POLYPEPTIDES

Abstract

The present disclosure relates to a class of engineered polypeptides having a binding affinity for high mobility group box 1 protein (HMGB1), and provides an HMGB1 binding polypeptide comprising the sequence EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20X.sub.21AFIYX.sub.26LED or a sequence having at least 93% identity thereto. The present disclosure also relates to the use of such an HMGB1 binding polypeptide as a therapeutic, prognostic and/or diagnostic agent.

Claims

1. A HMGB1 binding polypeptide, comprising an HMGB1 binding motif BM, which motif consists of an amino acid sequence selected from: i) TABLE-US-00034 (SEQ ID NO: 56) EX2X3X4AX6X7EIX10 XI1LPNLX16X17X18Q X20 X21AFIYX26LED wherein, independently of each other, X2 is selected from A, D, S and T; X3 is selected from E, R and W; X4 is selected from A, D and Q; X6 is selected from F and M; X7 is selected from E, H, W and Y; X10 is selected from I and L; X11 is selected from A and W; X16 is selected from N and T; X17 is selected from A, D, N and W; X18 is selected from A, E, Q, R, S, T and Y; X20 is selected from A and Q; X21 is selected from K, L and R; and X26 is selected from K and S; and ii) an amino acid sequence which has at least 93% identity to the sequence defined in i).

2. The HMGB1 binding polypeptide according to claim 1, wherein in sequence i) X2 is selected from A, D, S and T; X3 is selected from R and W; X4 is D; X6 is F; X7 is selected from E, Y and W; X10 is I; X11 is W; X16 is selected from N and T; X17 is D; X18 is selected from A, Q and R; X20 is Q; X21 is R; and X26 is selected from K and S.

3. The HMGB1 binding polypeptide according to claim 1, 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, 16 and 20-23.

4. The HMGB1 binding polypeptide according to claim 1, which binds to the A-box of HMGB1 such that the KD value of the interaction is at most 1×10.sup.−6M.

5. The HMGB1 binding polypeptide according to claim 1, wherein said HMGB1 binding motif forms part of a three-helix bundle protein domain.

6. The HMGB1 binding polypeptide according to claim 1, which comprises an amino acid sequence selected from: xi) TABLE-US-00035 (SEQ ID NO: 60) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an HMGB1 binding motif as defined in any one of claims 1-3; and xii) an amino acid sequence which has at least 89% identity to the sequence defined in xi).

7. The HMGB1 binding polypeptide according to claim 6, wherein sequence xi) is selected from the group consisting of SEQ ID NO:27-28.

8. A fusion protein or conjugate comprising a first moiety consisting of an HMGB1 binding polypeptide according to claim 1; and a second moiety consisting of a polypeptide having a desired biological activity.

9. The fusion protein or conjugate according to claim 8, comprising at least two HMGB1 binding polypeptide monomer units as said first and second moiety, respectively, wherein said first HMGB1 binding polypeptide monomer unit has affinity for the A-box of HMGB1, and wherein said second HMGB1 binding polypeptide monomer unit has affinity for the B-box of HMGB1.

10. A composition comprising an HMGB1 binding polypeptide according to claim 1 and at least one pharmaceutically acceptable excipient or carrier.

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. A method of treatment of an HMGB1 related disorder, comprising administering to a subject in need thereof an effective amount of an HMGB1 binding polypeptide, according to claim 1.

16. A method for determining the presence of HMGB1 in a subject, comprising the steps of: a) contacting the subject, or a sample isolated from the subject, with an HMGB1 binding polypeptide, according to claim 1, and b) obtaining a value corresponding to the amount of the HMGB1 binding polypeptide, that has bound in said subject or to said sample.

17. The method of claim 15, wherein said HMGB1 related disorder is inflammatory diseases, respiratory diseases, autoimmune diseases, infectious diseases, trauma, cardiovascular disease, neurodegenerative diseases, metabolic disorders, liver injury, and cancer.

18. The HMGB1 binding polypeptide according to claim 1, which binds to the A-box of HMGB1 such that the KD value of the interaction is at most 5×10.sup.−7M.

19. The HMGB1 binding polypeptide according to claim 1, which binds to the A-box of HMGB1 such that the KD value of the interaction is at most 1×10.sup.−7M.

20. The HMGB1 binding polypeptide according to claim 1, which binds to the A-box of HMGB1 such that the KD value of the interaction is at most 5×10.sup.−8M

21. The HMGB1 binding polypeptide according to claim 1, which binds to the A-box of HMGB1 such that the KD value of the interaction is at most 1×10.sup.−8M.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0225] FIG. 1 is a listing of the amino acid sequences of examples of selected HMGB1 A-box binding polypeptides and alanine/serine mutated variants thereof (SEQ ID NO:1-29); HMGB1 B-box binding polypeptides (SEQ ID NO:30-32); control polypeptides with affinity for an irrelevant target (Z03638, SEQ ID NO:33 and Z04726, SEQ ID NO:54); the albumin binding polypeptide PP013 (SEQ ID NO:34); A-box binding polypeptides in fusion with PP013 (SEQ ID NO:35-36); B-box binding polypeptide in fusion with PP013 (SEQ ID NO:37); homodimeric A-box binding polypeptides in fusion with PP013 (SEQ ID NO:43-45); heterodimeric A-box and B-box binding polypeptides in fusion with PP013 (SEQ ID NO:38-42, SEQ ID NO:46-48 and SEQ ID NO:55); control polypeptide (homodimeric Z03638) in fusion with PP013 (SEQ ID NO:49); full-length rat HMGB1 (SEQ ID NO:50); the A-box domain of human HMGB1, boxA (SEQ ID NO:51); the A-box domain of human HMGB1 with an N-terminal GST-tag, GST-boxA (SEQ ID NO:52); and the B-box domain of human HMGB1 with an N-terminal GST-tag, GST-boxB (SEQ ID NO:53). Listed amino acid sequences were used in selection, screening and/or characterization of the binding polypeptides of the disclosure. The deduced HMGB1 A-box or B-box binding motifs (BM) extend from residue 8 to residue 36 in sequences with SEQ ID NO:1-14, 16, 20-23 and 27-32. The amino acid sequences of the 49 amino acid residues long polypeptides (BMod) predicted to constitute the complete three-helix bundle within each of these Z variants extend from residue 7 to residue 55.

[0226] FIG. 2 shows binding of four different HMGB1 variants to Z-ABD polypeptides measured by Biacore, as described in Example 3. HMGB1(disulfide) (FIGS. 2A and 2E), HMGB1(red) (FIGS. 2B and 2F), boxA(disulfide) (FIGS. 2C and 2G) and GST-boxA(red) (FIGS. 2D and 2H), respectively, were injected over ZA3812 (FIG. 2A-D) and ZA3914 (FIG. 2E-H), respectively, captured on immobilized HSA. In FIG. 2A-C and FIG. 2E-G, the injected concentrations were 10 nM (black broken line), 40 nM (black solid line), 160 nM (gray broken line) and 640 nM (gray solid line), i.e. of HMGB1(disulfide), HMGB1(red) and boxA(disulfide), respectively, whereas in FIG. 2D and FIG. 2H, the injected concentrations were 2.5 nM (black broken line), 10 nM (black solid line), 40 nM (gray broken line) and 160 nM (gray solid line), i.e. of GST-boxA(red).

[0227] FIG. 3 shows inhibition of HMGB1-induced cell migration by the A-box binding polypeptide ZH 01 (FIG. 3A) and by the B-box binding polypeptides ZH 31 (FIG. 3B) and ZH 30 (FIG. 3C), respectively. Z04726 and ZZA4014 are negative control variants. The results are presented as cells/field measured by the number of cells on the lower side of the filter counted with a Leica DM LS2 microscope.

[0228] FIG. 4 shows analysis by the cAMP Hunter eXpress CXCR4 CHO-K1 GPCR Assay. A: Analysis of luminescent signal after HMGB1-CXCL12 binding to CXCR4. HMGB1 (200 nM-0.3 nM) was added together with CXCL12 (0.4 nM) and inhibited the luminescent signal after 30 min stimulation. B: Analysis of luminescent signal after ZZA4263 inhibition of the HMGB1-CXCL12 complex binding to CXCR4. ZZA4263 (1000 nM-0.0006 nM) or PBS was added together with HMGB1 (10 nM) and CXCL12 (0.4 nM) and incubated with the cells for 30 min. ZZA4263 enabled the luminescent signal to return (black squares), whereas the PBS negative control did not show any effect on the luminescent signal (grey dots).

[0229] FIG. 5A shows the PK profiles of ZZA4263 in mice after i.v. (solid dots) and i.p. (open dots) administration, respectively. FIG. 5B shows the PK profile of ZZA4263 in rat after i.v. administration.

[0230] FIG. 6 shows the evaluation of ZZA4263 in a CLP-induced sepsis model in mice, depicted as the effect of ZZA4263 on the survival rate (FIG. 6A) and Murine Sepsis Score (FIG. 6B), respectively.

EXAMPLES

Summary

[0231] The following Examples disclose the development of novel Z variant molecules targeting the human High Mobility Group Box 1 protein (HMGB1), based on phage display technology. The polypeptides described herein were sequenced, and their amino acid sequences are listed in FIG. 1 with the sequence identifiers SEQ ID NO:1-32. The Examples further describe the characterization of HMGB1 binding polypeptides, and demonstrate their in vitro and in vivo functionality. Furthermore, a number of fusion protein constructs were prepared in the context of a “formatting program”, in order to optimize the potencies of said polypeptides. The Examples describe the characterization of these formatted HMGB1 binding polypeptides and demonstrate their in vitro and in vivo functionality.

[0232] Different full-length HMGB1 proteins and fragments thereof used in the Examples described below are listed in Table 3. All HMGB1 target proteins were produced by HMGBiotech Srl, Milano, Italy. The HMGB1 proteins used correspond to the amino acid sequence of rat HMGB1. However, rat and human HMGB1 are identical in sequence within the A-box and the B-box domains. The alternative denotations in Table 3 follow the systematic nomenclature proposed by Antoine et al (2014) Mol Med 20:135-137).

TABLE-US-00011 TABLE 3 HMGB1 proteins and fragments thereof used in selection, screening and characterization of HMGB1 binding polypeptides SEQ ID Designation NO.sup.1 Description HMGB1 50 Fully reduced form of full-length (red) HMGB1; HMGB1C23hC45hC106h HMGB1 50 Disulfide linked form of full-length (disulfide) HMGB1; HMGB1C23-C45C106h boxA 51 Disulfide linked form of A-box (disulfide) domain; boxAC23-C45C106h boxA(red) 51 Reduced form of A-box domain GST-boxA 52 Reduced form of A-box domain (red) with N-terminal GST tag GST-boxB 53 Reduced form of B-box domain (red) with N-terminal GST tag b-HMGB1 50 Biotinylated, fully reduced form (red) of full-length HMGB1 b-boxA 51 Biotinylated, disulfide-linked (disulfide) form of A-box domain b-GST-boxA 52 Biotinylated, reduced form of A-box (red) domain with N-terminal GST tag b-GST-boxB 53 Biotinylated, reduced form of B-box (red) domain with N-terminal GST tag .sup.1Unmodified amino acid sequence, independent of redox status

Example 1

Selection and Screening of HMGB1 Binding Z Variants

Summary

[0233] In this Example, the reduced (i.e. thiol) forms of the A-box domain (boxA(red)) and the B-box domain (boxB(red)) of human HMGB1 were used as targets in phage display selections using a phage library of Z variants. Selected clones were DNA sequenced, produced in E. coli periplasmic fractions and assayed against full-length HMGB1, A-box or B-box in ELISA (enzyme-linked immunosorbent assay).

Materials and Methods

[0234] Biotinylation of target protein: Glutathione S-transferase (GST) fused target proteins GST-boxA(red) (SEQ ID NO:52) and GST-boxB(red) (SEQ ID NO:53), as well as boxA(disulfide) (SEQ ID NO:51) and HMGB1(disulfide) (SEQ ID NO:50), were biotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, cat. no. 21327) at a 10×molar excess, according to the manufacturer's recommendations. The reactions were performed at room temperature (RT) for 30-40 min. Buffer exchange to phosphate buffered saline (PBS; 10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) was performed both before and after biotinylation using PD-10 desalting columns (GE Healthcare, cat. no. 17-0851-01) according to the manufacturer's instructions.

[0235] Biotinylation of antibody: Anti-GST antibody (goat-polyclonal, Abcam, cat. no. ab6613) was biotinylated as described above.

[0236] Phage display selection of HMGB1 binding Z variants: A library of random variants of protein Z displayed on bacteriophage, constructed in phagemid pAY02592 essentially as described in Grönwall et al (2007) J Biotechnol 128:162-183, was used to select HMGB1 binding polypeptides. In this library, an albumin binding domain (“ABD”, i.e. domain GA3 of protein G from Streptococcus strain G148) is used as fusion partner to the Z variants. The library is denoted Zlib006Naive.II and has a size of 1.5×10.sup.10 library members (Z variants). Production of phage stock from phagemid library Zlib006Naive.II was essentially performed as described earlier (see e.g. PCT publication WO2017/072280) using E. coli RRIΔM15 cells (Rüther et al (1982) Nucleic Acids Res 10:5765-5772) from a glycerol stock of the library. The phage particles were precipitated in PEG/NaCl (polyethylene glycol/sodium chloride) from the supernatant twice, filtered and dissolved in PBS and glycerol as described in Grönwall et al supra. Phage stocks were stored at −80° C. before use.

[0237] Selections against GST-boxA(red), GST-boxB(red) and the corresponding biotinylated proteins, b-GST-boxA(red) and b-GST-boxB(red), were performed in four cycles. In tracks 1 and 2, Dynabeads® M-280 Streptavidin (SA beads, Invitrogen, cat. no. 11206D) were used to catch the b-GST-boxA(red):Z-variant and b-GST-boxB(red):Z-variant complexes. In tracks 3 and 4, SA beads coated with biotin-anti-GST antibody were used to catch the GST-boxA(red):Z-variant and GST-boxB(red):Z-variant complexes. As selection proceeded, the tracks were further divided according to target concentration and number and/or time of washes. Phage stock preparation and amplification of phage between selection cycles were performed essentially as described for selection against another biotinylated target in PCT publication WO2009/077175, with the following exceptions: 1) for amplification of phage particles between selection cycles 1 and 2, E. coli strain ER2738 (Lucigen, Middleton, Wis., USA) cells were grown to log phase in TSB supplemented with 10 μg/ml tetracycline and 2% glucose; 2) Infection was done during 30 min.

[0238] In order to reduce the amount of background binders, pre-selection was performed in each cycle using SA beads coated with biotin-GST, biotinylated as described previously for b-GST-boxA(red) and b-GST-boxB(red). Furthermore, in tracks 3 and 4, pre-selection was also performed using SA beads coated with biotin-anti-GST antibody. During pre-selection, the phage stock was incubated with coated beads end-over-end for 30-60 min at RT. All tubes and beads used in the pre-selections or selection were pre-blocked with PBS supplemented with 3% bovine serum albumin (BSA, Sigma, cat. no. A3059-100G) and 0.1% Tween20 (PBSTB).

