fc / SA beads), b-hFc and (Z00000) prepared essentially as described in WO2009 / 077175, respectively, as shown in Table 2. 2Preselection was performed by incubation of phage stocks with SA beads precoated with -Cys-biotin (Fc / Z00000 / SA beads), Protein A beads (Life technologies / Novex, Cat. No. 10002D) precoated with b-hFc (Fc / Protein A beads), or Dynabeads® His-Tag Isolation and Pulldown (IM beads, Life Technologies / Novex, Cat. No. 10104D) for >60 min at RT or overnight at 4° C. in cycles 1-4. b-hFc was incubated with beads (5, 8.5, or 10 μg b-hFc / mg Z00000 / SA beads, Protein A beads, or SA beads, respectively) for >1 h at RT, and then the beads were washed with 2×PBST (PBS supplemented with 0.1% Tween 20) before use in preselection. Exception 3: All tubes and beads used in the selection were preblocked with PBS supplemented with 3% BSA (bovine serum albumin, Sigma catalog number A3059) and 0.1% Tween 20. Exception 4: Selection was performed in RT solution, selection time was 140 min in the first cycle and 120 min in subsequent cycles. Exception 5: The following amounts of target protein and beads were used to capture target-phage complexes onto beads: 3.2 μg hIL-1R-I-Fc / mg Z00000 / SA beads, 4 μg hIL-1R-I-Fc / mg Protein A beads, 2 μg b-hIL-1R-I-Fc or 2 μg b-hIL-1R-I / mg SA beads, and 4.5 μg cIL-1R-I-His, respectively. 6 / mg IM beads. The type of beads used for each selection track is the same as that shown for pre-selection in Table 2. Exception 6: E. coli ER2738 strain cells (Lucigen, Middleton, WI, USA) grown in medium supplemented with 10 μg / ml tetracycline were used for phage infection. Exception 7: Log phase bacteria were infected with a 5-fold excess of M13K07 helper phage compared to bacteria. Exception 8: Amplification of phage particles after rounds 2 and 3 was performed essentially as described in WO2016 / 113246; however, in Example 5, tryptic soy broth + yeast extract (TSB-YE) medium supplemented with 100 μM IPTG, 25 μg / ml kanamycin, 100 μg / ml ampicillin, and 2% glucose was used in the overnight culture.

[0176] An overview of the selection strategy describing increasing stringency in the selection cycles obtained by decreasing the target concentration and increasing the number of washes is shown in Table 2.

[0177] [Table 2]

[0178] Washing was performed with PBST 0.1% for 1 min and elution was performed as per the protocol for bead-bound phage / target complexes eluted at pH 2 described in WO2009 / 077175.

[0179] Production of Z variants for ELISA: Z variants were produced essentially as described in WO2016 / 113246, except that the final volume of the pellet dissolved from 1 ml of medium was 1000 μl PBST 0.05% (PBS supplemented with 0.05% Tween 20).

[0180] The final supernatant of the periplasmic extract contained the Z variants as fusions with ABD (Z-ABD; ABD001, SEQ ID NO: 1660) designated as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (Gronwall et al. (2007) J Biotechnol, 128:162-183). Z###### refers to the individual 58 amino acid residue Z variants.

[0181] ELISA screening of Z variants: Binding of Z variants to human IL-1R-I was analyzed by ELISA essentially as described in WO2016 / 113246 using 1 nM hIL-1R-I-Fc as target and goat anti-human IgG-HRP (Southern Biotech, Catalog No. 2040-05) diluted 1:10,000 for detection. As a blank control, PBST 0.05% was added instead of Z-ABD periplasmic samples, and as a negative control, periplasmic extract with ABD001 (SEQ ID NO: 1660) was added instead of Z-ABD periplasmic samples. Control plates were assayed in a similar setup using 100 nM b-hFc instead of target protein and streptavidin-conjugated HRP (Thermo Scientific, Catalog No. N100) diluted 1:30,000 for detection.

[0182] Sequencing: In parallel with the ELISA screening, clones were selected for sequencing. PCR fragments amplified from single clones were sequenced and analyzed essentially as described in WO2009 / 077175.

[0183] Z variant EC 50Analysis: A selection of IL-1R-I binding Z variants were subjected to analysis of their response to a dilution series of hIL-1R-I-Fc using the ELISA described above. The target protein hIL-1R-I-Fc was serially diluted 1:10 from 100 nM to 0.01 nM. As background control, Z variants were assayed without added target protein. The resulting data were analyzed using GraphPad Prism 5 and nonlinear regression to obtain EC 50 The half effective concentration was calculated.

[0184] result Phage display selection of IL-1R-I binding Z variants: Individual clones were obtained after four cycles of phage display selection against human and cynomolgus IL-1R-I.

[0185] ELISA screening of Z variants: Clones obtained after 4 cycles of selection were individually produced in 96-well plates and screened for hIL-1R-I-Fc binding activity in an ELISA using 1 nM target protein. In parallel, an ELISA was performed to exclude Fc binding. Clones positive for hIL-1R-I were identified and no IL-1R-I positive clones were positive for Fc alone. No reaction was obtained for the ABD negative control. 45 identified Z variants showed ELISA screening results of >0.71 AU (8.5× control background).

[0186] Sequencing: In parallel with the ELISA screening, clones obtained after four cycles of selection were sequenced. Each variant was given a unique identification number, #####, and individual variants are referred to as Z#####. The amino acid sequences of a subset of the identified 58 amino acid residue long Z variants are listed in FIG. 1 and in the sequence listing as SEQ ID NOs: 1-19. The putative IL-1R-I binding motif spans from residue 8 to residue 36 in each sequence. The amino acid sequence of the 49 amino acid residue long polypeptide predicted to constitute a complete three-helix bundle within each of these Z variants spans from residue 7 to residue 55.

[0187] Z variant EC 50 Analysis: A subset of the Z variants described above (SEQ ID NOs: 1-19) were subjected to ELISA target titration using hIL-1R-I-Fc. The results were analyzed using EC 50 were used for the calculation of values ​​(Table 3).

[0188] [Table 3]

[0189] Example 2 Production and characterization of primary IL-1R-I binding Z variants This example describes a general procedure for the subcloning and production of His-tagged Z variants derived from primary phage selections, characterization of their target binding, target blocking, and melting temperature.

[0190] Materials and Methods His 6 Subcloning of tagged Z variants: DNA for each Z variant was amplified from the phage library vector pAY02592. 6 A subcloning strategy for the construction of tagged monomeric Z variant molecules was applied using standard molecular biology techniques. The Z gene fragment was subcloned into an expression vector, resulting in the coding sequence MGSSHHHHHHLQ-[Z#####]-VD.

[0191] Culture: E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing the gene fragments of each IL-1R-I binding Z variant. The resulting recombinant strains were cultivated at 30°C at a 50 ml scale using the EnPresso protocol (BioSilta) in medium supplemented with 50 μg / ml kanamycin, or at 37°C in 930 ml TSB-YE medium. To induce protein expression, IPTG was added at OD 600At approximately 10 (EnPresso) or 2 (TSB+YE), cultures were incubated for 16 hours (EnPresso) or 5 hours (TSB+YE). Cells were harvested by centrifugation.

[0192] His 6 Purification of tagged IL-1R-I binding Z variants: Approximately 1-2 g of each cell pellet was resuspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) supplemented with Benzonase® (Merck). After cell disruption, cell debris was removed by centrifugation and each supernatant was 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 Z variants were subsequently eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4). After IMAC purification, the buffer was desalted with PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH) using a PD-10 desalting column (GE Healthcare). 2 PO 4 , 8.1 mM Na 2 HPO 4 , pH 7.4). All Z variants were subjected to a second purification step. Each Z variant was loaded onto a 1 ml Resource15RPC column (GE Healthcare) pre-equilibrated with RPC solvent A (0.1% TFA, 10% ACN, 90% water). After column washing with RPC solvent A, bound proteins were eluted with a 20 ml linear gradient 0-50% RPC solvent B (0.1% TFA, 80% ACN, 20% water). The buffer was then desalted with PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH) using a PD-10 desalting column (GE Healthcare). 2 PO 4 , 8.1 mM Na 2 HPO 4 , pH 7.4).

[0193] Protein concentrations were determined by measuring absorbance at 280 nm using a NanoDrop® ND-1000 spectrophotometer (Saveen Werner AB) and the extinction coefficient of each protein. Samples with concentrations less than approximately 1 mg / ml were concentrated using Amicon Ultra-4, Ultracel-3K (Merck Millipore). Purity was analyzed by SDS-PAGE stained with Coomassie Blue, and the identity of each purified Z variant was confirmed using LC / MS analysis.

[0194] Biacore affinity analysis: His 6 Affinity of human and cynomolgus IL-1R-I for tagged Z variants (K D ) was determined using a Biacore2000 instrument (GE Healthcare). 6 were immobilized on a carboxylated dextran layer in different flow cells of a CM5 chip (GE Healthcare, Cat. No. BR100012). Immobilization was performed using amine coupling chemistry according to the manufacturer's protocol. Acetate (pH 4.5) (GE Healthcare, Cat. No. BR100350) was used for ligand dilution, and HBS-EP (GE Healthcare, Cat. No. BR100188) was used as the running buffer. One flow cell surface on the chip was activated and deactivated to serve as a blank during analyte injection. For kinetic experiments, HBS-EP was used as the running buffer, the flow rate was 50 μl / min, and the temperature was 25° C. Analytes (i.e., Z variants) were diluted in HBS-EP buffer to final concentrations of 40 nM and 10 nM, respectively, and injected over the target chip surface for 2 min, followed by 8 min of dissociation in the running buffer. Kinetic constants were calculated from the resulting sensorgrams using a 1:1 binding model in BiaEvaluation software 4.1 (GE Healthcare).

[0195] In vitro IL-1β neutralization assay: The TF-1 cell line proliferates in response to a number of different cytokines, including IL-1β. This cell line was used to assess the ability of primary IL-1R1 binders to inhibit IL-1β action. TF-1 cells were maintained in RPMI 1640 with 10% FCS (Gibco), penicillin-streptomycin (Lonza), and L-glutamine (Lonza) supplemented with 2 ng / ml rhGM-CSF (R&D Systems). Before use, cells were washed twice with RPMI 1640 without rhGM-CSF. Cells were then counted and cultured at 3 × 10 4 In a separate plate, serial dilutions of inhibitory binding agent (His 6 Tagged IL-1R-I binding Z variants (concentration range 400-0.1 nM) or the IL-1R-I binding protein Kineret (Anakinra, Sobi) (concentration range 0.3-0.0003 nM) were incubated in the presence of 0.6 nM IL-1β (PeproTech). Premixed complexes of Z variant polypeptides with IL-1β were transferred to wells containing TF-1 cells. Cells were incubated in a humidified 5% CO 2 Stimulation was performed for 72 hours at 37°C in ambient air. During the final 4 hours of incubation, 19 μl of 2-fold diluted CCK-8 (Fluka, Sigma Aldrich) was added per well to determine the proliferation response. Absorbance at 450 nm was measured using a microplate reader (Victor3, Perkin Elmer). Data on cell growth were evaluated by nonlinear regression for a 4-parameter dose-response curve and the median inhibitory concentration (IC 50 ) was determined using the GraphPadPrism program.

[0196] Circular dichroism (CD) spectroscopy: Purified His 6Tagged Z variants were diluted to 0.5 mg / ml in PBS. CD spectra from 250 to 195 nm were obtained for each diluted Z variant at 20 °C. Additionally, variable temperature measurements (VTM) were performed to determine the melting temperature (Tm). In VTM, absorbance at 221 nm was measured and the temperature was increased from 20 °C to 90 °C with a temperature gradient of 5 °C / min. To study the refolding ability of the Z variants, new CD spectra were obtained at 20 °C after the heating procedure. CD was measured using a 1 mm pathlength cell in a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB).

[0197] result His 6 Production of tagged Z variants: His 6 The tagged IL-1R-I binding Z variants were expressed as soluble gene products in E. coli. Purification yields of protein from approximately 1-2 g bacterial pellets were determined spectrophotometrically by measuring absorbance at 280 nm and ranged from approximately 3-25 mg for the different IL-1R-I binding Z variants before RPC purification. SDS-PAGE analysis of each final protein preparation showed that they contained mainly IL-1R-I binding Z variants. The exact identity and molecular weight of each Z variant was confirmed by HPLC-MS analysis.

[0198] Biacore affinity analysis: His 6 The interaction of tagged IL-1R-I binding Z variants with human and cynomolgus IL-1R-I was analyzed by injecting two concentrations of each Z variant over a surface containing immobilized IL-1R-I in a Biacore device. The affinity (K D ) are shown in Table 4. The strongest binder to human IL-1R-I (Z12967, SEQ ID NO: 9) bound 1.2×10 -9 M affinity and 3.5 × 10 binding to cynomolgus IL-1R-I -8 It bound with an affinity of M.

[0199] [Table 4]

[0200] In vitro IL-1β neutralization assay:His 6 The IL-1β inhibitory potential of tagged IL-1R-I binding Z variants was analyzed in a TF-1 cell assay. 50 The values ​​are shown in Table 5.

[0201] [Table 5]

[0202] CD analysis:His 6 The CD spectra determined for the tagged IL-1R-I binding Z variants showed that each had an α-helical structure at 20° C. This result was also confirmed in variable temperature measurements in which the melting temperatures were determined (Table 6). Reversible folding was observed for all IL-1R-I binding Z variants when the spectra measured before and after heating to 90° C. were overlaid.

