Patent Application
20210403533
The present disclosure relates to a class of engineered polypeptides having a binding affinity for interleukin-1 receptor type-I (in the following referred to as IL-1R-1). The present disclosure also relates to the use of such an IL-1R-I binding polypeptide as a therapeutic, prognostic and/or diagnostic agent.
The central role of the IL-1 system in the initiation and maintenance of inflammatory response, as well as its role in innate and adaptive branches of the immune system, makes it an attractive target for pharmaceutical intervention (Dinarello, 2009, Annu Rev Immunol, 27:519-50; Sims and Smith, 2010, Nat Rev Immunol.10(2):89-102). Since the IL-1 system in many cases plays the role of initiator and master cytokine in inflammatory response, many aspects of the progression of a pathological immune response can be modulated by such intervention. The biological activity of IL-1p can be pharmacologically inhibited by antibodies that prevent, or modulate its binding to the type IL-1 receptor IL-1R-1.
The biological activity of the IL-1 system can also be modulated by antagonizing the IL-1R. The recombinant form of endogenous IL-1 Ra denoted anakinra (Kineret®) is described in e.g. U.S. Pat. No. 6,599,873, EP 0343684 and EP 0541920. Anakinra has shown clinical efficacy in rheumatoid arthritis, cryopyrin-associated periodic syndromes (CAPS; Koné-Paut and Galeotti, 2014, Expert Rev Clin Immunol, 10(1):7-18), as well as in a large number of 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)).
The IL-1 receptor can also be antagonized by antibodies that bind to IL-1R-I subunit and prevent ligand-mediated IL-1 receptor activation and signaling. Several such antibodies are described in WO 2004/022718, which discloses IL-1R-I antagonists such as 15C4. Other examples of IL-1 receptor antagonizing antibodies binding to the IL-1R-I subunit are described in WO 2010/052505, which discloses IL-1R-I antagonists such as Antibody 9GL. Yet another example of an IL-1 receptor antagonizing antibody, denoted 2D8 is described in WO 2005/023872.
The unpredictable and chronic nature of inflammatory diseases, as well as a high unmet medical need, warrants the development of new modes of treatment. Since tissue penetration rate is negatively associated with the size of the molecule, relatively large antibody molecules inherently have poor tissue distribution and penetration capacity.
Thus, the use of antibodies, or other large molecule drugs, is not always optimal for therapy and there is continued need for provision of agents with a high affinity for the IL-1 receptor.
It is an object of the present disclosure to provide new IL-1R-I binding agents, which could for example be used for therapeutic, prognostic and diagnostic applications.
It is an object of the present disclosure to provide a molecule allowing for efficient therapy targeting various forms of inflammatory, autoinflammatory and autoimmune diseases.
It is furthermore an object of the present disclosure to provide a molecule suitable for prognostic and diagnostic applications.
These and other objects, which are evident to the skilled person from the present disclosure, are met by different aspects of the invention as claimed in the appended claims and as generally disclosed herein.
Thus, in the first aspect of the disclosure, there is provided an IL-1R-I binding polypeptide, comprising an IL-1R-I binding motif BM, which motif consists of an amino acid sequence selected from:
wherein, independently from each other,
The above definition of a class of sequence related, IL-1R-I binding polypeptides is based on a statistical analysis of a number of random polypeptide variants of a parent scaffold, that were selected for their interaction with IL-1R-I in several different selection experiments. The identified IL-1R-I binding motif, or “BM”, corresponds to the target binding region of the parent scaffold, which region constitutes two alpha helices within a three-helical bundle protein domain. In the parent scaffold, the varied amino acid residues of the two BM helices constitute a binding surface for interaction with the constant Fc part of antibodies. In the present disclosure, the random variation of binding surface residues and subsequent selection of variants have replaced the Fc interaction capacity with a capacity for interaction with IL-1R-I.
As the skilled person will realize, the function of any polypeptide, such as the IL-1R-I binding capacity of the polypeptide of the present disclosure, is dependent on the tertiary structure of the polypeptide. It is therefore possible to make minor changes to the sequence of amino acids in a polypeptide without affecting the function thereof. Thus, the disclosure encompasses modified variants of the IL-1R-I binding polypeptide, which have retained IL-1R-I binding characteristics.
In this way, also encompassed by the present disclosure is an IL-1R-I binding polypeptide comprising an amino acid sequence with 96% or greater identity to a polypeptide as defined in i). Thus, certain sequence variants of the sequence defined in i) are encompassed by the above definition under the proviso that within such sequence variants X5 is I or V. For example, it is possible that an amino acid residue belonging to a certain functional grouping of amino acid residues (e.g. hydrophobic, hydrophilic, polar etc) could be exchanged for another amino acid residue from the same functional group.
In some embodiments, such changes may be made in any position of the sequence of the IL-1R-I binding polypeptide as disclosed herein. In other embodiments, such changes may be made only in the non-variable positions, also denoted scaffold amino acid residues. In such cases, changes are not allowed in the variable positions, i.e. positions denoted with an “X” in sequence i).
The term “% identity”, as used throughout the specification, may for example be calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al, 1994, Nucleic Acids Research, 22: 4673-4680). A comparison is made over the window corresponding to the shortest of the aligned sequences. The shortest of the aligned sequences may in some instances 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 correspondences in the target sequence is reported as % identity. As used herein “Xa” and “Xm” are used to indicate amino acids in positions n and m in the sequence i) as defined above, wherein n and m are integers which indicate the position of an amino acid within said sequence as counted from the N-terminal end of said sequence. For example, X3 and X7 indicate the amino acid in position three and seven, respectively, from the N-terminal end of sequence i).
In embodiments according to the first aspect, there are provided polypeptides wherein Xn in sequence i) is independently selected from a group of possible residues according to Table 1. The skilled person will appreciate that Xn may be selected from any one of the listed groups of possible residues and that this selection is independent from the selection of amino acids in Xm, wherein n≠m. Thus, any of the listed possible residues in position Xn in Table 1 may be independently combined with any of the listed possible residues in any other variable position in Table 1.
The skilled person will appreciate that Table 1 is to be read as follows: In one embodiment according to the first aspect, there is provided a polypeptide wherein amino acid residue “Xn” in sequence i) is selected from “Possible residues”. Thus, Table 1 discloses several specific and individualized embodiments of the first aspect of the present disclosure. For example, in one embodiment according to the first aspect, there is provided a polypeptide wherein X4 in sequence i) is selected from A, D, E, I, K, O, S and V, and in another embodiment according to the first aspect, there is provided a polypeptide wherein X4 in sequence i) is selected from A, E, I, K, Q and V. For avoidance of doubt, the listed embodiments may be freely combined in yet other embodiments. For example, one such combined embodiment is a polypeptide in which X4 is selected from A, E, I, K, Q and V, while X6 is selected from H, Q, T and Y, and X1 is selected from H, R and Y.
In one embodiment of the present disclosure there is provided an IL-1R-I binding polypeptide, wherein in i)
In one embodiment of the present disclosure there is provided an IL-1R-I binding polypeptide, wherein in i)
In one embodiment of the present disclosure there is provided an IL-1R-I binding polypeptide, wherein in i)
In another embodiment of the present disclosure there is provided an IL-1R-I binding polypeptide, wherein said IL-1R-I binding motif consists of an amino acid sequence selected from a sequence wherein in i)
In another embodiment of the present disclosure there is provided an IL-1R-I binding polypeptide, wherein in i)
In a more specific embodiment defining a sub-class of IL-1R-I binding polypeptides, sequence i) fulfills at least five of the ten conditions I-X:
In some examples of an IL-1R-I binding polypeptide according to the first aspect, sequence i) fulfils at least six of the ten conditions I-X. More specifically, sequence i) may fulfil at least seven of the ten conditions I-X, such as at least eight of the ten conditions I-X, such as at least nine of the ten conditions I-X, such as all of the ten conditions I-X.
In some embodiments of an IL-1R-I binding polypeptide according to the first aspect, there is provided an IL-1R-I binding polypeptide, wherein X2X3X6 is VEQ or VEY. In some embodiments there is provided an IL-1R-I binding polypeptide, wherein X2X3X6 is IEQ or VEQ. In some embodiments X6X10 is selected from the group consisting of QF, QY, YF and YY. In some embodiments X6X10 is QF. In some embodiments X10X18 is selected from the group consisting of FK, FR, YK and YR. In some embodiments X10X18 is FK or FR. In some embodiments X18X25X28 is KRL or RRL. In some embodiments X5X7 is AM, IM or VM. In some embodiments X5X7 is IM or VM.
In one embodiment, X5 is selected from I and V. As demonstrated in the appended Example 4, the unintentional alteration in position X5 of the BM resulted in the finding of an IL-1R-I binding polypeptide (Z18557, SEQ ID NO:1205) displaying high affinity for IL-1R-I (see Table 11). The exchange of alanine in this position to valine or isoleucine allows for generation of IL-1R-I binding polypeptides with high affinity for IL-1R-I.
As described in detail in the experimental section to follow, the selection of IL-1R-I binding polypeptide variants has led to the identification of a number of individual IL-1R-I binding motif (BM) sequences. These sequences constitute individual embodiments of sequence i) according to this aspect. The sequences of individual IL-1R-I binding motifs correspond to amino acid positions 8-36 of the amino acid sequences listed as SEQ ID NOs 1-1632 and 1679 presented in
In one embodiment sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1205 and 1250-1632.
In one embodiment sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1206-1252 and 1679.
In another embodiment sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1253-1632. In another embodiment sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1253-1441 or the group consisting of SEQ ID NO:1442-1632.
In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO: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, such as from the group consisting of SEQ ID NO:268, 1205, 1252, 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 1571, such as from the group consisting of SEQ ID NO:1252, 1285, 1298, 1308, 1324, 1328, 1330, 1331, 1340, 1361, 1393, 1415, 1420 and 1421.
In one particular embodiment sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594 and 1679. In one embodiment sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1252, 1328, 1435 and 1679, such as the group consisting of SEQ ID NO:1252, 1328 and 1679; the group consisting of SEQ ID NO:1252, 1435, 1679; the group consisting of SEQ ID NO:1252 and 1679; or the group consisting of SEQ ID NO:1328 and 1435. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in sequence selected from the group consisting of SEQ ID NO:1252, 1328 and 1435.
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 a similar structural motif in the original domain. For example, without wishing to be bound by theory, the BM is thought to constitute two of the three helices of a three-helix bundle, and can therefore replace such a two-helix motif within any three-helix bundle. As the skilled person will realize, the replacement of two helices of the three-helix bundle domain by the two BM helices has to be performed so as not to affect the basic structure of the polypeptide. That is, the overall folding of the Ca backbone of the polypeptide according to this embodiment of the invention is substantially the same as that of the three-helix bundle protein domain of which it forms a part, e.g. having the same elements of secondary structure in the same order etc. Thus, a BM according to the present disclosure “forms part” of a three-helix bundle domain if the polypeptide according to this embodiment has the same fold as the original domain, implying that the basic structural properties are shared, those properties e.g. resulting in similar CD spectra. The skilled person is aware of other parameters that are relevant.
In particular embodiments, the IL-1R-I binding motif (BM) thus forms part of a three-helix bundle protein domain. For example, the BM may essentially constitute two alpha helices with an interconnecting loop, within said three-helix bundle protein domain. In particular embodiments, said three-helix bundle protein domain is selected from domains of bacterial receptor proteins. Non-limiting examples of such domains are the five different three-helical domains of Protein A from Staphylococcus aureus, such as domain B, and derivatives thereof. In some embodiments, the three-helical bundle protein domain is a variant of protein Z, which is derived from domain B of staphylococcal Protein A.
In some embodiments where the IL-1R-I binding polypeptide as 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 selected from:
wherein
[BM] is an IL-1R-I binding motif as defined herein;
Xa is selected from A and S;
Xb is selected from N and E;
Xc is selected from A, S and C;
Xd is selected from E, N and S;
Xe is selected from D, E and S;
Xf is selected from A and S; and
In some embodiments, said polypeptides may beneficially exhibit high structural stability, such as resistance to chemical modifications, to changes in physical conditions and to proteolysis, during production and storage, as well as in vivo.
As discussed above, polypeptides comprising minor changes as compared to the above amino acid sequences, which do not largely affect the tertiary structure and the function of the polypeptides, are also within the scope of the present disclosure. Thus, in some embodiments, sequence iv) has at least at least 93%, such as at least 95%, such as at least 97% identity to a sequence defined by iii). In particular embodiments, such minor changes as exemplified above may be present only in the flanking sequence, i.e. the parts of the sequence flanking the BM in iii) above. In other embodiments, minor changes can be present in the BM and/or in the flanking sequences.
In one embodiment, Xa in sequence iii) is A.
In one embodiment, Xa in sequence iii) is S.
In one embodiment, Xb in sequence iii) is N.
In one embodiment, Xb in sequence iii) is E.
In one embodiment, Xc in sequence iii) is A.
In one embodiment, Xc in sequence iii) is S.
In one embodiment, Xc in sequence iii) is C.
In one embodiment, Xd in sequence iii) is E.
In one embodiment, Xd in sequence iii) is N.
In one embodiment, Xd in sequence iii) is S.
In one embodiment, Xe in sequence iii) is D.
In one embodiment, Xe in sequence iii) is E.
In one embodiment, Xe in sequence iii) is S.
In one embodiment, XdXe in sequence iii) is selected from EE, ES, SE and SS.
In one embodiment, XdXe in sequence iii) is ES.
In one embodiment, XdXe in sequence iii) is SE.
In one embodiment, Xf in sequence iii) is A.
In one embodiment, Xf in sequence iii) is S.
In one embodiment, in sequence iii), Xa is A; Xb is N; Xc is A and Xf is A.
In one embodiment, in sequence iii), Xa is A; Xb is N; Xc is C and Xf is A.
In one embodiment, in sequence iii), Xa is S; Xb is E; Xc is S and Xf is S.
In one embodiment, in sequence iii), Xa is S; Xb is E; Xc is C and Xf is S.
In one embodiment, in sequence iii), Xa is A; Xb is N; Xc is A; XdXe is ND and Xf is A.
In one embodiment, in sequence iii), Xa is A; Xb is N; Xc is C; XdXe is ND and Xf is A.
In one embodiment, in sequence iii), Xa is S; Xb is E; Xc is S; XdXe is ND and Xf is S.
In one embodiment, in sequence iii), Xa is S; Xb is E; Xc is C; XdXe is ND and Xf is S.
In one embodiment, in sequence iii), Xa is A; Xb is N; Xc is A; XdXe is SE and Xf is A.
In one embodiment, in sequence iii), Xa is A; Xb is N; Xc is C; XdXe is SE and Xf is A.
In one embodiment, in sequence iii), Xa is S; Xb is E; Xc is S; XdXe is SE and Xf is S.
In one embodiment, in sequence iii), Xa is S; Xb is E; Xc is C; XdXe is SE and Xf is S.
In one embodiment, in sequence iii), Xa is A; Xb is N; Xc is A; XdXe is ES and Xf is A.
In one embodiment, in sequence iii), Xa is A; Xb is N; Xc is C; XdXe is ES and Xf is A.
In one embodiment, in sequence iii), Xa is S; Xb is E; Xc is S; XdXe is ES and Xf is S.
In one embodiment, in sequence iii), Xa is S; Xb is E; Xc is C; XdXe is ES and Xf is S.
In one embodiment, sequence iii) or iv) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-1638 and 1667-1670.
In yet a further embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-1638, 1667-1668 and 1670-1679; such as the group consisting of SEQ ID NO:20-1638 and 1670-1679. In one embodiment sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1206-1638 and 1670-1679, such as the group consisting of SEQ ID NO:1206-1632 and 1670-1679, such as the group consisting of SEQ ID NO:1210-1638 and 1670-1679, such as the group consisting of SEQ ID NO:1210-1632 and 1670-1679.
In one embodiment sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1205, 1250-1632 and 1670-1679.
In one embodiment sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1206-1252, 1672 and 1679.
In another embodiment sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1253-1632, 1670-1671 and 1673-1678. In another embodiment sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1253-1441, 1670-1671 and 1674-1678 or the group consisting of SEQ ID NO:1442-1632 and 1673.
