Polypeptide

Abstract

The present disclosure relates to a class of engineered polypeptides having a binding affinity for interleukin-6, and provides an IL-6 binding polypeptide comprising the sequence EEX.sub.3X.sub.4AWX.sub.7EIH X.sub.11 LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29. The present disclosure also relates to the use of such an IL-6 binding polypeptide as a therapeutic, prognostic and/or diagnostic agent.

Claims

1. An interleukin-6 (IL-6) binding polypeptide, comprising an IL-6 binding motif (BM), which motif consists of the amino acid sequence selected from: TABLE-US-00028 (SEQIDNO:1562) i)EEX.sub.3X.sub.4AWX.sub.7EIHX.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29 wherein, independently from each other, X.sub.3 is selected from A, F, H, K, Q, R, S, W and Y; X.sub.4 is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V and Y; X.sub.7 is selected from F, H, I, K, L, M, N, R, S, T, V, W and Y; X.sub.11 is selected from A, I, K, L, M, N, R, S, T and V; X.sub.16 is selected from N and T; X.sub.17 is selected from A, I, T and V; X.sub.18 is selected from D, E, G, H, K, N, Q, R, S and T; X.sub.20 is selected from I, L, M, R, T and V; X.sub.21 is selected from A, S, T and V; X.sub.25 is selected from I, M, Q, S, T, V and W; X.sub.26 is selected from K and S; X.sub.28 is selected from F, L, M and Y; and X.sub.29 is selected from D and R; and ii) an amino acid sequence which has at least 93% identity to the full-length sequence defined in i).

2. The IL-6 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:1-1551.

3. The IL-6 binding polypeptide according to claim 1, wherein said IL-6 binding motif forms part of a three-helix bundle protein domain.

4. The IL-6 binding polypeptide according to claim 1, which comprises a binding module BMod, the amino acid sequence of which is selected from: TABLE-US-00029 (SEQIDNO:1563) iii)K-[BM]-DPSQSX.sub.aX.sub.bLLX.sub.cEAKKLX.sub.dX.sub.eX.sub.fQ; wherein [BM] is an IL-6 binding motif as defined in claim 1 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 95% identity to the full length sequence defined by iii).

5. The IL-6 binding polypeptide according to claim 1, which comprises the amino acid sequence selected from: TABLE-US-00030 (SEQIDNO:1586) xi)VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an IL-6 binding motif as defined in claim 1; and xii) an amino acid sequence which has at least 96% identity to the full length sequence defined in xi).

6. The IL-6 binding polypeptide according to claim 5, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-1551.

7. The IL-6 binding polypeptide according to claim 1, which is capable of blocking the IL-6 dependent signaling via the cis-signaling pathway and/or the trans-signaling pathway.

8. The IL-6 binding polypeptide according to claim 7, wherein the half maximal inhibitory concentration of the blocking is at most 110.sup.6 M.

9. The IL-6 binding polypeptide according to claim 7, which is capable of blocking the interaction of IL-6/IL-6R with gp130.

10. The IL-6 binding polypeptide according to claim 1, which is capable of binding to IL-6 such that the EC50 value of the interaction is at most 110.sup.7 M or such that the K.sub.D value of the interaction is at most 110.sup.8 M.

11. A composition comprising the IL-6 binding polypeptide according to claim 1 and at least one pharmaceutically acceptable excipient or carrier.

12. The IL-6 binding polypeptide according to claim 1, wherein said IL-6 binding polypeptide modulates IL-6 function in vivo.

13. The IL-6 binding polypeptide according to claim 2, wherein sequence i) is the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-14 and SEQ ID NO:1512.

14. The IL-6 binding polypeptide according to claim 13, wherein sequence i) is the sequence from position 8 to position 36 in SEQ ID NO:1512.

15. A fusion protein or conjugate comprising a first moiety consisting of the IL-6 binding polypeptide according to claim 1; and a second moiety consisting of a polypeptide having a desired biological activity.

16. A polynucleotide encoding the IL-6 binding polypeptide according to claim 1.

17. A method of detecting IL-6, comprising contacting a sample suspected to contain IL-6 with the IL-6 binding polypeptide according to claim 1, and detecting the binding of the IL-6 binding polypeptide to IL-6 to indicate the presence of IL-6 in the sample.

18. A method of treatment of an IL-6 related disorder, comprising administering the IL-6 binding polypeptide according to claim 1 to a subject in need of treatment of an IL-6 related disorder that receives benefit from at least partial blocking of IL-6 signaling, wherein the IL-6 related disorder is rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, systemic juvenile idiopathic arthritis, vasculitis, psoriatic arthritis, psoriasis, a chronic inflammatory bowel disease, Crohn's disease, ulcerative colitis, Grave's disease, Behet's disease, uveitis, giant cell arteritis, multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, polymyositis, polymyalgia rheumatic, relapsing polychondritis, pancreatitis, peritonitis, nephritis, Sjgren's syndrome, adult Still's disease, or Castleman's disease.

19. The method of claim 18, wherein the blocking is of the cis- or the trans-signaling pathway.

20. The method of claim 18, wherein the IL-6 binding polypeptide blocks the interaction of IL-6/IL-6R with gp130.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A-VV is a listing of amino acid sequences of examples of IL-6 binding polypeptides of the present disclosure (SEQ ID NO:1-1551), control polypeptides (SEQ ID NO:1552-1553), the albumin binding domain (ABD) variant PP013 (SEQ ID NO:1554) as well as the amino acid sequences of human IL-6 (SEQ ID NO:1555) and murine IL-6 (SEQ ID NO:1556) used for selection, screening and/or characterization for illustration of the invention. In the IL-6 binding polypeptides of the present disclosure, the deduced IL-6 binding motifs (BM) extend from position 8 to position 36 in each sequence. The amino acid sequences of the 49 amino acid residues long polypeptides predicted to constitute the complete three-helix bundle within each of these Z variants extend from position 7 to position 55 (referred herein to as BMod).

(2) FIG. 2 shows the result of blocking of the interaction between hIL-6 and hIL-6R, assayed as described in Example 2. In contrast to the hIL-6R binding antibody tocilizumab (black), which was included for comparison, the tested IL-6 binding Z variants (gray) did not interfere with the binding of hIL-6 to its receptor hIL-6R.

(3) FIG. 3 shows the result of blocking of the binding of the hIL-6/hIL-6R complex to hgp130, assayed as described in Example 2. Concentration dependent blocking was seen for all tested primary IL-6 binding Z variants (gray) as well as for tocilizumab (black), which was included for comparison. The calculated IC50 value of each variant is shown in Table 3.

(4) FIG. 4 shows concentration dependent inhibition of IL-6 mediated trans-signaling in a gp130 expressing human umbilical vein endothelial cell (HUVEC) based system, assayed as described in Example 2. IL-6 binding Z variants C-terminally fused to the ABD variant PP013 (SEQ ID NO:1554) inhibited the trans-signaling, whereas the control Z variant Z03638 (SEQ ID NO:1552) in fusion with PP013 did not. Tocilizumab was included for comparison.

(5) FIG. 5 shows the result of blocking of the binding of the hIL-6/hIL-6R complex to hgp130, assayed as described in Example 7. Concentration dependent blocking was seen for all tested maturated IL-6 binding Z variants (gray) as well as for tocilizumab (black) and the primary binder Z06814 (SEQ ID NO:1512, broken line), which were both included for comparison. All maturated IL-6 binding Z variants showed more efficient blocking than the primary binders assayed in Example 2 (compare FIG. 3).

(6) FIG. 6 shows the result of the TF-1 cell neutralizing assay described in Example 7. Concentration dependent inhibition of IL-6 induced TF-1 cell proliferation was seen for all tested IL-6 binding Z variants (matured Z variants shown in black; primary binder Z06814 (SEQ ID NO:1512) shown as grey filled triangles) and for tocilizumab (grey filled squares), but not for the negative control antibody hIgG (grey filled circles).

(7) FIG. 7 shows the in vivo efficacy of the Z variant Z06814 (SEQ ID NO:1512) in fusion with the ABD variant PP013 (SEQ ID NO:1554) as scored by assaying IL-6 triggered serum amyloid-A (SAA) protein release in an anti-arthritic mouse model as described in Example 8. Four groups of mice were given 0 (filled squares), 0.025 (open dots), 2.5 (open triangles) or 25 (closed triangles) mg/kg body weight of the IL-6 binding Z06814-ABD fusion protein. As a control, mice were given 25 mg/kg of a control Z variant (Z04726; SEQ ID NO:1553) in fusion with ABD (referred to as Z.sub.control-ABD) (open squares). Mice were injected with hIL-6 and subsequently the levels of SAA protein were measured. Two additional control groups of mice received PBS (filled circles) and 25 mg/kg Z06814-ABD (crosses), respectively, but no subsequent IL-6 injection.

EXAMPLES

(8) Summary

(9) The following Examples disclose the development of novel Z variant molecules targeting interleukin 6 (IL-6). The Z variants were obtained using phage display technology. The genes encoding IL-6 binding polypeptides described herein were sequenced, and the corresponding amino acid sequences are listed in FIG. 1A-VV and denoted by the identifiers SEQ ID NO:1-1551.

Example 1

Selection and ELISA Screening of IL-6 Binding Z Variants

(10) In this Example, human (hIL-6) and murine IL-6 (mIL-6) were used as target proteins in phage display selections using a phage library of Z variants. The DNA of selected clones was sequenced and the clones were produced in E. coli periplasmic fractions and assayed against IL-6 in ELISA (enzyme-linked immunosorbent assay).

(11) Materials and Methods

(12) Biotinylation of the Target Proteins Human and Murine IL-6:

(13) hIL-6 and mIL-6 (Peprotech, cat. no. 200-06 and 216-16, respectively) were biotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, cat. no. 21327) at a 12 molar excess according to the manufacturer's recommendations. The reactions were performed at room temperature (RT) for 30 min. Next, buffer exchange to phosphate buffered saline (PBS, 10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) was performed using Slide-a-lyzer dialysis cassettes (Thermo Scientific, cat. no. 66333, 3,500 MWCO) according to the manufacturer's instructions.

(14) Phage Display Selection of IL-6 Binding Z Variants:

(15) A library of random variants of protein Z displayed on bacteriophage, constructed in phagemid pAY02592 essentially as described in Grnwall et al. (2007) J Biotechnol, 128:162-183, was used to select IL-6 binding Z variants. In this library, an albumin binding domain (abbreviated ABD and corresponding to GA3 of protein G from Streptococcus strain G148) was used as fusion partner to the Z variants. The library is denoted Zlib006Naive.II and has a size of 1.510.sup.10 library members (Z variants). E. coli RRIM15 cells (Ruther 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 [7 g/l dipotassium hydrogenphosphate, 1 g/l trisodium citrate dihydrate, 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/I 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 g/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 TSB-YE (Tryptic Soy Broth-Yeast Extract; 30 g/l TSB, 5 g/l yeast extract) supplemented with 100 M isopropyl--D-1-thiogalactopyranoside (IPTG) for induction of expression and with 25 g/ml kanamycin and 12.5 g/ml carbenicillin. Cells were grown at 30 C. for 22 h and the cells in the cultivation were pelleted by centrifugation at 15,900 g. 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 Grnwall et al., supra. Phage stocks were stored at 80 C. before use.

(16) Selection procedure and phage stock preparation were performed essentially as described for selection against another biotinylated target in WO2009/077175. In order to reduce the amount of background binders, pre-selection was performed by incubation of phage stock with SA-beads for 30 min at RT. All tubes and beads used in the selection were pre-blocked with PBS supplemented with 5% BSA. Selection was performed in PBS supplemented with 3% BSA and 0.1% Tween20 during 2 h at RT, followed by capture of target-phage complexes on Dynabeads M-280 Streptavidin (SA-beads, Invitrogen, cat. no. 11206D) using 1 mg beads per 1.6 g biotinylated hIL-6 or mIL-6. E. coli strain XL1-Blue (Agilent technologies, cat. no. 200268) was used for phage amplification.

(17) Selections against biotinylated hIL-6 and mIL-6 were performed in four cycles divided in four different final tracks: track (1) in cycle 1 was divided either in the second cycle or the fourth cycle, resulting in totally three tracks (1-1 to 1-3) in cycle 2, three tracks (1-1-1 to 1-3-1) in cycle 3 and four tracks (1-1-1-1 to 1-3-1-2) in cycle 4. After washes, bound phage were eluted from the selection tracks using 500 l 0.1 M glycine-HCl, pH 2.2, followed by immediate neutralization with 50 l 1 M Tris-HCl, pH 8.0, and 450 l PBS. An overview of the selection strategy and parameters used, describing the differences in the selection tracks in terms of lowered target concentration and increased number of washes, is shown in Table 2.