[0203] [Table 6]

[0204] Example 3 Design and construction of a first maturation library of IL-1R-I binding Z variants In this example, a maturation library was constructed. The library was used for the selection of further IL-1R-I binding Z variants. Selection from a maturation library is usually expected to yield binders with increased affinity (Orlova et al, (2006) Cancer Res 66(8):4339-48).

[0205] Materials and Methods Library design: The library was based primarily on human IL-1R-I binding Z variants derived from the primary selection described in Examples 1 and 2. The sequences of a subset of Z variants derived from the primary selection are listed in FIG. 1 and in the sequence listing as SEQ ID NOs: 1-19. In the new library, named Zlib006IL-1RI.I, 10 variable positions in the Z molecule scaffold were biased towards a constant amino acid residue, and three positions were held constant. Two oligonucleotides capable of incorporating a randomized set of trinucleotide building blocks generated by TRIM technology were ordered from Ella Biotech (Martinsried, Germany). These oligonucleotides represented helix 1 and nucleotide 2 of the Z molecule, respectively, and contained an annealing region of 18 overlapping nucleotides. The assembled double-stranded DNA sequence was: 5'-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN GCG NNN NNN GAG ATC NNN NNN CTG CCT AAC CTC ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3' (SEQ ID NO: 1680; designed codons are indicated as NNN). This sequence encodes partially randomized helices 1 and 2 of the Z variant amino acid sequence flanked by restriction sites XhoI and SacI. The design of each amino acid residue of the new library, including 10 variable amino acid positions (9, 10, 11, 13, 14, 17, 18, 25, 32, and 35) and three constant amino acid positions (24, 27, and 28) in the Z molecule scaffold, is shown in Table 7. The resulting theoretical library size was 9.7 × 10 8 A theoretical equal distribution of different amino acids was applied to each amino acid position, except for positions 9, 13, 17, 18, 32, and 35, which have a high distribution rate of some selected amino acids.

[0206] [Table 7]

[0207] Library construction: The two oligonucleotides were assembled, amplified, cloned into vector pAY02592, and transformed into E. coli ER2738 essentially as described for the mature library in WO2015 / 189430. Clones from the Zlib006IL-1RI.I library were sequenced (as described in WO2009 / 077175) to verify the contents of the constructed library compared to the library design and to evaluate the results.

[0208] Preparation of phage stocks: Phage stocks containing the phagemid library were prepared in two batches in incubator flasks. Cells from the glycerol stock containing the phagemid library were inoculated into 2 liters and 3 liters of TSB-YE medium supplemented with 2% glucose, 10 μg / ml tetracycline, and 100 μg / ml ampicillin, respectively. Cultures were grown at OD 600 Cultures were grown at 37°C until the β-terminal tail reached 0.5-0.9 in log phase. A 4-10 fold molar excess of M13K07 helper phage was used to infect the cultures and incubated at 37°C for 30 min. Batch 1 cultures 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 overnight at 30°C. Batch 2 cultures were supplemented with 0.1 mM IPTG followed by incubation at 37°C for 1 h. After addition of 25 μg / ml kanamycin, cultures were grown overnight at 30°C. For both batches, overnight grown cells were pelleted by centrifugation at 4,000 g, after which phage particles remaining in the medium were precipitated twice in PEG / NaCl, filtered, and dissolved in PBS and glycerol, as described in Gronwall et al., supra. Phage stocks were stored at -80°C until use in selections.

[0209] result Library construction: A new library, Zlib006IL-1RI.I, was designed based on a series of IL-1R-I binding Z variants with validated binding properties (Examples 1 and 2). The theoretical size of the designed library is 9.7×10 8 The actual library size, determined by titration after transformation into E. coli ER2738 cells, was 5.6 × 1 09 The quality of the library was tested by sequencing 192 transformants and comparing their actual sequences with the theoretical design. The content of the actual library compared with the designed library was shown to be satisfactory. Thus, the first maturation library of potential binders for IL-1R-I was successfully constructed.

[0210] Example 4 Selection and screening of Z variants from the first maturation library In this example, human and cynomolgus IL-1R-I were used as target proteins in phage display selections using a mature phage library of Z variants. DNA of selected clones was sequenced, and Z variants were produced in E. coli periplasmic fractions and assayed against IL-1R-I in ELISA and Biacore.

[0211] Materials and Methods Expression of target protein: Fc-fused cynomolgus IL-1R-I (cIL-1R-I-Fc, SEQ ID NO: 1666) was expressed using the Expi293 expression system (Thermo Fisher Scientific) essentially according to the manufacturer's protocol. Supernatants were harvested by centrifugation 7 days after transfection of the expression vector and stored at -70°C. Frozen supernatants from Expi293 cultures were thawed and filtered (0.22 μm). Supernatants containing cIL-1R-I-Fc were purified using affinity chromatography using a MabSelect SuRe column. The purified protein was buffer exchanged into PBS. Protein purity was analyzed by SDS-PAGE stained with Coomassie blue and molecular weight was analyzed using mass spectrometry (HPLC / MS or MALDI-TOF / MS).

[0212] Phage display selection of IL-1R-I binding Z variants: Biotinylated human target proteins b-hIL-1R-I-Fc and b-hIL-1R-I, and cynomolgus monkey target proteins cIL-1R-I-Fc (SEQ ID NO: 1666) and b-cIL-1R-I-Fc (cIL-1RI-Fc biotinylated as hIL-1R-I-Fc in Example 1) were used in phage selections with the new library of Z variant molecules described in Example 3. An overview of the selection strategy is shown in Table 8, describing the parallel selection tracks and the increase in overall stringency in the selection cycles obtained by decreasing the target concentration and increasing the number of washes. Selections were made for 4-5 cycles essentially as described in Example 1, with the following exceptions 1-6. Exception 1: Phage stocks were preselected for >60 min at RT by incubation with Fc / SA beads (selection tracks 1-1, 1-2, 1-4, 1-6, 1-8, 2-14, and 2-16), SA beads (selection tracks 1-3, 1-7, 1-9, 2-15, and 2-17), or Fc / Protein A beads (selection tracks 1-5), respectively. Exception 2: Selection times ranged from 30 to 100 min, as indicated for each track in Table 8. Exception 3: Target-phage complexes were captured onto beads using the following amounts of target protein and beads: 8 μg b-hIL-1R-I-Fc, 1 μg b-hIL-1R-I, or 4 μg cIL-1R-I-Fc / mg SA beads, and 5 μg cIL-1R-I-Fc / mg Protein A beads, respectively. In selection round 5, 2 mg SPHERO neutravidin beads (Spherotech catalog number NVM-20-5) were used for target-phage complex capture. Exception 4: In the second final wash step of tracks 2-14, 2-15, 2-17, 3-14, 3-15, and 3-17, respectively, 100x hIL-1R-I of the target concentration of each track was added to the wash buffer and washed for 15-20 min. Exception 5: After the bead washing procedure, the phage / target / bead complexes of selection tracks 4-1 to 4-13 were split in two. Half the samples were subjected to elution and the other half, denoted 4-1x to 4-13x, were subjected to an additional 66 h wash step before elution.Exception 6: As shown in Table 8, two wash rounds 4-4x and 4-6x of track 4 were subjected to a fifth selection round, resulting in tracks 5-1 and 5-2. E. coli XL-1 Blue cells (Agilent Technologies, Cat. No. 200268) grown in medium supplemented with 10 μg / ml tetracycline were used for phage infection, and a 10-fold excess of M13K07 helper phage compared to bacteria was infected into log-phase bacteria. Phage particles after rounds 1-3 were amplified essentially as described for rounds 2 and 3 in Example 1.

[0213] [Table 8] TIFF2025078711000019.tif233170

[0214] In the first selection cycle, nine selection tracks were performed; 1-1 to 1-9. In cycle 2, some tracks were split into 2, 3, or 6 tracks, and 2 tracks were pooled into one track, resulting in a total of 17 parallel tracks in cycles 2, 3, and 4; 2-1 to 2-17, 3-1 to 3-17, and 4-1 to 4-17, respectively. In cycle 5, two tracks were performed; 5-1 and 5-2. Batch 1 of Zlib006IL-1RI.I was used in selection tracks 1-1 to 1-5, and batch 2 was used in selection tracks 1-6 to 1-9, respectively. All selection tracks are shown in Table 8. The number of phage particles used for selection was typically 2000 times the number of eluted phage particles in the previous cycle.

[0215] Sequencing: Individual clones from cycle 4 or 5 of the different selection tracks were selected for sequencing. Amplification and sequence analysis of gene fragments was performed essentially as described in Example 1.

[0216] Production of Z variants for ELISA and Biacore screening: Z variants were produced essentially as described in Example 1, except that the culture volume was 1.2 ml and periplasmic extracts were clarified by filtration using 1.2 μm 96-well filter plates (Merck Millipore catalog number MSANLY50) after a freeze-thaw procedure. Biacore screened Z variants were diluted 5-fold in HBS-EP buffer.

[0217] ELISA screening of Z variants: Binding of Z variants to human was analyzed by ELISA essentially as described in Example 1, using 0.3 nM hIL-1R-I-Fc as the target protein. As a blank control, PBST 0.05% was added instead of Z-ABD periplasmic extract.

[0218] Z variant EC 50 Analysis: A selection of IL-1R-I binding Z variants were subjected to analysis of their response to a dilution series of hIL-1R-I-Fc using the ELISA described above. The target protein hIL-1R-I-Fc was serially diluted 1:10 from 100 nM to 0.01 nM. As background control, Z variants were assayed without added target protein. The resulting data were analyzed using GraphPad Prism 5 and nonlinear regression to obtain EC 50 Values ​​(half maximal effective concentrations) were calculated. Periplasmic extracts with Z12967 (SEQ ID NO: 9) were analyzed in parallel for signal comparison.

[0219] Biacore screening of Z variants: Binding of Z variants to human IL-1R-I was analyzed in a kinetic screening using a Biacore T200 instrument. Polyclonal goat anti-ABD antibodies (goat anti-ABD) were immobilized on a CM5 chip surface essentially as described for other proteins in Example 2. For kinetic screening, the analyte was injected in two steps. First, Z-ABD (ABD001, SEQ ID NO: 1660) periplasmic extract was injected over the surface at 5 μl / min for 1 min. As a second step, 100 nM hIL-1R-I was injected at 30 μl / min for 2 min, followed by dissociation in running buffer HBS-EP for 2 min. Glycine-HCl (pH 2.0) (cat. no. BR100355, GE Healthcare) was used for regeneration of the antibody surface between cycles. The temperature of the assay was 25° C. To perform kinetic analysis, the signal from 100 nM hIL-1R-I injected over a reference surface containing goat anti-ABD but no Z-ABD sample was subtracted from the sensorgram of Z-ABD binding to hIL-1R-I. D ) was calculated by subtracting 100 nM hIL-1R-I response from baseline using a 1:1 binding model in BiaEvaluation software 4.1 (GE Healthcare). Periplasmic extracts of Z12967 were included in the screening analysis for comparison. Periplasmic extracts of the four Z variants were also subjected to single cycle kinetics (SCK) assays using the same settings as above, but injecting five concentrations of hIL-1R-I, and evaluating the data using a 1:1 model for single cycle kinetics.

[0220] result Phage display selection of IL-1R-I binding Z variants: Individual clones were obtained after 4 or 5 cycles of phage display selection against human and cynomolgus IL-1R-I.

[0221] Sequencing: Clones obtained after 4 or 5 cycles of selection were sequenced. Each variant was given a unique identification number, #####, and individual variants are referred to as Z#####. The amino acid sequences of the 58 amino acid residue long Z variants are listed in FIG. 1 and in the sequence listing as SEQ ID NOs: 20-1209. The putative IL-1R-I binding motif spans from residue 8 to residue 36 in each sequence. The amino acid sequence of the 49 amino acid residue long polypeptide predicted to constitute a complete three-helix bundle within each of these Z variants spans from residue 7 to residue 55. In one sequenced Z variant (Z18557, SEQ ID NO: 1205), a valine residue was found to be present at position 12.

[0222] ELISA screening of Z variants: Clones obtained after 4 or 5 cycles of selection were individually produced in 96-well plates and screened for human IL-1R-I binding activity in ELISA. 98% of the assayed Z variants gave a positive signal 2-fold higher than the blank control or to 0.3 nM hIL-1R-I-Fc.

[0223] Z variant EC 50 Analysis: A subset of 115 Z variants showing results above 0.99 AU (16.5 x blank control) were subjected to ELISA target titration using hIL-1R-I-Fc. The results were analyzed using EC 50 The results for Z12967 (SEQ ID NO: 9) were 2.4 x 10 -10 It was M.

[0224] [Table 9] TIFF2025078711000021.tif196170

[0225] On average, the Z variants derived from this library (first maturation library) have an average EC 50 Compared to EC 50The value improved by 10 times.

[0226] Biacore Screening of Z Variants: A selection of IL-1R-I binding Z variants were subjected to Biacore kinetic screening. A single concentration of hIL-1R-I was injected over each Z-ABD captured from periplasmic extracts on a sensor chip surface containing anti-ABD antibodies. Calculated screening affinities are shown in Table 10. For Z16062 (SEQ ID NO: 266), the best K of the binders assayed was D Value (7.6×10 -9 M) was obtained. D is 2.5 x 10 -8 M. The four binders assayed in the SCK experiment had K values ​​that deviated from their respective screening kinetic affinities by 0–14%. D Got the value.

