IL-17A-BINDING POLYPEPTIDES
20170362290 · 2017-12-21
Inventors
- Fredrik Frejd (Stockholm, SE)
- Joachim Feldwisch (Tyresö, SE)
- Susanne Klint (Stockholm, SE)
- Lindvi Gudmundsdotter (Stockholm, SE)
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P1/02
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
C07K2318/20
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
International classification
Abstract
The present disclosure relates to a class of engineered polypeptides having a binding affinity for interleukin-17A (IL-17A), and provides an IL-17A binding polypeptide comprising the sequence EX.sub.2DX.sub.4AX.sub.6X.sub.7EIX.sub.10X.sub.11 LPNL X.sub.16X.sub.17X.sub.18QX.sub.20X.sub.21AFIX.sub.25 X.sub.26LX.sub.28X.sub.29. Also disclosed is the use of such an interleukin-17A binding polypeptide as a diagnostic, prognostic and/or therapeutic agent.
Claims
1. An IL-17A binding polypeptide, comprising an IL-17A binding motif BM, which motif consists of an amino acid sequence selected from: i) EX.sub.2DX.sub.4AX.sub.6X.sub.7EIX.sub.10X.sub.11LPNL X.sub.16X.sub.17X.sub.18QX.sub.20X.sub.21AFIX.sub.25 X.sub.26LX.sub.28X.sub.29 wherein, independently from each other, X.sub.2 is selected from A, H, M and Y; X.sub.4 is selected from A, D, E, F, K, L, M, N, Q, R, S and Y; X.sub.6 is selected from A, Q and W; X.sub.7 is selected from F, I, L, M, V, W and Y; X.sub.10 is selected from A and W; X.sub.11 is selected from A, D, E, F, G, L, M, N, Q, S, T and Y; X.sub.16 is selected from N and T; X.sub.17 is selected from H, W and Y; X.sub.18 is selected from A, D, E, H and V; X.sub.20 is selected from A, G, Q, S and W; X.sub.21 is selected from A, D, E, F, H, K, N, R, T, V, W and Y; X.sub.25 is selected from A, D, E, G, H, I, L, M, N, Q, R, S, T and V; X.sub.26 is selected from K and S; X.sub.28 is selected from I, L, N and R; and X.sub.29 is selected from D and R; and ii) an amino acid sequence which has at least 89% identity to the sequence defined in i).
2. An IL-17A binding polypeptide according to claim 1, wherein, in sequence i), X.sub.2 is selected from A, H and M; X.sub.4 is selected from A, D, E, F, L, M, N, Q, R and Y; X.sub.11 is selected from A, D, E, F, G, L, M, N, S, T and Y; X.sub.18 is selected from A, D, E and V; X.sub.20 is selected from A, G, Q and W; X.sub.21 is selected from E, F, H, N, R, T, V, W and Y; X.sub.25 is selected from A, D, E, G, H, I, L, N, Q, R, S, T and V; and X.sub.28 is selected from I, N and R.
3. An IL-17A binding polypeptide according to claim 2, wherein, in sequence i), X.sub.16 is T; X.sub.17 is W; X.sub.21 is selected from E, F, H, W, T and Y; X.sub.25 is selected from A, D, E, G, H, I, L, N, Q, R, S and T; X.sub.26 is K; and X.sub.29 is D.
4. An IL-17A binding polypeptide according to claim 1, wherein sequence i) is the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-1216.
5. An IL-17A binding polypeptide according to claim 1, which comprises an amino acid sequence binding module, BMod, selected from: iii) K-[BM]-DPSQS X.sub.aX.sub.bLLX.sub.c EAKKL X.sub.dX.sub.eX.sub.fQ; wherein [BM] is an IL-17A binding motif as defined in any one of claims 1-4 provided that X.sub.29 is D; X.sub.a is selected from A and S; X.sub.b is selected from N and E; X.sub.c is selected from A, S and C; X.sub.d is selected from E, N and S; X.sub.e is selected from D, E and S; X.sub.f is selected from A and S; and iv) an amino acid sequence which has at least 85% identity to a sequence defined by iii).
6. An IL-17A binding polypeptide according to claim 5, wherein said sequence iii) is the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-1216.
7. An IL-17A binding polypeptide according to claim 1, which comprises an amino acid sequence selected from: vii) YA-[BMod]-AP; wherein [BMod] is an IL-17A binding module as defined in any one of claims 5-6; and viii) an amino acid sequence which has at least 86% identity to a sequence defined by vii).
8. An IL-17A binding polypeptide according to claim 1, which comprises an amino acid sequence selected from: TABLE-US-00028 xiii) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an IL-17A binding motif as defined in any one of claims 1-4; and xiv) an amino acid sequence which has at least 86% identity to the sequence defined in xiii).
9. An IL-17A binding polypeptide according to claim 8, wherein sequence xiii) is selected from SEQ ID NO:1-1216.
10. An IL-17A binding polypeptide according to claim 1, which comprises an amino acid sequence selected from: TABLE-US-00029 xv) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an IL-17A binding motif as defined in any one of claims 1-4; and xvi) an amino acid sequence which has at least 86% identity to the sequence defined in xv).
11. An IL-17A binding polypeptide according to claim 10, wherein sequence xv) is selected from SEQ ID NO:1217-1222.
12. An IL-17A binding polypeptide according to claim 1, which is capable of binding to IL-17A 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.
13. A Fusion protein or conjugate comprising a first moiety consisting of an IL-17A binding polypeptide according to claim 1; and a second moiety consisting of a polypeptide having a desired biological activity.
14. A Complex, comprising at least one IL-17A binding polypeptide according to claim 1 or at least one fusion protein or conjugate according to claim 13, and at least one antibody or an antigen binding fragment thereof.
15. A Polynucleotide encoding a polypeptide according to claim 1.
16. A Composition comprising an IL-17A binding polypeptide, fusion protein, conjugate or complex according to claim 1 and at least one pharmaceutically acceptable excipient or carrier.
17. (canceled)
18. A medicament, diagnostic agent or prognostic agent comprising an IL-17A binding polypeptide, fusion protein, conjugate or complex or composition according to claim 1.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
Summary
[0296] The following Examples disclose the development of novel Z variant molecules targeted to interleukin 17A (IL-17A) based on phage display technology. The IL-17A binding polypeptides described herein were sequenced, and their amino acid sequences are listed in
Example 1
Selection and Screening of IL-17A Binding Z Variants
Materials and Methods
[0297] Biotinylation of target protein: Human IL-17A (hIL-17A Peprotech cat. no. 200-17; SEQ ID NO:1226) and murine IL-17A (mIL-17A Peprotech cat. no. 210-17; SEQ ID NO:1227) 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 Scentific, cat. no. 21327) at a 20× molar excess. Subsequent buffer exchange to phosphate buffered saline (PBS,10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) was performed using a dialysis cassette (Slide-a-lyzer 3.5 K, 3500 MWCO, Thermo Scentific, cat. no. 66333) according to the manufacturer's instructions.
[0298] Phage display selection of IL-17A binding Z variants: A library of random variants of protein Z displayed on bacteriophage, constructed in phagemid pAY02592 essentially as described in Grönwall et al. (2007) J Biotechnol, 128:162-183, was used to select IL-17A binding Z variants. In this library, an albumin binding domain (abbreviated ABD and corresponding to GA3 of protein G from Streptococcus strain G148) is used as fusion partner to the Z variants. The library is denoted Zlib006Naive.II and has a size of 1.5×10.sup.10 library members (Z variants). E. coli RRIΔM15 cells (Rüther et al., (1982) Nucleic Acids Res 10:5765-5772) from a glycerol stock containing the phagemid library Zlib006Naive.II, were inoculated in 20 l of a defined proline free medium [3 g/l KH.sub.2PO.sub.4, 2 g/l K.sub.2HPO.sub.4, 0.02 g/l uracil, 6.7 g/l YNB (Difco™ Yeast Nitrogen Base w/o amino acids, Becton Dickinson), 5.5 g/l glucose monohydrate, 0.3 g/l L-alanine, 0.24 g/l L-arginine monohydrochloride, 0.11 g/l L-asparagine monohydrate, 0.1 g/l L-cysteine, 0.3 g/l L-glutamic acid, 0.1 g/l L-glutamine, 0.2 g/l glycine, 0.05 g/l L-histidine, 0.1 g/l L-isoleucine, 0.1 g/l L-leucine, 0.25 g/l L-lysine monohydrochloride, 0.1 g/l L-methionine, 0.2 g/l L-phenylalanine, 0.3 g/l L-serine, 0.2 g/l L-threonine, 0.1 g/l L-tryptophane, 0.05 g/l L-tyrosine, 0.1 g/l L-valine], supplemented with 100 μg/ml ampicillin. The cultivations were grown at 37° C. in a fermenter (Belach Bioteknik, BR20). When the cells reached an optical density at 600 nm (OD.sub.600) of 0.75, approximately 2.6 l of the cultivation was infected using a 10× molar excess of M13K07 helper phage (New England Biolabs, cat. no. N0315S). The cells were incubated for 30 min, whereupon the fermenter was filled up to 20 l with cultivation medium [2.5 g/l (NH.sub.4).sub.2SO.sub.4; 5.0 g/l Yeast Extract (Merck 1.03753.0500); 25 g/l Peptone (Scharlau 07-119); 2 g/l K.sub.2HPO.sub.4; 3 g/l KH.sub.2PO.sub.4; 1.25 g/l Na.sub.3C.sub.6H.sub.5O.sub.7.2 H.sub.2O; 0.1 ml/l Breox FMT30 antifoaming agent] supplemented with 100 μM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for induction of expression and with 50 μg/ml ampicillin, 12.5 μg/ml carbenicillin, 25 μg/ml kanamycin, 35 ml/l of 1.217 M MgSO.sub.4 and 10 ml of a trace element solution [129 mM FeCl.sub.3; 36.7 mM ZnSO.sub.4; 10.6 mM CuSO.sub.4; 78.1 mM MnSO.sub.4; 94.1 mM CaCl.sub.2, dissolved in 1.2 M HCl]. A glucose-limited fed-batch cultivation was started where a 600 g/l glucose solution was fed to the reactor (15 g/h in the start, 40 g/h at the end of the fermentation after 17 h). Through the automatic addition of 25% NH.sub.4OH, the pH was controlled at 7. Air was supplemented (10 l/min) and the stirrer was set to keep the dissolved oxygen level above 30%. The cells in the cultivation were removed by tangential flow filtration.
[0299] The phage particles were precipitated from the supernatant twice in PEG/NaCl (polyethylene glycol/sodium chloride), filtered and dissolved in PBS and glycerol as described in Grönwall et al., supra. Phage stocks were stored at −80° C. before use.
[0300] Selections against biotinylated hIL-17A (b-hIL-17A) or biotinylated mIL-17A (b-mIL-17A) were performed in four cycles divided in six different tracks (Table 2). Phage stock preparation, selection procedure and amplification of phage between selection cycles were performed essentially as described in WO2009/077175 for selection against another target with the following exceptions. Exception 1: PBS supplemented with 3% bovine serum albumin (BSA, Sigma, cat. no. A3059) and 0.1% Tween20 (Acros Organics, cat. no. 233362500) was used as selection buffer. Exception 2: pre-selection was performed in cycles 1-3 by incubation of phage stock with Dynabeads® M-280 Streptavidin (SA beads, Invitrogen, cat. no. 11206D). Exception 3: all tubes and beads used in the selections were pre-blocked with PBS supplemented with 5% BSA and 0.1% Tween20. Exception 4: selections were performed in solution at RT and the time for selection was 200 min in the first cycle and 120 min in the following cycles. Exception 5: this was followed by catching of target-phage complexes on SA beads using 1 mg beads per 6.4 μg b-hIL-17A or b-mIL-17A. Exception 6: E. coli strain XL1-Blue cells (Agilent Technologies, cat. no. 200268) grown in medium supplemented with 10 μg/ml tetracycline (exception 7) were used for infection. Exception 8: tryptone yeast extract plates (15 g/l agar, 10 g/l tryptone water (Merck), 5 g/l yeast extract, 3 g/l NaCl, 2% glucose) supplemented with 0.1 g/l ampicillin and 0.01 g/l tetracycline were used for spreading of bacteria. Exception 9: a 10× excess of M13K07 helper phage compared to bacteria was allowed to infect log phase bacteria.
[0301] An overview of the selection strategy, describing an increased stringency in subsequent cycles obtained using a lowered target concentration and an increased number of washes, is shown in Table 2.
TABLE-US-00005 TABLE 2 Overview of the selection from a primary library Phage stock Target Higher amount Selection from library or concentration Number of of phage into Cycle track selection track Target (nM) washes selection track 1 1 Zlib006Naive.II hIL-17A 50 2 — 2 1-1 1 hIL-17A 25 4 Yes 2 1-2 1 hIL-17A 25 4 — 2 1-3 1 hIL-17A 5 5 — 2 1-4 1 mIL-17A 50 4 — 3 1-1-1 1-1 hIL-17A 12.5 6 Yes 3 1-2-1 1-2 hIL-17A 12.5 6 — 3 1-3-1 1-3 hIL-17A 1 8 — 3 1-4-1 1-4 hIL-17A 12.5 6 — 3 1-4-2 1-4 mIL-17A 5 8 — 4 1-1-1-1 1-1-1 hIL-17A 5 8 Yes 4 1-2-1-1 1-2-1 hIL-17A 5 8 — 4 1-3-1-1 1-3-1 hIL-17A 0.25 12 — 4 1-3-1-2 1-3-1 hIL-17A 0.05 15 — 4 1-4-1-1 1-4-1 mIL-17A 5 8 — 4 1-4-2-1 1-4-2 hIL-17A 0.5 12 —
[0302] Track 1 was divided in the second to the fourth cycles, resulting in a total of four tracks (1-1 to 1-4) in cycle 2, five tracks (1-1-1 to 1-4-2) in cycle 3 and six tracks (1-1-1-1 to 1-4-2-1) in cycle 4. The number of phage particles used for selections was about 2000 times the number of eluted phage particles in the previous cycle, but a higher amount was used in selection tracks 1-1, 1-1-1 and 1-1-1-1.
[0303] Washes were performed for 1 min using PBST 0.1% (PBS supplemented with 0.1% Tween-20), and elution was carried out as described in WO2009/077175.
[0304] Production of Z variants for ELISA: The Z variants were produced by inoculating single colonies from the selections into 1 ml TSB-YE medium supplemented with 100 μg/ml ampicillin and 0.1 mM IPTG in deep-well plates (Nunc, cat. no. 278752). The plates were incubated for 18-24 h at 37° C. Cells were pelleted by centrifugation, re-suspended in 400 μl PBST 0.05% and frozen at −80° C. to release the periplasmic fraction of the cells. Frozen samples were thawed in a water bath and the freeze-thawing procedure was repeated six times. 400 μl PBST 0.05% was added to the thawed samples and cells were pelleted by centrifugation.
[0305] The final supernatant of the periplasmic extract contained the Z-variants as fusions to ABD, expressed as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (Grönwall et al., supra). Z##### refers to individual 58 amino acid residue Z variants.
[0306] ELISA screening of Z variants: The binding of Z variants to human IL-17A was analyzed in ELISA assays. Half-area 96-well ELISA plates (Costar, cat. no. 3690) were coated at 4° C. overnight with 2 μg/ml of an anti-ABD goat antibody (produced in-house) diluted in coating buffer (50 mM sodium carbonate, pH 9.6; Sigma, cat. no. C3041). The antibody solution was poured off and the wells were washed in water and blocked with 100 μl of PBSC (PBS supplemented with 0.5% casein) for 1 to 3 h at RT. The blocking solution was discarded and 50 μl periplasmic solutions were added to the wells and incubated for 1.5 h at RT under slow agitation. As a blank control, PBST 0.05% was added instead of the periplasmic sample. The supernatants were poured off and the wells were washed 4 times with PBST 0.05%. Next, 50 μl of b-hIL-17A at a concentration of 6.5 nM in PBSC was added to each well. The plates were incubated for 1.5 h at RT followed by washes as described above. Streptavidin conjugated HRP (Thermo Scientific, cat. no. N100) diluted 1:30,000 in PBSC, was added to the wells and the plates were incubated for 1 h. After washing as described above, 50 μl ImmunoPure TMB substrate (Thermo Scientific, cat. no. 34021) was added to the wells and the plates were treated according to the manufacturer's recommendations. The absorbance at 450 nm was measured using a Victor.sup.3 multi-well plate reader (Perkin Elmer).
[0307] Sequencing: In parallel with the ELISA screening, all clones were picked for sequencing. PCR fragments were amplified from single colonies, sequenced and analyzed essentially as described in WO2009/077175.
Results
[0308] Phage display selection of IL-17A binding Z variants: Individual clones were obtained after four cycles of phage display selections against b-hIL-17A and b-mIL-17A.
[0309] ELISA screening of Z variants: The clones obtained after four cycles of selection were expanded in 96-well plates and screened for hIL-17A binding activity in ELISA. 42 Z variants were found to give a response of 4× the blank control or higher (0.43-3.2 AU) against hIL-17A at a concentration of 6.5 nM. No response was obtained for the blank control.
[0310] 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
Example 2
Production of Monomeric IL-17A Binding Z Variants
[0311] This Example describes the general procedure for subcloning and production of His-tagged Z variants and Z variants in fusion with ABD, which are used throughout in characterization experiments below.
Materials and Methods
[0312] Subcloning of Z variants with a His-tag: The DNA of respective Z variant was amplified from the 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 (essentially as described in WO2009/077175 for Z variants binding another target). The Z gene fragments were subcloned into the expression vector pAY01448 resulting in the encoded sequence MGSSHHHHHHLQ-[Z#####]-VD.
[0313] Subcloning of Z variants in fusion with ABD: The DNA of respective Z variant was amplified from the library vector pAY02592. A PCR was performed using suitable primer pairs and the resulting gene fragments were cleaved with restriction enzymes PstI and AccI and ligated into the expression vector pAY03362 digested with the same enzymes, resulting in an ABD fusion protein, wherein the ABD variant is PP013 (SEQ ID NO:1224). The constructs encoded by the expression vectors were MGSSLQ-[Z#####]-VDSS-PP013.
[0314] Cultivation: E. coli BL21(DE3) cells (Novagen) were transformed with plasmids containing the gene fragment of each respective IL-17A binding Z variant and cultivated at 37° C. in 800 or 1000 ml of TSB-YE medium supplemented with 50 μg/ml kanamycin. In order to induce protein expression, IPTG was added to a final concentration of 0.2 mM at OD.sub.600=2 and the cultivation was incubated at 37° C. for another 5 h. The cells were harvested by centrifugation.
[0315] Purification of IL-17A binding Z variants with a His.sub.6-tag: Approximately 2-5 g of each cell pellet was re-suspended in 30 ml of binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) supplemented with Benzonase® (Merck) to a concentration of 15 U/ml. 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 IL-17A binding Z variants were subsequently eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4).
