IL-IR-I BINDING POLYPEPTIDE
20210403533 · 2021-12-30
Inventors
- Joachim Feldwisch (Tyreso, SE)
- Malin Lindborg (Saltsjo-Boo, SE)
- Joakim Nilsson (Danderyd, SE)
- Erik Nordling (Danderyd, SE)
- Robert Svensson (Stockholm, SE)
Cpc classification
C07K2318/20
CHEMISTRY; METALLURGY
C07K2319/31
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
C07K14/79
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present disclosure relates to a class of engineered polypeptides having a binding affinity for interleukin-1 receptor type-I (IL-1R-I) which comprise the binding motif (BM) EX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7EIX.sub.10X.sub.11LPNLX.sub.16RX.sub.18QYX.sub.21AFIX.sub.25X.sub.26LX.sub.28D. The present disclosure also relates to the use of such an IL-1R-I binding polypeptide as a therapeutic, prognostic and/or diagnostic agent.
Claims
1. IL-1R-1 binding polypeptide, comprising an IL-1R-1 binding motif BM, which motif consists of an amino acid sequence selected from: i) TABLE-US-00049 (SEQ ID NO: 1686) EX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7EIX.sub.10X.sub.11LPNLX.sub.16RX.sub.18QYX.sub.21AFIX.sub.25X.sub.26LX.sub.28D wherein, independently from each other, X.sub.2 is selected from A, I, L, T and V; X.sub.3 is selected from E and Y; X.sub.4 is selected from A, E, I, K, Q, R, T, V and Y; X.sub.5 is selected from I and V; X.sub.6 is selected from Q and Y; X.sub.7 is selected from F and M; X.sub.10 is selected from F and Y; X.sub.11 is selected from A, D, E, F, G, H, K, L, Q, R, S, T, V and Y; X.sub.16 is selected from N and T; X.sub.18 is selected from K and R; X.sub.21 is selected from T and V; X.sub.25 is selected from I and R; X.sub.26 is selected from K and S, and X.sub.28 is selected from F and L, and ii) an amino acid sequence which has at least 93% identity to the sequence defined in i) provided that X.sub.5 is I or V.
2. (canceled)
3. IL-1R-1 binding polypeptide according to claim 1, wherein sequence i) fulfills at least five, such as at least six, at least seven, at least eight, at least nine or all of the ten conditions I-X: I. X.sub.3 is E; II. X.sub.5 is selected from I and V; III. X.sub.6 is selected from Q and Y; IV. X.sub.7 is selected from F and M; V. X.sub.10 is selected from F and Y; VI. X.sub.18 is selected from K and R; VII. X.sub.21 is T; VIII. X.sub.25 is R; IX. X.sub.26 is selected from K and S; and X. X.sub.28 is selected from F and L.
4.-6. (canceled)
7. IL-1R-1 binding polypeptide according to claim 1, wherein said IL-1R-1 binding motif forms part of a three-helix bundle protein domain, wherein said IL-1R-1 binding motif preferably essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.
8. (canceled)
9. IL-1R-1 binding polypeptide according to claim 1, which comprises an amino acid sequence selected from: xix) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO:1721) wherein [BM] is an IL-1R-1 binding motif as defined in claim 1; xx) an amino acid sequence which in the sequences flanking the BM has at least 89% identity to the sequence defined in xix); xxi) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO:1709) wherein [BM] is an IL-1R-1 binding motif as defined in claim 1; and xxii) an amino acid sequence which in the sequences flanking the BM has at least 89% identity to the sequence defined in xxi).
10. (canceled)
11. IL-1R-1 binding polypeptide according to claim 9, 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.
12. IL-1R-1 binding polypeptide according to claim 9 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.
13. IL-1R-1 binding polypeptide according to claim 9, 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.
14. IL-1R-1 binding polypeptide according to claim 1, which is capable of blocking the interaction of IL-1R-1 with IL-1 cytokines, such as the interaction of IL-1R-1 with IL-1α and/or IL-1β.
15. IL-1R-1 binding polypeptide according to claim 1, which is capable of binding to IL-1R-1 such that the K.sub.D value of the interaction is at most 1×10.sup.−6 M, such as at most 1×10.sup.−7 M, such as at most 1×10.sup.−8 M, such as at most 1×10.sup.−9 M, such as at most 9×10.sup.−10.
16.-17. (canceled)
18. Fusion protein or conjugate comprising a first moiety consisting of an IL-1R-1 binding polypeptide according to claim 1; and a second moiety consisting of a polypeptide having a desired biological activity.
19.-21. (canceled)
22. Fusion protein or conjugate according to claim 18, 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.
23. Fusion protein or conjugate according to claim 18, wherein said second moiety is selected from the group consisting of the albumin binding domain of streptococcal protein G or a derivative thereof; serum albumin; an Fc portion of an antibody, and transferrin.
24. Fusion protein or conjugate according to claim 23, wherein said second moiety is an Fc portion of an antibody, such as an IgG1 Fc or an IgG4 Fc.
25. (canceled)
26. Fusion protein or conjugate according to claim 18, 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:1648-1654 and 1734-1737.
27.-29. (canceled)
30. Composition comprising an IL-1R-1 binding polypeptide, fusion protein, or conjugate according to claim 1 and at least one pharmaceutically acceptable excipient or carrier.
31.-39. (canceled)
40. IL-1R-1 binding polypeptide, comprising an IL-1R-1 binding motif BM, which motif consists of an amino acid sequence selected from: i) a 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, and ii) an amino acid sequence which has at least 96% identity to the sequence defined in i) provided that X.sub.5 is I or V.
41. IL-1R-1 binding polypeptide according to claim 40, 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.
42. IL-1R-1 binding polypeptide according to claim 40, 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.
43. Method of treatment of an IL-1R-1 related disorder, comprising administering to a subject in need thereof an effective amount of an IL-1R-1 binding polypeptide according to claim 1.
44. Method of treatment of an IL-1R-1 related disorder, comprising administering to a subject in need thereof an effective amount of an IL-1R-1 binding polypeptide according to claim 1, wherein said IL-1R-1 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.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0279]
[0280]
[0281]
EXAMPLES
Summary
[0282] 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
Example 1
Selection and Screening of IL-1R-I Binding Z Variants
[0283] 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
[0284] 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.
[0285] Expression of his-tagged target protein: Expression of cynomolgus IL-1R-I-His.sub.6(cIL-1R-I-His.sub.6, 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-His.sub.6 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).
[0286] 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 1.5×10.sup.10 library members (Z variants). The construction in the phage library vector pAY02592 and the phage stock production was previously described in WO 2016/113246.
[0287] 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-His.sub.6 (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).sub.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-His.sub.6 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.
[0288] 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.
TABLE-US-00014 TABLE 2 Overview of the phage display selection from a naive library. Phage stock Target Number Selection from library or Preselection concentration of Cycle track selection track beads Target (nM) washes 1 1-1 Zlib006Naive.II Fc/Z00000/SA hIL-1R-I-Fc 100 2 1 1-2 Zlib006Naive.II Fc/protein A hIL-1R-I-Fc 100 2 1 1-3 Zlib006Naive.II Fc/SA b-hIL-1R-I-Fc 100 2 1 1-4 Zlib006Naive.II SA b-hIL-1R-I 100 2 2 2-1 1-1 Fc/Z00000/SA hIL-1R-I-Fc 67 4 2 2-2 1-1 Fc/Z00000/SA hIL-1R-I-Fc 33 4 2 2-3 1-2 Fc/protein A hIL-1R-I-Fc 67 4 2 2-5 1-3 Fc/SA b-hIL-1R-I-Fc 67 4 2 2-7 1-4 SA b-hIL-1R-I- 67 4 2 2-9 1-4 IM cIL-1R-I-His.sub.6 67 4 3 3-1 2-1 Fc/Z00000/SA hIL-1R-I-Fc 44 6 3 3-2 2-2 Fc/Z00000/SA hIL-1R-I-Fc 11 6 3 3-3 2-3 Fc/protein A hIL-1R-I-Fc 44 6 3 3-5 2-5 Fc/SA b-hIL-1R-I-Fc 44 6 3 3-7 2-7 SA b-hIL-1R-I 44 6 3 3-9 2-9 SA b-hIL-1R-I 44 6 4 4-1 3-1 Fc/Z00000/SA hIL-1R-I-Fc 30 8 4 4-2 3-2 Fc/Z00000/SA hIL-1R-I-Fc 3.7 8 4 4-3 3-3 Fc/protein A hIL-1R-I-Fc 30 8 4 4-5 3-5 Fc/SA b-hIL-1R-I-Fc 30 8 4 4-7 3-7 SA b-hIL-1R-I 30 8 4 4-9 3-9 IM cIL-1R-I-His.sub.6 30 8
[0289] 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.
[0290] 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).
[0291] 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.
[0292] 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.
[0293] 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.
[0294] EC.sub.50 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 EC.sub.50 values (the half maximal effective concentration) were calculated.
Results
[0295] 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.
[0296] 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).
[0297] 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
[0298] EC.sub.50 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 EC.sub.50 values (Table 3).
TABLE-US-00015 TABLE 3 Calculated EC.sub.50 values from ELISA titration analysis. Z variant SEQ ID NO EC.sub.50 (M) Z12884 13 6.4 × 10.sup.−10 Z12891 19 2.2 × 10.sup.−9 Z12895 12 2.3 × 10.sup.−10 Z12905 7 7.3 × 10.sup.−9 Z12910 11 2.3 × 10.sup.−10 Z12911 18 3.2 × 10.sup.−10 Z12912 10 3.9 × 10.sup.−10 Z12915 5 4.3 × 10.sup.−9 Z12917 15 3.3 × 10.sup.−10 Z12920 14 6.6 × 10.sup.−9 Z12922 3 2.9 × 10.sup.−9 Z12929 17 1.7 × 10.sup.−8 Z12933 2 1.5 × 10.sup.−9 Z12941 4 4.6 × 10.sup.−10 Z12961 16 2.9 × 10.sup.−10 Z12964 1 3.9 × 10.sup.−10 Z12967 9 1.7 × 10.sup.−10 Z12974 8 2.1 × 10.sup.−10 Z12975 6 2.6 × 10.sup.−10
Example 2
Production and Characterization of Primary IL-1R-1 Binding Z Variants
[0299] 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.
Materials and Methods
[0300] Subcloning of Z variants with a His.sub.6 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 His.sub.6 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.
[0301] 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.
[0302] Purification of IL-1R-I binding Z variants with a His.sub.6 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 KH.sub.2PO.sub.4, 8.1 mM Na.sub.2HPO.sub.4, 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 KH.sub.2PO.sub.4, 8.1 mM Na.sub.2HPO.sub.4, pH 7.4) using PD-10 desalting columns (GE Healthcare).
[0303] 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.
[0304] Biacore affinity analysis: Affinities (K.sub.D) for human and cynomolgus IL-1R-I were determined for His.sub.6-tagged Z variants using a Biacore 2000 instrument (GE Healthcare). hIL-1R-I or cIL-1R-I-His.sub.6 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).
[0305] 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 3×10.sup.4 cells per well. In separate plates, serial dilutions of the inhibitory binders; His.sub.6-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% CO.sub.2 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 (IC.sub.50) was determined using GraphPadPrism program.
[0306] Circular dichroism (CD) spectroscopy analysis: Purified His.sub.6-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.
Results
[0307] Production of His.sub.6-tagged Z variants: The IL-1R-I binding Z variants with a His.sub.6 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.
[0308] Biacore affinity analysis: The interactions of His.sub.6-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 (K.sub.D) 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.sup.−9 M and to cynomolgus IL-1R-I with an affinity of 3.5×10.sup.−8 M.
TABLE-US-00016 TABLE 4 Affinities for His.sub.6-Z polypeptides binding to IL-1R-I. Z variant SEQ ID NO Human IL-1R-I K.sub.D (M) Z12884 13 1.8 × 10.sup.−8 Z12895 12 5.5 × 10.sup.−9 Z12911 18 7.1 × 10.sup.−9 Z12917 15 8.0 × 10.sup.−9 Z12922 3 9.8 × 10.sup.−9 Z12967 9 1.2 × 10.sup.−9 Z12974 8 1.5 × 10.sup.−8 Z12975 6 8.9 × 10.sup.−8
[0309] In vitro IL-1β neutralization assay: The IL-1β inhibition ability of His.sub.6-tagged IL-1R-I-binding Z variants was analyzed in a TF-1 cell assay. The resulting IC.sub.50 values are presented in Table 5.