In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO: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, 1662 and 1670-1679, such as from the group consisting of SEQ ID NO:268, 1205, 1252, 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, 1571, 1670-1672, 1674, and 1676-1679, such as from the group consisting of SEQ ID NO:1252, 1285, 1298, 1308, 1324, 1328, 1330, 1331, 1340, 1361, 1393, 1415, 1420, 1421, 1670-1672, 1674, 1676-1677 and 1679.
In one particular embodiment sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594 and 1670-1679. In one embodiment sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1252, 1328, 1435, 1672, 1675-1676 and 1679 such as the group consisting of SEQ ID NO:1252, 1328 and 1679; the group consisting of SEQ ID NO:1252, 1435 and 1679; or the group consisting of SEQ ID NO:1328 and 1435. In one embodiment, sequence i) corresponds to the sequence from position 7 to position 55 in sequence selected from the group consisting of SEQ ID NO:1252, 1328 and 1435.
In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1672, 1675-1676 and 1679, such as the group consisting of SEQ ID NO:1672 and 1675-1676.
Also, in a further embodiment, there is provided an IL-1R-I binding polypeptide, which comprises an amino acid sequence selected from:
wherein [BMod] is an IL-1R-I binding module as defined herein; and
vi) an amino acid sequence which has at least 90% identity to a sequence defined in v).
Alternatively, there is provided an IL-1R-I binding polypeptide, which comprises an amino acid sequence selected from:
wherein [BMod] is an IL-1R-I binding module as defined herein; and
viii) an amino acid sequence which has at least 90% identity to a sequence defined in vii).
In one embodiment there is provided an IL-1R-I binding polypeptide, which comprises an amino acid selected from the group consisting of
wherein [BM] is an IL-1R-I binding motif as defined above and Xc is selected from A and C; and
x) an amino acid sequence which has at least 90% identity to a sequence defined in ix).
In another embodiment, there is provided an IL-1R-I binding polypeptide, which comprises an amino acid selected from the group consisting of
wherein [BM] is an IL-1R-I binding motif as defined above and Xc is selected from A, S and C; and
xii) an amino acid sequence which has at least 90% identity to a sequence defined in xi).
In another embodiment, there is provided an IL-1R-I binding polypeptide, which comprises an amino acid selected from the group consisting of
wherein [BM] is an IL-1R-I binding motif as defined above and Xc is selected from A, S and C;
xiv) an amino acid sequence which has at least 90% identity to a sequence defined in xiii).
In yet another embodiment, there is provided an IL-1R-I binding polypeptide, which comprises an amino acid selected from the group consisting of
wherein [BM] is an IL-1R-I binding motif as defined above and Xc is selected from A, S and C, Xd is selected from E, N and S, and Xe is selected from D, E and S;
xvi) and an amino acid sequence which has at least 90% identity to a sequence defined in xv).
In yet another embodiment, there is provided an IL-1R-I binding polypeptide, which comprises an amino acid selected from the group consisting of
wherein [BM] is an IL-1R-I binding motif as defined above and Xc is selected from A, S and C, Xd is selected from E, N and S, and Xe is selected from D, E and S;
xviii) and an amino acid sequence which has at least 90% identity to a sequence defined in xvii).
As discussed above, polypeptides comprising minor changes as compared to the above amino acid sequences, which do not largely affect the tertiary structure and the function of the polypeptide, also fall within the scope of the present disclosure. Thus, in some embodiments, sequence vi), viii), x), xii), xiv), xvi) or xviii) may for example 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 a sequence defined by v), vii), ix), xi), xiii), xv) and xvii), respectively. In particular embodiments, such minor changes as exemplified above can be present only in flanking sequences, i.e. the parts of the sequences flanking the BM in v), vii), ix), xi), xiii), xv) and xviii). In other embodiments, minor changes can be present in the BM and/or in the flanking sequences.
In some embodiments, the IL-1R-I binding motif may form part of a polypeptide comprising an amino acid sequence selected from:
wherein [BM] is an IL-1R-I binding motif as defined herein.
In one embodiment, said IL-1R-I binding polypeptide comprises an amino acid sequence which has at least 89% identity to any one of the sequences defined above.
In one embodiment, the IL-1R-I binding polypeptide comprises an amino acid sequence selected from:
wherein [BM] is an IL-1R-I binding motif as defined herein; and
xx) an amino acid sequence which has at least 89% identity to the sequence defined in xix).
In one embodiment, the IL-1R-I binding polypeptide comprises an amino acid sequence selected from:
wherein [BM] is an IL-1R-I binding motif as defined herein; and
xxii) an amino acid sequence which has at least 89% identity to the sequence defined in xxi).
In one particular embodiment, said IL-1R-I binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1667-1668 and 1670-1679.
In one embodiment, the IL-1R-I binding polypeptide comprises an amino acid sequence selected from:
xxiii) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO:1711) wherein [BM] is an IL-1R-I binding motif as defined herein; and
xxiv) an amino acid sequence which has at least 89% identity to the sequence defined in xxiii).
Again, polypeptides comprising minor changes as compared to the above amino acid sequences, which do not largely affect the tertiary structure and the function of the polypeptide, also fall within the scope of the present disclosure. Thus, in some embodiments, sequence xx), xxii) or xxiv) may for example 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 a sequence defined by xix), xxi) or xxiii) respectively. Once again, in particular embodiments such minor changes as exemplified above can be present only in the sequences flanking the BM as set out in xix), xxi) or xxiii). In other embodiments, minor changes can be present in the BM and/or in the flanking sequences.
Sequence xix)-xxiv) in such IL-1R-I binding polypeptide may be selected from the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-1638, 1667-1668 and 1670-1679 as presented in
In one embodiment of the IL-1R-I binding polypeptide according to this aspect, sequence xix), xxi) or xxiii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-1632, 1667-1668 and 1670-1679; such as the group consisting of SEQ ID NO:20-1638, 1667-1668 and 1670-1679. In one embodiment sequence xix), xxi) or xxiii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1206-1632, 1667-1668 and 1670-1679, such as the group consisting of SEQ ID NO:1210-1632, 1667-1668 and 1670-1679.
In one embodiment sequence xvii), xix) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1205, 1250-1632 and 1670-1679.
In one embodiment sequence xix), xxi) or xxiii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1206-1252, 1672 and 1679.
In another embodiment sequence xix), xxi) or xxiii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1253-1632, 1670-1671 and 1673-1678. In another embodiment sequence xix), xxi) or xxiii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1253-1441, 1670-1671 and 1674-1678 or the group consisting of 1442-1632 and 1673.
In one embodiment sequence xix), xxi) or xxiii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO: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, 1662 and 1670-1679, such as from the group consisting of SEQ ID NO:268, 1205, 1252, 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, 1571, 1670-1672, 1674 and 1676-1679, such as from the group consisting of SEQ ID NO:1252, 1285, 1298, 1308, 1324, 1328, 1330, 1331, 1340, 1361, 1393, 1415, 1420, 1421, 1670-1672, 1674, 1676-1677 and 1679.
In one particular embodiment sequence xix), xxi) or xxiii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594 and 1670-1679. In one embodiment sequence xix), xxi) or xxiii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1252, 1328, 1435 1672, 1675-1676 and 1679, such as the group consisting of SEQ ID NO:1252, 1328 and 1679; the group consisting of SEQ ID NO:1252, 1435 and 1679; or the group consisting of SEQ ID NO:1328 and 1435. In one embodiment, sequence xix), xxi) or xxiii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1252, 1328 and 1435.
In one embodiment, sequence xix), xxi) or xxiii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1672, 1675-1676 and 1679, such as from the group consisting of SEQ ID NO:1672, 1675 and 1676.
In one embodiment, said IL-1R-I binding polypeptide has a melting temperature of above 45° C.
Binding of a polypeptide as defined herein to IL-1R-I may interfere with signaling 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 which is capable of blocking IL-1R-I dependent signaling.
The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a substance for inhibiting a specific quantifiable biological or biochemical function. This quantitative measure indicates how much of a particular substance is needed to inhibit a specific biological function by 50% and is commonly used in the art. In one particular embodiment, there is provided an IL-1R-I binding polypeptide as defined herein capable of blocking IL-1R-I signaling such that the half maximal inhibitory concentration (IC50) of the blocking is at most 1×10−7 M, such as at most 5×10−8 M, such as at most 2×10−8 M, such as at most 1×10−8 M, such as at most 5×10−9 M, such as at most 2×10−9 M, such as at most 1×10−9 M, such as at most 5×10−10 M, such as at most 4×10−10 M, such as at most 3×10−10 M. In one embodiment, the half maximal inhibitory concentration (IC50) of the blocking is between 1×10−15 M and 1×10−7 M, such as between 1×10−15 M and 1×10−8 M, such as between 1×10−15 M and 1×10−9 M, such as between 1×10−15 M and 1×10−10 M, such as between 1×10−15 M and 1×10−11 M, such as between 1×10−15 M and 1×10−12 M.
The terms “IL-1R-I binding” and “binding affinity for IL-1R-I” as used in this specification refer to a property of a polypeptide which may be tested for example by ELISA, Biolayer Interferometry (BLI) or the use of surface plasmon resonance (SPR) technology. For example as described in the examples below, IL-1R-I binding affinity may be tested in an experiment in which samples of the polypeptide are captured on antibody-coated ELISA plates and biotinylated IL-1R-I is added followed by streptavidin-conjugated HRP. TMB substrate is added and the absorbance at 450 nm is measured using a multi-well plate reader, such as Victor3 (Perkin Elmer). The skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide for IL-1R-I. If a quantitative measure is desired, for example to determine the EC50 value (the half maximal effective concentration) for the interaction, ELISA may also be used. The response of the polypeptide against a dilution series of biotinylated IL-1R-I is measured using ELISA as described above. The skilled person may then interpret the results obtained by such experiments, and EC50 values may be calculated from the results using for example GraphPad Prism 5 and non-linear regression.
IL-1R-I binding affinity may also be tested in an experiment in which IL-1R-I, or a fragment thereof, is immobilized on a sensor chip of a surface plasmon resonance (SPR) instrument, and the sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on a sensor chip of the instrument, and a sample containing IL-1R-I, or a fragment thereof, is passed over the chip. The skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide for IL-1R-I. If a quantitative measure is desired, for example to determine a KD value for the interaction, surface plasmon resonance methods may also be used. Binding values may for example be defined in a Biacore (GE Healthcare) or ProteOn XPR 36 (Bio-Rad) instrument. IL-1R-I is suitably immobilized on a 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. KD values may then be calculated from the results using for example the 1:1 Langmuir binding model of the BIAevaluation 4.1 software, or other suitable software, provided by the instrument manufacturer.
Thus, in one embodiment, there is provided an IL-1R-I binding polypeptide as defined herein, which is capable of binding to IL-1R-I such that the EC50 value of the interaction is at most 1×10−7 M, such as at most 5×10−8 M, such as at most 1×10−8 M, such as at most 5×10−9 M, such as at most 1×10−9 M, such as at most 5×10−10 M, such as at most 4×10−10 M, such as at most 3×10−10 M, such as at most 2×10−10 M. In one embodiment, the EC50 value of the interaction is between 1×10−15 M and 1×10−7 M, such as between 1×10−15 M and 1×10−8 M, such as between 1×10−15 M and 1×10−9 M, such as between 1×10−15 M and 5×10−10 M, such as between 1×10−15 M and 1×10−10 M, such as between 1×10−15 M and 5×10−11 M, such as between 1×10−15 M and 1×10−11 M, such as between 1×10−15 M and 5×10−12 M, such as between 1×10−15 M and 1×10−12 M.
In one particular embodiment, there is provided an IL-1R-I binding polypeptide which is capable of binding to IL-1R-I such that the KD value of the interaction with IL-1R-I is at most 1×10−6 M, such as at most 1×10−7 M, such as at most 5×10−8 M, such as at most 4×10−8 M, such as at most 3×10−8 M, such as at most 2×10−8 M, such as at most 1×10−8 M, such as at most 7×10−9 M, such as at most 5×10−9 M, such as at most 4×10−9 M, such as at most 3×10−9 M, such as at most 2×10−9 M, such as at most 1×10−9 M, such as at most 9×10−10 M, such as at most 7×10−10 M, such as at most 5×10−10 M. In one embodiment, the KD value of the interaction with IL-1R-I is between 1×10−15 M and 1×10−6 M, between 1×10−15 M and 1×10−7 M, such as between 1×10−15 M and 1×10−8 M, such as between 1×10−15 M and 1×10−9 M, such as between 1×10−15 M and 1×10−10 M, such as between 1×10−15 M and 1×10−11 M, such as between 1×10−15 M and 1×10−12 M.
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.
The 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.
The skilled person will understand that various modifications and/or additions can be made to an IL-1R-I binding polypeptide according to any aspect disclosed herein in order to tailor the polypeptide to a specific application without departing from the scope of the present disclosure.
For example, in one embodiment there is provided an IL-1R-I binding polypeptide as described herein, which polypeptide has been extended by and/or comprises additional amino acids at the C terminus and/or N terminus. Such a polypeptide should be understood as a polypeptide having one or more additional amino acid residues at the very first and/or the very last position in the polypeptide chain. Thus, an IL-1R-I binding polypeptide may comprise any suitable number of additional amino acid residues, for example at least one additional amino acid residue. Each additional amino acid residue may individually or collectively be added in order to, for example, improve production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide. Such additional amino acid residues may comprise one or more amino acid residues added for the purpose of chemical coupling. One example of this is the addition of a cysteine residue. Additional amino acid residues may also provide a “tag” for purification or detection of the polypeptide, such as a His6 tag, a (HisGlu)3 tag (“HEHEHE” tag), a “myc” (c-myc) tag or a “FLAG” tag for interaction with antibodies specific to the tag or for immobilized metal affinity chromatography (IMAC) in the case of a His6-tag.
The further amino acids as discussed above may be coupled to the IL-1R-I binding polypeptide by means of chemical conjugation (using known organic chemistry methods) or by any other means, such as expression of the IL-1R-I binding polypeptide as a fusion protein or joined in any other fashion, either directly or via a linker, for example an amino acid linker.
The further amino acids as discussed above may for example comprise one or more polypeptide domain(s). A further polypeptide domain may provide the IL-1R-I binding polypeptide with another function, such as for example yet another binding function, an enzymatic function, a toxic function, a fluorescent signaling function or combinations thereof.
A further polypeptide domain may moreover provide another IL-1R-I binding moiety with the same IL-1R-I binding function. Thus, in a further embodiment, there is provided an IL-1R-I binding polypeptide in a multimeric form. Said multimer is understood to comprise at least two IL-1R-I binding polypeptides as disclosed herein as monomer units, the amino acid sequences of which may be the same or different. Multimeric forms of the polypeptides may comprise a suitable number of domains, each having an IL-1R-I binding motif, and each forming a monomer within the multimer. These domains may have the same amino acid sequence, but alternatively, they may have different amino acid sequences. In other words, the IL-1R-I binding polypeptide of the invention may form homo- or heteromultimers, for example homo- or heterodimers. In one embodiment, there is provided an IL-1R-I binding polypeptide, wherein said monomeric units are covalently coupled together. In one embodiment, there is provided an IL-1R-I binding polypeptide, wherein said monomeric units are non-covalently coupled together. In another embodiment, said IL-1R-I binding polypeptide monomer units are expressed as a fusion protein. In one embodiment, there is provided an IL-1R-I binding polypeptide in dimeric form. One example of an IL-1R-I binding polypeptide which will form a disulphide bonded dimer after production is the polypeptide having the sequence set out in SEQ ID NO:1658. The hinge region of IgG1 Fc (disclosed in
Additionally, “heterogenic” fusion polypeptides or proteins, or conjugates, in which an IL-1R-I binding polypeptide as described herein, or a multimer thereof, constitutes a first domain, or first moiety, and the second and further moieties have other functions than binding to IL-1R-1, are also contemplated and fall within the ambit of the present disclosure. The second and further moiety/moieties of the fusion polypeptide or conjugate in such a protein suitably also have a desired biological activity.