[0227] [Table 10] TIFF2025078711000023.tif42170

[0228] Example 5 Generation and characterization of IL-1R-I binding Z variants from the first maturation library This example shows His 6 The general procedure for subcloning and production of tagged and ABD-fused Z variants (derived from the first maturation library phage selection), their characterization of IL-1R-I binding, blocking, and melting temperatures are described.

[0229] Materials and Methods His 6 Subcloning of tagged Z variants: DNA for each Z variant was amplified from the phage library vector pAY02592 and tagged with an N-terminal His using standard molecular biology techniques essentially as described in Example 2. 6 The tag was used for subcloning.

[0230] Subcloning of Z variants in fusion with ABD: Using standard molecular biology techniques, positions 1 and 2 of the N-terminal sequence of each Z variant were mutated to amino acid residues A and E, respectively. The resulting novel Z variants were subcloned into an expression vector containing the ABD variant, thereby obtaining the coding sequence [Z#####]-ASGS-ABD (ABD variant was PP013 (SEQ ID NO: 1661)). Z##### refers to the individual 58 amino acid residue long Z variants.

[0231] Culture: E. coli T7E2 cells (GeneBridges) were transformed with the plasmids containing the gene fragments of each IL-1R-I binding Z variant. The resulting recombinant strains were cultivated at 30°C in medium supplemented with 50 μg / ml kanamycin at a 50 ml scale using the EnPresso protocol (BioSilta). To induce protein expression, IPTG was added at OD 600 A final concentration of 0.2 mM was added at approximately 10. After induction, the cultures were incubated for 16 hours. Cells were harvested by centrifugation.

[0232] His 6 Purification of tagged IL-1R-I binding Z variants: Essentially as described in Example 2, but the majority of samples were purified without buffer exchange between the IMAC and RPC purification steps. During RPC purification, RPC solvent A was changed to 100% in water with 0.1% TFA, and the linear gradient was 0-60% RPC solvent B in 18 ml.

[0233] Purification of IL-1R-I binding Z variants fused to ABD: Approximately 2 g of each cell pellet was resuspended in TST buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween 20, pH 8.0) supplemented with Benzonase® (Merck). After cell disruption and clarification by centrifugation, each supernatant was applied to a gravity-flow column with 1 ml agarose immobilized with anti-ABD ligand (homemade). TST buffer and 5 mM NH 4After washing with 0.1 M HAc buffer, the ABD-fusion Z variants were eluted with 0.1 M HAc. The buffer containing the eluate was desalted with PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH) using a PD-10 desalting column (GE Healthcare). 2 PO 4 , 8.1 mM Na 2 HPO 4 , pH 7.4).

[0234] Protein concentrations were determined by measuring absorbance at 280 nm using a NanoDrop® ND-1000 spectrophotometer (Saveen Werner AB) and the extinction coefficient of the respective protein. Purity was analyzed by SDS-PAGE stained with Coomassie Blue, and the identity of each purified Z variant was confirmed using LC / MS analysis.

[0235] Biacore dynamic analysis:His 6 The kinetic constants (k on and k off ) and affinity (K D ) was determined using a Biacore2000 instrument (GE Healthcare). Experiments were performed essentially as described in Example 2 using 100, 10, and 1 nM Z variants. Analyte injection time was 3 min, followed by 15 min dissociation, and 2x5s glycine, pH 3.0, supplemented with 0.5 M NaCl (GE Healthcare, Cat. No. BR100357) was used for surface regeneration between cycles. Kinetic constants were calculated from the resulting sensorgrams of each Z variant at two or three concentrations using a 1:1 binding model in BiaEvaluation software 4.1 (GE Healthcare). His 6 Tagged Z12967 was included in the assay for comparison.

[0236] In vitro IL-1β neutralization assay:His 6The tagged IL-1R-I specific Z variants were tested for their inhibitory potential in the TF-1 cell assay. The assay was performed as described in Example 2. Data on cell growth were evaluated by nonlinear regression on a four-parameter dose-response curve and IC 50 Values ​​were determined using the GraphPadPrism program. 6 Tagged Z12967 was included for comparison.

[0237] Circular dichroism (CD) spectroscopy: CD was measured using His 6 Tagged Z variants were analyzed.

[0238] result His 6 Subcloning with tags: Z variants were subcloned with an N-terminal His 6 The plasmid pG211 was subcloned into a construct carrying the gene pG211.

[0239] His 6 Production of tagged Z variants: His 6 The tagged IL-1R-I binding Z variants were expressed as soluble gene products in E. coli. Purified protein yields were determined spectrophotometrically by measuring absorbance at 280 nm and ranged from approximately 1 to 7 mg protein / g pellet. SDS-PAGE analysis of each final protein preparation showed that they contained primarily IL-1R-I binding Z variants. The exact identity and molecular weight of each Z variant was confirmed by HPLC-MS analysis.

[0240] Biacore dynamic analysis:His 6 The interaction of tagged IL-1R-I binding Z variants with human and cynomolgus IL-1R-I was analyzed by injecting various concentrations of Z variants over a surface containing immobilized IL-1R-I in a Biacore device. Kinetic parameters for human IL-1R-I binding (K D , k on , and k off) are summarized in Table 11. The K of Z18557 (SEQ ID NO: 1205), the strongest binder to human IL-1R-I, is D is 1.5 × 10 for human IL-1R-I -10 , 7.6 × 10 for cynomolgus IL-1R-I -9 The K of Z12967 against human IL-1R-I was M. D is 5.1 x 10 -10 It was M.

[0241] [Table 11]

[0242] In vitro IL-1β neutralization assay:His 6 The IL-1β inhibitory potential of tagged IL-1R-I binding Z variants was analyzed in a TF-1 cell assay. 50 The values ​​are shown in Table 12. Z18557 (SEQ ID NO: 1205) showed the highest inhibitory potency, with an IC 50 The IC value of Z12967 was less than 0.5 nM. 50 was 5.2 nM.

[0243] [Table 12]

[0244] CD analysis:His 6 The CD spectra determined for the tagged IL-1R-I binding Z variants showed that each had an α-helical structure at 20° C. This result was also confirmed in variable temperature measurements in which the melting temperatures were determined (Table 13). Reversible folding was observed for all IL-1R-I binding Z variants when the spectra measured before and after heating to 90° C. were overlaid.

[0245] [Table 13]

[0246] Example 6 Design and construction of two secondary maturation libraries of IL-1R-I binding Z variants In this example, two secondary maturation libraries were constructed and used for the selection of further IL-1R-I binding Z variants.

[0247] Materials and Methods Library design: The libraries were primarily based on the sequences of the human IL-1R-I binding Z variants described in Examples 4 and 5. In the new libraries, named Zlib006IL-1RI.II and Zlib006IL-1RI.III, 10 variable positions in the Z molecule scaffold were biased towards constant amino acid residues, and 3 positions were held constant, following a strategy based primarily on Z variants (SEQ ID NOs: 19-1209) derived from selections from the first maturation library. In addition, in both libraries, a new amino acid position (position 12 of the 58aaZ variant sequence) was included for variation, resulting in a total of 11 variable positions. Position 12 of Z18557 (SEQ ID NO: 1205), characterized in Example 5, was a valine. This library design resulted in the amino acids A, V, and I at position 12.

[0248] For Zlib006IL-1RI.II, a DNA linker was generated using split pool synthesis containing the following sequence ordered from DNA2.0 (Menlo Park, CA, USA): 5'-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN NNN NNN NNN GAG ATC NNN NNN CTG CCT AAC CTC ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3' (sequence number 1681, designed codons are denoted as NNN). The design of each amino acid residue in the new library, including 11 variable amino acid positions (9, 10, 11, 12, 13, 14, 17, 18, 25, 28, and 35) and three constant amino acid positions (24, 27, and 32) in the Z molecule scaffold, is shown in Table 14. The resulting theoretical library size was 2.2×1 07 A theoretical equal distribution of different amino acids was applied within each amino acid position, except for positions 10, 11, 12, 13, 17, 28, and 35, which have a high distribution rate of some of the selected amino acids.

[0249] For Zlib006IL-1RI.III, a library was constructed using TRIM technology similar to Zlib006IL-1RI.I described in Example 3. The DNA sequence 5'-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN NNN NNN NNN GAG ATC NNN NNN CTG CCT AAC CTC ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3 (SEQ ID NO: 1682, designed codons are indicated as NNN) was ordered from Ella Biotech (Martinsried, Germany). The design of each amino acid residue in the new library, including 11 variable amino acid positions (9, 10, 11, 12, 13, 14, 17, 18, 25, 32, and 35) and three constant amino acid positions (24, 27, and 28) in the Z molecule scaffold, is shown in Table 15. The resulting theoretical library size is 1.0×10 8 A theoretical equal distribution of different amino acids was applied within each amino acid position, except for positions 10, 11, 12, 32, and 35, which have a high distribution rate of some of the selected amino acids.

[0250] [Table 14]

[0251] [Table 15]

[0252] Library construction and phage stock preparation: Zlib006IL-1RI.II and Zlib006IL-1RI.III were constructed as described in WO2016 / 113246 (Example 4) and Example 3, respectively. For both libraries, E. coli XL-1 Blue was used for library transformation. Clones from the library of Z variants were sequenced (as described in Example 3) to verify the content of the constructed libraries compared to the library design and to evaluate the results.

[0253] Phage stocks containing the phagemid library were prepared in infiltration flasks. Cells from glycerol stocks containing the phagemid library were inoculated into 0.5 liter or 1 liter of TSB-YE medium, respectively, essentially as described for Zlib006IL-1RI.I batch 2 in Example 3. Cultures were incubated at OD 600 Infection was performed using a 10-20 fold molar excess of M13K07 helper phage at 0.8-0.9. Phage stocks were prepared as described in Example 3.

[0254] result Library construction: New libraries were designed based on a series of IL-1R-I binding Z variants selected from Zlib006IL-1RI.I with validated binding properties (Examples 4 and 5). The theoretical sizes of Zlib006IL-1RI.II and Zlib006IL-1RI.III are 2.2×10 7 Variants and 1.0×10 8 The actual library sizes, determined by titration after transformation of E. coli XL-1 Blue cells, were 1.2 × 10 9 Transformants and 7.0 × 10 9 It was a transformant.

[0255] The quality of the libraries was tested by sequencing 192 and 96 transformants derived from Zlib006IL-1RI.II and Zlib006IL-1RI.III, respectively, and comparing the actual sequences with the theoretical design. The content of the actual libraries compared to the designed library was shown to be satisfactory. Thus, two maturation libraries of potential binders to IL-1R-I were successfully constructed.

[0256] Example 7 Selection of Z variants from the second maturation library In this embodiment, human and cynomolgus IL-1R-I were used as target proteins in phage display selections using two secondary maturation phage libraries of Z variants.

[0257] Materials and Methods Phage display selection of IL-1R-I binding Z variants: Biotinylated human target proteins b-hIL-1R-I-Fc and b-hIL-1R-I, and cynomolgus monkey target protein b-cIL-1R-I-Fc were used in phage selections with the new library of Z variant molecules described in Example 6. An overview of the selection strategy is shown in Table 16, describing the parallel selection tracks and the overall increase in stringency over the selection cycles obtained by decreasing the target concentration and increasing the number of washes.

[0258] [Table 16] TIFF2025078711000030.tif192170

[0259] Selection was performed for 4 cycles essentially as described in Example 4, with the following exceptions 1-3: Exception 1: Pre-selection was performed in selection rounds 1 and 2 by incubation of phage stocks with SA beads (b-hIL-1R-I track) or Fc / SA beads (b-IL-1RI-Fc track) for more than 60 min at RT. Exception 2: In the second final wash step of tracks 2-1, 2-4, 2-9, 2-11, 2-12, 3-1, 3-4, 3-9, 3-11, and 3-12, respectively, hIL-1R-I was added to the wash buffer at a concentration 100-fold higher than the target concentration for the respective track and washed for 15-20 min. Exception 3: In the fourth selection round, tracks 4-1, 4-4, and 4-6 were split in two before the second final wash step. For one, hIL-1R-I was added to the wash buffer at a concentration 100-fold higher than the target concentration for the respective track and a second final wash was performed overnight, whereas for the other, a 1-hour wash with PBST was applied for the second final wash.

[0260] In the first selection cycle, 12 selection tracks were performed; 1-1 to 1-12. In cycle 2, two tracks were split into three and six tracks were pooled into two tracks, resulting in a total of 12 parallel tracks in cycles 2, 3, and 4; 2-1 to 2-12, 3-1 to 3-12, and 4-1 to 4-12, respectively. All selection tracks are shown in Table 16. The number of phage particles used for selection was typically 2000 times the number of eluted phage particles in the previous cycle.

[0261] result Phage display selection of IL-1R-I binding Z variants: Individual clones were obtained after four cycles of phage display selection against human and cynomolgus IL-1R-I.

[0262] Example 8 Screening of Z variants from the first and second maturation libraries In this example, DNA of selected clones from Examples 4 and 7 was sequenced and Z variants were produced in E. coli periplasmic fractions and assayed against IL-1R-I in ELISA and Biacore.

[0263] Materials and Methods Sequencing of potential binders: Individual clones from cycle 4 of selection using the three maturation libraries Zlib006IL-1RI.I, Zlib006IL-1RI.II, and Zlib006IL-1RI.III were selected for sequencing. Amplification and sequence analysis of gene fragments was performed essentially as described in Example 1.