TABLE-US-00017 TABLE 5 IC.sub.50 values for His.sub.6-Z polypeptides from a TF-1 cell assay. Z variant SEQ ID NO IC.sub.50 (nM) Z12884 13 27 Z12895 12 7.1 Z12911 18 30 Z12917 15 11 Z12922 3 66 Z12967 9 1.1 Z12974 8 60 Z12975 6 35 Kineret — 0.05
[0310] CD analysis: The CD spectra determined for the IL-1R-I binding Z variants with a His.sub.6 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.
TABLE-US-00018 TABLE 6 Melting temperatures (Tm). Z variant SEQ ID NO Tm (° C.) Z12911 18 64 Z12967 9 58 Z12922 3 47 Z12974 8 49 Z12975 6 51 Z12884 13 49 Z12917 15 56 Z12895 12 55
Example 3
Design and Construction of a First Matured Library of IL-1R-1 Binding Z Variants
[0311] 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).
Materials and Methods
[0312] 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
TABLE-US-00019 TABLE 7 Design of first maturation library Zlib006IL-1RI.I. Amino acid Amino acid randomization No. of position in the (percentage when uneven different Z variant molecule distribution) amino acids 9 A(30%), E, F, H, I, K, L, Q, R, S, 14 T, V, W, Y 10 A, D, E, F, H, I, K, L, Q, R, S, T, 15 V, W, Y 11 A, D, E, F, H, I, K, L, N, Q, R, S, 16 T, V, W, Y 13 A, F, H, I, L, Q(20%), S, T, V, W, 11 Y(20%) 14 A, D, E, F, H, I, K, L, M, Q, R, S, 16 T, V, W, Y 17 F, W(20%), Y 3 18 A(11%), D, E, F, G, H, I, K, L, N, 17 Q, R, S, T, V, W, Y 24 R 1 25 H, K, R, S 4 27 Y 1 28 T 1 32 A, I, L, R(70%) 4 35 F(80%), L(20%) 2
[0313] 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.
[0314] 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 OD.sub.600 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.
Results
[0315] 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 9.7×10.sup.8 Z variants. The actual size of the library, determined by titration after transformation to E. coli ER2738 cells, was 5.6×10.sup.9 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.
Example 4
Selection and Screening of Z Variants from the First Matured Library
[0316] 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.
Materials and Methods
[0317] 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).
[0318] 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.
TABLE-US-00020 TABLE 8 Overview of selections from the first matured library. Phage stock Target Number Selection from library or concentration Selection of Cycle track selection track Target (nM) time (min) washes 1 1-1 Zlib006IL-1RI.I b-hIL-1R-I-Fc 50 70 4 1 1-2 Zlib006IL-1RI.I b-hIL-1R-I-Fc 10 70 4 1 1-3 Zlib006IL-1RI.I b-hIL-1R-I 50 70 4 1 1-4 Zlib006IL-1RI.I b-cIL-1R-I-Fc 100 70 2 1 1-5 Zlib006IL-1RI.I cIL-1R-I-Fc 100 70 2 1 1-6 Zlib006IL-1RI.I b-hIL-1R-I-Fc 10 45 5 1 1-7 Zlib006IL-1RI.I b-hIL-1R-I 10 45 5 1 1-8 Zlib006IL-1RI.I b-hIL-1R-I-Fc 10 45 5 1 1-9 Zlib006IL-1RI.I b-hIL-1R-I 10 45 5 2 2-1 1-1 b-hIL-1R-I-Fc 20 60 6 2 2-2 1-1 b-hIL-1R-I-Fc 5 60 6 2 2-3 1-2 b-hIL-1R-I-Fc 4 60 8 2 2-4 1-2 b-hIL-1R-I-Fc 4 60 12 2 2-5 1-3 b-hIL-1R-I 20 60 6 2 2-6 1-3 b-hIL-1R-I 10 60 6 2 2-7 1-3 b-hIL-1R-I 10 60 10 2 2-8 1-4 b-cIL-1R-I-Fc 50 60 4 2 2-9 1-5 cIL-1R-I-Fc 50 60 4 2 2-10 1-1 b-cIL-1R-I-Fc 100 60 2 2 2-11 1-1 b-cIL-1R-I-Fc 50 60 6 2 2-12 1-1 cIL-1R-I-Fc 100 60 2 2 2-13 1-1 cIL-1R-I-Fc 50 60 6 2 2-14 1-8 b-hIL-1R-I-Fc 2 30 10* 2 2-15 1-9 b-hIL-1R-I 2 30 10* 2 2-16 1-9 b-cIL-1R-I-Fc 25 30 3 2 2-17 1-6/1-7 pool b-hIL-1R-I 2 30 10* 3 3-1 2-1 b-hIL-1R-I-Fc 5 60 8 3 3-2 2-2 b-hIL-1R-I-Fc 0.5 60 8 3 3-3 2-3 b-hIL-1R-I-Fc 1 60 12 3 3-4 2-4 b-hIL-1R-I-Fc 1 60 24 3 3-5 2-5 b-hIL-1R-I 5 60 8 3 3-6 2-6 b-hIL-1R-I 1 60 8 3 3-7 2-7 b-hIL-1R-I 2 60 20 3 3-8 2-8 b-cIL-1R-I-Fc 25 60 6 3 3-9 2-9 cIL-1R-I-Fc 25 60 6 3 3-10 2-10 b-hIL-1R-I-Fc 25 60 6 3 3-11 2-11 b-hIL-1R-I-Fc 5 60 8 3 3-12 2-12 b-hIL-1R-I-Fc 25 60 6 3 3-13 2-13 b-hIL-1R-I-Fc 5 60 8 3 3-14 2-14 b-hIL-1R-I-Fc 0.5 40 16* 3 3-15 2-15 b-hIL-1R-I 0.5 40 16* 3 3-16 2-16 b-hIL-1R-I 2 40 9 3 3-17 2-17 b-hIL-1R-I 0.4 40 16* 4 4-1 3-1 b-hIL-1R-I-Fc 1 40 10** 4 4-2 3-2 b-hIL-1R-I-Fc 0.05 40 10** 4 4-3 3-3 b-hIL-1R-I-Fc 0.2 40 16** 4 4-4 3-4 b-hIL-1R-I-Fc 0.2 40 36** 4 4-5 3-5 b-hIL-1R-I 1 40 10** 4 4-6 3-6 b-hIL-1R-I 0.1 40 10** 4 4-7 3-7 b-hIL-1R-I 0.4 40 30** 4 4-8 3-8 b-cIL-1R-I-Fc 12.5 40 8** 4 4-9 3-9 cIL-1R-I-Fc 50 40 8** 4 4-10 3-10 b-cIL-1R-I-Fc 50 40 6** 4 4-11 3-11 b-cIL-1R-I-Fc 10 40 12** 4 4-12 3-12 cIL-1R-I-Fc 50 40 6** 4 4-13 3-13 cIL-1R-I-Fc 10 40 12** 4 4-14 3-14 b-hIL-1R-I-Fc 0.1 40 20 4 4-15 3-15 b-hIL-1R-I 0.1 40 20 4 4-16 3-16 b-cIL-1R-I-Fc 5 40 16 4 4-17 3-17 b-hIL-1R-I 0.2 40 16 5 5-1 4-4x b-hIL-1R-I-Fc 50 100 3 5 5-2 4-6x b-hIL-1R-I 50 100 3 *Addition of hlL-1R-I in the second final wash step. **Selection track divided into two after washes. One part submitted to an extra wash, over the weekend, before elution.
[0319] 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.
[0320] 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.
[0321] 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.
[0322] 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.
[0323] EC.sub.50 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 EC.sub.50 values (the half maximal effective concentration) were calculated. A periplasmic extract with Z12967 (SEQ ID NO:9) was analyzed in parallel for signal comparison.
[0324] 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 (K.sub.D) 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.
Results
[0325] 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.
[0326] 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
[0327] 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.
[0328] EC.sub.50 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 EC.sub.50 values (Table 9). The result for Z12967 (SEQ ID NO:9) was 2.4×10.sup.−10 M.
TABLE-US-00021 TABLE 9 Calculated EC.sub.50 values from ELISA titration analysis. SEQ ID Z variant NO EC.sub.50 (M) Z15811 21 2.5 × 10.sup.−10 Z15813 23 3.5 × 10.sup.−10 Z15824 34 2.3 × 10.sup.−10 Z15840 49 2.5 × 10.sup.−10 Z15862 71 2.1 × 10.sup.−10 Z15876 1206 4.2 × 10.sup.−10 Z15886 94 2.6 × 10.sup.−10 Z15897 105 2.5 × 10.sup.−10 Z15923 131 2.4 × 10.sup.−10 Z15927 135 2.2 × 10.sup.−10 Z15929 137 2.4 × 10.sup.−10 Z15932 1207 2.3 × 10.sup.−10 Z15949 154 2.5 × 10.sup.−10 Z15953 158 2.1 × 10.sup.−10 Z15960 165 2.3 × 10.sup.−10 Z15961 166 2.1 × 10.sup.−10 Z15975 1209 2.4 × 10.sup.−10 Z15978 182 4.8 × 10.sup.−10 Z15987 191 2.3 × 10.sup.−10 Z15996 200 2.5 × 10.sup.−10 Z15997 201 2.5 × 10.sup.−10 Z15999 203 2.4 × 10.sup.−10 Z16000 204 2.4 × 10.sup.−10 Z16003 207 2.8 × 10.sup.−10 Z16004 208 2.5 × 10.sup.−10 Z16007 211 2.2 × 10.sup.−10 Z16008 212 2.3 × 10.sup.−10 Z16011 215 2.2 × 10.sup.−10 Z16023 227 2.1 × 10.sup.−10 Z16034 238 2.3 × 10.sup.−10 Z16041 245 2.4 × 10.sup.−10 Z16042 246 2.4 × 10.sup.−10 Z16043 247 2.3 × 10.sup.−10 Z16046 250 2.7 × 10.sup.−10 Z16048 252 2.5 × 10.sup.−10 Z16049 253 2.4 × 10.sup.−10 Z16050 254 2.3 × 10.sup.−10 Z16051 255 2.5 × 10.sup.−10 Z16053 257 2.5 × 10.sup.−10 Z16054 258 2.4 × 10.sup.−10 Z16058 262 2.3 × 10.sup.−10 Z16070 273 2.3 × 10.sup.−10 Z16072 275 2.2 × 10.sup.−10 Z16084 287 2.2 × 10.sup.−10 Z16089 291 2.4 × 10.sup.−10 Z16092 293 2.4 × 10.sup.−10 Z16093 294 2.1 × 10.sup.−10 Z16097 298 2.0 × 10.sup.−10 Z16102 302 2.8 × 10.sup.−10 Z16106 306 2.3 × 10.sup.−10 Z16116 316 2.1 × 10.sup.−10 Z16117 317 2.1 × 10.sup.−10 Z16121 321 2.3 × 10.sup.−10 Z16122 322 2.9 × 10.sup.−10 Z16127 325 2.1 × 10.sup.−10 Z16135 333 2.5 × 10.sup.−10 Z16141 339 2.8 × 10.sup.−10 Z16143 341 2.1 × 10.sup.−10 Z16155 351 2.9 × 10.sup.−10 Z16156 352 2.4 × 10.sup.−10 Z16161 357 2.5 × 10.sup.−10 Z16162 358 2.7 × 10.sup.−10 Z16163 359 1.7 × 10.sup.−10 Z16164 360 6.8 × 10.sup.−10 Z16165 361 2.2 × 10.sup.−10 Z16168 364 2.8 × 10.sup.−10 Z16169 365 1.8 × 10.sup.−10 Z16173 369 3.6 × 10.sup.−10 Z16178 374 2.4 × 10.sup.−10 Z16206 402 4.5 × 10.sup.−10 Z16207 403 2.2 × 10.sup.−10 Z16219 415 2.4 × 10.sup.−10 Z16234 429 5.4 × 10.sup.−10 Z16236 431 2.4 × 10.sup.−10 Z16241 436 4.9 × 10.sup.−10 Z16243 438 2.2 × 10.sup.−10 Z16245 440 2.2 × 10.sup.−10 Z16246 441 3.9 × 10.sup.−10 Z16255 449 1.7 × 10.sup.−10 Z16263 457 2.6 × 10.sup.−10 Z16264 458 3.0 × 10.sup.−10 Z16277 470 2.6 × 10.sup.−10 Z16301 494 2.2 × 10.sup.−10 Z16302 495 2.8 × 10.sup.−10 Z16305 498 2.2 × 10.sup.−10 Z16312 504 2.4 × 10.sup.−10 Z16319 511 3.0 × 10.sup.−10 Z16321 513 2.7 × 10.sup.−10 Z16329 519 4.5 × 10.sup.−10 Z16341 531 1.7 × 10.sup.−10 Z16363 550 2.0 × 10.sup.−10 Z16377 562 2.1 × 10.sup.−10 Z16378 563 2.0 × 10.sup.−10 Z16388 573 2.9 × 10.sup.−10 Z16405 590 3.6 × 10.sup.−10 Z16678 853 2.4 × 10.sup.−10 Z16681 856 2.3 × 10.sup.−10 Z16688 863 2.7 × 10.sup.−10 Z16749 921 2.7 × 10.sup.−10 Z16771 943 2.4 × 10.sup.−10 Z17244 1147 4.2 × 10.sup.−10 Z17255 1158 2.5 × 10.sup.−10 Z17257 1160 4.8 × 10.sup.−10 Z17269 1171 2.7 × 10.sup.−10 Z17270 1172 3.0 × 10.sup.−10 Z17274 1176 2.1 × 10.sup.−10 Z17278 1180 2.7 × 10.sup.−10 Z17279 1181 2.8 × 10.sup.−10 Z17280 1182 2.2 × 10.sup.−10 Z17288 1190 4.2 × 10.sup.−10 Z17292 1194 2.0 × 10.sup.−10 Z17296 1198 3.2 × 10.sup.−10 Z17297 1199 2.4 × 10.sup.−10 Z18557 1205 1.9 × 10.sup.−10
[0329] On average, the Z variants originating from this library (first matured library) displayed an improvement in EC.sub.50 values by a factor 10 compared with the average EC.sub.50 for the Z variants originating from the primary selection.