[0239] Selection was performed in solution in PBS supplemented with 10% fetal bovine serum (FBS; Gibco, cat. no. 10108-165) and 0.1% Tween20 (Acros Organics, cat. no. 233362500) and 2 mM dithiothreitol (DTT; Acros Organics, cat. no. 165680250) at RT. The time for selection was approximately 120 min followed by wash with PBS supplemented with 0.1% Tween20 (PBST 0.1%) and catch of target-phage complexes on SA beads or SA beads coated with biotin-anti-GST antibody using 1 mg beads per 4-8 μg b-GST-boxA(red) and b-GST-boxB(red), respectively, and 0.4 μg GST-boxA(red) and GST-boxB(red), respectively.

[0240] In the final selection cycle, log phase bacteria were infected with eluate and diluted before spreading onto TBAB plates (30 g/l tryptose blood agar base, Oxoid, cat. no. CM0233B) supplemented with 0.2 g/l ampicillin in order to form single colonies to be used in ELISA screening.

[0241] An overview of the selection strategy, describing an increased stringency in successive cycles with a lowered target concentration and an increased number of washes, is shown in Table 4. Unless noted otherwise, washes were performed for 1 min using PBST 0.1%. Elution was carried out as described in WO2009/077175.

TABLE-US-00012 TABLE 4 Overview of the selection against HMGB1 using a primary library Phage stock Proteins from library used in Target Number Duration Selection or selection pre- Target conc. of of last Cycle track track selection protein (nM) washes wash (h) 1 1 Zlib006Naive.II b-GST b-GST- 100 2 boxA(red) 1 2 Zlib006Naive.ll b-GST b-GST- 100 2 boxB(red) 1 3 Zlib006Naive.II b-GST, b- GST- 100 2 anti-GST boxA(red) antibody 1 4 Zlib006Naive.II b-GST, b- GST- 100 2 anti-GST boxB(red) antibody 2 1-1 1 b-GST b-GST- 50 4 boxA(red) 2 1-2 1 b-GST b-GST- 10 4 boxA(red) 2 2-1 2 b-GST b-GST- 50 4 boxB(red) 2 2-2 2 b-GST b-GST- 10 4 boxB(red) 2 3-1 3 b-GST, b- GST- 50 4 anti-GST boxA(red) 2 3-2 3 b-GST, b- GST- 10 4 anti-GST boxA(red) 2 4-1 4 b-GST, b- GST- 50 4 anti-GST boxB(red) 2 4-2 4 b-GST, b- GST- 10 4 anti-GST boxB(red) 3 1-1-1 1-1 b-GST b-GST- 25 6 boxA(red) 3 1-2-1 1-2 b-GST b-GST- 1 6 boxA(red) 3 2-1-1 2-1 b-GST b-GST- 25 6 boxB(red) 3 2-2-1 2-2 b-GST b-GST- 1 6 boxB(red) 3 3-1-1 3-1 b-GST, b- GST- 25 6 anti-GST boxA(red) 3 3-2-1 3-2 b-GST, b- GST- 1 6 anti-GST boxA(red) 3 4-1-1 4-1 b-GST, b- GST- 25 6 anti-GST boxB(red) 3 4-2-1 4-2 b-GST, b- GST- 1 6 anti-GST boxB(red) 4 1-1-1-1 1-1-1 b-GST b-GST- 12.5 10 boxA(red) 4 1-1-1-2 1-1-1 b-GST b-GST- 2.5 30 1 boxA(red) 4 1-2-1-1 1-2-1 b-GST b-GST- 0.1 10 boxA(red) 4 1-2-1-2 1-2-1 b-GST b-GST- 0.05 30 1 boxA(red) 4 2-1-1-1 2-1-1 b-GST b-GST- 12.5 10 boxB(red) 4 2-1-1-2 2-1-1 b-GST b-GST- 2.5 30 1 boxB(red) 4 2-2-1-1 2-2-1 b-GST b-GST- 0.1 10 boxB(red) 4 2-2-1-2 2-2-1 b-GST b-GST- 0.05 30 1 boxB(red) 4 3-1-1-1 3-1-1 b-GST, b- GST- 12.5 10 anti-GST boxA(red) 4 3-1-1-2 3-1-1 b-GST, b- GST- 2.5 30 1 anti-GST boxA(red) 4 3-2-1-1 3-2-1 b-GST, b- GST- 0.1 10 anti-GST boxA(red) 4 3-2-1-2 3-2-1 b-GST, b- GST- 0.05 30 1 anti-GST boxA(red) 4 4-1-1-1 4-1-1 b-GST, b- GST- 12.5 10 anti-GST boxB(red) 4 4-1-1-2 4-1-1 b-GST, b- GST- 2.5 30 1 anti-GST boxB(red) 4 4-2-1-1 4-2-1 b-GST, b- GST- 0.1 10 anti-GST boxB(red) 4 4-2-1-2 4-2-1 b-GST, b- GST- 0.05 30 1 anti-GST boxB(red)

[0242] Production of Z variants for ELISA: The Z variants were produced and the periplasmic fraction of each individual variant was prepared as described in WO2017/072280. The final supernatant of the periplasmic extract contained the Z variants as fusions to ABD, expressed as AQHDEALE-[ZH ##]-VDYV-[ABD]-YVPG (Grönwall et al supra). ZH ## refers to individual, 58 amino acid residue Z variants.

[0243] Sequencing: In parallel with the ELISA screening, all clones were sequenced. PCR fragments were amplified from single colonies, sequenced and analyzed essentially as described in WO2009/077175

[0244] ELISA screening of Z variants against reduced A-box and B-box: The binding of Z variants to HMGB1 was analyzed in ELISA assays. Half-area 96-well ELISA plates (Greiner, cat. no. 675061) were coated at 4° C. overnight with 2 μg/ml of an anti-ABD goat antibody (produced in-house) diluted in coating buffer (50 mM sodium carbonate, pH 9.6; Sigma, cat. no. C3041). The antibody solution was poured off and the wells were washed in water and blocked with 100 μl of PBSC (PBS supplemented with 0.5% casein; Sigma, cat. no. C8654) for 1-3 h at RT. The blocking solution was discarded and 50 μl periplasmic solutions, diluted 1:16 with PBST 0.05%, were added to the wells and incubated for 1.5 to 2.5 h at RT under slow agitation. As a negative control, periplasmic ABD was added. The supernatants were poured off and the wells were washed 4 times with PBST 0.05%. Then, 50 μl of either b-GST-boxA(red), at a concentration of 2 nM, or b-GST-boxB(red), at a concentration of 100 nM, in PBSC+1-2 mM DTT, was added to each well of A-box binding Z-variants or B-box binding Z-variant, respectively. The plates were incubated for 1 h at RT followed by washes as described above. Streptavidin conjugated HRP (Thermo Scientific, cat. no. N100) diluted 1:30,000 in PBSC+1-2 mM DTT, was added to the wells and the plates were incubated for approximately 45 min. After washing as described above, 50 μl ImmunoPure TMB substrate (Thermo Scientific, cat. no. 34021) was added to the wells and the plates were treated according to the manufacturer's recommendations. The absorbance at 450 nm was measured using a multi-well plate reader, Victor3 (Perkin Elmer).

[0245] ELISA screening of Z variants against the disulfide form of A-box and against HMGB1: To find out if selected A-box binding Z variants could also bind the disulfide form of HMGB1, A-box binding variants were screened in the same setup as described above against b-boxA(disulfide) at a concentration of 1000 nM and b-HMGB1(disulfide) at a concentration of 100 nM in PBSC.).

[0246] ELISA analysis of selectivity for each HMGB1 domain: In a similar setup as described above, 100 nM of the HMGB1 domain not used as target in the particular selection track was used to identify unselective Z variants (i.e. b-GST-boxB(red) was used to screen Z-variants from b-GST-boxA(red) tracks and b-GST-boxA(red) was used to screen Z-variants from b-GST-boxB(red) tracks.

[0247] EC50 analysis of Z variants: A sub-group of HMGB1 binding Z variants was subjected to an analysis of their response against a dilution series of b-GST-boxA(red) or b-GST-boxB(red) following the procedure described above. The Z variants ZH 01 (SEQ ID NO:1), ZH 02 (SEQ ID NO:2) and ZH 03 (SEQ ID NO:3) were diluted 1:16 in PBST 0.05% and target protein b-GST-boxA(red) was diluted in PBSC (PBS with 0.5% casein (Sigma cat no. C-8654)) with 0.5-1 mM DTT and added at a concentration of 1 μM and diluted stepwise 1:5 down to 60 μM. The Z variants ZH 30 (SEQ ID NO:30) and ZH 31 (SEQ ID NO:31) were diluted 1:16 in PBST 0.05% and b-GST-boxB(red) was diluted in PBSC without DTT present and added at a concentration of 7 μM and diluted stepwise 1:5 down to 90 μM. As a background control, all Z variants were also assayed with no target protein added. Periplasm containing the ABD moiety only was used as a negative control. In the same assay, the selectivity of the Z variants was tested by incubating periplasm samples with the opposite HMGB1 domain (i.e. b-GST-boxA(red) or b-GST-boxB(red)) than the one used as target in selection, added at a concentration of 1000 nM. Data were analyzed using GraphPad Prism 5 and non-linear regression, and EC50 values (the half maximal effective concentration) were calculated.

Results

[0248] Phage display selection of HMGB1 binding Z variants: Individual clones were obtained after four cycles of phage display selections against the reduced forms of the A-box and the B-box domains of HMGB1, i.e. boxA(red) and boxB(red), respectively.

[0249] Sequencing: Sequencing was performed for clones obtained after four cycles of selection. Each variant was given a unique identification number ##, and individual variants are referred to as ZH ##. The amino acid sequences of the 58 amino acid residues long Z variants are listed in FIG. 1 and in the sequence listing as SEQ ID NO:1-3 and 30-32. The deduced HMGB1 binding motifs extend from residue 8 to residue 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 residue 7 to residue 55.

[0250] ELISA screening of Z variants against reduced A-box and B-box: The clones obtained after four cycles of selection were produced in 96-well plates and screened for b-GST-boxA(red) or b-GST-boxB(red) binding activity in ELISA. ZH 01, ZH 02 and ZH 03 were found to give an average response of 0.7, 0.2 and 0.6 AU, respectively (corresponding to at least 3× the blank control) against b-GST-boxA(red) at a target concentration of 2 nM. ZH 30 and ZH 31 were found to give responses of 0.14 and 0.47 AU, respectively (corresponding to at least 2.3×the blank control) against b-GST-boxB(red) at a concentration of 100 nM.

[0251] ELISA screening of Z variants against the disulfide form of A-box and against HMGB1: The A-box binding Z variants ZH 01, ZH 02, and ZH 03 were all found to bind to b-boxA(disulfide), giving responses of 3.06, 2.02 and 1.17 AU, respectively (corresponding to at least 3×the blank control) at a target concentration of 1000 nM. In the same way, ZH 01, ZH 02 and ZH 03 showed binding to b-HMGB1(disulfide) with a response of 3.07, 1.17 and 2.96 AU, respectively (corresponding to at least 3×the blank control) at a target concentration of 100 nM.

[0252] ELISA analysis of selectivity for each HMGB1 domain: No significant binding was detected to either biotin, GST or the HMGB1 domain not used as target in selection. These results indicate that the selected Z variants are specific to boxA(red) or boxB(red), respectively.

[0253] EC50 analysis of Z variants: A subset of Z variants having the highest ELISA values in the ELISA screening experiments described above was selected and subjected to a target titration in ELISA format. Periplasm samples were incubated with a serial dilution of either b-GST-boxA(red) or b-GST-boxB(red). Obtained values were analyzed and EC50 values calculated (Table 5).

TABLE-US-00013 TABLE 5 Calculated EC50 values from ELISA titration analysis Designation SEQ ID NO Target EC50 (M) ZH01 1 boxA(red) 2.2 × 10.sup.−9 ZH02 3 boxA(red) 5.6 × 10.sup.−9 ZH03 2 boxA(red) 2.7 × 10.sup.−9 ZH30 30 boxB(red) 6.9 × 10.sup.−9 ZH31 31 boxB(red) 9.9 × 10.sup.−9

Example 2

Production of Monomeric HMGB1 Binding Z Variants

Summary

[0254] This Example describes the general procedure for subcloning and production of His-tagged Z variants and Z variants in fusion with an albumin binding domain, which are used throughout the characterization experiments that follow.

Materials and Methods

[0255] Subcloning of Z variants with a His.sub.6-tag: DNA encoding each Z variant 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 and essentially as described in PCT publication WO2009/077175. The Z gene fragments were subcloned into an expression vector, resulting in the encoded sequence MGSSHHHHHHLQ-[ZH ##]-VD.

[0256] Subcloning of Z variants in fusion with ABD: The N-terminal amino acids in positions 1 and 2 (V and D, respectively) of the Z variants ZH 01, ZH 03 and ZH 31 were mutated to the amino acid residues A and E, respectively, using standard molecular biology techniques. The resulting new Z variants, ZH 27 (SEQ ID NO:27), ZH 28 (SEQ ID NO:28) and ZH 32 (SEQ ID NO:32), were subcloned into an expression vector containing the ABD variant PP013 (SEQ ID NO:34). The constructs encoded by the expression vectors were in the format [ZH ##]-ASGS-PP013 and denoted ZA3812 (SEQ ID NO:35), ZA3914 (SEQ ID NO:36) and ZA3975 (SEQ ID NO:37).

[0257] Expression of HMGB1 binding Z variants: Production of Z variants was accomplished essentially as follows: E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing the gene fragment of each respective HMGB1 binding Z variant and cultivated at 37° C. in 980 ml of TSB-YE medium supplemented with 50 μg/ml kanamycin. In order to induce protein expression, isopropyl-β-D-1-thiogalactopyranoside (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.

[0258] Purification of HMGB1 binding Z variants with a His.sub.6-tag: Approximately 1-2 g of each cell pellet was re-suspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) supplemented with Benzonase® (Merck, cat. no. 1.01654.0001). After cell disruption, cell debris was removed by centrifugation and each supernatant was applied on a 1 ml His GraviTrap IMAC column (GE Healthcare, cat. no. 11-0033-99). Contaminants were removed by washing with wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4) and the Z variants were subsequently eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4). Z variants were subjected to a second purification step by reverse phase chromatography (RPC), for which each Z variant was loaded onto a 1 ml Resource 15RPC column (GE Healthcare), previously equilibrated with RPC solvent A (0.1% trifluoroacetic acid (TFA), 10% acetonitrile (ACN), 90% water). After column wash with RPC solvent A, bound proteins were eluted with a linear gradient of 0-60% RPC solvent B (0.1% TFA, 80% ACN, 20% water) for 18 ml. The buffer was then exchanged to DPBS (Corning, cat. no. 21-031-CVR) using PD-10 desalting columns.

[0259] Protein concentrations were determined by measuring the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer (Saveen Werner AB) and the extinction coefficient of the respective protein. In batches where endotoxin levels exceeded 10 EU/mg, an endotoxin removal step was applied using a 1 ml EndoTrap red column (Hyglos, cat. no. 321063) according to supplier's recommendations. Samples with a concentration less than approximately 1 mg/ml were concentrated using Am icon Ultra-4, Ultracel-3K (Merck Millipore). The purity was analyzed by SDS-PAGE stained with Coomassie Blue, endotoxin level was analyzed with Endosafe PTS (Charles River) and the identity of each purified Z variant was confirmed using LC/MS analysis.