[0316] Purification of IL-17A binding Z variants in fusion with ABD: Approximately 1-2 g of each cell pellet was re-suspended in 30 ml 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 by sonication 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. To fractions eluted in the anti-ABD agarose affinity chromatography purification step, acetonitrile (ACN) was added to a final concentration of 10% and the sample was loaded on 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. Fractions containing pure ABD-fused Z variants were identified by SDS-PAGE analysis and pooled. After the RPC purification, the buffer of the pool 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 columns (GE Healthcare). Finally, the ABD fused Z variants were purified on 1 ml EndoTrap® red columns (Hyglos) to ensure low endotoxin content.
[0317] Protein concentrations were determined by measuring the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer 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.
Results
[0318] Cultivation and purification: The IL-17A binding Z variants with a His.sub.6-tag as well as the Z variants fused at their C-terminus to ABD, were expressed as soluble gene products in E. coli. The amount of purified protein from approximately 1-5 g bacterial pellet was determined spectrophotometrically by measuring the absorbance at 280 nm and ranged from approximately 5 mg to 20 mg for the different IL-17A binding Z variants. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the IL-17A binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.
Example 3
Assessment of Blocking Ability of Primary IL-17A Binding Z Variants
[0319] Normal human dermal fibroblasts (NHDF) produce IL-6 upon stimulation with IL-17A (Chang and Dong 2007, Cell Research 17:435-440) and the amount of released IL-6 to the supernatant is correlated to the concentration of added IL-17A. Thus, blocking of IL-17A leads to a reduction of IL-6 in the supernatant that can be quantified. In this Example, the assay was used to evaluate the blocking ability of IL-17A binding Z variants Z06260 (SEQ ID NO:1200), Z06282 (SEQ ID NO:1206) and Z06455 (SEQ ID NO:1214), alone or in fusion with ABD (PP013; SEQ ID NO:1224).
Materials and Methods
[0320] NHDF assay with His.sub.6-tagged Z-variants: NHDF cells (Lonza, cat. no. CC-2511) were cultured in fibroblast basal medium (Lonza, cat. no. CC-3132) supplied with growth promoting factors (Lonza, cat. no. CC-5034). On the day before the experiment, 10.sup.5 cells were seeded in 100 μl per well into 96-well culture plates (Greiner, cat. no. 655180). On the day of the experiment, dilutions of the IL-17A specific Z variants His.sub.6-Z06260, His.sub.6-Z06282 and His.sub.6-Z06455 were prepared in a separate 96-well plate. The Z variants were titrated in three-fold steps from 3130 nM to 0.2 nM in medium containing 0.031 nM hIL-17A. A standard curve of hIL-17A was also prepared (0.002-31 nM) as well as controls containing medium with 0.031 nM hIL-17A or medium alone. The medium in the plate with the overnight cultured NHDF cells was discarded. Test samples, standard curve samples and controls were transferred to the cell-containing plate. The NHDF cells were stimulated for 18-24 h at 37° C. and the IL-6 content in the supernatants was subsequently quantified using the IL-6 specific ELISA described below.
[0321] NHDF assay with His.sub.6-tagged and ABD fused Z variants: NHDF cells were prepared as described above. On the day of the experiment, dilutions of the IL-17A specific Z variant constructs His.sub.6-Z06282, His.sub.6-Z06455, Z06260-ABD, Z06282-ABD and Z06455-ABD were prepared in a separate 96-well plate. The Z variant constructs were titrated in four-fold steps from 780 nM to 0.2 nM in medium containing 0.031 nM IL-17A and 8 μM HSA. Preparation of a standard IL-17A curve and controls, as well as downstream analysis, was performed as described above.
[0322] IL-6 ELISA: 96-well half area plates (Costar, cat. no. 3690) were coated with the anti-IL-6 monoclonal antibody MAB206 (R&D systems) at a concentration of 4 μg/ml in PBS (50 μl/well) and incubated overnight at 4° C. On the next day, the plates were rinsed twice in tap water and blocked with PBS+2% BSA (Sigma) for 2 h. An IL-6 standard (R&D systems, cat. no. 206-IL-50), titrated in a 2-fold dilution series (0.2-50 ng/ml) and supernatants from the cell assay plate were added to the coated ELISA plates (50 μl/well) and incubated for 1.5 h at RT. The plates were washed 4 times in an automated ELISA washer and 0.25 μg/ml (50 μl/well) of biotinylated anti-IL-6 polyclonal antibody (R&D systems, BAF206) was added. After incubation for 1 h, the plate was washed and 50 μl of streptavidin-HRP (Thermo Fisher, cat. no. N100) diluted 8000 times was added to each well. After one additional hour of incubation and subsequent washing, the plate was developed with 50 μl TMB (Thermo Fisher, cat. no. 34021) per well and the reactions were stopped with 50 μl 2M H.sub.2SO.sub.4. The absorbance at 450 nm was measured in a 96-well plate reader (Victor.sup.3) and the concentration of IL-6 in each sample was calculated. The results are presented as the percentage of maximum IL-6 production, calculated as:
100−[(ABS.sub.IL-17A blocker−ABS.sub.background)/(ABS.sub.Max IL-6 prod−ABS.sub.background)]×100
Results
[0323] Results from the first NHDF assay showed that all three Z variants His.sub.6-Z06260, His.sub.6-Z06282 and His.sub.6-Z06455 blocked IL-17A induced IL-6 production in a dose dependent manner (
[0324] The NHDF assay was repeated with the same Z variants fused to ABD as well as inclusion of HSA in the assay to ascertain retained binding to the antigen when mimicking in vivo conditions. In vivo, ABD will bind to albumin and convey longer circulation half-life. The results showed that all three binders in fusion with ABD blocked IL-17A induced IL-6 production equally well or better than the His.sub.6-tagged Z variants (
TABLE-US-00006 TABLE 3 IC50 values for primary Z-variants blocking IL-17A induced IL-6 production in the presence of HSA Analyte SEQ ID NO of Z variant IC50 (nM) Z06260-ABD 1200 8 His.sub.6-Z06282 1206 16 Z06282-ABD 1206 15 His.sub.6-Z06455 1214 67 Z06455-ABD 1214 15
Example 4
Design and Construction of a First Maturated Library of IL-17A Binding Z Variants
[0325] In this Example, a maturated library was designed and constructed. The library was used for selection of further IL-17A binding Z variants. Selections from maturated libraries are usually expected to result in binders with increased affinity (Orlova et al., (2006) Cancer Res 66(8):4339-48). In this study, randomized single stranded linkers were generated using split-pool DNA synthesis, enabling incorporation of defined codons in desired positions in the synthesis.
Materials and Methods
[0326] Library design: The library was based on the sequences of the IL-17A binding Z variants identified as described in Example 1. In the new library, 13 variable positions in the Z molecule scaffold were biased towards certain amino acid residues, according to a strategy based on the Z variant sequences defined in SEQ ID NO:1200-1216. A DNA linker was generated using split-pool synthesis containing the following 147 bp 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 GCG NNN NNN GAG ATC NNN NNN TTA CCT AAC TTA 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:1248; randomized codons are denoted NNN), comprising a coding sequence for a partially randomized helix 1 and 2 in the corresponding amino acid sequence, flanked by restriction sites for XhoI and SacI. The theoretical distributions of amino acid residues in the new library including 13 variable Z positions (9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32 and 35) in the Z molecule scaffold are given in Table 4. The resulting theoretical library size is 6.1×10.sup.9 variants.
TABLE-US-00007 TABLE 4 Library design, first maturation Amino acid position in Randomization No of the Z variant (amino acid amino molecule abbreviations) acids Proportion 9 A, H, M, W, Y 5 ⅕ 10 A, D (60%), G, L, N 5 1/10, 6/10 (D) 11 D, E, F, N, Q R, Y 7 1/7 13 A, E, Q, W 4 ¼ 14 F, I, L, M, V, W 6 ⅙ 17 A, F, Q, W 4 ¼ 18 A, D, E, L, M, S, T 7 1/7 24 A, H, R, T, W, Y 6 ⅙ 25 A, D, H, R, V 5 ⅕ 27 A, G, Q, R, S, W 6 ⅙ 28 F, H, K, N, R, S, T, V, Y 9 1/9 32 A, G, H, I, Q, R, S, V 8 ⅛ 35 I, L, N, R 4 ¼
[0327] Library construction: The library was amplified using AmpliTaq Gold polymerase (Applied Biosystems, cat. no. 4311816) during 12 cycles of PCR, and pooled products were purified with QIAquick PCR Purification Kit (QIAGEN, cat. no. 28106) according to the supplier's recommendations. The purified pool of randomized library fragments was digested with restriction enzymes XhoI and SacI-HF (New England Biolabs, cat. no. R0146L and cat. no. R3156M, respectively) and concentrated using the QIAquick PCR Purification Kit. Subsequently, the product was subjected to gel electrophoresis using a preparative 2.5% agarose gel (Nuisieve GTC agarose, Cambrex, Invitrogen) and purified from said gel using QIAGEN Gel Extraction Kit (QIAGEN, cat. no. 28706) according to the supplier's recommendations.
[0328] The phagemid vector pAY02592 (essentially as pAffi1 described in Grönwall et al., supra) was restricted with the same enzymes, purified using phenol/chloroform extraction and ethanol precipitation. The restricted fragments and the restricted vector were ligated in a molar ratio of 5:1 with T4 DNA ligase (Fermentas, cat. no. EL0011) for 2 h at RT, followed by overnight incubation at 4° C. The ligated DNA was recovered by phenol/chloroform extraction and ethanol precipitation, followed by dissolution in 10 mM Tris-HCl, pH 8.5. Thus, the resulting library in vector pAY02592 encoded Z variants each fused to an albumin binding domain (ABD) derived from streptococcal Protein G.
[0329] The ligation reactions (approximately 160 ng DNA/transformation) were electroporated into electrocompetent E. coli ER2738 cells (Lucigen, Middleton, Wis., USA, 50 μl). Immediately after electroporation, approximately 1 ml of recovery medium (supplied with E. coli ER2738 cells) was added. The transformed cells were incubated at 37° C. for 60 min. Samples were taken for titration and for determination of the number of transformants. The cells were thereafter pooled and cultivated overnight at 37° C. in 1 l of TSB-YE medium, supplemented with 2% glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin. The cells were pelleted for 15 min at 4,000 g and resuspended in a PBS/glycerol solution (approximately 40% glycerol). The cells were aliquoted and stored at −80° C. Clones from the library of Z variants were sequenced in order to verify the content and to evaluate the outcome of the constructed library vis-à-vis the library design. Sequencing was performed as described in Example 1 and the amino acid distribution was verified.
[0330] Preparation of phage stock: Phage stock containing the phagemid library was prepared in a 20 l fermenter (Belach Bioteknik). Cells from a glycerol stock containing the phagemid library were inoculated in 10 l of TSB-YE supplemented with 1 g/l glucose, 100 mg/l ampicillin and 10 mg/l tetracycline. When the cells reached an optical density at 600 nm (OD.sub.600) of 0.52, approximately 1.7 l of the cultivation was infected using a 5× molar excess of M13K07 helper phage. The cells were incubated for 30 min, whereupon the fermenter was filled up to 10 l with complex fermentation medium [2.5 g/l (NH.sub.4).sub.2SO.sub.4; 5.0 g/l yeast extract; 30 g/l tryptone, 2 g/l K.sub.2HPO.sub.4; 3 g/l KH.sub.2PO.sub.4, 1.25 g/l; Na.sub.3C.sub.6H.sub.5O.sub.7.2 H.sub.2O; Breox FMT30 antifoaming agent 0.1 ml/l]. The following components were added: 10 ml carbenicillin 25 mg/ml; 5 ml kanamycin 50 mg/ml; 1 ml 1 M IPTG; 17.5 ml/l 1.217 M MgSO.sub.4 and 5 ml of a trace element solution [129 mM FeCl.sub.3; 36.7 mM ZnSO.sub.4; 10.6 mM CuSO.sub.4; 78.1 mM MnSO.sub.4; 94.1 mM CaCl.sub.2, dissolved in 1.2 M HCl]. A glucose limited fed-batch cultivation was started where a 600 g/l glucose solution was fed to the reactor (3.5 g/h at the start, 37.5 g/h after 20 h continuing until the end of the cultivation). Through the automatic addition of 25% NH.sub.4OH, the pH was controlled at pH 7. Air was supplemented (5 l/min) and the stirrer was set at 500 rpm. After 24 h of fed-batch cultivation the OD.sub.600 was 19.4. The cells in the cultivation were pelleted by centrifugation at 15,900 g. The phage particles were precipitated from the supernatant twice in PEG/NaCl, filtered and dissolved in PBS and glycerol as described in Example 1. Phage stocks were stored at −80° C. until use in selection.
Results
[0331] Library construction: The new library was designed based on a set of IL-17A binding Z variants with verified binding properties (Example 1 and 3). The theoretical size of the designed library was 6.1×10.sup.9 Z variants. The actual size of the library, determined by titration after transformation to E. coli ER2738 cells, was 4.5×10.sup.9 transformants.
[0332] The library quality was tested by sequencing of 96 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 maturated library of potential binders to IL-17A was thus successfully constructed.
Example 5
Selection, Screening and Characterization of Z Variants from the First Maturated Library
Materials and Methods
[0333] Phage display selection of IL-17A binding Z variants: The target proteins hIL-17A and mIL-17A were biotinylated as described in Example 1. Phage display selections, using the new library of Z variant molecules constructed as described in Example 4, were performed in four cycles against hIL-17A and mIL-17A essentially as described in Example 1, with the following exceptions. Exception 1: PBST 0.1% was used as selection buffer. At selection, fetal calf serum (FCS, Gibco, cat. no. 10108-165) and human serum albumin (HSA, Albucult, Novozymes, cat. no. 230-005) were added to the selection buffer to a final concentration of 10% and 1.5 μM, respectively. Exception 2: a pre-selection step was performed in cycle 1 by incubating the phage stock with SA beads. Exception 3: all tubes and beads used in the selection were pre-blocked with PBS supplemented with 3% BSA and 0.1% Tween20. Exception 4: the time for selection was 140, 70, 60 and 50 min in cycles 1, 2, 3 and 4, respectively.
[0334] An overview of the selection strategy, describing an increased stringency in subsequent cycles obtained by using a lowered target concentration and an increased number of washes, is shown in Table 5.
TABLE-US-00008 TABLE 5 Overview of the selection from the first maturated library Phage stock Target Number Number Number of Selection from library or conc. of 1 min of 10 min overnight Elution Cycle track selection track Target (nM) washes washes washes at 1 1 Zlib006IL- hIL-17A 25 2 — — pH 5.5 17A.I 1 2 Zlib006IL- hIL-17A 12.5 3 — — pH 2.2 17A.I 1 3 Zlib006IL- hIL-17A 25 2 — — pH 2.2 17A.I 2 1-1 1 hIL-17A 12.5 6 — — pH 5.5 2 2-1 2 hIL-17A 2.5 10 — — pH 2.2 2 2-2 2 hIL-17A 5 6 — — pH 2.2 2 3-1 3 hIL-17A 7.5 10 — — pH 2.2 2 3-2 3 hIL-17A 12.5 6 — — pH 2.2 2 3-3 3 mIL-17A 12.5 6 — — pH 2.2 3 1-1-1 1-1 hIL-17A 2.5 10 — — pH 5.5 3 2-1-1 2-1 hIL-17A 0.05 15 1 — pH 2.2 3 2-2-1 2-2 hIL-17A 0.5 15 1 — pH 2.2 3 3-1-1 3-1 hIL-17A 0.75 15 1 — pH 2.2 3 3-2-1 3-2 hIL-17A 2.5 10 — — pH 2.2 3 3-3-1 3-3 hIL-17A 5 10 — — pH 2.2 4 1-1-1-1a 1-1-1 hIL-17A 0.05 20 — — pH 5.5 4 1-1-1-1b 1-1-1 hIL-17A 0.05 20 — 1 pH 5.5 4 1-1-1-2a 1-1-1 hIL-17A 1 12 — — pH 5.5 4 1-1-1-2b 1-1-1 hIL-17A 1 12 — 1 pH 5.5 4 2-1-1-1a 2-1-1 hIL-17A 0.0025 30 — — pH 2.2 4 2-1-1-1b 2-1-1 hIL-17A 0.0025 30 — 1 pH 2.2 4 2-1-1-2a 2-1-1 hIL-17A 0.025 20 — — pH 2.2 4 2-1-1-2b 2-1-1 hIL-17A 0.025 20 — 1 pH 2.2 4 2-2-1-1a 2-2-1 hIL-17A 0.005 30 — — pH 2.2 4 2-2-1-1b 2-2-1 hIL-17A 0.005 30 — 1 pH 2.2 4 2-2-1-2a 2-2-1 hIL-17A 0.05 20 — — pH 2.2 4 2-2-1-2b 2-2-1 hIL-17A 0.05 20 — 1 pH 2.2 4 3-1-1-1a 3-1-1 hIL-17A 0.025 30 — — pH 2.2 4 3-1-1-1b 3-1-1 hIL-17A 0.025 30 — 1 pH 2.2 4 3-1-1-2a 3-1-1 hIL-17A 0.25 20 — — pH 2.2 4 3-1-1-2b 3-1-1 hIL-17A 0.25 20 — 1 pH 2.2 4 3-2-1-1a 3-2-1 hIL-17A 0.05 20 — — pH 2.2 4 3-2-1-1b 3-2-1 hIL-17A 0.05 20 — 1 pH 2.2 4 3-2-1-2a 3-2-1 hIL-17A 1 12 — — pH 2.2 4 3-2-1-2b 3-2-1 hIL-17A 1 12 — 1 pH 2.2 4 3-3-1-1a 3-3-1 mIL-17A 0.05 20 — — pH 2.2 4 3-3-1-1b 3-3-1 mIL-17A 0.05 20 — 1 pH 2.2 4 3-3-1-2a 3-3-1 mIL-17A 1 15 — — pH 2.2 4 3-3-1-2b 3-3-1 mIL-17A 1 15 — 1 pH 2.2
[0335] Tracks 1-3 in cycle 1 were split in the second to fourth cycles, resulting in a total of six tracks (1-1 to 3-3) in cycle 2, six tracks (1-1-1 to 3-3-1) in cycle 3 and 24 tracks (1-1-1-1 a to 3-3-1-2b) in cycle 4. Washes were performed using PBST 0.1% for 1 min. However, for track 1-3 in cycle 3, one of the washes lasted for 10 min.