[0330] 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.sup.−9 M) of the assayed binders. The K.sub.D of Z2967 was 2.5×10.sup.−8 M. The four binders assayed in a S1K experiment got K.sub.D values that deviated from their respective screening kinetics affinities by 0-14%.
TABLE-US-00022 TABLE 10 Calculated K.sub.D values from Biacore kinetic screening. SEQ ID Z variant NO K.sub.D (M) Z15824 34 9.2 × 10.sup.−9 Z15840 49 1.1 × 10.sup.−8 Z15864 73 1.6 × 10.sup.−8 Z15873 82 2.3 × 10.sup.−8 Z15876 1206 8.3 × 10.sup.−9 Z15882 90 9.0 × 10.sup.−9 Z15889 97 1.5 × 10.sup.−8 Z15897 105 1.2 × 10.sup.−8 Z15908 116 1.7 × 10.sup.−8 Z15947 152 1.1 × 10.sup.−8 Z15953 158 1.1 × 10.sup.−8 Z15972 177 1.2 × 10.sup.−8 Z15986 190 9.5 × 10.sup.−9 Z16009 213 2.0 × 10.sup.−8 Z16010 214 1.2 × 10.sup.−8 Z16011 215 1.1 × 10.sup.−8 Z16012 216 1.2 × 10.sup.−8 Z16013 217 1.8 × 10.sup.−8 Z16014 218 1.1 × 10.sup.−8 Z16015 219 1.5 × 10.sup.−8 Z16016 220 1.2 × 10.sup.−8 Z16017 221 1.8 × 10.sup.−8 Z16018 220 1.6 × 10.sup.−8 Z16019 223 1.5 × 10.sup.−8 Z16020 224 2.0 × 10.sup.−8 Z16021 225 9.6 × 10.sup.−9 Z16022 226 1.5 × 10.sup.−8 Z16023 227 9.2 × 10.sup.−9 Z16024 228 1.4 × 10.sup.−8 Z16025 229 1.9 × 10.sup.−8 Z16026 230 1.6 × 10.sup.−8 Z16027 231 1.4 × 10.sup.−8 Z16028 232 1.9 × 10.sup.−8 Z16029 233 2.1 × 10.sup.−8 Z16030 234 1.7 × 10.sup.−8 Z16031 235 2.0 × 10.sup.−8 Z16032 236 2.6 × 10.sup.−8 Z16033 237 1.9 × 10.sup.−8 Z16034 238 9.1 × 10.sup.−9 Z16035 239 1.7 × 10.sup.−8 Z16036 240 1.1 × 10.sup.−8 Z16037 241 1.4 × 10.sup.−8 Z16038 242 8.9 × 10.sup.−9 Z16039 243 1.4 × 10.sup.−8 Z16040 244 2.0 × 10.sup.−8 Z16041 245 1.0 × 10.sup.−8 Z16042 246 9.7 × 10.sup.−9 Z16043 247 8.3 × 10.sup.−9 Z16044 248 1.7 × 10.sup.−8 Z16045 249 2.0 × 10.sup.−8 Z16046 250 1.8 × 10.sup.−8 Z16047 251 1.9 × 10.sup.−8 Z16048 252 1.1 × 10.sup.−8 Z16049 253 1.4 × 10.sup.−8 Z16050 254 1.3 × 10.sup.−8 Z16051 255 1.5 × 10.sup.−8 Z16052 256 1.2 × 10.sup.−8 Z16053 257 1.1 × 10.sup.−8 Z16054 258 2.9 × 10.sup.−8 Z16055 259 1.2 × 10.sup.−8 Z16056 260 1.3 × 10.sup.−8 Z16057 261 3.3 × 10.sup.−8 Z16058 262 1.4 × 10.sup.−8 Z16059 263 1.6 × 10.sup.−8 Z16060 264 1.9 × 10.sup.−8 Z16061 265 1.2 × 10.sup.−8 Z16062 266 7.6 × 10.sup.−9 Z16063 267 1.2 × 10.sup.−8 Z16065 268 7.8 × 10.sup.−9
Example 5
Production and Characterization of IL-1R-I Binding Z Variants from the First Matured Library
[0331] This Example describes the general procedure for subcloning and production of His.sub.6-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.
Materials and Methods
[0332] Subcloning of Z variants with a His.sub.6 tag: The DNA of the respective Z variant was amplified from the phage library vector pAY02592 and was subcloned with an N-terminal His.sub.6 tag using standard molecular biology techniques essentially as described in Example 2.
[0333] 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.
[0334] 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.
[0335] Purification of IL-1R-I binding Z variants with a His.sub.6 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.
[0336] 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 NH.sub.4Ac 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 KH.sub.2PO.sub.4, 8.1 mM Na.sub.2HPO.sub.4, pH 7.4) using PD-10 desalting columns (GE Healthcare).
[0337] 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.
[0338] Biacore kinetic analysis: The kinetic constants (k.sub.on and k.sub.off) and affinities (K.sub.D) for human and cynomolgus IL-1R-I were determined for His.sub.6-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). His.sub.6-tagged Z12967 was included in the assay for comparison.
[0339] In vitro IL-1β neutralization assay: His.sub.6-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 IC.sub.50 values were determined using GraphPadPrism program. His.sub.6-tagged Z12967 was included for comparison.
[0340] Circular dichroism (CD) spectroscopy analysis: CD was analyzed for His.sub.6-tagged Z variants, as described in Example 2.
Results
[0341] Subcloning with His.sub.6 tag: Z variants were subcloned into constructs with an N-terminal His.sub.6.
[0342] Production of His.sub.6-tagged Z variants: The IL-1R-I binding Z variants with a His.sub.6 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.
[0343] Biacore kinetic analysis: The interactions of His.sub.6-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 (K.sub.D, k.sub.on and k.sub.off) 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 K.sub.D of 1.5×10.sup.−10 against human IL-1R-I and 7.6×10.sup.−9 M against cynomolgus IL-1R-1. The K.sub.D of Z12967 to human IL-1R-I was 5.1×10.sup.−10 M.
TABLE-US-00023 TABLE 11 Kinetic parameters and affinities for His.sub.6-Z polypeptides binding to human IL-1R-I. Z variant SEQ ID NO k.sub.on (1/Ms) k.sub.off (1/s) K.sub.D (M) Z15862 71 2.8 × 10.sup.6 1.9 × 10.sup.−3 6.7 × 10.sup.−10 Z15876 1206 2.1 × 10.sup.6 1.3 × 10.sup.−3 6.2 × 10.sup.−10 Z15927 135 2.4 × 10.sup.6 1.2 × 10.sup.−3 5.1 × 10.sup.−10 Z15953 158 6.0 × 10.sup.6 1.5 × 10.sup.−3 2.4 × 10.sup.−10 Z15961 166 4.3 × 10.sup.6 2.0 × 10.sup.−3 4.6 × 10.sup.−10 Z16007 211 2.3 × 10.sup.6 2.1 × 10.sup.−3 9.2 × 10.sup.−10 Z16023 227 5.3 × 10.sup.6 1.5 × 10.sup.−3 2.9 × 10.sup.−10 Z16043 247 2.5 × 10.sup.6 1.1 × 10.sup.−3 4.4 × 10.sup.−10 Z16062 266 2.6 × 10.sup.6 1.1 × 10.sup.−3 4.4 × 10.sup.−10 Z16065 268 6.9 × 10.sup.6 1.3 × 10.sup.−3 1.9 × 10.sup.−10 Z16072 275 4.6 × 10.sup.6 2.2 × 10.sup.−3 4.7 × 10.sup.−10 Z16093 294 5.6 × 10.sup.6 1.4 × 10.sup.−3 2.5 × 10.sup.−10 Z16097 298 3.1 × 10.sup.6 1.5 × 10.sup.−3 4.8 × 10.sup.−10 Z16116 316 2.6 × 10.sup.6 2.3 × 10.sup.−3 8.8 × 10.sup.−10 Z16117 317 4.0 × 10.sup.6 2.7 × 10.sup.−3 6.9 × 10.sup.−10 Z16127 325 6.7 × 10.sup.6 1.9 × 10.sup.−3 2.9 × 10.sup.−10 Z16143 341 3.1 × 10.sup.6 1.6 × 10.sup.−3 5.3 × 10.sup.−10 Z16163 359 3.5 × 10.sup.6 2.0 × 10.sup.−3 5.5 × 10.sup.−10 Z16165 361 2.1 × 10.sup.6 3.6 × 10.sup.−3 1.7 × 10.sup.−9 Z16169 365 2.6 × 10.sup.6 1.0 × 10.sup.−3 3.9 × 10.sup.−10 Z16219 415 2.5 × 10.sup.6 5.6 × 10.sup.−3 2.2 × 10.sup.−9 Z16245 440 2.7 × 10.sup.6 3.1 × 10.sup.−3 1.2 × 10.sup.−9 Z16255 449 3.0 × 10.sup.6 3.1 × 10.sup.−3 1.0 × 10.sup.−9 Z16301 494 3.1 × 10.sup.6 2.1 × 10.sup.−3 6.6 × 10.sup.−10 Z16341 531 3.0 × 10.sup.6 1.6 × 10.sup.−3 5.2 × 10.sup.−10 Z16363 550 3.2 × 10.sup.6 1.8 × 10.sup.−3 5.7 × 10.sup.−10 Z16377 562 2.1 × 10.sup.6 1.1 × 10.sup.−3 5.4 × 10.sup.−10 Z16378 563 6.6 × 10.sup.6 3.6 × 10.sup.−3 5.5 × 10.sup.−10 Z16681 856 3.6 × 10.sup.6 5.0 × 10.sup.−3 1.4 × 10.sup.−9 Z17274 1176 1.1 × 10.sup.7 2.4 × 10.sup.−3 2.2 × 10.sup.−10 Z17280 1182 2.4 × 10.sup.6 5.3 × 10.sup.−3 2.2 × 10.sup.−9 Z17292 1194 1.3 × 10.sup.7 5.0 × 10.sup.−3 3.8 × 10.sup.−10 Z18557 1205 4.4 × 10.sup.6 6.7 × 10.sup.−4 1.5 × 10.sup.−10
[0344] In vitro IL-1β neutralization assay: The IL-1β inhibition ability of His.sub.6-tagged IL-1R-1-binding Z variants was analyzed in a TF-1 cell assay. The resulting IC.sub.50 values are presented in Table 12. Z18557 (SEQ ID NO:1205) displayed the best inhibition capacity with an IC.sub.50 value below 0.5 nM. The IC.sub.50 of Z12967 was 5.2 nM.