[0260] Purification of HMGB1 binding Z variants in fusion with ABD: Approximately 5 g of each cell pellet was re-suspended in TST-buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween20, pH 8.0) supplemented with Benzonase® (Merck). After cell disruption, clarification by centrifugation and passing through a 0.45 μm filter, each supernatant was applied on a gravity flow column with 5 ml agarose immobilized with an anti-ABD ligand (produced in-house). After washing with TST-buffer and 5 mM NH.sub.4Ac pH 5.5 buffer, the ABD fused Z variants were eluted with 0.1 M HAc. The eluates were then subjected to a second purification step by RPC. 10% ACN was added to each eluate before loaded on a 3 ml Resource RPC column (GE Healthcare, cat. no. 17-1182-01) equilibrated with solvent A (10% ACN, 0.1% TFA, 90% milli-Q-water) and eluted using a gradient of 0-60% solvent B (80% ACN, 0.1% TFA, 20% milli-Q-water) for 54 ml. Eluted fractions were analyzed by SDS-PAGE and HPLC-MS and pooled. The buffer of the eluate was exchanged to DPBS using PD-10 desalting columns.

[0261] Endotoxin removal and determination of protein concentrations, as well as analysis of purity and identity, was performed as described above for Z variants with a His.sub.6-tag.

Results

[0262] Production of HMGB1 binding Z variants: The HMGB1 binding Z variants with His.sub.6-tag or in fusion with ABD were successfully cloned and expressed as soluble gene products in E. coli. Each DNA construct was verified by DNA sequencing. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the HMGB1 binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.

Example 3

Characterization of Primary HMGB1 Binding Z Variants

SUMMARY

[0263] In this Example, Z variants were characterized in vitro in terms of secondary structure, stability, binding profile and functional properties.

[0264] Biacore was used to characterize the interactions of the Z variants with full-length HMGB1 as well as with the individual A-box and B-box domains separately. The melting temperature and secondary structure content were analyzed by circular dichroism (CD) spectroscopy. Furthermore, the ability of Z variants to functionally block HMGB1-induced mechanisms was investigated using an in vitro cell migration assay.

Materials and Methods

[0265] Circular dichroism (CD) spectroscopy analysis: The His.sub.6-Z variants produced in Example 2 were diluted to 25 μM in PBS or H.sub.2O. 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, absorbance was measured at 221 nm while the temperature was raised from 20° C. to 80° C. (in some cases to 90° C.), with a temperature slope of 5° C./min. A new CD spectrum was obtained at 20° C. after the heating procedure, in order to study the refolding ability of the Z variants. The CD measurements were performed on a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) using a cell with an optical path length of 1 mm.

[0266] Biacore kinetic and specificity analysis: Kinetic constants (k.sub.a and k.sub.d) and affinities (K.sub.D) for HMGB1(red), HMGB1(disulfide), boxA(disulfide) and GST-boxA(red) were determined for the A-box binding Z-ABD polypeptides ZA3812 (SEQ ID NO:35) and ZA3914 (SEQ ID NO:36) (see Example 2), as well as for the HMGB1(red) interaction with the B-box binding polypeptide ZA3975, using a Biacore 2000 instrument (GE Healthcare).

[0267] The experiment was performed as a capture assay, in which human serum albumin (HSA, Novozymes, cat. no. 230-005) was immobilized on the carboxylated dextran layer of one CM5 chip surface (GE Healthcare, cat. no. BR100012) to immobilization levels of approximately 3000 RU. The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using HBS-EP (GE Healthcare, cat. no. BR100188) as running buffer. In a first set of experiments, Z-ABD polypeptides were analyzed under non-reducing conditions. One flow cell surface was activated and deactivated for use as blank during ligand and analyte injections. In the kinetic experiment, HBS-EP was used as running buffer and the flow rate was 30 μl/min. The ligand, i.e. the Z-ABD polypeptide, was diluted in HBS-EP buffer to a concentration of 30 nM and injected during 2 min. The interaction between HSA and ABD had a very slow off rate, resulting in very low dissociation during the following measurement.

[0268] The analyte, i.e. HMGB1(disulfide) or boxA(disulfide), was diluted in HBS-EP buffer within a concentration range of 2.5 to 640 nM and injected during 6 min, followed by dissociation in running buffer for 12 min. After dissociation, the surfaces were regenerated with one injection of 10 mM HCl.

[0269] In a second set of experiments, ZA3812 and ZA3914 were analyzed under reducing conditions where 2 mM DTT was included in the sample and running buffer. Analyses were performed as described above, except that HMGB1(red) and GST-boxA(red) were used as targets.

[0270] In a third experiment, ZA3975 was analyzed for binding to HMGB1(red) under reduced conditions (1.5 mM DTT included in the sample and running buffer). The ligand ZA3975 was diluted in HBS-EP buffer to a concentration of 100 nM and injected during 4 min. The analyte, HMGB1(red), was diluted in HBS-EP buffer to a concentration within the range of 8.3 to 675 nM and injected during 4 min, followed by dissociation in running buffer for 20 min. After dissociation, the surfaces were regenerated with three injections of 10 mM HCl.

[0271] Kinetic constants were calculated from sensorgrams using the BiaEvaluation software 4.1 (GE Healthcare). The Langmuir 1:1 model was used for fitting of all data except for HMGB1(disulfide), where a model for heterogeneous ligand parallel reactions better represented the experimental data and bulk was set to zero. Double referencing was applied to all data prior to kinetic fit, i.e. responses obtained from a blank surface and from a blank cycle where buffer was injected instead of analyte were subtracted.

[0272] In vitro cell migration assay: Mouse 3T3 fibroblast cells were used to assess the ability of His.sub.6-tagged Z variants to inhibit migration of cells in response to HMGB1 in modified Boyden Chambers (Neuroprobe Inc.). Mouse 3T3 fibroblasts were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco) with 10% fetal bovine serum (FBS, Gibco), 1% L-glutamine and antibiotics (Lonza). Prior to use, cells were centrifuged and the pellet was resuspended in DMEM (without FBS) without further washing. Polycarbonate filters (Neuroprobe Inc.; #PFA8) with 8 μm pores were coated with human fibronectin (Roche). Serum-free DMEM (negative control), DMEM containing 1 nM HMGB1(red) (positive control), and DMEM containing 1 nM HMGB1(red) plus Z variants ZH 01 (SEQ ID NO:1) and ZH 31 (SEQ ID NO:31) at a concentration of 10 or 100 ng/ml, were placed in the lower wells. A control polypeptide, Z04726 (SEQ ID NO:54), binding an irrelevant target, was used as negative control. boxA(red) (SEQ ID NO:51), a known competitor of HMGB1, was used at 1 nM concentration as positive control for inhibition of HMGB1 induced cell migration. Fibronectin-coated filters were placed so as to separate the lower and the upper wells, and 50000 3T3 cells were placed in the upper wells. The chambers were kept at 37° C. in a 5% CO.sub.2, humidified incubator for 3 h. The cells were then fixed with 100% ethanol (Sigma) and stained 20 minutes with Giemsa Stain (Sigma). The cells on the lower side of the filter were counted with a Leica DM LS2 microscope at 400× magnification.

[0273] ZH 30 was tested in a migration assay by performing an extended titration to allow IC50 calculations with 0.2 nM HMGB1(red) and lower concentrations of Z variants (0.1 ng/ml, 0.3 ng/ml, 1 ng/ml). A control polypeptide, ZZA4014 (SEQ ID NO:49), binding an irrelevant target and cloned and produced as described in Example 4, was used as negative control in this set up.

Results

[0274] CD analysis: The CD spectra determined for five HMGB1 binding Z variants with a His.sub.6 tag showed that all variants have an α-helical structure at 20° C. as judged from the typical minima at 208 and 222 nm. Reversible folding was seen for all Z variants when spectra measured before and after heating to 80-90° C. were superimposed. The melting temperatures (Tm) are summarized in Table 6.

TABLE-US-00014 TABLE 6 Melting temperatures (Tm) Z variant SEQ ID NO Tm (° C.) ZH01 1 49 ZH02 2 58 ZH03 3 54 ZH30 30 53 ZH31 31 57

[0275] Biacore kinetic and specificity analysis: The interactions of the A-box binding Z-ABD polypeptides ZA3812 (SEQ ID NO:35) and ZA3914 (SEQ ID NO:36) with four different HMGB1 variants were analyzed in a Biacore instrument by injecting various concentrations of HMGB1 variants over a surface containing ZA3812 and ZA3914, respectively, captured on immobilized HSA. Both ZA3812 and ZA3914 showed binding to all four tested HMGB1 variants. In addition, the interaction of the B-box binding Z-ABD polypeptide ZA3975 (SEQ ID NO:37) was analyzed for binding to HMGB1(red) in the same manner.

[0276] A summary of the estimated (based on data from four concentrations within the range 2.5 to 640 nM of injected HMGB1 variant for ZA3812 and ZA3914; or from two concentrations, 8.3 and 25 nM, for ZA3975) kinetic parameters (k.sub.a and k.sub.d) and affinity constants (K.sub.D) for binding of ZA3812, ZA3914 and ZA3975 to HMGB1 variants, obtained using a 1:1 interaction model or a heterogeneous ligand parallel reactions model, is given in Table 7. Resulting sensorgrams for the A-box binding polypeptides are displayed in FIGS. 2A and 2B.

TABLE-US-00015 TABLE 7 Kinetic parameters and affinity constants Z-ABD k.sub.a1 k.sub.d1 k.sub.a2 k.sub.d2 K.sub.D1 K.sub.D2 variant Analyte (1/Ms) (1/s) (1/Ms) (1/s) (M) (M) ZA3812 HMGB1(disulfide) 1.6 × 10.sup.5 1.8 × 10.sup.−2 1.3 × 10.sup.4 3.5 × 10.sup.−5 1.1 × 10.sup.−7 2.7 × 10.sup.−9 ZA3812 boxA(disulfide) 1.5 × 10.sup.5 2.1 × 10.sup.4  1.4 × 10.sup.−9 ZA3812 HMGB1(red) 5.0 × 10.sup.5 4.0 × 10.sup.−4 .sup. 8.1 × 10.sup.−10 ZA3812 GST-boxA(red) 7.2 × 10.sup.4 7.6 × 10.sup.−5 1.1 × 10.sup.−9 ZA3914 HMGB1(disulfide) 1.8 × 10.sup.5 2.5 × 10.sup.−2 1.4 × 10.sup.4 9.5 × 10.sup.−5 1.4 × 10.sup.−7 6.8 × 10.sup.−9 ZA3914 boxA(disulfide) 1.7 × 10.sup.5 1.5 × 10.sup.−4 0.9 × 10.sup.−9 ZA3914 HMGB1(red) 1.8 × 10.sup.5 1.1 × 10.sup.−3 6.1 × 10.sup.−9 ZA3914 GST-boxA(red) 1.2 × 10.sup.5 1.0 × 10.sup.−4 0.8 × 10.sup.−9 ZA3975 HMGB1(red) 2.5 × 10.sup.6 2.8 × 10.sup.−4 .sup. 1.1 × 10.sup.−10

[0277] In vitro cell migration assay: ZH 01, binding to HMGB1 A-box, as well as ZH 31 and ZH 30, both binding to HMGB1 B-box, were shown to inhibit HMGB1 induced cell migration, whereas no inhibition was seen for the negative control Z variants Z04726 and ZZA4014 (FIG. 3A-3C).

Example 4

Design and Production of Homo- and Heterodimers in Fusion with ABD

Summary

[0278] Five different heterodimeric polypeptides were designed, in which the A-box binding Z variant ZH 01 (SEQ ID NO:1) and the B-box binding Z variant ZH 31 (SEQ ID NO:31) were combined into a trispecific polypeptide targeting two different domains of HMGB1 and albumin. The order of Z variants with respect to one another and to ABD was varied. Different linkers were also evaluated. Furthermore, three homodimeric polypeptides based on ZH 01 were constructed. This Example describes the general procedure for subcloning and production of the formatted homo- and heterodimeric polypeptides in fusion with ABD, which are used throughout the characterization experiments that follow.

Materials and Methods

[0279] Subcloning of formatted polypeptides in fusion with ABD: The N-terminus of respective Z variant was cloned with either VD or AE in amino acid positions 1 and 2, as described previously for monomeric Z variants. The subcloning of the homo- and heterodimeric constructs was performed using standard molecular biology techniques. The Z variants were subcloned into an expression vector containing DNA encoding the ABD variant PP013 (SEQ ID NO:34), The constructs encoded by the expression vectors were [ZH ##]-GAP(G.sub.4S).sub.3TS-[ZH ##]-ASGS-PP013, [ZH ##]-GAP(G.sub.4S)TS-[ZH ##]-ASGS-PP013 and [ZH ##]-ASGS-PP013-GT(G.sub.4S)-[ZH ##], where the linkers GAP(G.sub.4S).sub.3TS, GAP(G.sub.4S)TS and GT(G.sub.4S) are referred to in the following as L1, L2 and L3, respectively. An overview of the designed homo- and heterodimeric polypeptides is shown in Table 8.

TABLE-US-00016 TABLE 8 Homo- and heterodimeric polypeptides SEQ A-box B-box ID binding binding Designation NO Z Z Format ZZA4010 38 ZH01 ZH31 Z(boxA)-L1-Z(boxB)-ABD ZZA4011 39 ZH01 ZH31 Z(boxA)-L2-Z(boxB)-ABD ZZA4012 40 ZH01 ZH31 Z(boxB)-L1-Z(boxA)-ABD ZZA4013 41 ZH01 ZH31 Z(boxB)-L2-Z(boxA)-ABD ZAZ4015 42 ZH01 ZH31 Z(boxA)-ABD-L3-Z(boxB) ZZA4053 43 ZH27 — Z(boxA)-L1-Z(boxA)-ABD ZZA4054 44 ZH27 — Z(boxA)-L2-Z(boxA)-ABD ZAZ4057 45 ZH27 — Z(boxA)-ABD-L3-Z(boxB) ZZA4261 46 ZH27 ZH32 Z(boxA)-L2-Z(boxB)-ABD ZZA4262 47 ZH27 ZH32 Z(boxB)-L1-Z(boxA)-ABD ZZA4263 48 ZH27 ZH32 Z(boxB)-L2-Z(boxA)-ABD .sup. ZZA4014.sup.1 49 — — Z03638-L2-Z3638-ABD .sup.1negative control

[0280] Production of homo- and heterodimeric polypeptides: E. coli T7E2 cells were transformed with plasmids containing the gene fragment of each respective homo- and heterodimeric polypeptide. The cultivation and purification were carried out essentially as described for Z-ABD polypeptides in Example 2.

Results

[0281] Production of homo- and heterodimeric polypeptides: The polypeptides were successfully cloned and expressed as soluble gene products in E. coli. Each DNA construct was verified by DNA sequencing. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the expected polypeptide. The correct identity and molecular weight of each polypeptide were confirmed by HPLC-MS analysis.

Example 5

Characterization of Homo- and Heterodimeric Polypeptides

Summary

[0282] In this Example, the homo- and heterodimeric polypeptides produced as described in Example 4 were characterized in vitro in terms of binding profile and functional properties. The profile of the homo- and heterodimeric polypeptides' binding to HMGB1 was analyzed using an A200® biosensor (Attana, Stockholm, Sweden). Furthermore, the ability of the polypeptides to functionally block HMGB1-induced mechanisms was investigated using the in vitro cell migration assay described for Z-ABD polypeptides in Example 3.