Thus, in a second aspect of the present disclosure, there is provided a fusion protein or a conjugate, comprising a first moiety consisting of an IL-1R-1 binding polypeptide according to the first aspect, and a second moiety consisting of a polypeptide having a desired biological activity. In another embodiment, said fusion protein or conjugate may additionally comprise further moieties, comprising desired biological activities that can be either the same or different from the biological activity of the second moiety.
Thus, in one embodiment, said IL-1R-I binding polypeptide comprises an IL-1R-I binding motif BM as set out in sequence i) or ii); a binding module BMod as set out in sequence iii) or iv), or an amino acid sequence as set out in any one of sequences v)-xxiv). In particular, said IL-1R-I binding motifs may correspond to an amino acid sequence from position 8 to 36; said binding modules may correspond to an amino acid sequence from position 7 to position 55; and said sequence xix), xxi), or xxiii) may be selected from the group consisting of sequences selected from SEQ ID NO:s shown in
Non-limiting examples of a desired biological activity comprise a therapeutic activity, a binding activity, and an in vivo half-life increasing activity.
In one embodiment, said desired biological activity is an in vivo half-life increasing activity such that said second moiety increases in vivo half-life of the fusion protein or conjugate.
An in vivo half-life increasing activity should in this context be understood as an activity which increases the in vivo half-life of the fusion protein or conjugate. The half-life of a first moiety (and possibly additional moieties) is increased by fusion or conjugation to a second moiety having an in vivo half-life increasing activity. It will be appreciated that the corresponding fusion protein or conjugate may comprise a first IL-1R-I binding moiety as described above, a second moiety having an in vivo half-life increasing activity, and optionally one or more additional moieties, such as an additional IL-1R-I binding moiety. For example, said fusion protein or conjugate may comprise two IL-1R-I binding polypeptides and a second moiety which increases in vivo half-life compared to the fusion or conjugate comprising the two IL-1R-I binding polypeptides only. Examples of fusion proteins comprising an IL-1R-I binding polypeptide and a half-life extending moiety have been produced and experimentally tested (Example 10). In one embodiment, said fusion protein or conjugate comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1639-1657.
Half-life extension of the resulting fusion protein or conjugate may be accomplished by a second moiety having an inherently long in vivo half-life. A polypeptide that increases the hydrodynamic size of the fusion protein or conjugate may for example impose an increased half-life upon the fusion protein or conjugate. Alternatively, the second moiety may consist of a natural protein having a long in vivo half-life, such as serum albumin or the Fc portion of an antibody.
In one embodiment, said second moiety comprises one of the albumin binding domain of streptococcal protein G or a derivative thereof; albumin; an Fc portion of an antibody, and transferrin. In one embodiment, said fusion protein or conjugate comprises a sequence selected from the group consisting of SEQ ID NO:1639-1658.
In one embodiment, said 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 may be used as a half-life extending moiety is the Fc part set out in Uniprot, accession number P01861, amino acid residues 99-327. In one embodiment, said fusion protein or conjugate comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1646-1654.
In one embodiment, said Fc portion of an antibody is a mutated Fc which relative to an unmutated form of Fc displays improved affinity to FcRn and/or reduced effector response. Thus, said Fc portion is a variant of a naturally occurring Fc in which one or more mutations have been introduced. For example, said fusion protein or conjugate comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1734-1737.
In another non-limiting example, wherein said second moiety has in vivo half-life increasing activity, said second moiety is transferrin. In particular embodiments of the fusion protein or conjugate disclosed herein, there is provided a polypeptide selected from the group consisting of SEQ ID NO:1657.
In one particular embodiment, the in vivo half-life increasing activity is an albumin binding activity. Non-limiting examples of moieties providing such an albumin binding activity are an albumin binding camelid Ig VHH domain and an albumin binding engineered single Ig VH domain.
In one embodiment, said albumin binding activity is provided by the albumin binding domain of the peptostreptococcal albumin binding (PAB) protein from Finegoldia magna, or a derivative thereof.
In one embodiment, said albumin binding activity is provided by the albumin binding domain of streptococcal protein G, or a derivative thereof. Examples of such an albumin binding domain are polypeptides having an amino acid sequence as set out in SEQ ID NO:1659-1661. Preferably, the albumin binding domain is as set out in SEQ ID NO:1661. In one embodiment, said fusion protein or conjugate comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1639-1645.
In another non-limiting example, said in vivo half-life increasing activity is provided by a polyethylene glycol (PEG) or a hydroxyethyl starch (HES).
For example, said fusion protein or conjugate, comprising at least one further moiety, may comprise [IL-1R-I binding polypeptide]-[half-life increasing moiety]-[moiety with affinity for selected target]. It is to be understood that the three moieties in this example may be arranged in any order from the N- to the C-terminal of the polypeptide.
Another non-limiting example is a binding activity which allow the fusion protein to display a modified tissue distribution compared to the unfused IL-1R-I binding polypeptide. In one embodiment the binding activity is binding to the transferrin receptor. Such a binding activity could infer that the conjugate or fusion protein is transferred across the blood brain barrier and into the brain tissue (Yu et al, 2011, Sci Transl Med 3(84):84ra44). In a preferred embodiment, such binding activity is provided by an antibody fragment.
Another non-limiting example of a binding activity is an activity which acts to block a biological activity.
In one embodiment, there is provided an IL-1R-I binding polypeptide, fusion protein or conjugate wherein the binding activity acts to block a biological activity.
With regard to the description above of fusion proteins or conjugates incorporating an IL-1R-I binding polypeptide according to the disclosure, it is to be noted that the designation of first, second and further moieties is made for clarity reasons to distinguish between an IL-1R-I binding polypeptide or polypeptides according to the invention on the one hand, and moieties 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 designations first and second monomer units are made for clarity reasons to distinguish between said units. Thus, for example, said first moiety (or monomer unit) may without restriction appear at the N-terminal end, in the middle, or at the C-terminal end of the fusion protein or conjugate.
The skilled person is aware that the construction of a fusion protein often involves the use of linkers between the functional moieties to be fused, and there are different kinds of linkers with different properties, such as flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers. Linkers have been used to for example increase stability or improve folding of fusion proteins, to increase expression, improve biological activity, enable targeting and alter pharmacokinetics of fusion proteins. Thus, in one embodiment, the polypeptide according to any aspect disclosed herein further comprises at least one linker, such as at least one linker selected from flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers.
In one embodiment, said linker is arranged between a first moiety consisting of an IL-1R-I binding polypeptide as defined herein and a second moiety consisting of a polypeptide having a desired biological activity. In another embodiment, said linker is arranged within said first moiety. For example, one or more linker(s) may be arranged between monomeric units of the polypeptide as defined herein. In yet another embodiment, linkers may be arranged within said first moiety and between said first moiety and said second moiety.
Flexible linkers are often used in the art when the joined domains require a certain degree of movement or interaction, and may be particularly useful in some embodiments. Such linkers are generally composed of small, non-polar (for example G) or polar (for example S or T) amino acids. Some flexible linkers primarily consist of stretches of G and S residues, for example (GGGGS)p. Adjusting the copy number “p” allows for optimization of linker in order to achieve appropriate separation between the functional moieties or to maintain necessary inter-moiety interaction. Apart from G and S linkers, other flexible linkers are known in the art, such as G and S linkers containing additional amino acid residues, such as T and A, to maintain flexibility, as well as polar amino acid residues to improve solubility. In one embodiment, said linker is a flexible linker comprising at least one amino acid residue(s) selected from the group consisting of glycine, serine and alanine. The skilled person is aware of other suitable linkers.
In one embodiment, said linker is selected from GS, VDSS, VDGS, VEGS, ASGS, (GGGGS)2 (SEQ ID NO:1683), AS(GGGGS)2 (SEQ ID NO:1684) and ((KEAAA)3KELAA)2 (SEQ ID NO:1685). In one particular embodiment, said linker is AS(GGGGS)2 (SEQ ID NO:1684) or ((KEAAA)3KELAA)2 (SEQ ID NO:1685). In one embodiment, said fusion protein or conjugate comprising a linker comprises a polypeptide having an amino acid sequence as set out in SEQ ID NO:1639-1658. In particular, an exemplary fusion protein or conjugate comprising the linker AS(GGGGS)2 is set out in the amino acid sequence of SEQ ID NO:1646-1658, such as SEQ ID NO:1648 and 1657.
In further aspects of the present disclosure, there is provided a polynucleotide encoding an IL-1R-I binding polypeptide or a fusion protein as described herein; an expression vector comprising said polynucleotide; and a host cell comprising said expression vector.
Also encompassed by this disclosure is a method of producing a polypeptide or fusion protein as described above, comprising culturing said host cell under conditions permissive of expression of said polypeptide from its expression vector, and isolating the polypeptide.
The IL-1R-I binding polypeptide of the present disclosure may alternatively be produced by non-biological peptide synthesis using amino acids and/or amino acid derivatives having protected reactive side-chains, the non-biological peptide synthesis comprising
It should be understood that the IL-1R-I binding polypeptide according to the present disclosure may be useful as a therapeutic, diagnostic or prognostic agent in its own right or as a means for targeting other therapeutic or diagnostic agents, with e.g. direct or indirect effects on IL-1R-I. A direct therapeutic effect may for example be accomplished by inhibiting IL-1R-I signaling.
Thus, in another aspect, there is provided a composition comprising an IL-1R-I binding polypeptide, fusion protein or conjugate as described herein and at least one pharmaceutically acceptable excipient or carrier. In one embodiment, said 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 combination are immune response modifying agents and anti-cancer agents as described herein. In one embodiment, said composition comprises a fusion protein or conjugate wherein said desired biological activity is an in vivo half-life increasing activity such that said second moiety increases in vivo half-life of the fusion protein or conjugate. Examples of moieties providing such half-life increasing activities are disclosed elsewhere herein.
Hence, in another aspect of the present disclosure, there is provided an IL-1R-I binding polypeptide, fusion protein, conjugate or composition as described herein for use as a medicament, a prognostic agent or a diagnostic agent. In one embodiment, said IL-1R-I binding polypeptide is provided for use as a medicament.
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 to modulate IL-1R-I function in vivo. As used herein, the term “modulate” refers to changing the activity, such as rendering IL-1R-I function hypomorph, partially inhibiting or fully inhibiting IL-1R-I function.
In one embodiment, there is provided an IL-1R-I binding polypeptide, fusion protein or conjugate or composition as described herein, for use, in the treatment, prognosis or diagnosis of an IL-1R-I related disorder.
As used herein, the term “IL-1R-I related disorder” refers to any disorder, disease or condition in which IL-1R-I plays a regulatory role in the signaling pathway. Thus, in one embodiment, there is provided an IL-1R-I binding polypeptide, fusion protein or conjugate or composition as described herein, for use in the treatment of an IL-1R-I related disorder, such as a disorder selected from the group consisting of inflammatory disease, auto-inflammatory syndrome, autoimmune disease, infectious disease, cardiovascular disease, ischaemic disease, cancer and diabetes. In one embodiment, said IL-1R-I related disorder is selected from the group consisting of inflammatory disease, autoinflammatory syndrome and autoimmune disease.
In one embodiment, said IL-1R-I related disorder is selected from the group consisting of 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 syndrome; Muckle Wells Syndrome; macrophage activation syndrome; Behget's disease; uveitis; acne vulgaris; pyoderma gangrenosum; gout; type 2 diabetes, new-onset diabetes; dry eye syndrome; hidradenitis suppurativa; neutrophilic dermatoses, in particular cytophagic histiocytic panniculitis, Weber-Christian disease, and neutrophilic panniculitis; cardiovascular disease, myocardial infarction, stroke; liver failure, kidney failure; acute lung injury; pseudogout, calcium-pyrophosphate deposition disorder, chondrocalcinosis, deficiency of the IL-1 receptor antagonist (DIRA), deficiency of the IL-36 receptor antagonist (DITRA), ADAM2 deficiency (DADA2), pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome, pyoderma gangrenosum, acne and suppurative hidradenitis (PASH) syndrome, PAPA and suppurative hidradenitis (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, haemochromatosis-related arthritis, periarticular calcinosis, osteoarthritis, inflammatory osteoarthritis, hand osteoarthritis, pustular psoriasis such as generalised pustular psoriasis (GPP), palmoplantar pustulosis (PPP), acrodermatitis continua of Hallopeau (ACH); Blau syndrome; Sweet syndrome; various vasculitides such as giant-cell arteritis (GCA), polymyalgia rheumatica (PMR), Takayasu 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), indolent myeloma, Waldenstrom's macroglobulinemia, and multiple myeloma; tumor-induced cachexia; solid tumor growth; neonatal disorders such as bronchopulmonary dysplasia (prophylaxis), necrotising enterocolitis (NEC), retinopathy of prematurity (ROP), cerebral palsy due to perinatal cerebral ischemia, and infant respiratory distress syndrome (IRDS); Whipple's disease; traumatic brain injury; refractory epilepsy; systemic inflammatory response syndrome (SIRS); cutaneous lupus; Jessner-Kanof disease; amyotrophic lateral sclerosis; systemic sclerosis (scleroderma); septic shock; acute pancreatitis; chronic recurrent multifocal osteomyelitis, non-bacterial osteitis (NBO), synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome, and Majeed syndrome; relapsing polychondritis; idiopathic recurrent pericarditis (IRP); myocarditis; Erdheim-Chester disease; juvenile xantogranuloma; islet-cell transplantation; haemodialysis-induced systemic inflammation; graft-versus-host disease; ANCA-associated glomerulonephritis and recurrent glomerulonephritis; Cogan syndrome; autoimmune inner-ear disease; chronic granulomatous disease (CGD); Castleman's disease; cardiac failure; diastolic cardiac failure; antisynthetase syndrome; acute ACL injury; acute haemorrhagic leukoencephalitis; AA amyloidosis; Di George syndrome; generalised fatigue; chronic fatigue syndrome (CFS); gulf-war illness (GWI); and narcolepsy.
In another embodiment, said IL-1R-I related 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.
The skilled person will appreciate that said IL-1R-I binding polypeptide, fusion protein or conjugate or a composition comprising an anti-IL-1R-I binding polypeptide, fusion protein or conjugate as described herein may 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, there is provided an IL-1R-I binding polypeptide, fusion protein or conjugate or a composition as described herein for oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, duodenal or suppository administration. In one embodiment, said administration is oral, intravenous, subcutaneous or duodenal administration and in one particular embodiment, said administration is oral administration.
As used herein, the term “subject” refers to a mammalian subject, such as a human subject.
Non-limiting examples of indications where topical administration routes could be especially relevant in IL-1R-I mediated disease include ocular diseases such as dry eye disease, uveitis, scleritis, and ulcerative keratitis; dermatological diseases such as ichtyosis and generalized pustular psoriasis; pulmonary diseases such as adult respiratory distress syndrome, bronchopulmonary dysplasia, chronic obstructive pulmonary disease, asbestosis, and silicosis.
In some embodiments it may be beneficial that the polypeptide, fusion protein, conjugate or composition for use as described above, is administered repeatedly within a period of time, for example within a 24 hour period from disease onset. In one particular embodiment wherein said IL-1R-I binding polypeptide, fusion protein, conjugate or composition is administered repeatedly within a 24 hour period from disease onset, such as at least two times within a 24 hour period from disease onset, such as at least three times within a 24 hour period from disease onset, to a subject in need thereof. In yet a particular embodiment, said IL-1R-I binding polypeptide, fusion protein, conjugate or composition is administered continuously during a limited time period such as during 24 hours from disease onset. Repeated or continuous administration may be preferred when distribution to the brain is required, such as in acute treatment of stroke.
In some embodiments wherein said fusion protein or conjugate comprises a second moiety having an in vivo half-life increasing activity, said fusion protein or conjugate, or composition comprising such a fusion protein or conjugate, is administered once weekly to a subject in need thereof.
In a related aspect, there is provided a method of treatment of an IL-1R-I related disorder, comprising administering to a subject in need thereof an effective amount of an IL-1R-I binding polypeptide, fusion protein, conjugate or a composition as described herein. In a more specific embodiment of said method, the IL-1R-I binding polypeptide, fusion protein, conjugate or composition as described herein modulates IL-1R-I function in vivo.