TABLE-US-00024 TABLE 12 IC.sub.50 values for His.sub.6-Z polypeptides from a TF-1 cell assay. Z variant SEQ ID NO IC.sub.50 (nM) Z15862 71 6.6 Z15876 1206 2.4 Z15927 135 2.5 Z15953 158 3.7 Z15961 166 2.9 Z16007 211 5.9 Z16023 227 3.8 Z16043 247 2.5 Z16062 266 1.8 Z16065 268 1.7 Z16072 275 4.3 Z16093 294 3.3 Z16097 298 3.4 Z16116 316 4.1 Z16117 317 8.5 Z16127 325 4.1 Z16143 341 4.8 Z16163 359 1.8 Z16165 361 11.5 Z16169 365 0.76 Z16219 415 24.2 Z16245 440 8.2 Z16255 449 8.5 Z16301 494 4.5 Z16341 531 3.9 Z16363 550 8.1 Z16377 562 3.1 Z16378 586 32.7 Z16681 856 20.1 Z17274 1176 4.6 Z17280 1182 5.4 Z17292 1194 13.2 Z18557 1205 <0.5
[0345] CD analysis: The CD spectra determined for the IL-1R-I binding Z variants with a His.sub.6 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.
TABLE-US-00025 TABLE 13 Melting temperatures (Tm). Z variant SEQ ID NO Tm (° C.) Z15876 1206 55 Z15927 135 56 Z15953 158 56 Z15961 166 59 Z16023 227 60 Z16043 247 54 Z16062 266 54 Z16065 268 59 Z16093 294 52 Z16097 298 56 Z16116 316 59 Z16117 317 63 Z16127 325 59 Z16163 359 57 Z16169 365 48 Z16245 440 63 Z16341 531 59 Z16377 562 52 Z16681 856 68 Z17280 1182 62 Z18557 1205 60
Example 6
Design and Construction of Two Second Matured Libraries of IL-1R-I Binding Z Variants
[0346] In this Example, two second matured libraries were constructed. The libraries were used for selections of additional IL-1R-I binding Z variants.
Materials and Methods
[0347] 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.
[0348] 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 2.2×10.sup.7 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.
[0349] 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 1.0×10.sup.8 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.
TABLE-US-00026 TABLE 14 Zlib006IL-1RI.II library design, second maturation. Amino acid Amino acid randomization No. of position in the (percentage when uneven different Z variant molecule distribution) amino acids 9 A, E, I, L, T, V 6 10 E(50%), I, L, R, V, Y 6 11 A, E(27%), I, K, Q, R, T, V, Y 9 12 A(50%), I, V 3 13 A, I, L, Q(50%), V, Y 6 14 F, M, Q, W, Y 5 17 F(70%), Y(30%) 2 18 A, D, E, F, G, H, I, K, L, Q, R, 16 S, T, V, W, Y 24 R 1 25 K, R 2 27 Y 1 28 I, T(71.4%), V 3 32 R 1 35 F(25%), I(12.5%), L(50%), 4 Y(12.5%)
TABLE-US-00027 TABLE 15 Zlib006IL-1RI.III library design, second maturation. Amino acid Amino acid randomization No. of position in the (percentage when uneven different Z variant molecule distribution) amino acids 9 A, E, I, L, T, V 6 10 E(60%), I, V 3 11 A, D, E(15%), F, H, I, K, L, Q, 15 R, S, T, V, W, Y 12 A(50%), I(20%), V(30%) 3 13 A, E, F, H, I, K, L, Q, R, S, 14 T, V, W, Y 14 A, E, F, H, I, K, L, Q, R, S, 14 T, V, W, Y 17 F, I, L, W, Y 5 18 A, D, E, F, G, H, I, K, L, Q, 16 R, S, T, V, W, Y 24 R 1 25 K, R 2 27 Y 1 28 T 1 32 I(10%), R(90%) 2 35 F(30%), L(70%) 2
[0350] 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.
[0351] 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 OD.sub.600 of 0.8-0.9. Phage stocks were prepared as described in Example 3.
Results
[0352] 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 2.2×10.sup.7 and 1.0×10.sup.8 Z variants, respectively. The actual sizes of the libraries, determined by titration after transformation to E. coli XL-1 Blue cells, were 1.2×10.sup.9 and 7.0×10.sup.9 transformants, respectively.
[0353] 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.
Example 7
Selection of Z Variants from the Second Matured Libraries
[0354] 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.
Materials and Methods
[0355] 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.
TABLE-US-00028 TABLE 16 Overview of selections from the two second matured libraries. Phage stock from Target Selection library or selection concentration Selection Number of Cycle track track Target (nM) time (min) washes 1 1-1 Zlib006IL-1RI.II b-hIL-1R-I-Fc 10 45 5 1 1-2 Zlib006IL-1RI.II b-hIL-1R-I 10 45 5 1 1-3 Zlib006IL-1RI.II b-cIL-1R-I-Fc 25 45 3 1 1-4 Zlib006IL-1RI.III b-hIL-1R-I-Fc 20 45 5 1 1-5 Zlib006IL-1RI.III b-hIL-1R-I 20 45 5 1 1-6 Zlib006IL-1RI.III b-cIL-1R-I-Fc 50 45 3 1 1-7 Zlib006IL-1RI.II b-hIL-1R-I-Fc 10 45 5 1 1-8 Zlib006IL-1RI.II b-hIL-1R-I 10 45 5 1 1-9 Zlib006IL-1RI.II b-cIL-1R-I-Fc 25 45 3 1 1-10 Zlib006IL-1RI.III b-hIL-1R-I-Fc 20 45 5 1 1-11 Zlib006IL-1RI.III b-hIL-1R-I 20 45 5 1 1-12 Zlib006IL-1RI.III b-cIL-1R-I-Fc 50 45 3 2 2-1 1-1 b-hIL-1R-I-Fc 1 30 10* 2 2-2 1-2 b-hIL-1R-I 2 30 10 2 2-3 1-2 b-hIL-1R-I 1 30 10 2 2-4 1-2 b-hIL-1R-I 1 30 10* 2 2-5 1-3 b-hIL-1R-I 10 30 8 2 2-6 1-4 b-hIL-1R-I-Fc 4 30 8 2 2-7 1-5 b-hIL-1R-I 4 30 8 2 2-8 1-5 b-hIL-1R-I 2 30 10 2 2-9 1-5 b-hIL-1R-I 2 30 10* 2 2-10 1-6 b-hIL-1R-I 20 30 6 2 2-11 1-7/1-8/1-9 pool b-hIL-1R-I 1 30 10* 2 2-12 1-10/1-11/1-12 pool b-hIL-1R-I 2 30 10* 3 3-1 2-1 b-hIL-1R-I-Fc 0.4 40 16* 3 3-2 2-2 b-hIL-1R-I 0.4 40 15 3 3-3 2-3 b-hIL-1R-I 0.1 40 20 3 3-4 2-4 b-hIL-1R-I 0.1 40 16* 3 3-5 2-5 b-cIL-1R-I-Fc 5 40 12 3 3-6 2-6 b-hIL-1R-I-Fc 0.8 40 12 3 3-7 2-7 b-hIL-1R-I 0.8 40 12 3 3-8 2-8 b-hIL-1R-I 0.2 40 20 3 3-9 2-9 b-hIL-1R-I 0.2 40 16* 3 3-10 2-10 b-cIL-1R-I-Fc 10 40 9 3 3-11 2-11 b-hIL-1R-I 0.4 40 16* 3 3-12 2-12 b-hIL-1R-I 0.4 40 16* 4 4-1 3-1 b-hIL-1R-I-Fc 0.125 40 20** 4 4-2 3-2 b-hIL-1R-I 0.125 40 20 4 4-3 3-3 b-hIL-1R-I 0.05 40 30 4 4-4 3-4 b-hIL-1R-I 0.05 40 20** 4 4-5 3-5 b-hIL-1R-I 1 40 16 4 4-6 3-6 b-hIL-1R-I-Fc 0.16 40 16* 4 4-7 3-7 b-hIL-1R-I 0.16 40 16 4 4-8 3-8 b-hIL-1R-I 0.1 40 30 4 4-9 3-9 b-hIL-1R-I 0.1 40 20** 4 4-10 3-10 b-hIL-1R-I 2 40 16 4 4-11 3-11 b-hIL-1R-I 0.2 40 16 4 4-12 3-12 b-hIL-1R-I 0.2 40 16 *Addition of hIL-1R-I in the second final wash step. **Selection track divided into two before second final wash step. One part submitted to an over-night wash in the presence of hIL-1R-I and one part submitted to a 1 h wash in PBST.
[0356] 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.
[0357] 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.
Results
[0358] 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.
Example 8
Screening of Z Variants from the First and Second Matured Libraries
[0359] 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.
Materials and Methods
[0360] 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.
[0361] 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.
[0362] 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.
[0363] EC.sub.50 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 EC.sub.50 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.
[0364] 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.
Results
[0365] 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
[0366] 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.
[0367] EC.sub.50 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 EC.sub.50 values (Table 17). The results for Z12967, Z16065 and Z16218 were 2.6×10.sup.−10, 2.1×10.sup.−10 and 2.2×10.sup.−10 M, respectively.
TABLE-US-00029 TABLE 17 Calculated EC.sub.50 values from ELISA titration analysis. SEQ ID Z variant NO EC.sub.50 (M) Z15840 49 2.3 × 10.sup.−10 Z15862 71 2.5 × 10.sup.−10 Z15876 1206 1.9 × 10.sup.−10 Z15934 1208 1.7 × 10.sup.−10 Z15945 150 2.3 × 10.sup.−10 Z15975 1209 1.9 × 10.sup.−10 Z16062 266 2.1 × 10.sup.−10 Z16183 379 2.3 × 10.sup.−10 Z16208 404 2.2 × 10.sup.−10 Z16603 782 2.2 × 10.sup.−10 Z16606 785 2.3 × 10.sup.−10 Z18754 1252 3.5 × 10.sup.−10 Z18757 1321 3.0 × 10.sup.−10 Z18758 1281 2.4 × 10.sup.−10 Z18759 1314 2.4 × 10.sup.−10 Z18760 1435 1.7 × 10.sup.−10 Z18763 1431 2.6 × 10.sup.−10 Z18766 1274 3.3 × 10.sup.−10 Z18769 1339 2.9 × 10.sup.−10 Z18770 1284 2.8 × 10.sup.−10 Z18771 1341 2.5 × 10.sup.−10 Z18774 1389 2.6 × 10.sup.−10 Z18776 1418 2.2 × 10.sup.−10 Z18777 1326 2.6 × 10.sup.−10 Z18785 1323 2.5 × 10.sup.−10 Z18786 1320 2.4 × 10.sup.−10 Z18790 1439 2.6 × 10.sup.−10 Z18799 1423 2.3 × 10.sup.−10 Z18803 1327 2.5 × 10.sup.−10 Z18804 1329 2.7 × 10.sup.−10 Z18805 1437 2.7 × 10.sup.−10 Z18806 1271 2.7 × 10.sup.−10 Z18808 1342 4.0 × 10.sup.−10 Z18810 1278 2.9 × 10.sup.−10 Z18811 1405 2.8 × 10.sup.−10 Z18813 1375 2.0 × 10.sup.−10 Z18814 1368 2.7 × 10.sup.−10 Z18819 1276 2.9 × 10.sup.−10 Z18820 1417 3.3 × 10.sup.−10 Z18824 1264 2.8 × 10.sup.−10 Z18825 1275 3.5 × 10.sup.−10 Z18826 1356 2.4 × 10.sup.−10 Z18827 1318 2.4 × 10.sup.−10 Z18831 1308 2.9 × 10.sup.−10 Z18832 1340 1.5 × 10.sup.−10 Z18834 1361 2.5 × 10.sup.−10 Z18836 1430 2.3 × 10.sup.−10 Z18838 1337 2.4 × 10.sup.−10 Z18843 1309 2.8 × 10.sup.−10 Z18846 1331 1.9 × 10.sup.−10 Z18866 1324 3.1 × 10.sup.−10 Z18874 1421 2.2 × 10.sup.−10 Z18875 1420 2.9 × 10.sup.−10 Z18877 1301 3.5 × 10.sup.−10 Z18878 1302 3.7 × 10.sup.−10 Z18879 1390 3.4 × 10.sup.−10 Z18881 1355 2.8 × 10.sup.−10 Z18882 1311 3.1 × 10.sup.−10 Z18884 1403 2.3 × 10.sup.−10 Z18886 1432 2.9 × 10.sup.−10 Z18887 1386 2.1 × 10.sup.−10 Z18888 1422 2.3 × 10.sup.−10 Z18889 1344 2.4 × 10.sup.−10 Z18890 1352 2.5 × 10.sup.−10 Z18904 1587 3.8 × 10.sup.−10 Z18908 1591 2.4 × 10.sup.−10 Z18912 1251 1.4 × 10.sup.−10 Z18917 1509 2.2 × 10.sup.−10 Z18920 1270 2.4 × 10.sup.−10 Z18966 1272 2.5 × 10.sup.−10 Z18967 1388 3.1 × 10.sup.−10 Z18968 1472 2.0 × 10.sup.−10 Z18971 1559 3.5 × 10.sup.−10 Z18982 1553 2.5 × 10.sup.−10 Z19012 1588 2.4 × 10.sup.−10 Z19053 1526 2.4 × 10.sup.−10 Z19054 1528 2.5 × 10.sup.−10 Z19056 1586 2.2 × 10.sup.−10 Z19057 1558 2.2 × 10.sup.−10 Z19059 1240 3.5 × 10.sup.−10 Z19060 1228 2.2 × 10.sup.−10 Z19072 1554 1.6 × 10.sup.−10 Z19075 1618 1.5 × 10.sup.−10 Z19078 1248 2.2 × 10.sup.−10 Z19085 1564 2.1 × 10.sup.−10 Z19090 1540 2.0 × 10.sup.−10 Z19115 1489 2.3 × 10.sup.−10 Z19117 1631 2.1 × 10.sup.−10 Z19118 1579 2.3 × 10.sup.−10 Z19123 1241 2.6 × 10.sup.−10 Z19151 1594 2.3 × 10.sup.−10 Z19158 1571 1.5 × 10.sup.−10 Z19159 1364 2.1 × 10.sup.−10 Z19171 1593 2.3 × 10.sup.−10 Z19172 1551 1.6 × 10.sup.−10 Z19180 1393 2.0 × 10.sup.−10 Z19181 1549 1.8 × 10.sup.−10 Z19182 1628 1.7 × 10.sup.−10 Z19183 1235 2.2 × 10.sup.−10 Z19302 1419 2.6 × 10.sup.−10 Z19304 1273 2.7 × 10.sup.−10 Z19305 1261 2.6 × 10.sup.−10
[0368] 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 K.sub.D values of Z12967, Z16065 and Z16218 were 2.7×10.sup.−8, 7.5×10.sup.−9 and 1.3×10.sup.−8 M (average of duplicates), respectively.