Materials and Methods

[0283] Binding analysis: The Attana A200® biosensor was used to characterize interactions between the dimeric polypeptides (SEQ ID NO:38-48) and HMGB1(red) under reducing conditions and/or HMGB1(disulfide) under non-reducing conditions. The monomeric ZA3812 (SEQ ID NO:35) was included for comparison. The experiment was performed as a capture assay, in which HSA was immobilized on Attana's sensor chip Low-NonSpecific-Binding (LNB). Immobilization was performed using amine coupling. The instrument has two sensor surfaces and HSA was immobilized on both surfaces. Around 110 Hz of HSA was stably immobilized on the surfaces.

[0284] In the kinetic experiment, HBS-EP+/−2 mM DTT was used as running buffer and the flow rate was 25 μl/min at 22° C. In each cycle, one polypeptide was tested against one concentration of HMGB1. The ligand, i.e. the HMGB1 binding polypeptide, was diluted in HBS-EP (+/−DTT) buffer to a concentration of 0.5 μg/ml and injected for 84 s over the HSA surface in channel A. The interaction between HSA and ABD had a very slow off-rate, resulting in a very low dissociation during the measurement. The analyte HMGB1(reduced) was diluted in HBS-EP+2 mM DTT within a concentration range of 1.33 to 21.4 μg/ml, and the analyte HMGB1(disulfide) was diluted in HBS-EP buffer within a concentration range of 0.62 to 10 μg/ml. Both analytes were subsequently injected for 84 s over both channel A and B, where channel B was used as a blank surface for curve subtraction. Buffer injections were also made for surface subtractions. After dissociation, the surfaces were regenerated with a 30 s injection of 100 mM HCl followed by a 30 s injection of 20 mM NaOH. Data was collected by Attester Software and subsequently processed in the Evaluation Software. To calculate the kinetic parameters (k.sub.a and k.sub.d) and affinity constant (K.sub.D), the experimental data were fitted using a 1:1 or a 1:2 binding model.

[0285] In vitro cell migration assay: The migration assay was performed essentially as described for monomeric Z variants in Example 3. One monomeric Z-ABD polypeptide, ZA3812 (SEQ ID NO:35); five heterodimeric polypeptides, ZZA4010 (SEQ ID NO:38), ZZA4011 (SEQ ID NO:39), ZZA4012 (SEQ ID NO:40), ZZA4013 (SEQ ID NO:41) and ZAZ4015 (SEQ ID NO:42); and three homodimeric polypeptides, ZZA4053 (SEQ ID NO:43), ZZA4054 (SEQ ID NO:44) and AZ4057 (SEQ ID NO:45), were tested in a migration assay by performing an extended titration, to allow IC50 calculations with 0.2 nM HMGB1 and reducing doses of polypeptides (0.1 nM, 0.3 nM, 1 nM). ZZA4014 (SEQ ID NO:49), binding an irrelevant target, was included as a negative control.

Results

[0286] Binding analysis of dimeric polypeptides targeting HMGB1: The interactions of a set of dimeric polypeptides, as well as one monomeric polypeptide, with HMGB1(reduced) and/or HMGB1(disulfide) were analyzed using an Attana A200® biosensor instrument by injecting various concentrations of HMGB1 over a surface containing captured polypeptides in fusion with ABD bound by immobilized HSA. All tested polypeptides showed binding to both HMGB1 redox forms. A summary of the approximate (based on data from five concentrations within the range 0.62 to 21 nM, of injected HMGB1) kinetic parameters (k.sub.a and k.sub.d) and affinity constant (K.sub.D), obtained by fitting the experimental data using a 1:1 or a 1:2 binding model, are given in Table 9.

TABLE-US-00017 TABLE 9 Summary of kinetic parameters of HMGB1-binding polypeptides Average k.sub.d.sup.1 Average Kp SEQ k.sub.d.sup.1 K.sub.D HMGB1 HMGB1 ID HMGB1 HMGB1 (disulfide) (disulfide) Designation NO (red) (s.sup.−1) (red) (M) (s.sup.−1) (M) ZA3812 35 1.3 × 10.sup.−2 5.1 × 10.sup.−8 4.8 × 10.sup.−4 1.3 × 10.sup.−7 ZZA4010 38 5.5 × 10.sup.−4 4.4 × 10.sup.−9 1.0 × 10.sup.−3 7.0 × 10.sup.−8 ZZA4011 39 4.9 × 10.sup.−4 4.2 × 10.sup.−9 1.1 × 10.sup.−3 1.1 × 10.sup.−7 ZZA4012 40 4.6 × 10.sup.−4 3.0 × 10.sup.−9 4.5 × 10.sup.−4 1.8 × 10.sup.−8 ZZA4013 41 4.2 × 10.sup.−4 3.2 × 10.sup.−9 6.6 × 10.sup.−4 3.2 × 10.sup.−8 ZAZ4015 42 4.0 × 10.sup.−4 2.0 × 10.sup.−8 1.3 × 10.sup.−3 3.5 × 10.sup.−8 ZZA4053 43 1.3 × 10.sup.−2 9.5 × 10.sup.−8 6.2 × 10.sup.−4 9.7 × 10.sup.−8 ZZA4054 44 1.3 × 10.sup.−2 1.5 × 10.sup.−7 6.3 × 10.sup.−4 9.3 × 10.sup.−8 ZAZ4057 45 9.6 × 10.sup.−3 2.1 × 10.sup.−8 6.5 × 10.sup.−4 3.1 × 10.sup.−8 ZZA4261 46 n.a. n.a. 1.4 × 10.sup.−3 1.1 × 10.sup.−8 ZZA4262 47 n.a. n.a. 9.8 × 10.sup.−4 5.4 × 10.sup.−9 ZZA4263 48 n.a. n.a. 9.5 × 10.sup.−4 7.6 × 10.sup.−9 .sup.1Relevant K.sub.d (the most represented in %) n.a. Not analyzed

[0287] In vitro cell migration assay with dimeric polypeptides: Homo- and heterodimeric polypeptides were analyzed for their ability to inhibit HMGB1 induced cell migration. IC50 values were calculated from experimental data and are shown in Table 10. With this experimental set up, the lowest detection limit for IC50 values is 0.1 nM. The negative homodimeric control polypeptide ZZA4014 (SEQ ID NO:49) had no effect on HMGB1 induced migration.

TABLE-US-00018 TABLE 10 Summary of IC50 values polypeptides inhibiting HMGB1 induced cell migration Designation SEQ ID NO IC50 (nM) ZA3812  35 ≤0.1 ZZA4010 38 0.1-0.3 ZZA4011 39 ≤0.1 ZZA4012 40 ≤0.1 ZZA4013 41 0.2 ZAZ4015 42 >1 ZZA4053 43 0.3-1   ZZA4054 44 >1 ZAZ4057 45 >1

Example 6

Alanine Scan of HMGB1-Binding Z Variant

Summary

[0288] In this Example, alanine scanning mutagenesis was used to analyze the individual contribution from residues in ZH 01 to the interaction with HMGB1. Each mutated Z variant was analyzed by circular dichroism (CD) spectroscopy by measuring the melting temperature and secondary structure content to ensure preserved structure and stability properties.

Materials and Methods

[0289] Construction of single-point mutation variants of ZH 01 for alanine scan: Single-point mutations of ZH 01 (SEQ ID NO:1) with codon substitution to alanine at residues 9, 10, 11, 13, 14, 17, 18, 24, 27, 28, 32 or 35 were designed. Residue A25 in ZH 01 was mutated to a serine. Synthesis and cloning of the designed Z variants (SEQ ID NO:14-26; FIG. 1) were ordered from DNA2.0 (Newark, Calif.). The Z gene fragments were subcloned into an expression vector resulting in the encoded sequence MGSSHHHHHHLQ-[ZH ##]-VD.

[0290] Cultivation of mutated Z variants with His.sub.6-tag: E. coli T7E2 cells were transformed with plasmids containing the gene fragment of each respective mutated Z variant. The resulting recombinant strains were cultivated in media supplemented with 50 μg/ml kanamycin at 30° C. in 50 ml scale using the EnPresso protocol (BioSilta). In order to induce protein expression, IPTG was added to a final concentration of 0.2 mM at OD.sub.600≈10. After induction, the cultivations were incubated for 16 h. The cells were harvested by centrifugation.

[0291] Purification of mutated Z variants with a His.sub.6-tag: The purification and verification of Z variants were performed as described in Example 2.

[0292] Circular dichroism (CD) spectroscopy analysis: The measurements were performed essentially as described in Example 3.

[0293] Binding analysis of mutated Z variants: A binding analysis was performed using a Biacore 2000 instrument. All alanine mutated Z variants, together with the original Z variant ZH 01, were analyzed against GST-boxA(red), HMGB1(red), boxA(disulfide) and HMGB1(disulfide). The immobilizations and Biacore analyses were performed essentially as described in Example 3. However, in these analyses, the different targets were directly coupled to separate flow cells of different CM5 chip surfaces and a flow rate of 30 μl/min was used. The ligand immobilization levels on the surfaces were 1410 RU for GST-boxA(red), 1380 RU for HMGB1(red), 540 RU for boxA(disulfide) and 2900 RU for HMGB1(disulfide). The analytes, i.e. the Z variants, were each diluted in HBS-EP buffer (or in HBS-EP+2 mM DTT for use in analysis with reduced targets GST-boxA(red) and HMGB1(red)), within a concentration range of 50 to 1350 nM and injected for 3 min, followed by dissociation in running buffer for 35 min. No regeneration buffer was used in the analyses. Instead, the dissociation time was enough for all binders to dissociate from the chip surfaces. HBS-EP was used as running buffer for targets boxA(disulfide) and HMGB1(disulfide) while HBS-EP+2 mM DTT was used for reduced targets GST-boxA(red) and HMGB1(red). The dissociation constants were calculated as in Example 3.

Results

[0294] Production of mutated Z variants: The mutated Z variants with His.sub.6-tag were successfully cloned and expressed as soluble gene products in E. coli. Each DNA construct was verified by DNA sequencing. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the desired Z variant. The correct identity and molecular weight of each mutated Z variant were confirmed by HPLC-MS analysis.

[0295] CD analysis: The CD spectra and melting temperatures (Tm) for 13 mutated Z variants with His.sub.6-tag was analyzed as described in Example 3. The secondary structure contents and refolding properties showed to be unaffected by the introduction of a point mutation. However, the thermal stability was affected to various extents, resulting in a shift in melting temperature with +/−10° C. compared to the original Z variant ZH 01. The melting temperatures (Tm) are summarized in Table 11.

TABLE-US-00019 TABLE 11 Melting temperatures (Tm) of mutated Z variants Designation SEQ ID NO Mutation Tm (° C.) ZH01 1 — 49 ZH14 14 D9A 59 ZH15 15 W10A 44 ZH16 16 D11A 50 ZH17 17 F13A 43 ZH18 18 W14A 41 ZH19 19 IVA 42 ZH20 20 W18A 55 ZH21 21 D24A 53 ZH22 22 A25S 46 ZH23 23 Q27A 47 ZH24 24 R28A 47 ZH25 25 Y32A 56 ZH26 26 E35A 56

[0296] Binding analysis of mutated Z variants: The interaction of HMGB1 with ZH 01 and the 13 mutated variants thereof was analyzed in a Biacore instrument by injecting various concentrations of the Z variants over surfaces containing immobilized GST-boxA(red), HMGB1(red), boxA(disulfide) and HMGB1(disulfide), respectively. A summary of the dissociation constants from the experiments, which were obtained by using a 1:1 interaction model, is given in Table 12.

TABLE-US-00020 TABLE 12 Dissociation constants for binding of Z variants to GST- boxA(red), HMGB1(red), boxA(disulfide) and HMGB1(disulfide) GST- boxA(di- HMGB1(di- Z SEQ boxA(red) HMGB1(red) sulfide) sulfide) variant ID NO Mutation K.sub.D (M) K.sub.D (M) K.sub.D (M) K.sub.D (M) ZH01 1 — 2.5 × 10.sup.−8 1.2 × 10.sup.−8 3.8 × 10.sup.−7 1.7 × 10.sup.−7 ZH14 14 D9A 3.6 × 10.sup.−8 1.8 × 10.sup.−8 7.7 × 10.sup.−7 1.5 × 10.sup.−7 ZH15 15 W10A n.d. 5.7 × 10.sup.−7 n.d. 1.2 × 10.sup.−5 ZH16 16 D11A 2.1 × 10.sup.−8 1.0 × 10.sup.−8 6.6 × 10.sup.−7 1.3 × 10.sup.−7 ZH17 17 F13A n.d. n.d. n.d. 6.8 × 10.sup.−6 ZH18 18 W14A 3.7 × 10.sup.−7 2.9 × 10.sup.−7 n.d. 2.2 × 10.sup.−6 ZH19 19 I17A n.d. n.d. n.d. 3.1 × 10.sup.−6 ZH20 20 W18A 1.3 × 10.sup.−7 9.0 × 10.sup.−8 5.0 × 10.sup.−5 1.5 × 10.sup.−6 ZH21 21 D24A 1.1 × 10.sup.−7 6.4 × 10.sup.−8 1.5 × 10.sup.−6 6.7 × 10.sup.−7 ZH22 22 A25S 1.7 × 10.sup.−8 9.0 × 10.sup.−9 3.3 × 10.sup.−7 1.9 × 10.sup.−7 ZH23 23 Q27A 6.7 × 10.sup.−8 4.1 × 10.sup.−8 7.9 × 10.sup.−7 8.7 × 10.sup.−7 ZH24 24 R28A n.d. 6.8 × 10.sup.−7 n.d. 7.8 × 10.sup.−6 ZH25 25 Y32A n.d. n.d. n.d. 7.8 × 10.sup.−6 ZH26 26 E35A 2.2 × 10.sup.−7 1.1 × 10.sup.−7 n.d. 2.4 × 10.sup.−6 n.d. not determined due to low response.

Example 7

Design and Construction of a Maturation Library of HMGB1 A-Box Binding Z Variants

Summary

[0297] In this Example, a new library was designed based on the HMGB1 A-box binding variant ZH 01, as well as on the result from the alanine scan described in Example 6. The maturation library contained approximately 1×10.sup.10 individual clones.

Materials and Methods

[0298] Design of an affinity maturation HMGB1 A-box library: A new library was designed, in which 13 positions of the Z variant molecules were biased towards amino acid residues based on the sequence of ZH 01 (SEQ ID NO:1). Each position was randomized allowing amino acid residues A, D, E, F, H, I, K, L, N, Q, R, S, T, Y, V, W (excluding C, G, M and P in all positions), with the exception of position 24 in the Z molecule in which amino acid G was also allowed. The amino acid residues based on the sequence of the HMGB1 binding Z variant ZH 01 were spiked at a higher proportion to generate an average mutation frequency of approximately four mutations per molecule. The randomization frequency in each position was also normalized with the results from the alanine scan described above, resulting in less mutations in positions important for HMGB1 binding or for thermal stability and more mutations in positions of less importance (Table 13).