(18) TABLE-US-00008 TABLE 2 Overview of the strategy for primary selection Phage stock Target from library concen- Number Selection or selection tration of Cycle track track Target (nM) washes 1 1 Zlib006Naive.II hIL-6 100 2 2 1-1 1 hIL-6 50 5 2 1-2 1 hIL-6 10 5 2 1-3 1 mIL-6 100 4 3 1-1-1 1-1 hIL-6 25 6 3 1-2-1 1-2 hIL-6 2 8 3 1-3-1 1-3 hIL-6 25 6 4 1-1-1-1 1-1-1 hIL-6 10 8 4 1-2-1-1 1-2-1 hIL-6 0.5 12 4 1-3-1-1 1-3-1 mIL-6 10 8 4 1-3-1-2 1-3-1 hIL-6 0.5 12

(19) Sequencing:

(20) PCR fragments were amplified from single colonies using a standard PCR program and the primers AFFI-21 (5-tgcttccggctcgtatgttgtgtg; SEQ ID NO:1557) and AFFI-22 (5-cggaaccagagccaccaccgg; SEQ ID NO:1558). Sequencing of amplified fragments was performed using the biotinylated oligonucleotide AFFI-72 (5-biotin-cggaaccagagccaccaccgg; SEQ ID NO:1559) and a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), used in accordance with the manufacturer's recommendations. The sequencing reactions were purified by binding to magnetic streptavidin coated beads (Detach Streptavidin Beads, Nordiag, cat. no. 2012-01) using a Magnatrix 8000 (Magnetic Biosolution) instrument and analyzed on an ABI PRISM 3130xl Genetic Analyzer (PE Applied Biosystems).

(21) Production of Z Variants for ELISA:

(22) Sequenced Z variants were produced by inoculating single colonies from the selections in 1 ml TSB-YE medium supplemented with 100 g/ml ampicillin and 0.1 mM IPTG in deep-well plates (Nunc, cat. no. 278752). The plates were incubated for 24 h at 37 C. Cells were pelleted by centrifugation, re-suspended in 200 l PBST 0.05% (PBS supplemented with 0.05% Tween-20), frozen at 80 C. and thawed in a water bath to release the periplasmic fraction of the cells. The freeze-thawing procedure was repeated five times. The samples were diluted with PBST 0.05% to a total of 800 l and cells were pelleted by centrifugation. The supernatant of the periplasmic extract contained the Z variants as fusions to ABD, expressed as AQHDEALE-[Z #####]-VDYV4ABDFYVPG (Grnwall et al., supra). Z ##### refers to individual, 58 amino acid residue Z variants.

(23) ELISA Analysis of Z Variants:

(24) The binding of Z variants to IL-6 was analyzed in an ELISA assay. Half-area 96-well ELISA plates (Costar, cat. no. 3690) were coated at 4 C. overnight with 2 g/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 blocked with 100 l of PBSC (PBS supplemented with 0.5% casein; Sigma, cat. no. C8654) for 1.5 h at RT. The blocking solution was discarded and 50 l periplasmic solution was added to the wells and incubated for 1.5 h at RT under slow shaking. The supernatants were poured off and the wells were washed 4 times with PBST 0.05%. Next, 50 l of biotinylated hIL-6 at a concentration of 7.7 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) was diluted 1:30 000 in PBSC and added to the wells followed by 45 min incubation. After washing as described above, 50 l ImmunoPure TMB substrate (Thermo Scientific, cat. no. 34021) was added to the wells and the plates were treated according to the manufacturer's recommendations. A Z variant binding a specific irrelevant protein was used as a positive control by assaying against that specific irrelevant protein, and as a negative control by assaying against hIL-6. As blank control, PBST 0.05% was added instead of the periplasmic sample. Absorbance was measured at 450 nm using a multi-well plate reader (Victor.sup.3, Perkin Elmer).

(25) Results

(26) Phage Display Selection of IL-6 Binding Z Variants:

(27) Individual clones were prepared after four cycles of phage display selections against biotinylated hIL-6 and mIL-6.

(28) Sequencing:

(29) Sequencing was performed for clones picked at random from selection round four. Each Z variant was given a unique identification number #####, and individual variants are referred to as Z #####. The amino acid sequences of 58 residues long Z variants are listed in FIG. 1A-VV and in the sequence listing as SEQ ID NO:1503-1551. The deduced IL-6 binding motifs (BM) extend from position 8 to position 36 in each sequence. The amino acid sequences of the 49 amino acid residues long polypeptides predicted to constitute the complete three-helix bundle within each of these Z variants (BMod) extend from position 7 to position 55.

(30) ELISA Analysis of Z Variants:

(31) The clones obtained after four cycles of selection were produced in 96-well plates and screened for hIL-6 binding activity in ELISA. All clones giving a response with signals corresponding to at least 3 the negative control were considered as positive IL-6 binders. The control molecule specific for an irrelevant protein gave a positive signal for the specific protein, whereas no signal was obtained against hIL-6.

Example 2

Production and In Vitro Characterization of IL-6 Binding Z Variants

(32) In this Example, a subset of Z variants were subcloned, produced and functionally assessed in competition ELISAs and cell assays. Two different

(33) ELISA assays were applied to investigate if the IL-6 binding Z variants were able to block the specific interaction between IL-6 and IL-6R or between IL-6 and the gp130 receptor, respectively. The potency of the Z variant polypeptides was assessed using two different cell assays, mimicking the classical cis-signaling pathway and trans-signaling pathway, respectively. Finally, circular dichroism (CD) spectroscopy was performed for a subset of the Z variants in order to investigate their secondary structure and determine their melting temperatures, Tm.

(34) Materials and Methods

(35) Subcloning of Z Variants:

(36) The DNA of 13 IL-6 binding Z variants, Z06777 (SEQ ID NO:1503), Z06779 (SEQ ID NO:1504), Z06789 (SEQ ID NO:1505), Z06791 (SEQ ID NO:1506), Z06792 (SEQ ID NO:1507), Z06799 (SEQ ID NO:1508), Z06802 (SEQ ID NO:1509), Z06805 (SEQ ID NO:1510), Z06809 (SEQ ID NO:1511), Z06814 (SEQ ID NO:1512), Z06829 (SEQ ID NO:1513), Z06834 (SEQ ID NO:1514), Z06844 (SEQ ID NO:1515) was amplified from the library vector pAY02592. A subcloning strategy for construction of monomeric Z variant molecules with an 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.

(37) A subset of five IL-6 binding Z variants, Z06789, Z06799, Z06809, Z06814 and Z06829, and two control Z variants binding an irrelevant target, Z03638 (SEQ ID NO:1552) and Z04726 (SEQ ID NO:1553), were subcloned in fusion with the ABD variant PP013 (SEQ ID NO:1554). The constructs encoded by the expression vectors were MGSSLQ-[Z #####]-VDGS-PP013.

(38) Cultivation and Purification:

(39) E. coli BL21(DE3) cells (Novagen) were transformed with plasmids containing the gene fragment of each respective IL-6 binding Z variant and cultivated at 37 C. in 800 or 1000 ml of TSB-YE medium supplemented with 50 g/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.

(40) Purification of IL-6 Binding Z Variants with a His.sub.6-Tag:

(41) Protein purification was performed under either native or denatured conditions.

(42) The purification under native conditions was performed as follows:

(43) Approximately 2-5 g of each cell pellet was resuspended in 10 ml PBS. After cell disruption by sonication, cell debris was removed by centrifugation and each supernatant was applied on 2 ml Talon cobolt columns (Clontech, cat. no. 635504) equilibrated with 20 ml wash buffer (46.6 mM Na.sub.2HPO.sub.4, 3.4 mM NaH.sub.2PO.sub.4, and 300 mM NaCl, pH 7.0). Contaminants were removed by washing with wash buffer, and the IL-6 binding Z variants were subsequently eluted with elution buffer (50 mM NaH.sub.2PO.sub.4, 100 mM NaCl, 30 mM HAc and 70 mM NaAc, pH 5.0).

(44) The purification under denatured conditions was performed as follows: Approximately 2-5 g of each cell pellet was resuspended in 10 ml lysis buffer (7 M guanidinium hydrochloride, 47 mM Na.sub.2HPO.sub.4, 2.65 mM NaH.sub.2PO.sub.4, 10 mM Tris-HCl, 104 mM NaCl, pH 8.0) followed by incubation at 37 C., 150 rpm for 2 h. The washing and elution steps were performed as for the native purification but using different buffers (wash buffer: 6 M guanidinium hydrochloride, 47 mM Na.sub.2HPO.sub.4, 3.4 mM NaH.sub.2PO.sub.4, 300 mM NaCl, pH 8.0; elution buffer: 6 M urea, 0.1 M NaCl, 29.6 mM HAc, 70.4 mM NaAc and 50 mM NaH.sub.2PO.sub.4, pH 5.0). Purified Z variants were buffer exchanged to PBS using PD-10 columns (GE Healthcare) according to the manufacturer's protocol.

(45) Protein concentrations were determined by measuring the absorbance at 280 nm using the extinction coefficient of the respective protein. The purity of the IL-6 binding Z variants was analyzed by SDS-PAGE stained with Coomassie Blue.

(46) Purification of IL-6 Binding Z Variants in Fusion with ABD:

(47) Approximately 2.5 g of each cell pellet was re-suspended in 20 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 anti-ABD agarose (WO2014/064237). 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. Buffer exchange 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) was performed using PD-10 columns (GE Healthcare). Next, the ABD fused Z variants were purified on 1 ml Detoxi-Gel Endotoxin Removing Columns (Pierce, cat. no. 20344) to ensure low endotoxin content. Protein concentrations were determined by measuring the absorbance at 280 nm, using 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-ABD variant was confirmed using LC/MS analysis.

(48) Analysis of Binding Site:

(49) A first assay was employed to evaluate the interference of the IL-6 binding Z variants with the interaction between hIL-6 and the human IL-6R (hIL-6R). In this experiment, half area 96-well ELISA plates were coated with anti-IL-6R capture antibody (R&D Systems) at a concentration of 2 g/ml. Plates were incubated overnight at 4 C. and then washed twice in tap water. Next, the plates were blocked for 1 h in PBSC, and hIL-6R (R&D Systems) was added at a concentration of 250 ng/ml. Plates were incubated for 1.5 h at RT and then washed 4 times with 200 l 0.05% Tween/PBS. In separate plates, serial dilutions (concentration range 500-0.5 nM) of the 13 His.sub.6-tagged Z variant polypeptides were titrated with 2.5 nM of biotinylated hIL-6. The IL-6R antibody tocilizumab (Roche) was prepared in the same way and included for comparison. Each pre-mixed complex of Z variant and biotinylated hIL-6 was then transferred to wells containing hIL-6R. Plates were incubated for another 1.5 h and then washed four times. A 1:8000 dilution of streptavidin-HRP (Thermo Scientific) was added and the plates were incubated for 1 h. Plates were washed a final four times with 0.05% Tween/PBS and TMB substrate (Thermo Scientific) was added for 15 min before the reaction was stopped with 2 M H.sub.2SO.sub.4. The absorbance was measured at 450 nm using a microplate reader (Victor.sup.3, Perkin Elmer).

(50) A second assay was employed to evaluate the interference of the IL-6 binding Z variants on the interaction between human gp130 (hgp130) and the hIL-6/hIL-6R complex. In this experiment, half area 96-well ELISA plates were coated with Fc-fused hgp130 (hgp130-Fc) at a concentration of 4 g/ml. Plates were incubated overnight at 4 C. and then washed twice in tap water. Next, the plates were blocked for 1 h in PBSC. Plates were incubated for 1.5 h at RT and then washed 4 times with 200 l 0.05% Tween/PBS. In separate plates, serial dilutions (concentration range 500-0.5 nM) of the 13 His.sub.6-tagged Z variant polypeptides were titrated with fixed concentration of hIL-6/hIL-6R (0.5 nM and 5 nM, respectively).

(51) The IL-6R binding antibody tocilizumab (Roche) was prepared in the same way and included for comparison. The pre-mixed complex of each Z variant polypeptide and hIL-6/hIL-6R was then transferred to wells containing hgp130. Plates were incubated for 1.5 h and then washed 4 times. A biotinylated anti-IL-6R antibody (R&D Systems) was added and the plates were incubated for another 1.5 h followed by washing. A 1:8000 dilution of streptavidin-HRP (Thermo Scientific) was added and the plates were incubated for 1 h. Next, plates were washed four times and TMB substrate (Thermo Scientific) was added for 15 min before the reaction was stopped with 2 M H.sub.2SO.sub.4. The absorbance was measured at 450 nm using a microplate reader (Victor.sup.3, Perkin Elmer).