[0264] Production of Z variants for ELISA and Biacore screening: Z variants were produced essentially as described in Example 4, except that periplasmic extracts were prepared by heat treatment (82° C., 20 min) followed by clarification by filtration. Z variants screened in Biacore were diluted 5-fold in HBS-EP buffer.

[0265] ELISA screening of Z variants: Binding of Z variants to human IL-1R-I was analyzed by ELISA essentially as described in Example 1, using 0.2 nM hIL-1R-I-Fc as target protein. Periplasmic extracts of ABD001 were used as negative control.

[0266] Z variant EC 50 Analysis: Selection of IL-1R-I binding was subjected to analysis of response to a dilution series of hIL-1R-I-Fc using the ELISA described above. The target protein hIL-1R-I-Fc was serially diluted 1:10 from 20 to 0.002 nM. As background control, the Z variant was assayed without added target protein. The resulting data was analyzed using GraphPad Prism5 and nonlinear regression to obtain EC 50Values ​​(half maximal effective concentrations) were calculated. Periplasmic extracts with Z12967, Z16065, and Z16218 (corresponding to SEQ ID NOs: 9, 268, and 414, respectively) were analyzed in parallel for signal comparison. Periplasmic extracts of ABD001 were used as negative controls.

[0267] Biacore screening of Z variants: Binding of Z variants to human IL-1R-I was analyzed in a kinetic screen using a Biacore2000 instrument essentially as described in Example 4, except that Z-ABD (ABD001, SEQ ID NO: 1660) periplasmic extract was injected for 2 min. Periplasmic extracts of Z12967, Z16065, and Z16218 were included in duplicate in the screening analysis for comparison. Periplasmic extracts with ABD (ABD001, SEQ ID NO: 1660) were included as a negative control.

[0268] result Sequencing: Clones obtained after four cycles of selection were sequenced. Each variant was given a unique identification number, #####, and individual variants are referred to as Z#####. The amino acid sequences of the 58 amino acid residue long Z variants are listed in FIG. 1 and in the sequence listing as SEQ ID NOs: 1210-1632. The putative IL-1R-I binding motif spans from residue 8 to residue 36 in each sequence. The amino acid sequence of the 49 amino acid residue long polypeptide predicted to constitute a complete three-helix bundle within each of these Z variants spans from residue 7 to residue 55.

[0269] ELISA screening of Z variants: Clones obtained after four cycles of selection were individually produced in 96-well plates and screened for human IL-1R-I binding activity in ELISA. 99.5% of the assayed Z variants gave a positive signal 2-fold higher than the blank control or to 0.2 nM hIL-1R-I-Fc.

[0270] Z variant EC 50Analysis: A subset of 102 Z variants showing results above 0.74 AU (8.2x blank control) were subjected to ELISA target titration using hIL-1R-I-Fc. The results were analyzed using EC 50 The results for Z12967, Z16065, and Z16218 were 2.6 × 10 -10 , 2.1×10 -10 , and 2.2 × 10 -10 It was M.

[0271] [Table 17] TIFF2025078711000032.tif144170

[0272] Biacore screening of Z variants: A subset of 188 IL-1R-I binding Z variants was subjected to Biacore kinetic screening. A single concentration of hIL-1R-I was injected over each Z-ABD captured from periplasmic extracts on a sensor chip surface containing anti-ABD antibodies. Calculated screening affinities are shown in Table 18. K for Z12967, Z16065, and Z16218 D The values ​​are 2.7×10 -8 , 7.5×10 -9 , and 1.3 × 10 -8 M (mean of duplicates).

[0273] Z16065 and binding motif BM X 5 Z18814, which has an amino acid sequence that differs only at position X, has three times the affinity of Z16065. 5 The highest binding affinity (K D ) (Table 11). Z18814 has X 5 It has an isoleucine residue at position X. 5 Several Z variants with valine or isoleucine at position 1 were found to have high affinity for IL-1R-I.

[0274] [Table 18] TIFF2025078711000034.tif144170

[0275] Example 9 Generation and characterization of Z variants derived from the first and second maturation libraries This example illustrates the His fragments derived from the first and second maturation library phage selections. 6 General procedures for subcloning and production of tagged Z variants, characterization of their target binding, target blocking, and melting temperature are described.

[0276] Materials and Methods His 6 Subcloning of tagged Z variants: A subset of the Z variants screened in Example 8 was selected for subcloning. DNA for each Z variant was amplified from the phage library vector pAY02592 and tagged with an N-terminal His tag using standard molecular biology techniques essentially as described in Example 2. 6 The tag was used for subcloning. Culture: E. coli T7E2 cells (GeneBridges) were transformed with the plasmids containing the gene fragments of each IL-1R-I binding Z variant. The resulting recombinant strains were cultivated at 30°C in medium supplemented with 50 μg / ml kanamycin at a 50 ml scale using the EnPresso protocol (BioSilta). To induce protein expression, IPTG was added at OD 600 A final concentration of 0.2 mM was added at approximately 10. After induction, the cultures were incubated for 16 hours. Cells were harvested by centrifugation.

[0277] His 6Purification of tagged IL-1R-I binding Z variants: Approximately 2 / 3 of the IL-1R-I binding Z variants were purified as described in Example 2, but without buffer exchange between the IMAC and RPC purification steps. The linear gradient during RPC purification was also changed to 18 ml of 0-60% RPC solvent B. The remaining 1 / 3 of the IL-1R-I binding Z variants were only IMAC purified, followed by a final buffer exchange to PBS. Purification was essentially performed as described in the first part of Example 2. Approximately 1-2 g of each cell pellet was used.

[0278] Biacore dynamic analysis:His 6 The kinetic constants (k on and k off ) and affinity (K D ) was determined using a Biacore2000 instrument (GE Healthcare). Experiments were performed essentially as described in Example 5 using 48, 12, 3, and 0.75 nM Z variants. Analyte injection times were 3 min, followed by 6 min dissociation, and 2x5s glycine (pH 3.0) supplemented with 0.5 M NaCl was used for surface regeneration between cycles. Kinetic constants were calculated from the resulting sensorgrams of each Z variant at three or four concentrations using a 1:1 binding model in BiaEvaluation software 4.1 (GE Healthcare). His 6 Tagged Z12967 (SEQ ID NO: 9), Z16065 (SEQ ID NO: 268), Z16163 (SEQ ID NO: 359), and Z18557 (SEQ ID NO: 1205) were included in the assay for comparison.

[0279] In vitro IL-1β neutralization assay:His 6 The tagged IL-1R-I specific Z variants were tested for their inhibitory potential in the TF-1 cell assay. The assay was performed as described in Example 2. Data on cell growth were evaluated by nonlinear regression on a four-parameter dose-response curve and IC 50Values ​​were determined using the GraphPadPrism program. 6 Tagged Z12967, Z16065, Z16163, and Z18557 were included for comparison.

[0280] Circular dichroism (CD) spectroscopy:His 6 CD for tagged Z variants was analyzed as described in Example 2.

[0281] result His 6 Production of tagged Z variants: His 6 The tagged IL-1R-I binding Z variants were expressed as soluble gene products in E. coli. The purified protein yields were determined spectrophotometrically by measuring absorbance at 280 nm and ranged from approximately 0.4 to 7 mg protein / g pellet for IL-1R-I binding Z variants that were both IMAC-purified and RPC-purified. For IL-1R-I binding Z variants that were only IMAC-purified, the purified protein yields ranged from approximately 4 to 15 mg protein / g pellet. SDS-PAGE analysis of each final protein preparation showed that they contained mainly IL-1R-I binding Z variants. The exact identity and molecular weight of each Z variant was confirmed by HPLC-MS analysis.

[0282] Biacore dynamic analysis:His 6 The interaction of tagged IL-1R-I binding Z variants with human and cynomolgus IL-1R-I was analyzed by injecting various concentrations of Z variants over a surface containing immobilized IL-1R-I in a Biacore device. Kinetic parameters for human IL-1R-I binding (K D , k on , and k off The calculated values ​​of K for Z12967, Z16065, Z16163, and Z18557 are summarized in Table 19. D The values ​​are 8.2×10 -10 , 5.1×10 -10 , 7.6×10 -10 , and 1.4 × 10-10 The K of cynomolgus monkey IL-1R-I binding was M. D The values ​​are 2.0 × 10 for Z18754, Z18760, and Z18800, respectively. -9 , 6.7×10 -9 , and 3.3 × 10 -9 It was M.

[0283] [Table 19] TIFF2025078711000036.tif34170

[0284] In vitro IL-1β neutralization assay:His 6 The IL-1β inhibitory potential of tagged IL-1R-I binding Z variants was analyzed in a TF-1 cell assay. 50 The values ​​are shown in Table 20. The IC of Z12967, Z16065, Z16163, and Z18557 50 The values ​​were 7.4, 3.3, 3.3, and 0.6 nM, respectively.

[0285] [Table 20]

[0286] CD analysis:His 6 The CD spectra determined for the tagged IL-1R-I binding Z variants showed that each had an α-helical structure at 20° C. This result was also confirmed in variable temperature measurements in which the melting temperatures were determined (Table 21). Reversible folding was observed for all IL-1R-I binding Z variants when the spectra measured before and after heating to 90° C. were overlaid.

[0287] BM's X 5The thermal stability and α-helical content of IL-1R-I binding Z variants (Z18912, Z18920, Z18968, Z19072, Z19075, Z19143, Z19147, Z19151, Z19158, Z19159, Z19170, Z19172, Z19180, Z19181, and Z19182) bearing valine or isoleucine residues at positions X 5 The results were not substantially different from the Z variant, which has an alanine residue at 1.

[0288] [Table 21]

[0289] Example 10 Production and characterization of IL-1R-I binding fusion proteins This example describes the general steps for DNA construction and production of different IL-1R-I binding protein variants fused to different in vivo half-life extending fusion partners.

[0290] Materials and Methods DNA constructs: DNA encoding a series of IL-1R-I binding Z variants (see below) fused to human IgG1 Fc (SEQ ID NO: 1662), albumin binding polypeptide variant (PP013; SEQ ID NO: 1661), human albumin (SEQ ID NO: 1663), or human transferrin (SEQ ID NO: 1664), optionally via different linkers (Table 22), were codon-optimized for expression in E. coli or Chinese Hamster Ovary (CHO) cells and synthesized by Invitrogen GeneArt gene synthesis services at Thermo Fisher Scientific. Genes were cloned into expression vectors for subsequent expression in E. coli or CHO cells.

[0291] The fusion proteins were based on the binding motifs of IL-1R-I binding Z variants a) Z12967 (SEQ ID NO: 9), b) Z12895 (SEQ ID NO: 12), c) Z18831 (SEQ ID NO: 1308), d) Z18846 (SEQ ID NO: 1331), e) Z18754 (SEQ ID NO: 1252), f) Z19151 (SEQ ID NO: 1594), g) Z18874 (SEQ ID NO: 1421), h) Z18760 (SEQ ID NO: 1435), i) Z18800 (SEQ ID NO: 1328), j) Z18898 (SEQ ID NO: 1285), and k) Z19147 (SEQ ID NO: 1307), as well as the different fusion partners described above. IL-1R-I binding Z variants a)-k) contained different scaffold mutations (positions 1, 2, 23, 52, and / or 53) or deletions combined with mutations (deletion at positions 1-3; ΔV1D2A3, mutations at positions 52 and 53) (see Table 22 below). Mutated Z variants are listed in FIG. 1 as SEQ ID NOs: 1667-1668 and 1670-1679. The resulting fusion polypeptides were PSI0405 (SEQ ID NO: 1639), PSI0407 (SEQ ID NO: 1640), PSI0411 (SEQ ID NO: 1641), PSI0412 (SEQ ID NO: 1642), PSI0415 (SEQ ID NO: 1643), PSI0416 (SEQ ID NO: 1644), PSI0417 (SEQ ID NO: 1645), PSI0534 (SEQ ID NO: 1646), PSI0535 (SEQ ID NO: 1647), PSI0536 (SEQ ID NO: 1648), PSI0537 (SEQ ID NO: 1649), PSI0538 (SEQ ID NO: 1650), PSI0539 (SEQ ID NO: 1651), PSI0540 (SEQ ID NO: 1652), PSI0541 (SEQ ID NO: 1653), PSI0542 (SEQ ID NO: 1654), PSI0543 (SEQ ID NO: 1655), PSI0544 (SEQ ID NO: 1656), PSI0545 (SEQ ID NO: 1657), PSI0546 (SEQ ID NO: 1658), PSI0547 (SEQ ID NO: 1659), PSI0548 (SEQ ID NO: 1660), PSI0549 (SEQ ID NO: 1661), PSI0549 (SEQ ID NO: 1662), PSI0540 (SEQ ID NO: 1663), PSI0541 (SEQ ID NO: 1664), PSI0542 (SEQ ID NO: 1665), PSI0543 (SEQ ID NO: 1666), (SEQ ID NO: 1648), PSI0537 (SEQ ID NO: 1649), PSI0538 (SEQ ID NO: 1650), PSI0539 (SEQ ID NO: 1651), PSI0574 (SEQ ID NO: 1652), PSI0575 (SEQ ID NO: 1653), PSI0576 (SEQ ID NO: 1654), PSI0580 (SEQ ID NO: 1655), PSI0581 (SEQ ID NO: 1656), and PSI0582 (SEQ ID NO: 1657) (see Table 22).