TABLE-US-00021 TABLE 13 The design of HMGB1 A-box maturation library. The percentages of the amino acids used in each of the 13 randomized library positions are indicated Position in BM 2 3 4 6 7 10 11 17 18 20 21 25 28 Position in full length Z variant Codon 9 10 11 13 14 17 18 24 25 27 28 32 35 A Ala 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 53.0 3.1 1.3 1.3 1.9 C Cys 0 0 0 0 0 0 0 0 0 0 0 0 0 D Asp 53.0 1.3 53.0 1.3 1.9 1.3 1.9 72.0 3.1 3.1 1.3 1.3 1.9 E Glu 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 3.1 1.3 1.3 72.0 F Phe 3.1 1.3 3.1 81.0 1.9 1.3 1.9 1.8 3.1 3.1 1.3 1.3 1.9 G Gly 0 0 0 0 0 0 0 1.8 0 0 0 0 0 H His 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 3.1 1.3 1.3 1.9 I Ile 3.1 1.3 3.1 1.3 1.9 81.0 1.9 1.8 3.1 3.1 1.3 1.3 1.9 K Lys 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 3.1 1.3 1.3 1.9 L Leu 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 3.1 1.3 1.3 1.9 M Met 0 0 0 0 0 0 0 0 0 0 0 0 0 N Asn 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 3.1 1.3 1.3 1.9 P Pro 0 0 0 0 0 0 0 0 0 0 0 0 0 Q Gln 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 53.0 1.3 1.3 1.9 R Arg 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 3.1 81.0 1.3 1.9 S Ser 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 3.1 1.3 1.3 1.9 T Thr 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 3.1 1.3 1.3 1.9 V Val 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 3.1 1.3 1.3 1.9 W Trp 3.1 81.0 3.1 1.3 72.0 1.3 72.0 1.8 3.1 3.1 1.3 1.3 1.9 Y Tyr 3.1 1.3 3.1 1.3 1.9 1.3 1.9 1.8 3.1 3.1 1.3 81.0 1.9

[0299] Two oligonucleotides, one forward and one reverse complementary, with complementary 3′-ends were generated using TRIM technology. These oligos were ordered from Ella Biotech GmbH (Martinsried, Germany).

[0300] The construction of the library was performed essentially as described earlier (e.g. PCT publication WO2017/072280) in a vector denoted pAY03894, but with the exception that after transformation, the cells were pooled and cultivated in 3 l medium TSB-YE medium, supplemented with 10 μg/ml tetracycline and 100 μg/ml ampicillin. Furthermore, the vector pAY03894 differs from the earlier described library vector in three amino acid positions as follows: Y5F, N52S and D53E, thus including these mutations in the expressed protein Z variants displayed on the phage. The library quality and the distribution of amino acids were verified by sequencing as described in WO2009/077175.

[0301] Preparation of phage stock: Cells from a glycerol stock containing the phagemid library were inoculated into 10 l cultivation 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; 0.1 ml/l Breox FMT30 antifoaming agent, supplemented with 25 μg/ml carbenicillin, 10 μg/ml tetracycline, 5 ml/l of 1.217 M MgSO.sub.4 and 10 ml of a trace element solution [194 mM FeCl.sub.3; 55 mM ZnSO.sub.4; 10.6 mM CuSO.sub.4; 62.5 mM MnSO.sub.4; 47 mM CaCl.sub.2, dissolved in 1.2 M HCl]. pH was controlled to 7 through the automatic addition of 25% NH.sub.4OH, air was supplemented (10 l/min), and the stirrer was set to keep the dissolved oxygen level above 30%. When the cells reached an optical density at 600 nm (OD.sub.600) of 0.50, the cultivation was infected using a 10× molar excess of M13K07 helper phage. The cells were incubated for 30 min. Then expression was induced by the addition of IPTG to a concentration of 100 μM. 1 h after induction, the cultivation was supplemented with 25 μg/ml kanamycin, and a glucose-limited fed-batch cultivation was started where a 600 g/l glucose solution was fed to the reactor (15 g/h at the start, 40 g/h after 20 h and to the end of the cultivation). The cultivation was harvested 24 h after the addition of helper phages. The cells in the cultivation were removed by centrifugation (15,900×g, 50 min). The phage particles were precipitated from the supernatant twice in PEG/NaCl, filtered and dissolved in PBS and glycerol as described in Example 1. Phage stocks were stored at −80° C. until use in selection.

Results

[0302] Library construction: The new library was designed based on the HMGB1 binding variant ZH 01 (see Examples 1-3) as well as on the result from the alanine scan described in Example 6. The theoretical size of the designed library was 4.7×10.sup.7Z variants. The actual size of the library, determined by titration after transformation to E. coli. ER2738 cells, was 1.3×10.sup.10 transformants. The library quality was tested by sequencing of 96 transformants and by comparing their actual sequences with the theoretical design. Sequence analysis of individual library members verified a distribution of codons in accordance with the theoretical design.

Example 8

Selection and Screening of Affinity Matured HMGB1 A-Box Binding Z Variants

Summary

[0303] In this Example GST-boxA(red), biotinylated HMGB1(red), alternating targets HMGB1(disulfide) and HMGB1(red), and biotinylated boxA(disulfide) were used as targets in phage display selections using an HMGB1 A-box maturation phage library of Z variants. Selected clones were DNA sequenced, produced in E. coli periplasmic fractions and assayed against different target proteins in ELISA and Biacore.

Materials and Methods

[0304] Biotinylation of proteins: GST-boxA(red), HMGB1(red), boxA(disulfide) and a mouse monoclonal anti-HMGB1(C-terminal part) antibody (DPH1.1, HMGBiotech cat. no. HM-904) were biotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin as described in Example 1.

[0305] Phage display selection of HMGB1 A-box binding Z variants: Phage display selection was performed using a phage stock of the newly produced A-box maturation library. Selections against biotinylated GST-boxA(red), biotinylated HMGB1(red), alternating targets HMGB1(disulfide) and HMGB1(red), and biotinylated boxA(disulfide) were performed in four cycles. In track 1, selection was performed in solution and SA beads were used to catch the b-GST-boxA(red):Z-variant complexes. In track 2, selection was performed on solid phase where SA beads were used to catch the HMGB1(red) prior to selection. In track 3, an alternating target strategy was used. Selection was either performed in solution where SA beads with pre-immobilized b-anti-HMGB1 antibody were used to catch the HMGB1(disulfide) or on solid phase where SA beads were used to catch the HMGB1(red) prior to selection. In track 4 with descendants, selection was performed on solid phase where SA beads were used to catch the b-boxA(disulfide) prior to selection.

[0306] As selection proceeded, the tracks were further divided according to target concentration and number and/or time of washes. Phage stock preparation, selection procedure and amplification of phage between selection cycles were performed essentially as described in Example 1. The selection buffer consisted of either PBST 0.1% supplemented with 10% FBS and 2 mM DTT (tracks 1, 2 and track 3 (in cycles using HMGB1(red)) or PBST 0.1% supplemented with 10% FBS and 1.5 μM HSA (tracks 3 (in cycles using HMGB1(disulfide)) and 4). No pre-selection was performed. The washing steps were performed either during 1 min, 1 h, 4 h or overnight and with or without addition of a non-biotinylated version of the target used during the selection. An overview of the selection strategy, describing an increased stringency in subsequent cycles, using a lowered target concentration and an increased number of washes, is shown in Table 14.

TABLE-US-00022 TABLE 14 Overview of the selection against HMGB1 using the A-box maturation library Phage Conc. of stock from Target Number Duration free target Selection library or conc. of of last in last Cycle track selection track Target protein (nM) washes wash (h) wash (nM) 1 1 A-box matlib b-GST-boxA(red) 100 2 — — 1 2 A-box matlib b-HMGB1(red) 100 2 — — 1 3 A-box matlib HMGB1(disulfide) 10 2 — — 1 4 A-box matlib b-boxA(disulfide) 100 2 — — 2 1-1 1 b-GST-boxA(red) 10 5 1 — 2 2-1 2 b-HMGB1(red) 10 5 1 — 2 3-1 3 b-HMGB1(red) 1 5 1 — 2 3-2 3 HMGB1(disulfide) 1 5 1 — 2 4-1 4 b-boxA(disulfide) 10 4 — — 3 1-1-1 1-1 b-GST-boxA(red) 1 6 1 100 3 2-1-1 2-1 b-HMGB1(red) 1 6 1 100 3 3-1-1 3-1 HMGB1(disulfide) 0.2 6 — — 3 3-2-1 3-2 HMGB1(disulfide) 0.2 6 — — 3 4-1-1 4-1 b-boxA(disulfide) 1 6 1 100 4 1-1-1-1 1-1-1 b-GST-boxA(red) 1 10 4 100 4 1-1-1-2 1-1-1 b-GST-boxA(red) 1 10 ON 100 4 2-1-1-1 2-1-1 b-HMGB1(red) 1 6 4 100 4 2-1-1-2 2-1-1 b-HMGB1(red) 1 6 ON 100 4 3-1-1-1 3-1-1 b-HMGB1(red) 1 6 4 100 4 3-1-1-2 3-1-1 b-HMGB1(red) 1 6 ON 100 4 3-2-1-1 3-2-1 HMGB1(disulfide) 0.2 6 4 — 4 3-2-1-2 3-2-1 HMGB1(disulfide) 0.2 6 ON — 4 4-1-1-1 4-1-1 b-boxA(disulfide) 1 6 4 100 4 4-1-1-2 4-1-1 b-boxA(disulfide) 1 6 ON 100 ON = overnight

[0307] Production of Z variants in periplasmic fractions: The Z variants were produced and the periplasmic fraction of each individual variant was prepared as described in Example 1, with the exception that periplasmic fractions were obtained by heating the bacterial suspensions to 70° C. during 15 min. The final supernatant of the periplasmic extract contained the Z variants as fusions to ABD, expressed as AQHDEALE-[ZH ##]-VDYV-[ABD]-YVPG (SEQ ID NO:131).

[0308] ELISA screening of Z variants: The binding of Z variants to HMGB1 was analyzed in ELISA assays. Each Z variant was analyzed against the target used in the selection that it was discovered in. The ELISA was performed essentially as described in Example 1. When GST-boxA(red) or HMGB1(red) were used as target, PBSC+0.5 mM DTT was used for dilution of reagents in the two last steps to ensure reduced conditions during the binding event. Target concentrations used in the assay were 2 nM of b-GST-boxA(red), 10 nM of b-HMGB1(red), 100 nM HMGB1(disulfide) and 10 nM b-boxA(disulfide), respectively. As negative control, a periplasmic fraction containing the fusion protein ABD with no Z fusion partner, i.e. AQHDEALEVDYV-[ABD]-YVPG (SEQ ID NO:132), was used. Periplasm samples containing the primary HMGB1 binding Z variant ZH 01 (SEQ ID.NO:1) in fusion with ABD was included on each plate and analyzed as positive control. The absorbance at 450 nm was measured using a multi-well plate reader, EnSpire (Perkin Elmer).

[0309] Sequencing: In parallel with the ELISA screening, all clones were sequenced. PCR fragments were amplified from single colonies, sequenced and analyzed as described in Example 1.

[0310] Biacore screening of Z variants: The binding of Z variants to boxA(disulfide) was analyzed in an affinity screening using a Biacore 2000 instrument. A polyclonal goat anti-ABD antibody (goat anti-ABD) was immobilized on CM5 chip surfaces basically as described for other proteins in Example 3. For the kinetic screening, analytes were injected in two steps. First, a Z-ABD (where ABD is GA3) periplasmic extract was injected over the surface at 5 μl/min for 10 min. As a second step, 200 nM boxA(disulfide) was injected at 20 μl/min for 5 min, followed by 10 min of dissociation in running buffer HBS-EP. Glycine-HCl pH 2.0 (GE Healthcare, cat. no. BR100355) was used for regeneration of the antibody surfaces between the cycles. The temperature of the assay was 25° C. Before performing the kinetic analyses, the signal from 200 nM boxA(disulfide) injected over a reference surface containing goat anti-ABD but no Z-ABD sample was subtracted from the sensorgram of Z-ABD binding to boxA(disulfide). Rough screening affinities (K.sub.D) were calculated from the reference subtracted 200 nM boxA(disulfide) response using a 1:1 binding model of the BiaEvaluation software. A periplasmic extract of ZH 01 was included in the screening analysis for comparison.

Results

[0311] Phage display selection of HMGB1 binding Z variants: Individual clones were obtained after four cycles of phage display selections against GST-boxA(red), biotinylated HMGB1(red), alternating targets HMGB1(disulfide) and HMGB1(red), and biotinylated boxA(disulfide).

[0312] ELISA screening of Z variants: Clones obtained after four cycles of selection were produced in 96-well plates and screened against target used in each selection, respectively. Z variants found to give an average response corresponding to 2×the negative control or higher were considered as positive clones and further subjected to DNA-sequencing.

[0313] Sequencing: Sequencing was performed for clones showing a positive response in the ELISA screen against target used in each selection. Each Z variant was given a unique identification number ##, and individual Z variants are referred to as ZH ##. Resulting unique amino acid sequences of the 58 amino acid residues long Z variants are listed in FIG. 1 and in the sequence listing as SEQ ID NO:4-13.

[0314] Biacore screening of Z variants: Seven new unique Z variants giving the highest response in ELISA-screen, ZH 04 (SEQ ID NO:4), ZH 05 (SEQ ID NO:5), ZH 06 (SEQ ID NO:6), ZH 07 (SEQ ID NO:7), ZH 08 (SEQ ID NO:8), ZH 09 (SEQ ID NO:9) and ZH 10 (SEQ ID NO:10) were submitted to a Biacore affinity screening together with ZH 01 (SEQ ID NO:1) and ZH 29 (SEQ ID NO:29; its binding motif is identical to that of ZH 01, but the sequence comprises scaffold amino acid variations V1A, D2E, Y5F, N52S and D53E). A single concentration of boxA(disulfide) was injected over each Z-ABD captured from periplasmic extracts on a sensor chip surface containing an anti-ABD antibody. The calculated screening affinities are presented in Table 15.

TABLE-US-00023 TABLE 15 Calculated approximate K.sub.D values from Biacore affinity screening Z variant SEQ ID NO K.sub.D (M) ZH04 4 5.4 × 10.sup.−8 (Note 1) ZH05 5 5.4 × 10.sup.−8 ZH06 6 7.1 × 10.sup.−8 ZH07 7 4.4 × 10.sup.−8 (Note 2) ZH29 29 3.6 × 10.sup.−8 (Note 3) ZH08 8 3.1 × 10.sup.−8 ZH09 9 9.1 × 10.sup.−8 ZH10 10 4.7 × 10.sup.−8 .sup. ZH01.sup.4 1 4.7 × 10.sup.−9 1) Average K.sub.D from 2 separate measurements 2) Average K.sub.D from 8 separate measurements 3) Average K.sub.D from 3 separate measurements 4) Differs in scaffold residues in positions 1 (V), 2 (D), 5 (Y), 52 (N) and 53 (D)

Example 9

Design and Construction of a Maturation Library of HMGB1 B-Box Binding Z Variants

Summary

[0315] In this Example, a new library was designed based on the HMGB1 B-box binding variants ZH 30 and ZH 31.

Materials and Methods

[0316] Design of an affinity maturation HMGB1 B-box library: The design of the library was based on the amino acid sequences of the HMGB1 B-box binding Z variants ZH 30 (SEQ ID NO:30) and ZH 31 (SEQ ID NO:31), identified and characterized as described in Example 1 and Example 3. In the new library, 13 variable positions in the Z molecule scaffold were either conserved to certain amino acid residues or randomized according to a strategy based on the sequences of the two Z variants. The design for each amino acid residue of the new library, including six variable amino acid positions (11, 18, 24, 25, 32, and 35) and seven constant amino acid positions (9, 10, 13, 14, 17, 27, and 28) in the Z molecule, are displayed in Table 16. The resulting theoretical library size was 3.4×10.sup.7 variants. In the theoretical design, an even distribution of the different amino acids was applied within each amino acid position.