(52) In Vitro Neutralization Assays:

(53) A first assay, evaluating the classical signaling pathway, used the TF-1 cell line that proliferates in response to human IL-6, TNF and GM-CSF. TF-1 cells were cultured in RPMI1640 with L-glut (Lonza) supplemented with 10% FCS (Gibco), Pen-Strep (Lonza) and 2 ng/ml rhGM-CSF (R&D Systems). Prior to use, cells were washed twice in RPMI1640 in the absence of rhGM-CSF. Cells were then counted and dispensed into 96 well flat bottomed plates at a density of 410.sup.4 cells per well. In separate plates, serial dilutions of the inhibitory compounds (IL-6 binding Z variants, with a His.sub.6-tag (concentration range 1000-0.1 nM) or in fusion with the ABD variant PP013 (SEQ ID NO:1554, concentration range 200-0.007 nM)), and the IL-6R binding antibody tocilizumab (Roche; concentration range 200-0.007 nM) were incubated in the presence of 0.099 nM rhIL-6 (R&D Systems, UK). In addition, the ABD-fused variants were incubated with or without 9 M rhHSA (Novozymes). The pre-mixed complexes of the Z variant polypeptides and hIL-6 were then transferred to wells containing TF-1 cells which were incubated for 72 h at 37 C. in a humidified 5% CO.sub.2 atmosphere. During the last four hours of incubation 10 l of CCK-8 (Fluke, Sigma Aldrich) was added per well to determine the number of proliferating cells. The absorbance was measured at 450 nm (Abs450) using a microplate reader (Victor.sup.3, Perkin Elmer). The data on cell growth was assessed by non-linear regression to a four-parameter dose-response curve, and the half maximal inhibitory concentration (IC50) was determined using GraphPadPrism program. The inhibition of IL-6-dependent proliferation of TF-1 cells by the inhibitory molecules was as Abs450 minus control wells that contained cells but no hIL-6.

(54) To address the trans-signaling pathway, a second assay was used. Herein, human umbilical vein endothelial cells (HUVECs) were stimulated with hIL-6 and soluble hIL-6R and the readout was the production of monocyte chemoattractant protein-1 (MCP-1). HUVECs (Lonza) were grown in EGM-2 bullet kit media (Lonza) and passaged in culture no more than eight times. Cells were grown until 75% confluence before use. Cells were detached using trypsin/EDTA (Lonza), resuspended and washed once in fresh medium. Next, cells were counted and dispensed into 96 well flat bottom plates at a density of 210.sup.4 cells per well. Cells were cultured overnight at 37 C. in a humidified 5% CO.sub.2 atmosphere. In separate plates, serial dilutions (100-0.0015 nM) of the IL-6 binding Z variants Z06789, Z06799, Z06809, Z06814 and Z06829 in fusion with the ABD variant PP013 (SEQ ID NO:1554), PP013-fused negative control Z03638 (SEQ ID NO:1552) binding a different target, and serial dilutions (200-0.003 nM) of tocilizumab (Roche) were incubated in the presence of recombinant hIL-6 (10 ng/ml; 0.5 nM) and soluble hIL-6R at a fixed concentration of 200 ng/ml (5.6 nM) with or without 9 M rhHSA (Novozymes). The pre-mixed solutions with the test molecules and hIL-6/hIL-6R were then transferred to wells containing HUVECs, which were incubated for 24 h at 37 C. in a humidified 5% CO.sub.2 atmosphere. Cell free supernatant was collected after the incubation period and human MCP-1 levels were determined by sandwich ELISA using the MCP-1 Duoset ELISA development system (R&D Systems).

(55) MCP-1 ELISA:

(56) Half area 96-well ELISA plates were coated with anti-MCP-1 capture antibody (R&D Systems) at a concentration of 1 g/ml. Plates were incubated overnight at 4 C., washed twice in tap water and blocked for 1 h in PBSC. Plates were then washed four times with 4200 l 0.05% Tween/PBS before standards and samples were added. Plates were incubated for 2 h at RT and washed before addition of 0.1 g/ml biotinylated anti-MCP-1 antibody (R&D Systems). Plates were then incubated for another 1.5 h, then washed four times. Next, a 1:8000 dilution of streptavidin-HRP (Thermo Scientific) was added and the plates were incubated for 1 h. The plates were washed a final four times and TMB substrate (Thermo Scientific) was added for 20 min before the reaction was stopped with 2 M H.sub.2SO.sub.4. The absorbance was measured at 450 nm using a microplate reader (Victor.sup.3, Perkin Elmer).

(57) CD Analysis:

(58) A subset of the purified His.sub.6-tagged Z variants was 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. 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.

(59) Results

(60) Cultivation and Purification:

(61) The 13 IL-6 binding Z variants (SEQ ID NO:1503-1515), constructed with an N-terminal His.sub.6 tag, were produced in E. coli. The amount of IMAC-purified protein from approximately 2-5 g bacterial pellets, determined spectrophotometrically by measuring the absorbance at 280 nm, ranged from approximately 10 mg to 20 mg for the different IL-6 binding Z variants. 2 mg to 12 mg were obtained from approximately 2.5 g bacterial pellet of the five Z variants fused to the ABD variant PP013 (SEQ ID NO:1554). SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the IL-6 binding Z variant. The correct identity and molecular weight of each IL-6 binding Z variant was confirmed by HPLC-MS analysis.

(62) Analysis of Binding Site:

(63) The ability of the 13 tested IL-6 binding His.sub.6-tagged Z variants to block the interactions between either (i) hIL-6 and hIL-6R or (ii) hgp130 and preformed hIL-6/hIL-6R complex was investigated in two separate competitive ELISA experiments. None of the 13 Z variants showed any significant effect when tested for blocking of the hIL-6/hIL-6R interaction (FIG. 2). However, all Z variants showed a clear concentration-dependent blocking of the interaction between pre-formed hIL-6/hIL-6R and hgp130, i.e. the trans-signaling-resembling interaction (FIG. 3). The calculated 1050 value for each Z variant is shown in Table 3. The antibody tocilizumab, included for comparison, showed a blocking effect in both experiments.

(64) TABLE-US-00009 TABLE 3 IC50 values for primary Z variants blocking the hIL-6/hIL-6R interaction with hgp130 SEQ ID NO of Z variant Z variant: IC50 (M) His.sub.6-Z06777 1503 4.6 10.sup.8 His.sub.6-Z06779 1504 3.6 10.sup.8 His.sub.6-Z06789 1505 6.2 10.sup.9 His.sub.6-Z06791 1506 2.3 10.sup.8 His.sub.6-Z06792 1507 1.7 10.sup.8 His.sub.6-Z06799 1508 3.7 10.sup.9 His.sub.6-Z06802 1509 2.7 10.sup.8 His.sub.6-Z06805 1510 4.2 10.sup.8 His.sub.6-Z06809 1511 3.3 10.sup.9 His.sub.6-Z06814 1512 1.6 10.sup.9 His.sub.6-Z06829 1513 1.1 10.sup.8 His.sub.6-Z06834 1514 5.3 10.sup.8 His.sub.6-Z06844 1515 8.7 10.sup.8

(65) In Vitro Neutralization Assays:

(66) Two different cell assays were used for investigating the ability of the IL-6 binding Z variants to block IL-6 dependent signaling in the classical signaling pathway and the trans-signaling pathway, respectively. The first assay, evaluating the classical signaling pathway, employed the TF-1 cell line that proliferates in response to human IL-6, TNF and GM-CSF. The direct signaling of IL-6 to cell surface IL-6 receptor, in conjunction with a signaling receptor sub-unit called gp130, is termed cis-signaling. This assay showed that all 13 variants were capable of blocking IL-6 dependent growth of the TF-1 cells. The calculated 1050 values for His.sub.6-tagged Z variants and Z variants recombinantly fused to the ABD variant PP013 (SEQ ID NO:1554), as well as for the hIL-6R binding antibody tocilizumab included for comparison, are shown in Table 4.

(67) TABLE-US-00010 TABLE 4 IC50 values for primary Z variants blocking the IL-6 dependent growth of TF-1 cells SEQ ID NO of Z variant Z variant: IC50 (M) His.sub.6-Z06777 1503 7.2 10.sup.8 His.sub.6-Z06779 1504 3.0 10.sup.8 His.sub.6-Z06789 1505 8.6 10.sup.9 His.sub.6-Z06791 1506 5.2 10.sup.9 His.sub.6-Z06792 1507 5.3 10.sup.8 His.sub.6-Z06799 1508 3.9 10.sup.9 His.sub.6-Z06802 1509 4.3 10.sup.8 His.sub.6-Z06805 1510 7.7 10.sup.8 His.sub.6-Z06809 1511 2.0 10.sup.8 His.sub.6-Z06814 1512 1.6 10.sup.9 His.sub.6-Z06829 1513 2.8 10.sup.8 His.sub.6-Z06834 1514 6.1 10.sup.8 His.sub.6-Z06844 1515 1.1 10.sup.7 Z06789-ABD 1505 ~1 10.sup.7 Z06799-ABD 1508 2.4 10.sup.9 Z06809-ABD 1511 1.0 10.sup.8 Z06814-ABD 1512 .sup.8.0 10.sup.10 Z06829-ABD 1513 1.5 10.sup.8 tocilizumab .sup.3.0 10.sup.10

(68) To investigate if also the trans-signaling pathway could be blocked in a cell based system, a second assay using gp130 expressing human umbilical vein endothelial cells (HUVECs) was performed. Incubation of HUVECs with preformed hIL-6/hIL-6R complexes results in IL-6 trans-signaling dependent secretion of Monocyte Chemoattractant Protein-1 (MCP-1), allowing for analysis of any trans-signaling blocking capabilities of IL-6 binding Z variants. In this assay, five Z variants recombinantly fused to the ABD variant PP013 (SEQ ID NO:1554) were analyzed in the presence of HSA. The hIL-6R binding antibody tocilizumab was included for comparison. All five investigated Z variants were shown to inhibit trans-signaling (FIG. 4). One variant, Z06814-ABD, was shown to be more potent than tocilizumab and exhibited an approximate IC50 value of 1 nM compared to 5 nM for tocilizumab.

(69) CD Analysis:

(70) The CD spectra determined for seven Z variants showed that each had an -helical structure at 20 C. The melting temperatures (Tm) determined through variable temperature measurements are shown in Table 5.

(71) TABLE-US-00011 TABLE 5 Melting temperatures for a selection of Z variants Z variant SEQ ID NO: Tm ( C.) His.sub.6-Z06779 1504 51 His.sub.6-Z06789 1505 52 His.sub.6-Z06792 1507 48 His.sub.6-Z06799 1508 44 His.sub.6-Z06809 1511 51 His.sub.6-Z06814 1512 49 His.sub.6-Z06829 1513 40

Example 3

Design and Construction of a First Maturated Library of IL-6 Binding Z Variants

(72) In this Example, a maturated library was constructed. The library was used for selections of new IL-6 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 synthesis, enabling incorporation of defined codons in desired positions in the synthesis.

(73) Materials and Methods

(74) Library Design:

(75) The library was based on a selection of sequences of the IL-6 binding Z variants described in Example 1 and 2. In the new library, 12 variable positions in the Z molecule scaffold were biased towards certain amino acid residues and one position was kept constant, according to a strategy based on the Z variant sequences defined in SEQ ID NO:1503-1551. Using split-pool synthesis, a DNA linker of 147 bp was generated, encoding a partially randomized helix 1 and 2 of the Z variant amino acid sequence. Thus, 5-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN GCG TGG NNN GAG ATC NNN NNN CTG CCT AAC CTC ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3 (SEQ ID NO:1560, randomized codons denoted NNN) flanked by restriction sites XhoI and SacI, was ordered from DNA 2.0 (Menlo Park, Calif., USA). The theoretical distributions of amino acid residues in the new library including 12 variable Z positions (9, 10, 11, 14, 17, 18, 24, 25, 27, 28, 32 and 35) in the Z molecule scaffold are given in Table 6. The resulting theoretical library size is 3.610.sup.9 variants.

(76) TABLE-US-00012 TABLE 6 Library design, first maturation Amino acid Randomization No of position (amino acid amino in Z abbreviations) acids Proportion 9 E (70%), H, Q, T, 4 1/10, 7/10 (E) 10 A, H, Q, R, 4 1/4 11 A, D, E, H, K, Q, R, S, T, V 10 1/10 13 W 1 1/1 14 A, F, H, L, R, S, T, W, Y 9 1/9 17 H (70%), Q, S, T 4 1/10, 7/10 (H) 18 A, H, I, K, L, M, S, T, V 9 1/9 24 A, H, I, T, V 5 1/5 25 D, E, H, N, Q, R, S, T 8 1/8 27 A, H, I, L, M, R, T, V 8 1/8 28 A, E, H, S, T, V 6 1/6 32 A, H, I, M, Q, S, T, V, W 9 1/9 35 F, L, M, Y 4 1/4

(77) Library Construction:

(78) 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) and concentrated using a PCR Purification Kit (Qiagen, cat. no. 28106). Subsequently, the product was run on a preparative 2.5% agarose gel (Nuisieve GTC agarose, Cambrex, Invitrogen) and purified using a QIAGEN gel extraction Kit (QIAGEN, cat. no. 28706) according to the supplier's recommendations.

(79) The phagemid vector pAY02592 (essentially as pAffi1 described in Grnwall et al, supra) was restricted with the same enzymes and purified using phenol/chloroform extraction and ethanol precipitation. The restricted fragments and 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.

(80) The ligation reactions (approximately 160 ng DNA/transformation) were electroporated into electrocompetent E. coli ER2738 cells (50 l, Lucigen, Middleton, Wis., USA). Immediately after electroporation, approximately 1 ml of recovery medium (supplied with the 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. Next, the cells were pooled and cultivated overnight at 37 C. in 11 of TSB-YE medium, supplemented with 2% glucose, 10 g/ml tetracycline and 100 g/ml ampicillin. The cells were pelleted for 7 min at 4,000 g and resuspended in a PBS/glycerol solution (approximately 40% glycerol), 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--vis the library design. Sequencing was performed as described in Example 1 and the amino acid distribution was verified.