[0292] [Table 22] TIFF2025078711000040.tif50170

[0293] Cultivation and purification of fusion proteins: E. coli cells were transformed with expression vectors containing gene fragments corresponding to IL-1R-I binding fusion proteins (see Table 22) and then cultured in bioreactors using fed-batch technology or in percolation flasks, after which the proteins were expressed and the cells were harvested by centrifugation. The cell pellets were stored at -20°C. IL-1R-I binding Z variants (see Table 22) were also expressed using the ExpiCHO Expression System (Thermo Fisher Scientific) essentially according to the manufacturer's protocol. The supernatants were harvested by centrifugation 12 days after transfection of the expression vectors and stored at -70°C.

[0294] Frozen E. coli cell pellets were resuspended and then disrupted by sonication, followed by centrifugation to remove cell debris, followed by filtration (0.22 μm). Frozen supernatants from ExpiCHO cultures were thawed and filtered (0.22 μm). Each supernatant containing an IL-1R-I binding Z variant was purified using conventional chromatographic methods, including affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography. Z variants used in animal studies were also subjected to endotoxin removal purification using Detoxi-Gel endotoxin removal columns (Pierce, Cat. No. 20344). Purified Z variants were exchanged into PBS buffer, which was also the formulation buffer used in subsequent experiments unless otherwise stated. The purity of the IL-1R-I-binding Z variants was analyzed by SDS-PAGE stained with Coomassie blue, and the molecular weight of each purified Z variant was analyzed using mass spectrometry (HPLC / MS or MALDI-TOF / MS).

[0295] The IL-1R-I binding fusion protein PSI0589 (SEQ ID NO: 1658) (dimeric Z18754 variant linked at the hinge region by a disulfide) was obtained by removal of the IgG1 Fc part from PSI0536 (see Table 22). This was done by incubation of purified PSI0536 with IdeS protease (FabRICATOR, Genovis AB). The cleavage products were then purified using conventional chromatographic methods (such as ion exchange and affinity chromatography). The purified dimeric Z18754 variant was subjected to buffer exchange into PBS, which was also the formulation buffer used in the subsequent experiments. Purity was analyzed by SDS-PAGE stained with Coomassie Blue and molecular weight was analyzed using mass spectrometry (HPLC / MS or MALDI-TOF / MS).

[0296] Biolayer Interferometry (BLI) Kinetic Analysis: Binding of four IL-1R-I binding fusion proteins to human and cynomolgus IL-1R-I was analyzed using BLI. Fc-fused IL-1R-I binding Z variants designated PSI0536, PSI0537, PSI0534, and PSI0535 (SEQ ID NOs: 1648, 1649, 1646, 1647) were loaded onto a Protein A sensor (Pall, ForteBio Cat. No. 18-5010) at 0.5 μg / ml for 150 seconds in each cycle using an OctetRED96 instrument (Pall, ForteBio). These sensors were subsequently exposed to different increasing concentrations of human IL-1R-I (R&D systems catalogue no. 269-1R / CF) ranging from 0.15 to 5 nM or cynomolgus IL-1R-I (SEQ ID NO: 1665) ranging from 2.5 to 80 nM. Association was recorded for 300 s, dissociation was recorded for 300 s (human) or 90 s (cynomolgus), and spectrograms were recorded and analyzed according to the 1:1 Langmuir model using a global fit (Octet System Data Analysis 8.2 software, Pall, ForteBio). Each sensor was referenced against a sensor with the same ligand loaded on the sensor but exposed to buffer only. After each association / dissociation cycle, all sensors were regenerated with 3 pulses of 10 mM glycine (pH 2.5) for 10 s each. All steps were performed at 25° C. with a shaking speed of 1000 rpm, and all dilutions of fusion proteins (Z-Fc fusion proteins) and analytes (human or cynomolgus IL-1R-I) were in 1× kinetic buffer (Pall, ForteBio).

[0297] result Culture and purification: All IL-1R-I binding fusion proteins (Table 22) were expressed at high levels in E. coli or CHO cells as soluble proteins. Purification yielded highly pure protein preparations that were analyzed by SDS-PAGE stained with Coomassie Blue. The exact identity and molecular weight of each Z variant was confirmed by mass spectrometry.

[0298] BLI kinetic analysis: Four Z-Fc fusion protein variants (SEQ ID NOs: 1648, 1649, 1646, 1647) were analyzed for their ability to bind human and cynomolgus IL-1R-I loaded onto a Protein A sensor using BLI technology on an OctetRED96 instrument. All four variants bound to IL-1R-I with B-max values ​​ranging from 0.46 to 0.72 nm (human) or 0.31 to 0.37 nm (cynomolgus) according to a 1:1 model. Kinetic data (association rate constants (k on ), dissociation rate constant (k off ), and the dissociation constant K D ) are shown in Table 23.

[0299] [Table 23]

[0300] Example 11: In vitro pharmacological activity analysis using cell-based assays Materials and Methods The inhibitory effect of an IL-1R-I binding fusion protein on IL-1β-induced IL-6 production in normal human dermal fibroblast (NHDF) cells was monitored.

[0301] Cells were seeded 3 days prior to treatment with proteins. Proteins (IL-1R-I binding fusion proteins or anakinra) were diluted in serum-free growth medium in the presence of 9 μM recombinant human serum albumin (rHSA) to a starting concentration of 100 nM, followed by 9 serial dilutions 1:4 to obtain a concentration range of proteins from 100 nM to 0.38 pM. IL-1R-I binding fusion proteins or anakinra were tested in the presence of a challenge dose of 3.4 pM IL-1β, and cells were incubated with proteins for 22 hours at 37°C, after which the medium was harvested. After dilution of the harvested medium 41-fold, IL-6 content was analyzed using a human IL-6 ELISA kit (R&D Systems) according to the manufacturer's recommended conditions. Data were analyzed using XLfit, and IC50 Values ​​were calculated from concentration-response curves.

[0302] His produced as described in Example 9 6 The inhibitory effect of tagged IL-1R-I binding Z variants on IL-1β-induced IL-6 production in NHDF cells was similarly monitored and IC 50 values ​​were calculated.

[0303] Chemically Synthesized Protein: A chemically synthesized version of SEQ ID NO: 1672, designated PSI0558, was ordered from BACHEM AG. The activity of PSI0558 was evaluated according to the methods described above.

[0304] result IL-1β-induced IL-6 release from NHDF cells was reduced in a concentration-dependent manner by IL-1R-I binding fusion proteins and anakinra. Each IL-1R-I binding fusion protein was tested in duplicate, and data from both experiments in this assay are shown in Table 24 along with the historical mean values ​​(n>20) for anakinra, and one of the two experiments is also shown in Figure 2.

[0305] [Table 24]

[0306] Several IL-1R-I binding fusion proteins were tested in the same manner, but only once. The results from this experiment are shown in Table 25.

[0307] [Table 25]

[0308] His previously demonstrated in the TF-1 cell assay (Example 9, Table 20) 6 The IL-1β inhibitory capacity of the tagged IL-1R-I binding Z variants was confirmed in this NHDF assay (results not shown).

[0309] Chemically synthesized PSI0558 IC 50 The value was 690 pM.

[0310] Example 12 In vitro pharmacological activity analysis using whole blood assays The inhibitory effect of IL-1R-I binding fusion proteins on IL-1β-induced IL-6 production in whole blood from individual donors was measured.

[0311] Materials and Methods Blood was drawn from healthy donors 1-3 hours prior to treatment with IL-1R-I binding fusion proteins (SEQ ID NO: 1648-1649). Proteins (IL-1R-I binding fusion proteins or anakinra) were diluted in serum-free RPMI (ThermoFisher Scientific, RPMI1640 medium, GlutaMAX™ Supplement, Cat. No. 61870010) to a starting concentration of 500 nM, followed by 10 serial dilutions 1:3, thereby resulting in a concentration range of proteins from 500 nM to 25 pM. IL-1R-I binding fusion polypeptides or anakinra were tested in the presence of a challenge dose of 100 pM IL-1β. Blood was incubated with the proteins for 21-23 hours, after which plasma was collected. The collected plasma was diluted two-fold and the IL-6 content was analyzed using a human IL-6 kit (V-PLEX human IL-6 kit human interleukin-6, MSD) according to the manufacturer's recommended conditions.

[0312] Calculate IL-6 concentrations (pg / ml) using the standard curve (provided in the kit), analyze the data using XLfit, and derive the IC from the concentration-response curve. 50 values ​​were calculated.

[0313] result IL-1β-induced IL-6 release in human whole blood was reduced in a concentration-dependent manner by IL-1R-I binding fusion proteins and anakinra. IL-1R-I binding fusion proteins were tested in blood from 5-6 individual healthy donors and compared to anakinra data from 10 individual blood donors (Figure 3). IC values ​​for SEQ ID NO:1648 (PSI0536), SEQ ID NO:1649 (PSI0537), and anakinra were 50 The median values ​​were 0.32, 1.25, and 0.31 nM, respectively.

[0314] Example 13 In vivo pharmacokinetics of IL-IR-1 binding fusion protein in rats In this example, the pharmacokinetics of an IL-1R-1 binding fusion protein (SEQ ID NOs: 1646-1651) was evaluated in a single dose study in male rats.

[0315] Materials and Methods Fusion Proteins: IL-1R-I binding fusion proteins PSI0534, PSI0535, PSI0536, PSI0537, PSI0538, and PSI0539 (see Table 22, SEQ ID NOs:1646-1651) were evaluated in this study. All six test articles were made up as a solution of 25 mM sodium phosphate and 125 mM sodium chloride, pH 7.0.

[0316] Survival phase: The pharmacokinetic properties of the six fusion proteins were investigated in male Sprague-Dawley rats. For each test item, three rats were injected iv into the lateral tail vein with a single dose of 15 mg / kg (2.5 ml / kg) and another three rats were injected sc into the neck with a single dose of 30 mg / kg (5 ml / kg). Blood samples for serum preparation were collected before administration, 5 and 20 min after iv administration, and 1, 4, 8, 24, 48, 72, and 96 h after administration, or 20 min after sc administration, 1, 4, 8, 24, 48, 72, 96, and 120 h after administration.

[0317] Quantitative ELISA: Fusion protein levels in rat serum samples were determined by enzyme-linked immunosorbent assay (ELISA), which uses a quantitative sandwich enzyme immunoassay technique.

[0318] Briefly; goat polyclonal anti-Z antibody (homemade, Affibody AB), which recognizes the Z variant domain of IL-1R-I binding fusion protein, was coated onto a microplate. Unbound polyclonal antibody was washed away and casein was added as a blocking agent to reduce non-specific binding to the plastic surface. After removal of unbound casein by a second washing step, standards and samples were pipetted into the wells and any fusion protein present was bound to the immobilized antibody. After washing away any unbound material, HRP-labeled swine anti-rabbit antibody (Dako, Cat. No. P0399) was added. After washing to remove any unbound anti-rabbit HRP reagent, substrate solution was added to the wells and color was developed in proportion to the amount of fusion protein bound in the first step. Color development was stopped and color intensity was measured.

[0319] Pharmacokinetic Analysis: Pharmacokinetic analysis was based on median serum concentration versus time data from each dose group. Maximum observed concentrations (C max ) and the time to reach maximum serum concentration (t max ) were obtained directly from the bioanalytical data. Other pharmacokinetic parameters (i.e., clearance (CL), apparent clearance after sc administration (CL / F), apparent volume of distribution at steady state (V ss ), mean residence time (MRT), and terminal half-life (t 1 / 2z) was estimated by noncompartmental analysis using Phoenix WinNonlin software version 6.3 (Pharsight Corp., USA). Estimation of the terminal phase slope after iv administration was based on four data points from 24 to 96 h. Estimation of the terminal phase slope after sc administration was based on five data points from 24 to 120 h. Subcutaneous bioavailability (F) was calculated using Microsoft Excel.

[0320] result No significant differences in pharmacokinetics were observed among the six tested fusion proteins. The pharmacokinetics after iv administration were characterized by a clearance of approximately 2.4 ml / h kg and a volume of distribution of approximately 140 ml / kg, which translated into a mean residence time of approximately 60 hours (Table 26).

[0321] [Table 26]

[0322] Subcutaneous bioavailability averaged 50%, with peak levels observed 24 hours after dosing, and serum levels then declined with half-lives ranging from 45 to 59 hours (Table 27).

[0323] [Table 27]

[0324] Example 14 In vivo pharmacokinetics of IL-1R-I binding fusion protein in monkeys Materials and Methods Fusion Protein: The IL-1R-I binding fusion protein designated PSI0536 (SEQ ID NO: 1648) was constituted in a solution of 25 mM sodium phosphate and 125 mM sodium chloride, pH 7.0.

[0325] Survival Phase: The pharmacokinetic properties of PSI0536 were investigated in naive male cynomolgus monkeys. Three monkeys were injected iv into the tail vein with a single dose of 5 mg / kg (1 ml / kg) and three monkeys were injected sc into the back with a single dose of 10 mg / kg (1 ml / kg). Blood samples for serum preparation were collected pre-dose, 5 and 20 min after iv administration, and 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 240, and 504 h after administration, or 20 min after sc administration, and 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 240, and 504 h after administration.

[0326] Quantitative ELISA: PSI0536 in monkey serum was quantified according to the ELISA described in Example 13.

[0327] Pharmacokinetic Analysis: Pharmacokinetic parameters were determined based on individual serum concentration versus time data using WinNonlin software as described in Example 13. Estimation of terminal slope after iv administration was based on seven data points from 72 to 504 hours. Estimation of terminal slope after sc administration was based on eight data points from 48 to 504 hours. Subcutaneous bioavailability (F) was calculated using Microsoft Excel.