TABLE-US-00024 TABLE 16 The design of HMGB1 B-box maturation library Amino acid position in the Z No of variant Randomization amino Pro- molecule (amino acid abbreviations) acids portion 9 A 1 1/1 10 W 1 1/1 11 A, D, E, F, G, H, I, K, L, M, 18  1/18 N, Q, R, S, T, V, W, Y 13 E 1 1/1 14 Q 1 1/1 17 W 1 1/1 18 A, D, E, F, G, H, I, K, L, M, 18  1/18 N, Q, R, S, T, V, W, Y 24 A, D, E, F, G, H, I, K, L, M, 18  1/18 N, Q, R, S, T, V, W, Y 25 A, D, E, F, G, H, I, K, L, M, 18  1/18 N, Q, R, S, T, V, W, Y 27 F 1 1/1 28 Q 1 1/1 32 A, D, E, F, G, H, I, K, L, M, 18  1/18 N, Q, R, S, T, V, W, Y 35 A, D, E, F, G, H, I, K, L, M, 18  1/18 N, Q, R, S, T, V, W, Y

[0317] Construction of an HMGB1 B-box maturation library: A maturation library for HMGB1 B-box binding Z variants is constructed in analogy to what is described above in Example 7, but using the design described in this Example. Two oligonucleotides, one forward and one reverse complementary, with complementary 3′-ends are generated, for example as described in Example 7. The two oligonucleotides are annealed and extended by PCR, using outer primers, to yield one gene fragment covering 147 bp corresponding to partially randomized helix 1 and 2 and flanked by restriction sites XhoI and SacI. The oligonucleotides are for example ordered from Ella Biotech GmbH. Construction of the library is performed essentially as described in Example 7, in a vector that is electroporated into E. coli that is subsequently cultivated. The library quality and distribution of amino acids are verified by sequencing as described in WO2009/077175.

[0318] Preparation of phage stock: Cells from a glycerol stock containing the phagemid library are cultivated and phage stock is produced and prepared as described in for example WO2017/072280 or Example 1 above. Phage stocks are stored at −80° C. until used in selection.

Expected Results

[0319] Design of an affinity maturation HMGB1 B-box library: The new library is designed based on the previously selected HMGB1 B-box binding variants ZH 30 and ZH 31. The theoretical size of the designed library is 3.4×10.sup.7Z variants.

[0320] Construction of an HMGB1 B-box maturation library: A library for maturation of HMGB1 B-box binding Z variants is constructed according to the design presented in this Example. The actual size of the library, determined by titration after transformation to E. coli. ER2738 cells, is for example 5×10.sup.8 transformants. The library quality is tested by sequencing of 96 transformants and by comparing their actual sequences with the theoretical design. Sequence analysis of individual library members verifies a distribution of codons in accordance with the theoretical design.

[0321] Preparation of phage stock: A phage stock of the newly constructed affinity maturation library of HMGB1 B-box binding Z variants is produced and stored at −80° C. until use in selection.

Example 10

Selection, Screening and Characterization of Affinity Matured HMGB1 B-Box Binding Z Variants

Summary

[0322] This Example describes the selection, screening and general procedure for subcloning and production of B-box binding Z variants, formatted as monomers and homo- or heterodimers, in fusion with ABD, to be used throughout in characterization experiments below.

Materials and Methods

[0323] Biotinylation of proteins: GST-boxB(red), HMGB1(red), and a mouse monoclonal anti-HMGB1(C-terminal part) antibody are biotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin as described in Example 1.

[0324] Phage display selection of HMGB1 B-box binding Z variants: Phage display selection is performed using a phage stock of the newly produced HMGB B-box affinity maturation library. For example, selections against biotinylated GST-boxB(red), biotinylated HMGB1(red), alternating targets HMGB1(disulfide) and HMGB1(red), and HMGB1(disulfide) are performed in four cycles. Selections are for example done as described in Example 8 with the proteins described above. SA beads are used to catch biotinylated proteins and the mouse monoclonal anti-HMGB1 antibody is used to catch non-biotinylated HMGB1 proteins.

[0325] As selections proceed, the tracks are further divided according to target concentration and number and/or time of washes. Phage stock preparation, selection procedure and amplification of phage between selection cycles are performed essentially as described in Example 1 or 8. The selection buffer used is either PBST 0.1% supplemented with 10% FBS and 2 mM DTT (in cycles using GST-boxB(red) or HMGB1(red)) or PBST 0.1% supplemented with 10% FBS and 1.5 μM HSA (in cycles using HMGB1(disulfide)). The washing steps are performed as described in Example 8 with or without addition of a non-biotinylated version of the target used during the selection.

[0326] Production of Z variants in periplasmic fractions: The Z variants are produced, and the periplasmic fraction of each individual variant is prepared as described in Example 1. The final supernatant of the periplasmic extract contains the respective Z variant as a fusion to ABD (GA3), expressed as AQHDEALE-[ZH ##]-VDYV-[ABD]-YVPG.

[0327] Sequencing: In parallel with the ELISA screening, all clones are sequenced. PCR fragments are amplified from single colonies, sequenced and analyzed as described in Example 1.

[0328] Screening of Z variants: The binding of Z variants to HMGB1 may for example be analyzed in ELISA assays against relevant proteins as described in Example 1 and/or in Biacore screening as described in Example 8.

[0329] Production and analysis of individual HMGB1 B-box binding Z variants: Z variants considered positive in screening assays are cloned as monomers, homodimers and/or heterodimers comprising for example a second B-box binding Z-variant or an A-box binding Z variant described within the scope of this invention, as well as in fusion with ABD. Production is performed as described in Example 2 and the obtained polypeptides are assayed for binding against relevant HMGB1 proteins using for example Biacore and ELISA, and further characterized in different in vitro and in vivo assays as described in Example 3.

Results

[0330] Phage display selections against for example biotinylated GST-boxB(red), biotinylated HMGB1(red), alternating targets HMGB1(disulfide) and HMGB1(red), and HMGB1(disulfide) are expected to generate new B-box binding Z variants with improved binding properties. Z variants with higher affinity for the B-box of HMGB1 are expected to show enhanced HMGB1 inhibiting potency in in vitro and in vivo models, alone or for example in combination with an A-box binding Z-variant.

Example 11

Binding Specificity Study

Summary

[0331] The high-mobility group box (HMGB) family includes four members: HMGB1, HMGB2, HMGB3 and HMGB4. HMGB1 shows 84%, 81% and 42% identity at the amino acid sequence level to HMGB2, HMGB3 and HMGB4, respectively. HMGB1 also shows 86% identity to the SP100 nuclear antigen. In this example, SPR was used to analyze the target binding affinity of ZZA4263 for HMGB1(red), HMGB1(disulfide), HMGB2, HMGB3 and SP100 as well as for tumor necrosis factor (TNF), interleukin-6 (IL-6) and interleukin-1β (IL-1β), the latter three being involved in the HMGB1 signaling pathway.

Materials and Methods

[0332] HSA (Albucult®, Novozyme) was diluted to 10 μg/ml in acetate, pH 4.0, and immobilized onto a CM5 sensor chip using a Biacore 2000 instrument. Immobilization was performed by amine chemistry, using amine coupling kit, according to the manufacturer's instructions. The first surface of each chip was activated and deactivated for use as a reference cell (blank surface) during analyte injections. HBS-EP was used as running buffer, supplemented with 1.5 mM DTT when analyzing the binding to HMGB1(red). ZZA4263 was injected at a concentration of 100 nM for 4 minutes over the HSA and the reference sensor chip surfaces at a constant flow rate of 30 μl/min. This was followed by 4 minutes' injection of 0, 8.3, 25, 75, 225 and 675 nM of HMGB1(red), HMGB1(disulfide), HMGB2 (Biorbyt, cat. no. orb180287), HMGB3 (Biorbyt, cat. no. orb180288), SP100 (Biorbyt, cat. no. orb169341), TNF (R&D Systems, cat. no. 210-TA), IL-6 (R&D Systems, cat. no. 206-IL) and IL-1β (R&D Systems, cat. no. 201-LB/CF), respectively. The proteins were allowed to dissociate for 20 minutes before the chip surfaces were regenerated by two 10 second pulses using 10 mM HCl. The reference surface and the buffer sample were subtracted from all curves. The buffer sample was subtracted to adjust for the dissociation of ZZA4263 from HSA. All curves were fitted to a kinetic 1:1 Langmuir model in the BIA Evaluation software to estimate the binding properties (k.sub.a, k.sub.d and K.sub.D).

Results

[0333] ZZA4263 was injected and captured on a surface with immobilized HSA followed by concentration series of HMGB1(red), HMGB1(disulfide), HMGB2, HMGB3, SP100, TNF, IL-6 and IL-1β. The highly homologous human proteins HMGB2, HMGB3, SP100 all bind to ZZA4263, although with 17-550 times lower affinity compared to HMGB1(red). A summary of the kinetic parameters is presented in Table 17. No significant binding was detected to any of the other tested proteins involved in the HMGB1 signaling pathway (TNF, IL-6 and IL-1β). Thus, ZZA4263 shows high specificity to HMGB1 and homologous proteins.

TABLE-US-00025 TABLE 17 Kinetic constants for ZZA4263 binding to HMGB1 and homologous proteins k.sub.a k.sub.d K.sub.D Analyte (1/Ms) (1/s) (M) HMGB1(red) 1.3 × 10.sup.6 1.5 × 10.sup.−4 .sup. 1.2 × 10.sup.−10 HMGB1 (disulfide) 4.9 × 10.sup.4 5.4 × 10.sup.−4 1.1 × 10.sup.−8 HMGB2 1.8 × 10.sup.5 3.6 × 10.sup.−4 2.0 × 10.sup.−9 HMGB3 2.0 × 10.sup.4 1.3 × 10.sup.−3 6.6 × 10.sup.−8 SP100 nuclear antigen 5.7 × 10.sup.3 2.2 × 10.sup.−4 3.8 × 10.sup.−8

Example 12

Inhibitory Effect of ZZA4263 in CXCR4 In Vitro Cell Assay

Summary

[0334] Blocking of the HMGB1-CXCL12 complex binding to CXCR4 is thought to be beneficial in tissue injury, where an excessive recruitment of leukocytes is supported by HMGB1 (Schiraldi et al (2012) J Exp Med 209:551-63). This Example describes analysis by the cAMP Hunter eXpress CXCR4 CHO-K1 GPCR assay, which includes cells that overexpress the receptor CXCR4. Forskolin-induced release of cAMP activates an enzymatic reaction that produces a luminescent signal, which is inhibited upon HMGB1-CXCL12 binding to CXCR4. The ability of the HMGB1-binding polypeptide ZZA4263 to reverse the inhibition of the signal, via blocking of HMGB1-CXCL12 binding to CXCR4, was demonstrated.

Materials and Methods

[0335] cAMP Hunter eXpress CXCR4 CHO-K1 GPCR Assay kit with reagents was obtained from DiscoverX (cat no 95-0081E2CP2L). cAMP CHO-K1 CXCR4 eXpress cells (DiscoverX, cat no: 95-0081E2) were thawed and seeded into a white 96-well plate, with clear bottom, in 100 μl AssayComplete Cell Plate 2 Reagent. The cells were cultured overnight at 37° C., 5% CO.sub.2.

[0336] On the day of the experiment, dilution of HMGB1 (HMGBiotech, cat no: HM001), CXCL12 (DiscoverX, cat no: 92-1011) and ZZA4263 (SEQ ID NO: 48) was performed in a separate dilution plate. First, a serial dilution of HMGB1 (200 nM-0.3 nM) in Cell Assay Buffer with forskolin and tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Sigma-Aldrich) was performed and incubated with CXCL12 at RT for 15 min. Second, a serial dilution of ZZA4263 (1000 nM-0.0006 nM) in PBS was performed and incubated with HMGB1 (10 nM) and CXCL12 (0.4 nM) in Cell Assay Buffer with forskolin and TCEP at RT for 15 min. Cell medium was removed from cells and 30 μl Cell Assay Buffer was added together with 15 μl HMGB1, CXCL12 and ZZA4263 dilutions. Cells were incubated at 37° C., 5% CO.sub.2 for 30 min. cAMP standard was serially diluted and added to the plate after the incubation. All Cell Assay Buffer was removed from the cells and 45 μl of fresh Cell Assay Buffer was added to each well. Subsequently, 15 μl cAMP Antibody Reagent as well as 60 μl cAMP Working Detection Solution including cAMP Lysis Buffer, Substrate Reagent 1, Substrate Reagent 2 and cAMP Solution D were added to each well. After 1 h of incubation, 60 μl cAMP Solution A was added to each well and the cell plate was incubated for additional 3 h in RT. The luminescence was measured in a 96-well plate reader (Enspire).

Results

[0337] ZZA4263 was investigated for its capacity to block the HMGB1-CXCL12 complex binding to CXCR4, in the cAMP Hunter eXpress CXCR4 CHO-K1 GPCR Assay. Firstly, as demonstrated by FIG. 4A, HMGB1 was shown to bind to CXCR4 in the presence of CXCL12 and to inhibit the luminescent signal in a dose-dependent manner. Secondly, as demonstrated by FIG. 4B, ZZA4263 was shown to inhibit the binding of the HMGB1-CXCL12 complex to CXCR4 in a dose dependent manner, where release of the inhibition enables the luminescent signal to return. The PBS negative control did not show any inhibition.

Example 13

Pharmacokinetic Study of ZZA4263 in Mouse and Rat

Summary

[0338] This Example describes the pharmacokinetic (PK) profiles of ZZA4263 obtained in mice and rats after intravenous (i.v.) or intraperitoneal (i.p) administration (mouse only). ZZA4263 was administered at a dose of 1.6 mg/kg in both species. Blood samples were collected up to three weeks post dose and analyzed using an antibody based sandwich PK-ELISA.

Materials and Methods

[0339] PK study in mouse: Six C57BL/6J mice (Charles River) were divided into two groups for i.p. or i.v. administration. ZZA4263 was prepared in PBS and administered as a bolus injection through tail vein or i.p. at 1.6 mg/kg (83 nmol/kg) at a slow and steady rate. The dosing volume administered was 5 and 20 ml/kg for i.v. and i.p. respectively. Serum samples were collected from tail vein of each mouse at time points 0 (predose), 5 min, 8 h, 48 h and 120 h. To prepare sera, the blood samples were left at RT for at least 30 min before centrifugation at 2000×g for 5 min. Sera were extracted and transferred into pre-labelled test tubes and stored at −20° C.

[0340] PK study in rat: Six female Sprague Dawley (SD) rats were divided into two groups for crosswise sampling. ZZA4263 was administered as a bolus injection through tail vein at 1.6 mg/kg (83 nmol/kg) at a slow and steady rate. The dosing volume administered was 1 ml/kg. Serum samples (0.2-0.3 ml) were collected from tail vein of each rat at time points 0 (predose), 30 min, 3 h, 24 h, 120 h, 216 h, 336 h and 456 h for group 1 and at time points 0 (predose), 5 min, 1 h, 8 h, 72 h, 168 h, 264 h, 408 h and 504 h for group 2. To prepare sera, the blood samples were left at RT for at least 30 min before centrifugation at 2000×g for 5 min. Sera were extracted and transferred into pre-labelled test tubes and stored at −20° C.