(81) Preparation of Phage Stock:

(82) 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 (Tryptic Soy Broth-Yeast Extract; 30 g/l TSB, 5 g/l yeast extract) 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 0.64, approximately 1.1 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.7.2H.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 isopropyl-8-D-1-thiogalactopyranoside (IPTG); 17.5 ml/l of 300 g/l MgSO.sub.4 and 5 ml of a trace element solution [35 g/l FeCl.sub.3.6H.sub.2O; 10.56 g/l ZnSO.sub.4.7H.sub.2O; 2.64 g/l CuSO.sub.4.5H.sub.2O; 13.2 g/l MnSO.sub.4.H.sub.2O; 13.84 g/l CaCl.sub.2.2H.sub.2O, 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 in the start, 37.5 g/h after 20 h and until the end of the cultivation). The pH was controlled at pH 7 through the automatic addition of 25% NH.sub.4OH. 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 22. The cells in the cultivation were pelleted by centrifugation at 15,900 g. The phage particles were precipitated twice from the supernatant 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.

(83) Results

(84) Library Construction:

(85) The new library was designed based on a set of IL-6 binding Z variants with verified binding properties (Example 1 and 2). The theoretical size of the designed library was 3.610.sup.9 Z variants. The actual size of the library, determined by titration after transformation to E. coli ER2738 cells, was 3.510.sup.9 transformants.

(86) The library quality was tested by sequencing of 192 transformants and by comparing their actual sequences with the theoretical design. The contents of the actual library compared to the designed library were shown to be satisfactory. A maturated library of potential binders to IL-6 was thus successfully constructed.

Example 4

Selection, Screening and Characterization of Z Variants from the First Maturated Library

(87) Materials and Methods

(88) Phage Display Selection of Matured IL-6 Binding Z Variants:

(89) The target proteins hIL-6 (R&D Systems, cat. no. 206-IL/CF) and mIL-6 (Abnova, cat. no. P4346 II6) were biotinylated as described in Example 1. Phage display selections, using the new library of Z variant molecules described in Example 3, were performed in four cycles against hIL-6 and mIL-6 essentially as described in Example 1 with the following exceptions. 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 M, respectively. All tubes and beads used in the selection were pre-blocked with PBST 0.1% supplemented with 3% BSA. In cycle 1A, a pre-selection step was performed by incubation of phage stock with SA-beads. The selection volume was 2 ml in cycle 1 for all tracks. For capture of phage-target complexes, 1 mg beads per 4 g biotinylated hIL-6 or mIL-6 was used.

(90) The six tracks (1-6) in cycle 1 were divided either in the second cycle or the third cycle, resulting in totally seven tracks (1-1 to 6-2) in cycle 2, twelve tracks (1-1-1 to 6-2-1) in cycle 3 and twelve tracks (1-1-1-1 to 6-2-1-1) in cycle 4.

(91) The bound phage particles were eluted using two different procedures; 1) 500 l 0.1 M glycine-HCl, pH 2.2, followed by immediate neutralization with 50 l 1 M Tris-HCl, pH 8.0, and 450 l PBS, or 2) 500 l of 100 mM sodium phosphate and 150 mM sodium chloride, pH 5.5 and neutralization with 500 l PBS.

(92) 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 7.

(93) TABLE-US-00013 TABLE 7 Overview of the selection strategy for the first maturation Phage stock from library or Target Number Selection selection concentration of Elution Cycle track track Target (nM) washes at 1 1 Zlib006IL- hIL-6 50 2 pH 2.2 6.I 1 2 Zlib006IL- hIL-6 25 2 pH 2.2 6.I 1 3 Zlib006IL- hIL-6 10 3 pH 2.2 6.I 1 4 Zlib006IL- hIL-6 50 2 pH 5.45 6.I 1 5 Zlib006IL- hIL-6 25 2 pH 5.45 6.I 1 6 Zlib006IL- mIL-6 100 2 pH 2.2 6.I 2 1-1 1 hIL-6 25 8 pH 2.2 2 2-1 2 hIL-6 10 8 pH 2.2 2 3-1 3 hIL-6 2.5 12 pH 2.2 2 4-1 4 hIL-6 25 8 pH 5.45 2 5-1 5 hIL-6 10 8 pH 5.45 2 6-1 6 mIL-6 100 4 pH 2.2 2 6-2 6 mIL-6 50 6 pH 2.2 3 1-1-1 1-1 hIL-6 5 12 pH 2.2 3 1-1-2 1-1 hIL-6 1.25 15 pH 2.2 3 2-1-1 2-1 hIL-6 1.25 12 pH 2.2 3 2-1-2 2-1 hIL-6 0.5 15 pH 2.2 3 3-1-1 3-1 hIL-6 0.5 20 pH 2.2 3 3-1-2 3-1 hIL-6 0.05 20 pH 2.2 3 4-1-1 4-1 hIL-6 5 12 pH 5.45 3 4-1-2 4-1 hIL-6 1.25 15 pH 5.45 3 5-1-1 5-1 hIL-6 2.5 12 pH 5.45 3 5-1-2 5-1 hIL-6 1 15 pH 5.45 3 6-1-1 6-1 mIL-6 50 11 pH 2.2 3 6-2-1 6-2 mIL-6 25 11 pH 2.2 4 1-1-1-1 1-1-1 hIL-6 0.5 16 pH 2.2 4 1-1-2-1 1-1-2 hIL-6 0.05 20 pH 2.2 4 2-1-1-1 2-1-1 hIL-6 0.1 16 pH 2.2 4 2-1-2-1 2-1-2 hIL-6 0.025 20 pH 2.2 4 3-1-1-1 3-1-1 hIL-6 0.025 30 pH 2.2 4 3-1-2-1 3-1-2 hIL-6 0.0025 30 pH 2.2 4 4-1-1-1 4-1-1 hIL-6 1 16 pH 5.45 4 4-1-2-1 4-1-2 hIL-6 0.1 20 pH 5.45 4 5-1-1-1 5-1-1 hIL-6 0.2 16 pH 5.45 4 5-1-2-1 5-1-2 hIL-6 0.05 20 pH 5.45 4 6-1-1-1 6-1-1 mIL-6 10 12 pH 2.2 4 6-2-1-1 6-2-1 mIL-6 1 16 pH 2.2

(94) Amplification of Phage Particles:

(95) Amplification of phage particles between selection cycle 1 and 2 was performed essentially as described in Example 1, with the following exceptions. E. coli ER2738 was used for phage amplification and M13K07 helper phage was used in 5 excess. The amplification of phage particles between the selection cycles 2 and 4 was done by infection of bacteria in solution according to the following. After infection of log phase E. coli ER2738 with phage particles, TSB supplemented with 2% glucose, 10 g/ml tetracycline and 100 g/ml ampicillin was added, followed by incubation with rotation for 30 min at 37 C. Thereafter, the bacteria were infected with M13K07 helper phage. The infected bacteria were pelleted by centrifugation, re-suspended in TSB-YE medium supplemented with 100 M IPTG, 25 g/ml kanamycin and 100 g/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. Finally, the phage particles were re-suspended in selection buffer before entering the next selection cycle. In the last 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. CMO233B) supplemented with 0.2 g/l ampicillin in order to form single colonies for use in ELISA screening.

(96) Sequencing of Potential Binders:

(97) Individual clones from the different selection tracks were picked for sequencing. All clones run in the ELISA screening were sequenced. Amplification of gene fragments and sequence analysis of gene fragments were performed essentially as described in Example 1.

(98) ELISA Screening of Z Variants:

(99) Single colonies containing Z variants (expressed as Z variant ABD fusion proteins) were randomly picked from the selected clones of the IL-6 maturated library and cultivated as described in Example 1. Preparation of the periplasmic supernatants was performed as in Example 1 but with six freeze thawing cycles. ELISA screenings were performed essentially as described in Example 1 using biotinylated hIL-6 at a concentration of 0.58 nM. The periplasmic fraction of the primary IL-6 binder Z06814 was used as a positive control. A negative control was created by using periplasm containing ABD only.

(100) ELISA EC50 Analysis of Human IL-6 Binders:

(101) A selection of IL-6 binders was subjected to an analysis of the response against a dilution series of biotinylated hIL-6 using ELISA as described above. Biotinylated protein was added at a concentration of 25 nM and diluted stepwise 1:3 down to 11 pM. All Z variants were also assayed without added target protein as a background control. Periplasm samples containing the primary IL-6 binder Z06814 (SEQ ID. NO:1512) were included and analyzed as a positive control. As a negative control, periplasm containing ABD only was assayed against biotinylated hIL-6. Two binders originating from the selection against mIL-6, Z11612 (SEQ ID NO:151) and Z11616 (SEQ ID NO:152) were subjected to an analysis of the response against a dilution series of biotinylated mIL-6 using ELISA as described above. Biotinylated protein was added at a concentration of 227 nM and diluted stepwise 1:3 down to 104 pM. Obtained values were analyzed using GraphPad Prism 5 and non-linear regression.

(102) Results

(103) Phage Display Selection of Maturated IL-6 Binding Z Variants:

(104) Selection was performed in totally 12 parallel tracks containing four cycles each. The different selection tracks differed in target concentration, target type (hIL-6 or mIL-6), selection time, wash conditions and the pH of the elution buffer.

(105) Sequencing:

(106) Randomly picked clones were sequenced. Each individual Z variant was given an identification number, Z #####, as described in Example 1. In total, 809 new unique Z variant molecules were identified.

(107) The amino acid sequences of 58 residues long Z variants are listed in FIG. 1A-VV and in the sequence listing as SEQ ID NO:7, SEQ ID NO:15-89 and SEQ ID NO:151-871. The deduced IL-6 binding motifs (BM) extend from position 8 to position 36 in each sequence. The amino acid sequences of the 49 amino acid residues long polypeptides predicted to constitute the complete three-helix bundle within each of these Z variants (BMod) extend from position 7 to position 55.

(108) ELISA Screening of Z Variants:

(109) Clones obtained after four selection cycles were produced in 96-well plates and screened for hIL-6 binding activity using ELISA. All randomly picked clones were analyzed. 796 of the 809 unique Z variants were found to give a response of 3 the negative control or higher (0.3-2.1 AU) against hIL-6 at a concentration of 0.58 nM. Clones from all selection tracks using hIL-6 as selection target showed positive signals. The negative controls had absorbencies of 0.078-0.102 AU. The average response of the blank controls of a representative set of plates was 0.087 AU.

(110) ELISA EC50 Analysis of IL-6 Binders:

(111) A subset of Z variants was selected based on the result in the ELISA experiment described above (highest ELISA value normalized against the positive control on each plate, respectively) and subjected to a target titration in ELISA format. Periplasm samples were incubated with a serial dilution of biotinylated hIL-6 or mIL-6. A periplasm sample with the primary binder Z06814 (SEQ ID NO:1512) was also assayed against hIL-6 as a positive control. Obtained values were analyzed and their respective EC50 values were calculated (Tables 8 and 9).

(112) TABLE-US-00014 TABLE 8 Calculated EC50 values against hIL-6 Z variant SEQ ID NO EC50 ELISA (M) Z11213 15 1.5 10.sup.10 Z11214 16 1.3 10.sup.10 Z11215 17 1.4 10.sup.10 Z11217 18 1.3 10.sup.10 Z11222 19 1.4 10.sup.10 Z11251 20 1.4 10.sup.10 Z11277 21 1.2 10.sup.10 Z11278 22 1.5 10.sup.10 Z11283 23 1.8 10.sup.10 Z11300 24 1.9 10.sup.10 Z11321 25 1.4 10.sup.10 Z11329 26 1.4 10.sup.10 Z11351 27 1.7 10.sup.10 Z11380 28 1.7 10.sup.10 Z11384 29 1.5 10.sup.10 Z11433 30 2.1 10.sup.10 Z11472 31 1.8 10.sup.10 Z11552 32 1.2 10.sup.9 Z11632 7 1.9 10.sup.10 Z11642 33 2.3 10.sup.10 Z11644 34 2.4 10.sup.10 Z11674 35 2.6 10.sup.10 Z11698 36 2.0 10.sup.10 Z11711 37 3.5 10.sup.10 Z11723 38 2.5 10.sup.10 Z11781 39 2.9 10.sup.10 Z11784 40 2.7 10.sup.10 Z11788 41 2.5 10.sup.10 Z11789 42 2.4 10.sup.10 Z11791 43 3.4 10.sup.10 Z11794 44 2.2 10.sup.10 Z11802 45 2.6 10.sup.10 Z11803 46 2.8 10.sup.10 Z11805 47 3.4 10.sup.10 Z11814 48 2.5 10.sup.10 Z11815 49 2.8 10.sup.10 Z11817 50 2.5 10.sup.10 Z11818 51 2.4 10.sup.10 Z11819 52 2.5 10.sup.10 Z11823 53 2.1 10.sup.10 Z11824 54 2.3 10.sup.10 Z11833 55 2.9 10.sup.10 Z11835 56 2.9 10.sup.10 Z11836 57 3.6 10.sup.10 Z11860 58 1.8 10.sup.10 Z11861 59 2.2 10.sup.10 Z11862 60 1.9 10.sup.10 Z11865 61 2.3 10.sup.10 Z11866 62 2.5 10.sup.10 Z11871 63 1.7 10.sup.10 Z11872 64 2.8 10.sup.10 Z11874 65 3.1 10.sup.10 Z11875 66 2.1 10.sup.10 Z11881 67 1.7 10.sup.10 Z11882 68 1.4 10.sup.10 Z11883 69 2.1 10.sup.10 Z11890 70 1.9 10.sup.10 Z11892 71 2.3 10.sup.10 Z11893 72 2.0 10.sup.10 Z11895 73 2.8 10.sup.10 Z11896 74 2.9 10.sup.10 Z11897 75 3.0 10.sup.10 Z11901 76 1.7 10.sup.10 Z11903 77 2.3 10.sup.10 Z11904 78 2.4 10.sup.10 Z11905 79 3.1 10.sup.10 Z11906 80 2.5 10.sup.10 Z11907 81 3.6 10.sup.10 Z11912 82 1.8 10.sup.10 Z11918 83 3.2 10.sup.10 Z11922 84 2.7 10.sup.10 Z11923 85 2.1 10.sup.10 Z11929 86 2.6 10.sup.10 Z11933 87 1.6 10.sup.10 Z11937 88 3.0 10.sup.10 Z11939 89 2.1 10.sup.10 Z06814 1512 2.9 10.sup.10