[0369] Z18814, which has an amino acid sequence that only differs from Z16065 in position X.sub.5 of the binding motif BM, has three times higher affinity than Z-6065. Z16065 has the highest binding affinity (K.sub.D) among the IL-1R-I binding Z variants (Table 11) with an alanine residue in position X.sub.5. Z18814 has an isoleucine residue in position X.sub.5. Several Z variants with valine or isoleucine in position X.sub.5 were found to have high affinity to IL-1R-1.
TABLE-US-00030 TABLE 18 Calculated KD values from Biacore kinetic screenina. Biacore SEQ screening Z variant ID NO K.sub.D (M) Z15840 49 6.9 × 10.sup.−9 Z15862 71 1.3 × 10.sup.−8 Z15876 1206 5.7 × 10.sup.−9 Z15932 1207 5.5 × 10.sup.−9 Z15934 1208 9.4 × 10.sup.−9 Z15945 150 9.6 × 10.sup.−9 Z15975 1209 8.9 × 10.sup.−9 Z16062 266 6.4 × 10.sup.−9 Z16183 379 6.5 × 10.sup.−9 Z16191 387 6.3 × 10.sup.−9 Z16208 404 1.1 × 10.sup.−8 Z16397 582 1.3 × 10.sup.−8 Z16440 624 1.3 × 10.sup.−8 Z16603 782 9.1 × 10.sup.−9 Z16606 785 9.6 × 10.sup.−9 Z16737 910 1.2 × 10.sup.−8 Z18754 1252 2.5 × 10.sup.−9 Z18756 1366 2.9 × 10.sup.−9 Z18757 1321 3.0 × 10.sup.−9 Z18758 1281 4.8 × 10.sup.−9 Z18759 1314 3.4 × 10.sup.−9 Z18760 1435 3.0 × 10.sup.−9 Z18762 1415 2.1 × 10.sup.−9 Z18763 1431 5.2 × 10.sup.−9 Z18766 1274 3.1 × 10.sup.−9 Z18767 12353 5.1 × 10.sup.−9 Z18769 1339 2.7 × 10.sup.−9 Z18770 1284 2.6 × 10.sup.−9 Z18771 1341 3.0 × 10.sup.−9 Z18774 1389 3.0 × 10.sup.−9 Z18776 1418 2.8 × 10.sup.−9 Z18777 1326 3.8 × 10.sup.−9 Z18782 1333 2.7 × 10.sup.−9 Z18783 1257 3.4 × 10.sup.−9 Z18784 1433 3.7 × 10.sup.−9 Z18785 1323 2.7 × 10.sup.−9 Z18786 1320 4.8 × 10.sup.−9 Z18790 1439 3.0 × 10.sup.−9 Z18794 1424 3.7 × 10.sup.−9 Z18796 1352 3.6 × 10.sup.−9 Z18798 1268 3.2 × 10.sup.−9 Z18799 1423 2.6 × 10.sup.−9 Z18800 1328 1.9 × 10.sup.−9 Z18803 1327 3.1 × 10.sup.−9 Z18804 1329 3.3 × 10.sup.−9 Z18805 1437 3.7 × 10.sup.−9 Z18806 1271 4.1 × 10.sup.−9 Z18807 1400 3.8 × 10.sup.−9 Z18808 1342 4.2 × 10.sup.−9 Z18810 1278 3.1 × 10.sup.−9 Z18811 1405 4.5 × 10.sup.−9 Z18813 1375 2.0 × 10.sup.−9 Z18814 1368 2.4 × 10.sup.−9 Z18817 1343 3.5 × 10.sup.−9 Z18819 1276 3.0 × 10.sup.−9 Z18820 1417 3.3 × 10.sup.−9 Z18821 1269 3.5 × 10.sup.−9 Z18823 1282 2.4 × 10.sup.−9 Z18824 1264 3.3 × 10.sup.−9 Z18825 1275 4.9 × 10.sup.−9 Z18826 1356 3.5 × 10.sup.−9 Z18827 1318 4.3 × 10.sup.−9 Z18828 1296 2.6 × 10.sup.−9 Z18830 1295 2.9 × 10.sup.−9 Z18831 1308 3.0 × 10.sup.−9 Z18832 1340 1.6 × 10.sup.−9 Z18833 1297 3.8 × 10.sup.−9 Z18834 1361 2.4 × 10.sup.−9 Z18836 1430 2.9 × 10.sup.−9 Z18838 1337 3.3 × 10.sup.−9 Z18839 1440 3.1 × 10.sup.−9 Z18841 1359 2.5 × 10.sup.−9 Z18843 1309 3.1 × 10.sup.−9 Z18844 1349 4.0 × 10.sup.−9 Z18846 1331 2.1 × 10.sup.−9 Z18847 1363 3.0 × 10.sup.−9 Z18848 1304 3.0 × 10.sup.−9 Z18849 1299 3.0 × 10.sup.−9 Z18850 1367 2.6 × 10.sup.−9 Z18851 1291 3.6 × 10.sup.−9 Z18853 1298 2.3 × 10.sup.−9 Z18856 1330 2.9 × 10.sup.−9 Z18860 1265 6.2 × 10.sup.−9 Z18861 1369 3.3 × 10.sup.−9 Z18863 1362 2.4 × 10.sup.−9 Z18866 1324 2.7 × 10.sup.−9 Z18868 1391 2.7 × 10.sup.−9 Z18870 1428 3.0 × 10.sup.−9 Z18871 1332 3.5 × 10.sup.−9 Z18872 1410 2.6 × 10.sup.−9 Z18873 1306 3.1 × 10.sup.−9 Z18874 1421 2.3 × 10.sup.−9 Z18875 1420 2.7 × 10.sup.−9 Z18876 1286 3.7 × 10.sup.−9 Z18877 1301 2.7 × 10.sup.−9 Z18878 1302 3.8 × 10.sup.−9 Z18879 1390 6.2 × 10.sup.−9 Z18881 1355 3.2 × 10.sup.−9 Z18882 1311 2.6 × 10.sup.−9 Z18884 1403 2.7 × 10.sup.−9 Z18885 1294 3.9 × 10.sup.−9 Z18886 1432 3.9 × 10.sup.−9 Z18887 1386 2.9 × 10.sup.−9 Z18888 1422 2.6 × 10.sup.−9 Z18889 1344 3.4 × 10.sup.−9 Z18890 1352 3.1 × 10.sup.−9 Z18893 1353 2.1 × 10.sup.−9 Z18894 1312 2.5 × 10.sup.−9 Z18895 1411 2.6 × 10.sup.−9 Z18896 1436 3.5 × 10.sup.−9 Z18898 1285 2.5 × 10.sup.−9 Z18904 1587 3.4 × 10.sup.−9 Z18908 1591 3.6 × 10.sup.−9 Z18909 1303 2.7 × 10.sup.−9 Z18910 1416 3.1 × 10.sup.−9 Z18911 1441 2.8 × 10.sup.−9 Z18912 1251 3.6 × 10.sup.−9 Z18916 1536 4.1 × 10.sup.−9 Z18917 1509 7.1 × 10.sup.−9 Z18919 1350 2.8 × 10.sup.−9 Z18920 1270 3.2 × 10.sup.−9 Z18922 1533 4.9 × 10.sup.−9 Z18923 1482 9.0 × 10.sup.−9 Z18925 1479 1.1 × 10.sup.−8 Z18927 1372 3.4 × 10.sup.−9 Z18938 1370 4.5 × 10.sup.−9 Z18941 1409 3.3 × 10.sup.−9 Z18942 1483 1.5 × 10.sup.−8 Z18948 1414 4.3 × 10.sup.−9 Z18952 1290 3.0 × 10.sup.−9 Z18956 1625 1.4 × 10.sup.−8 Z18958 1283 2.8 × 10.sup.−9 Z18959 1357 2.9 × 10.sup.−9 Z18966 1272 2.9 × 10.sup.−9 Z18967 1388 3.2 × 10.sup.−9 Z18968 1472 2.4 × 10.sup.−9 Z18971 1559 5.5 × 10.sup.−9 Z18982 1553 5.2 × 10.sup.−9 Z18989 1592 4.5 × 10.sup.−9 Z19012 1588 4.5 × 10.sup.−9 Z19031 1600 3.1 × 10.sup.−9 Z19043 1505 3.4 × 10.sup.−9 Z19053 1526 8.7 × 10.sup.−9 Z19054 1528 7.0 × 10.sup.−9 Z19056 1586 6.9 × 10.sup.−9 Z19057 1558 4.2 × 10.sup.−9 Z19059 1240 9.2 × 10.sup.−9 Z19060 1228 1.0 × 10.sup.−8 Z19072 1554 5.2 × 10.sup.−9 Z19075 1618 4.8 × 10.sup.−9 Z19078 1248 9.5 × 10.sup.−9 Z19079 1236 1.4 × 10.sup.−8 Z19085 1564 4.9 × 10.sup.−9 Z19090 1540 3.0 × 10.sup.−9 Z19094 1231 1.8 × 10.sup.−8 Z19099 1548 3.8 × 10.sup.−9 Z19107 1566 6.7 × 10.sup.−9 Z19115 1489 6.1 × 10.sup.−9 Z19117 1631 4.2 × 10.sup.−9 Z19118 1579 8.4 × 10.sup.−9 Z19123 1241 1.1 × 10.sup.−8 Z19127 1213 9.8 × 10.sup.−9 Z19135 1247 1.6 × 10.sup.−8 Z19141 1506 1.7 × 10.sup.−8 Z19143 1471 1.4 × 10.sup.−9 Z19144 1374 3.1 × 10.sup.−9 Z19146 1434 2.9 × 10.sup.−9 Z19147 1307 2.4 × 10.sup.−9 Z19151 1594 2.7 × 10.sup.−9 Z19153 1310 3.1 × 10.sup.−9 Z19158 1571 1.5 × 10.sup.−9 Z19159 1364 1.9 × 10.sup.−9 Z19160 1379 3.8 × 10.sup.−9 Z19166 1305 3.5 × 10.sup.−9 Z19168 1500 4.6 × 10.sup.−9 Z19169 1402 2.7 × 10.sup.−9 Z19170 1334 2.0 × 10.sup.−9 Z19171 1593 3.0 × 10.sup.−9 Z19172 1551 4.5 × 10.sup.−9 Z19180 1393 .sup. 7.9 × 10.sup.−10 Z19181 1549 9.7 × 10.sup.−9 Z19182 1628 2.8 × 10.sup.−9 Z19183 1235 1.2 × 10.sup.−8 Z19302 1419 3.2 × 10.sup.−9 Z19303 1262 4.0 × 10.sup.−9 Z19304 1273 4.5 × 10.sup.−9 Z19305 1261 3.8 × 10.sup.−9
Example 9
Production and Characterization of Z Variants from the First and Second Matured Libraries
[0370] This Example describes the general procedure for subcloning and production of His.sub.6-tagged Z variants originating from the first and second maturation library phage selections, characterization of their target binding, target blocking and melting points.