In one embodiment, said IL-1R-I related disorder is selected from the group as defined above in connection with the previous aspect.
In some embodiments of said method of treatment, it may be beneficial that the polypeptide, fusion protein, conjugate or composition as described above, is administered repeatedly within a period of time, for example within a 24 hour period from disease onset. In one particular embodiment wherein said IL-1R-I binding polypeptide, fusion protein, conjugate or composition is administered repeatedly within a 24 hour period from disease onset, such as at least two times within a 24 hour period from disease onset, such as at least three times within a 24 hour period from disease onset, to a subject in need thereof. In yet a particular embodiment, said IL-1R-I binding polypeptide, fusion protein, conjugate or composition is administered continuously during a limited time period, such as within 24 hours from disease onset. Repeated or continuous administration may be preferred when distribution to the central nervous system is required, such as in acute treatment of stroke.
In other embodiments, of said method of treatment, said fusion protein or conjugate comprises a second moiety having an in vivo half-life increasing activity, or said composition comprises a fusion protein or conjugate comprising a second moiety having an in vivo half-life increasing activity. In such methods, said fusion protein, conjugate or composition may be administered once weekly to a subject in need thereof.
It may be beneficial to administer a therapeutically effective amount of an IL-1R-I binding polypeptide, fusion protein or conjugate or composition as described herein and at least one second drug substance, such as an immune response modulating agent as described above or an anti-cancer agent.
As used herein, the term “co-administration” encompasses both concomitant and sequential administration. Thus, in one embodiment there is provided a method as defined above further comprising co-administration of an immune response modulating agent as described above. In another embodiment there is provided a method as defined above further comprising co-administration of an anti-cancer agent as described above.
In another aspect of the present disclosure, there is provided a method of detecting IL-1R-I, comprising providing a sample suspected to contain IL-1R-I, contacting said sample with an IL-1R-I binding polypeptide, fusion protein, conjugate or a composition as described herein, and detecting the binding of the IL-1R-I binding polypeptide, fusion protein, conjugate or composition to indicate the presence of IL-1R-I in the sample. In one embodiment, said method further comprises an intermediate washing step for removing non-bound polypeptide, fusion protein, conjugate or composition, after contacting the sample.
While the 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 may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to any particular embodiment contemplated, but that the invention will include all embodiments falling within the scope of the appended claims.
The following Examples disclose the development of novel Z variant molecules targeted to interleukin 1 receptor I (IL-1R-1) based on phage display technology. The IL-1R-I binding polypeptides described herein were sequenced, and their amino acid sequences are listed in
In this Example, human and cynomolgus IL-1R-I (hIL-1R-I and cIL-1R-1, respectively) were used as target proteins in phage display selections using a phage library of Z variants. The DNA of selected clones was sequenced, the Z variants were produced in E. coli as periplasmic fractions and assayed against IL-1R-I in ELISA (enzyme-linked immunosorbent assay).
Materials and methods
Biotinylation of target protein: Human IL-1R-1-Fc (hIL-1 RI-Fc Creative BioMart cat. no. IL1 RI-771H) and human IL-1R-I (hIL-1 RI, RnD Systems cat. no. 269-1R/CF) were biotinylated according to the manufacturer's recommendations at room temperature (RT) for 30 min using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, cat. no. 21327) at a 10×molar excess. Prior to the biotinylation, a buffer exchange to phosphate buffered saline (PBS, 10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) was performed for hIL-1R-1-Fc using a dialysis cassette (Slide-a-lyzer 3.5 K, 3500 MWCO, Thermo Scientific, cat. no. 66333), according to the manufacturer's instructions, and hIL-1R-I was dissolved in 100 mM phosphate buffer pH 6.5. After the biotinylation, a buffer exchange to PBS was performed for both proteins, as described above.
Expression of his-tagged target protein: Expression of cynomolgus IL-1R-I-His6(cIL-1R-I-His6, SEQ ID NO:1665) was performed using the FreeStyle 293-F expression system (Thermo Fisher Scientific), essentially according to the manufacturer's protocol. Supernatants were harvested by centrifugation 5 days after transfection of expression vectors and stored at −70° C. The frozen supernatant from the FreeStyle 293-F cultures were thawed and filtrated (0.22 μm). The supernatant, containing the cIL-1R-I-His6 was purified using affinity chromatography with an IMAC column. The purified protein was buffer exchanged to PBS. The purity of the protein was analyzed by SDS-PAGE stained with Coomassie Blue and the molecular weight was analyzed using mass spectrometry (HPLC/MS or MALDI-TOF/MS).
Phage display selection of IL-1R-I binding Z variants: A library of random variants of protein Z displayed on bacteriophage in fusion to a wild type albumin binding domain (abbreviated ABD001, SEQ ID NO:1660) was used to select IL-1R-I binding Z variants. The library, which is denoted Zlib006Naive.II, has a size of 1.5×1010 library members (Z variants). The construction in the phage library vector pAY02592 and the phage stock production was previously described in WO 2016/113246.
Selections against hIL-1R-I-Fc, biotinylated hIL-1R-1-Fc (b-hIL-1R-1-Fc), biotinylated hIL-1R-I (b-hIL-1R-1) and cIL-1R-I-His6 (SEQ ID NO:1665) were performed in four selection cycles. In the first cycle, four selection tracks were run in parallel; 1-1 to 1-4. The selection tracks 1-1 and 1-4 were divided into two parts in cycle two, resulting in a total of six parallel tracks in round 2, 3 and 4; 2-1 to 2-9, 3-1 to 3-9 and 4-1 to 4-9, respectively, as presented in Table 2. The number of phage particles used for selections was more than 2000 times the number of eluted phage particles in the previous cycle, but a lower amount was used in selection tracks 4-1 to 4-5. Phage stock preparation, selection procedure and amplification of phage between selection cycles were performed essentially as described in WO 2009/077175 with the following exceptions 1-8. 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% Tween20 (Acros Organics, cat. no. 233362500) was used as selection buffer. Exception 2: pre-selection was performed in cycles 1-4 at >60 min at RT or overnight at 4° C. by incubation of phage stock with Dynabeads® M-280 Streptavidin (SA beads, Life technologies/Invitrogen, cat. no. 11206D), SA beads pre-coated with biotinylated human IgG1-Fc (b-hFc, Jackson Immuno Research cat. no. 009-060-008) (Fc/SA beads), SA beads pre-coated with b-hFc and (Z00000)2-Cys-biotin, prepared essentially as described in WO 2009/077175 (Fc/Z00000/SA beads), protein A beads (Life technologies/Novex, cat. no. 10002D) pre-coated with b-hFc (Fc/protein A beads) or Dynabeads® His-Tag Isolation and Pulldown (IM beads, Life Technologies/Novex, cat. no. 10104D), respectively, as presented in Table 2. b-hFc was incubated with the beads (5, 8.5 or 10 μg b-hFc per mg Z00000/SA beads, protein A beads or SA beads, respectively) for >1 h at RT, then the beads were washed with 2×PBST (PBS supplemented with 0.1% Tween20) before use in pre-selection. Exception 3: all tubes and beads used in the selections were pre-blocked with PBS supplemented with 3% BSA (bovine serum albumin, Sigma cat. no. A3059) and 0.1% Tween20. Exception 4: selections were performed in solution at RT and the time for selection was 140 min in the first cycle and 120 min in the following cycles. Exception 5: target-phage complexes were captured on beads using the following amount of target protein and beads: 3.2 μg hIL-1R-1-Fc per mg Z00000/SA beads, 4 μg hIL-1R-1-Fc per mg protein A beads, 2 μg b-hIL-1R-1-Fc or 2 μg b-hIL-1R-I per mg SA beads and 4.5 μg cIL-1R-I-His6 per mg IM beads, respectively. The bead type used for each selection track is the same as presented for pre-selection in Table 2. Exception 6: E. coli strain ER2738 cells (Lucigen, Middleton, Wis., USA) grown in medium supplemented with 10 μg/ml tetracycline were used for phage infection. Exception 7: a 5× excess of M13K07 helper phage compared to bacteria was allowed to infect log phase bacteria. Exception 8: amplification of phage particles after round 2 and 3 was performed essentially as described in WO 2016/113246; Example 5, but using Tryptic Soy Broth+Yeast Extract (TSB-YE) medium supplemented with 100 μM IPTG, 25 μg/ml kanamycin, 100 μg/ml ampicillin and 2% glucose in the overnight culture.
An overview of the selection strategy, describing an increased stringency in the selection cycles obtained by using a lowered target concentration and an increased number of washes, is shown in Table 2.
Washes were performed for 1 min using PBST 0.1%, and elution was carried out as in the protocol for bead bound phage/target complexes eluted at pH 2 described in WO 2009/077175.
Production of Z variants for ELISA: The Z variants were produced essentially as described in WO 2016/113246 with the exception that the final volume of pellet dissolving from the 1 ml culture was 1000 μl PBST 0.05% (PBS supplemented with 0.05% Tween20).
The final supernatant of the periplasmic extract contained the Z variants as fusions to ABD (Z-ABD; ABD001, SEQ ID NO:1660), expressed as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (Gronwall et al. (2007) J Biotechnol, 128:162-183). Z##### refers to individual 58 amino acid residue Z variants.
ELISA screening of Z variants: The binding of Z variants to human IL-1R-I was analyzed in ELISA, essentially as described in WO 2016/113246, using 1 nM hIL-1R-1-Fc as target, and using a goat anti-human IgG-HRP (Southern Biotech, cat. no. 2040-05) diluted 1:10,000 for detection. As blank control, PBST 0.05% was added instead of the Z-ABD periplasmic sample and as negative control, a periplasmic extract with ABD001 (SEQ ID NO:1660) was added instead of the Z-ABD periplasmic sample. Control plates were assayed in a similar setup using 100 nM b-hFc instead of target protein and streptavidin conjugated HRP (Thermo Scientific, cat. no. N100) diluted 1:30,000 for detection.
Sequencing: In parallel with the ELISA screening, the clones were picked for sequencing. PCR fragments, amplified from single colonies, were sequenced and analyzed essentially as described in WO 2009/077175.
EC50 analysis of Z variants: A selection of IL-1R-I binding Z variants was subjected to an analysis of response against a dilution series of hIL-1R-1-Fc using ELISA as described above. The target protein hIL-1R-1-Fc was diluted stepwise 1:10 from 100 to 0.01 nM. As a background control, the Z variants were assayed with no target protein added. Obtained data was analyzed using GraphPad Prism 5 and non-linear regression, and the EC50 values (the half maximal effective concentration) were calculated.
Phage display selection of IL-1R-I binding Z variants: Individual clones were obtained after four cycles of phage display selections against human and cynomolgus IL-1R-1.
ELISA screening of Z variants: The clones obtained after four cycles of selection were produced individually in 96-well plates and were screened for hIL-1R-1-Fc binding activity in ELISA using 1 nM target protein. In parallel, an ELISA to exclude Fc binding was run. Clones positive to hIL-1R-I were identified and none of the IL-1R-I positive clones were positive to Fc solely. No response was obtained for the ABD negative control. 45 identified Z variants displayed an ELISA screening result that was more than 0.71 AU (8.5× the control background).
Sequencing: In parallel with ELISA screening, sequencing was performed for clones obtained after four cycles of selection. 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 residues long Z variants are listed in
EC50 analysis of Z variants: A subset of said Z variants (SEQ ID NO:1-19) was subjected to an ELISA target titration using hIL-1R-I-Fc. Obtained results were used for calculation of EC50 values (Table 3).
This Example describes the general procedure for subcloning and production of His-tagged Z variants originating from the primary phage selection, characterization of their target binding, target blocking and melting points.
Subcloning of Z variants with a His6 tag: The DNA of respective Z variant was amplified from the phage library vector pAY02592. A subcloning strategy for construction of monomeric Z variant molecules with N-terminal His6 tag was applied using standard molecular biology techniques. The Z gene fragments were subcloned into an expression vector resulting in the encoded sequence MGSSHHHHHHLQ-[Z#####]-VD.
Cultivation: E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing the gene fragment of each respective IL-1R-I binding Z variant. The resulting recombinant strains were cultivated in media supplemented with 50 μg/ml kanamycin, either at 30° C. in 50 ml scale using the EnPresso protocol (BioSilta), or at 37° C. in 930 ml TSB-YE medium. In order to induce protein expression, IPTG was added to a final concentration of 0.2 mM at OD600≈10 (EnPresso) or 2 (TSB+YE). After induction, the cultivations were incubated for 16 h (EnPresso) or 5 h (TSB+YE). The cells were harvested by centrifugation.
Purification of IL-1R-I binding Z variants with a His6 tag: 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 on 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 the IMAC purification, the buffer was exchanged to PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) using PD-10 desalting columns (GE Healthcare). All Z variants were subjected to a second purification step. Each Z variant was loaded on a 1 ml Resource 15RPC column (GE Healthcare), previously equilibrated with RPC solvent A (0.1% TFA, 10% ACN, 90% water). After column wash with RPC solvent A, bound proteins were eluted with a linear gradient 0-50% RPC solvent B (0.1% TFA, 80% ACN, 20% water) for 20 ml. The buffer was then exchanged to PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) using PD-10 desalting columns (GE Healthcare).
Protein concentrations were determined by measuring the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer (Saveen Werner AB) and the extinction coefficient of the respective protein. Samples with a concentration less than approximately 1 mg/ml were concentrated using Amicon Ultra-4, Ultracel-3K (MerckMillipore). The purity was analyzed by SDS-PAGE stained with Coomassie Blue and the identity of each purified Z variant was confirmed using LC/MS analysis.
Biacore affinity analysis: Affinities (KD) for human and cynomolgus IL-1R-I were determined for His6-tagged Z variants using a Biacore 2000 instrument (GE Healthcare). hIL-1R-I or cIL-1R-I-His6 was immobilized on the carboxylated dextran layer in different flow cells of a CM5 chip (GE Healthcare, cat. no. BR100012). The 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 running buffer. One flow cell surface on the chip was activated and deactivated for use as blank during analyte injections. In the kinetic experiment, HBS-EP was used as running buffer, the flow rate was 50 μl/min and the temperature was 25° C. The analytes, i.e. the Z variants, were each diluted in HBS-EP buffer to final concentrations of 40 nM and 10 nM, and were injected over the target chip surfaces for 2 min, followed by dissociation in running buffer for 8 min. Kinetic constants were calculated from the obtained sensorgrams using a 1:1 binding model, in the BiaEvaluation software 4.1 (GE Healthcare).
In vitro IL-1β neutralization assay: The TF-1 cell line proliferates in response to many different cytokines including IL-1β. This cell line was used to assess the ability of primary IL-1R1 binders to inhibit IL-1β actions. TF-1 cells were maintained in RPM11640 with L-glutamine (Lonza) supplemented with 10% FCS (Gibco), Penicillin-Streptomycin (Lonza) and 2 ng/ml rhGM-CSF (R&D Systems). Prior to use, cells were washed twice in RPM11640 without rhGM-CSF. Cells were then counted and dispensed into 96 well flat bottomed plates at a density of 3×104 cells per well. In separate plates, serial dilutions of the inhibitory binders; His6-tagged IL-1R-I binding Z variants, with a concentration range of 400-0.1 nM, or the IL-1R-I binding protein Kineret (anakinra, Sobi), with a concentration range of 0.3-0.0003 nM, were incubated in the presence of 0.6 nM IL-1β (PeproTech). The pre-mixed complexes of the Z variant polypeptides and IL-13 were transferred to wells containing TF-1 cells. The cells were stimulated for 72 h at 37° C. in a humidified 5% CO2 atmosphere. During the last four hours of incubation, 19 μl of 2× diluted CCK-8 (Fluka, Sigma Aldrich), was added per well to determine the proliferative responses. The absorbance was measured at 450 nm using a microplate reader (Victor3, Perkin Elmer). The data on cell growth was assessed by non-linear regression to a four-parameter dose-response curve, and the half maximal inhibitory concentration (IC50) was determined using GraphPadPrism program.