(113) TABLE-US-00015 TABLE 9 Calculated EC50 values against mIL-6 Z variant SEQ ID NO EC50 ELISA (M) Z11612 151 9.3 10.sup.9 Z11616 152 7.7 10.sup.9

Example 5

Design and Construction of a Second Maturated Library of IL-6 Binding Z Variants

(114) In this Example, a second maturated library was constructed essentially as described in Example 4. The library was used for selections of IL-6 binding Z variants.

(115) Materials and Methods

(116) Library Design:

(117) The library was primarily based on a selection of sequences of the human IL-6 binding Z variants described in Example 4. In the new library, 13 variable positions in the Z molecule scaffold were biased towards certain amino acid residues, according to a strategy mainly based on the Z variants from the first maturation, i.e. sequences defined in SEQ ID NO:7, SEQ ID NO:15-89 and SEQ ID NO:151-871. Randomized double stranded linkers were generated by the Colibra technology, which enables incorporation of randomized sets of trinucleotide building blocks using ligations and restrictions of the subsequently built up double stranded DNA. A library of double-stranded DNA, 5-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN GCT NNN NNN GAG ATC NNN NNN CTG CCG AAC CTG ACC NNN NNN CAG NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3 (SEQ ID NO:1561, randomized codons are denoted NNN) encoding a partially randomized helix 1 and 2 of the Z variant amino acid sequence, flanked by restriction sites XhoI and SacI, was ordered from Isogenica (Essex, UK). The theoretical distributions of amino acid residues in the new library, including eight variable amino acid positions (10, 11, 14, 18, 24, 25, 27 and 32) and five constant amino acid positions (9, 13, 17, 28, and 35) in the Z molecule scaffold are given in Table 10. The resulting theoretical library size was 2.610.sup.7 variants.

(118) Library Construction and Phage Stock Preparation:

(119) The library was constructed essentially as described in Example 3. Phage stock of the library was prepared as described in Example 3.

(120) TABLE-US-00016 TABLE 10 Library design, second maturation Amino acid No of position amino in Z Allowed amino acids acids Proportion 9 E 1 1/1 10 A, D, E, F, H, I, K, L, N, Q, R, 1 1/16 S, T, V, W, Y 11 A, D, E, F, G, H, I, K, L, M, N, 17 1/18 Q, R, S, T, V, W, Y 13 W 1 1/1 14 A, D, E, F, H, I, K, L, M, N, Q, 7 1/17 R, S, T, V, W, Y 17 H 1 1/1 18 A, D, E, F, G, H, I, K, L, N, Q, 17 1/17 R, S, T, V, W, Y 24 I, L, V 1 1/3 25 A, D, E, F, G, H, I, K, L, N, Q, 10 1/17 R, S, T, V, W, Y 27 I, M, V 1 1/3 28 A 12 1/1 32 S, T 16 1/2 35 F 1 1/1
Results

(121) Library Construction and Phage Stock Preparation:

(122) The new library was designed based on a set of IL-6 binding Z variants with verified binding properties (Example 4). The theoretical size of the designed library was 2.610.sup.7Z variants. The actual size of the library, determined by titration after transformation to E. coli ER2738 cells, was 1.810.sup.9 transformants.

(123) The library quality was tested by sequencing of 192 transformants and by comparing their actual sequences with the theoretical design. The contents of the actual library compared to the theoretical library were shown to be satisfying. A maturated library of potential binders to IL-6 was thus successfully constructed.

Example 6

Selection, Screening and Characterization of Z Variants from the Second Maturated Library

(124) Materials and Methods

(125) Second Phage Display Selection of Maturated IL-6 Binding Z Variants:

(126) The target protein hIL-6 was biotinylated as described in Example 4. Phage display selections, using the second maturated library of Z variant molecules described in Example 5 were performed against hIL-6 essentially as described in Example 4, with the following exceptions. The selection volume was 4 ml in cycle 1 for all tracks. In cycle 2, selection tracks 1-2 and 1-3 were handled in one common tube and not separated until after the last 1 min wash, whereupon they were treated separately. Also in cycle 4, each set of the selection tracks 1-1-1-1 to 1-1-1-3, 1-1-1-4 to 1-1-1-6, 1-1-2-1 to 1-1-2-3 and 1-1-2-4 to 1-1-2-6, respectively, were handled in a common tube and split into three separate tubes after the last 1 min wash and thereafter treated separately using the different washing strategies outlined in Table 11. The bound phage particles were eluted using glycine-HCl, pH 2.2, as described in Example 1. The amplification of phage particles between the selection cycles was performed essentially as described in Example 1.

(127) An overview of the selection strategy and parameters used, describing the differences in the selection tracks in terms of lowered target concentration and an increased number of washes, is shown in Table 11.

(128) Sequencing of Potential Binders:

(129) Individual clones from the different selection tracks were picked for sequencing. All clones subjected to the ELISA screening were sequenced. Amplification of gene fragments and sequence analysis of gene fragments were performed essentially as described in Example 1.

(130) ELISA Screening of Z Variants:

(131) 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-6 second maturated library and cultivated as described in Example 1. Preparation of the periplasmic supernatants and ELISA screenings were performed essentially as described in Example 1 and freeze thawing was performed in 150 l PBST 0.05% and repeated 8 times. Biotinylated hIL-6 was used at a concentration of 0.25 nM. The periplasmic fraction of the IL-6 binder Z06814 (SEQ ID NO:1512) was used in duplicate as positive control on each ELISA plate. As a negative control, periplasm containing ABD only was assayed against biotinylated hIL-6.

(132) TABLE-US-00017 TABLE 11 Overview of the selection strategy for the second maturation Phage Number stock of 4 h from washes Number Number library or Target Number Number with of of over Selection selection concentration of 1 min of 4 h unlabeled overnight weekend Cycle track track (nM) washes washes Z06814 washes washes 1 1 Zlib006IL- 50 5 6A.II 1 2 Zlib006IL- 10 5 6A.II 2 1-1 1 10 15 2 1-2 1 5 15 2 1-3 1 5 15 1 2 2-1 2 2.5 12 3 1-1-1 1-1 1 5 1 3 1-1-2 1-1 1 5 1 3 1-2-1 1-2 0.5 15 3 1-3-1 1-3 0.5 15 3 2-1-1 2-1 0.5 12 3 2-1-2 2-1 0.1 12 4 1-1-1-1 1-1-1 1 5 1 4 1-1-1-2 1-1-1 1 5 1 4 1-1-1-3 1-1-1 1 5 1 4 1-1-1-4 1-1-1 1 5 1 4 1-1-1-5 1-1-1 1 5 1 4 1-1-1-6 1-1-1 1 5 1 4 1-1-2-1 1-1-2 1 5 1 4 1-1-2-2 1-1-2 1 5 1 4 1-1-2-3 1-1-2 1 5 1 4 1-1-2-4 1-1-2 1 5 1 4 1-1-2-5 1-1-2 1 5 1 4 1-1-2-6 1-1-2 1 5 1 4 1-2-1-1 1-2-1 0.5 15 4 1-2-1-2 1-2-1 0.5 15 1 4 1-3-1-1 1-3-1 0.5 15 4 2-1-1-1 2-1-1 0.5 12 4 2-1-2-1 2-1-2 0.1 12

(133) ELISA EC50 Analysis of IL-6 Binders:

(134) A selection of IL-6 binders was subjected to an analysis of the response against a dilution series of biontinylated hIL-6 using ELISA as described in Example 2. Biotinylated protein was added at a concentration of 5 nM and diluted stepwise 1:3 down to 83 fM. As a background control, all Z variants were also assayed without added target protein. Periplasm samples containing the primary IL-6 binder Z06814 (SEQ ID NO:1512) as well as maturated binder Z11632 (SEQ ID NO:7) were included as positive controls. As a negative control, periplasm containing ABD only was assayed against biotinylated hIL-6. Obtained values were analyzed using Graph Pad Prism 5 and non-linear regression.

(135) Results

(136) Second Phage Display Selection of Maturated IL-6 Binding Z Variants:

(137) Selection was performed in 17 parallel tracks in total, each track containing four cycles. The selection tracks differed in target concentration, selection time and wash conditions as outlined in Table 11.

(138) Sequencing of Potential Binders:

(139) Randomly picked clones were sequenced. Each individual Z variant was given an identification number, Z #####, as described in Example 1. In total, 707 new unique Z variant molecules were identified. The amino acid sequences of 58 residues long Z variants are listed in FIG. 1A-VV and in the sequence listing as SEQ ID NO:1-6, SEQ ID NO:8-14, SEQ ID NO:90-150 and SEQ ID NO:872-1502. The deduced IL-6 binding motifs (BM) extend from position 8 to position 36 in each sequence. The amino acid sequences of the 49 amino acid residues long polypeptides predicted to constitute the complete three-helix bundle within each of these Z variants (BMod) extend from position 7 to position 55.

(140) ELISA Screening of Z Variants:

(141) Clones obtained after four selection cycles were produced in 96-well plates and screened for human IL-6 binding activity using ELISA. All randomly picked clones were analyzed. 705 of the 707 unique Z variants were found to give a response of 3 the negative controls or higher (0.3-2.3 AU) against hIL-6 at a concentration of 0.25 nM. Positive signals were shown for clones originating from all selection tracks. The average response of the negative controls on the plates was 0.085 AU.

(142) ELISA EC50 Analysis of IL-6 Binders:

(143) A subset of Z variants was selected based on the result in the ELISA experiment described above. All Z variants exhibiting an absorbance of over 1.6 AU or a response over 1.7 after normalizing the response against the average response of the duplicate positive control Z06814 (SEQ ID NO:1512) on each plate were subjected to a target titration in ELISA format as described in Example 4. Periplasm samples with the maturated binder Z11632 (SEQ ID NO:7) as well as the primary binder Z06814 (SEQ ID NO:1512) were also assayed as positive controls. Obtained values were analyzed and their respective EC50 values were calculated (Table 12).