Materials and Methods
[0371] Subcloning of Z variants with a His.sub.6 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 His.sub.6 tag using standard molecular biology techniques essentially as described in Example 2.
[0372] 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 OD.sub.600≈10. After induction, the cultivations were incubated for 16 h. The cells were harvested by centrifugation.
[0373] Purification of IL-1R-I binding Z variants with a His.sub.6 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.
[0374] Biacore kinetic analysis: The kinetic constants (k.sub.on and k.sub.off) and affinities (K.sub.D) for human and cynomolgus IL-1R-I were determined for His.sub.6-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). His.sub.6-tagged Z12967 (SEQ ID NO:9), Z16065 (SEQ ID NO:268), Z16163 (SEQ ID NO:359) and Z18557 (SEQ ID NO:1205) were included in the assay for comparison.
[0375] In vitro IL-1β neutralization assay: His.sub.6-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 IC.sub.50 values were determined using GraphPadPrism program. His.sub.6-tagged Z12967, Z16065, Z16163 and Z18557 were included for comparison.
[0376] Circular dichroism (CD) spectroscopy analysis: CD was analyzed for His.sub.6-tagged Z variants, as described in Example 2.
Results
[0377] Production of His.sub.6-tagged Z variants: The IL-1R-I binding Z variants with a His.sub.6 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.
[0378] Biacore kinetic analysis: The interactions of His.sub.6-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 (K.sub.D, k.sub.on and k.sub.off) for the human IL-1R-I binding is given in Table 19. The K.sub.D values of Z12967, Z16065, Z16163 and Z18557 were 8.2×10.sup.−10, 5.1×10.sup.−10, 7.6×10.sup.−10 and 1.4×10.sup.−10 M, respectively. The K.sub.D values of cynomolgus IL-1R-1 binding were 2.0×10.sup.−9, 6.7×10.sup.−9 and 3.3×10.sup.−9 M for Z18754, Z18760 and Z18800, respectively.
TABLE-US-00031 TABLE 19 Kinetic parameters and affinities for His.sub.6-Z polypeptides binding to human IL-1R-I. Z variant SEQ ID NO k.sub.on (1/Ms) k.sub.off (1/s) K.sub.D (M) Z15876 1206 5.2 × 10.sup.6 4.1 × 10.sup.−3 8.0 × 10.sup.−10 Z15932 1207 1.8 × 10.sup.7 1.1 × 10.sup.−2 6.4 × 10.sup.−10 Z15934 1208 1.4 × 10.sup.7 1.1 × 10.sup.−2 7.5 × 10.sup.−10 Z15975 1209 4.1 × 10.sup.7 2.9 × 10.sup.−2 7.1 × 10.sup.−10 Z18754 1252 7.0 × 10.sup.7 6.0 × 10.sup.−3 8.6 × 10.sup.−11 Z18760 1435 1.3 × 10.sup.7 2.7 × 10.sup.−3 2.1 × 10.sup.−10 Z18762 1415 4.2 × 10.sup.7 5.2 × 10.sup.−3 1.3 × 10.sup.−10 Z18769 1339 1.9 × 10.sup.7 3.1 × 10.sup.−3 1.6 × 10.sup.−10 Z18770 1284 3.0 × 10.sup.7 5.0 × 10.sup.−3 1.7 × 10.sup.−10 Z18799 1423 2.6 × 10.sup.7 4.5 × 10.sup.−3 1.8 × 10.sup.−10 Z18800 1328 1.8 × 10.sup.7 2.5 × 10.sup.−3 1.4 × 10.sup.−10 Z18813 1375 2.0 × 10.sup.7 3.9 × 10.sup.−3 2.0 × 10.sup.−10 Z18814 1368 3.0 × 10.sup.7 4.4 × 10.sup.−3 1.5 × 10.sup.−10 Z18817 1343 2.8 × 10.sup.7 4.8 × 10.sup.−3 1.7 × 10.sup.−10 Z18823 1283 4.7 × 10.sup.6 1.8 × 10.sup.−3 3.8 × 10.sup.−10 Z18831 1308 4.0 × 10.sup.7 5.3 × 10.sup.−3 1.3 × 10.sup.−10 Z18832 1340 5.9 × 10.sup.7 7.0 × 10.sup.−3 1.2 × 10.sup.−10 Z18834 1361 5.8 × 10.sup.7 8.0 × 10.sup.−3 1.4 × 10.sup.−10 Z18846 1331 4.7 × 10.sup.7 5.1 × 10.sup.−3 1.1 × 10.sup.−10 Z18850 1367 1.3 × 10.sup.7 2.3 × 10.sup.−3 1.7 × 10.sup.−10 Z18853 1298 2.2 × 10.sup.7 3.0 × 10.sup.−3 1.4 × 10.sup.−10 Z18856 1330 4.6 × 10.sup.7 5.7 × 10.sup.−3 1.2 × 10.sup.−10 Z18863 1362 2.2 × 10.sup.7 4.8 × 10.sup.−3 2.2 × 10.sup.−10 Z18866 1324 3.6 × 10.sup.7 4.2 × 10.sup.−3 1.2 × 10.sup.−10 Z18874 1421 4.9 × 10.sup.7 5.2 × 10.sup.−3 1.1 × 10.sup.−10 Z18875 1420 2.4 × 10.sup.7 2.8 × 10.sup.−3 1.2 × 10.sup.−10 Z18882 1311 1.2 × 10.sup.7 3.0 × 10.sup.−3 2.5 × 10.sup.−10 Z18888 1422 1.8 × 10.sup.7 2.9 × 10.sup.−3 1.6 × 10.sup.−10 Z18893 1353 8.6 × 10.sup.6 2.4 × 10.sup.−3 2.7 × 10.sup.−10 Z18898 1285 6.7 × 10.sup.7 4.4 × 10.sup.−3 6.6 × 10.sup.−11 Z18912 1251 9.2 × 10.sup.7 1.9 × 10.sup.−2 2.1 × 10.sup.−10 Z18920 1270 3.6 × 10.sup.7 5.5 × 10.sup.−3 1.5 × 10.sup.−10 Z18968 1472 3.0 × 10.sup.7 8.4 × 10.sup.−3 2.8 × 10.sup.−10 Z19072 1554 4.3 × 10.sup.7 1.8 × 10.sup.−2 4.1 × 10.sup.−10 Z19075 1618 1.4 × 10.sup.7 6.4 × 10.sup.−3 4.5 × 10.sup.−10 Z19143 1471 1.7 × 10.sup.7 3.9 × 10.sup.−3 2.2 × 10.sup.−10 Z19147 1307 2.8 × 10.sup.7 4.3 × 10.sup.−3 1.5 × 10.sup.−10 Z19151 1594 2.8 × 10.sup.7 7.0 × 10.sup.−3 2.5 × 10.sup.−10 Z19158 1571 2.7 × 10.sup.7 4.4 × 10.sup.−3 1.6 × 10.sup.−10 Z19159 1364 2.7 × 10.sup.7 5.0 × 10.sup.−3 1.9 × 10.sup.−10 Z19170 1334 2.1 × 10.sup.7 4.3 × 10.sup.−3 2.0 × 10.sup.−10 Z19172 1551 4.9 × 10.sup.7 2.3 × 10.sup.−2 4.6 × 10.sup.−10 Z19180 1393 2.2 × 10.sup.7 3.1 × 10.sup.−3 1.4 × 10.sup.−10 Z19181 1549 2.0 × 10.sup.7 1.6 × 10.sup.−2 8.2 × 10.sup.−10 Z19182 1628 2.1 × 10.sup.7 5.5 × 10.sup.−3 2.6 × 10.sup.−10
[0379] In vitro IL-1β neutralization assay: The IL-1β inhibition ability of His.sub.6-tagged IL-1R-1-binding Z variants was analyzed in a TF-1 cell assay. The resulting IC.sub.50 values are presented in Table 20. The IC.sub.50 values of Z12967, Z16065, Z16163 and Z18557 were 7.4, 3.3, 3.3 and 0.6 nM, respectively.
TABLE-US-00032 TABLE 20 IC.sub.50 values for His.sub.6-Z polypeptides from a TF-1 cell assay. Z variant SEQ ID NO IC.sub.50 (nM) Z15934 1208 6.4 Z15975 1209 3.3 Z18754 1252 0.6 Z18760 1435 0.9 Z18813 1375 1.2 Z18814 1368 0.4 Z18831 1308 0.4 Z18832 1340 0.5 Z18834 1361 0.5 Z18846 1331 0.2 Z18874 1421 0.3 Z18912 1251 1 Z18968 1472 1.4 Z19072 1554 2.4 Z19159 1364 1.2 Z19172 1551 2 Z19181 1549 4.6 Z19182 1628 1.5
[0380] CD analysis: The CD spectra determined for the IL-1R-I binding Z variants with a His.sub.6 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.
[0381] The thermo-stability and the a-helical content of the IL-1R-I binding Z variants having a valine or isoleucine residue in position X.sub.5 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 X.sub.5.
TABLE-US-00033 TABLE 21 Melting temperatures (Tm). Z variant SEQ ID NO Tm (° C.) Z15876 1206 55 Z15932 1207 60 Z15934 1208 62 Z15975 1209 59 Z18754 1252 63 Z18760 435 57 Z18762 1415 62 Z18769 1339 59 Z18770 1284 59 Z18799 1423 60 Z18800 1328 67 Z18813 1375 61 Z18814 1368 61 Z18817 1343 60 Z18823 1283 52 Z18831 1308 58 Z18832 1340 58 Z18834 1361 58 Z18846 1331 60 Z18850 1367 62 Z18853 1298 59 Z18856 1330 61 Z18863 1362 57 Z18866 1324 61 Z18874 1421 61 Z18875 1420 61 Z18882 1311 53 Z18888 1422 61 Z18893 1353 60 Z18898 1285 59 Z18912 1251 53 Z18920 1270 56 Z18968 1472 62 Z19072 1554 63 Z19075 1618 56 Z19143 1471 56 Z19147 1307 57 Z19151 1594 59 Z19158 1571 55 Z19159 1364 54 Z19170 1334 52 Z19172 1551 59 Z19180 1393 58 Z19181 1549 61 Z19182 1628 62
Example 10
Production and Characterization of IL-1R-I Binding Fusion Proteins
[0382] 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.
Materials and Methods
[0383] 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.