Circular dichroism (CD) spectroscopy analysis: Purified His6-tagged Z variants were diluted to 0.5 mg/ml in PBS. For each diluted Z variant, a CD spectrum at 250-195 nm was obtained at 20° C. In addition, a variable temperature measurement (VTM) was performed to determine the melting temperature (Tm). In the VTM, the absorbance was measured at 221 nm while the temperature was raised from 20 to 90° C., with a temperature slope of 5° C./min. A new CD spectrum was obtained at 20° C. after the heating procedure in order to study the refolding ability of the Z variants. The CD measurements were performed on a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) using a cell with an optical path length of 1 mm.
Production of His6-tagged Z variants: The IL-1R-I binding Z variants with a His6 tag were expressed as soluble gene products in E. coli. The amount of purified protein from approximately 1-2 g bacterial pellet was determined spectrophotometrically by measuring the absorbance at 280 nm and ranged from approximately 3 to 25 mg for the different IL-1R-I binding Z variants before RPC purification. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the IL-1R-I binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.
Biacore affinity analysis: The interactions of His6-tagged IL-1R-1-binding Z variants with human and cynomolgus IL-1R-I were analyzed in a Biacore instrument by injecting two concentrations of each Z variant over surfaces containing immobilized IL-1R-1. The calculated affinities (KD) for human IL-1R-I are presented in Table 4. The strongest binder to human IL-1R-I (Z12967, SEQ ID NO:9) bound to human IL-1R-I with an affinity of 1.2×10−9 M and to cynomolgus IL-1R-I with an affinity of 3.5×10−8 M.
In vitro IL-1β neutralization assay: The IL-1β inhibition ability of His6-tagged IL-1R-I-binding Z variants was analyzed in a TF-1 cell assay. The resulting IC50 values are presented in Table 5.
CD analysis: The CD spectra determined for the IL-1R-I binding Z variants with a His6 tag showed that each had an a-helical structure at 20° C. This result was also verified in the variable temperature measurements, wherein the melting temperatures were determined (Table 6). Reversible folding was seen for all the IL-1R-I binding Z variants when overlaying spectra measured before and after heating to 90° C.
In this Example, a matured library was constructed. The library was used for selection of further IL-1R-I binding Z variants. Selections from matured libraries are usually expected to result in binders with increased affinity (Orlova et al., (2006) Cancer Res 66(8):4339-48).
Library design: The library was primarily based on human IL-1R-I binding Z variants from the primary selection described in Example 1 and 2. Sequences of a subset of the Z variants derived from the primary selection are listed in
Library construction: The two oligonucleotides were assembled, amplified, cloned into vector pAY02592, and transformed into E. coli ER2738, essentially as described for matured libraries in WO 2015/189430. Clones from the Zlib0061L-1 RI.I library were sequenced (as described in WO 2009/077175) in order to verify the content and to evaluate the outcome of the constructed library vis-à-vis the library design.
Preparation of phaqe stock: A phage stock containing the phagemid library was prepared in shake flasks in two batches. Cells from a glycerol stock containing the phagemid library were inoculated in 2 and 3 l, respectively, of TSB-YE medium, supplemented with 2% glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin. The cultivations were grown at 37° C. until OD600 reached log-phase 0.5-0.9. The cultivations were infected using a 4-10×molar excess of M13K07 helper phage and were incubated for 30 minutes at 37° C. Batch 1 cultivations were pelleted by centrifugation, dissolved in TSB-YE medium, supplemented with 100 μg/ml ampicillin, 25 μg/ml kanamycin and 0.1 mM IPTG and grown at 30° C. overnight. To batch 2 cultivation, 0.1 mM IPTG was added, followed by an incubation of 1 h at 37° C. After addition of 25 μg/ml kanamycin, the cultures were grown at 30° C. overnight. For both batches, the overnight grown cells were pelleted by centrifugation at 4,000 g and the phage particles remaining in the medium were thereafter precipitated twice in PEG/NaCl, filtered and dissolved in PBS and glycerol as described in Grönwall et al., supra. Phage stocks were stored at −80° C. until use in selection.
Library construction: The new library Zlib0061L-1 RI.I was designed based on a set of IL-1R-I binding Z variants with verified binding properties (Example 1 and 2). The theoretical size of the designed library was 9.7×108 Z variants. The actual size of the library, determined by titration after transformation to E. coli ER2738 cells, was 5.6×109 transformants. The library quality was tested by sequencing 192 transformants and by comparing their actual sequences with the theoretical design. The contents of the actual library compared to the designed library were shown to be satisfactory. A first matured library of potential binders to IL-1R-I was thus successfully constructed.
In this Example, human and cynomolgus IL-1R-I were used as target proteins in phage display selections using a matured phage library of Z variants. The DNA of selected clones was sequenced, the Z variants were produced in E. coli periplasmic fractions and assayed against IL-1R-I in ELISA and Biacore.
Expression of target protein: Expression of Fc fused cynomolgus IL-1R-I (cIL-1R-I-Fc, SEQ ID NO:1666) was performed 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 expression vectors and stored at −70° C. The frozen supernatant from the Expi293 culture were thawed and filtrated (0.22 μm). The supernatant containing the cIL-1R-I-Fc was purified using affinity chromatography with a MabSelect SuRe column. The purified protein was buffer exchanged to PBS. The purity of the protein was analyzed by SDS-PAGE stained with Coomassie Blue and the molecular weight was analyzed using mass spectrometry (HPLC/MS or MALDI-TOF/MS).
Phage display selection of IL-1R-1 binding Z variants: The biotinylated human target proteins b-hIL-1R-I-Fc and b-hIL-1R-1, and the cynomolgus 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 using the new library of Z variant molecules described in Example 3. An overview of the selection strategy, describing the parallel selection tracks and an overall increased stringency in the selection cycles obtained by using a lowered target concentration and an increased number of washes, is shown in Table 8. The selections were performed in four to five cycles essentially as described in Example 1, with the following exceptions 1-6. Exception 1: pre-selection was performed for >60 min at RT by incubation of phage stocks 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 track 1-5), respectively. Exception 2: the time for selection was 30-100 min as presented for each track in Table 8 Exception 3: target-phage complexes were captured on beads using the following amount 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 per mg SA beads, respectively, and 5 μg cIL-1R-I-Fc per mg protein A beads. In selection round 5, 2 mg SPHERO neutravidin beads (Spherotech cat. no. 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, hIL-1R-I was added to the wash buffer in 100 times higher concentration than the target concentration of the respective track and the wash was run for 15-20 min. Exception 5: the phage/target/bead complexes of selection tracks 4-1 to 4-13 were divided into two after the bead wash procedure. Half of the samples was submitted to elution and the other half, denoted 4-1× to 4-13×, was submitted to an extra 66 h, wash step before elution. Exception 6: two selection rounds four tracks, 4-4× and 4-6×, were submitted to a fifth selection round, resulting in tracks 5-1 and 5-2, as presented in Table 8. 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× excess of M13K07 helper phage compared to bacteria was allowed to infect log phase bacteria. Amplification of phage particles after round 1 to 3 was performed essentially as described for round 2 and 3 in Example 1.
In the first selection cycle, nine selection tracks were run; 1-1 to 1-9. In cycle two, some tracks were divided into two, three or six tracks and two tracks were pooled into one track, resulting in a total of 17 parallel tracks in cycle 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 run; 5-1 and 5-2. In selection tracks 1-1 to 1-5, batch 1 of Zlib0061L-1R1.I was used, and in 1-6 to 1-9, batch 2 was used, respectively. All selection tracks are presented in Table 8. The number of phage particles used for selections was typically more than 2,000 times the number of eluted phage particles in the previous cycle.
Sequencing: Individual clones from cycle 4 or 5 of the different selection tracks were picked for sequencing. Amplification and sequence analysis of gene fragments were performed essentially as described in Example 1.
Production of Z variants for ELISA and Biacore screening: Z variants were produced essentially as described in Example 1 with the exception that the culture volume was 1.2 ml and that the periplasmic extracts were clarified by filtration using 1.2 μm 96 well filter plates (MerckMillipore cat. no. MSANLY50) after the freeze-thawing procedure. Z variants screened in Biacore were diluted five times in HBS-EP buffer.
ELISA screening of Z variants: The binding of Z variants to human was analyzed in ELISA, essentially as described in Example 1, using 0.3 nM hIL-1R-1-Fc as target protein. As blank control, PBST 0.05% was added instead of the Z-ABD periplasmic extract.
EC50 analysis of Z variants: A selection of IL-1R-I binding Z variants was subjected to an analysis of response against a dilution series of hIL-1R-1-Fc using ELISA as described above. The target protein hIL-1R-1-Fc was diluted stepwise 1:10 from 100 to 0.01 nM. As a background control, the Z variants were assayed with no target protein added. Obtained data was analyzed using GraphPad Prism 5 and non-linear regression, and the EC50 values (the half maximal effective concentration) were calculated. A periplasmic extract with Z12967 (SEQ ID NO:9) was analyzed in parallel for signal comparison.
Biacore screening of Z variants: The binding of Z variants to human IL-1R-I was analyzed in a kinetic screening using a Biacore T200 instrument. A polyclonal goat anti-ABD antibody (goat anti-ABD) was immobilized on CM5 chip surfaces basically as described for other proteins in Example 2. For the kinetic screening, analytes were injected in two steps. First, a 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 2 min of dissociation in running buffer HBS-EP. Glycine-HCl pH 2.0 (cat. no. BR100355, GE Healthcare) was used for regeneration of the antibody surfaces between the cycles. The temperature of the assay was 25° C. Before performing the kinetic analyses, 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. Rough screening affinities (KD) were calculated from the reference subtracted 100 nM hIL-1R-I response using a 1:1 binding model of the BiaEvaluation software 4.1 (GE Healthcare). A periplasmic extract of Z12967 was included in the screening analysis for comparison. Periplasmic extracts of four Z variants were also submitted to a single cycle kinetics (SCK) assay, using the same setup as above, but injecting five hIL-1R-I concentrations and evaluating the data with a 1:1 model for single cycle kinetics.
Phage display selection of IL-1R-I binding Z variants: Individual clones were obtained after four or five cycles of phage display selections against human and cynomolgus IL-1R-I.
Sequencing: Sequencing was performed for clones obtained after four or five cycles of selection. 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 residues long Z variants are listed in
ELISA screening of Z variants: Clones obtained after four or five cycles of selection were produced individually in 96-well plates and were screened for human IL-1R-I binding activity in ELISA. 98% of the assayed Z variants gave a positive signal of 2× the blank control or higher against 0.3 nM hIL-1R-I-Fc.
EC50 analysis of Z variants: A subset of 115 Z variants displaying results over 0.99 AU (16.5× the blank control) was subjected to ELISA target titration using hIL-1R-I-Fc. Obtained results were used for calculation of EC50 values (Table 9). The result for Z12967 (SEQ ID NO:9) was 2.4×10−10 M.
On average, the Z variants originating from this library (first matured library) displayed an improvement in EC50 values by a factor 10 compared with the average EC50 for the Z variants originating from the primary selection.
Biacore screening of Z variants: A selection of IL-1R-I binding Z variants was submitted to a 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 an anti-ABD antibody. The calculated screening affinities are presented in Table 10. Z16062 (SEQ ID NO:266) got the best K value (7.6×10−9 M) of the assayed binders. The KD of Z2967 was 2.5×10−8 M. The four binders assayed in a S1K experiment got KD values that deviated from their respective screening kinetics affinities by 0-14%.
This Example describes the general procedure for subcloning and production of His6-tagged and ABD-fused Z variants (originating from the first maturation library phage selection), characterization of their IL-1R-I binding, blocking and melting points.
Subcloning of Z variants with a His6 tag: The DNA of the respective Z variant was amplified from the phage library vector pAY02592 and was subcloned with an N-terminal His6 tag using standard molecular biology techniques essentially as described in Example 2.
Subcloning of Z variants in fusion with ABD: The N terminal sequence of respective Z variant was mutated, in position 1 and 2 to amino acid residues A and E respectively, using standard molecular biology techniques. The resulting new Z variants were subcloned into an expression vector containing an ABD variant, giving the encoding sequence [Z#####]-ASGS-ABD, where the ABD variant was PP013 (SEQ ID NO:1661). Z##### refers to individual, 58 amino acid residue long Z variants.
Cultivation: E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing the gene fragment of each respective IL-1R-I binding Z variant. The resulting recombinant strains were cultivated in media supplemented with 50 μg/ml kanamycin at 30° C. in 50 ml scale using the EnPresso protocol (BioSilta). In order to induce protein expression, IPTG was added to a final concentration of 0.2 mM at OD600≈10. After induction, the cultivations were incubated for 16 h. The cells were harvested by centrifugation.
Purification of IL-1R-I binding Z variants with a His6 tag: Purification was performed essentially as described in Example 2, but without the buffer exchange between the IMAC and RPC purification steps for the majority of the samples. During RPC purification, the RPC solvent A was changed to 0.1% TFA in 100% water and the linear gradient was 0-60% RPC solvent B for 18 ml.
Purification of IL-1R-I binding Z variants with ABD fusion: Approximately 2 g of each cell pellet was re-suspended in TST-buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween20, pH 8.0) supplemented with Benzonase® (Merck). After cell disruption and clarification by centrifugation, each supernatant was applied on a gravity flow column with 1 ml agarose immobilized with an anti-ABD ligand (produced in-house). After washing with TST-buffer and 5 mM NH4Ac pH 5.5 buffer, the ABD fused Z variants were eluted with 0.1 M HAc. The buffer of the eluate was exchanged to PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) using PD-10 desalting columns (GE Healthcare).
Protein concentrations were determined by measuring the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer (Saveen Werner AB) and the extinction coefficient of the respective protein. The purity was analyzed by SDS-PAGE stained with Coomassie Blue and the identity of each purified Z variant was confirmed using LC/MS analysis.
Biacore kinetic analysis: The kinetic constants (kon and koff) and affinities (KD) for human and cynomolgus IL-1R-I were determined for His6-tagged Z variants using a Biacore 2000 instrument (GE Healthcare). The experiment was run essentially as described in Example 2, using 100, 10 and 1 nM of the Z variants. The analyte injection time was 3 min followed by 15 min dissociation and 2×5 s glycine pH 3.0 (GE Healthcare, cat. no. BR100357), supplemented with 0.5 M NaCl, was used for regeneration of the surfaces between the cycles. Kinetic constants were calculated from the obtained sensorgrams of two or three concentrations of the respective Z variant, using a 1:1 binding model in the BiaEvaluation software 4.1 (GE Healthcare). His6-tagged Z12967 was included in the assay for comparison.
In vitro IL-1β neutralization assay: His6-tagged IL-1R-I specific Z variants were tested for their inhibitory capacity in the TF-1 cell assay. The assay was run as described in Example 2. The data on cell growth was assessed by non-linear regression to a four-parameter dose-response curve, and IC50 values were determined using GraphPadPrism program. His6-tagged Z12967 was included for comparison.
Circular dichroism (CD) spectroscopy analysis: CD was analyzed for His6-tagged Z variants, as described in Example 2.
Subcloning with His6 tag: Z variants were subcloned into constructs with an N-terminal His6.
Production of His6-tagged Z variants: The IL-1R-I binding Z variants with a His6 tag were expressed as soluble gene products in E. coli. The amount of purified protein was determined spectrophotometrically by measuring the absorbance at 280 nm and ranged from approximately 1 to 7 mg protein per g pellet. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the IL-1R-I binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.
Biacore kinetic analysis: The interactions of His6-tagged IL-1R-1-binding Z variants with human and cynomolgus IL-1R-I were analyzed in a Biacore instrument by injecting various concentrations of the Z variants over a surface containing immobilized IL-1R-1. A summary of the calculated kinetic parameters (KD, kon and koff) for the human IL-1R-I binding is given in Table 11. The strongest binder to human IL-1R-1, Z18557 (SEQ ID NO:1205), got a KD of 1.5×10−10 against human IL-1R-I and 7.6×10−9 M against cynomolgus IL-1R-1. The KD of Z12967 to human IL-1R-I was 5.1×10−10 M.