[0341] Quantification by ELISA: 96-well half area plates were coated with mouse anti-Z monoclonal antibody (2 μg/ml) in PBS (50 μl/well) and incubated overnight at 4° C. Plates were washed in PBST and blocked with PBSC for 1.5 h. ZZA4263 standard and serum samples were added to the plates (50 μl/well) and incubated for 1.5 h at RT. The ZZA4263 standard was diluted at 10 different concentrations between 1.3-50 μM in PBSC+1% mouse or rat serum pool. Serum samples were diluted 100× (minimal required dilution) in PBSC and titrated in 1:2 steps in PBSC+1% mouse or rat serum pool. Two quality control samples in PBSC+1% mouse or rat serum pool to a concentration of 40 and 4 μM, respectively, were included on each plate. Following washing with PBST, a goat anti-ABD polyclonal antibody (2 μg/ml; 50 μl/well) was added. After incubation for 1 h, the plates were washed and anti-goat IgG-HRP (100 ng/ml; 50 μl/well) added to each well. After one additional hour of incubation and subsequent washing, the plates were developed TMB (50 μl/well) and the reactions were stopped with 2M H.sub.2SO.sub.4 (50 μl/well). The absorbance at 450 nm was measured in a 96-well plate reader (Victor3). Data analysis was performed using Graph Pad Prism and Excel software.

Results

[0342] The PK profiles of ZZA4263 in mice after i.v. and i.p. administration, respectively, are shown in FIG. 5A. The bioavailability after i.p. administration was 67% of i.v. administration and the terminal half-life appears to be the same when comparing the two different administration routes. The PK profile of ZZA4263 in rat after i.v. administration is shown in FIG. 5B and the estimated t½ was 25-36 h.

Example 14

In Vivo Evaluation of ZZA4263 in a CLP-Induced Sepsis Model

Summary

[0343] The Cecal Ligation and Puncture (CLP) model is a standard animal model of microbial sepsis. The study described in this Example primarily assessed the capacity of the HMGB-1 binding polypeptide ZZA4263 to reduce mortality in CLP-induced sepsis in mice.

Materials and Methods

[0344] The animal study in C57BL mice was performed at Pharmaseed Ltd. (Israel) in compliance with The Israel Animal Welfare Act and following The Israel Board for Animal Experiments Ethics Committee approval # IL-19-1-49. On day 1, sepsis was induced following a modification of a previously published method of CLP (Ruiz et al (2016), Intensive Care Medicine Experimental 4:22-35). Sham operated control mice (n=10) were anesthetized and underwent laparotomy without puncture or cecal ligation. Sepsis induced mice were divided into groups of twenty. CLP is a harsh procedure and animals that either died or showed severe symptoms with low chance of recovery were excluded on humane grounds before treatment initiation (or before second dose). 0.14 mg/kg ZZA4263, 1.4 mg/kg ZZA4263, 25 mg/kg imipenem/cilastatin (antibiotics positive control) and vehicle (PBS), respectively, were administered i.p. to mice on day 2, 3, 4 and 5 (first dose 24 h after the surgery). Sham-operated mice did not receive any treatment. Until termination of the study on day 7, mice were monitored with regard to morbidity and mortality (twice daily), Murine Sepsis Score (MSS; twice daily), body weight (once daily) and body temperature (twice daily).

Results

[0345] The effect of the HMGB1 binding polypeptide ZZA4263 in the treatment of sepsis was assessed in the CLP model in mice. ZZA4263 demonstrated dose dependent increased survival rate (FIG. 6A) and lower MSS, i.e. reduced severity of sepsis (FIG. 6B), compared to vehicle. Both dose groups that received ZZA4263 showed a lower MSS compared to the group receiving positive control imipenem/cilastatin. The higher dose group, i.e. 1.4 mg/kg of ZZA4263, also showed an increased survival rate compared to imipenem/cilastatin. All mice subjected to CLP suffered from pronounced weight loss during the whole study period, but no significant differences were observed between the treatment groups. Sham operated mice lost weight after the surgery, but their body weight recovered to baseline by the end of the study. Body temperature was comparable in all groups. The temperature remained stable during the study except for animals that were in near-death condition for which the body temperature dropped drastically.

Itemized List of Embodiments

[0346] 1. HMGB1 binding polypeptide, comprising an HMGB1 binding motif BM, which motif consists of an amino acid sequence selected from:

i)

TABLE-US-00026 (SEQ ID NO: 56) EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10X.sub.11LPNLX.sub.16 X.sub.17X.sub.18QX.sub.20X.sub.21AFIYX.sub.26LED
wherein, independently of each other, [0347] X.sub.2 is selected from A, D, S and T; [0348] X.sub.3 is selected from E, R and W; [0349] X.sub.4 is selected from A, D and Q; [0350] X.sub.6 is selected from F and M; [0351] X.sub.7 is selected from E, H, W and Y; [0352] X.sub.10 is selected from I and L; [0353] X.sub.11 is selected from A and W; [0354] X.sub.16 is selected from N and T; [0355] X.sub.17 is selected from A, D, N and W; [0356] X.sub.18 is selected from A, E, Q, R, S, T and Y; [0357] X.sub.20 is selected from A and Q; [0358] X.sub.21 is selected from K, L and R; and [0359] X.sub.26 is selected from K and S;
and
ii) an amino acid sequence which has at least 93% identity to the sequence defined in i).

[0360] 2. HMGB1 binding polypeptide according to item 1, wherein in sequence i) [0361] X.sub.2 is selected from A, D, S and T; [0362] X.sub.3 is selected from E, R and W; [0363] X.sub.4 is selected from D and Q; [0364] X.sub.6 is selected from F and M; [0365] X.sub.7 is selected from E, H, W and Y; [0366] X.sub.10 is selected from I and L; [0367] X.sub.11 is W; [0368] X.sub.16 is selected from N and T; [0369] X.sub.17 is selected from D, N and W; [0370] X.sub.18 is selected from A, E, Q, R, T and Y; [0371] X.sub.20 is Q; [0372] X.sub.21 is selected from K, L and R; and [0373] X.sub.26 is selected from K and S.

[0374] 3. HMGB1 binding polypeptide according to any preceding item, wherein in sequence i) [0375] X.sub.2 is selected from A, D, S and T; [0376] X.sub.3 is selected from E, R and W; [0377] X.sub.4 is selected from D and Q; [0378] X.sub.6 is selected from F and M; [0379] X.sub.7 is selected from E, H, W and Y; [0380] X.sub.10 is selected from I and L; [0381] X.sub.11 is W; [0382] X.sub.16 is selected from N and T; [0383] X.sub.17 is selected from D, N and W; [0384] X.sub.18 is selected from A, E, Q, R and T; [0385] X.sub.20 is Q; [0386] X.sub.21 is selected from K, L and R; and [0387] X.sub.26 is selected from K and S.
4. HMGB1 binding polypeptide according to any preceding item, wherein in sequence i) [0388] X.sub.2 is selected from A and D; [0389] X.sub.3 is selected from E and W; [0390] X.sub.4 is selected from D and Q; [0391] X.sub.6 is selected from F and M; [0392] X.sub.7 is selected from E, H and W; [0393] X.sub.10 is selected from I and L; [0394] X.sub.11 is W; [0395] X.sub.16 is selected from N and T; [0396] X.sub.17 is selected from D, N and W; [0397] X.sub.18 is selected from A, E, and T; [0398] X.sub.20 is Q; [0399] X.sub.21 is selected from K, L and R; and [0400] X.sub.26 is selected from K and S.

[0401] 5. HMGB1 binding polypeptide according to any one of items 1-3, wherein in sequence i) [0402] X.sub.2 is selected from A, D, S and T; [0403] X.sub.3 is selected from R and W; [0404] X.sub.4 is D; [0405] X.sub.6 is F; [0406] X.sub.7 is selected from E, Y and W; [0407] X.sub.10 is I; [0408] X.sub.11 is W; [0409] X.sub.16 is selected from N and T; [0410] X.sub.17 is D; [0411] X.sub.18 is selected from A, Q and R; [0412] X.sub.20 is Q; [0413] X.sub.21 is R; and [0414] X.sub.26 is selected from K and S.

[0415] 6. HMGB1 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:1-14, 16 and 20-23.

[0416] 7. HMGB1 binding polypeptide according to item 6, 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.

[0417] 8. HMGB1 binding polypeptide according to item 7, 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:1-10.

[0418] 9. HMGB1 binding polypeptide according to item 8, 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:1-3.

[0419] 10. HMGB1 binding polypeptide according to any one of items 6-7, 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:4-13.

[0420] 11. HMGB1 binding polypeptide according to any one of items 7-9, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1.

[0421] 12. HMGB1 binding polypeptide according to any preceding item, which binds to the A-box of HMGB1 such that the K.sub.D value of the interaction is at most 1×10.sup.−6 M, for example at most 5×10.sup.−7 M, for example at most 1×10.sup.−7 M, for example at most 5×10.sup.−8 M, for example at most 1×10.sup.−8 M.

[0422] 13. HMGB1 binding polypeptide according to any preceding item, which binds to the A-box of HMGB1 such that the half maximal effective concentration of the interaction, in a suitable assay of the effect, is at most 1×10.sup.−6 M, for example at most 5×10.sup.−7 M, for example at most 1×10.sup.−7 M, for example at most 5×10.sup.−8 M, for example at most 1×10.sup.−8 M.

[0423] 14. HMGB1 binding polypeptide according to any preceding item, which binds to the HMGB1 A-box region in its reduced state.

[0424] 15. HMGB1 binding polypeptide according to any preceding item, which binds to the HMGB1 A-box region in its oxidized state.

[0425] 16. HMGB1 binding polypeptide, comprising an HMGB1 binding motif BM, which motif consist of an amino acid sequence selected from:

iii)

TABLE-US-00027 (SEQ ID NO: 57) EAWX.sub.4AEQEIW X.sub.11LPNLX.sub.16X.sub.17X.sub.18 QF QAFIX.sub.25X.sub.26LX.sub.28D [0426] wherein each of X.sub.4, X.sub.11, X.sub.17, X.sub.18, X.sub.25, and X.sub.28 are, independently of each other, selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, [0427] W and Y; [0428] X.sub.16 is selected from N and T; and [0429] X.sub.26 is selected from K and S;
and
iv) an amino acid sequence which has at least 93% identity to the sequence defined in iii).

[0430] 17. HMGB1 binding polypeptide according to item 16, wherein in sequence iii) [0431] X.sub.4 is selected from L and S; [0432] X.sub.11 is selected from D and Q; [0433] X.sub.16 is selected from N and T; [0434] X.sub.17 is selected from E and L; [0435] X.sub.18 is selected from A and Q; [0436] X.sub.25 is selected from L and M; [0437] X.sub.26 is selected from K and S; and [0438] X.sub.28 is selected from I and L.

[0439] 18. HMGB1 binding polypeptide according to any one of items 16-17, wherein sequence iii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:30 and SEQ ID NO:31.

[0440] 19. HMGB1 binding polypeptide according to item 18, wherein sequence iii) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:31.

[0441] 20. HMGB1 binding polypeptide according to any one of items 16-19, which binds t to the B-box of HMGB1 such that the K.sub.D value of the interaction is at most 1×10.sup.−6 M, for example at most 5×10.sup.−7 M, for example at most 1×10.sup.−7 M, for example at most 5×10.sup.−8 M, for example at most 1×10.sup.−8 M.

[0442] 21. HMGB1 binding polypeptide according to item 20, which binds to the B-box of HMGB1 such that the half maximal effective concentration of the interaction, in a suitable assay of the effect, is at most 1×10.sup.−6 M, for example at most 5×10.sup.−7 M, for example at most 1×10.sup.−7 M, for example at most 5×10.sup.−8 M, for example at most 1×10.sup.−8 M.

[0443] 22. HMGB1 binding polypeptide according to any preceding item, wherein said HMGB1 binding motif forms part of a three-helix bundle protein domain.

[0444] 23. HMGB1 binding polypeptide according to item 22, wherein said HMGB1 binding motif essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.

[0445] 24. HMGB1 binding polypeptide according to item 23, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.

[0446] 25. HMGB1 binding polypeptide according to item 24, wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.

[0447] 26. HMGB1 binding polypeptide according to any preceding item, which comprises a binding module BMod, the amino acid sequence of which is selected from:

v)

TABLE-US-00028 (SEQ ID NO: 58) K-[BM]-DPSQSX.sub.aX.sub.bLLX.sub.c EAKKLX.sub.dX.sub.eX.sub.fQ;
wherein [0448] [BM] is an HMGB1 binding motif as defined in any one of items 1-11 and 16-19; [0449] X.sub.a is selected from A and S; [0450] X.sub.b is selected from N and E; [0451] X.sub.c is selected from A, S and C; [0452] X.sub.d is selected from E, N and S; [0453] X.sub.e is selected from D, E and S; and [0454] X.sub.f is selected from A and S; and
vi) an amino acid sequence which has at least 93% identity to a sequence defined in v).

[0455] 27. HMGB1 binding polypeptide according to item 26, wherein sequence v) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-14, 16, 20-23 and 27-29.

[0456] 28. HMGB1 binding polypeptide according to item 27, wherein sequence v) 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 29.

[0457] 29. HMGB1 binding polypeptide according to item 28, wherein sequence v) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-14.

[0458] 30. HMGB1 binding polypeptide according to item 29, wherein sequence v) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-10.

[0459] 31. HMGB1 binding polypeptide according to item 30, wherein sequence v) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-3.

[0460] 32. HMGB1 binding polypeptide according to any one of items 27-29, wherein sequence v) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:4-13.

[0461] 33. HMGB1 binding polypeptide according to any one of items 27-31, wherein sequence v) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1.

[0462] 34. HMGB1 binding polypeptide according to any one of items 27-28, wherein sequence v) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:29.

[0463] 35. HMGB1 binding polypeptide according to item 26, wherein sequence v) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:30-32.

[0464] 36. HMGB1 binding polypeptide according to item 35, wherein sequence v) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:31.

[0465] 37. HMGB1 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:

[0466] vii) YA-[BMod]-AP;

[0467] wherein [BMod] is an HMGB1 binding module as defined in any one of items 26-36; and

[0468] viii) an amino acid sequence which has at least 90% identity to a sequence defined in vii).

[0469] 38. HMGB1 binding polypeptide according to item 37, which comprises an amino acid sequence selected from:

[0470] ix)

TABLE-US-00029 (SEQ ID NO: 59) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is an HMGB1 binding motif as defined in any one of items 1-11 and 16-19; and

[0471] x) an amino acid sequence which has at least 89% identity to the sequence defined in ix).

[0472] 39. HMGB1 binding polypeptide according to item 38, wherein sequence ix) is selected from the group consisting of SEQ ID NO:1-3 and 14-26.

[0473] 40. HMGB1 binding polypeptide according to item 39, wherein sequence ix) is selected from the group consisting of SEQ ID NO:1-3.

[0474] 41. HMGB1 binding polypeptide according to item 40, wherein sequence ix) is SEQ ID NO:1.

[0475] 42. HMGB1 binding polypeptide according to item 38, wherein sequence ix) is selected from the group consisting of SEQ ID NO:30-31.

[0476] 43. HMGB1 binding polypeptide according to item 42, wherein sequence ix) is SEQ ID NO:31.

[0477] 44. HMGB1 binding polypeptide according to item 37, which comprises an amino acid sequence selected from:

[0478] xi)

TABLE-US-00030 (SEQ ID NO: 60) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,
wherein [BM] is an HMGB1 binding motif as defined in any one of items 1-11 and 16-19; and

[0479] xii) an amino acid sequence which has at least 89% identity to the sequence defined in xi).