[0336] After the last wash in cycle 4, the target-phage complexes on SA beads from all tracks were divided in two equal parts. Bound phage particles from the first part were immediately eluted, while the second part was subjected to an overnight wash before elution of phage particles. The bound phage particles were eluted using two different procedures: 1) with glycine-HCl, pH 2.2, as in Example 1, or 2) 500 μl of 100 mM sodium phosphate, 150 mM sodium chloride, pH 5.5 and neutralization with 500 μl PBS.
[0337] Amplification of phage particles: Amplification of phage particles between selection cycle 1 and 2 was performed essentially as described in Example 1, with the following three exceptions. Exception 1: E. coli ER2738 was used for phage amplification. Exception 2: M13K07 helper phage were used in 5× excess. Exception 3: the amplification of phage particles between the selection cycles 2 and 4 was performed as follows: after infection of log phase E. coli ER2738 with phage particles, TSB supplemented with 2 glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin was added and followed by incubation with rotation for 30 min at 37° C. Next, the bacteria were infected with M13K07 helper phage. The infected bacteria were pelleted by centrifugation, re-suspended in TSB-YE medium supplemented with 100 μM IPTG, 25 μg/ml kanamycin and 100 μg/ml ampicillin, and grown overnight at 30° C. The overnight cultures were centrifuged, and phage particles in the supernatant were precipitated twice with PEG/NaCl buffer. Lastly, phages were re-suspended in selection buffer before entering the next selection cycle.
[0338] In the final selection cycle, log phase bacteria were infected with eluate and diluted before spreading onto TBAB plates (30 g/l tryptose blood agar base, Oxoid cat. no. CM0233B) supplemented with 0.2 g/l ampicillin in order to form single colonies to be used in ELISA screening.
[0339] Sequencing of potential binders: Individual clones from the different selection tracks were picked for sequencing. Amplification of gene fragments and sequence analysis of gene fragments were performed essentially as described in Example 1.
[0340] ELISA screening of Z variants: Single colonies containing Z variants (expressed as Z variant ABD fusion proteins as described in Example 1) were randomly picked from the selected clones of the maturated library, and grown in cultivations essentially as described in Example 1. Preparation of the periplasmic supernatants and ELISA screenings were also performed essentially as described in Example 1 with the following two exceptions. Exception 1: biotinylated hIL-17A was used at a concentration of 0.4 nM. Exception 2: the periplasmic fraction of Z variant Z06282 (SEQ ID NO:1206) from the primary selection was used as a positive control.
[0341] EC50 analysis of Z variants: A selection of IL-17A binding Z variants was subjected to an analysis of response against a dilution series of b-hIL-17A using ELISA as described above. Biotinylated target protein was added at a concentration of 6 nM and diluted stepwise 1:3 down to 8 pM. As a background control, the Z variants were also assayed with no target protein added. Periplasm samples containing the primary IL-17A binder Z06282 (SEQ ID NO:1206) were included as a positive control. Data were analyzed using GraphPad Prism 5 and non-linear regression, and EC50 values (the half maximal effective concentration) were calculated.
Results
[0342] Phage display selection of maturated IL-17A binding Z variants: Selection was performed in a total of 24 parallel tracks containing four cycles each. The different selection tracks differed in target concentration, target species origin (human IL-17A or murine IL-17A), selection time, wash conditions and the pH of the elution buffer. Clones originating from the selection tracks using only human IL-17 and elution at pH 2.2 were shown to have the best performance in ELISA screen.
[0343] Sequencing: Randomly picked clones were sequenced. Each individual Z variant was given an identification number, Z#####, as described in Example 1. In total, 932 new unique Z variant molecules were identified. For the 494 best performing variants in the ELISA screen below, the amino acid sequences of the 58 amino acid residues long Z variants are listed in
[0344] ELISA screening of Z variants: Clones obtained after four selection cycles were produced in 96-well plates and screened for human IL-17A binding activity using ELISA. All randomly picked clones were analyzed. 494 of the 932 unique Z variants were found to give a response of 2× the blank control or higher (0.15-2.0 AU) against hIL-17A at a concentration of 0.4 nM. Positive signals were shown for clones originating from all selection tracks. The blank controls had absorbances of 0.055-0.075 AU.
[0345] EC50 analysis of Z variants: A subset of Z variants was selected based on the result of the ELISA screening experiment described above (absorbance over 1.25 AU) or based on variation in amino acid sequence, and subjected to a target titration in ELISA format. Periplasm samples were incubated with a serial dilution of b-hIL-17A ranging from 6 nM to 8 pM. A periplasm sample containing Z06282 (SEQ ID NO:1206) identified in the primary selection was included as a positive control. Obtained values were analyzed and the respective EC50 values were calculated (Table 6).
TABLE-US-00009 TABLE 6 Calculated EC50 values from ELISA titration analysis SEQ EC50 Z variant ID NO (M) Z10210 36 1.1 × 10.sup.−9 Z10241 11 3.4 × 10.sup.−10 Z10255 37 4.6 × 10.sup.−10 Z10257 38 6.7 × 10.sup.−10 Z10433 28 9.4 × 10.sup.−10 Z10459 39 4.7 × 10.sup.−10 Z10462 12 3.4 × 10.sup.−10 Z10465 40 5.0 × 10.sup.−10 Z10470 41 4.7 × 10.sup.−10 Z10483 42 4.0 × 10.sup.−10 Z10508 2 3.8 × 10.sup.−10 Z10529 43 6.4 × 10.sup.−10 Z10532 1 3.6 × 10.sup.−10 Z10534 13 4.1 × 10.sup.−10 Z10550 44 7.9 × 10.sup.−10 Z10565 45 3.8 × 10.sup.−10 Z10566 14 5.5 × 10.sup.−10 Z10675 15 3.6 × 10.sup.−10 Z10676 46 4.0 × 10.sup.−10 Z10690 47 5.9 × 10.sup.−10 Z10703 48 7.5 × 10.sup.−10 Z10708 49 4.1 × 10.sup.−10 Z10710 50 5.4 × 10.sup.−10 Z10718 16 5.1 × 10.sup.−10 Z10728 51 5.3 × 10.sup.−10 Z10745 52 6.8 × 10.sup.−10 Z10756 53 6.4 × 10.sup.−10 Z10759 54 6.7 × 10.sup.−10 Z10775 55 5.1 × 10.sup.−10 Z10778 56 4.7 × 10.sup.−10 Z10779 57 5.7 × 10.sup.−10 Z10800 58 6.4 × 10.sup.−10 Z10807 59 6.0 × 10.sup.−10 Z10844 60 7.5 × 10.sup.−10 Z10857 61 4.5 × 10.sup.−10 Z10858 62 5.2 × 10.sup.−10 Z10859 31 6.9 × 10.sup.−10 Z10863 3 4.5 × 10.sup.−10 Z10914 63 5.1 × 10.sup.−10 Z06282 1206 5.0 × 10.sup.−9
Example 6
Design and Construction of a Second Maturated Library of IL-17A Binding Z Variants
[0346] In this Example, a second maturated library was constructed essentially as described in Example 4. The library was used for selections of additional IL-17A binding Z variants.
Materials and Methods
[0347] Library design: The library was based on the sequences of IL-17A binding Z variants selected and characterized in Example 5. In the new library, the 13 positions in the Z molecule scaffold that were varied in the maturation library described in Examples 4 and 5 were biased towards certain amino acid residues, according to a strategy based on the Z variant sequences of the 37 top performing variants in the EC50 analysis (Table 6). A DNA linker was generated using split-pool synthesis and ordered from DNA 2.0. It contained the following 147 bp, encoding partially randomized helix 1 and 2 of the amino acid sequence: 5′-AA ATA AAT CTC GAG GTA GAT GCC/GCA AAA TAC GCC AAA GAA/GAG NNN NNN NNN GCG NNN NNN GAG ATC/ATT NNN NNN TTA/CTG CCT/CCC AAC TTA/CTC ACC NNN NNN CAA/CAG NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3′ (SEQ ID NO:1249; randomized codons are denoted NNN) flanked by restriction sites XhoI and SacI. The theoretical distributions of amino acid residues in the new library, including six variable amino acid positions (11, 14, 18, 25, 28 and 32) and seven constant amino acid positions (9, 10, 13, 17, 24, 27 and 35) in the Z molecule scaffold are given in Table 7. The resulting theoretical library size is 3.9×10.sup.6 variants.
[0348] Library construction: The library was constructed essentially as described in Example 4 with the following exception: the cells were cultivated overnight in 0.5 l of TSB-YE medium, supplemented with 2% glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin.
[0349] Preparation of phage stock: Phage stock containing the phagemid library was prepared in shake flasks. Cells from a glycerol stock containing the phagemid library were inoculated in 0.5 l 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 0.6 and then approximately 83 ml of the cultivation was infected using a 5× molar excess of M13K07 helper phage and incubated for 40 min at 37° C. The cells in the cultivation were pelleted by centrifugation, dissolved in TSB-YE medium, supplemented with 100 μg/ml ampicillin, 25 μg/ml kanamycin and 0.1 mM IPTG and grown at 30° C. for 18 h. After cultivation, the 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 Example 1. Phage stocks were stored at −80° C. until use in selection.
TABLE-US-00010 TABLE 7 Library design, second maturation Amino acid position in Randomization No of the Z variant (amino acid amino molecule abbreviations) acids Proportion 9 A 1 1/1 10 D 1 1/1 11 A, D, E, F, G, H, I, K, L, M, 17 1/17 N, Q, R, S, T V, W 13 A 1 1/1 14 A, F, I, L, M, V, Y 7 1/7 17 A 1 1/1 18 A, D, E, F, G, H, I, K, L, M, 17 1/17 N, Q, R, S, V, W, Y 24 W 1 1/1 25 A, D (50%), E, F, I, L, M, V, 10 1/18, W, Y ½ (0) 27 W 1 1/1 28 A, D, E, F, G, H, I, L, M, Q, 12 1/12 W, Y 32 A, D, E, F, G, H, K, L, M, N, 16 1/16 Q, R, S, T, W, Y 35 R 1 1/1
Results
[0350] Library construction: The new library was designed based on a set of IL-17A binding Z variants with verified binding properties (Example 5). The theoretical size of the designed library was 3.9×10.sup.6 Z variants. The actual size of the library, determined by titration after transformation to E. coli. ER2738 cells, was 1.4×10.sup.9 transformants.
[0351] The library quality was tested by sequencing of 96 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 maturated library of potential binders to IL-17A was thus successfully constructed.
Example 7
Selection, Screening and Characterization of Z Variants from the Second Maturated Library
Materials and Methods
[0352] Phage display selection of IL-17A binding Z variants: The target protein hIL-17A was biotinylated as described in Example 1. Phage display selections, using the second maturated library of Z variant molecules constructed as described in Example 6, were performed against hIL-17A essentially as described in Example 5 with the following exceptions. Exception 1: selections were performed in solution or at solid phase in RT. Exception 2: the time for selection was 60 min, 10 min or 1 min in cycle 1 and 30 min, 4 min or 10 sec in cycles 2, 3 and 4. Selection in solution was followed by catching of target-phage complexes on SA beads as described in Example 1. Exception 3: during selection on solid phase, the target was caught on SA beads prior to selection.
[0353] An overview of the selection strategy, summarizing the differences between selection in solution and selection on solid phase, as well as the increased stringency in subsequent cycles obtained by using a lowered target concentration and an increased number of washes, is shown in Table 8.
[0354] Tracks 1-6 in cycle 1 were divided in the second to fourth cycles, resulting in total of seven tracks (1-1 to 6-1) in cycle 2, eight tracks (1-1-1 to 6-1-1) in cycle 3 and 16 tracks (1-1-1-1 to 6-1-1-1) in cycle 4. Washes were performed using PBST 0.1% during 1 min. However, one of the washes lasted for 15 min in tracks 2-1-1, 3-1-1 and 5-1-1 in cycle 3.
[0355] After the last wash in cycle 4, the target-phage complexes on SA beads from five of the tracks (1-1-1-1, 1-2-1-2, 3-1-1-1, 4-1-1-1 and 5-1-1-2) were divided in two equal parts. Bound phage particles from the first part were immediately eluted, while the second part was subjected to a wash during approximately 65 h before the elution of phage particles. The bound phage were eluted using glycine-HCl, pH 2.2, as described in Example 1.
[0356] The amplification of phage particles between selection cycles was performed essentially as described in Example 1.
TABLE-US-00011 TABLE 8 Overview of the selection from the second maturated library Phage stock Target Selection Number Number Number Selection from library or conc. time of 1 min of 15 min of ~65 h Selection Cycle track selection track (pM) (min) washes washes washes method 1 1 Zlib006IL- 12500 60 3 — — solution 17A.II 1 2 Zlib006IL- 12500 60 3 — — solution 17A.II 1 3 Zlib006IL- 1250 60 3 — — solution 17A.II 1 4 Zlib006IL- 12500 10 3 — — solid 17A.II phase 1 5 Zlib006IL- 2500 10 3 — — solid 17A.II phase 1 6 Zlib006IL- 2500 1 3 — — solid 17A.II phase 2 1-1 1 5000 30 6 — — solution 2 1-2 1 2500 30 10 — — solution 2 2-1 2 1000 30 10 — — solution 2 3-1 3 100 30 10 — — solution 2 4-1 4 5000 4 6 — — solid phase 2 5-1 5 250 4 10 — — solid phase 2 6-1 6 250 0.17 10 — — solid phase 3 1-1-1 1-1 500 30 15 — — solution 3 1-2-1 1-2 50 30 15 — — solution 3 2-1-1 2-1 25 30 14 1 — solution 3 3-1-1 3-1 5 30 14 1 — solution 3 4-1-1 4-1 500 4 15 — — solid phase 3 4-1-2 4-1 50 4 15 — — solid phase 3 5-1-1 5-1 25 4 14 1 — solid phase 3 6-1-1 6-1 5 0.17 15 — — solid phase 4 1-1-1-1 1-1-1 50 30 20 — — solution 4 1-1-1-1X 1-1-1 50 30 20 — 1 solution 4 1-1-1-2 1-1-1 5 30 20 — — solution 4 1-2-1-1 1-2-1 25 30 20 — — solution 4 1-2-1-2 1-2-1 5 30 20 — — solution 4 1-2-1-2X 1-2-1 5 30 20 — 1 solution 4 2-1-1-1 2-1-1 0.5 30 20 — — solution 4 3-1-1-1 3-1-1 0.05 30 20 — — solution 4 3-1-1-1X 3-1-1 0.05 30 20 — 1 solution 4 4-1-1-1 4-1-1 50 4 20 — — solid phase 4 4-1-1-1X 4-1-1 50 4 20 — 1 solid phase 4 4-1-2-1 4-1-2 25 4 20 — — solid phase 4 5-1-1-1 5-1-1 2.5 4 20 — solid phase 4 5-1-1-2 5-1-1 0.5 4 20 — — solid phase 4 5-1-1-2X 5-1-1 0.5 4 20 — 1 solid phase 4 6-1-1-1 6-1-1 0.5 0.17 20 — — solid phase
[0357] Sequencing of potential binders: Individual clones from the different selection tracks were picked for sequencing. Amplification and sequence analysis of gene fragments were performed essentially as described in Example 1.
[0358] ELISA screening of Z variants: Single colonies containing Z variants (expressed as Z variant ABD fusion proteins as described in Example 1) were randomly picked from the selected clones of the IL-17A second maturated library and grown in cultivations as described in Example 1. Preparation of the periplasmic supernatants and ELISA screenings were performed essentially as described in Example 1 with the following two exceptions. Exception 1: biotinylated hIL-17A was used at a concentration of 0.2 or 0.33 nM. Exception 2: the periplasmic fraction of Z variant Z10241 (SEQ ID NO:11), identified in selections from the first maturated library, was used as a positive control and the blank control was created by exchanging the periplasmic step with addition of PBST 0.05%.
[0359] EC50 analysis of Z variants: A selection of IL-17A binding Z variants was subjected to an analysis of the response against a dilution series of b-hIL-17A using ELISA as described in Example 5. Biotinylated protein was added at a concentration of 10 nM and diluted stepwise 1:2 eight times followed by one 1:5 dilution down to 8 pM. As a background control, the Z variants were also assayed with no target protein added. A periplasm sample containing the previously maturated Z variant Z10241 (SEQ ID NO:11) was included as positive control. Data were analyzed using GraphPad Prism 5 and non-linear regression and EC50 values were calculated.
Results
[0360] Phage display selection of IL-17A binding Z variants from a second maturated library: Selection was performed in a total of 16 parallel tracks containing four cycles each. The selection tracks differed in target concentration, selection time, wash conditions and if the selection was performed in solution or on solid phase.
[0361] Sequencing: Randomly picked clones were sequenced. Each individual Z variant was given an identification number, Z#####, as described in Example 1. In total, 759 new unique Z variant molecules were identified.
[0362] For the 704 best performing variants in the ELISA screen below, the amino acid sequences of the 58 amino acid residues long Z variants are listed in
[0363] ELISA screening of Z variants: Clones obtained after four selection cycles were produced in 96-well plates and screened for hIL-17A binding activity using ELISA. All randomly picked clones were analyzed. 704 of the 759 unique Z variants were found to give a response of 2× the blank controls or higher (0.22-2.2 AU) against hIL-17A at a concentration of 0.2 or 0.33 nM. Positive signals were collected for clones originating from all selection tracks. The average response of the blank controls was 0.11 AU, based on a representative set of plates.
[0364] EC50 analysis of Z variants: A subset of IL-17A binding Z variants was selected based on the result in the ELISA experiment described above (Z variants with absorbances over the response of the positive control Z10241, SEQ ID NO:11) and subjected to a target titration in ELISA format as described in Example 5. Obtained values were analyzed and their respective EC50 values were calculated (Table 9).
TABLE-US-00012 TABLE 9 Calculated EC50 values from ELISA titration analysis SEQ EC50 Z variant ID NO (M) Z12059 17 5.3 × 10.sup.−10 Z12060 5 5.1 × 10.sup.−10 Z12073 18 4.3 × 10.sup.−10 Z12078 8 5.2 × 10.sup.−10 Z12081 6 5.6 × 10.sup.−10 Z12115 20 4.2 × 10.sup.−10 Z12163 9 5.0 × 10.sup.−10 Z12180 21 3.7 × 10.sup.−10 Z12211 22 4.6 × 10.sup.−10 Z12212 64 7.8 × 10.sup.−10 Z12256 23 4.7 × 10.sup.−10 Z12264 10 3.9 × 10.sup.−10 Z12275 24 4.2 × 10.sup.−10 Z12285 65 9.1 × 10.sup.−10 Z12439 66 6.1 × 10.sup.−10
Example 8
In Vitro Characterization of a Subset of Maturated IL-17A Binding Z Variants
Materials and Methods
[0365] Subcloning and production of Z variants with an N-terminal His.sub.6-tag were performed as described in Example 2. One additional variant, His.sub.6-Z15167 (SEQ ID NO:4), was created by site directed mutagenesis of His.sub.6-Z10532 (SEQ ID NO:1) resulting in substitution of D in position 25 with A. Production of His.sub.6-Z15167 was performed as described above for other Z variants.