In vitro IL-1β neutralization assay: The IL-1β inhibition ability of His6-tagged IL-1R-1-binding Z variants was analyzed in a TF-1 cell assay. The resulting IC50 values are presented in Table 12. Z18557 (SEQ ID NO:1205) displayed the best inhibition capacity with an IC50 value below 0.5 nM. The IC50 of Z12967 was 5.2 nM.
CD analysis: The CD spectra determined for the IL-1R-I binding Z variants with a His6 tag showed that each had an a-helical structure at 20° C. This result was also verified in the variable temperature measurements, wherein melting temperatures were determined (Table 13). Reversible folding was seen for all the IL-1R-I binding Z variants when overlaying spectra measured before and after heating to 90° C.
In this Example, two second matured libraries were constructed. The libraries were used for selections of additional IL-1R-I binding Z variants.
Library design: The libraries were primarily based on sequences of human IL-1R-I binding Z variants described in Example 4 and 5. In the new libraries, denoted Zlib0061L-1 RI.II and Zlib0061L-1 RI.III, 10 variable positions in the Z molecule scaffold were biased towards certain amino acid residues and three positions were kept constant, respectively, according to a strategy mainly based on Z variants (SEQ ID NO:19-1209) from the selection from the first matured library. In addition, in both libraries, a new amino acid position was included for variation; position 12 of the 58 aa Z variant sequence, resulting in 11 variable positions, in total. Position 12 of Z18557 (SEQ ID NO:1205), characterized in Example 5, was valine. The library design allowed for the amino acids A, V and I in position 12.
For Zlib0061L-1 RI.II, a DNA linker was generated using split-pool synthesis containing the following sequence ordered from DNA 2.0 (Menlo Park, Calif., 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′ (SEQ ID NO:1681, designed codons are denoted NNN). The design for each amino acid residue of the new library, including eleven 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, are displayed in Table 14. The resulting theoretical library size was 2.2×107 variants. An even theoretical distribution of the different amino acids was applied within each amino acid position, except from position 10, 11, 12, 13, 17, 28 and 35, where a higher portion of selected amino acids was applied.
For Zlib0061L-1 RI.III, the library was constructed similarly as Zlib0061L-1 RI.I, described in Example 3, using the TRIM technology. 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 denoted NNN) was ordered from Ella Biotech (Martinsried, Germany). The design for each amino acid residue of the new library, including eleven 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, are displayed in Table 15. The resulting theoretical library size was 1.0×108 variants. An even theoretical distribution of the different amino acids was applied within each amino acid position, except from position 10, 11, 12, 32 and 35, where a higher portion of selected amino acids was applied.
Library construction and phage stock preparation: Zlib0061L-1 RI.II and Zlib0061L-1RI.III were constructed as described in WO 2016/113246 (Example 4) and in Example 3, respectively. For both libraries, E. coli XL-1 Blue was used for library transformation. Clones from the libraries of Z variants were sequenced (as described in Example 3) in order to verify the content and to evaluate the outcome of the constructed library vis-à-vis the library design.
Phage stocks containing the phagemid libraries were prepared in shake flasks. Cells from glycerol stocks containing the phagemid library were inoculated in 0.5 or 1 l, respectively, of TSB-YE medium, essentially as described for Zlib0061L-1 RI.I batch 2 in Example 3. The cultivations were infected using a 10-20× molar excess of M13K07 helper phage at an OD600 of 0.8-0.9. Phage stocks were prepared as described in Example 3.
Library construction: The new libraries were designed based on a set of IL-1R-I binding Z variants, selected from Zlib0061L-1 RI.I, with verified binding properties (Examples 4 and 5). The theoretical sizes of Zlib0061L-1 RI.II and Zlib0061L-1 RI.III were 2.2×107 and 1.0×108 Z variants, respectively. The actual sizes of the libraries, determined by titration after transformation to E. coli XL-1 Blue cells, were 1.2×109 and 7.0×109 transformants, respectively.
The library qualities were tested by sequencing of 192 and 96 transformants from Zlib0061L-1 RI.II and Zlib0061L-1 RI.III, respectively, and by comparing their actual sequences with the theoretical design. The contents of the actual library compared to the designed library were shown to be satisfactory. Two matured libraries of potential binders to IL-1R-I were thus successfully constructed.
In this Example, human and cynomolgus IL-1R-I were used as target proteins in phage display selections using two second matured phage libraries of Z variants.
Phage display selection of IL-1R-I binding Z variants: The biotinylated human target proteins b-hIL-1R-I-Fc and b-hIL-1R-I, and the cynomolgus target protein b-IL-1R-I-Fc, were used in phage selections using the new libraries of Z variant molecules described in Example 6. An overview of the selection strategy, describing the parallel selection tracks and an overall increased stringency in the selection cycles obtained by using a lowered target concentration and an increased number of washes, is shown in Table 16.
The selections were performed in four cycles essentially as described in Example 4, with the following exceptions 1-3. Exception 1: pre-selection was performed in selection round 1 and 2 for >60 min at RT by incubation of phage stocks with SA beads (b-hIL-1R-I tracks) or Fc/SA beads (b-IL-i RI-Fc tracks). Exception 2: in the second final wash step of tracks 2-i, 2-4, 2-9, 2-11, 2-12, 3-i, 3-4, 3-9, 3-i11 and 3-12, respectively, hIL-1R-I was added to the wash buffer in 100 times higher concentration than the target concentration of the respective track and the wash was run for 15-20 min. Exception 3: In the fourth selection round, the tracks 4-i, 4-4 and 4-6 were divided into two before the second final wash step. For one part, hIL-1R-I was added to the wash buffer in 100 times higher concentration than the target concentration of the respective track and the second final wash was run overnight. For the other part, a 1 h wash in PBST was applied for the second final wash.
In the first selection cycle, twelve selection tracks were run; 1-1 to 1-12. In cycle two, two tracks were divided into three, and six tracks were pooled into two tracks, resulting in a total of 12 parallel tracks in cycle 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 presented in Table 16. The number of phage particles used for selections was typically more than 2000 times the number of eluted phage particles in the previous cycle.
Phage display selection of IL-1R-I binding Z variants: Individual clones were obtained after four cycles of phage display selections against human and cynomolgus IL-1R-1.
In this Example, DNA of selected clones from Example 4 and Example 7 was sequenced, the Z variants were produced in E. coli periplasmic fractions and assayed against IL-1R-I in ELISA and Biacore.
Sequencing of potential binders: Individual clones from cycle 4 of selections using the three matured libraries Zlib0061L-1 RI.I, Zlib0061L-1 RI.II and Zlib0061L-1 RI.III were picked for sequencing. Amplification and sequence analysis of gene fragments were performed essentially as described in Example 1.
Production of Z variants for ELISA and Biacore screening: Z variants were produced essentially as described in Example 4 with the exception that the periplasmic extracts were prepared by heat-treatment (82° C., 20 min) followed by clarification by filtration. Z variants screened in Biacore were diluted five times in HBS-EP buffer.
ELISA screening of Z variants: The binding of Z variants to human IL-1R-I was analyzed in ELISA, essentially as described in Example 1, using 0.2 nM hIL-1R-1-Fc as target protein. As negative control, a periplasmic extract of ABD001 was used.
EC50 analysis of Z variants: A selection of IL-1R-I binding was subjected to an analysis of response against a dilution series of hIL-1R-1-Fc using ELISA as described above. The target protein hIL-1R-1-Fc was diluted stepwise 1:10 from 20 to 0.002 nM. As a background control, the Z variants were assayed with no target protein added. Obtained data was analyzed using GraphPad Prism 5 and non-linear regression, and the EC50 values (the half maximal effective concentration) were calculated. Periplasmic extracts with Z12967, Z16065 and Z16218 (corresponding to SEQ ID:9, 268 and 414, respectively) were analyzed in parallel for signal comparison. As negative control, a periplasmic extract of ABD001 was used.
Biacore screening of Z variants: The binding of Z variants to human IL-1R-I was analyzed in a kinetic screening, using a Biacore 2000 instrument, essentially as described in Example 4, but with the exception that Z-ABD (ABD001, SEQ ID NO:1660) periplasmic extracts were injected during 2 min. Periplasmic extracts of Z12967, Z16065 and Z16218 were included in the screening analysis in duplicates for comparison. A periplasmic extract with ABD (ABD001, SEQ ID NO:1660) was included as negative control.
Sequencing: Sequencing was performed for clones obtained after four cycles of selection. 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 residues long Z variants are listed in
ELISA screening of Z variants: Clones obtained after four cycles of selection were produced individually in 96-well plates and were screened for human IL-1R-I binding activity in ELISA. 99.5% of the assayed Z variants gave a positive signal of 2× the blank control or higher against 0.2 nM hIL-1R-I-Fc.
EC50 analysis of Z variants: A subset of 102 Z variants displaying results over 0.74 AU (8.2× the blank control) was subjected to ELISA target titration using hIL-1R-I-Fc. Obtained results were used for calculation of EC50 values (Table 17). The results for Z12967, Z16065 and Z16218 were 2.6×10−10, 2.1×10−10 and 2.2×10−10 M, respectively.
Biacore screening of Z variants: A subset of 188 IL-1R-I binding Z variants was submitted to a 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 an anti-ABD antibody. The calculated screening affinities are presented in Table 18. The KD values of Z12967, Z16065 and Z16218 were 2.7×10−8, 7.5×10−9 and 1.3×10−8 M (average of duplicates), respectively.
Z18814, which has an amino acid sequence that only differs from Z16065 in position X5 of the binding motif BM, has three times higher affinity than Z-6065. Z16065 has the highest binding affinity (KD) among the IL-1R-I binding Z variants (Table 11) with an alanine residue in position X5. Z18814 has an isoleucine residue in position X5. Several Z variants with valine or isoleucine in position X5 were found to have high affinity to IL-1R-1.
7.9 × 10−10
This Example describes the general procedure for subcloning and production of His6-tagged Z variants originating from the first and second maturation library phage selections, characterization of their target binding, target blocking and melting points.
Subcloning of Z variants with a His6 tag: A subset of Z variants screened in Example 8 was chosen for subcloning. The DNA of the respective Z variant was amplified from the phage library vector pAY02592 and was subcloned with an N-terminal His6 tag using standard molecular biology techniques essentially as described in Example 2.
Cultivation: E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing the gene fragment of each respective IL-1R-I binding Z variant. The resulting recombinant strains were cultivated in media supplemented with 50 μg/ml kanamycin at 30° C. in 50 ml scale using the EnPresso protocol (BioSilta). In order to induce protein expression, IPTG was added to a final concentration of 0.2 mM at OD600≈10. After induction, the cultivations were incubated for 16 h. The cells were harvested by centrifugation.
Purification of IL-1R-I binding Z variants with a His6 tag: Approximately 2/3 of the IL-1R-I binding Z variants were purified as described in Example 2 but without the buffer exchange between the IMAC and RPC purification steps. The linear gradient during RPC purification was also changed to 0-60% RPC solvent B for 18 ml. The remaining 1/3 of the IL-1R-I binding Z variants were only IMAC purified before the 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.
Biacore kinetic analysis: The kinetic constants (kon and koff) and affinities (KD) for human and cynomolgus IL-1R-I were determined for His6-tagged Z variants using a Biacore 2000 instrument (GE Healthcare). The experiment was run essentially as described in Example 5, using 48, 12, 3 and 0.75 nM of the Z variants. The analyte injection time was 3 min followed by 6 min dissociation and 2×5 s glycine pH 3.0, supplemented with 0.5 M NaCl, was used for regeneration of the surfaces between the cycles. Kinetic constants were calculated from the obtained sensorgrams of three or four concentrations of the respective Z variant, using a 1:1 binding model in the BiaEvaluation software 4.1 (GE Healthcare). His6-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.
In vitro IL-1β neutralization assay: His6-tagged IL-1R-I specific Z variants were tested for their inhibitory capacity in the TF-1 cell assay. The assay was run as described in Example 2. The data on cell growth was assessed by non-linear regression to a four-parameter dose-response curve, and IC50 values were determined using GraphPadPrism program. His6-tagged Z12967, Z16065, Z16163 and Z18557 were included for comparison.
Circular dichroism (CD) spectroscopy analysis: CD was analyzed for His6-tagged Z variants, as described in Example 2.
Production of His6-tagged Z variants: The IL-1R-I binding Z variants with a His6 tag were expressed as soluble gene products in E. coli. The amount of purified protein was determined spectrophotometrically by measuring the absorbance at 280 nm and ranged from approximately 0.4 to 7 mg protein per g pellet for IL-1R-I binding Z variants which were both IMAC and RPC purified. The amount of purified protein ranged from approximately 4 to 15 mg protein per g pellet for IL-1R-I binding Z variants which were only IMAC purified. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the IL-1R-I binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.
Biacore kinetic analysis: The interactions of His6-tagged IL-1R-1-binding Z variants with human and cynomolgus IL-1R-I were analyzed in a Biacore instrument by injecting various concentrations of the Z variants over a surface containing immobilized IL-1R-1. A summary of the calculated kinetic parameters (KD, kon and koff) for the human IL-1R-I binding is given in Table 19. The KD values of Z12967, Z16065, Z16163 and Z18557 were 8.2×10−10, 5.1×10−10, 7.6×10−10 and 1.4×10−10 M, respectively. The KD values of cynomolgus IL-1R-1 binding were 2.0×10−9, 6.7×10−9 and 3.3×10−9 M for Z18754, Z18760 and Z18800, respectively.
In vitro IL-1β neutralization assay: The IL-1β inhibition ability of His6-tagged IL-1R-1-binding Z variants was analyzed in a TF-1 cell assay. The resulting IC50 values are presented in Table 20. The IC50 values of Z12967, Z16065, Z16163 and Z18557 were 7.4, 3.3, 3.3 and 0.6 nM, respectively.
CD analysis: The CD spectra determined for the IL-1R-I binding Z variants with a His6 tag showed that each had an a-helical structure at 20° C. This result was also verified in the variable temperature measurements, wherein melting temperatures were determined (Table 21). Reversible folding was seen for all the IL-1R-I binding Z variants when overlaying spectra measured before and after heating to 90° C.
The thermo-stability and the a-helical content of the IL-1R-I binding Z variants having a valine or isoleucine residue in position X5 of the BM (Z18912, Z18920, Z18968, Z19072, Z19075, Z19143, Z19147, Z19151, Z19158, Z19159, Z19170, Z19172, Z19180, Z19181 and Z19182) were not substantially different from the Z variants having an alanine residue in position X5.
This Example describes the common steps for DNA construction and production of different IL-1R-I binding proteins variants fused to different in vivo half-life extending fusion partners.
DNA construction: DNA encoding a set of IL-1R-I binding Z variants (see below) fused to human IgG1 Fc (SEQ ID NO:1662), an 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 for expression in Chinese hamster ovary (CHO) cells and synthesized by the Invitrogen GeneArt Gene Synthesis service at Thermo Fisher Scientific. The genes were cloned in expression vectors for subsequent expression in E. coli or in CHO cells.
The fusion proteins were based on the binding motifs of the 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) and the different fusions partners described above. The IL-1R-I binding Z variants a)-k) contained different scaffold mutations, (positions 1, 2, 23, 52 and/or 53) or a deletion combined with mutations (deletion at positions 1-3; AV1 D2A3, mutations in positions 52 and 53), see Table 22 below. The mutated Z variants are listed in
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
Cultivation and purification of fusion proteins: E. coli cells were transformed with expression vectors containing the gene fragments corresponding to the IL-1R-I binding fusion proteins, see Table 22, and then cultivated in bioreactors using fed-batch techniques or in shake flasks, followed by protein expression and harvest of cells by centrifugation. Cell pellets were stored at −20° C. Expression of IL-1R-I binding Z variants, see Table 22, were also performed using the ExpiCHO expression system (Thermo Fisher Scientific), essentially according to the manufacturer's protocol. Supernatants were harvested by centrifugation 12 days after transfection of expression vectors and stored at −70° C.