[0480] 45. HMGB1 binding polypeptide according to item 44, wherein sequence xi) is selected from the group consisting of SEQ ID NO:27-28.

[0481] 46. HMGB1 binding polypeptide according to item 45, wherein sequence xi) is SEQ ID NO:27.

[0482] 47. HMGB1 binding polypeptide according to item 44, wherein sequence xi) is SEQ ID NO:32.

[0483] 48. HMGB1 binding polypeptide according to any one of items 1-36, which comprises an amino acid sequence selected from:

[0484] xiii) FA-[BMod]-AP;

wherein [BMod] is an HMGB1 binding module as defined in any one of items 26-36; and

[0485] xiv) an amino acid sequence which has at least 90% identity to a sequence defined in xiii).

[0486] 49. HMGB1 binding polypeptide according to item 48, which comprises an amino acid sequence selected from:

[0487] xv)

TABLE-US-00031 (SEQ ID NO: 61) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
wherein [BM] is an HMGB1 binding motif as defined in any one of items 1-11 and 16-19; and

[0488] xvi) an amino acid sequence which has at least 89% identity to the sequence defined in xv).

[0489] 50. HMGB1 binding polypeptide according to item 49, wherein sequence xv) is selected from the group consisting of SEQ ID NO:4-13 and 29.

[0490] 51. HMGB1 binding polypeptide according to item 48, which comprises an amino acid sequence selected from:

[0491] xvii)

TABLE-US-00032 (SEQ ID NO: 62) AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is an HMGB1 binding motif as defined in any one of items 1-11 and 16-19; and

[0492] xviii) an amino acid sequence which has at least 89% identity to the sequence defined in xvii).

[0493] 52. HMGB1 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:

TABLE-US-00033 (SEQ ID NO: 63) ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK; (SEQ ID NO: 64) ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 65) ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK; (SEQ ID NO: 66) ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK; (SEQ ID NO: 67) AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK; (SEQ ID NO: 68) VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 69) AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK; (SEQ ID NO: 70) VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; (SEQ ID NO: 71) VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK; (SEQ ID NO: 72) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 73) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP; (SEQ ID NO: 74) AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 75) AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP; (SEQ ID NO: 76) AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; (SEQ ID NO: 77) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 78) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP; (SEQ ID NO: 79) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 80) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP; (SEQ ID NO: 81) AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK; (SEQ ID NO: 82) AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; (SEQ ID NO: 83) AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; (SEQ ID NO: 84) AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP; (SEQ ID NO: 85) AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK; (SEQ ID NO: 86) AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; (SEQ ID NO: 87) AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK; (SEQ ID NO: 88) AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP; (SEQ ID NO: 89) AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK; (SEQ ID NO: 90) AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK; (SEQ ID NO: 91) AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK; (SEQ ID NO: 92) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 93) VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; (SEQ ID NO: 94) VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 95) VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK; (SEQ ID NO: 96) VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; (SEQ ID NO: 97) VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; (SEQ ID NO: 98) VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK; (SEQ ID NO: 99) VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; (SEQ ID NO: 100) VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK; (SEQ ID NO: 101) VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK; (SEQ ID NO: 102) VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK; (SEQ ID NO: 103) VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK; (SEQ ID NO: 104) VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; (SEQ ID NO: 105) AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and (SEQ ID NO: 106) ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is an HMGB1 binding motif as defined in any one of items 1-11 and 16-19.

[0494] 53. HMGB1 binding polypeptide according to any preceding item, wherein said HMGB1 is human HMGB1.

[0495] 54. HMGB1 binding polypeptide according to any preceding item which comprises additional amino acids at the C-terminal and/or N-terminal end.

[0496] 55. HMGB1 binding polypeptide according to item 54, wherein said additional amino acid(s) improve(s) production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide.

[0497] 56. HMGB1 binding polypeptide according to any preceding item in multimeric form, comprising at least two HMGB1 binding polypeptide monomer units, whose amino acid sequences may be the same or different.

[0498] 57. HMGB1 binding polypeptide according to item 56, wherein a first HMGB1 binding polypeptide monomer unit is an HMGB1 binding polypeptide according to any one of items 1-15, and wherein a second HMGB1 binding polypeptide monomer unit is an HMGB1 binding polypeptide according to any one of items 16-21.

[0499] 58. HMGB1 binding polypeptide according to any one of items 56-57, wherein a first HMGB1 binding polypeptide monomer unit has affinity for the A-box of HMGB1, and wherein a second HMGB1 binding polypeptide monomer unit has affinity for the B-box of HMGB1.

[0500] 59. HMGB1 binding polypeptide according to any one of items 56-58, wherein said HMGB1 binding polypeptide monomer units are covalently coupled together.

[0501] 60. HMGB1 binding polypeptide according to item 59, wherein the HMGB1 binding polypeptide monomer units are expressed as a fusion protein.

[0502] 61. HMGB1 binding polypeptide according to any one of items 56-60, in dimeric form.

[0503] 62. Fusion protein or conjugate comprising [0504] a first moiety consisting of an HMGB1 binding polypeptide according to any preceding item; and [0505] a second moiety consisting of a polypeptide having a desired biological activity.

[0506] 63. Fusion protein or conjugate according to item 62, wherein said desired biological activity is a therapeutic activity.

[0507] 64. Fusion protein or conjugate according to item 62, wherein said desired biological activity is a binding activity.

[0508] 65. Fusion protein or conjugate according to item 62, wherein said desired biological activity is an enzymatic activity.

[0509] 66. Fusion protein or conjugate according to item 64, wherein said binding activity is albumin binding activity which increases in vivo half-life of the fusion protein or conjugate.

[0510] 67. Fusion protein or conjugate according to item 66, wherein said second moiety comprises the albumin binding domain of streptococcal protein G or a derivative thereof.

[0511] 68. Fusion protein or conjugate according to item 64, wherein said binding activity acts to block a biological activity.

[0512] 69. Fusion protein or conjugate according to item 63, wherein the second moiety is a therapeutically active polypeptide.

[0513] 70. Fusion protein or conjugate according to any one of items 62-65 and 68-69, wherein the second moiety is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.

[0514] 71. Fusion protein or conjugate according to any one of items 62-65 and 68-69, wherein the second moiety is selected from the group consisting of an antibody and an antigen binding fragment thereof.

[0515] 72. Fusion protein or conjugate according to item 71, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments, Fab′ fragments, F(ab′).sub.2 fragments, Fc fragments, Fv fragments, single chain Fv (scFv) fragments, (scFv).sub.2 and domain antibodies.

[0516] 73. Fusion protein or conjugate according to item 72, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments and scFv fragments.

[0517] 74. Fusion protein or conjugate according to item 73, wherein said at least one antibody or antigen binding fragment thereof is a full-length antibody.

[0518] 75. Fusion protein according to any one of items 62-74, wherein the second moiety further comprises a linker.

[0519] 76. HMGB1 binding polypeptide, fusion protein or conjugate according to any preceding item, further comprising a label.

[0520] 77. HMGB1 binding polypeptide, fusion protein or conjugate according to item 76, wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides, radioactive particles and pretargeting recognition tags.

[0521] 78. HMGB1 binding polypeptide, fusion protein or conjugate according to item 77, comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the HMGB1 binding polypeptide via a thiol group of a cysteine residue or an amine group of a lysine residue.

[0522] 79. HMGB1 binding polypeptide, fusion protein or conjugate according to item 78, which comprises a pretargeting recognition tag forming part of a complementary pair of pretargeting moieties, for example selected from stept(avidin)/biotin, oligonucleotide/complementary oligonucleotide such as DNA/complementary DNA, RNA/complementary RNA, phosphorothioate nucleic acid/complementary phosphorothioate nucleic acid and peptide nucleic acid/complementary peptide nucleic acid and morpholinos/complementary morpholinos.

[0523] 80. A polynucleotide encoding a polypeptide according to any one of items 1-75.

[0524] 81. Expression vector comprising a polynucleotide according to item 80.

[0525] 82. Host cell comprising an expression vector according to item 81.

[0526] 83. Method of producing a polypeptide according to any one of items 1-75, comprising [0527] culturing a host cell according to item 82 under conditions permissive of expression of said polypeptide from said expression vector, and [0528] isolating said polypeptide.

[0529] 84. Composition comprising an HMGB1 binding polypeptide, fusion protein or conjugate according to any one of items 1-79 and at least one pharmaceutically acceptable excipient or carrier.

[0530] 85. HMGB1 binding polypeptide, fusion protein or conjugate according to any one of items 1-79 or a composition according item 84 for oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration, such as for topical administration.

[0531] 86. HMGB1 binding polypeptide, fusion protein or conjugate according to any one of items 1-79 or a composition according to item 84 for use as a medicament, a diagnostic agent and/or a prognostic agent.

[0532] 87. HMGB1 binding polypeptide, fusion protein, conjugate or composition for use according to item 86 as a medicament.

[0533] 88. HMGB1 binding polypeptide, fusion protein, conjugate or composition for use according to item 86 as a diagnostic agent and/or a prognostic agent.

[0534] 89. HMGB1 binding polypeptide, fusion protein, conjugate or composition for use as a medicament according to item 87, wherein said polypeptide, fusion protein, conjugate or composition modulates HMGB1 function in vivo.

[0535] 90. HMGB1 binding polypeptide, fusion protein, conjugate or composition for use according to any one of items 86-89 in the treatment, prognosis or diagnosis of an HMGB1 related disorder.

[0536] 91. HMGB1 binding polypeptide, fusion protein, conjugate or composition for use according to item 90, wherein said HMGB1 related disorder is selected from the group consisting of inflammatory diseases, respiratory diseases, autoimmune diseases, infectious diseases, trauma, cardiovascular disease, neurodegenerative diseases, metabolic disorders, liver injury and cancers.

[0537] 92. HMGB1 binding polypeptide, fusion protein, conjugate or composition for use according to any one of items 90-91, wherein said HMGB1 related disorder is selected from the group consisting of arthritis (such as rheumatoid arthritis, collagen-induced arthritis, crystal-induced arthritis ankylosing spondylitis), atherosclerosis, hepatitis, inflammatory bowel disease, chronic inflammatory anemia, myositis, pancreatitis, pulmonary fibrosis, pulmonary inflammation, hepatic ischemia-reperfusion injury, drug-induced liver intoxication (such as acetaminophen/paracetamol intoxication), acute or chronic liver failure, nonalcoholic fatty liver disease, liver fibrosis, cirrhosis, hemolytic uremic syndrome, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, glomerulonephritis, juvenile idiopathic arthritis, antineutrophilic cytoplasmatic antibody (ANCA)-associated vasculitis, systemic vasculitis scleroderma, Sjogren syndrome, Behcet's disease, cancer (such as breast cancer, colorectal cancer, hepatocellular carcinoma, lung cancer, pancreatic cancer, renal cell carcinoma, melanoma and mesothelioma), ischemia/reperfusion, stroke, ischemic brain injury, chemical toxemia, traumatic brain injury, neuroinflammation, epileptogenesis, cognitive dysfunctions, atherosclerosis, gastric ulcer, hyperoxia, sepsis, endotoxemia, hemorrhagic shock, cerebrovascular disease, myocardial infarction, heart failure, Alzheimer's disease, multiple sclerosis, epilepsy, diabetes, obesity, transplant rejection, chronic kidney disease, sciatica and neuropathic pain.

[0538] 93. HMGB1 binding polypeptide, fusion protein, conjugate or composition for use according to item 92, wherein said HMGB1 related disorder is selected from the group consisting of cerebrovascular diseases, chronic inflammatory anemia, acute liver failure, drug-induced liver intoxication, hemolytic uremic syndrome, systemic lupus erythematosus, mesothelioma, lung cancer, stroke, sepsis, sciatica and neuropathic pain, such as selected from the group consisting of drug-induced liver intoxication, sepsis, sciatica and neuropathic pain.

[0539] 94. Method of treatment of an HMGB1 related disorder, comprising administering to a subject in need thereof an effective amount of an HMGB1 binding polypeptide, fusion protein or conjugate according to any one of items 1-79 or a composition according to item 84.

[0540] 95. Method according to item 94, wherein said HMGB1 related disorder is selected from the group consisting of inflammatory diseases, respiratory diseases, autoimmune diseases, infectious diseases, trauma, cardiovascular disease, neurodegenerative diseases, metabolic disorders, liver injury and cancers.

[0541] 96. Method according to item 95, wherein said HMGB1 related disorder is selected from the group consisting of arthritis (such as rheumatoid arthritis, collagen-induced arthritis, crystal-induced arthritis ankylosing spondylitis), atherosclerosis, hepatitis, inflammatory bowel disease, chronic inflammatory anemia, myositis, pancreatitis, pulmonary fibrosis, pulmonary inflammation, hepatic ischemia-reperfusion injury, drug-induced liver intoxication (such as acetaminophen/paracetamol intoxication), acute or chronic liver failure, nonalcoholic fatty liver disease, liver fibrosis, cirrhosis, hemolytic uremic syndrome, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, glomerulonephritis, juvenile idiopathic arthritis, antineutrophilic cytoplasmatic antibody (ANCA)-associated vasculitis, systemic vasculitis scleroderma, Sjogren syndrome, Behcet's disease, cancer (such as breast cancer, colorectal cancer, hepatocellular carcinoma, lung cancer, pancreatic cancer, renal cell carcinoma, melanoma and mesothelioma), ischemia/reperfusion, stroke, ischemic brain injury, chemical toxemia, traumatic brain injury, neuroinflammation, epileptogenesis, cognitive dysfunctions, atherosclerosis, gastric ulcer, hyperoxia, sepsis, endotoxemia, hemorrhagic shock, cerebrovascular disease, myocardial infarction, heart failure, Alzheimer's disease, multiple sclerosis, epilepsy, diabetes, obesity, transplant rejection, chronic kidney disease, sciatica and neuropathic pain.

[0542] 97. Method according to item 96, wherein said HMGB1 related disorder is selected from the group consisting of cerebrovascular diseases, chronic inflammatory anemia, acute liver failure, drug-induced liver intoxication, hemolytic uremic syndrome, systemic lupus erythematosus, mesothelioma, lung cancer, stroke, sepsis, sciatica and neuropathic pain, such as selected from the group consisting of drug-induced liver intoxication, sepsis, sciatica and neuropathic pain.

[0543] 98. Method of detecting HMGB1 in vitro, comprising providing a sample suspected to contain HMGB1, contacting said sample with an HMGB1 binding polypeptide, fusion protein or conjugate according to any one of items 1-79 or a composition according to item 84, and detecting the binding of the HMGB1 binding polypeptide, fusion protein, conjugate or composition to indicate the presence of HMGB1 in the sample.

[0544] 99. Method for determining the presence of HMGB1 in a subject, comprising the steps of: [0545] a) contacting the subject, or a sample isolated from the subject, with an HMGB1 binding polypeptide, fusion protein or conjugate according to any one of items 1-79 or a composition according to item 84, and [0546] b) obtaining a value corresponding to the amount of the HMGB1 binding polypeptide, fusion protein, conjugate or composition that has bound in said subject or to said sample.

[0547] 100. Method according to item 99, further comprising a step of comparing said value to a reference.

[0548] 101. Method according to any one of items 99-100, wherein said subject is a mammalian subject, such as a human subject.

[0549] 102. Method according to any one of items 99-101, wherein the method is performed in vivo.