[0366] 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.
[0367] IL-17 binding ELISA screening: 96-well half area plates (Costar, 3690) were coated overnight at 4° C. with hIL-17A at 1 μg/ml in PBS in a volume of 50 μl/well. On the day of analysis, the plate was rinsed twice in tap water and then blocked with PBS+2% BSA (Sigma) for 2 h. Z10241 (SEQ ID NO:11) was used as a standard and was titrated in a 3-fold dilution series (300-0.005 ng/ml) and the other Z variants were added in four different dilutions to the coated ELISA plate (50 μl/well) and incubated for 1.5 h at RT. The plate was washed 4 times in an automated ELISA washer and 2 μg/ml (50 μl/well) of a goat anti-Z antibody was added. After 1 h of incubation, the plate was washed and 50 μl of anti-goat IgG-HRP (DAKO) diluted 5000 times was added per well. The plate was developed after another 1 h incubation, washed with 50 μl TMB (Thermo Fisher, 34021) per well and the reaction was stopped with 50 μl 2 M H.sub.2SO.sub.4. The plates were read in a multi label reader (Victor.sup.3, Perkin Elmer).
[0368] Biacore kinetic analysis: Kinetic constants (k.sub.on and k.sub.off) and affinities (K.sub.D) for human IL-17A were determined for 27 His.sub.6-tagged Z variants. The hIL-17A was immobilized in the flow cell on the carboxylated dextran layer of a CM5 chip surface (GE Healthcare, cat. no. BR100012). The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v surfactant P20, GE Healthcare, cat. no. BR100188) 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 and the flow rate was 50 μl/min. The analytes, i.e. the Z variants, were each diluted in HBS-EP buffer to final concentrations of 100 nM, 20 nM and 4 nM and injected for 4 min, followed by dissociation in running buffer for 8 min. After 8 min dissociation, the surfaces were regenerated with two injections of 10 mM HCl. Kinetic constants were calculated from the sensorgrams using the Langmuir 1:1 model of BiaEvaluation software 4.1 (GE Healthcare).
[0369] Biacore binding specificity analysis: The interactions of three His.sub.6-tagged IL-17A binding Z variants (Z10508 (SEQ ID NO:2), Z10532 (SEQ ID NO:1) and Z15167 (SEQ ID NO:4)) with hIL-17A (SEQ ID NO:1226), hIL-17F (SEQ ID NO:1228; R&D Systems, cat. no. 1335-IL/CF) and the human IL-17A/F heterodimer (R&D Systems, cat. no. 5194-IL-025/CF) were analyzed in a Biacore 2000 instrument. The three different IL-17 variants were immobilized in the flow cells on the carboxylated dextran layer of a CM5 chip surface. The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using HBS-EP as running buffer. One flow cell surface on the chip was activated and deactivated for use as blank during analyte injections. In the binding experiment, HBS-EP was used as running buffer and the flow rate was 50 μl/min. The analytes, i.e. the Z variants, were each diluted in HBS-EP running buffer to final concentrations of 4, 20, 100 and 500 nM and injected for 4 min. After 15 min of dissociation, the surfaces were regenerated with four injections of 10 mM HCl. The results were analyzed using the BiaEvaluation software.
[0370] Blocking of IL-17A induced IL-6 production in the NHDF assay: The NHDF assay and quantification by the IL-6 specific ELISA was performed essentially as described in Example 3. In brief, on the day before the experiment, 5000 cells were seeded per well into a half area 96-well culture plates in 100 μl. On the day of the experiment, dilutions of 36 IL-17A specific Z variants were prepared in a separate 96-well plate. The Z variants were titrated in three-fold steps from 4690 nM to 0.08 nM in medium containing 0.9 nM hIL-17A. A standard curve of hIL-17A (6.2-0.0001 nM) was prepared, as well as controls containing medium with 0.9 nM hIL-17A or medium alone.
Results
[0371] CD analysis: The CD spectra determined for the IL-17A binding Z variants with a His.sub.6 tag showed that each had a a-helical structure at 20° C. The results of the variable temperature measurements, wherein melting temperatures (Tm) were determined, are shown in Table 10. Reversible folding was seen for all the IL-17A binding Z variants when overlaying spectra measured before and after heating to 90° C.
[0372] Analysis of IL-17A binding capacity by ELISA: Purified His.sub.6-tagged Z variant molecules from the first and second maturation round were screened for their capacity to bind IL-17A in an ELISA assay. The results are shown in
[0373] Biacore kinetic analysis: The interactions of 28 His.sub.6-tagged IL-17A-binding Z variants with hIL-17A were analyzed in a Biacore instrument by injecting various concentrations of the Z variants over a surface containing immobilized IL-17A. A summary of the kinetic parameters (K.sub.D, k.sub.a (k.sub.on) and k.sub.d (k.sub.off)) for binding of the Z variants to hIL-17A using a 1:1 interaction model is given in Table 11.
TABLE-US-00013 TABLE 10 Melting temperatures (Tm) SEQ ID NO Analyte of Z variant Tm (° C.) His6-Z10241 11 48 His6-Z10433 28 56 His6-Z10462 12 51 His6-Z10508 2 54 His6-Z10532 1 57 His6-Z10534 13 41 His6-Z10566 14 51 His6-Z10675 15 52 His6-Z10681 29 52 His6-Z10718 16 48 His6-Z10722 30 51 His6-Z10859 31 52 His6-Z10863 3 51 His6-Z12059 17 50 His6-Z12060 5 52 His6-Z12073 18 49 His6-Z12077 19 52 His6-Z12078 8 51 His6-Z12081 6 50 His6-Z12115 20 51 His6-Z12163 9 54 His6-Z12180 21 51 His6-Z12192 32 45 His6-Z12211 22 49 His6-Z12256 23 47 His6-Z12264 10 54 His6-Z12275 24 46 His6-Z12283 25 45 His6-Z12289 33 51 His6-Z12344 26 49 His6-Z12481 27 49 His6-Z12498 34 48 His6-Z12522 35 51 His6-Z12634 7 50 Z10241-His6 11 47 His6-Z15167 4 57 Z10199 1217 56
[0374] Biacore binding specificity analysis: The binding of three His.sub.6-tagged IL-17A binding Z variants (Z10508, Z10532 and Z15167) to hIL-17A, hIL-17F and hIL-17A/F were tested in a Biacore instrument by injecting the Z variants over surfaces containing the IL-17 variants. The ligand immobilization levels on the surfaces were 657 RU, 977 RU and 770 RU of IL-17A, IL-17F and IL-17A/F, respectively. All tested Z variants showed binding to human IL-17A and weaker binding to IL-17A/F, whereas no binding to IL-17F could be detected. The resulting curves, from which responses from a blank surface were subtracted, are displayed in
[0375] Blocking of IL-17A induced IL-6 production in the NHDF assay: Purified His.sub.6-tagged Z variants from the first and second maturation rounds were screened for their capacity to block IL-17A induced IL-6 production in the NHDF assay. Results from the NHDF assay showed that all tested maturated binders had an increased IL-17A specific blocking capacity compared to the primary binder His.sub.6-Z06282. A graph displaying typical inhibition profiles of a selection of binders from the first maturation library are shown in
TABLE-US-00014 TABLE 11 Kinetic parameters for binding of Z variants to hIL-17A SEQ ID NO Analyte of Z variant k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M) His6-Z10241 11 5.2 × 10.sup.5 1.2 × 10.sup.−3 2.2 × 10.sup.−9 His6-Z10433 28 4.7 × 10.sup.5 3.1 × 10.sup.−3 6.4 × 10.sup.−9 His6-Z10508 2 5.1 × 10.sup.5 2.0 × 10.sup.−3 4.0 × 10.sup.−9 His6-Z10532 1 6.2 × 10.sup.5 1.5 × 10.sup.−3 2.4 × 10.sup.−9 His6-Z10566 14 6.1 × 10.sup.5 5.7 × 10.sup.−3 9.3 × 10.sup.−9 His6-Z10675 15 5.7 × 10.sup.5 1.1 × 10.sup.−3 1.9 × 10.sup.−9 His6-Z10681 29 6.7 × 10.sup.5 3.5 × 10.sup.−3 5.2 × 10.sup.−9 His6-Z10859 31 5.6 × 10.sup.5 6.8 × 10.sup.−3 1.2 × 10.sup.−8 His6-Z10863 3 8.3 × 10.sup.5 2.5 × 10.sup.−3 3.0 × 10.sup.−9 His6-Z12060 5 7.5 × 10.sup.5 3.3 × 10.sup.−3 4.3 × 10.sup.−9 His6-Z12073 18 4.9 × 10.sup.5 1.2 × 10.sup.−3 2.3 × 10.sup.−9 His6-Z12077 19 6.5 × 10.sup.5 2.0 × 10.sup.−3 3.1 × 10.sup.−9 His6-Z12078 8 4.5 × 10.sup.5 8.1 × 10.sup.−4 1.8 × 10.sup.−9 His6-Z12081 6 6.2 × 10.sup.5 3.8 × 10.sup.−3 6.2 × 10.sup.−9 His6-Z12163 9 7.3 × 10.sup.5 1.6 × 10.sup.−3 2.2 × 10.sup.−9 His6-Z12192 32 4.6 × 10.sup.5 5.6 × 10.sup.−3 1.2 × 10.sup.−8 His6-Z12211 22 5.8 × 10.sup.5 1.4 × 10.sup.−3 2.4 × 10.sup.−9 His6-Z12256 23 3.5 × 10.sup.5 1.2 × 10.sup.−3 3.4 × 10.sup.−9 His6-Z12264 10 6.0 × 10.sup.5 5.5 × 10.sup.−4 .sup. 9.3 × 10.sup.−10 His6-Z12275 24 5.2 × 10.sup.5 1.1 × 10.sup.−3 2.0 × 10.sup.−9 His6-Z12283 25 5.5 × 10.sup.5 1.8 × 10.sup.−3 3.3 × 10.sup.−9 His6-Z12289 33 6.2 × 10.sup.5 3.3 × 10.sup.−3 5.3 × 10.sup.−9 His6-Z12344 26 7.1 × 10.sup.5 6.6 × 10.sup.−4 .sup. 9.2 × 10.sup.−10 His6-Z12481 27 5.2 × 10.sup.5 8.9 × 10.sup.−4 1.7 × 10.sup.−9 His6-Z12498 34 6.0 × 10.sup.5 2.3 × 10.sup.−3 3.8 × 10.sup.−9 His6-Z12522 35 7.1 × 10.sup.5 4.4 × 10.sup.−3 6.2 × 10.sup.−9 His6-Z12634 7 6.9 × 10.sup.5 4.3 × 10.sup.−3 6.3 × 10.sup.−9 His6-Z15167 4 1.3 × 10.sup.6 1.4 × 10.sup.−3 1.1 × 10.sup.−9
TABLE-US-00015 TABLE 12 IC50 values for maturated Z-variants SEQ ID NO IC50 Analyte of Z variant: (nM) His6-Z10241 11 5.3 His6-Z10433 28 14 His6-Z10462 12 6.6 His6-Z10508 2 5.7 His6-Z10532 1 6.4 His6-Z10534 13 6.5 His6-Z10566 14 9.6 His6-Z10675 15 5.3 His6-Z10681 29 25 His6-Z10718 16 28 His6-Z10722 30 35 His6-Z10859 31 26 His6-Z10863 3 8.5 His6-Z12059 17 3.7 His6-Z12060 5 7.9 His6-Z12073 18 4.0 His6-Z12077 19 5.5 His6-Z12078 8 4.5 His6-Z12081 6 8.1 His6-Z12115 20 3.0 His6-Z12163 9 7.0 His6-Z12180 21 4.3 His6-Z12192 32 28 His6-Z12211 22 2.9 His6-Z12256 23 4.4 His6-Z12264 10 4.4 His6-Z12275 24 4.8 His6-Z12283 25 6.9 His6-Z12289 33 18 His6-Z12344 26 4.8 His6-Z12481 27 3.0 His6-Z12498 34 11 His6-Z12522 35 10 His6-Z12634 7 9.8 Z10241-His6 11 3.3
Example 9
Production of IL-17 Binding Z-ABD-Z Polypeptides
[0376] IL-17A is a homodimeric cytokine that possesses two receptor binding sites. It was speculated that a polypeptide comprising two moieties of an IL-17A binding Z variant of the present disclosure would block IL-17A more efficiently than a polypeptide comprising one such moiety. This Example describes the general procedure for subcloning and production of polypeptides comprising two Z variants in fusion with the albumin binding domain variant PP013 (SEQ ID NO:1224) in the general format Z-[L1]-ABD-[L2]-Z where [L1] and [L2] are linkers separating the Z and ABD moieties.
Materials and Methods
[0377] Subcloning of Z-[L1]-ABD-[L2]-Z polypeptides with different [L1] and [L2] linker lengths: The DNA of Z06282 (SEQ ID NO:1206), Z10241 (SEQ ID NO:11) and Z10532 (SEQ ID NO:1) were amplified from the library vector pAY02592 by PCR using Pfu Turbo DNA polymerase (Agilent Technologies, cat. no. 600254) together with suitable primer pairs. The Z-[L1]-ABD-[L2]-Z constructs were generated by ligation of DNA encoding each moiety into the expression vector pET26b(+) (Novagen, Madison, Wis.) in three subsequent cloning steps using T4 DNA ligase (Fermentas, cat. no. EL0011). DNA encoding the three moieties were separated by DNA encoding hybridized linkers with different number of repeats of (GGGGS).sub.n, flanked by restriction enzyme sites. The constructs encoded by the expression vectors were Z#####-[GAP-(G.sub.4S).sub.n-TS]-PP013-[GT-(G.sub.4S).sub.n-PR]-Z#####, where each n individually is 1-4, and as further specified in Table 13.
TABLE-US-00016 TABLE 13 Dimeric Z variants fused to ABD via different [L1] and [L2] lengths SEQ Designation ID NO Z-[L1]-ABD-[L2]-Z polypeptide ZAZ3174 1233 Z06282-[GAP-(G.sub.4S).sub.4-TS]-PP013-[GT-(G.sub.4S).sub.4- PR]-Z06282 ZAZ3175 1234 Z06282-[GAP-(G.sub.4S).sub.3-TS]-PP013-[GT-(G.sub.4S).sub.3- PR]-Z06282 ZAZ3176 1235 Z06282-[GAP-(G.sub.4S).sub.2-TS]-PP013-[GT-(G.sub.4S).sub.2- PR]-Z06282 ZAZ3236 1240 Z06282-[GAP-G.sub.4S-TS]-PP013-[GT-G.sub.4S- PR]-Z06282 ZAZ3237 1241 Z06282-[GAP-G.sub.4S-TS]-PP013-[GT-(G.sub.4S).sub.2- PR]-Z06282 ZAZ3220 1236 Z10241-[GAP-(G.sub.4S).sub.4-TS]-PP013-[GT-(G.sub.4S).sub.4- PR]-Z10241 ZAZ3221 1237 Z10532-[GAP-(G.sub.4S).sub.4-TS]-PP013-[GT-(G.sub.4S).sub.4- PR]-Z10532 Restriction sites (I)-(IV) in DNA encoding Z#####-[(I)-(G.sub.4S)n-(II)]-PP013-[(III)-(G.sub.4S)n-(IV)]-Z##### are cleavable with restriction enzymes AscI (I), SpeI (II), KpnI (III) and SacII (IV), respectively
[0378] Subcloning of Z-[L1]-ABD-[L2]-Z polypeptides with a minimal [L1] linker: DNA encoding additional dimeric Z variants comprising Z06282 (SEQ ID NO:1206) but with a minimal linker [L1] were generated using standard molecular biology techniques. The constructs encoded by the expression vectors were Z06282-VDGS-PP013-GT-(G.sub.4S).sub.n-PR-Z06282 and as further specified in Table 14.
[0379] The genes encoding Z12876 (SEQ ID NO:1218), Z14253 (SEQ ID NO:1219), Z14254 (SEQ ID NO:1220) and Z14255 (SEQ ID NO:1221) (corresponding to Z10241 (SEQ ID NO:11), Z10532 (SEQ ID NO:1), Z10508 (SEQ ID NO:2) and Z10863 (SEQ ID NO:3), respectively, but starting with the amino acid residues AE instead of VD) were amplified by PCR using Pfu Turbo DNA polymerase together with suitable primer pairs. The Z-[L1]-ABD-[L2]-Z constructs were generated by ligation of each fragment into the expression vector pET26b(+) in three subsequent cloning steps using T4 DNA ligase. DNA encoding the three moieties were separated by linkers further modified by site-directed mutagenesis using standard molecular biology techniques. The constructs encoded by the expression vectors were Z####-[VDGS]-PP013-[GT-G.sub.4S-PK]-Z#### and Z#####-[ASGS]-PP013-[GT-G.sub.4S]-Z#####, and as further specified in Table 14.
TABLE-US-00017 TABLE 14 Dimeric Z variants fused to ABD via a minimal [L1] linker SEQ ID Designation NO Z-[L1]-ABD-[L2]-Z polypeptide Mutations ZAZ3234 1238 Z06282-[VDGS]-PP013- GT-G.sub.4S-PR-Z06282 ZAZ3235 1239 Z06282-[VDGS]-PP013- GT-(G.sub.4S).sub.2-PR-Z06282 ZAZ3269 1242 Z12876-[VDGS]-PP013- R117K [GT-G.sub.4S-PK]-Z12876 ZAZ3270 1243 Z12876-[ASGS]-PP013- V59A, D60S, [GT-G.sub.4S]-Z12876 Δ116P, Δ117R ZAZ3363 1244 Z14253-[ASGS]-PP013- V59A, D60S, [GT-G.sub.4S]-Z14253 Δ116P, Δ117R ZAZ3364 1245 Z14254-[ASGS]-PP013- V59A, D60S, [GT-G.sub.4S]-Z14254 Δ116P, Δ117R ZAZ3365 1246 Z14255-[ASGS]-PP013- V59A, D60S, [GT-G.sub.4S]-Z14255 Δ116P, Δ117R ZAZ3422 1247 Z15166-[ASGS]-PP013- D25A in both [GT-G.sub.4S]-Z15166 Z14253 moieties in ZAZ3363 Δdeletion of the indicated amino acid residue
[0380] Cultivation: E. coli BL21(DE3) cells (Novagen) were transformed with plasmids containing the gene fragment of each respective Z-ABD-Z polypeptide and cultivated at 37° C. in 800 or 1000 ml of TSB-YE medium supplemented with 50 μg/ml kanamycin. In order to induce protein expression, IPTG was added to a final concentration of 0.2 mM at OD.sub.600=2 and the cultivation was incubated at 37° C. for another 5 h. The cells were harvested by centrifugation.