[0328] result The pharmacokinetics of PSI0536 (SEQ ID NO: 1648) following iv administration was characterized by low clearance (1.07 ml / h kg) and a small volume of distribution (117 ml / kg), which translated into a mean residence time of 109 hours (Table 28).

[0329] [Table 28]

[0330] The mean subcutaneous bioavailability of PSI0536 was 73.9%. Peak levels were observed at an average of 18.7 hours post-dose. Serum levels then declined with a half-life of 77.3 hours (Table 29).

[0331] [Table 29]

[0332] conclusion Assuming a minimum target serum concentration of 100 nM or even 200 nM, the combination of high subcutaneous bioavailability with low clearance facilitates a once weekly subcutaneous dosing regimen of 10 mg / kg PSI0536 in monkeys.

[0333] Example 15 Production and characterization of IL-1-RI binding fusion proteins with mutated Fc portions Four IL-1-RI binding fusion proteins of IL-1R-I binding polypeptide variant Z18754 (SEQ ID NO: 1252) with mutation variants of the Fc part of IgG1 or IgG4 were produced. In the IgG1 Fc part, one mutation (N297A) was introduced to abolish the effector function due to interaction with FcγR3A. Fc-containing polypeptides produced in mammalian cells can produce such effector function in the absence of mutation. Furthermore, in one fusion protein (SEQ ID NO: 1735), two other residues, designated LS, were introduced to increase affinity for FcRn (WO2009 / 086320), and in another fusion protein (SEQ ID NO: 1736), three mutations, designated YTE, were introduced to increase affinity for FcRn (WO02060919). A fusion protein with IgG4 Fc (a subtype known to induce low effector response) of Z18754 (SEQ ID NO: 1252) was produced (SEQ ID NO: 1737). A mutation known as S228P (van der Neut Kolfschoten M. et al, 2007, Science 317(5844):1554-7) was further introduced into the hinge region of this fusion protein. This mutation eliminates arm exchange that may occur in the IgG4 subclass of antibodies.

[0334] Materials and Methods Fusion proteins PSI0653-PSI0656 (SEQ ID NOs: 1734-1737) (see Table 30 for details) were produced in CHO cells according to Example 10.

[0335] The inhibitory effect of IL-1R-I binding fusion proteins on IL-1β-induced IL-6 production in normal human dermal fibroblast (NHDF) cells was monitored as shown in Example 11.

[0336] Affinity for FCγR3A was analyzed using biolayer interferometry essentially as disclosed in Example 10, except that biotinylated FCγR3A (Sino Biological) was immobilized on a streptavidin biosensor (Pall / Fortebio) for kinetics and proteins were tested at concentrations of 100 and 1000 nM. In addition, human IgG (GammaNorm, Octapharma) was used as a control in the measurements.

[0337] Human biotinylated FcRn (Immunitrack ApS) was immobilized on a biotin CAPture chip (GE Healthcare) and affinity for FcRn was assessed using Biacore essentially as described in Example 9, except that analytes were tested at five concentrations ranging from 2.5 to 200 nM.

[0338] [Table 30]

[0339] result Culture and purification: All IL-1-RI binding fusion proteins with mutated Fc moieties (Table 30) were expressed at high levels in CHO cells as soluble proteins. Purification yielded highly pure protein preparations that were analyzed by SDS-PAGE stained with Coomassie Blue. The exact identity and molecular weight of each Z variant was confirmed by mass spectrometry.

[0340] In vitro pharmacological activity: IL-1β-induced IL-6 release from NHDF cells was reduced in a concentration-dependent manner by IL-1R-I binding fusion proteins. IL-1R-I binding fusion proteins were tested once. The results from this experiment are shown in Table 31.

[0341] [Table 31]

[0342] FCγR3A affinity analysis: Human IgG bound to FCγR3A with an affinity of 57 nM at a concentration of 1000 nM. Fusion protein PSI0653-PSI0655 (SEQ ID NO: 1734-1736) did not bind at any concentration. PSI0656 (SEQ ID NO: 1737) bound weakly at 1000 nM but not at 100 nM.

[0343] Biacore FcRn affinity analysis: The affinity to FcRn was in the order SEQ ID NO:1735>SEQ ID NO:1736>SEQ ID NO:1734=SEQ ID NO:1737.

[0344] Conclusion: The mutated Fc variants of the fusion protein (SEQ ID NO: 1734:1737) were equivalent to the previously produced protein in terms of quality and activity (see Example 10). Removal of the glycan binding site eliminated binding to FcγR3A, thus removing the effector function of the IgG1 Fc portion in SEQ ID NO: 1734-1736. SEQ ID NO: 1737, which contains the IgG4 Fc, had a lower affinity for FcγR3A than would be expected for that subclass of Fc. The FcRn affinity-enhancing mutations in SEQ ID NO: 1735 and 1736 increased affinity, with SEQ ID NO: 1735 having the greatest increase.