Frozen E. coli cell pellets were resuspended and then disrupted by sonication and the cell debris subsequently removed by centrifugation followed by filtration (0.22 μm). The frozen supernatants from the ExpiCHO cultures were thawed and filtrated (0.22 μm). Each supernatant, containing the IL-1R-I binding Z variants was purified using conventional chromatography methods, such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. Z variants for use in animal studies were also subjected to an endotoxin removal purification using Detoxi-Gel Endotoxin Removing Columns (Pierce, cat.no. 20344). Purified Z variants were buffer exchanged to PBS and, unless otherwise stated, PBS was also the formulation buffer used in subsequent experiments. 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).
The IL-1R-I binding fusion protein PS10589 (SEQ ID NO:1658), a dimeric Z18754 variant linked in the hinge region by disulfides, was obtained by removing the IgG1 Fc part from PS10536, see Table 22. This was achieved by incubation of purified PS10536 with the IdeS protease (FabRICATOR, Genovis AB). The cleavage products were then purified using conventional chromatography methods such as ion exchange chromatography and affinity chromatography. The purified dimeric Z18754 variant was subjected to buffer exchange into PBS, which was also the formulation buffer used in subsequent experiments. The purity was analyzed by SDS-PAGE stained with Coomassie Blue and the molecular weight was analyzed using mass spectrometry (HPLC/MS or MALDI-TOF/MS).
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. The Fc fused IL-1R-I binding Z variants denoted PS10536, PS10537, PS10534 and PS10535 (SEQ ID NO:1648, 1649, 1646, 1647) were loaded onto a Protein A sensor (Pall, ForteBio cat. no. 18-5010) at 0.5 μg/ml for 150 s in each cycle using an OctetRED96 instrument (Pall, ForteBio). Subsequently, these sensors were exposed to different and increasing concentrations of human IL-1R-I (R&D systems cat. no. 269-1R/CF) ranging from 0.15 to 5 nM or cynomolgus IL-1R-I (SEQ ID NO:I665) ranging from 2.5 to 80 nM. Association was recorded for 300 s and dissociation for 300 s (human) or 90 s (cynomolgus) and spectrograms were recorded and analyzed according to a 1:1 Langmuir model using global fit (Octet System Data Analysis 8.2 software, Pall, ForteBio). Each sensor was referenced to a sensor with the same ligand loaded onto it but exposed to buffer only. Following each association/dissociation cycle, all sensors were regenerated by three pulses of 10 mM glycine at pH 2.5 for 10 s each. All steps were carried out at 25° C. at a shaking speed of 1000 rpm and all dilutions of fusion proteins (Z-Fc fusion proteins) and analytes (human or cynomolgus IL-1R-1) were in 1×kinetics buffer (Pall, ForteBio).
Cultivation and purification: All the IL-1R-I binding fusion proteins (Table 22) were expressed to high levels in E. coli or CHO cells as soluble proteins. Purification resulted in protein preparations with high purity, which was analyzed by SDS-PAGE stained with Coomassie Blue. The correct identity and molecular weight of each Z variant were confirmed by mass spectrometry analysis.
BLI kinetic analysis: Four Z-Fc fusion protein variants (SEQ ID NO: 1648, 1649, 1646, 1647) were analyzed for the ability to bind human and cynomolgus IL-1R-I loaded onto protein A sensor using BLI technology on an OctetRED96 instrument. All four variants bound IL-1R-I according to a 1:1 model with B-max values ranging from 0.46 to 0.72 nm (human) or 0.31 to 0.37 nm (cynomolgus). The kinetic data; association rate constant (kon), dissociation rate constant (koff) and dissociation constant KD for the Z-Fc fusion protein variants are presented in Table 23.
The inhibitory effect of IL-1R-I binding fusion proteins on IL-1β induced IL-6 production in Normal Human Dermal Fibroblasts (NHDF) cells was monitored.
Cells were seeded three days prior to treatment with proteins. Proteins (IL-1R-I binding fusion proteins or anakinra) were diluted to a starting concentration of 100 nM and subsequently serially 1:4 nine times resulting in a concentration range of 100 nM to 0.38 μM in serum-free growth medium in the presence of 9 μM recombinant human serum albumin (rHSA). The IL-1R-I binding fusion proteins or anakinra were tested in presence of a challenge dose of 3.4 pM IL-1β and the cells were incubated for 22 hours with proteins at 37° C., followed by harvesting of medium. Harvested medium was diluted 41× before IL-6 content was analyzed using a human IL-6 ELISA kit (R&D Systems) according to manufacturer's recommendations. Data was analyzed using XLfit and IC50 values were calculated from concentration-response curves.
The inhibitory effect of His6-tagged IL-1R-I binding Z variants, produced as described in Example 9, on IL-1β induced IL-6 production in NHDF cells was similarly monitored and IC50 values calculated.
Chemically synthesized protein: A chemically synthesized version of SEQ ID NO:1672, denoted PS10558, was ordered from BACHEM AG. The activity of PS10558 was assessed according to the method set out above.
The IL-1β induced IL-6 release from NHDF cells was reduced in a concentration-dependent manner by the IL-1R-I binding fusion proteins as well as by anakinra. Each IL-1R-I binding fusion protein was tested twice and the data from both experiments along with historical average of anakinra (n>20) in this assay are presented in Table 24 and one of the two experiments is also presented in
Some IL-1R-I binding fusion proteins were tested in the same manner but only one time. The results from this experiment are presented in Table 25.
The IL-1β inhibition ability of His6-tagged IL-1R-I-binding Z variants, previously demonstrated in a TF-1 cell assay (Example 9, Table 20), was confirmed in this NHDF assay (results not shown).
The IC50 value of the chemically synthesized PS10558 was 690 μM.
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.
Blood from healthy donors was collected 1-3 hours prior to treatment with the IL-1R-I binding fusion proteins (SEQ ID NO:1648-1649). Proteins (IL-1R-I binding fusion proteins or anakinra) were diluted to starting concentration of 500 nM in serum free RPMI (ThermoFisher Scientific, RPMI 1640 Medium, GlutaMAX™ Supplement, Cat. No.: 61870010) and subsequently serially diluted 1:3 in a ten-step dilution series resulting in a concentration range of 500 nM to 25 pM. The IL-1R-I binding fusion polypetides or anakinra were tested in presence of a challenge dose of 100 pM IL-1β. The blood was incubated for 21-23 hours with proteins, followed by harvesting of plasma. Harvested plasma was diluted 2× and IL-6 content was analyzed by using a human IL-6 kit (V-PLEX Human IL-6 Kit Human interleukin-6, MSD) according to manufacturer's recommendations.
IL-6 concentrations (pg/ml) were calculated using a standard curve (provided by the kit) and data was analyzed using XLfit and IC50 values were calculated from the concentration-response curves.
The IL-1β induced IL-6 release in human whole blood was reduced in a concentration-dependent manner by the IL-1R-I binding fusion proteins as well as by anakinra. IL-1R-I binding fusion proteins were tested in blood from five to six individual healthy donors and compared to anakinra data from ten individual blood donors (
In this Example, the pharmacokinetics of IL-1R-1-binding fusion proteins (SEQ ID NO:1646-1651) were evaluated in a single dose study in male rats.
Fusion proteins: The IL-1R-I binding fusion proteins PS10534, PS10535, PS10536, PS10537, PS10538 and PS10539 (see Table 22, SEQ ID NO:1646-1651) were evaluated in this study. All six test items were constituted as a solution in 25 mM sodium phosphate and 125 mM sodium chloride, pH 7.0.
In-life phase: The pharmacokinetic properties of the six fusion proteins were investigated in male Sprague-Dawley rats. For each test item, three rats were given an i.v. single dose of 15 mg/kg (2.5 ml/kg) injected in the lateral tail vein and another three rats were given a s.c. single dose of 30 mg/kg (5 ml/kg) injected in the neck region. Blood samples for serum preparation were collected pre dosing and at 5 and 20 min, 1, 4, 8, 24, 48, 72 and 96 hrs post i.v. dosing, or 20 min, 1, 4, 8, 24, 48, 72, 96 and 120 hrs post s.c. dosing.
Quantitative ELISA: Determination of fusion protein levels in rat serum samples was performed by enzyme-linked immunosorbent assay (ELISA). The ELISA assay employed the quantitative sandwich enzyme immunoassay technique.
In brief; a goat polyclonal anti-Z antibody recognizing the Z variant domain of the IL-1R-I binding fusion protein (produced in house, Affibody AB) was coated onto a microplate. Unbound polyclonal antibody was washed away and casein was added as blocking agent to reduce unspecific binding to the plastic surface. After removal of unbound casein by a second wash step, standards and samples were pipetted to the wells and any fusion protein present was bound to the immobilized antibody. After washing away any unbound substances, a HRP labled Swine anti-rabbit antibody (Dako, cat. no. P0399) was added. Following a wash to remove any unbound anti-rabbit HRP reagent, a substrate solution was added to the wells and color developed in proportion to the amount of fusion protein bound in the initial step. The color development was stopped and the intensity of the color was measured.
Pharmacokinetic analysis: The pharmacokinetic analysis was based on median serum concentration versus time data from each dose group. The observed maximum concentration (Cmax) and the time to maximum serum concentration (tmax) were taken directly from the bioanalytical data. Other pharmacokinetic parameters, i.e. clearance (CL), apparent clearance following s.c. administration (CL/F), apparent volume of distribution at steady-state (Vss), mean residence time (MRT) and terminal half-life (t1/2z), were estimated by non-compartmental analysis using Phoenix WinNonlin software version 6.3 (Pharsight Corp., USA). Estimation of the terminal slope following i.v. dosing was based on four data points, from 24 to 96 hrs. Estimation of the terminal slope following s.c. dosing was based on five data points, from 24 to 120 hrs. Calculation of the subcutaneous bioavailability (F) was performed using Microsoft Excel.
There was no significant difference in the pharmacokinetics between the six tested fusion proteins. The pharmacokinetics following i.v. administration was characterized by a clearance in the order of 2.4 ml/h·kg and a volume of distribution of about 140 ml/kg, translating into a mean residence time of ca 60 hrs (Table 26).
The subcutaneous bioavailability was on average 50% and peak levels were observed at 24 hrs after dosing. The serum levels then declined with a half-life ranging from 45 to 59 hrs (Table 27).
Fusion proteins: The IL-1R-I binding fusion protein denoted PS10536 (SEQ ID NO:1648) was constituted as a solution in 25 mM sodium phosphate and 125 mM sodium chloride, pH 7.0.
In-life phase: The pharmacokinetic properties of PS10536 were investigated in naive male Cynomolgus monkeys. Three monkeys were given an i.v. single dose of 5 mg/kg (1 ml/kg) injected in the tail vein and another three monkeys were given a s.c. single dose of 10 mg/kg (1 ml/kg) injected in the dorsal region. Blood samples for serum preparation were collected pre dosing and at 5 and 20 min, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 240 and 504 hrs post i.v. dosing, or 20 min, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 240 and 504 hrs post s.c. dosing.
Quantitative ELISA: Quantification of PS10536 in serum from monkeys was performed according to the ELISA described in Example 13.
Pharmacokinetic analysis: Pharmacokinetic parameters were based on individual serum concentration versus time data and determined using WinNonlin software as described in Example 13. Estimation of the terminal slope following i.v. dosing was based on seven data points, from 72 to 504 hrs. Estimation of the terminal slope following s.c. dosing was based on eight data points, from 48 to 504 hrs. Calculation of the subcutaneous bioavailability (F) was performed using Microsoft Excel.
The pharmacokinetics of PS10536 (SEQ ID NO:1648) following i.v. administration was characterized by a low clearance (1.07 ml/h·kg) and a small volume of distribution (117 ml/kg), translating into a mean residence time of 109 hrs (Table 28).
The mean subcutaneous bioavailability of PS10536 was 73.9%. Peak levels were observed at an average of 18.7 hrs after dosing. The serum levels then declined with a half-life of 77.3 hrs (Table 29).
Assuming a minimum target serum concentration of 100 nM or even 200 nM, the high subcutaneous bioavailability in combination with the low clearance facilitates a once weekly subcutaneous dose regimen of 10 mg/kg PS10536 in monkey.
Four IL-1-R-I binding fusion proteins of the IL-1R-I binding polypeptide variant Z18754 (SEQ ID NO:1252) with mutated variants of the Fc portion of IgG1 or IgG4 were produced. In the IgG1 Fc portion, one mutation [N297A] was introduced in order to abolish effector functions through interaction with FCγR3A. Fc-containing polypeptides produced in mammalian cells might otherwise cause such effector functions. In addition, in one fusion protein (SEQ ID NO:1735) two other residues were introduced, denoted LS (WO2009/086320), to increase affinity to FcRn, and in another fusion protein (SEQ ID NO:1736) three mutations denoted YTE (WO02060919) were introduced to increase affinity to FcRn. One fusion protein of Z18754 (SEQ ID NO:1252) with an IgG4 Fc a subtype, which is known to elicit low effector responses, was produced (SEQ ID NO:1737). One mutation, known as S228P (van der Neut Kolfschoten M. et al., 2007, Science 317(5844):1554-7), was furthermore introduced in the hinge region of this fusion protein. This mutation abolishes the arm exchange that may take place for the IgG4 subclass of antibodies.
The fusion proteins PS10653-PS10656 (SEQ ID NO:1734-1737), see Table 30 for specifications, were produced in CHO cells according to Example 10.
The inhibitory effect of IL-1R-I binding fusion proteins on IL-1β induced IL-6 production in Normal Human Dermal Fibroblasts (NHDF) cells was monitored as set out in Example 11.
The affinity towards FCγR3A was analysed with Biolayer Interferometry essentially as disclosed in Example 10, with the exception that biotinylated FCγR3A (Sino Biological) was immobilized on a Streptavidin biosensor for kinetics (Pall/Fortebio) and the proteins were tested using concentrations of 100 and 1000 nM. In addition Human IgG (GammaNorm, Octapharma) was used as control in the measurements.
The affinity towards FcRn was assessed using Biacore essentially as described in Example 9 with the exception that human biotinylated FcRn (Immunitrack ApS) was immobilized on a Biotin CAPture chip (GE Healthcare) and the analytes were tested in five concentrations, ranging from 2.5-200 nM.
Cultivation and purification: All the IL-1-R-I binding fusion proteins with mutated Fc portions (Table 30) were expressed at high levels in CHO cells as soluble proteins. Purification resulted in protein preparations with high purity, which were analyzed by SDS-PAGE stained with Coomassie Blue. The correct identity and molecular weight of each Z variant were confirmed by mass spectrometry analysis.
In vitro pharmacological activity: The IL-1β induced IL-6 release from NHDF cells was reduced in a concentration-dependent manner by the IL-1R-1 binding fusion proteins. The IL-1R-1 binding fusion proteins were tested one time. The results from this experiment are accounted for in Table 31.
FCγR3A affinity analysis: Human IgG bound to FCγR3A with an affinity of 57 nM at 1000 nM concentration. The fusion proteins PS10653-PS10655 (SEQ ID NO 1734-1736) showed no binding at either concentration. PS10656 (SEQ ID NO 1737) showed a weak binding at 1000 nM but no binding at 100 nM.
Biacore FcRn affinity analysis: The affinity towards FcRn were in the order SEQ ID NO 1735>SEQ ID NO 1736>SEQ ID NO 1734=SEQ ID NO 1737.
Conclusion: The mutated Fc variants of the fusion proteins (SEQ ID NO:1734:1737) were equal to the previously produced proteins in terms of quality and activity (see Example 10). The removal of glycan attachment site removed the effector function of the IgG1 Fc portion in SEQ ID NO:1734-1736 as binding to FCγR3A was abolished. The IgG4 Fc containing SEQ ID NO:1737 had low affinity towards FCγR3A as expected for an Fc of that subclass. The FcRn affinity enhancing mutations in SEQ ID NO:1735 and 1736 displayed increased affinity, with SEQ ID NO:1735 showing the greatest increase.
1. IL-1R-I binding polypeptide, comprising an IL-1R-I binding motif BM, which motif consists of an amino acid sequence selected from:
wherein, independently from each other,
2. IL-1R-I binding polypeptide, wherein said IL-1R-I binding motif consists of an amino acid sequence selected from a sequence wherein in i)
3. IL-1R-I binding polypeptide according to any one of the preceding items, wherein sequence i) fulfills at least five of the ten conditions I-X:
4. IL-1R-I binding polypeptide according to item 3, wherein sequence i) fulfills at least six of the ten conditions I-X.
5. IL-1R-I binding polypeptide according to item 4, wherein sequence i) fulfills at least seven of the ten conditions I-X.