[0381] Purification of IL-17A binding Z-ABD-Z polypeptides: Cell pellets were re-suspended in TST buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween 20, pH 8.0) supplemented with Benzonase® (Merck). After cell disruption by sonication and clarification by centrifugation, each supernatant was applied onto a column packed with agarose and 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 Z-ABD-Z polypeptides were eluted with 0.1 M HAc. Acetonitrile (ACN) was added to eluted fractions to a final concentration of 10% and the samples were loaded on SOURCE 15RPC columns (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 from RPC solvent A to RPC solvent B (0.1% TFA, 80% ACN, 20% water). Fractions containing pure Z-ABD-Z polypeptides were identified by SDS-PAGE analysis and pooled. After the RPC purification, the buffer of the pool 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 Sephadex G-25 columns (GE Healthcare). Finally, the Z-ABD-Z variants were purified on EndoTrap® red columns (Hyglos) to ensure low endotoxin content.
[0382] Protein concentrations were determined by measuring the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer and the extinction coefficient of the respective protein. Purity was analyzed by SDS-PAGE stained with Coomassie Blue, and the identity of each purified Z-ABD-Z variant was confirmed by HPLC-MS analysis.
Results
[0383] Cultivation and purification: The IL-17A binding Z variants in fusion with ABD were expressed as soluble gene products in E. coli. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the desired IL-17A binding Z-ABD-Z polypeptide. The correct identity and molecular weight of each Z-ABD-Z polypeptide were confirmed by HPLC-MS analysis.
Example 10
Solubility of Z-ABD-Z Polypeptides
[0384] The solubility of three Z-ABD-Z polypeptides in physiological buffer was investigated by consecutive concentrations of the samples using ultrafiltration, followed by concentration measurements by absorbance readings at 280 nm and visual inspection of the samples.
Materials and Methods
[0385] The Z-ABD-Z polypeptides ZAZ3363 (SEQ ID NO:1244), ZAZ3364 (SEQ ID NO:1245) and ZAZ3422 (SEQ ID NO:1247) were diluted in PBS, pH 7.4, to 2.5 mg/ml. 12 Amicon Ultra centrifugal filter units with a cut-off of 3 kDa (Millipore, cat. no. UFC800324) were prerinsed with PBS by centrifugation at 4000 g for 10 min in a swinging bucket rotor centrifuge. The concentrators were emptied, and 4 ml of each Z-ABD-Z polypeptide were added to a first set of three different centrifugal filter units. Centrifugation was performed at 4000 g, 20° C., for 13-16 min, resulting in approximately 1 ml concentrate. A 20 μl sample was removed from each concentrate (UF sample 1) for further analysis and the rest of the sample volumes were transferred to a second set of three centrifugal filter units. The centrifugation and sample removal were repeated three times with spinning times of 8-10 min, 7 min and 13 min, respectively, (UF samples 2, 3 and 4, respectively). Absorbance readings were performed using a NanoDrop® ND-1000 Spectrophotometer and by diluting UF samples 1-4 in PBS 3, 6, 12 and 24 times, respectively. The concentrations were calculated using the theoretical extinction coefficient 1 Abs280=0.612 mg/ml (the same for all three Z-ABD-Z polypeptides). Absorbance readings of the undiluted filtrates were also performed.
Results
[0386] Concentrations determined by absorbance readings at 280 nm after each centrifugal step are shown in Table 15. No aggregates were detected by visual inspection of the concentrates. Thus, the solubility of ZAZ3363, ZAZ3364 and ZAZ3422 were determined to be at least 60 mg/ml in PBS, pH 7.4. Absorbance readings of the undiluted filtrates showed concentrations very close to 0 mg/ml.
TABLE-US-00018 TABLE 15 Concentration after consecutive concentration of Z-ABD-Z samples Total time of Z-ABD-Z SEQ Concentration (mg/ml) centrifugation polypeptide ID NO UF1 UF2 UF3 UF4 (min) ZAZ3363 1244 9.6 21 36 64 44 ZAZ3364 1245 8.2 21 38 60 43 ZAZ3322 1247 9.3 20 35 64 44
Example 11
Biacore Binding Cross-Species Analysis
Materials and Methods
[0387] The interaction of the Z-ABD-Z polypeptides ZAZ3363 (SEQ ID NO:1244), ZAZ3364 (SEQ ID NO:1245) and ZAZ3365 (SEQ ID NO:1246) with hIL-17A and cynomolgus monkey IL-17A (cIL-17A, SEQ ID NO:1229; Evitria, custom order) as well as the interaction of ZAZ3220 (SEQ ID NO:1236) with hIL-17A and rhesus monkey IL-17A (rmIL-17-A, SEQ ID NO:1230; Cusabio, cat. no. CSB-EP011597MOV) were analyzed in a Biacore 2000. HSA was immobilized in the flow cell on the carboxylated dextran layer of a CM5 chip surface. The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using HBS-EP as running buffer. The HSA immobilization levels on the surfaces were 953 RU (used for ZAZ3363, ZAZ3364 and ZAZ3365) and 493 RU (used for ZAZ3220), respectively. One flow cell surface on the chip was activated and deactivated for use as blank during analyte injections. In the binding experiments, HBS-EP was used as running buffer and the flow rate was 30 μl/min. ZAZ3363, ZAZ3364 and ZAZ3365 were diluted in HBS-EP running buffer to a final concentration of 200 nM and injected for 5 min, followed by injections of the IL-17A variants. ZAZ3220 was diluted in HBS-EP running buffer to a final concentration of 500 nM and injected for 4 min followed by injections of the IL-17A variants. hIL-17A and cIL-17A were each diluted in HBS-EP running buffer to final concentrations of 2.5, 10 and 40 nM and injected for 5 min over surfaces with ZAZ3363, ZAZ3364 and ZAZ3365 captured on HSA. After 10 min of dissociation, the surface was regenerated with two injections of 10 mM HCl. hIL-17A and rmIL-17A were diluted in HBS-EP running buffer to final concentrations of 0.1, 0.3, 0.9, 2.7, and 8.1 nM and 2.7, 8.1, 24.3, 72 and 216 nM, respectively, and injected for 6 min over surfaces with ZAZ3220 captured on HSA. After 5 min of dissociation, the surface was regenerated with two injections of 10 mM HCl. The results were analyzed using the BiaEvaluation software.
Results
[0388] Binding of hIL-17A and cIL-17A to ZAZ3363, ZAZ3364 and ZAZ3365, and hIL-17A and rmIL-17A to ZAZ3220 were tested in a Biacore instrument by injecting the Z-ABD-Z polypeptides over a surface containing HSA followed by injections of the IL-17A variants. All tested Z variants showed binding to the tested IL-17A variants, i.e. ZAZ3363, ZAZ3364 and ZAZ3365 showed binding to human and cynomolgus monkey IL-17A (
Example 12
Biacore Binding Specificity Analysis
[0389] In this Example, the specificities of two Z-ABD-Z polypeptides were tested by analysing their potential interaction with a set of proteins of the IL-17 family, with other interleukins and with other abundant plasma proteins. The same set of analyte proteins were also analyzed for their potential interaction with the ABD variant PEP07843.
Materials and Methods
[0390] The interaction of ZAZ3363 (SEQ ID NO:1244) or ZAZ3422 (SEQ ID NO:1247) with panels of 24 or 12 different proteins (specified in Table 16), respectively, were analyzed in a Biacore 2000 instrument. HSA and the ABD variant PEP07843 (SEQ ID NO:1225, corresponding to PP013 (SEQ ID NO:1224) with an N-terminal extension of GSS) were immobilized on a CM5 chip surface and a blank surface was prepared as described in Example 11. The ligand immobilization levels on the surfaces were 1315 RU and 99 RU of HSA and PEP07843, respectively, for the chip used in ZAZ3363 runs, and 791 RU and 100 RU of HSA and PEP07843, respectively, for the chip used in ZAZ3422 runs. In the binding experiment, HBS-EP was used as running buffer and the flow rate was 30 μl/min. ZAZ3363 and ZAZ3422 were diluted in HBS-EP or HBS-EP supplemented with 500 mM NaCl to a final concentration of 200 nM and injected for 7 min followed by injections of the analytes. The analytes, i.e. the panels of 24 or 12 different proteins, were each diluted in HBS-EP or HBS-EP supplemented with 500 mM NaCl to final concentrations of from 0.4 to 250 nM and injected for 5 min. After 7 (ZAZ3363) or 10 (ZAZ3422) min of dissociation, the surfaces were regenerated with four (ZAZ3363) or five (ZAZ3422) injections of 10 mM NaOH and two injections of 10 mM HCl. The results were analyzed using the BiaEvaluation software.
TABLE-US-00019 TABLE 16 Analyte proteins tested with ZAZ3363 and ZAZ3422 Second injection First injection Analyte ZAZ3363 ZAZ3422 conc. Buffer for (200 nM) (200 nM) Analyte protein Cat. No. (nM) sample dilution Yes Yes hIL-17A.sup.1 200-17 2, 10, 50 HBS-EP and HBS-EP + NaCl Yes Yes hIL-17A/F.sup.2 5194-IL-025/CF 50, 250 HBS-EP + NaCl Yes Yes hIL-17B.sup.2 1248-IB-025/CF 50, 250 HBS-EP + NaCl Yes Yes hIL-17C.sup.2 1234-IL-025/CF 50, 250 HBS-EP Yes Yes hIL-17D.sup.2 1504-IL-025/CF 50, 250 HBS-EP + NaCl Yes Yes hIL-17E.sup.2 1258-IL-025/CF 50, 250 HBS-EP Yes Yes hIL-17F.sup.2 1335-IL/CF 50, 250 HBS-EP + NaCl Yes Yes hIL-1beta.sup.1 200-1B 50, 250 HBS-EP Yes Yes hIL-6.sup.2 206IL/CF 50, 250 HBS-EP Yes Yes hIL23.sup.2 1290-IL 50, 250 HBS-EP Yes Yes hGM-CSF.sup.2 215-GM/CF 50, 250 HBS-EP Yes Yes IgG ATC L04AC07 50, 250 HBS-EP (RoActemra).sup.3 Yes No IgA.sup.4 P80-102 50, 250 HBS-EP Yes No IL-17RA.sup.2 177-IR 50, 250 HBS-EP Yes No IL-1R1.sup.2 269-1R/CF 50, 250 HBS-EP Yes No Alpha-2-HS- PRO-1644 50, 250 HBS-EP glycoprotein (AHSG Human HEK).sup.5 Yes No Haptoglobin PRO-567 50, 250 HBS-EP Human (seems to be beta chain).sup.5 Yes No Alpha-1- PRO-529 50, 250 HBS-EP antitrypsin (SERPINA1 Human).sup.5 Yes No Human a-2 10952-H08B 50, 250 HBS-EP macroglobulin.sup.6 Yes No Human 10870-H08H 50, 250 HBS-EP Hemopexin/ HPX Protein.sup.6 Yes No AMBP/Alpha 1 13141-H08H1 50, 250 HBS-EP microglobulin.sup.6 Yes No Beta-2- 11976-H08H 50, 250 HBS-EP microglobulin/ B2M.sup.6 Yes No Transthyretin/ 12091-H08H 50, 250 HBS-EP TTR/ Prealbumin/ PALB Protein.sup.6 Yes No holo-Transferrin.sup.7 T4132 50, 250 HBS-EP Yes No IL-17RA.sup.2 177-IR 50, 250 HBS-EP Yes No IL-1R1.sup.2 269-1R/CF 50, 250 HBS-EP Suppliers: .sup.1Peprotech; .sup.2R&D Systems; .sup.3Roche/Apoteket AB; .sup.4Bethyl; .sup.5ProSpec; .sup.6Sino Biological Inc; .sup.7Sigma
Results
[0391] The specificity of ZAZ3363 and ZAZ3422 was tested in a Biacore instrument by investigating their interaction with 24 or 12 analyte proteins, respectively. The Z-ABD-Z polypeptides were injected over surfaces containing HSA followed by injection of the analyte proteins. The only interactions detected for ZAZ3363 and ZAZ3422 were strong binding by hIL-17A and weaker binding by hIL-17A/F, as expected and in line with the results presented in Example 8. The analyte proteins were also assessed for their potential interaction with the ABD variant PEP07843, but no interactions were detected. Thus, the Z-ABD-Z polypeptides appear to be highly specific.
Example 13
Kinetic Measurements of Z-ABD-Z, IL-17A and HSA Complexes using KinExA®
[0392] Technical limitations of SPR to accurately determine kinetic parameters for high affinity interactions and the higher complexity of determining the affinity between two dimeric targets by SPR warranted the use of Kinetic Exclusion Assay (KinExA®) technology to further analyze the binding of Z-ABD-Z polypeptides in complex with HSA to IL-17A, as well as the binding of Z-ABD-Z polypeptides alone, or in complex with IL-17A, to HSA. The KinExA® measures the equilibrium binding affinity and kinetics between unmodified molecules in solution phase (Darling and Brault, 2004. Assay and Drug Dev Tech 2(6):647-657)
Materials and Methods
[0393] The Z-ABD-Z polypeptides ZAZ3220 (SEQ ID NO:1236), ZAZ3363 (SEQ ID NO:1244) and ZAZ3422 (SEQ ID NO:1247), as well as HSA (Novozymes, cat. no. 230-005), human IL-17A (Peprotech, cat. no. 200-17), biotinylated (as described in Example 1) human IL-17A, mouse monoclonal anti-HSA antibody (Abcam, cat. no. 10241) and an in house produced goat polyclonal anti-Z antibody were sent to Sapidyne Instruments Inc (Boise, Id., USA) who performed the KinExA® mesurements and analysis.
[0394] Binding of Z-ABD-Z/HSA to IL-17A: For determination of the affinity, i.e. K.sub.D, the respective Z-ABD-Z polypeptide, in complex with HSA, were used as a constant binding partner (CBP) and IL-17A was used as titrant. Data analysis was performed using the KinExA® Pro software and applying a least squares analysis to fit the optimal solutions for the K.sub.D and the Active Binding site Concentration (ABC) to a curve representative of a 1:1 reversible bi-molecular interaction.
[0395] For determination of k.sub.a, the direct binding curve analysis was applied, using the same immobilized IL-17A as the capture reagent for kinetic experiments as for equilibrium experiments. The amount of free Z-ABD-Z/HSA in the sample was measured pre-equilibrium, yielding data points that monitored the decrease in free Z-ABD-Z/HSA as the sample moved toward equilibrium.
[0396] Binding of Z-ABD-Z and Z-ABD-Z/IL-17A to HSA: The Z-ABD-Z polypeptide ZAZ3363 was further analyzed for binding to HSA in the presence or absence of IL-17A. For determination of K.sub.D, ZAZ3363, free or in complex with IL-17A, was used as a constant binding partner (CBP) and HSA was used as titrant. Data analysis was performed using the KinExA® Pro software as described above.
[0397] For determination of k.sub.a, the direct binding curve analysis was applied, using the same immobilized HSA as the capture reagent for kinetic experiments as for equilibrium experiments. The amount of ZAZ3363/IL-17A, in the sample was measured pre-equilibrium, yielding data points that monitored the decrease in free ZAZ3363/IL-17A as the sample moved toward equilibrium.
Results
[0398] In a first set of KinExA® measurements, the Z-ADB-Z polypeptides ZAZ3220, ZAZ3363 and ZAZ3422, respectively, in complex with HSA were shown to bind IL-17A with an exceptionally high affinity, with K.sub.D values in the subpicomolar to femtomolar range. The calculated kinetic parameters when assuming a monovalent binding between these Z-ABD-Z polypeptides and dimeric IL-17A are shown in Table 17.
[0399] In a second set of KinExA® measurements, the interaction between ZAZ3363, free or in complex with IL-17A, and HSA was measured. The calculated kinetic parameters from these analyses are shown in Table 18. The affinity of ZAZ3363 for HSA was not significantly affected by the presence of IL-17A.
[0400] To summarize, measurements using KinExA® technology indicated an exceptionally high affinity of Z-ABD-Z polypeptides for IL-17A. The measured K.sub.D of <0.33 pM is superior to the reported affinities for two of the clinically most advanced comparators secukinumab (K.sub.D=122 pM; WO2006/013107 and WO2012/125680) and ixekizumab (K.sub.D=2 pM; WO2007/070750). In addition, simultaneous binding to both IL-17A and albumin was demonstrated, i.e. both binding functions are intact in the Z-ABD-Z fusion protein.
TABLE-US-00020 TABLE 17 Kinetic parameters for Z-ABD-Z polypeptides binding to IL-17A SEQ ka.sup.a [*] kd K.sub.D [*] CBP ID NO (M.sup.−1s.sup.−1) (s.sup.−1) (M) ZAZ3220/HSA 1236 1.35 × 10.sup.7 1.51 × 10.sup.−7 <6.5 × 10.sup.−14 [1.25 × 10.sup.7- [**] 1.46 × 10.sup.7] ZAZ3363/HSA 1244 4.78 × 10.sup.6 1.55 × 10.sup.−6 3.23 × 10.sup.−13 [5.64 × 10.sup.6- [1.54 × 10.sup.−13- 4.04 × 10.sup.6] 5.54 × 10.sup.−13] ZAZ3422/HSA 1247 7.80 × 10.sup.6 6.08 × 10.sup.−7 7.85 × 10.sup.−14 [6.37 × 10.sup.6- [1.63 × 10.sup.−14- 9.56 × 10.sup.6] 1.73 × 10.sup.−13] [*] 95% confidence interval [**] 95% confidence interval for K.sub.D was not resolved
TABLE-US-00021 TABLE 18 Kinetic parameters for a Z-ABD-Z polypeptide binding to HSA SEQ ka [*] kd K.sub.D [*] CBP ID NO (M.sup.−1s.sup.−1) (s.sup.−1) (M) ZAZ3363 1244 n.d. n.d. 4.85 × 10.sup.−11 [3.23 × 10.sup.−11- 6.87 × 10.sup.−11] ZAZ3363/IL-17A 1244 1.21 × 10.sup.6 3.56 × 10.sup.−5 2.94 × 10.sup.−11 [9.35 × 10.sup.5- [1.72 × 10.sup.−11- 1.50 × 10.sup.6] 4.64 × 10.sup.−11] [*] 95% confidence interval
Example 14
Characterization of Z-ABD-Z Polypeptides in the NHDF Assay
Materials and Methods
[0401] Blocking of IL-17A induced IL-6 production in NHDF assay: NHDF cells (Lonza, cat. no. CC-2511) were cultured in fibroblast basal medium (Lonza, cat. no CC-3132) supplied with growth promoting factors (Lonza, cat. no. CC-5034). On the day before the experiment, 5000 cells per well were seeded into half area 96 well culture plates (Greiner, cat. no. 675180) in 100 μl. On the day of the experiment, dilutions of IL-17A specific Z-ABD-Z polypeptides with different linker lengths between the Z and ABD moieties (ZAZ3174 (SEQ ID NO:1233), ZAZ3175 (SEQ ID NO:1234), ZAZ3176 (SEQ ID NO:1235), ZAZ3220 (SEQ ID NO:1236), ZAZ3221 (SEQ ID NO:1237), ZAZ3234 (SEQ ID NO:1238), ZAZ3235 (SEQ ID NO:1239), ZAZ3236 (SEQ ID NO:1240), ZAZ3237 (SEQ ID NO:1241), ZAZ3269 (SEQ ID NO:1242) and ZAZ3270 (SEQ ID NO:1243)) were prepared in a separate 96-well plate. The Z-ABD-Z polypeptides were titrated in three-fold steps from 190 nM to 0.003 nM in medium containing 0.9 nM hIL-17A and 8 μM HSA. A standard IL-17A curve was also prepared (6.2-0.0001 nM), as well as controls containing medium with 0.9 nM IL-17A or medium alone. The medium in the plate with NHDF cells cultured overnight was discarded and 100 μl/well of the sample was transferred to the cell plate, which was placed in an incubator at 37° C. for 18-24 h. The next day, the IL-6 content in supernatants was quantified using IL-6 specific ELISA as described in Example 3. Additional Z-ABD-Z polypeptides (ZAZ3363 (SEQ ID NO:1244), ZAZ3364 (SEQ ID NO:1245), ZAZ3365 (SEQ ID NO:1246) and ZAZ3422 (SEQ ID NO:1247)) were analyzed in subsequent NHDF assays essentially as described above, but using an NHDF cell line from ATCC (cat. no. PSC-201-012). The blocking capacities of these Z-ABD-Z polypeptides were also analyzed after incubation of the polypeptides at 40° C. for 2 and 4 weeks.