[0384] 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
TABLE-US-00034 TABLE 22 Fusion proteins and expression systems Fusion Expression SEQ ID NO proteins Construct system 1639 PSI0405 Z12967[V1A; D2E; N52S; D53E]-VEGS-ABD E. coli 1640 PSI0407 Z12895[V1A; D2E; N52S; D53E]-VEGS-ABD E. coli 1641 PSI0411 AS-ABD-GS-Z12967[V1A; D2E; N52S; D53E] E. coli 1642 PSI0412 AS-ABD-(G.sub.4S).sub.2- E. coli Z12967[V1A; D2E; N52S; D53E] 1643 PSI0415 AS-ABD-Z12967[V1A; D2E; N52S; D53E] E. coli 1644 PSI0416 AS-ABD-Z12967[ΔV1D2A3; N52S; D53E] E. coli 1645 PSI0417 AS-ABD-((KEAAA).sub.3KELAA).sub.2- E. coli Z12967[V1A; D2E; N52S; D53E] 1646 PSI0534 Z18831[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- E. coli IgG1 Fc 1647 PSI0535 Z18846[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- E. coli IgG1 Fc 1648 PSI0536 Z18754[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- E. coli IgG1 Fc 1649 PSI0537 Z19151[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- E. coli IgG1 Fc 1650 PSI0538 Z18874[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- E. coli IgG1 Fc 1651 PSI0539 Z18760[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- E. coli IgG1 Fc 1652 PSI0574 Z18800[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- E. coli IgG1 Fc 1653 PSI0575 Z18898[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- E. coli IgG1 Fc 1654 PSI0576 Z19147[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- E. coli IgG1 Fc 1655 PSI0580 Z18754[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- CHO Albumin 1656 PSI0581 Z18754[V1A; D2E; T23N; N52S; D53E]- CHO AS(G.sub.4S).sub.2-Albumin 1657 PSI0582 Z18754[V1A; D2E; N52S; D53E]-AS(G.sub.4S).sub.2- CHO Transferrin 1658 PSI0589 PSI0536[amino acids 1-86], dimeric
[0385] 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.
[0386] 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).
[0387] 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).
[0388] 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).
Results
[0389] 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.
[0390] 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 (k.sub.on), dissociation rate constant (k.sub.off) and dissociation constant K.sub.D for the Z-Fc fusion protein variants are presented in Table 23.
TABLE-US-00035 TABLE 23 Kinetic parameters for IL-1R-I binding fusion proteins Fusion proteins and Human IL-1R-I Cynomolgus IL-1R-I SEQ ID NO K.sub.D (M) k.sub.on (1/Ms) k.sub.off (1/s) K.sub.D (M) k.sub.on (1/Ms) k.sub.off (1/s) PSI0536 (SEQ 7.5 × 10.sup.−10 8.99 × 10.sup.5 6.79 × 10.sup.−4 1.4 × 10.sup.−8 1.70 × 10.sup.6 2.34 × 10.sup.−2 ID NO: 1648) PSI0537 (SEQ 1.2 × 10.sup.−9 8.41 × 10.sup.5 1.02 × 10.sup.−3 3.1 × 10.sup.−8 1.78 × 10.sup.6 5.48 × 10.sup.−2 ID NO: 1649) PSI0534 (SEQ 1.6 × 10.sup.−9 4.04 × 10.sup.5 6.42 × 10.sup.−4 2.8 × 10.sup.−8 1.08 × 10.sup.6 2.99 × 10.sup.−2 ID NO: 1646) PSI0535 (SEQ 7.2 × 10.sup.−10 5.83 × 10.sup.5 4.21 × 10.sup.−4 1.1 × 10.sup.−8 1.49 × 10.sup.6 1.66 × 10.sup.−2 ID NO: 1647)
Example 11
In Vitro Pharmacological Activity Analysis Usinq a Cell-Based Assay
Materials and Methods
[0391] 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.
[0392] 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 IC.sub.50 values were calculated from concentration-response curves.
[0393] The inhibitory effect of His.sub.6-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 IC.sub.50 values calculated.
[0394] 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.
Results
[0395] 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
TABLE-US-00036 TABLE 24 In vitro IC.sub.50 values Denotation SEQ ID NO IC.sub.50 (pM) PSI0536 1648 66; 45 PSI0537 1649 260; 200 PSI0534 1646 140; 110 PSI0535 1647 80; 50 PSI0538 1650 87; 70 PSI0539 1651 240; 110 anakinra Average 70
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.
TABLE-US-00037 TABLE 25 In vitro IC.sub.50 values Denotation SEQ ID NO IC.sub.50 (pM) PSI0589 1658 1.8 PSI0582 1657 2.0 PSI0580 1655 1500 PSI0581 1656 1800
[0396] The IL-1β inhibition ability of His.sub.6-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).
[0397] The IC.sub.50 value of the chemically synthesized PS10558 was 690 μM.
Example 12
In Vitro Pharmacological Activity Analysis Using a Whole Blood Assay
[0398] 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.
Materials and Methods
[0399] 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.
[0400] IL-6 concentrations (pg/ml) were calculated using a standard curve (provided by the kit) and data was analyzed using XLfit and IC.sub.50 values were calculated from the concentration-response curves.
Results
[0401] 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 (
Example 13
In Vivo Pharmacokinetics of IL-IR-1 Binding Fusion Proteins in Rats
[0402] 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.
Material and Methods
[0403] 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.
[0404] 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.
[0405] 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.
[0406] 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.
[0407] Pharmacokinetic analysis: The pharmacokinetic analysis was based on median serum concentration versus time data from each dose group. The observed maximum concentration (C.sub.max) and the time to maximum serum concentration (t.sub.max) 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 (V.sub.ss), mean residence time (MRT) and terminal half-life (t.sub.1/2z), 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.
Results
[0408] 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.Math.kg and a volume of distribution of about 140 ml/kg, translating into a mean residence time of ca 60 hrs (Table 26).
TABLE-US-00038 TABLE 26 Pharmacokinetic parameter estimates following an intravenous single dose administration, 15 mg/kg, to male rats Parameter PSI0534 PSI0535 PSI0536 PSI0537 PSI0538 PSI0539 CL (ml/h .Math. kg) 2.83 2.40 2.68 2.06 2.36 2.03 V.sub.ss (ml/kg) 159 149 143 136 146 113 MRT (hrs) 56.4 62.1 53.5 65.9 61.6 55.5 t.sub.1/2z (hrs) 48.7 52.1 44.7 55.1 50.4 48.3
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).
TABLE-US-00039 TABLE 27 Pharmacokinetic parameter estimates following a subcutaneous single dose administration, 30 mg/kg, to male rats Parameter PSI0534 PSI0535 PSI0536 PSI0537 PSI0538 PSI0539 F (%) 41.5 49.6 46.7 60.3 47.2 52.2 C.sub.max (nM) 828 866 948 1115 796 886 t.sub.max (hrs) 24 24 24 24 24 24 CL/F (ml/h .Math. kg) 6.81 5.70 6.05 4.69 5.98 5.42 V.sub.z/F (ml/kg) 445 457 403 372 509 424 t.sub.1/2z (hrs) 45.3 55.6 46.1 55.1 59.0 54.3 MRT (hrs) 72.4 87.3 74.5 87.4 91.7 86.2
Example 14
In Vivo Pharmacokinetics of an IL-1R-I Binding Fusion Protein in Monkeys
Material and Methods
[0409] 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.
[0410] 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.
[0411] Quantitative ELISA: Quantification of PS10536 in serum from monkeys was performed according to the ELISA described in Example 13.
[0412] 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.
Results
[0413] The pharmacokinetics of PS10536 (SEQ ID NO:1648) following i.v. administration was characterized by a low clearance (1.07 ml/h.Math.kg) and a small volume of distribution (117 ml/kg), translating into a mean residence time of 109 hrs (Table 28).
TABLE-US-00040 TABLE 28 Mean (±SD) pharmacokinetic parameter estimates of PSI0536 following an intravenous single dose of 5.04 (±0.01) mg/kg to three male monkeys Parameter Estimate CL (ml/h .Math. kg) 1.07 ± 0.04 V.sub.ss (ml/kg) 117 ± 3 MRT (hrs) 109 ± 1 t.sub.1/2z (hrs) 92.7 ± 3.7
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).
TABLE-US-00041 TABLE 29 Mean (+SD) pharmacokinetic parameter estimates of PSI0536 following a subcutaneous single dose of 10.1 (+0.1) mg/kg to three male monkeys Parameter Estimate F (%) 73.9 ± 5.5 C.sub.max (nM) 779 ± 159 t.sub.max (hrs) 18.7 ± 7.5 CL/F (ml/h .Math. kg) 1.46 ± 0.10 V.sub.z/F (ml/kg) 163 ± 23 t.sub.1/2z (hrs) 77.3 ± 6.3 MRT (hrs) 115 ± 14
Conclusion
[0414] 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.
Example 15
Production and Characterization of IL-1-1R-I Binding Fusion Proteins with Mutations in the Fc Portion
[0415] 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.
Material and Methods
[0416] The fusion proteins PS10653-PS10656 (SEQ ID NO:1734-1737), see Table 30 for specifications, were produced in CHO cells according to Example 10.
[0417] 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.
[0418] 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.
[0419] 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.
TABLE-US-00042 TABLE 30 Fusion proteins and expression systems SEQ ID Fusion Expression NO proteins Construct system 1734 PSI0653 Z18754[N297A]-IgG1 Fc CHO 1735 PSI0654 Z18754[N297A]-LS-IgG1 Fc CHO 1736 PSI0655 Z18754[N297A]-YTE-IgG1 Fc CHO 1737 PSI0656 Z18754[S228P]-IgG4 Fc CHO
Results
[0420] 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.
[0421] 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.
TABLE-US-00043 TABLE 31 In vitro IC.sub.50 values SEQ ID NO: Fusion proteins IC.sub.50 (pM) 1734 PSI0653 4.3 1735 PSI0654 2.9 1736 PSI0655 5.9 1737 PSI0656 8.4
[0422] 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.
[0423] 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.
[0424] 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.
Itemized Listing of Embodiments
[0425] 1. IL-1R-I binding polypeptide, comprising an IL-1R-I binding motif BM, which motif consists of an amino acid sequence selected from:
TABLE-US-00044 i) (SEQ ID NO: 1686) EX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7EIX.sub.10X.sub.11LPNLX.sub.16RX.sub.18QYX.sub.21AFIX.sub.25X.sub.26LX.sub.28D
wherein, independently from each other, [0426] X.sub.2 is selected from A, D, E, F, H, I, L, Q, S, T and V; [0427] X.sub.3 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; [0428] X.sub.4 is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W and Y; [0429] X.sub.5 is selected from A, I and V; [0430] X.sub.6 is selected from F, H, I, Q, R, T, V and Y; [0431] X.sub.7 is selected from A, D, E, F, G, H, I, L, M, Q, S, T, V, W and Y; [0432] X.sub.10 is selected from F and Y; [0433] X.sub.11 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y; [0434] X.sub.16 is selected from N and T; [0435] X.sub.18 is selected from K, R and S; [0436] X.sub.21 is selected from Q, T and V; [0437] X.sub.25 is selected from I, M, R, V and Y; [0438] X.sub.26 is selected from K and S, and [0439] X.sub.28 is selected from F, I, L and M, [0440] and [0441] ii) an amino acid sequence which has at least 96% identity to the sequence defined in i) provided that X.sub.5 is I or V.
[0442] 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) [0443] X.sub.2 is selected from A, I, L, T and V; [0444] X.sub.3 is selected from E and Y; [0445] X.sub.4 is selected from A, E, I, K, Q, R, T, V and Y; [0446] X.sub.5 is selected from I and V; [0447] X.sub.6 is selected from Q and Y; [0448] X.sub.7 is selected from F and M; [0449] X.sub.10 is selected from F and Y; [0450] X.sub.11 is selected from A, D, E, F, G, H, K, L, Q, R, S, T, V and Y; [0451] X.sub.16 is selected from N and T; [0452] X.sub.18 is selected from K and R; [0453] X.sub.21 is selected from T and V; [0454] X.sub.25 is selected from I and R; [0455] X.sub.26 is selected from K and S, and [0456] X.sub.28 is selected from F and L, [0457] and an amino acid sequence which has at least 93% identity to the sequence defined in i).
[0458] 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: [0459] I. X.sub.3 is E; [0460] II. X.sub.5 is selected from I and V; [0461] III. X.sub.6 is selected from Q and Y; [0462] IV. X.sub.7 is selected from F and M; [0463] V. X.sub.10 is selected from F and Y; [0464] VI. X.sub.18 is selected from K and R; [0465] VII. X.sub.21 is T; [0466] VIII. X.sub.25 is R; [0467] IX. X.sub.26 is selected from K and S; and [0468] X. X.sub.28 is selected from F and L.
[0469] 4. IL-1R-I binding polypeptide according to item 3, wherein sequence i) fulfills at least six of the ten conditions I-X.