(144) TABLE-US-00018 TABLE 12 Calculated EC50 values from ELISA titration analysis of Z variants from the second maturation as well as positive controls Z06814 and Z11632 Z variant SEQ ID NO EC50 ELISA (M) Z14521 90 2.3 10.sup.10 Z14524 91 2.7 10.sup.10 Z14525 92 2.5 10.sup.10 Z14538 93 3.2 10.sup.10 Z14547 94 2.5 10.sup.10 Z14550 95 2.7 10.sup.10 Z14551 96 2.6 10.sup.10 Z14556 97 2.8 10.sup.10 Z14559 98 2.3 10.sup.10 Z14596 99 2.4 10.sup.10 Z14609 100 3.0 10.sup.10 Z14614 101 3.8 10.sup.10 Z14620 102 3.1 10.sup.10 Z14630 6 2.5 10.sup.10 Z14634 103 2.3 10.sup.10 Z14645 104 2.5 10.sup.10 Z14651 105 2.4 10.sup.10 Z14662 106 2.7 10.sup.10 Z14673 107 2.3 10.sup.10 Z14700 8 2.4 10.sup.10 Z14706 108 2.3 10.sup.10 Z14710 109 2.6 10.sup.10 Z14712 9 2.6 10.sup.10 Z14720 110 2.2 10.sup.10 Z14722 111 3.2 10.sup.10 Z14731 112 2.1 10.sup.10 Z14746 113 2.8 10.sup.10 Z14765 114 2.7 10.sup.10 Z14767 115 2.4 10.sup.10 Z14782 116 2.4 10.sup.10 Z14783 117 2.4 10.sup.10 Z14784 118 2.5 10.sup.10 Z14788 119 3.0 10.sup.10 Z14829 120 2.7 10.sup.10 Z14861 4 2.3 10.sup.10 Z14862 10 2.2 10.sup.10 Z14867 121 2.6 10.sup.10 Z14868 122 2.8 10.sup.10 Z14878 123 2.9 10.sup.10 Z14888 124 2.2 10.sup.10 Z14929 125 2.1 10.sup.10 Z14944 126 1.9 10.sup.10 Z14976 1 1.6 10.sup.10 Z14984 5 1.6 10.sup.10 Z14990 127 2.5 10.sup.10 Z14992 128 2.8 10.sup.10 Z15003 129 3.0 10.sup.10 Z15015 2 1.8 10.sup.10 Z15024 130 3.1 10.sup.10 Z15025 131 2.1 10.sup.10 Z15031 132 2.1 10.sup.10 Z15036 11 2.4 10.sup.10 Z15042 133 2.2 10.sup.10 Z15053 134 2.4 10.sup.10 Z15057 135 2.3 10.sup.10 Z15067 136 2.2 10.sup.10 Z15079 137 2.1 10.sup.10 Z15082 138 2.2 10.sup.10 Z15097 139 2.2 10.sup.10 Z15102 140 2.1 10.sup.10 Z15110 12 2.0 10.sup.10 Z15111 141 2.3 10.sup.10 Z15117 142 2.1 10.sup.10 Z15122 3 1.7 10.sup.10 Z15126 13 1.5 10.sup.10 Z15129 143 2.1 10.sup.10 Z15140 144 2.1 10.sup.10 Z15141 145 2.3 10.sup.10 Z15142 14 1.6 10.sup.10 Z15145 146 2.3 10.sup.10 Z15151 147 1.9 10.sup.10 Z15159 148 1.8 10.sup.10 Z15162 149 1.8 10.sup.10 Z15164 150 2.0 10.sup.10 Z06814 1512 3.2 10.sup.10 Z11632 7 2.5 10.sup.10

Example 7

Subcloning, Production and Characterization of a Subset of IL-6 Binding Z Variants

(145) In this Example, a subset of affinity-matured IL-6 binding Z variants were produced and functionally assessed by SPR, ELISA, cell-based assays and CD. SPR was used for measuring the kinetic parameters of Z variants interacting with IL-6. Competition ELISA was applied to investigate the binding mode of Z variants to human IL-6 protein. A TF-1 cell-based assay was applied to assess the ability of Z variants to block IL-6 dependent signaling. CD was used to investigate the secondary structure of the Z variants and determine their melting temperatures.

(146) Materials and Methods

(147) Subcloning of Z Variants into Expression Vectors:

(148) The DNA of 14 IL-6 binding Z variants, Z11632 (SEQ ID NO:7), Z14630 (SEQ ID NO:6), Z14700 (SEQ ID NO:8), Z14712 (SEQ ID NO:9), Z14861 (SEQ ID NO:4), Z14862 (SEQ ID NO:10), Z14976 (SEQ ID NO:1), Z14984 (SEQ ID NO:5), Z15015 (SEQ ID NO:2), Z15036 (SEQ ID NO:11), Z15110 (SEQ ID NO:12), Z15122 (SEQ ID NO:3), Z15126 (SEQ ID NO:13) and Z15142 (SEQ ID NO:14), was amplified from the library vector pAY02592. The subcloning was performed as described in Example 2. The Z gene fragments were subcloned into the expression vector pAY01448 resulting in the encoded sequence MGSSHHHHHHLQ-[Z #####]-VD (SEQ ID NO: 1595).

(149) Protein Expression and Purification Under Denatured Conditions:

(150) E. coli Rosetta cells (Novagen) were transformed with plasmids containing the gene fragment of each respective IL-6 binding Z variant and cultivated at 37 C. in 100 ml of TSB-YE medium supplemented with 50 g/ml kanamycin. Expression was induced at OD.sub.600=0.8 by addition of IPTG at a final concentration of 1 mM and the cultures were incubated at 25 C. for another 16-20 h. The cells were harvested by centrifugation.

(151) Protein purification was performed under denatured conditions essentially as described in Example 2. Protein concentrations were determined by measuring the absorbance at 280 nm, using the extinction coefficient of the respective protein. The purity of the IL-6 binding Z variants was analyzed by SDS-PAGE stained with Coomassie Blue.

(152) Protein Expression and Purification Under Native Conditions:

(153) E. coli BL21 (DE3) cells (NEB, cat. no. C25271) were transformed with plasmids containing gene fragments of matured variants Z11632 (SEQ ID NO:7), Z14630 (SEQ ID NO:6), Z14700 (SEQ ID NO:8), Z14712 (SEQ ID NO:9), Z14861 (SEQ ID NO:4), Z14862 (SEQ ID NO:10), Z14976 (SEQ ID NO:1), Z14984 (SEQ ID NO:5), Z15015 (SEQ ID NO:2), Z15036 (SEQ ID NO:11), Z15110 (SEQ ID NO:12), Z15122 (SEQ ID NO:3), Z15142 (SEQ ID NO:14), of the primary Z variant Z06814 (SEQ ID NO:1512), as well as of the control Z variant Z04726 (SEQ ID NO:1553). Transformed bacterial cells were cultivated at 37 C. in 1000 ml of LB medium supplemented with 50 g/ml kanamycin. In order to induce protein expression, IPTG was added to a final concentration of 0.1 mM at OD.sub.600=0.8 and the cultures were incubated at 25 C. for 17 h. The cells were harvested by centrifugation at 4 C. and 8000 rpm for 30 min. Supernatants were discarded and cell pellets re-suspended in 10 ml PBS. After cell disruption by sonication, cell debris was removed by centrifugation and each supernatant was applied on 2 ml Ni-NTA columns (QIAGEN, cat. no. 30410) equilibrated with 20 ml wash buffer (20 mM NaH.sub.2PO.sub.4, 10 mM NaCl, 20 mM imidazole, pH 6.0). Contaminants were removed by washing with wash buffer, and Z variants were eluted with elution buffer (20 mM NaH.sub.2PO.sub.4, 10 mM NaCl, 250 mM imidazole, pH 6.0). The eluents were subjected to purification on an ion exchange column (Life Technologies, cat. no. 4481317), and Z variants were eluted by an increasing salt concentration. Buffer solutions of eluents were then changed to PBS (10 mM Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4, 137 mM NaCl, 2.7 mM KCl) using a VIVASPIN 6 column (Sartorius, cat. no. VS0691). The purity of Z variants was analyzed by SDS-PAGE stained with Coomassie Blue.

(154) ProteOn Kinetic Analysis:

(155) Kinetic constants (k.sub.on and k.sub.off) and affinities (K.sub.D) for human IL-6 were determined for 6 His.sub.6-tagged Z variants purified under denatured conditions. The IL-6 binding variants Z06814 (SEQ ID NO:1512), Z14861 (SEQ ID NO:4), Z014976 (SEQ ID NO:1), Z14984 (SEQ ID NO:5), Z15015 (SEQ ID NO:2), and Z15122 (SEQ ID NO:3) were diluted to 5 g/ml in 10 mM NaAc buffer, pH 4.5, and immobilized separately on GLC chip (Bio-Rad, cat. no. 176-5011). The immobilization was performed using amine coupling chemistry according to the manufacturer's recommendations and PBST 0.05% was used as running buffer. PBST 0.05% was also used as running buffer in the kinetic experiment using a flow rate was 60 l/min. The analyte hIL-6 was diluted in the PBST 0.05% running buffer to final concentrations of 50 nM, 12.5 nM, 3.1 nM, 0.78 nM, 0.19 nM and 0 nM and injected in triplicate for 3 min, followed by dissociation in running buffer for 90 min. After dissociation, the surfaces were regenerated with HCl supplemented with 0.05% Tween 20. Kinetic constants were calculated from the sensorgrams using a 1:1 model in Bio-Rad manager Software (Bio-Rad).

(156) Analysis of Binding Site:

(157) The interference of 14 maturated IL-6 binding Z variants (purified under denatured conditions) with the interaction between human gp130 (hgp130) and the hIL-6/hIL-6R complex was assessed as described in Example 2. The primary binder Z06814 and the hIL-6R binding antibody tocilizumab were included for comparison.

(158) TF-1 Cell-Based Assay:

(159) TF-1 cells were cultured in RPMI1640 with L-glutamine (HyClone, cat. no. SH30027) supplemented with 10% FBS (HyClone, cat. no. SH30919.03), Pen-Strep (HyClone, cat. no. 15140-163) and 2 ng/ml rhGM-CSF (R&D Systems, cat. no. 215-GM-010). Prior to use, cells were washed twice in RPMI-1640 in absence of rhGM-CSF. Cells were then counted and dispensed into a 96-well plate (Corning, cat. no. 3596) at a density of 410.sup.4 cells per well. In separate plates, serial dilutions (concentration range 10-0.00061 nM) of Z variants (purified under native conditions), tocilizumab (Roche) and control IgG (Jackson Immunoresearch, cat. no. Jac-009-000-003) were incubated in the presence of 0.099 nM rhIL-6 (R&D Systems, cat. no. 206-IL/CF). These pre-mixtures were then transferred to wells containing TF-1 cells, which were incubated for 72 h at 37 C. in a humidified 5% CO.sub.2 atmosphere. During the last four hours of incubation, 10 l of WST (DoGen, cat. no. EZ3000) were included per well. The absorbance was measured at 450 nM using a Victor X3 plate reader (Perkin Elmer). Relative cell viability was calculated by dividing the absorbance of each well by the mean absorbance of IL-6-treated wells in each plate. The data was assessed by non-linear regression to a four-parameter dose-response curve, and the half-maximal inhibitory concentration (IC50) was determined using Graphpad Prism software.

(160) CD Analysis:

(161) CD analysis was performed as described in Example 2 using Z variants purified under native conditions.

(162) Results

(163) ProteOn Kinetic Analysis:

(164) The interactions of 6 His.sub.6-tagged IL-6-binding Z variants with human IL-6 were analyzed in a ProteOn instrument by injecting various concentrations of the hIL-6 over surfaces containing different immobilized Z variants. The ligand immobilization levels of the surfaces were between 100-220 RU each. 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 hIL-6 to the Z variants using a 1:1 interaction model is given in Table 13.

(165) TABLE-US-00019 TABLE 13 Kinetic parameters for binding of hIL-6 to Z variants SEQ k.sub.on k.sub.off K.sub.D Z variant ID NO: (M.sup.1s.sup.1) (s.sup.1) (M) His.sub.6-Z06814 1512 4.3 10.sup.5 8.8 10.sup.5 2.0 10.sup.10 His.sub.6-Z14861 4 3.6 10.sup.5 6.3 10.sup.5 1.7 10.sup.10 His.sub.6-Z14976 1 3.1 10.sup.5 2.7 10.sup.5 8.8 10.sup.11 His.sub.6-Z14984 5 3.0 10.sup.5 7.4 10.sup.5 2.5 10.sup.10 His.sub.6-Z15015 2 4.3 10.sup.5 7.2 10.sup.5 1.7 10.sup.10 His.sub.6-Z15122 3 3.1 10.sup.5 3.9 10.sup.5 1.2 10.sup.10

(166) Analysis of Binding Site:

(167) All maturated IL-6 binding Z variants showed a clear concentration-dependent blocking of the trans-signaling resembling interaction between preformed hIL-6/hIL-6R complexes and hgp130 (FIG. 5). Each maturated Z variant showed a higher blocking capacity than both the primary binder Z06814 and tocilizumab, i.e. which would correspond to IC50 values less than 1.6 nM.

(168) TF-1 Cell-Based Assay:

(169) A TF-1 cell-based assay was conducted to evaluate the efficacy and potency of IL-6 binding Z variants in the classical signaling pathway. This assay showed that all affinity-matured IL-6 binding Z variants were capable of blocking IL-6 dependent growth of the TF-1 cells (FIG. 6). The calculated IC50 values for Z variants and for the hIL-6R binding antibody tocilizumab are shown in Table 14.

(170) TABLE-US-00020 TABLE 14 IC50 values for matured Z variants blocking the IL-6 dependent growth of TF-1 cells Z variant SEQ ID NO: IC50 (M) His.sub.6-Z11632 7 1.2 10.sup.10 His.sub.6-Z14630 6 8.7 10.sup.11 His.sub.6-Z14700 8 1.0 10.sup.10 His.sub.6-Z14712 9 2.2 10.sup.10 His.sub.6-Z14861 4 1.9 10.sup.10 His.sub.6-Z14862 10 4.3 10.sup.10 His.sub.6-Z14976 1 4.2 10.sup.11 His.sub.6-Z14984 5 2.7 10.sup.10 His.sub.6-Z15015 2 2.7 10.sup.11 His.sub.6-Z15036 11 1.8 10.sup.10 His.sub.6-Z15110 12 9.3 10.sup.10 His.sub.6-Z15122 3 8.5 10.sup.11 His.sub.6-Z15142 14 5.1 10.sup.10 His.sub.6-Z06814 1512 1.3 10.sup.10 tocilizumab N/A 4.1 10.sup.10

(171) CD Analysis:

(172) The CD spectra determined for 10 matured Z variants showed that each one had an -helical structure at 20 C. The melting temperatures (Tm) determined by variable temperature measurement are shown in Table 15.