[0345] Table of embodiments 1. An IL-1R-I binding polypeptide comprising an IL-1R-I binding motif BM, said motif comprising: i)EX 2 X 3 X 4 X5 X 6 X 7 EIX 10 X 11 LPNLX 16 RX 18 QYX 21 AFIX 25 X 26 LX 28 D (SEQ ID NO: 1686) (Independently of each other, X 2 is selected from A, D, E, F, H, I, L, Q, S, T, and V; X 3 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 4 is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 5 is selected from A, I, and V; X 6 is selected from F, H, I, Q, R, T, V, and Y; X 7 is selected from A, D, E, F, G, H, I, L, M, Q, S, T, V, W, and Y; X 10 is selected from F and Y; X 11 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y; X 16 is selected from N and T; X 18 is selected from K, R, and S; X 21 is selected from Q, T, and V; X 25 is selected from I, M, R, V, and Y; X 26 is selected from K and S, and X 28 is selected from F, I, L, and M; and ii) X 5is I or V, comprising an amino acid sequence selected from amino acid sequences having at least 96% identity to the sequence defined in i). 2. The IL-1R-I binding motif is i) X 2 is selected from A, I, L, T, and V; X 3 is selected from E and Y; X 4 is selected from A, E, I, K, Q, R, T, V, and Y; X 5 is selected from I and V; X 6 is selected from Q and Y; X 7 is selected from F and M; X 10 is selected from F and Y; X 11 is selected from A, D, E, F, G, H, K, L, Q, R, S, T, V, and Y; X 16 is selected from N and T; X 18 is selected from K and R; X 21 is selected from T and V; X 25 is selected from I and R; X 26 is selected from K and S, and X 28 is selected from F and L, and An IL-1R-I binding polypeptide consisting of an amino acid sequence selected from the amino acid sequences having at least 93% identity to the sequence defined under i). 3. The sequence i) satisfies the following 10 conditions I to X: IX 3 is E; II.X 5 is selected from I and V; III.X 6is selected from Q and Y; IV.X 7 is selected from F and M; VX 10 is selected from F and Y; VI.X 18 is selected from K and R; VII.X 21 is T; VIII.X 25 is R; IX.X 26 is selected from K and S; and XX 28 is selected from F and L 13. The IL-1R-I binding polypeptide of any one of the preceding claims, wherein the IL-1R-I binding polypeptide satisfies at least five of the following criteria: 4. An IL-1R-I-binding polypeptide according to item 3, in which sequence i) satisfies at least six of the ten conditions I to X. 5. An IL-1R-I binding polypeptide according to item 4, in which sequence i) satisfies at least seven of the ten conditions I to X. 6. An IL-1R-I binding polypeptide according to item 5, in which sequence i) satisfies at least 8 of the 10 conditions I to X. 7. An IL-1R-I-binding polypeptide according to item 6, in which sequence i) satisfies at least 9 of the 10 conditions I to X. 8. An IL-1R-I-binding polypeptide according to item 7, in which sequence i) satisfies all of the ten conditions I to X. 9.X 2 X 3 X 6 9. An IL-1R-I binding polypeptide according to any one of items 1 to 8, wherein is VEQ or VEY. 10.X 2 X 3 X 6 9. An IL-1R-I binding polypeptide according to any one of items 1 to 8, wherein is IEQ or VEQ. 11.X 6 X 109. The IL-1R-I binding polypeptide according to any one of items 1 to 8, wherein is selected from the group consisting of QF, QY, YF, and YY. 12.X 6 X 10 9. An IL-1R-I-binding polypeptide according to any one of items 1 to 8, wherein is QF. 13.X 10 X 18 is selected from the group consisting of FK, FR, YK, and YR. 14.X 10 X 18 9. An IL-1R-I-binding polypeptide according to any one of items 1 to 8, wherein is FK or FR. 15.X 18 X 25 X 28 9. The IL-1R-I-binding polypeptide according to any one of items 1 to 8, wherein is KRL or RRL. 16.X 5 X 7 9. An IL-1R-I-binding polypeptide according to any one of items 1 to 8, wherein is IM or VM. 17. An IL-1R-I-binding polypeptide according to any of the preceding items, wherein sequence i) corresponds to the sequence of positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 1632 and 1679 (such as the group consisting of SEQ ID NOs: 20 to 1632 and 1679). 18. An IL-1R-I-binding polypeptide according to any of the preceding items, wherein sequence i) corresponds to the sequence at positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1206 to 1632 and 1679 (such as the group consisting of SEQ ID NOs: 1210 to 1632 and 1679). 19. An IL-1R-I-binding polypeptide according to any of the preceding items, wherein sequence i) corresponds to the sequence at positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594, and 1679 (such as the group consisting of SEQ ID NOs: 1252, 1328, 1435, and 1679). 20. An IL-1R-I-binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from positions 8 to 36 in SEQ ID NO: 1252. 21. An IL-1R-I-binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from positions 8 to 36 in SEQ ID NO: 1328. 22. An IL-1R-I-binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from positions 8 to 36 in SEQ ID NO: 1435. 23. An IL-1R-I binding polypeptide according to any of the preceding items, wherein said IL-1R-I binding motif forms part of a three-helix bundle protein domain. 24. An IL-1R-I binding polypeptide according to item 23, wherein the IL-1R-I binding motif essentially forms part of two helices together with an interconnecting loop within the three-helix bundle protein domain. 25. An IL-1R-I binding polypeptide according to item 24, wherein the three-helix bundle protein domain is selected from bacterial receptor domains. 26. An IL-1R-I binding polypeptide according to item 25, wherein the three-helix bundle protein domain is selected from the domain of protein A from Staphylococcus aureus or a derivative thereof. 27. A binding module BMod, the amino acid sequence of which is iii) K-[BM]-DPSQSX a X b LLX c EAKKLX d X e X f Q; (SEQ ID NO: 1687) ( [BM] is an IL-1R-I binding motif as defined in any one of items 1 to 22; X a is selected from A and S; X b is selected from N and E; X c is selected from A, S, and C; X d is selected from E, N, and S; X e is selected from D, E, and S; X f is selected from A and S); and iv) an amino acid sequence having at least 91% identity to the sequence defined in iii); 4. An IL-1R-I binding polypeptide according to any of the preceding items, selected from: 28. An IL-1R-I-binding polypeptide according to any of the preceding items, wherein sequence iii) corresponds to a sequence from positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 1638, 1667 to 1668, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 20 to 1638 and 1670 to 1679). 29. The IL-1R-I-binding polypeptide according to item 28, wherein sequence iii) corresponds to a sequence from positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1206 to 1632 and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 1210 to 1632 and 1670 to 1679). 30. The IL-1R-I-binding polypeptide according to item 28, wherein the sequence iii) corresponds to a sequence from positions 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 1252, 1328, 1435, 1672, 1675 to 1676, and 1679). 31. An IL-1R-I-binding polypeptide according to item 30, wherein sequence iii) corresponds to the sequence from positions 7 to 55 in SEQ ID NO: 1672. 32. An IL-1R-I-binding polypeptide according to item 30, wherein sequence iii) corresponds to the sequence from positions 7 to 55 in SEQ ID NO: 1675. 33. An IL-1R-I-binding polypeptide according to item 30, wherein sequence iii) corresponds to the sequence from positions 7 to 55 in SEQ ID NO: 1676. 34.ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK SEQ ID NO:1695; ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK SEQ ID NO:1696; ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK SEQ ID NO:1697; ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK SEQ ID NO:1698; AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK SEQ ID NO:1699; VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK SEQ ID NO:1700; AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK SEQ ID NO:1701; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK SEQ ID NO:1702; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK SEQ ID NO:1703; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK SEQ ID NO:1704; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP SEQ ID NO:1705; AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK SEQ ID NO:1706; AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP SEQ ID NO:1707; AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK SEQ ID NO:1708; AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK SEQ ID NO:1709; AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP SEQ ID NO:1710; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK SEQ ID NO:1711; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP SEQ ID NO:1712; AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK SEQ ID NO:1713; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK SEQ ID NO:1714; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP SEQ ID NO:1715; AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK SEQ ID NO:1716; AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK SEQ ID NO:1717; AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP SEQ ID NO:1718; AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK SEQ ID NO:1719; AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK SEQ ID NO:1720; VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK SEQ ID NO:1721; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK SEQ ID NO:1722; VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK SEQ ID NO:1723; VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK SEQ ID NO:1724; VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK SEQ ID NO:1725; VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK SEQ ID NO:1726; VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK SEQ ID NO:1727; VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK SEQ ID NO:1728; VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK SEQ ID NO:1729; VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK SEQ ID NO:1730; AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK SEQ ID NO: 1731, and ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK SEQ ID NO: 1732, comprising an amino acid sequence selected from [BM] is an IL-1R-I binding motif as defined in any one of items 1 to 22. 35.xix) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1721) (wherein [BM] is an IL-1R-I binding motif as defined in any one of items 1 to 22, VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1721)); and xx) an amino acid sequence selected from the amino acid sequences having at least 89% identity to a sequence defined in xix) in a sequence adjacent to the BM; 36.xxi) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO: 1709) (wherein [BM] is an IL-1R-I binding motif as defined in any one of items 1 to 22, AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO: 1709)); and xxii) an amino acid sequence having at least 89% identity to the sequence defined in xxi) in the sequence adjacent to the BM 35. The IL-1R-I-binding polypeptide according to any one of items 1 to 34, comprising an amino acid sequence selected from: 37. An IL-1R-I-binding polypeptide according to item 35 or 36, wherein sequence xix) or xxi) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 1632, 1667 to 1668, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 20 to 1632, 1667 to 1668, and 1670 to 1679). 38. The IL-1R-I-binding polypeptide according to item 37, wherein xix) or xxi) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1206 to 1632, 1667 to 1668, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 1210 to 1632, 1667 to 1668, and 1670 to 1679). 39. An IL-1R-I-binding polypeptide according to item 38, wherein sequence xix) or xxi) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 1252, 1328, 1435, 1672, 1675 to 1676, and 1679). 40. The IL-1R-I-binding polypeptide according to item 39, wherein sequence xxi) corresponds to the sequence from positions 1 to 58 in SEQ ID NO: 1672. 41. An IL-1R-I-binding polypeptide according to item 39, wherein sequence xxi) corresponds to the sequence from positions 1 to 58 in SEQ ID NO: 1675. 42. The IL-1R-I-binding polypeptide according to item 39, wherein the sequence xxi) corresponds to the sequence from positions 1 to 58 in SEQ ID NO: 1676. 43. An IL-1R-I binding polypeptide according to any of the preceding items, which is capable of blocking IL-1R-I dependent signalling. 44. Half-maximal inhibitory concentration (IC 50 ) is up to 1×10 -7 M (maximum 1 × 10 -8 M, etc., up to 1×10 -9 M, etc., up to 5 x 10 -10 44. The IL-1R-I binding polypeptide according to item 43, wherein said polypeptide is selected from the group consisting of IL-1R-I-binding polypeptides having the following structure: 45. An IL-1R-I binding polypeptide according to item 43 or 44, which is capable of blocking the interaction of IL-1R-I with IL-1 cytokines (such as the interaction of IL-1R-I with IL-1α and / or IL-1β). 46. ​​EC of Interaction 50 The maximum value is 1×10 -7 M (maximum 1 × 10 -8 M, etc., up to 5 x 10 -9 M, etc., up to 1×10 -10 M, etc., up to 5 x 10 -10 M, etc., up to 2 x 10 -10 IL-1R-I binding polypeptide according to any of the preceding items, capable of binding to IL-1R-I such that the polypeptide is 47. K for Interaction D The maximum value is 1×10 -6 M (maximum 1 × 10 -7 M, etc., up to 1×10 -8 M, etc., up to 1×10 -9 M, etc., up to 9 x 10 -10 IL-1R-I binding polypeptide according to any of the preceding items, which is capable of binding to IL-1R-I such that the IL-1R-I is 48. An IL-1R-I-binding polypeptide according to any one of items 43 to 47, wherein said IL-1R-I is human IL-1R-I or cynomolgus IL-1R-I (such as human IL-1R-I). 49. An IL-1R-I binding polypeptide according to any of the preceding items, comprising additional amino acids at the C-terminus and / or N-terminus. 50. An IL-1R-I binding polypeptide according to item 49, wherein said further amino acids improve the production, purification, in vivo or in vitro stabilization, coupling or detection of the polypeptide. 51. An IL-1R-I binding polypeptide according to any of the preceding items in the form of a multimer comprising at least two IL-1R-I binding polypeptide monomer units, the amino acid sequences of which may be identical or different. 52. An IL-1R-I binding polypeptide according to item 51, wherein the IL-1R-I binding polypeptide monomer units are covalently linked to each other. 53. An IL-1R-I binding polypeptide according to item 52, wherein the IL-1R-I binding polypeptide monomer unit is expressed as a fusion protein. 54. An IL-1R-I binding polypeptide in dimeric form according to any one of items 51 to 53. 55.- A first part consisting of an IL-1R-I binding polypeptide according to any of the preceding items; and a second portion consisting of a polypeptide having a desired biological activity A fusion protein or conjugate comprising: 56. The fusion protein or conjugate according to item 55, wherein the desired biological activity is a therapeutic activity. 57. The fusion protein or conjugate according to item 55, wherein the desired biological activity is binding activity. 58. The fusion protein or conjugate according to item 55, wherein the desired biological activity is a binding activity that modifies the tissue distribution of the fusion protein or conjugate. 59. The fusion protein or conjugate according to item 57, wherein the desired biological activity is an in vivo half-life increasing activity, such that the second portion increases the in vivo half-life of the fusion protein or conjugate. 60. The fusion protein or conjugate according to item 59, wherein said in vivo half-life increasing activity is albumin binding activity. 61. A fusion protein or conjugate according to item 60, in which said albumin binding activity is obtained by the albumin binding domain of streptococcal protein G or a derivative thereof. 62. The fusion protein or conjugate according to item 61, wherein the second part comprises a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1659 to 1661 (such as SEQ ID NO: 1661). 63. The fusion protein or conjugate according to item 59, wherein the second moiety is serum albumin. 64. The fusion protein or conjugate according to item 59, wherein the second portion is an Fc portion of an antibody (such as IgG1 Fc or IgG4 Fc). 65. The fusion protein or conjugate according to item 64, wherein the Fc is a mutated Fc that exhibits improved FcRn and / or reduced effector response compared to the unmutated form of the Fc. 66. The fusion protein or conjugate according to item 58 or 59, wherein the second moiety is transferrin. 67. The fusion protein or conjugate according to any one of items 59 to 65, comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1639 to 1658 and 1734 to 1737 (such as the group consisting of SEQ ID NOs: 1646 to 1654 and 1734 to 1737). 68. The fusion protein or conjugate according to item 57, wherein said binding activity acts to block biological activity. 69. The fusion protein or conjugate according to any one of items 55 to 68, further comprising at least one linker, said linker being optionally selected from the group consisting of a flexible amino acid linker, a fixed amino acid linker, and a cleavable amino acid linker. 70. The fusion protein or conjugate according to item 69, wherein the linker is disposed between the first portion and the second portion. 71. The fusion protein or conjugate according to item 69, wherein the linker is disposed within the first portion. 72. The fusion protein or conjugate according to item 69 or 70, wherein the linker is a flexible linker comprising at least one amino acid residue selected from the group consisting of glycine, serine, and alanine. 73. The linker is GS, VDSS, VDGS, VEGS, ASGS, (GGGGS) 2 (SEQ ID NO: 1683), AS(GGGGS) 2 (SEQ ID NO: 1684), and ((KEAAA)3KELAA) 2 73. The fusion protein or conjugate according to item 72, selected from the group consisting of: (SEQ ID NO: 1685). 74. The linker is AS(GGGGS) 2 (SEQ ID NO: 1684) and ((KEAAA) 3 KELAA) 2 74. The fusion protein or conjugate according to item 73, selected from: (SEQ ID NO: 1685). 75. A polynucleotide encoding a polypeptide or a fusion protein according to any one of items 1 to 74. 76. An expression vector comprising the polynucleotide according to item 75. 77. A host cell comprising an expression vector according to item 76. 78.- Cultivating the host cell according to item 77 under conditions allowing expression of said polypeptide from said expression vector, and - isolating said polypeptide. 78. A method for producing a polypeptide according to any one of items 1 to 77, comprising: 79. A composition comprising an IL-1R-I binding polypeptide, fusion protein, or conjugate according to any one of items 1 to 74, and at least one pharma- ceutically acceptable excipient or carrier. 80. The composition according to item 79, further comprising at least one additional active agent (such as an agent selected from an immune response modifier and an anti-cancer agent). 81. The composition according to item 79 or 80, wherein the fusion protein is a fusion protein according to any one of items 59 to 67. 