Results
[0402] Z-ABD-Z polypeptides were investigated for their capacity to block IL-17A induced IL-6 production in the NHDF assay. First, it was seen that the Z-ABD-Z format increased the blocking capacity significantly compared to the monomeric His.sub.6-Z format, as shown in
TABLE-US-00022 TABLE 19 Calculated IC50 values from NHDF assays Incubation Z-ABD-Z SEQ at 40° C. IC50 polypeptide ID NO (weeks) (nM) ZAZ3174 1233 n.a. 0.63 ZAZ3175 1234 n.a. 0.56 ZAZ3176 1235 n.a. 0.50 ZAZ3220 1236 n.a. 0.27 ZAZ3221 1237 n.a. 0.39 ZAZ3234 1238 n.a. 0.66 ZAZ3235 1239 n.a. 0.60 ZAZ3236 1240 n.a. 0.54 ZAZ3237 1241 n.a. 0.49 ZAZ3269 1242 n.a. 0.42 ZAZ3270 1243 n.a. 0.41 ZAZ3363 1244 0 0.38 ZAZ3363 1244 2 0.25 ZAZ3363 1244 4 0.49 ZAZ3364 1245 0 0.20 ZAZ3364 1245 2 0.40 ZAZ3364 1245 4 0.47 ZAZ3365 1246 0 0.45 ZAZ3365 1246 2 0.34 ZAZ3365 1246 4 0.55 ZAZ3422 1247 0 0.38 ZAZ3422 1247 2 0.45 ZAZ3422 1247 4 0.42
Example 15
In Vivo Neutralization of hIL-17A
[0403] Human IL-17A is able to bind to and stimulate the mouse IL-17 receptor, leading to elevation and subsequent secretion of mouse KC (CXCL1) chemokine.
Materials and Methods
[0404] In vivo neutralization of hIL-17A in the KC mouse model: Dose ranging experiments were performed to identify the optimal dose of human IL-17A for induction of mouse KC. These experiments revealed that a 150 μg/kg dose of human IL-17A induced a suitable level of KC in mouse serum collected 2 h after IL-17A administration. ZAZ3174 (SEQ ID NO:1233) and ZAZ3220 (SEQ ID NO:1236) were analyzed in this model at two different occasions. In the first experiment, ZAZ3174 was administered s.c. to mice at three different doses; 0.25, 2.5 and 25 mg/kg, 9 h prior to a subcutaneous injection of human IL-17A. The mice were sacrificed 2 h post administration of human IL-17A, and KC levels were determined by ELISA using a commercially available kit according to the manufacturer's instruction (KC Quantikine; R&D Systems, cat no. D1700). In the second experiment, ZAZ3220 was administered s.c. to mice at three different doses, 0.05, 0.1, 0.4 mg/kg, 9 h prior to a subcutaneous injection of human IL-17A. The mice were sacrificed 2 h post administration of human IL-17A, and KC levels were determined by ELISA as above.
Results
[0405] In vivo neutralization of hIL-17A in the KC mouse model: The assayed Z-ABD-Z polypeptides block the ability of human IL-17A to stimulate the mouse IL-17 receptor, leading to inhibition of an elevation of mouse KC, in a dose dependent manner. ZAZ3174 at a dose of 2.5 mg/kg under the conditions described, blocked the induction of KC completely, as shown in
Example 16
In Vivo Pharmacokinetics of Z-ABD-Z Polypeptides in Rats
[0406] This Example describes two separate experiments in which the pharmacokinetic parameters in rat were determined for two different Z-ABD-Z polypeptides following subcutaneous (s.c.) and/or intravenous (i.v.) injections.
Materials and Methods
[0407] In a first experiment, ZAZ3220 (SEQ ID NO:1236) formulated in PBS was administered i.v. (n=3) to Sprague Dawley (SD) male rats (Charles River, Germany) at a dose of 1.2 mg/kg corresponding to 57 nmol/kg. Blood samples were collected from the tail vein of each rat at time points 0.08, 8, 24, 48, 72, 120, 168, 240, 336, 408 and 504 h after administration.
[0408] In a second experiment, ZAZ3363 (SEQ ID NO:1244) formulated in PBS was administered i.v. (n=5) or s.c. (n=6) to SD male rats at a dose of 1.2 mg/kg corresponding to 64 nmol/kg. Blood samples were collected from the tail vein of each rat at time points 0, 0.08, 0.5, 1, 3, 8, 24, 72, 120, 168, 216, 264, 336, 408, 456 and 504 h post i.v. administration or at time points 0, 0.08, 0.5, 1, 3, 8, 24, 72, 120 and 168 h post s.c. administration.
[0409] Serum was prepared from the blood samples and stored at −20° C. until analysis. Quantification of ZAZ3220 and ZAZ3363 in serum from rats was performed using a PK-ELISA.
[0410] PK-ELISA: 96-well, half-area plates (50 μl/well) were coated with 4 μg/well of goat anti-Z Ig (produced in-house) in coating buffer (Sigma, cat. no. C3041) overnight at 4° C. The next day, the plates were blocked with PBS+0.5% casein for 1.5 h. Individual rat serum, minimally diluted 10× in PBS-casein+10% rat serum pool (assay matrix) was added to the plates and titrated in 2-fold dilution series. Included on one plate was standard of each Z-ABD-Z polypeptide (titrated between 300 ng/ml and 3 pg/ml) and on each plate four controls of each Z-ABD-Z polypeptide diluted to concentration within the linear range of the assay. Standard and controls were diluted in assay matrix. The diluted standard, controls and samples were prepared in separate plates and transferred to the coated ELISA plates. The plates were incubated for 1.5 h at RT followed by 1.5 h incubation with a custom made detection rabbit anti-ABD Ig (4 μg/ml, CUV002) and 1 h incubation with donkey anti-rabbit IgG-HRP (Jackson Immunoresearch, cat. no. 711-035-152). The reaction was developed with TMB (Thermo Fisher) and the development reaction was stopped after 15 min with 2 M H.sub.2SO.sub.4. The absorbance was read at 450 nm in an ELISA reader (Victor.sup.3, Perkin Elmer). The concentrations of the respective Z-ABD-Z variant in samples were calculated from the standard curve using GraphPad Prism5 and a four parameter logistic (4-PL) curve-fit.
[0411] Pharmacokinetic analysis: The pharmacokinetic analysis was based on individual rat serum concentration versus time. Terminal half-life (T½) and bioavailability was estimated using Microsoft Excel and GraphPad Prism and applying a two compartment model.
Results
[0412] Mean serum concentration-time profiles of the Z-ABD-Z polypeptides ZAZ3220 and ZAZ3363 following single administrations of 1.2 mg/kg to SD rats are shown in
TABLE-US-00023 TABLE 20 Mean pharmacokinetic parameters of Z-ABD-Z polypeptides following a single i.v. or s.c. administration to SD rats Pharmakokinetic ZAZ3220 ZAZ3363 ZAZ3363 parameter i.v. i.v. s.c. Cmax (nmol/L) 1905 1870 330 Tmax (h) 0.08 0.08 24 AUC.sub.0-t (h*nmol/L) 46369 42757 19351 t.sub.1/2 elimination (h) 49 49 45 MRT (h) 35 33 47 CL (L/h/kg) 0.003 0.003 0.003 F (%) n.a. n.a. 45 Cmax: Peak serum concentration; Tmax: Time to reach the peak serum concentration; AUC.sub.0-t: Area under the concentration-time curve from time zero to last quantifiable concentration; T.sub.1/2: Half-life; MRT: Mean residence time; CL: Clearance; F: Percentage absolute bioavailability, calculated as F = [(Mean AUC.sub.s.c. × Dose.sub.i.v.)/(Mean AUC.sub.i.v. × Dose.sub.s.c.)] × 100
Example 17
Topical Administration of Z Variant to Eyes of Rabbits
[0413] Local administration to the eye is beneficial in ophthalmologic disorders, e.g. uveitis or dry eye diseases. Topical administration enables extremely high local drug concentrations with a minimal risk of systemic side effects. In this Example, the possibility to topically administer Z variant molecules to eyes was investigated in rabbits. The concentration of a Z variant molecule and a control IgG was measured in the anterior chamber (aqueous humor), in the vitreous humor and in serum after four repeated topical dosings.
Materials and Methods
[0414] Production of Z variant: The Z variant Z10199 (SEQ ID NO:1217) derived from Z06282 (SEQ ID NO:1206) but starting AE was subcloned without any tag but with the C-terminal addition of amino acid residues VD. Expression was performed essentially as described in Example 2, and purification was carried out by anion exchange and reverse phase chromatography. The sample was buffer exchanged to PBS pH 7.2 and endotoxins were removed using an EndoTrap® red 10 column (Hyglos).
[0415] Animal study: 48 nmol (1 drop, 50 μl) of Z10199 and ZAZ3174 (SEQ ID NO:1233), respectively, was administered topically to each eye of rabbits (n=2 per molecule) at time points 0, 1, 2 and 3 hours. A control human IgG antibody (Xolair; Novartis) was administered in the same way to two different rabbits. As a negative control, one rabbit was administered PBS only. After each administration, the eyelids were held closed during approximately 30 s to keep the sample on the cornea. After 4 h, vitreous humor, aqueous humor and serum were collected. The samples were centrifuged to remove tissue debris and aggregates and the levels of Z10199, ZAZ3174 and antibody, respectively, were quantified by ELISA.
[0416] Quantification by ELISA: The concentration of Z10199 and ZAZ3174, respectively, in the collected samples was analyzed by a sandwich ELISA using an in-house produced polyclonal goat anti-protein Z immunoglobulin for capture, an in-house produced polyclonal rabbit anti-protein Z immunoglobulin as primary antibody and goat anti-rabbit IgG-HRP (Dako cat. no. P0448) as secondary antibody. Detection was performed by incubation with ImmunoPure TMB for 15 min at RT and the reaction was stopped by addition of 2 M H.sub.2SO.sub.4. Absorption at 450 nm was measured with the microplate reader Victor.sup.3. The concentration of Z10199 was calculated from a standard curve prepared with the same molecule and using GraphPad prism5 and a non-linear regression formula.
[0417] The concentration of IgG in the collected samples was analyzed by an IgG ELISA kit (Abcam 100547) and performed as described by the manufacturer, using a standard curve provided and analysis by non-linear regression as above.
Results
[0418]
Example 18
Pharmacokinetic Analysis of Duodenal Administration of ZAZ3363 Formulations in Rat
Materials and Methods
[0419] Test item: ZAZ3363 (SEQ ID NO:1244) was formulated in 1) OAF1: 0.12 M sodium chenodeoxycholate (Sigma, cat. no. C8261) and 0.12 M propyl gallate (Sigma, cat. no. P3130), pH 7.4; 2) OAF2: 50 mg/ml sodium caprate (Sigma, cat. no. C4151); or 3) 50 mM sodium phosphate (PBS), pH 7.0, at a concentration of 50 mg/ml (OAF1 and OAF2) or 100 mg/ml (PBS).
[0420] NHDF cell assay: The activity of ZAZ3363 (SEQ ID NO:1244) formulated in OAF1, OAF2 or PBS was verified in the IL-17 dependent NHDF cell assay described in Example 14.
[0421] Duodenal administration: Male Sprague Dawley rats (Charles River) were anesthetized with isofluorane, and a small incision was made to localise the duodenum. An indwelling catheter (R-DUOD, AgnTho's, Sweden) was inserted surgically into the duodenum 20 mm from its origin in an area of minimal vasculature. The catheter was tunnelled subcutaneously to the back of the animals. Abdominal musculature was closed with sutures, while the abdominal skin incision and sub scapular exteriorization site was closed with stainless steel wound clips. The animals were left to recover for 5-6 days before being taken to the study. Post-operative analgesia was administered s.c. prior to surgery (carprofen 5 mg/kg and buprenorphine 0.05 mg/kg). Carprofen was administered once daily also on the two days following surgery. Additional doses of buprenorphine were administered when necessary. Antibiotics (enrofloxacin 0.3 mg/ml) was administered in the drinking water during the recovery period (3-5 days), as well as during the experiment. To mask the bitter taste of enrofloxacin, one piece of sugar was added to 500 ml of drinking water. To ensure a maintained patency, the duodenal catheter was flushed daily with sterile water (0.2-0.5 ml).
[0422] ZAZ3363 was administered directly into duodenum at a dose of 5.4 μmol/kg body weight formulated in OAF1 or OAF2 (n=2) or at 10.8 μmol/kg formulated in PBS (n=2) in a volume of 0.5 ml at time-point zero. Blood samples were taken under isoflurane anesthesia at 1, 3, 8, 24, 72, 120 and 168 h after administration and serum was prepared by standard procedures. The concentration of ZAZ3363 in serum was measured by the quantitative PK-ELISA described in Example 16, but titrating the standard of ZAZ3363 between 70 and 1 ng/ml.
[0423] Pharmacokinetic analysis: The pharmacokinetic analysis was based on individual rat serum concentration versus time. Terminal half-life (T½) and bioavailability was estimated using Microsoft Excel and GraphPad Prism.
Results
[0424] Activity of formulated ZAZ3363: The activity of ZAZ3363 formulated in OAF1 and OAF2, respectively, was compared to formulation in PBS in the NHDF cell assay.
[0425] Intraduodenal uptake of ZAZ3363: Intestinal uptake of OAF1, OAF2 and PBS formulated ZAZ3363 was examined in a rat model of intraduodenal administration (i.d.). The experiment showed an increased uptake with OAF1 and OAF2 formulations compared to PBS (
TABLE-US-00024 TABLE 21 Bioavailability and T½ after intraduodenal administration of ZAZ3363 in three different formulations Formulation T½ (h) Bioavailability (%) PBS 43 0.0007 +/− 0.0002 OAF1 43 0.2 +/− 0.02 OAF2 50 0.8 +/− 1.2
Example 19
Characterization of Anti-IL-17A/Anti-TNF Complexes in the NHDF Assay
Materials and Methods
[0426] Production of complexes and control antibody: Two different complexes targeting IL-17A and TNF were constructed, as well as an antibody with affinity for TNF. The antibody denoted “Ada”, having the same CDR sequences and specificity as the commercially available monoclonal antibody adalimumab, was constructed using the heavy chain (HC) and light chain (LC) sequences HC.sub.Ada (SEQ ID NO:1231) and LC.sub.Ada (SEQ ID NO:1232). The IL-17A targeting Z variant Z14253 (SEQ ID NO:1219) moiety was genetically fused, via a flexible 15 residue (GGGGS).sub.3 linker, to the C-termini of HC.sub.Ada or LC.sub.Ada, resulting in the complexes HC.sub.Ada-Z14253 and LC.sub.Ada-Z14253, respectively. A schematic of the constructed complexes is shown in
[0427] Blocking of IL-17A induced IL-6 production in NHDF assay: The NHDF assay was performed essentially as described in Example 14, titrating the study samples HC.sub.Ada-Z14253 and LC.sub.Ada-Z14253 and their comparators in three-fold steps from 63 nM to 0.003 nM in a medium containing 0.9 nM rhIL-17A. The comparators were ZAZ3363 (SEQ ID NO:1244), the anti-TNF antibody Ada, as well as a negative control Z variant Z04726 (SEQ ID NO:1223; targeting taq polymerase) recombinantly fused to the ABD variant PP013 (SEQ ID NO:1224) via a VDSS linker (denoted Z04726-PP013), as described in Example 2. A standard IL-17A curve was also prepared (6.2-0.0001 nM) as well as controls containing medium with 0.9 nM IL-17A or medium alone. The IL-6 content in supernatants was quantified using the IL-6 specific ELISA described in Example 3.
[0428] Blocking of TNF or TNF/IL-17A induced IL-8 production in NHDF assay: NHDF (Lonza, cat. no. CC-2511) cells were cultured in fibroblast basal medium (Lonza, cat. no. CC-3132) supplied with growth promoting factors (Lonza, cat. no CC-5034). On the day before the experiment, 5000 cells per well were seeded into a half area 96 well culture plates (Greiner, cat. no 675180) in 100 μl. On the day of the experiment, dilutions of HC.sub.Ada-Z14253 and LC.sub.Ada-Z14253 and the comparators described above were prepared in a separate 96-well plate. The complexes and comparators were titrated in three-fold steps from 5 nM to 0.0007 nM in medium containing 0.1 nM human TNF (R&D Systems, cat. no. 2210-TA/CF) or a mixture of 0.05 nM and 0.1 nM rhIL-17A. Controls containing medium with 0.1 nM TNF or a mixture of 0.05 nM and 0.1 nM rhIL-17A or medium alone were also prepared. The medium in the plate with the overnight cultured NHDF cells was discarded and 100 μl/well of the sample was transferred to the cell plate. The plate was placed in an incubator at 37° C. for 18-24 h. The next day, the IL-8 content in supernatants was quantified using an IL-8 specific ELISA.