(173) TABLE-US-00021 TABLE 15 Melting temperatures for a subset of matured Z variants Z variant SEQ ID NO: Tm ( C.) His.sub.6-Z11632 7 51 His.sub.6-Z14630 6 57 His.sub.6-Z14700 8 53 His.sub.6-Z14712 9 55 His.sub.6-Z14861 4 48 His.sub.6-Z14862 10 53 His.sub.6-Z15015 2 56 His.sub.6-Z15036 11 59 His.sub.6-Z15110 12 50 His.sub.6-Z15142 14 53

Example 8

In Vivo Activity of IL-6 Binding Z Variants in Fusion with ABD

(174) A Serum Amyloid A (SAA) mouse model was used in order to explore the in vivo blocking effect of the IL-6 binding Z variants in fusion with ABD. The acute phase protein SAA is secreted from liver cells and can be induced by the proinflammatory cytokines IL-1, IL-6 and TNF. Due to the sequence homology of the human and mouse cytokines, the human variants are able to act on their corresponding mouse receptors and induce a murine SAA response. Note, that the human TNF protein is only able to interact with murine TNFRII (not murine TNFRI).

(175) Materials and Methods

(176) The IL-6 targeting Z variant Z06814 (SEQ ID NO:1512) and a control Z variant Z04726 (SEQ ID NO:1535) binding an irrelevant target, were cloned and produced in fusion with the ABD variant PP013 (SEQ ID NO:1554) as described in Example 2. Four groups of Balb/c mice (n=8) were injected subcutaneously (s.c.) with various doses (0, 0.025, 2.5 or 25 mg/kg body weight) of Z06814-ABD 9 h prior to intraperitoneal (i.p.) administration of hIL-6 at 5 g/kg (R&D Systems). A fifth group of mice (n=8) received 25 mg/kg of Z04726-ABD. Two additional control groups of mice received PBS (n=4) and 25 mg/kg of Z06814-ABD (n=8), respectively, but no subsequent IL-6 injection. After 20 h, the blood was taken by cardiac puncture and serum was collected.

(177) Serum was assessed for the content of murine SAA by ELISA (Tridelta) according to the manufacturer's instructions. In brief, diluted serum samples were added to SAA-precoated plates together with anti-SAA-HRP. The plates were incubated for 1 h and then washed four times. TMB substrate was added for 20 min and the reaction was stopped with stop solution. The absorbance was measured at 450 nm using a microplate reader (Victor.sup.3, Perkin Elmer).

(178) Results

(179) The anti-arthritic efficacy of the Z variant Z06814 (SEQ ID NO:1512) was assessed in vivo using a mouse model for IL-6 triggered serum amyloid-A (SAA) protein release. Four groups of mice were given 0, 0.025, 2.5 or 25 mg/kg body weight of the IL-6 binding Z06814-ABD fusion protein or 25 mg/kg of a control Z04726-ABD fusion protein 9 h before an injection of 5 g/kg body weight of hIL-6. After an additional 22 h, the levels of SAA protein were measured and compared between the different groups. In animals receiving either no Z06814-ABD or 25 mg/kg of the control Z04726-ABD fusion, SAA protein levels in serum increased to levels of approximately 500-600 g/ml. Control animals given PBS only (and no hIL-6) exhibited levels in the range of 16-64 g/ml. In animals given Z06814-ABD, significantly lower SAA protein levels were measured in a dose-dependent manner (FIG. 7). For the group given the highest dose of Z06814-ABD (25 mg/kg body weight), SAA protein levels were as low as for animals given no hIL-6 injection.

ITEMIZED LISTING OF EMBODIMENTS

(180) 1. IL-6 binding polypeptide, comprising an IL-6 binding motif BM, which motif consists of an amino acid sequence selected from:

(181) TABLE-US-00022 (SEQIDNO:1562) i)EEX.sub.3X.sub.4AWX.sub.7EIHX.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20X.sub.21AFIX.sub.25X.sub.26LX.sub.28X.sub.29
wherein, independently from each other,

(182) X.sub.3 is selected from A, F, H, K, Q, R, S, W and Y;

(183) X.sub.4 is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V and Y;

(184) X.sub.7 is selected from F, H, I, K, L, M, N, R, S, T, V, W and Y;

(185) X.sub.11 is selected from A, I, K, L, M, N, R, S, T and V;

(186) X.sub.16 is selected from N and T;

(187) X.sub.17 is selected from A, I, T and V;

(188) X.sub.18 is selected from D, E, G, H, K, N, Q, R, S and T;

(189) X.sub.20 is selected from I, L, M, R, T and V;

(190) X.sub.21 is selected from A, S, T and V;

(191) X.sub.25 is selected from I, M, Q, S, T, V and W;

(192) X.sub.26 is selected from K and S;

(193) X.sub.28 is selected from F, L, M and Y; and

(194) X.sub.29 is selected from D and R;

(195) and

(196) ii) an amino acid sequence which has at least 93% identity to the sequence defined in i).

(197) 2. IL-6 binding polypeptide according to item 1, wherein in sequence i):

(198) X.sub.3 is selected from A, H, K, Q, R and Y;

(199) X.sub.4 is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V and Y;

(200) X.sub.7 is selected from F, H, I, K, L, M, N, R, T, V, W and Y;

(201) X.sub.11 is selected from A, I, K, L, N, S, T and V;

(202) X.sub.16 is T;

(203) X.sub.17 is selected from A, I, T and V;

(204) X.sub.18 is selected from D, E, H, K, N, Q, R, S and T;

(205) X.sub.20 is selected from I, L, M, R and V;

(206) X.sub.21 is selected from A, S and V;

(207) X.sub.25 is selected from I, Q, S, T, V and W;

(208) X.sub.26 is K;

(209) X.sub.28 is selected from F, L, M and Y; and

(210) X.sub.29 is D.

(211) 3. IL-6 binding polypeptide according to item 1 or 2, wherein sequence i) fulfills at least six of the eleven conditions I-XI: I. X.sub.3 is selected from K and R; II. X.sub.11 is selected from A and L; III. X.sub.16 is T; IV. X.sub.17 is selected from I and V; V. X.sub.18 is selected from D and E; VI. X.sub.20 is M; VII. X.sub.21 is A; VIII. X.sub.25 is selected from S and T; IX. X.sub.26 is K; X. X.sub.28 is F; and XI. X.sub.29 is D.

(212) 4. IL-6 binding polypeptide according to item 3, wherein sequence i) fulfills at least seven of the eleven conditions I-XI.

(213) 5. IL-6 binding polypeptide according to item 4, wherein sequence i) fulfills at least eight of the eleven conditions I-XI.

(214) 6. IL-6 binding polypeptide according to item 5, wherein sequence i) fulfills at least nine of the eleven conditions I-XI.

(215) 7. IL-6 binding polypeptide according to item 6, wherein sequence i) fulfills at least ten of the eleven conditions I-XI.

(216) 8. IL-6 binding polypeptide according to item 7, wherein sequence i) fulfills all of the eleven conditions I-XI.

(217) 9. IL-6 binding polypeptide according to any preceding item, wherein X.sub.17X.sub.20X.sub.21 is selected from VMA and IMA.

(218) 10. IL-6 binding polypeptide according to any one of items 1-8, wherein X.sub.20X.sub.21X.sub.28 is MAF.

(219) 11. IL-6 binding polypeptide according to any one of items 1-8, wherein X.sub.17X.sub.20X.sub.28 is selected from VMF and IMF.

(220) 12. IL-6 binding polypeptide according to any one of items 1-8, wherein X.sub.17X.sub.21X.sub.28 is selected from VAF and IAF.

(221) 13. IL-6 binding polypeptide according to any preceding item, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-1551.

(222) 14. IL-6 binding polypeptide according to item 13, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-1502.

(223) 15. IL-6 binding polypeptide according to item 14, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15-89 and SEQ ID NO:151-871.

(224) 16. IL-6 binding polypeptide according to item 14, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-6, SEQ ID NO:8-14, SEQ ID NO:90-150 and SEQ ID NO:872-1502.

(225) 17. IL-6 binding polypeptide according to item 13, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-152 and SEQ ID NO:1503-1515.

(226) 18. IL-6 binding polypeptide according to item 17, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-150 and SEQ ID NO:1503-1515.

(227) 19. IL-6 binding polypeptide according to item 17, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-152.

(228) 20. IL-6 binding polypeptide according to item 18 or 19, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-150.

(229) 21. IL-6 binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15-89 and SEQ ID NO:151-152.

(230) 22. IL-6 binding polypeptide according to item 20, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-6, SEQ ID NO:8-14 and SEQ ID NO:90-150.

(231) 23. IL-6 binding polypeptide according to item 18, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-14 and SEQ ID NO:1503-1515.

(232) 24. IL-6 binding polypeptide according to item 23, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-14 and SEQ ID NO:1512.

(233) 25. IL-6 binding polypeptide according to item 24, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-14.

(234) 26. IL-6 binding polypeptide according to any one of items 16, 22 and 25, wherein sequence i corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-5.

(235) 27. IL-6 binding polypeptide according to item 24, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1512.

(236) 28. IL-6 binding polypeptide according to any preceding item, wherein said IL-6 binding motif forms part of a three-helix bundle protein domain.

(237) 29. IL-6 binding polypeptide according to item 28, wherein said IL-6 binding motif essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.

(238) 30. IL-6 binding polypeptide according to item 29, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.

(239) 31. IL-6 binding polypeptide according to item 30, wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.

(240) 32. IL-6 binding polypeptide according to any preceding item, which comprises a binding module BMod, the amino acid sequence of which is selected from:

(241) TABLE-US-00023 (SEQIDNO:1563) iii)K-[BM]-DPSQSX.sub.aX.sub.bLLX.sub.cEAKKLX.sub.dX.sub.eX.sub.fQ;
wherein

(242) [BM] is an IL-6 binding motif as defined in any one of items 1-27

(243) provided that X.sub.29 is D;

(244) X.sub.a is selected from A and S;

(245) X.sub.b is selected from N and E;

(246) X.sub.c is selected from A, S and C;

(247) X.sub.d is selected from E, N and S;

(248) X.sub.e is selected from D, E and S;

(249) X.sub.f is selected from A and S; and iv) an amino acid sequence which has at least 91% identity to a sequence defined by iii).

(250) 33. IL-6 binding polypeptide according to any one of items 1-31, which comprises a binding module BMod, the amino acid sequence of which is selected from:

(251) TABLE-US-00024 (SEQIDNO:1564) v)K-[BM]-QPEQSX.sub.aX.sub.bLLX.sub.cEAKKLX.sub.dX.sub.eX.sub.fQ,
wherein

(252) [BM] is an IL-6 binding motif as defined in any one of items 1-27

(253) provided that X.sub.29 is R;

(254) X.sub.a is selected from A and S;

(255) X.sub.b is selected from N and E;

(256) X.sub.c is selected from A, S and C;

(257) X.sub.d is selected from E, N and S;

(258) X.sub.e is selected from D, E and S;

(259) X.sub.f is selected from A and S; and vi) an amino acid sequence which has at least 91% identity to a sequence defined by v).

(260) 34. IL-6 binding polypeptide according to any one of items 1-32, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-1551.

(261) 35. IL-6 binding polypeptide according to item 34, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-1502.

(262) 36. IL-6 binding polypeptide according to item 35, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15-89 and SEQ ID NO:151-871.

(263) 37. IL-6 binding polypeptide according to item 35, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-6, SEQ ID NO:8-14, SEQ ID NO:90-150 and SEQ ID NO:872-1502.

(264) 38. IL-6 binding polypeptide according to item 34, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-152 and SEQ ID NO:1503-1515.

(265) 39. IL-6 binding polypeptide according to item 38, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-150 and SEQ ID NO:1503-1515.

(266) 40. IL-6 binding polypeptide according to item 35 or 38, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-152.

(267) 41. IL-6 binding polypeptide according to item 39 or 40, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-150.

(268) 42. IL-6 binding polypeptide according to item 40, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15-89 and SEQ ID NO:151-152.

(269) 43. IL-6 binding polypeptide according to item 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-6, SEQ ID NO:8-14 and SEQ ID NO:90-150.

(270) 44. IL-6 binding polypeptide according to item 39, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-14 and SEQ ID NO:1503-1515.

(271) 45. IL-6 binding polypeptide according to item 44, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-14 and SEQ ID NO:1512.

(272) 46. IL-6 binding polypeptide according to item 45, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-14.

(273) 47. IL-6 binding polypeptide according to any one of items 37, 43 and 46, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-5.

(274) 48. IL-6 binding polypeptide according to item 45, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1512.

(275) 49. IL-6 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from: vii) YA-[BMod]-AP, wherein [BMod] is an IL-6 binding module as defined in any one of items 32-48; and viii) an amino acid sequence which has at least 90% identity to a sequence defined by vii).