[0470] 5. IL-1R-I binding polypeptide according to item 4, wherein sequence i) fulfills at least seven of the ten conditions I-X.
[0471] 6. IL-1R-I binding polypeptide according to item 5, wherein sequence i) fulfills at least eight of the ten conditions I-X.
[0472] 7. IL-1R-I binding polypeptide according to item 6, wherein sequence i) fulfills at least nine of the ten conditions I-X.
[0473] 8. IL-1R-I binding polypeptide according to item 7, wherein sequence i) fulfills all of the ten conditions I-X.
[0474] 9. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X.sub.2X.sub.3X.sub.6 is VEQ or VEY.
[0475] 10. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X.sub.2X.sub.3X.sub.6 is IEQ or VEQ.
[0476] 11. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X.sub.6X.sub.10 is selected from the group consisting of QF, QY, YF and YY.
[0477] 12. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X.sub.6X.sub.10 is QF.
[0478] 13. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X.sub.10X.sub.18 is selected from the group consisting of FK, FR, YK and YR.
[0479] 14. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X.sub.10X.sub.18 is FK or FR.
[0480] 15. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X.sub.18X.sub.25X.sub.28 is KRL or RRL.
[0481] 16. IL-1R-I binding polypeptide according to any one of items 1-8, wherein X.sub.5X.sub.7 is IM or VM.
[0482] 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.
[0483] 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.
[0484] 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.
[0485] 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.
[0486] 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.
[0487] 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.
[0488] 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.
[0489] 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.
[0490] 25. IL-1R-I binding polypeptide according to item 24, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.
[0491] 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.
[0492] 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:
TABLE-US-00045 iii) (SEQ ID NO: 1687) K-[BM]-DPSQSX.sub.aX.sub.bLLX.sub.cEAKKLX.sub.dX.sub.eX.sub.fQ; [0493] wherein [0494] [BM] is an IL-1R-I binding motif as defined in any one of items 1-22; [0495] X.sub.a is selected from A and S; [0496] X.sub.b is selected from N and E; [0497] X.sub.c is selected from A, S and C; [0498] X.sub.d is selected from E, N and S; [0499] X.sub.e is selected from D, E and S; [0500] X.sub.f is selected from A and S; and [0501] iv) an amino acid sequence which has at least 91% identity to a sequence defined by iii).
[0502] 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.
[0503] 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.
[0504] 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.
[0505] 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.
[0506] 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.
[0507] 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.
[0508] 34. IL-1R-I binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
TABLE-US-00046 SEQ ID NO: 1695 ADNNFNK-[BM]DPSQSANLLSEAKKLNESQAPK; SEQ ID NO: 1696 ADNKFNK-[BM]DPSQSANLLAEAKKLNDAQAPK; SEQ ID NO: 1697 ADNKFNK-[BM]DPSVSKEILAEAKKLNDAQAPK; SEQ ID NO: 1698 ADAQQNNFNK-[BM]DPSQSTNVLGEAKKLNESQAPK; SEQ ID NO: 1699 AQHDE-[BM]DPSQSANVLGEAQKLNDSQAPK; SEQ ID NO: 1700 VDNKFNK-[BM]DPSQSANLLAEAKKLNDAQAPK; SEQ ID NO: 1701 AEAKYAK-[BM]DPSESSELLSEAKKLNKSQAPK; SEQ ID NO: 1702 VDAKYAK-[BM]DPSQSSELLAEAKKLNDAQAPK; SEQ ID NO: 1703 VDAKYAK-[BM]DPSQSSELLAEAKKLNDSQAPK; SEQ ID NO: 1704 AEAKYAK-[BM]DPSQSSELLSEAKKLNDSQAPK; SEQ ID NO: 1705 AEAKYAK-[BM]DPSQSSELLSEAKKLNDSQAP; SEQ ID NO: 1706 AEAKFAK-[BM]DPSQSSELLSEAKKLNDSQAPK; SEQ ID NO: 1707 AEAKFAK-[BM]DPSQSSELLSEAKKLNDSQAP; SEQ ID NO: 1708 AEAKYAK-[BM]DPSQSSELLAEAKKLNDAQAPK; SEQ ID NO: 1709 AEAKYAK-[BM]DPSQSSELLSEAKKLSESQAPK; SEQ ID NO: 1710 AEAKYAK-[BM]DPSQSSELLSEAKKLSESQAP; SEQ ID NO: 1711 AEAKFAK-[BM]DPSQSSELLSEAKKLSESQAPK; SEQ ID NO: 1712 AEAKFAK-[BM]DPSQSSELLSEAKKLSESQAP; SEQ ID NO: 1713 AEAKYAK-[BM]DPSQSSELLAEAKKLSEAQAPK; SEQ ID NO: 1714 AEAKYAK-[BM]DPSQSSELLSEAKKLESSQAPK; SEQ ID NO: 1715 AEAKYAK-[BM]DPSQSSELLSEAKKLESSQAP; SEQ ID NO: 1716 AEAKYAK-[BM]DPSQSSELLAEAKKLESAQAPK; SEQ ID NO: 1717 AEAKYAK-[BM]DPSQSSELLSEAKKLSDSQAPK; SEQ ID NO: 1718 AEAKYAK-[BM]DPSQSSELLSEAKKLSDSQAP; SEQ ID NO: 1719 AEAKYAK-[BM]DPSQSSELLAEAKKLSDSQAPK; SEQ ID NO: 1720 AEAKYAK-[BM]DPSQSSELLAEAKKLSDAQAPK; SEQ ID NO: 1721 VDAKYAK-[BM]DPSQSSELLSEAKKLNDSQAPK; SEQ ID NO: 1722 VDAKYAK-[BM]DPSQSSELLAEAKKLNDAQAPK; SEQ ID NO: 1723 VDAKYAK-[BM]DPSQSSELLSEAKKLSESQAPK; SEQ ID NO: 1724 VDAKYAK-[BM]DPSQSSELLAEAKKLSEAQAPK; SEQ ID NO: 1725 VDAKYAK-[BM]DPSQSSELLSEAKKLESSQAPK; SEQ ID NO: 1726 VDAKYAK-[BM]DPSQSSELLAEAKKLESAQAPK; SEQ ID NO: 1727 VDAKYAK-[BM]DPSQSSELLSEAKKLSDSQAPK; SEQ ID NO: 1728 VDAKYAK-[BM]DPSQSSELLAEAKKLSDSQAPK; SEQ ID NO: 1729 VDAKYAK-[BM]DPSQSSELLAEAKKLSDAQAPK; SEQ ID NO: 1730 VDAKYAK-[BM]DPSQSSELLAEAKKLNKAQAPK; SEQ ID NO: 1731 AEAKYAK-[BM]DPSQSSELLAEAKKLNKAQAPK, and SEQ ID NO: 1732 ADAKYAK-[BM]DPSQSSELLSEAKKLNDSQAPK,
wherein [BM] is an IL-1R-I binding motif as defined in any one of items 1-22.
[0509] 35. IL-1R-I binding polypeptide according to any one of items 1-34, which comprises an amino acid sequence selected from:
TABLE-US-00047 xix) (SEQ ID NO: 1721) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK
[0510] wherein [BM] is an IL-1R-I binding motif as defined in any one of items 1-22; and
[0511] xx) an amino acid sequence which in the sequences flanking the BM has at least 89% identity to the sequence defined in xix).
[0512] 36. IL-1R-I binding polypeptide according to any one of items 1-34, which comprises an amino acid sequence selected from:
TABLE-US-00048 xxi) (SEQ ID NO: 1709) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
[0513] wherein [BM] is an IL-1R-I binding motif as defined in any one of items 1-22; and
[0514] xxii) an amino acid sequence which in the sequences flanking the BM has at least 89% identity to the sequence defined in xxi).
[0515] 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.
[0516] 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.
[0517] 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.
[0518] 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.
[0519] 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.
[0520] 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.
[0521] 43. IL-1R-I binding polypeptide according to any preceding item, which is capable of blocking IL-1R-I dependent signaling.
[0522] 44. IL-1R-I binding polypeptide according to item 43, wherein the half maximal inhibitory concentration (IC.sub.50) of the blocking is at most 1×10.sup.−7 M, such as at most, 1×10.sup.−8 M, such as at most 1×10.sup.−9 M, such as at most 5×10.sup.−10 M.
[0523] 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.
[0524] 46. IL-1R-I binding polypeptide according to any preceding item which is capable of binding to IL-1R-I such that the EC.sub.50 value of the interaction is at most 1×10.sup.−7 M, such as at most 1×10.sup.−8 M, such as at most 5×10.sup.−9 M, such as at most 1×10.sup.−10 M such as at most 5×10.sup.−10 M, such as at most 2×10.sup.−10 M.
[0525] 47. IL-1R-I binding polypeptide according to any preceding item which is capable of binding to IL-1R-I such that the K.sub.D value of the interaction is at most 1×10.sup.−6 M, such as at most 1×10.sup.−7 M, such as at most 1×10.sup.−8 M, such as at most 1×10.sup.−9 M, such as at most 9×10.sup.−10.
[0526] 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.
[0527] 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.
[0528] 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.
[0529] 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.
[0530] 52. IL-1R-I binding polypeptide according to item 51, wherein said IL-1R-I binding polypeptide monomer units are covalently coupled together.
[0531] 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.
[0532] 54. IL-1R-I binding polypeptide according to any one of items 51-53, in dimeric form.
[0533] 55. Fusion protein or conjugate comprising [0534] a first moiety consisting of an IL-1R-I binding polypeptide according to any preceding item; and [0535] a second moiety consisting of a polypeptide having a desired biological activity.
[0536] 56. Fusion protein or conjugate according to item 55, wherein said desired biological activity is a therapeutic activity.
[0537] 57. Fusion protein or conjugate according to item 55, wherein said desired biological activity is a binding activity.
[0538] 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.
[0539] 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.
[0540] 60. Fusion protein or conjugate according to item 59, said in vivo half-life increasing activity is an albumin binding activity.
[0541] 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.
[0542] 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.
[0543] 63. Fusion protein or conjugate according to item 59, wherein said second moiety is serum albumin.
[0544] 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.
[0545] 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.
[0546] 66. Fusion protein or conjugate according to item 58 or 59, wherein said second moiety is transferrin.
[0547] 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.
[0548] 68. Fusion protein or conjugate according to item 57, wherein said binding activity acts to block a biological activity.
[0549] 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.
[0550] 70. Fusion protein or conjugate according item 69, wherein said linker is arranged between said first moiety and said second moiety.
[0551] 71. Fusion protein or conjugate according item 69, wherein said linker is arranged within said first moiety.
[0552] 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.
[0553] 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).sub.2 (SEQ ID NO:1683), AS(GGGGS).sub.2 (SEQ ID NO:1684) and ((KEAAA).sub.3KELAA).sub.2 (SEQ ID NO:1685).
[0554] 74. Fusion protein or conjugate according to item 73, wherein said linker is selected from a AS(GGGGS).sub.2 (SEQ ID NO:1684) and ((KEAAA).sub.3KELAA).sub.2 (SEQ ID NO:1685).
[0555] 75. A polynucleotide encoding a polypeptide or a fusion protein according to any one of items 1-74.
[0556] 76. Expression vector comprising a polynucleotide according to item 75.
[0557] 77. Host cell comprising an expression vector according to item 76.
[0558] 78. Method of producing a polypeptide according to any one of items 1-77, comprising [0559] culturing a host cell according to item 77 under conditions permissive of expression of said polypeptide from said expression vector, and [0560] isolating said polypeptide.
[0561] 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.
[0562] 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.
[0563] 81. Composition according to item 79 or 80, wherein said fusion protein is a fusion protein according to any one of claims 59-67.
[0564] 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.
[0565] 83. IL-1R-I binding polypeptide, fusion protein, conjugate or composition according to item 82 for use as a medicament.
[0566] 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.
[0567] 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.
[0568] 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.
[0569] 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.
[0570] 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.
[0571] 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.
[0572] 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.
[0573] 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.
[0574] 92. Fusion protein, conjugate or composition for use according to item
[0575] 91, wherein said fusion protein, conjugate or composition is administered once weekly to a subject in need thereof.
[0576] 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.
[0577] 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.
[0578] 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.
[0579] 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.
[0580] 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.
[0581] 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.
[0582] 99. Method according to item 97, wherein said fusion protein, conjugate or composition is administered once weekly to a subject in need thereof.