82. An IL-1R-I binding polypeptide, fusion protein or conjugate according to any one of items 1 to 74 or a composition according to any one of items 79 to 81 for use as a medicament, diagnostic agent and / or prognostic agent. 83. The IL-1R-I binding polypeptide, fusion protein, conjugate or composition according to item 82 for use as a medicament. 84. An IL-1R-I binding polypeptide, fusion protein, conjugate or composition for use according to item 83, wherein said polypeptide, fusion protein, conjugate or composition modulates IL-1R-I function in vivo. 85. An IL-1R-I binding polypeptide, fusion protein, conjugate or composition according to item 82 for use as a diagnostic and / or prognostic agent. 86. An IL-1R-I binding polypeptide, fusion protein, conjugate or composition according to any one of items 82 to 85 for use in the treatment, prognosis or diagnosis of an IL-1R-I associated disorder. 87. The IL-1R-I binding polypeptide, fusion protein, conjugate, or composition for use according to item 86, wherein the IL-1R-I-related disorder is selected from the group consisting of inflammatory diseases, autoinflammatory syndromes, autoimmune diseases, infectious diseases, cardiovascular diseases, ischemic diseases, cancer, and diabetes. 88. The above IL-1R-I-related disorders include: Familial Mediterranean Fever (FMF); Cryopyrin-Associated Periodic Syndrome (CAPS); TNF Receptor-Associated Periodic Syndrome (TRAPS); Hyper IgD Syndrome (HIDS); Periodic Fever; Aphthous Stomatitis; Pharyngitis; Adenitis (PFAPA); Rheumatoid Arthritis (RA), Juvenile RA, Juvenile Idiopathic Arthritis, Systemic Juvenile Idiopathic Arthritis; Adult-Onset Still's Disease; Schnitzler's Syndrome; Muckle-Wells Syndrome; Macrophage Activation Syndrome; Behçet's Disease; Uveitis; Acne Vulgaris; Pyoderma Gangrenosum; Gout; Type 2 Glycolytics; Diabetic disease, incipient diabetes mellitus; dry eye syndrome; sweat gland abscess; neutrophilic dermatoses, especially histiocytic panniculitis, Weber-Christian disease, and neutrophilic panniculitis; cardiovascular disease, myocardial infarction, stroke; liver failure, renal failure; acute lung injury; pseudogout, calcium pyrophosphate deposition disease, chondrocalcinosis, IL-1 receptor antagonist deficiency (DIRA), IL-36 receptor antagonist deficiency (DITRA), ADAM2 deficiency (DADA2), septic arthritis, pyoderma gangrenosum-acne (PAPA) syndrome, pyoderma gangrenosum-acne-suppurative hidradenitis ( PASH syndrome, PAPA·Hidradenitis Suppurativa (PAPASH) syndrome, Autoinflammatory syndrome with lymphedema (AISLE), Autoinflammatory syndrome with phospholipase C-gamma-2 mutations, NALP12-associated periodic syndrome (NAPS12), Mevalonate kinase deficiency (MKD), Psoriatic arthritis, Reactive arthritis, Ankylosing spondylitis, Hemochromatosis-related arthritis, Periarticular calcification, Osteoarthritis, Inflammatory osteoarthritis, Osteoarthritis, Pustular psoriasis (Generalized pustular psoriasis (GPP), Pustulosis pallidum plantaris (PPP), Acrodermatitis continua of Alobeau (AC) H), etc.); Blau syndrome; Sweet syndrome; various vasculitis (such as giant cell arteritis (GCA), polymyalgia rheumatica (PMR), Takayasu's arteritis, Kawasaki disease, urticarial vasculitis, and Henoch-Schönlein purpura (HSP)); neutrophilic urticaria and idiopathic cold urticaria; lichen planus; polymyositis, dermatomyositis, juvenile dermatomyositis, and inclusion body myositis; various stages of myeloma (such as smoldering myeloma (SMM), asymptomatic myeloma, Waldenström's macroglobulinemia, and multiple myeloma); neoplastic cachexia; solid tumor growth;Neonatal disorders (bronchopulmonary dysplasia (prophylaxis), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP), cerebral palsy due to perinatal cerebral ischemia, and infantile respiratory distress syndrome (IRDS)); Whipple's disease; traumatic brain injury; refractory epilepsy; systemic inflammatory response syndrome (SIRS); cutaneous lupus; Jessner-Kanoff disease; amyotrophic lateral sclerosis; systemic sclerosis (scleroderma); septic shock; acute pancreatitis; chronic relapsing multifocal osteomyelitis, nonbacterial osteitis (NBO), synovitis, acne, pustulosis, bone 88. The IL-1R-I binding polypeptide, fusion protein, conjugate, or composition for use according to item 86 or 87, selected from the group consisting of: inflammatory bowel syndrome, inflammatory bowel disease, chronic granulomatous disease (CGD), Castleman's disease, heart failure, diastolic heart failure, antisynthase syndrome, acute ACL injury, acute hemorrhagic leukoencephalitis, AA amyloidosis, DiGeorge syndrome, generalized fatigue, chronic fatigue syndrome (CFS), Gulf War illness (GWI), and narcolepsy. 89. The IL-1R-I binding polypeptide, fusion protein, conjugate, or composition for use according to item 87, wherein the IL-1R-I associated disorder is cancer (such as a cancer selected from the group consisting of multiple myeloma, colon cancer, breast cancer, lung cancer, head and neck cancer, melanoma, and prostate cancer). 90. The IL-1R-I binding polypeptide, fusion protein, conjugate or composition for use according to any one of items 82 to 89, wherein said IL-1R-I binding polypeptide, fusion protein, conjugate or composition is administered to a subject in need thereof repeatedly within 24 hours from disease onset (such as at least twice within 24 hours from disease onset, such as at least three times within 24 hours from disease onset, such as continuously for 24 hours from disease onset). 91. A fusion protein, conjugate or composition for use according to any one of items 82 to 84, 86 to 89, wherein the fusion protein or conjugate is a fusion protein or conjugate according to any one of items 58 to 67 and the composition is a composition according to item 81. 92. The fusion protein, conjugate, or composition for use according to item 91, wherein the fusion protein, conjugate, or composition is administered once a week to a subject in need thereof. 93. A method for treating an IL-1R-I associated disorder, comprising administering to a subject in need thereof an effective amount of an IL-1R-I binding polypeptide, fusion protein, or conjugate according to any one of items 1 to 74 or a composition according to any one of items 79 to 81. 94. The method according to item 93, wherein the IL-1R-I associated disorder is selected from the group consisting of inflammatory diseases, autoinflammatory syndromes, autoimmune diseases, infectious diseases, cardiovascular diseases, ischemic diseases, cancer, and diabetes. 95. The method according to item 93 or 94, wherein said IL-1R-I associated disorder is selected from the group defined in item 88. 96. The method according to item 94, wherein the IL-1R-I associated disorder is cancer (such as a cancer selected from the group consisting of multiple myeloma, colon cancer, breast cancer, lung cancer, head and neck cancer, melanoma, and prostate cancer). 97. The method according to any one of items 93 to 96, wherein the IL-1R-I binding polypeptide, fusion protein, or conjugate, or composition is administered to a subject in need thereof repeatedly within 24 hours from disease onset (such as at least twice within 24 hours from disease onset, such as at least three times within 24 hours from disease onset, such as continuously for 24 hours from disease onset). 98. The method according to any one of items 93 to 96, wherein the fusion protein or conjugate is a fusion protein or conjugate according to any one of items 59 to 67 and the composition is a composition according to item 81. 99. The method according to item 97, wherein the fusion protein, conjugate, or composition is administered to a subject in need thereof once a week. The present invention can be summarized as follows. 1. An IL-1R-I binding polypeptide comprising an IL-1R-I binding motif BM, said motif comprising: i)EX 2 X 3 X 4 X 5 X 6 X 7 EIX 10 X 11 LPNLX 16 RX 18 QYX 21 AFIX 25 X 26 LX 28 D (SEQ ID NO: 1686) (Independently of each other, X 2 is selected from A, I, L, T, and V; X 3 is selected from E and Y; X 4 is selected from A, E, I, K, Q, R, T, V, and Y; X 5 is selected from I and V; X 6 is selected from Q and Y; X 7 is selected from F and M; X 10 is selected from F and Y; X 11 is selected from A, D, E, F, G, H, K, L, Q, R, S, T, V, and Y; X 16 is selected from N and T; X 18 is selected from K and R; X 21 is selected from T and V; X 25 is selected from I and R; X 26 is selected from K and S, and X 28 is selected from F and L; and ii) an amino acid sequence having at least 93% identity to the sequence defined in i); IL-1R-I binding polypeptide, comprising an amino acid sequence selected from: 2. An IL-1R-I binding polypeptide comprising an IL-1R-I binding motif BM, said motif comprising: i)EX 2 X 3 X 4 X 5 X 6 X 7 EIX 10 X 11 LPNLX 16 RX 18 QYX 21 AFIX 25 X 26 LX 28 D (SEQ ID NO: 1686) (Independently of each other, X 2 is selected from A, E, F, H, I, L, Q, S, T, and V; X 3 is selected from A, D, E, F, H, I, K, L, Q, R, S, T, V, W, and Y; X 4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 5 is selected from A, I, and V; X 6 is selected from H, Q, R, and Y; X 7 is selected from A, D, E, F, G, H, I, L, M, Q, S, V, W, and Y; X 10 is selected from F and Y; X 11 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, and Y; X 16 is selected from N and T; X 18 is selected from K and R; X 21 is selected from T and V; X 25 is selected from I and R; X 26 is selected from K and S, and X 28 is selected from F and L; and ii) X 5 an amino acid sequence having at least 96% identity to the sequence defined in i), provided that IL-1R-I binding polypeptide, comprising an amino acid sequence selected from: 3. The sequence i) satisfies the following 10 conditions I to X: IX 3 is E; II.X 5 is selected from I and V; III.X 6 is selected from Q and Y; IV.X 7 is selected from F and M; VX 10 is selected from F and Y; VI.X 18 is selected from K and R; VII.X 21 is T; VIII.X 25 is R; IX.X 26 is selected from K and S; and XX 28 is selected from F and L 3. An IL-1R-I-binding polypeptide according to item 1 or 2, which satisfies at least five (such as at least six, at least seven, at least eight, at least nine, or all) of the above. 4. An IL-1R-I-binding polypeptide according to any of the preceding items, wherein sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 1632 and 1679 (such as the group consisting of SEQ ID NOs: 20 to 1632 and 1679). 5. An IL-1R-I-binding polypeptide according to item 4, wherein sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1206 to 1632 and 1679 (such as the group consisting of SEQ ID NOs: 1210 to 1632 and 1679). 6. The IL-1R-I-binding polypeptide according to item 5, wherein the sequence i) corresponds to a sequence from positions 8 to 36 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594, and 1679 (such as the group consisting of SEQ ID NOs: 1252, 1328, 1435, and 1679). 7. An IL-1R-I binding polypeptide according to any one of the preceding items, wherein the IL-1R-I binding motif forms part of a three-helix bundle protein domain, and preferably the IL-1R-I binding motif essentially forms part of two helices together with an interconnecting loop within the three-helix bundle protein domain. 8. An IL-1R-I binding polypeptide according to item 7, wherein the three-helix bundle protein domain is selected from bacterial receptor domains, preferably the domain of protein A from Staphylococcus aureus or a derivative thereof. 9.xix) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO: 1721), where [BM] is an IL-1R-I binding motif as defined in any one of the preceding items; and xx) an amino acid sequence having at least 89% identity to the sequence defined in xix) in the sequence adjacent to the BM 4. The IL-1R-I binding polypeptide of any one of the preceding claims, comprising an amino acid sequence selected from: 10.xxi) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO: 1709), where [BM] is an IL-1R-I binding motif as defined in any one of items 1 to 8; and xxii) an amino acid sequence having at least 89% identity to the sequence defined in xxi) in the sequence adjacent to the BM 9. The IL-1R-I-binding polypeptide according to any one of items 1 to 8, comprising an amino acid sequence selected from: 11. An IL-1R-I-binding polypeptide according to item 9 or 10, wherein sequence xix) or xxi) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 1632, 1667 to 1668, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 20 to 1632, 1667 to 1668, and 1670 to 1679). 12. The IL-1R-I-binding polypeptide according to item 11, wherein xix) or xxi) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1206 to 1632, 1667 to 1668, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 1210 to 1632, 1667 to 1668, and 1670 to 1679). 13. An IL-1R-I-binding polypeptide according to item 12, wherein sequence xix) or xxi) corresponds to a sequence at positions 1 to 58 in a sequence selected from the group consisting of SEQ ID NOs: 1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594, and 1670 to 1679 (such as the group consisting of SEQ ID NOs: 1252, 1328, 1435, 1672, 1675 to 1676, and 1679). 14. An IL-1R-I binding polypeptide according to any of the preceding items, which is capable of blocking the interaction of IL-1R-I with IL-1 cytokines (such as the interaction of IL-1R-I with IL-1α and / or IL-1β). 15. K of the interaction D The maximum value is 1×10 -6 M (maximum 1 × 10 -7 M, etc., up to 1×10 -8 M, etc., up to 1×10 -9 M, etc., up to 9 x 10 -10 IL-1R-I binding polypeptide according to any of the preceding items, which is capable of binding to IL-1R-I such that the IL-1R-I is 16. An IL-1R-I binding polypeptide according to item 14 or 15, wherein the IL-1R-I is human IL-1R-I or cynomolgus IL-1R-I (such as human IL-1R-I). 17. An IL-1R-I binding polypeptide according to any of the preceding items in the form of a multimer comprising at least two IL-1R-I binding polypeptide monomer units, the amino acid sequences of which may be identical or different. 18.- A first part consisting of an IL-1R-I binding polypeptide according to any of the preceding items; and a second portion consisting of a polypeptide having a desired biological activity A fusion protein or conjugate comprising: 19. The fusion protein or conjugate according to item 18, wherein the desired biological activity is a therapeutic activity. 20. The fusion protein or conjugate according to item 18, wherein the desired biological activity is binding activity. 21. The fusion protein or conjugate according to item 18, wherein the desired biological activity is a binding activity that modifies the tissue distribution of the fusion protein or conjugate. 22. The fusion protein or conjugate according to item 18, wherein the desired biological activity is an in vivo half-life enhancing activity, such that the second moiety increases the in vivo half-life of the fusion protein or conjugate. 23. The fusion protein or conjugate according to item 18, wherein the second moiety is selected from the group consisting of the albumin binding domain of streptococcal protein G or a derivative thereof; serum albumin; the Fc portion of an antibody, and transferrin. 24. The fusion protein or conjugate according to item 23, wherein the second portion is an Fc portion of an antibody (such as IgG1 Fc or IgG4 Fc). 25. The fusion protein or conjugate according to item 24, wherein the Fc is a mutated Fc that exhibits improved affinity for FcRn and / or a reduced effector response compared to the unmutated form of the Fc. 26. The fusion protein or conjugate according to any one of items 18 to 25, comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1639 to 1658 and 1734 to 1737 (such as the group consisting of SEQ ID NOs: 1648 to 1654 and 1734 to 1737). 27. A polynucleotide encoding the polypeptide or fusion protein according to any one of items 1 to 26. 28. An expression vector comprising the polynucleotide according to item 27. 29. A host cell comprising the expression vector according to item 28. 30. A composition comprising an IL-1R-I binding polypeptide, fusion protein, or conjugate according to any one of items 1 to 26, and at least one pharma- ceutically acceptable excipient or carrier. 31. The composition according to item 30, further comprising at least one additional active agent (such as an agent selected from an immune response modifying agent and an anti-cancer agent). 32. The composition according to item 30 or 31, wherein the fusion protein is a fusion protein according to any one of items 22 to 26. 33. An IL-1R-I binding polypeptide, fusion protein or conjugate according to any one of items 1 to 26 or a composition according to any one of items 30 to 32 for use as a medicament, diagnostic agent and / or prognostic agent. 34. An IL-1R-I binding polypeptide, fusion protein, conjugate or composition according to item 33 for use as a medicament. 35. An IL-1R-I binding polypeptide, fusion protein, conjugate or composition according to any one of items 33 to 34 for use in the treatment, prognosis or diagnosis of an IL-1R-I associated disorder. 36. The IL-1R-I binding polypeptide, fusion protein, conjugate or composition for use according to item 35, wherein the IL-1R-I associated disorder is selected from the group consisting of inflammatory diseases, autoinflammatory syndromes, autoimmune diseases, infectious diseases, cardiovascular diseases, ischemic diseases, cancer and diabetes. 37. The IL-1R-I binding polypeptide, fusion protein, conjugate or composition for use according to any one of items 33 to 36, wherein the IL-1R-I binding polypeptide, fusion protein, conjugate or composition is administered to a subject in need thereof repeatedly within 24 hours from disease onset (such as at least twice within 24 hours from disease onset, such as at least three times within 24 hours from disease onset, such as continuously for 24 hours from disease onset). 38. The fusion protein, conjugate or composition for use according to any one of items 33 to 37, wherein the fusion protein or conjugate is a fusion protein or conjugate according to any one of items 22 to 26 and the composition is a composition according to item 32. 39. The fusion protein, conjugate or composition for use according to item 38, wherein the fusion protein, conjugate or composition is administered once a week to a subject in need thereof.

Claims

[Claim 1] IL-1R-I binding polypeptide comprising an IL-1R-I binding motif BM, said motif comprising: i)︥ ２ ︸ ３ ︸ ４ ︸ ５ ︸ ６ ︸ ７ ︥︊ １０ ︸ １１ phuff. １６ ︹ １８ ﹁︸ ２１ 1.|.︸ ２５ ︸ ２６ ︹ ２８ ........................ (Independently of each other, X ２ is selected from A, I, L, T, and V; X ３ is selected from E and Y; X ４ is selected from A, E, I, K, Q, R, T, V, and Y; X ５ is selected from I and V; X ６ is selected from Q and Y; X ７ is selected from F and M; X １０ is selected from F and Y; X １１ is selected from A, D, E, F, G, H, K, L, Q, R, S, T, V, and Y; X １６ is selected from N and T; X １８ is selected from K and R; X ２１ is selected from T and V; X ２５ is selected from I and R; X ２６ is selected from K and S, and X ２８ is selected from F and L; and ii) an amino acid sequence having at least 93% identity to the sequence defined in i); An IL-1R-I binding polypeptide comprising an amino acid sequence selected from:

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