(276) 50. IL-6 binding polypeptide according to any one of items 1-48, which comprises an amino acid sequence selected from: ix) FN-[BMod]-AP; wherein [BMod] is an IL-6 binding module as defined in any one of items 32-48; and x) an amino acid sequence which has at least 90% identity to a sequence defined by ix).

(277) 51. IL-6 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:

(278) 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]-DPSQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK;
wherein [BM] is an IL-6 binding motif as defined in any one of items 1-27.

(279) 52. IL-6 binding polypeptide according to any one of items 1-49, which comprises an amino acid sequence selected from:

(280) TABLE-US-00026 xi)VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an IL-6 binding motif as defined in any one of items 1-27; and xii) an amino acid sequence which has at least 89% identity to the sequence defined in xi).

(281) 53. IL-6 binding polypeptide according to any one of items 1-49, which comprises an amino acid sequence selected from:

(282) TABLE-US-00027 xiii)AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an IL-6 binding motif as defined in any one of items 1-27; and xiv) an amino acid sequence which has at least 89% identity to the sequence defined in xiii).

(283) 54. IL-6 binding polypeptide according to item 52, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-1551.

(284) 55. IL-6 binding polypeptide according to item 54, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-1502.

(285) 56. IL-6 binding polypeptide according to item 55, wherein sequence xi) is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15-89 and SEQ ID NO:151-871.

(286) 57. IL-6 binding polypeptide according to item 55, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-6, SEQ ID NO:8-14, SEQ ID NO:90-150 and SEQ ID NO:872-1502.

(287) 58. IL-6 binding polypeptide according to item 54, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-152 and SEQ ID NO:1503-1515.

(288) 59. IL-6 binding polypeptide according to item 58, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-150 and SEQ ID NO:1503-1515.

(289) 60. IL-6 binding polypeptide according to item 55 or 58, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-152.

(290) 61. IL-6 binding polypeptide according to item 59 or 60, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-150.

(291) 62. IL-6 binding polypeptide according to item 60, wherein sequence xi) is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15-89 and SEQ ID NO:151-152.

(292) 63. IL-6 binding polypeptide according to item 61, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-6, SEQ ID NO:8-14 and SEQ ID NO:90-150.

(293) 64. IL-6 binding polypeptide according to item 59, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-14 and SEQ ID NO:1503-1515.

(294) 65. IL-6 binding polypeptide according to item 64, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-14 and SEQ ID NO:1512.

(295) 66. IL-6 binding polypeptide according to item 65, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-14.

(296) 67. IL-6 binding polypeptide according to any one of items 57, 63 and 66, wherein sequence xi) is selected from the group consisting of SEQ ID NO:1-5.

(297) 68. IL-6 binding polypeptide according to item 65, wherein sequence xi) is SEQ ID NO:1512.

(298) 69. IL-6 binding polypeptide according to any preceding item, which is capable of blocking the IL-6 dependent signaling via the cis-signaling pathway and/or the trans-signaling pathway.

(299) 70. IL-6 binding polypeptide according to item 69, wherein the half maximal inhibitory concentration (IC50) of the blocking is at most 110.sup.6 M, such as at most 110.sup.7 M, such as at most 110.sup.8 M, such as at most 110.sup.9 M, such as at most 110.sup.10 M.

(300) 71. IL-6 binding polypeptide according to item 69 or 70, which is capable of blocking the interaction of IL-6/IL-6R with gp130.

(301) 72. IL-6 binding polypeptide according to any preceding item, which is capable of binding to IL-6 such that the EC50 value of the interaction is at most 110.sup.7 M, such as at most 110.sup.8 M, such as at most 110.sup.9 M, such as at most 110.sup.10 M.

(302) 73. IL-6 binding polypeptide according to any preceding item, which is capable of binding to IL-6 such that the K.sub.D value of the interaction is at most 110.sup.8 M, such as at most 110.sup.9 M, such as at most 110.sup.10 M.

(303) 74. IL-6 binding polypeptide according to any preceding item which comprises additional amino acids at the C-terminal and/or N-terminal end.

(304) 75. IL-6 binding polypeptide according to item 74, wherein said additional amino acid(s) improve(s) production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide.

(305) 76. IL-6 binding polypeptide according to any preceding item in multimeric form, comprising at least two IL-6 binding polypeptide monomer units, whose amino acid sequences may be the same or different.

(306) 77. IL-6 binding polypeptide according to item 76, wherein said IL-6 binding polypeptide monomer units are covalently coupled together.

(307) 78. IL-6 binding polypeptide according to item 77, wherein the IL-6 binding polypeptide monomer units are expressed as a fusion protein.

(308) 79. IL-6 binding polypeptide according to item 78, in dimeric form.

(309) 80. Fusion protein or conjugate comprising

(310) a first moiety consisting of an IL-6 binding polypeptide according to any preceding item; and

(311) a second moiety consisting of a polypeptide having a desired biological activity.

(312) 81. Fusion protein or conjugate according to item 80, wherein said desired biological activity is a therapeutic activity.

(313) 82. Fusion protein or conjugate according to item 80, wherein said desired biological activity is a binding activity.

(314) 83. Fusion protein or conjugate according to item 82, wherein said binding activity is albumin binding activity which increases in vivo half-life of the fusion protein or conjugate.

(315) 84. Fusion protein or conjugate according to item 83, wherein said second moiety comprises the albumin binding domain of streptococcal protein G or a derivative thereof.

(316) 85. Fusion protein or conjugate according to item 82, wherein said binding activity acts to block a biological activity.

(317) 86. Fusion protein or conjugate according to item 80, wherein said desired biological activity is an enzymatic activity.

(318) 87. Fusion protein or conjugate according to item 81, wherein the second moiety is a therapeutically active polypeptide.

(319) 88. Fusion protein or conjugate according to item 87, wherein the second moiety is an immune response modifying agent.

(320) 89. Fusion protein or conjugate according to item 87, wherein the second moiety is an anti-cancer agent.

(321) 90. Fusion protein or conjugate according to any one of items 80, 81, 86, 88 and 89, wherein the second moiety is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.

(322) 91. IL-6 binding polypeptide, fusion protein or conjugate according to any preceding item, further comprising a label.

(323) 92. IL-6 binding polypeptide, fusion protein or conjugate according to item 91, wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and radioactive particles.

(324) 93. IL-6 binding polypeptide, fusion protein or conjugate according to any preceding item, comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the IL-6 binding polypeptide via a thiol group of a cysteine residue or an amine group of a lysine residue.

(325) 94. A polynucleotide encoding a polypeptide according to any one of items 1-79.

(326) 95. Expression vector comprising a polynucleotide according to item 94.

(327) 96. Host cell comprising an expression vector according to item 95.

(328) 97. Method of producing a polypeptide according to any one of items 1-79, comprising

(329) culturing a host cell according to item 96 under conditions permissive of expression of said polypeptide from said expression vector, and

(330) isolating said polypeptide.

(331) 98. Composition comprising an IL-6 binding polypeptide, fusion protein or conjugate according to any one of items 1-93 and at least one pharmaceutically acceptable excipient or carrier.

(332) 99. Composition according to item 98, further comprising at least one additional active agent, such as an agent selected from an immune response modifying agent and an anti-cancer agent.

(333) 100. IL-6 binding polypeptide, fusion protein or conjugate according to any one of items 1-93 or a composition according to any one of items 98-99 for oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration, such as for topical administration.

(334) 101. IL-6 binding polypeptide, fusion protein or conjugate according to any one of items 1-93 or a composition according to any one of items 98-99 for use as a medicament, a diagnostic agent or a prognostic agent.

(335) 102. IL-6 binding polypeptide, fusion protein or conjugate according to any one of items 1-93 or a composition according to any one of items 98-99 for use as a medicament.

(336) 103. IL-6 binding polypeptide, fusion protein, conjugate or composition for use according to item 102, wherein said polypeptide, fusion protein, conjugate or composition modulates IL-6 function in vivo.

(337) 104. IL-6 binding polypeptide, fusion protein, conjugate or composition for use according to any one of items 101-103, in the treatment, prognosis or diagnosis of an IL-6 related disorder.

(338) 105. IL-6 binding polypeptide, fusion protein, conjugate or composition for use according to any one of items 101-103, in the treatment of an IL-6 related disorder.

(339) 106. IL-6 binding polypeptide, fusion protein, conjugate or composition for use according to item 105, wherein said IL-6 related disorder is selected from the group consisting of inflammatory diseases, autoimmune diseases, infectious disease, cancer, diabetes, neurological disease and depression.

(340) 107. IL-6 binding polypeptide, fusion protein, conjugate or composition for use according to item 106, wherein said IL-6 related disorder is selected from the group consisting of inflammatory diseases and autoimmune diseases.

(341) 108. IL-6 binding polypeptide, fusion protein, conjugate or composition for use according to item 107, wherein said IL-6 related disorder is selected from the group consisting of rheumatoid arthritis (RA), juvenile RA, juvenile idiopathic arthritis or systemic juvenile idiopathic arthritis, vasculitis, psoriatic arthritis, psoriasis, ankylosing spondylitis, chronic inflammatory bowel disease such as Crohn's disease and ulcerative colitis; Grave's disease, Behcet's disease, uveitis, giant cell arteritis, multiple sclerosis (MS), systemic sclerosis, systemic lupus erythematosus (SLE), polymyositis, polymyalgia rheumatic, asthma, chronic obstructive pulmonary disease (COPD), relapsing polychondritis, pancreatitis, peritonitis, nephritis, Kawasaki's disease, Sjgren's syndrome and adult Still's disease.

(342) 109. IL-6 binding polypeptide, fusion protein, conjugate or composition for use according to item 106, wherein said IL-6 related disorder is cancer, such as a cancer selected from the group consisting of colitis associated cancer, renal cancer, kidney cancer, prostate cancer, malignant lymphoma, multiple myeloma, Castleman's disease, breast cancer and lung cancer.

(343) 110. IL-6 binding polypeptide, fusion protein, conjugate or composition for use according to item 106, wherein said IL-6 related disorder is selected from Alzheimer's disease, HIV, diabetes, sepsis, cachexia, myelodysplastic syndrome (MDS), liver cirrhosis, graft versus host disease, myocardial infarction, endometriosis and osteoporosis.

(344) 111. Method of treatment of an IL-6 related disorder, comprising administering to a subject in need thereof an effective amount of an IL-6 binding polypeptide, fusion protein or conjugate according to any one of items 1-93 or a composition according to any one of items 98-99.

(345) 112. Method according to item 111, wherein said IL-6 related disorder is selected from the group consisting of inflammatory diseases, autoimmune diseases, infectious disease, cancer, diabetes, neurological disease and depression.

(346) 113. Method according to item 112, wherein said IL-6 related disorder is selected from the group consisting of inflammatory diseases and autoimmune diseases.

(347) 114. Method according to item 113, wherein said IL-6 related disorder is selected from the group consisting of rheumatoid arthritis (RA), juvenile RA, juvenile idiopathic arthritis or systemic juvenile idiopathic arthritis, vasculitis, psoriatic arthritis, psoriasis, ankylosing spondylitis, chronic inflammatory bowel disease such as Crohn's disease and ulcerative colitis; Grave's disease, Behcet's disease, uveitis, giant cell arteritis, multiple sclerosis (MS), systemic sclerosis, systemic lupus erythematosus (SLE), polymyositis, polymyalgia rheumatic, asthma, chronic obstructive pulmonary disease (COPD), relapsing polychondritis, pancreatitis, peritonitis, nephritis, Kawasaki's disease, Sjgren's syndrome and adult Still's disease.

(348) 115. Method according to item 112, wherein said IL-6 related disorder is cancer, such as a cancer selected from the group consisting of selected from the group consisting of colitis associated cancer, renal cancer, kidney cancer, prostate cancer, malignant lymphoma, multiple myeloma, Castleman's disease, breast cancer and lung cancer.

(349) 116. Method according to item 112, wherein said IL-6 related disorder is selected from Alzheimer's disease, HIV, diabetes, sepsis, cachexia, myelodysplastic syndrome (MDS), liver cirrhosis, graft versus host disease, myocardial infarction, endometriosis and osteoporosis.

(350) 117. Method of detecting IL-6, comprising providing a sample suspected to contain IL-6, contacting said sample with an IL-6 binding polypeptide, fusion protein or conjugate according to any one of items 1-90 or a composition according to any one of items 98-99, and detecting the binding of the IL-6 binding polypeptide, fusion protein, conjugate or composition to indicate the presence of IL-6 in the sample.

(351) 118. Method for determining the presence of IL-6 in a subject, the method comprising the steps: contacting the subject, or a sample isolated from the subject, with an IL-6 binding polypeptide, fusion protein or conjugate according to any one of items 1-93 or a composition according to any one of items 98-99, and obtaining a value corresponding to the amount of the IL-6 binding polypeptide, fusion protein, conjugate or composition that has bound in said subject or to said sample.

(352) 119. Method according to item 118, further comprising a step of comparing said value to a reference.

(353) 120. Method according to item 118 or 119, wherein said subject is a mammalian subject, such as a human subject.

(354) 121. Method according to any one of items 118-120, wherein the method is performed in vivo.

(355) 122. Method according to any one of items 118-120, wherein the method is performed in vitro.