[0429] IL-8 ELISA: IL-8 was quantified by a DuoSet ELISA kit (R&D systems, cat. no. DY208). Half area plates (Costar, cat. no. 3690) were coated with the anti-IL-8 capture antibody, 4 μg/ml in PBS, 50 μl/well, overnight at 4° C. On the day of the analysis, the plate was rinsed twice in tap water and then blocked with PBS+1% BSA for 2 h. IL-8 standard (R&D Systems, cat. no. 890806), titrated in a 2-fold dilution series (20-0.01 ng/ml) and supernatants from the cell assay plate were added to the coated ELISA plate (50 μl/well) and incubated for 1.5 h at RT. The plate was washed 4 times in an automated ELISA washer and 20 ng/ml (50 μl/well) of biotinylated anti-IL-8 detection antibody was added. After another 1 h incubation, the plate was washed and 50 μl of streptavidin-HRP (Thermo Fisher, cat. no. N100) diluted 8000× was added per well. The plate was developed after one additional hour's incubation and washing, with 50 μl TMB (Thermo Fisher, cat. no 34021) per well, and the reaction was stopped with 50 μl 2 M H.sub.2SO.sub.4. The absorbances were read in a multi label reader (Victor.sup.3, Perkin Elmer).
Results
[0430] Blocking of IL-17A induced IL-6 production in NHDF assay: The two complexes HC.sub.Ada-Z14253 and LC.sub.Ada-Z14253 were studied with regard to their capacity to block IL-17A induced IL-6 production in the NHDF assay. Results from the NHDF assay are presented in
[0431] Blocking of TNF or TNF/IL-17A induced IL-8 production in NHDF assay: The two complexes HC.sub.Ada-Z14253 and LC.sub.Ada-Z14253 were studied with regard to their capacity to block TNF or a mixture of TNF/IL-17A induced IL-8 production in the NHDF assay. Results from the NHDF assay showed that HC.sub.Ada-Z14253 and LC.sub.Ada-Z14253 have similar inhibitory profiles as the anti-TNF antibody Ada with regard to specific TNF blocking capacity (
Example 20
In Vivo Pharmacokinetics of Z-ABD-Z Polypeptide in Monkeys
[0432] This Example describes a repeated dose study conducted over 10 days in cynomolgus monkeys administered with the Z-ABD-Z polypeptide ZAZ3363. The results were used for estimation of the half-life of ZAZ3363 in cynomolgus.
Materials and Methods
[0433] ZAZ3363 (SEQ ID NO:1244) was administered at 20 mg/kg (n=2; 1 male and 1 female) and 40 mg/kg (n=4; 2 male and 2 female) as a short i.v. infusion on days 1, 4, 7 and 10. Plasma samples for determination of ZAZ3363 concentration were collected in connection with the first dose on day 1 (at time points 0 (pre-dose), 5 min, 0.5, 1, 2, 6, 24 and 48 h after administration) and last dose on day 10 (at time points 0 (pre-dose), 5 min, 0.5, 1, 2, 6, 24, 48 h, 5, 7, 10, 12, 14 and 21 days after administration). Quantification of ZAZ3363 in plasma samples was carried out by LC-MS/MS based on peptides obtained after tryptic digestion of ZAZ3363. Concentration-time profiles were evaluated using both non-compartmental methods with separate analyses for day 1 and day 10 and compartmental methods evaluating the data for day 1 and day 10 combined for each animal.
Results
[0434] Mean plasma concentration-time profiles of ZAZ3363 following administration on day 1 and day 10 are shown in
Example 21
Oral Administration of Z-ABD-Z Polypeptide in Dogs
Materials and Methods
[0435] Preparation of capsules: ZAZ3363 (SEQ ID NO:1244) was formulated in OAF1 (see Example 18) at a concentration of 100 mg/ml. The formulation was lyophilized and filled in hard shell capsules subsequently enteric coated (performed by Catalent Pharma Solutions, Beinheim, France). Each capsule contained approximately 25 mg ZAZ3363.
[0436] Animal study: The animal study was performed at Huntingdon Life Science (Cambridgeshire, UK). Fasted beagle dogs (n=3; female individuals) each received six capsules containing ZAZ3363 (approximately 150 mg). Serum samples were taken at 0.5, 1, 2, 4, 6, 8, 12, 24 and 96 h after administration.
[0437] Quantification: The concentration of ZAZ3363 in serum samples was quantified using a PK sandwich ELISA essentially as described in Example 16, but using an in-house produced monoclonal anti-Z IgG in the first coating step.
Results
[0438] The individual dog serum concentrations versus time profiles of ZAZ3363 are shown in
Itemized Listing of Embodiments
[0439] 1. IL-17A binding polypeptide, comprising an IL-17A binding motif BM, which motif consists of an amino acid sequence selected from: [0440] i) EX.sub.2DX.sub.4AX.sub.6X.sub.7EIX.sub.10X.sub.11LPNL X.sub.16X.sub.17X.sub.18QX.sub.20X.sub.21AFIX.sub.25 X.sub.26LX.sub.28X.sub.29 [0441] wherein, independently from each other,
[0442] X.sub.2 is selected from A, H, M and Y;
[0443] X.sub.4 is selected from A, D, E, F, K, L, M, N, Q, R, S and Y;
[0444] X.sub.6 is selected from A, Q and W;
[0445] X.sub.7 is selected from F, I, L, M, V, W and Y;
[0446] X.sub.10 is selected from A and W;
[0447] X.sub.11 is selected from A, D, E, F, G, L, M, N, Q, S, T and Y;
[0448] X.sub.16 is selected from N and T;
[0449] X.sub.17 is selected from H, W and Y;
[0450] X.sub.18 is selected from A, D, E, H and V;
[0451] X.sub.20 is selected from A, G, Q, S and W;
[0452] X.sub.21 is selected from A, D, E, F, H, K, N, R, T, V, W and Y;
[0453] X.sub.25 is selected from A, D, E, G, H, I, L, M, N, Q, R, S, T and V;
[0454] X.sub.26 is selected from K and S;
[0455] X.sub.28 is selected from I, L, N and R; and
[0456] X.sub.29 is selected from D and R; [0457] and [0458] ii) an amino acid sequence which has at least 89% identity to the sequence defined in i).
[0459] 2. IL-17A binding polypeptide according to item 1, wherein, in sequence i),
[0460] X.sub.2 is selected from A, H and M;
[0461] X.sub.4 is selected from A, D, E, F, L, M, N, Q, R and Y;
[0462] X.sub.11 is selected from A, D, E, F, G, L, M, N, S, T and Y;
[0463] X.sub.18 is selected from A, D, E and V;
[0464] X.sub.20 is selected from A, G, Q and W;
[0465] X.sub.21 is selected from E, F, H, N, R, T, V, W and Y;
[0466] X.sub.25 is selected from A, D, E, G, H, I, L, N, Q, R, S, T and V; and
[0467] X.sub.28 is selected from I, N and R.
[0468] 3. IL-17A binding polypeptide according to item 2, wherein, in sequence i),
[0469] X.sub.16 is T;
[0470] X.sub.17 is W;
[0471] X.sub.21 is selected from E, F, H, W, T and Y;
[0472] X.sub.25 is selected from A, D, E, G, H, I, L, N, Q, R, S and T;
[0473] X.sub.26 is K; and
[0474] X.sub.29 is D.
[0475] 4. IL-17A binding polypeptide according to any preceding item, wherein sequence i) fulfills at least six of the eleven conditions I-XI: [0476] I. X.sub.2 is A; [0477] II. X.sub.4 is selected from D, E and Q; [0478] III. X.sub.6 is A; [0479] IV. X.sub.7 is selected from F and V; [0480] V. X.sub.16 is T; [0481] VI. X.sub.17 is W; [0482] VII. X.sub.18 is selected from A and D; [0483] VIII. X.sub.20 is W; [0484] IX. X.sub.26 is K; [0485] X. X.sub.28 is R; and [0486] XI. X.sub.29 is D.
[0487] 5. IL-17A binding polypeptide according to item 4, wherein sequence i) fulfills at least seven of the eleven conditions I-XI.
[0488] 6. IL-17A binding polypeptide according to item 5, wherein sequence i) fulfills at least eight of the eleven conditions I-XI.
[0489] 7. IL-17A binding polypeptide according to item 6, wherein sequence i) fulfills at least nine of the eleven conditions I-XI.
[0490] 8. IL-17A binding polypeptide according to item 7, wherein sequence i) fulfills at least ten of the eleven conditions I-XI.
[0491] 9. IL-17A binding polypeptide according to item 8, wherein sequence i) fulfills all of the eleven conditions I-XI.
[0492] 10. IL-17A binding polypeptide according to any preceding item, wherein X.sub.2X.sub.6, X.sub.2X.sub.10 or X.sub.6X.sub.10 is AA.
[0493] 11. IL-17A binding polypeptide according to any preceding item, wherein X.sub.2X.sub.17, X.sub.2X.sub.20, X.sub.6X.sub.17, X.sub.6X.sub.20, X.sub.10X.sub.17 or X.sub.10X.sub.20 is AW.
[0494] 12. IL-17A binding polypeptide according to any preceding item, wherein X.sub.2X.sub.28, X.sub.6X.sub.28 or X.sub.10X.sub.28 is AR.
[0495] 13. IL-17A binding polypeptide according to any preceding item, wherein X.sub.17X.sub.28 or X.sub.20X.sub.28 is WR.
[0496] 14. IL-17A binding polypeptide according to any preceding item, wherein X.sub.17X.sub.20 is WW.
[0497] 15. IL-17A binding polypeptide according to any preceding item, wherein sequence i) is the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-1216.
[0498] 16. IL-17A binding polypeptide according to item 15, wherein sequence i) is the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-66, 1200, 1206 and 1214.
[0499] 17. IL-17A binding polypeptide according to item 16, wherein sequence i) is the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-66.
[0500] 18. IL-17A binding polypeptide according to item 17, wherein sequence i) is the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-35.
[0501] 19. IL-17A binding polypeptide according to item 18, wherein sequence i) is the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-27.
[0502] 20. IL-17A binding polypeptide according to item 19, wherein sequence i) is the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-10.
[0503] 21. IL-17A binding polypeptide according to item 20, wherein sequence i) is the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-7.
[0504] 22. IL-17A binding polypeptide according to item 21, wherein sequence i) is the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-4.
[0505] 23. IL-17A binding polypeptide according to item 22, wherein sequence i) is the sequence from position 8 to position 36 in SEQ ID NO:1.
[0506] 24. IL-17A binding polypeptide according to any preceding item, wherein said IL-17A binding motif forms part of a three-helix bundle protein domain.
[0507] 25. IL-17A binding polypeptide according to item 24, wherein said IL-17A binding motif essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.
[0508] 26. IL-17A binding polypeptide according to item 25, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.
[0509] 27. IL-17A binding polypeptide according to item 26, wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.
[0510] 28. IL-17A binding polypeptide according to any preceding item, which comprises an amino acid sequence binding module, BMod, selected from: [0511] iii) K-[BM]-DPSQS X.sub.aX.sub.bLLX.sub.c EAKKL X.sub.dX.sub.eX.sub.fQ; [0512] wherein [0513] [BM] is an IL-17A binding motif as defined in any one of items 1-23 provided that X.sub.29 is D; [0514] X.sub.a is selected from A and S; [0515] X.sub.b is selected from N and E; [0516] X.sub.c is selected from A, S and C; [0517] X.sub.d is selected from E, N and S; [0518] X.sub.e is selected from D, E and S; [0519] X.sub.f is selected from A and S; and [0520] iv) an amino acid sequence which has at least 85% identity to a sequence defined by iii).
[0521] 29. IL-17A binding polypeptide according to any one of items 1-27, which comprises an amino acid sequence binding module, BMod, selected from: [0522] v) K-[BM]-QPEQS X.sub.aX.sub.bLLX.sub.c EAKKL X.sub.dX.sub.eX.sub.fQ; [0523] wherein [0524] [BM] is an IL-17A binding motif as defined in any one of items 1-23 provided that X.sub.29 is R; [0525] X.sub.a is selected from A and S; [0526] X.sub.b is selected from N and E; [0527] X.sub.c is selected from A, S and C; [0528] X.sub.d is selected from E, N and S; [0529] X.sub.e is selected from D, E and S; [0530] X.sub.f is selected from A and S; and [0531] vi) an amino acid sequence which has at least 85% identity to a sequence defined by v).
[0532] 30. IL-17A binding polypeptide according to item 28 or 29, wherein X.sub.a in sequence iii) or v) is A.
[0533] 31. IL-17A binding polypeptide according to item 28 or 29, wherein X.sub.a in sequence iii) or v) is S.
[0534] 32. IL-17A binding polypeptide according to any one of items 28-31, wherein X.sub.b in sequence iii) or v) is N.
[0535] 33. IL-17A binding polypeptide according to any one of items 28-31, wherein X.sub.b in sequence iii) or v) is E.
[0536] 34. IL-17A binding polypeptide according to any one of items 28-33, wherein X.sub.c in sequence iii) or v) is A.
[0537] 35. IL-17A binding polypeptide according to any one of items 28-33, wherein X.sub.c in sequence iii) or v) is S.
[0538] 36. IL-17A binding polypeptide according to any one of items 28-33, wherein X.sub.c in sequence iii) or v) is C.
[0539] 37. IL-17A binding polypeptide according to any one of items 28-36, wherein X.sub.d in sequence iii) or v) is E.
[0540] 38. IL-17A binding polypeptide according to any one of items 28-36, wherein X.sub.d in sequence iii) or v) is N.
[0541] 39. IL-17A binding polypeptide according to any one of items 28-36, wherein X.sub.d in sequence iii) or v) is S.
[0542] 40. IL-17A binding polypeptide according to any one of items 28-39, wherein X.sub.e in sequence iii) or v) is D.
[0543] 41. IL-17A binding polypeptide according to any one of items 28-39, wherein X.sub.e in sequence iii) or v) is E.
[0544] 42. IL-17A binding polypeptide according to any one of items 28-39, wherein X.sub.e in sequence iii) or v) is S.
[0545] 43. IL-17A binding polypeptide according to any one of items 37 and 39-42, wherein X.sub.dX.sub.e in sequence iii) or v) is selected from EE, ES, SD, SE and SS.
[0546] 44. IL-17A binding polypeptide according to item 43, wherein X.sub.dX.sub.e in sequence iii) or v) is ES.
[0547] 45. IL-17A binding polypeptide according to item 43, wherein X.sub.dX.sub.e in sequence iii) or v) is SE.
[0548] 46. IL-17A binding polypeptide according to item 43, wherein X.sub.dX.sub.e in sequence iii) or v) is SD.
[0549] 47. IL-17A binding polypeptide according to any one of items 28-46, wherein X.sub.f in sequence iii) or v) is A.
[0550] 48. IL-17A binding polypeptide according to any one of items 28-46, wherein X.sub.f in sequence iii) or v) is S.
[0551] 49. IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X.sub.a is A; X.sub.b is N; X.sub.c is A and X.sub.f is A.
[0552] 50. IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is A and X.sub.f is A.
[0553] 51. IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X.sub.a is A; X.sub.b is N; X.sub.c is C and X.sub.f is A.
[0554] 52. IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is S and X.sub.f is S.
[0555] 53. IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is S and X.sub.f is A.
[0556] 54. IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is A and X.sub.f is S.
[0557] 55. IL-17A binding polypeptide according to item 28 or 29, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is C and X.sub.f is S.
[0558] 56. IL-17A binding polypeptide according to item 49, wherein, in sequence iii) or v), X.sub.a is A; X.sub.b is N; X.sub.c is A; X.sub.dX.sub.e is ND and X.sub.f is A.
[0559] 57. IL-17A binding polypeptide according to item 50, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is A; X.sub.dX.sub.e is ND and X.sub.f is A.
[0560] 58. IL-17A binding polypeptide according to item 51, wherein, in sequence iii) or v), X.sub.a is A; X.sub.b is N; X.sub.c is C; X.sub.dX.sub.e is ND and X.sub.f is A.
[0561] 59. IL-17A binding polypeptide according to item 52, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is S; X.sub.dX.sub.e is ND and X.sub.f is S.
[0562] 60. IL-17A binding polypeptide according to item 53, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is S; X.sub.dX.sub.e is ND and X.sub.f is A.
[0563] 61. IL-17A binding polypeptide according to item 55, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is C; X.sub.dX.sub.e is ND and X.sub.f is S.
[0564] 62. IL-17A binding polypeptide according to item 49, wherein, in sequence iii) or v), X.sub.a is A; X.sub.b is N; X.sub.c is A; X.sub.dX.sub.e is SE and X.sub.f is A.
[0565] 63. IL-17A binding polypeptide according to item 50, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is A; X.sub.dX.sub.e is SE and X.sub.f is A.
[0566] 64. IL-17A binding polypeptide according to item 51, wherein, in sequence iii) or v), X.sub.a is A; X.sub.b is N; X.sub.c is C; X.sub.dX.sub.e is SE and X.sub.f is A.
[0567] 65. IL-17A binding polypeptide according to item 52, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is S; X.sub.dX.sub.e is SE and X.sub.f is S.
[0568] 66. IL-17A binding polypeptide according to item 54, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is A; X.sub.dX.sub.e is SE and X.sub.f is S.
[0569] 67. IL-17A binding polypeptide according to item 55, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is C; X.sub.dX.sub.e is SE and X.sub.f is S.
[0570] 68. IL-17A binding polypeptide according to item 49, wherein, in sequence iii) or v), X.sub.a is A; X.sub.b is N; X.sub.c is A; X.sub.dX.sub.e is ES and X.sub.f is A.
[0571] 69. IL-17A binding polypeptide according to item 50, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is A; X.sub.dX.sub.e is ES and X.sub.f is A.
[0572] 70. IL-17A binding polypeptide according to item 51, wherein, in sequence iii) or v), X.sub.a is A; X.sub.b is N; X.sub.c is C; X.sub.dX.sub.e is ES and X.sub.f is A.
[0573] 71. IL-17A binding polypeptide according to item 52, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is S; X.sub.dX.sub.e is ES and X.sub.f is S.
[0574] 72. IL-17A binding polypeptide according to item 55, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is C; X.sub.dX.sub.e is ES and X.sub.f is S.
[0575] 73. IL-17A binding polypeptide according to item 49, wherein, in sequence iii) or v), X.sub.a is A; X.sub.b is N; X.sub.c is A; X.sub.dX.sub.e is SD and X.sub.f is A.
[0576] 74. IL-17A binding polypeptide according to item 50, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is A; X.sub.dX.sub.e is SD and X.sub.f is A.
[0577] 75. IL-17A binding polypeptide according to item 51, wherein, in sequence iii) or v), X.sub.a is A; X.sub.b is N; X.sub.c is C; X.sub.dX.sub.e is SD and X.sub.f is A.
[0578] 76. IL-17A binding polypeptide according to item 52, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is S; X.sub.dX.sub.e is SD and X.sub.f is S.