6. IL-1R-I binding polypeptide according to item 5, wherein sequence i) fulfills at least eight of the ten conditions I-X.
7. IL-1R-I binding polypeptide according to item 6, wherein sequence i) fulfills at least nine of the ten conditions I-X.
8. IL-1R-I binding polypeptide according to item 7, wherein sequence i) fulfills all of the ten conditions I-X.
9. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X2X3X6 is VEQ or VEY.
10. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X2X3X6 is IEQ or VEQ.
11. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X6X10 is selected from the group consisting of QF, QY, YF and YY.
12. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X6X10 is QF.
13. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X10X18 is selected from the group consisting of FK, FR, YK and YR.
14. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X10X18 is FK or FR.
15. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X18X25X28 is KRL or RRL.
16. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X5X7 is IM or VM.
17. IL-1R-I binding polypeptide according to any preceding item, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-1632, and 1679, such as the group consisting of SEQ ID NO:20-1632, and 1679.
18. IL-1R-I binding polypeptide according to any preceding item, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1206-1632 and 1679, such as the group consisting of SEQ ID NO:1210-1632 and 1679.
19. IL-1R-I binding polypeptide according to any preceding item, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594 and 1679, such as the group consisting of SEQ ID NO:1252, 1328, 1435 and 1679.
20. IL-1R-I binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1252.
21. IL-1R-I binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1328.
22. IL-1R-I binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1435.
23. IL-1R-I binding polypeptide according to any preceding item, wherein said IL-1R-I binding motif forms part of a three-helix bundle protein domain.
24. IL-1R-I binding polypeptide according to item 23, wherein said IL-1R-I binding motif essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.
25. IL-1R-I binding polypeptide according to item 24, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.
26. IL-1R-I binding polypeptide according to item 25, wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.
27. IL-1R-I binding polypeptide according to any preceding item, which comprises a binding module BMod, the amino acid sequence of which is selected from:
28. IL-1R-I binding polypeptide according to any preceding item, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-1638, 1667-1668 and 1670-1679; such as the group consisting of SEQ ID NO:20-1638 and 1670-1679.
29. IL-1R-I binding polypeptide according to item 28, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1206-1632 and 1670-1679, such as the group consisting of SEQ ID NO:1210-1632 and 1670-1679.
30. IL-1R-I binding polypeptide according to item 28, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594 and 1670-1679, such as the group consisting of SEQ ID NO:1252, 1328, 1435, 1672, 1675-1676 and 1679.
31. IL-1R-I binding polypeptide according to item 30, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1672.
32. IL-1R-I binding polypeptide according to item 30, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1675.
33. IL-1R-I binding polypeptide according to item 30, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1676.
34. IL-1R-I binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
wherein [BM] is an IL-1R-I binding motif as defined in any one of items 1-22.
35. IL-1R-I binding polypeptide according to any one of items 1-34, which comprises an amino acid sequence selected from:
wherein [BM] is an IL-1R-I binding motif as defined in any one of items 1-22; and
xx) an amino acid sequence which in the sequences flanking the BM has at least 89% identity to the sequence defined in xix).
36. IL-1R-I binding polypeptide according to any one of items 1-34, which comprises an amino acid sequence selected from:
wherein [BM] is an IL-1R-I binding motif as defined in any one of items 1-22; and
xxii) an amino acid sequence which in the sequences flanking the BM has at least 89% identity to the sequence defined in xxi).
37. IL-1R-I binding polypeptide according to item 35 or 36, wherein sequence xix) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-1632, 1667-1668 and 1670-1679; such as the group consisting of SEQ ID NO:20-1632, 1667-1668 and 1670-1679.
38. IL-1R-I binding polypeptide according to item 37 wherein xix) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting SEQ ID NO:1206-1632, 1667-1668 and 1670-1679, such as the group consisting of SEQ ID NO:1210-1632, 1667-1668 and 1670-1679.
39. IL-1R-I binding polypeptide according to item 38, wherein sequence xix) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594 and 1670-1679, such as the group consisting of SEQ ID NO:1252, 1328, 1435, 1672, 1675-1676 and 1679.
40. IL-1R-I binding polypeptide according to item 39, wherein sequence xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:1672.
41. IL-1R-I binding polypeptide according to item 39, wherein sequence xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:1675.
42. IL-1R-I binding polypeptide according to item 39, wherein sequence xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:1676.
43. IL-1R-I binding polypeptide according to any preceding item, which is capable of blocking IL-1R-I dependent signaling.
44. IL-1R-I binding polypeptide according to item 43, wherein the half maximal inhibitory concentration (IC50) of the blocking is at most 1×10−7 M, such as at most, 1×10−8 M, such as at most 1×10−9 M, such as at most 5×10−10 M.
45. 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. IL-1R-I binding polypeptide according to any preceding item which is capable of binding to IL-1R-I such that the EC50 value of the interaction is at most 1×10−7 M, such as at most 1×10−8 M, such as at most 5×10−9 M, such as at most 1×10−10 M such as at most 5×10−10 M, such as at most 2×10−10 M.
47. IL-1R-I binding polypeptide according to any preceding item which is capable of binding to IL-1R-I such that the KD value of the interaction is at most 1×10−6 M, such as at most 1×10−7 M, such as at most 1×10−8 M, such as at most 1×10−9 M, such as at most 9×10−10.
48. IL-1R-I binding polypeptide according to any one of items 43-47, wherein said IL-1R-I is human IL-1R-I or cynomolgus IL-1R-I, such as human IL-1R-I.
49. IL-1R-I binding polypeptide according to any preceding item which comprises additional amino acids at the C-terminal and/or N-terminal end.
50. IL-1R-I binding polypeptide according to item 49, wherein said additional amino acid(s) improve(s) production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide.
51. IL-1R-I binding polypeptide according to any preceding item in multimeric form, comprising at least two IL-1R-I binding polypeptide monomer units, the amino acid sequences of which are the same or different.
52. IL-1R-I binding polypeptide according to item 51, wherein said IL-1R-I binding polypeptide monomer units are covalently coupled together.
53. IL-1R-I binding polypeptide according to item 52, wherein the IL-1R-I binding polypeptide monomer units are expressed as a fusion protein.
54. IL-1R-I binding polypeptide according to any one of items 51-53, in dimeric form.
55. Fusion protein or conjugate comprising
56. Fusion protein or conjugate according to item 55, wherein said desired biological activity is a therapeutic activity.
57. Fusion protein or conjugate according to item 55, wherein said desired biological activity is a binding activity.
58. Fusion protein or conjugate according to item 55, wherein said desired biological activity is a binding activity modifying tissue distribution of said fusion protein or conjugate.
59. Fusion protein or conjugate according to item 57, wherein said desired biological activity is an in vivo half-life increasing activity such that said second moiety increases in vivo half-life of the fusion protein or conjugate.
60. Fusion protein or conjugate according to item 59, said in vivo half-life increasing activity is an albumin binding activity.
61. Fusion protein or conjugate according to item 60, wherein said albumin binding activity is provided by the albumin binding domain of streptococcal protein G or a derivative thereof.
62. Fusion protein or conjugate according to item 61, wherein said second moiety comprises a polypeptide having an amino acid sequence as set out in SEQ ID NO:1659-1661, such as SEQ ID NO:1661.
63. Fusion protein or conjugate according to item 59, wherein said second moiety is serum albumin.
64. Fusion protein or conjugate according to item 59, wherein said second moiety is an Fc portion of an antibody, such as an IgG1 Fc or an IgG4 Fc.
65. Fusion protein or conjugate according to item 64, wherein said Fc is a mutated Fc which relative to an unmutated form of Fc displays improved affinity to FcRn and/or reduced effector response.
66. Fusion protein or conjugate according to item 58 or 59, wherein said second moiety is transferrin.
67. Fusion protein or conjugate according to any one of items 59-65, comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1639-1658 and 1734-1737, such as from the group consisting of SEQ ID NO:1646-1654 and 1734-1737.
68. Fusion protein or conjugate according to item 57, wherein said binding activity acts to block a biological activity.
69. Fusion protein or conjugate according to any one of items 55-68, further comprising at least one linker, said linker optionally being selected from the group consisting of flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers.
70. Fusion protein or conjugate according item 69, wherein said linker is arranged between said first moiety and said second moiety.
71. Fusion protein or conjugate according item 69, wherein said linker is arranged within said first moiety.
72. Fusion protein or conjugate according item 69 or 70, wherein said linker is a flexible linker comprising at least one amino acid residue(s) selected from the group consisting of glycine, serine and alanine.
73. Fusion protein or conjugate according to item 72, wherein said linker is selected from the group consisting of GS, VDSS, VDGS, VEGS, ASGS, (GGGGS)2 (SEQ ID NO:1683), AS(GGGGS)2 (SEQ ID NO:1684) and ((KEAAA)3KELAA)2 (SEQ ID NO:1685).
74. Fusion protein or conjugate according to item 73, wherein said linker is selected from a AS(GGGGS)2 (SEQ ID NO:1684) and ((KEAAA)3KELAA)2 (SEQ ID NO:1685).
75. A polynucleotide encoding a polypeptide or a fusion protein according to any one of items 1-74.
76. Expression vector comprising a polynucleotide according to item 75.
77. Host cell comprising an expression vector according to item 76.
78. Method of producing a polypeptide according to any one of items 1-77, comprising
79. Composition comprising an IL-1R-I binding polypeptide, fusion protein, or conjugate according to any one of items 1-74 and at least one pharmaceutically acceptable excipient or carrier.
80. Composition according to item 79, further comprising at least one additional active agent, such as an agent selected from an immune response modifying agent and an anti-cancer agent.
81. Composition according to item 79 or 80, wherein said fusion protein is a fusion protein according to any one of claims 59-67.
82. IL-1R-I binding polypeptide, fusion protein, or conjugate according to any one of items 1-74 or a composition according to any one of items 79-81 for use as a medicament, a diagnostic agent and/or a prognostic agent.
83. IL-1R-I binding polypeptide, fusion protein, conjugate or composition according to item 82 for use as a medicament.
84. 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. IL-1R-I binding polypeptide, fusion protein, conjugate or composition according to item 82 for use as a diagnostic agent and/or a prognostic agent.
86. IL-1R-I binding polypeptide, fusion protein, conjugate or composition for use according to any one of items 82-85, in the treatment, prognosis or diagnosis of an IL-1R-I related disorder.
87. IL-1R-I binding polypeptide, fusion protein, conjugate or composition for use according to item 86, wherein said IL-1R-I related disorder is selected from the group consisting of inflammatory disease, auto-inflammatory syndromes, autoimmune disease, infectious disease, cardiovascular disease, ischaemic disease, cancer and diabetes.
88. IL-1R-I binding polypeptide, fusion protein, conjugate or composition for use according to item 86 or 87, wherein said IL-1R-I related disorder is selected from the group consisting of 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 syndrome; Muckle Wells Syndrome; macrophage activation syndrome; Behget's disease; uveitis; acne vulgaris; pyoderma gangrenosum; gout; type 2 diabetes, new-onset diabetes; dry eye syndrome; hidradenitis suppurativa; neutrophilic dermatoses, in particular cytophagic histiocytic panniculitis, Weber-Christian disease, and neutrophilic panniculitis; cardiovascular disease, myocardial infarction, stroke; liver failure, kidney failure; acute lung injury; pseudogout, calcium-pyrophosphate deposition disorder, chondrocalcinosis, deficiency of the IL-1 receptor antagonist (DIRA), deficiency of the IL-36 receptor antagonist (DITRA), ADAM2 deficiency (DADA2), pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome, pyoderma gangrenosum, acne and suppurative hidradenitis (PASH) syndrome, PAPA and suppurative hidradenitis (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, haemochromatosis-related arthritis, periarticular calcinosis, osteoarthritis, inflammatory osteoarthritis, hand osteoarthritis, pustular psoriasis such as generalised pustular psoriasis (GPP), palmoplantar pustulosis (PPP), acrodermatitis continua of Hallopeau (ACH); Blau syndrome; Sweet syndrome; various vasculitides such as giant-cell arteritis (GCA), polymyalgia rheumatica (PMR), Takayasu 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), indolent myeloma, Waldenstrom's macroglobulinemia, and multiple myeloma; tumor-induced cachexia; solid tumor growth; neonatal disorders such as bronchopulmonary dysplasia (prophylaxis), necrotising enterocolitis (NEC), retinopathy of prematurity (ROP), cerebral palsy due to perinatal cerebral ischemia, and infant respiratory distress syndrome (IRDS); Whipple's disease; traumatic brain injury; refractory epilepsy; systemic inflammatory response syndrome (SIRS); cutaneous lupus; Jessner-Kanof disease; amyotrophic lateral sclerosis; systemic sclerosis (scleroderma); septic shock; acute pancreatitis; chronic recurrent multifocal osteomyelitis, non-bacterial osteitis (NBO), synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome, and Majeed syndrome; relapsing polychondritis; idiopathic recurrent pericarditis (IRP); myocarditis; Erdheim-Chester disease; juvenile xantogranuloma; islet-cell transplantation; haemodialysis-induced systemic inflammation; graft-versus-host disease; ANCA-associated glomerulonephritis and recurrent glomerulonephritis; Cogan syndrome; autoimmune inner-ear disease; chronic granulomatous disease (CGD); Castleman's disease; cardiac failure; diastolic cardiac failure; antisynthetase syndrome; acute ACL injury; acute haemorrhagic leukoencephalitis; AA amyloidosis; Di George syndrome; generalised fatigue; chronic fatigue syndrome (CFS); gulf-war illness (GWI); and narcolepsy.
89. IL-1R-I binding polypeptide, fusion protein, conjugate or composition for use according to item 87, wherein said IL-1R-I related 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. IL-1R-I binding polypeptide, fusion protein, conjugate or composition for use according to any one of items 82-89, wherein said IL-1R-I binding polypeptide, fusion protein, conjugate or composition is administered repeatedly within a 24 hour period from disease onset, such as at least two times within a 24 hour period from disease onset, such as at least three times within a 24 hour period from disease onset, such as continuously during 24 hours from disease onset, to a subject in need thereof.
91. Fusion protein, conjugate or composition for use according to any one of items 82-84, 86-89, wherein said fusion protein or conjugate is a fusion protein or conjugate according to any one of items 58-67 and said composition is a composition according to item 81.
92. Fusion protein, conjugate or composition for use according to item
91, wherein said fusion protein, conjugate or composition is administered once weekly to a subject in need thereof.
93. Method of treatment of an IL-1R-I related 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 1-74 or a composition according to any one of items 79-81.
94. Method according to item 93, wherein said IL-1R-I related disorder is selected from the group consisting inflammatory disease, auto-inflammatory syndrome, autoimmune disease, infectious disease, cardiovascular disease, ischaemic disease, cancer and diabetes.
95. Method according to item 93 or 94, wherein said IL-1R-I related disorder is selected from the group as defined in item 88.
96. Method according to item 94, wherein said IL-1R-I related disorder is cancer, such as a cancer selected from the group consisting of selected from the group consisting of multiple myeloma, colon cancer, breast cancer, lung cancer, head and neck cancer, melanoma and prostate cancer.
97. Method according to any one of items 93-96, wherein said IL-1R-I binding polypeptide, fusion protein or conjugate or composition is administered repeatedly within a 24 h period from disease onset, such as at least two times within a 24 hour period from disease onset, such as at least three times within a 24 hour period from disease onset, such as continuously during 24 hours from disease onset, to a subject in need thereof.
98. Method according to any one of claims 93-96, wherein said fusion protein or conjugate is a fusion protein or conjugate according to any one of items 59-67 and said composition is a composition according to item 81.
99. Method according to item 97, wherein said fusion protein, conjugate or composition is administered once weekly to a subject in need thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17164226.7 | Mar 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/058448 | 4/3/2018 | WO | 00 |