[0579] 77. IL-17A binding polypeptide according to item 54, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is A; X.sub.dX.sub.e is SD and X.sub.f is S.
[0580] 78. IL-17A binding polypeptide according to item 55, wherein, in sequence iii) or v), X.sub.a is S; X.sub.b is E; X.sub.c is C; X.sub.dX.sub.e is SD and X.sub.f is S.
[0581] 79. IL-17A binding polypeptide according to item 28, wherein said sequence iii) is the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-1216.
[0582] 80. IL-17A binding polypeptide according to item 79, wherein sequence iii) is the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-66, 1200, 1206 and 1214.
[0583] 81. IL-17A binding polypeptide according to item 80, wherein sequence iii) is the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-66.
[0584] 82. IL-17A binding polypeptide according to item 81, wherein sequence iii) is the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-35.
[0585] 83. IL-17A binding polypeptide according to item 82, wherein sequence iii) is the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-27.
[0586] 84. IL-17A binding polypeptide according to item 83, wherein sequence iii) is the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-10.
[0587] 85. IL-17A binding polypeptide according to item 84, wherein sequence iii) is the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-7.
[0588] 86. IL-17A binding polypeptide according to item 85, wherein sequence iii) is the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-4.
[0589] 87. IL-17A binding polypeptide according to item 86, wherein sequence iii) the sequence from position 7 to position 55 in SEQ ID NO:1.
[0590] 88. IL-17A binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from: [0591] vii) YA-[BMod]-AP; [0592] wherein [BMod] is an IL-17A binding module as defined in any one of items 28-87; and [0593] viii) an amino acid sequence which has at least 86% identity to a sequence defined by vii).
[0594] 89. IL-17A binding polypeptide according to any one of items 1-87, which comprises an amino acid sequence selected from: [0595] ix) FA-[BMod]-AP; [0596] wherein [BMod] is an IL-17A binding module as defined in any one of items 28-87; and [0597] x) an amino acid sequence which has at least 86% identity to a sequence defined by ix).
[0598] 90. IL-17A binding polypeptide according to any one of items 1-87, which comprises an amino acid sequence selected from: [0599] xi) FN-[BMod]-AP; [0600] wherein [BMod] is an IL-17A binding module as defined in any one of items 28-87; and [0601] xii) an amino acid sequence which has at least 86% identity to a sequence defined by xi).
[0602] 91. IL-17A binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
TABLE-US-00025 ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK; ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK; ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK; AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK; VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDAQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDAQAP; AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK; AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]DPSQSSELLAEAKKLSEAQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]DPSQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]DPSQSSELLAEAKKLESAQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]DPSQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]DPSQSSELLAEAKKLSDSQAPK; VDAKYAK-[BM]DPSQSSELLAEAKKLSDAQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]DPSQSSELLAEAKKLNKAQAPK; AEAKYAK-[BM]DPSQSSELLAEAKKLNKAQAPK; and ADAKYAK-[BM]DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is an IL-17A binding motif as defined in any one of items 1-23.
[0603] 92. IL-17A binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
TABLE-US-00026 xiii) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; [0604] wherein [BM] is an IL-17A binding motif as defined in any one of items 1-23; and [0605] xiv) an amino acid sequence which has at least 86% identity to the sequence defined in xiii).
[0606] 93. IL-17A binding polypeptide according to item 92, wherein sequence xiii) is selected from SEQ ID NO:1-1216.
[0607] 94. IL-17A binding polypeptide according to item 93, wherein sequence xiii) is selected from SEQ ID NO:1-66, 1200, 1206 and 1214. [0608] 95. IL-17A binding polypeptide according to item 94, wherein sequence xiii) is selected from SEQ ID NO:1-66.
[0609] 96. IL-17A binding polypeptide according to item 95, wherein sequence xiii) is selected from SEQ ID NO:1-35.
[0610] 97. IL-17A binding polypeptide according to item 96, wherein sequence xiii) is selected from SEQ ID NO:1-27.
[0611] 98. IL-17A binding polypeptide according to item 97, wherein sequence xiii) is selected from SEQ ID NO:1-10.
[0612] 99. IL-17A binding polypeptide according to item 98, wherein sequence xiii) is selected from SEQ ID NO:1-7.
[0613] 100. IL-17A binding polypeptide according to item 99, wherein sequence xiii) is selected from SEQ ID NO:1-4.
[0614] 101. IL-17A binding polypeptide according to item 100, wherein sequence xiii) is SEQ ID NO:1.
[0615] 102. IL-17A binding polypeptide according to any one of items 1-91, which comprises an amino acid sequence selected from:
TABLE-US-00027 xv) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; [0616] wherein [BM] is an IL-17A binding motif as defined in any one of items 1-23; and [0617] xvi) an amino acid sequence which has at least 86% identity to the sequence defined in xv).
[0618] 103. IL-17A binding polypeptide according to item 102, wherein sequence xv) is selected from SEQ ID NO:1217-1222.
[0619] 104. IL-17A binding polypeptide according to item 103, wherein sequence xv) is selected from SEQ ID NO:1218-1222.
[0620] 105. IL-17A binding polypeptide according to item 104, wherein sequence xv) is selected from SEQ ID NO:1219-1222.
[0621] 106. IL-17A binding polypeptide according to item 105, wherein sequence xv) is selected from SEQ ID NO:1219 and SEQ ID NO:1222.
[0622] 107. IL-17A binding polypeptide according to item 106, wherein sequence xv) is SEQ ID NO:1219.
[0623] 108. IL-17A binding polypeptide according to any preceding item, which is capable of binding to IL-17A 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.
[0624] 109. IL-17A binding polypeptide according to any preceding item, which is capable of binding to an IL-17A molecule selected from the group consisting of human IL-17A and murine IL-17A.
[0625] 110. IL-17A binding polypeptide according to item 109, which is capable of binding to human IL-17A.
[0626] 111. IL-17A binding polypeptide according to item 109, which is capable of binding to murine IL-17A.
[0627] 112. IL-17A binding polypeptide according to any one of items 109-111, which is capable of binding to human IL-17A and to murine IL-17A.
[0628] 113. IL-17A binding polypeptide according to any one of items 109, 110 and 112, wherein said human IL-17A comprises the amino acid sequence SEQ ID NO:1226 or an antigenically effective fragment thereof.
[0629] 114. IL-17A binding polypeptide according to any one of items 109, 111 and 112, wherein said murine IL-17A comprises the amino acid sequence SEQ ID NO:1227 or an antigenically effective fragment thereof.
[0630] 115. IL-17A binding polypeptide according to any preceding item which comprises additional amino acids at the C-terminal and/or N-terminal end.
[0631] 116. IL-17A binding polypeptide according to item 115, wherein said additional amino acid(s) improve(s) and/or simplify/simplifies production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide.
[0632] 117. IL-17A binding polypeptide according to any preceding item in multimeric form, comprising at least two IL-17A binding polypeptide monomer units, whose amino acid sequences may be the same or different.
[0633] 118. IL-17A binding polypeptide according to item 117, wherein said IL-17A binding polypeptide monomer units are covalently coupled together.
[0634] 119. IL-17A binding polypeptide according to item 118, wherein the IL-17A binding polypeptide monomer units are expressed as a fusion protein.
[0635] 120. IL-17A binding polypeptide according to any one of items 117-119, in dimeric form.
[0636] 121. Fusion protein or conjugate comprising [0637] a first moiety consisting of an IL-17A binding polypeptide according to any preceding item; and [0638] a second moiety consisting of a polypeptide having a desired biological activity.
[0639] 122. Fusion protein or conjugate according to item 121, wherein said desired biological activity is a therapeutic activity.
[0640] 123. Fusion protein or conjugate according to item 121, wherein said desired biological activity is a binding activity.
[0641] 124. Fusion protein or conjugate according to item 123, wherein said binding activity is albumin binding activity which increases in vivo half-life of the fusion protein or conjugate.
[0642] 125. Fusion protein or conjugate according to item 124, comprising two IL-17A binding polypeptides, each as defined in any one of items 1-116, with an albumin binding moiety between them.
[0643] 126. Fusion protein or conjugate according to item 125, which is capable of binding to IL-17A such that the K.sub.D value of the interaction is at most 1×10.sup.−10 M, such as at most 1×10.sup.−11 M, such as at most 1×10.sup.−12 M, such as at most 1×10.sup.−13 M.
[0644] 127. Fusion protein or conjugate according to any one of items 124-126, wherein said second moiety comprises the albumin binding domain of streptococcal protein G or a derivative thereof.
[0645] 128. Fusion protein or conjugate according to item 123, wherein said binding activity acts to inhibit a biological activity.
[0646] 129. Fusion protein or conjugate according to item 123, wherein said binding activity acts to stimulate a biological activity.
[0647] 130. Fusion protein or conjugate according to item 121, wherein said desired biological activity is an enzymatic activity.
[0648] 131. Fusion protein or conjugate according to item 122, wherein the second moiety is a therapeutically active polypeptide.
[0649] 132. Fusion protein or conjugate according to item 131, wherein the second moiety is an immune response modifying agent, for example an anti-inflammatory agent.
[0650] 133. Fusion protein or conjugate according to any one of items 121-122 and 130-132, wherein the second moiety is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines, and agonists, antagonists and inhibitors thereof.
[0651] 134. Fusion protein or conjugate according to item 131, wherein the second moiety is a toxic compound.
[0652] 135. Fusion protein or conjugate according to item 123, wherein said binding activity is binding to an immune response associated factor, for example an inflammation-associated factor.
[0653] 136. Complex, comprising at least one IL-17A binding polypeptide according to any one of items 1-117 or at least one fusion protein or conjugate according to any one of items 121-135, and at least one antibody or an antigen binding fragment thereof.
[0654] 137. Complex according to item 136, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments, Fab′ fragments, F(ab′).sub.2 fragments, Fc fragments, Fv fragments, single chain Fv fragments, (scFv).sub.2 and domain antibodies.
[0655] 138. Complex according to item 137, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments and scFv fragments.
[0656] 139. Complex according to item 138, wherein said at least one antibody or antigen binding fragment thereof is a full-length antibody.
[0657] 140. Complex according to any one of items 136-139, wherein said antibody or antigen binding fragment thereof is a monoclonal antibody or an antigen binding fragment thereof.
[0658] 141. Complex according to any one of items 136-140, wherein said antibody or antigen binding fragment thereof is selected from the group consisting of human antibodies, humanized antibodies and chimeric antibodies, and antigen-binding fragments thereof.
[0659] 142. Complex according to item 141, wherein said antibody or antigen binding fragment thereof is a human or humanized antibody, or an antigen binding fragment thereof.
[0660] 143. Complex according to any one of items 136-142, wherein said antibody or antigen binding fragment thereof has affinity for an antigen, such as selected from the group consisting of an antigen associated with an angiogenesis related disorder and an antigen associated with the immune response or with a disorder of the immune system.
[0661] 144. Complex according to item 143, wherein said antigen is associated with an angiogenesis related disorder.
[0662] 145. Complex according to item 143, wherein said antigen is associated with the immune response or with a disorder of the immune system, for example associated with inflammation.
[0663] 146. Complex according to any one of items 136-145, which is a fusion protein or a conjugate.
[0664] 147. Complex according to any one of items 136-146, wherein said IL-17A binding polypeptide is attached to the N-terminus or C-terminus of the heavy chain of said antibody or antigen binding fragment thereof.
[0665] 148. Complex according to any one of items 136-146, wherein said IL-17A binding polypeptide is attached to the N-terminus or C-terminus of the light chain of said antibody or antigen binding fragment thereof.
[0666] 149. Complex according to any one of items 136-146, wherein said IL-17A binding polypeptide is attached to the N-terminus and/or C-terminus of the light chain and heavy chain of said antibody or antigen binding fragment thereof.
[0667] 150. Complex according to any one of items 146-149, which is a fusion protein.
[0668] 151. IL-17A binding polypeptide, fusion protein, conjugate or complex according to any preceding item, further comprising at least one linker, for example selected from the group consisting of non-peptidic linkers, flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers.
[0669] 152. Fusion protein or conjugate according item 151, wherein said linker is arranged between said first moiety and said second moiety.
[0670] 153. Complex according to item 151, wherein said linker is arranged between said IL-17A binding polypeptide and said antibody or antigen binding fragment thereof.
[0671] 154. IL-17A binding polypeptide, fusion protein, conjugate or complex according any one of items 151-153, wherein said linker is a flexible linker comprising amino acid residues selected from the group consisting of glycine, serine and threonine.
[0672] 155. IL-17A binding polypeptide, fusion protein, conjugate or complex according to item 154, wherein said linker comprises a sequence with a general formula selected from
(G.sub.nS.sub.m).sub.p and (S.sub.nG.sub.m).sub.p,
wherein, independently,
[0673] n=1-7,
[0674] m=0-7,
[0675] n+m≦8 and
[0676] p=1-10.
[0677] 156. IL-17A binding polypeptide, fusion protein, conjugate or complex according to item 155, wherein n=1-5.
[0678] 157. IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 155-156, wherein m=0-5.
[0679] 158. IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 155-157, wherein p=1-5.
[0680] 159. IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 156-158, wherein n=4, m=1 and p=1-4.
[0681] 160. IL-17A binding polypeptide, fusion protein, conjugate or complex according to item 159, wherein said linker comprises a sequence selected from the group consisting of G.sub.4S, (G.sub.4S).sub.2, (G.sub.4S).sub.3 and (G.sub.4S).sub.4.
[0682] 161. IL-17A binding polypeptide, fusion protein, conjugate or complex according to item 160, wherein said linker comprises the sequence G.sub.4S.
[0683] 162. IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 155-159, wherein said general formula is GT(G.sub.nS.sub.m).sub.p.
[0684] 163. IL-17A binding polypeptide, fusion protein, conjugate or complex according to item 162, wherein said linker comprises the sequence GTG.sub.4S.
[0685] 164. IL-17A binding polypeptide, fusion protein, conjugate or complex according to any preceding item, further comprising a label.
[0686] 165. IL-17A binding polypeptide, fusion protein, conjugate or complex according to item 164, wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles.
[0687] 166. IL-17A binding polypeptide, fusion protein, conjugate or complex according to any preceding item, comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the IL-17A binding polypeptide via a thiol group of a cysteine residue or an amine group of a lysine residue.
[0688] 167. IL-17A binding polypeptide, fusion protein, conjugate or complex according to any preceding item, comprising one or more polyethylene glycol moieties.
[0689] 168. Polynucleotide encoding a polypeptide according to any one of items 1-163.
[0690] 169. Expression vector comprising a polynucleotide according to item 168.
[0691] 170. Host cell comprising an expression vector according to item 169.
[0692] 171. Method of producing a polypeptide according to any one of items 1-163, comprising [0693] culturing a host cell according to item 170 under conditions permissive of expression of said polypeptide from said expression vector, and [0694] isolating said polypeptide.
[0695] 172. Composition comprising an IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-167 and at least one pharmaceutically acceptable excipient or carrier.
[0696] 173. Composition according to item 172, further comprising at least one additional active agent, such as an agent selected from the group consisting of therapeutically active polypeptides, immune response modifying agents, anti-inflammatory agents and toxic compounds.
[0697] 174. IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-167 or a composition according to any one of items 172-173 for oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration, such as for oral administration or such as for topical administration.
[0698] 175. IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-167 or a composition according to any one of items 172-173 for use as a medicament, a diagnostic agent or a prognostic agent.
[0699] 176. IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 175, wherein said polypeptide, fusion protein, conjugate, complex or composition modulates IL-17A function in vivo.
[0700] 177. IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use according to any one of items 175-176, wherein said polypeptide, fusion protein, conjugate, complex or composition inhibits IL-17A signaling.
[0701] 178. IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use according to any one of items 175-177, wherein said polypeptide, fusion protein, conjugate, complex or composition blocks binding of IL-17A to at least one of its cognate receptors.
[0702] 179. IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use according to any one of items 175-178, in the treatment, diagnosis or prognosis of an IL-17A associated condition.
[0703] 180. IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 179, wherein said IL-17A associated condition is selected from the group consisting of inflammatory diseases, autoimmune diseases and cancer.
[0704] 181. IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 180, wherein said IL-17A associated condition is selected from the group consisting of inflammatory diseases and autoimmune diseases.
[0705] 182. IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 181, wherein said IL-17A associated condition is selected from the group consisting of inflammatory conditions, allergic conditions, hypersensitivity reactions, autoimmune diseases, severe infections and transplant rejections.
[0706] 183. IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 182, wherein said IL-17A associated condition is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, uveitis and dry eye disease.
[0707] 184. IL-17A binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 183, wherein said IL-17A associated condition is psoriasis.
[0708] 185. IL-17A binding polypeptide, fusion protein, conjugate or complex for use in prognosis according to item 179, wherein said IL-17A associated condition is cancer, such as a cancer selected from the group consisting of gastric cancers, colorectal cancers, non-small cell lung cancers, hepatocellular carcinomas and adenocarcinomas.
[0709] 186. Method of detecting IL-17A, comprising providing a sample suspected to contain IL-17A, contacting said sample with an IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-167 or a composition according to any one of items 172-173, and detecting the binding of the IL-17A binding polypeptide, fusion protein, conjugate, complex or composition to indicate the presence of IL-17A in the sample.
[0710] 187. Method for determining the presence of IL-17A in a subject, the method comprising the steps: [0711] contacting the subject, or a sample isolated from the subject, with an IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-167 or a composition according to any one of items 172-173, and [0712] obtaining a value corresponding to the amount of the IL-17A binding polypeptide, fusion protein, conjugate, complex or composition that has bound in said subject or to said sample.
[0713] 188. Method according to item 187, further comprising a step of comparing said value to a reference.
[0714] 189. Method according to item 187 or 188, wherein said subject is a mammalian subject, such as a human subject.
[0715] 190. Method according to any one of items 187-189, performed in vivo.
[0716] 191. Method according to any one of items 187-189, performed in vitro.
[0717] 192. Method of treatment of an IL-17A associated condition, comprising administering to a subject in need thereof an effective amount of an IL-17A binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-167 or a composition according to any one of items 172-173.
[0718] 193. Method according to item 192, wherein said IL-17A associated condition is selected from the group consisting of inflammatory diseases, autoimmune diseases and cancer.
[0719] 194. Method according to item 193, wherein said IL-17A associated condition is selected from the group consisting of inflammatory diseases and autoimmune diseases.
[0720] 195. Method according to item 194, wherein said IL-17A associated condition is selected from the group consisting of inflammatory conditions, allergic conditions, hypersensitivity reactions, autoimmune diseases, severe infections and transplant rejections.
[0721] 196. Method according to item 195, wherein said IL-17A associated condition is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, uveitis and dry eye disease.
[0722] 197. Method according to item 196, wherein said IL-17A associated condition is psoriasis.