EP4341280A1 - Polypeptides related to hmgb1 useful for promoting tissue regeneration, compositions comprising same, and uses thereof - Google Patents

Polypeptides related to hmgb1 useful for promoting tissue regeneration, compositions comprising same, and uses thereof

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Publication number
EP4341280A1
EP4341280A1 EP22728676.2A EP22728676A EP4341280A1 EP 4341280 A1 EP4341280 A1 EP 4341280A1 EP 22728676 A EP22728676 A EP 22728676A EP 4341280 A1 EP4341280 A1 EP 4341280A1
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European Patent Office
Prior art keywords
amino acids
hmgb1
sequence
injury
polypeptide
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EP22728676.2A
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German (de)
French (fr)
Inventor
Jagdeep Nanchahal
Alvaro VINALS GUITART
Wyatt YUE
Nicola BURGESS-BROWN
Tzung Yuan Lee
Ana Isabel ESPIRITO SANTO
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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Publication of EP4341280A1 publication Critical patent/EP4341280A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • This application also incorporates-by-reference each of the nucleotide sequences which are present in the text file named “220519_91203-B- PCT_Sequence_Listing_AWG.txt”, which is 78 kilobytes in size, and which was created on May 18, 2022 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is being filed as part of this application.
  • This invention concerns polypeptides related to HMGB1 that promote tissue regeneration without inducing deleterious inflammation and methods of treating acute and chronic conditions involving tissue injury by administering such a polypeptide to a subject in need of treatment for such a condition.
  • HMGB1 is a prototypical alarmin [9,10] and under physiological conditions has an essential role in transcription [11,12]
  • G Aiert On exposure to the appropriate activating factors, cells in G Aiert can rapidly enter Gi and effect tissue repair. If not required, stem cells in G Aiert revert back to Go after approximately 3 weeks [13], thereby ensuring that they are not exhausted and the niche is not depleted.
  • HMBG1 comprises two L-shaped Box domains, A and B, each containing three a- helices (I - III) connected by flexible regions ( Figure 1A).
  • the C-terminus of the protein is intrinsically disordered and contains a high proportion of carboxylic acid residues (Glu/Asp) comprising the acidic tail.
  • Glu/Asp carboxylic acid residues
  • the oxidation status of HMGB1 cysteine residues (Cys 22, Cys 44 in Box A and Cys 105 in Box B) is a key determinant of the extracellular activities of HMGB 1 and in turn is dependent on the mechanism of release.
  • HMGB1 passively released from the nuclei following injury or cell necrosis is the fully-reduced form (FR-HMGB1). It binds to CXCL12 and the heterocomplex signals via the cell surface receptor CXCR4 to transition stem and progenitor cells to GAi ert [8] . Partial oxidation in the local inflammatory environment results in the formation of the disulfide HMGB1 (DS-HMGB1) [15,16], which has a disulfide bond between Cys 22 and Cys 44.
  • DS-HMGB1 disulfide HMGB1
  • TLR-4 signaling by DS-HMGB1 results in production of several proinflammatory cytokines, including TNF [19], whilst TLR-2 signaling has been shown to be detrimental in multiple processes, including thrombosis and reperfusion injury [20], and autoimmune disorders [21]
  • DS-HMGB1 signaling via RAGE plays a key role in platelet activation and NET formation by neutrophils to promote thrombus formation [20,22-24]
  • Intracellular signaling via all three receptors converges to induce NF-kb activity [25] in a MyD88-dependent manner [26,27] Oxidation of all three cysteine residues through the action of extracellular reactive oxygen species results in sulfonyl-HMGB 1 (SO3), which is biologically inactive [14,19]
  • the disulfide bridge in Box A of DS-HMGB1 (Cys22-Cys44) is essential for TLR-4 signaling ( Figure IB and Figure 1C), initiating binding to TLR-4 but also has a relatively high dissociation rate.
  • MD-2 then binds to Box B with low affinity but very low dissociation rates, stabilizing the interaction [28]; the Phe-Cys-Ser-Glu (FCSE, 104-107) peptide in Box B is essential for this interaction [29]
  • the capacity of DS-HMGB1 to signal via TLR-4 has been overcome by substituting cysteines at positions 22, 44 and 105 with serine, resulting in an engineered form described as 3S-HMGB1 [14] Whilst the authors claimed that 3S-HMGB1 has enhanced regenerative properties compared to FR-HMGB1 [30], we found that in bone, blood and skeletal muscle injuries it was equivalent to FR-HMGB1.
  • This invention provides a polypeptide represented by the following formula: H 2 N-A-X-B-A-X-B-HOOC wherein each A represents consecutive amino acids, the sequence of which
  • (a) is identical to the sequence of the corresponding one to six amino acids preceding amino acid 90 in wild type human HMGB1, or
  • (b) differs from the sequence of (a) in respect to one or more amino acids
  • each A optionally has a methionine at the amino terminus, wherein each A may be the same or different; wherein each X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 - 162 of wild type human HMGB 1; wherein each B represents consecutive amino acids, the sequence of which (1) is a sequence of five or six amino acids
  • (a) is identical to the sequence of the corresponding one to six amino acids following amino acid 168 in wild type human HMGB1,
  • (b) is identical to the sequence of the corresponding one to six amino acids following amino acid 167 in wild type human HMGB1,
  • (c) differs at one or more positions from the sequence of the corresponding one to six amino acids following amino acid 168 in wild type human HMGB 1 , or
  • each B may be the same or different; and wherein each - represents a peptide bond between each of A and X, X and B, B and A, A and X, and X and B; with the proviso that in the B-A between the two Xs, the number of amino acids must be at least 12; and with the additional proviso that in the B at the carboxy terminal end of the polypeptide, the one to six consecutive amino acids of (2) may be absent.
  • This invention also provides a composition comprising a polypeptide in accordance with the invention and a carrier, and methods of treating a subject suffering from, or at risk for developing, a condition which would be alleviated by promoting regeneration of a tissue or cells that rely upon CXCR4 + cells for repair which comprise administering to the subject a polypeptide or a composition of the invention in an amount effective to promote regeneration of the tissue or cells and to have a therapeutic or prophylactic effect.
  • Figures 1A-1C show a schematic of the HMGB1 structure and locations of known immunogenic activities.
  • Figure 1A Structure of HMGB1 (PDB 2YRQ, conformer 1) showing the alpha helices of each Box domain.
  • Figure IB Structure from ( Figure 1A) coloured in PyMol according to known interactions with LPS, TLR-4 or RAGE. The regions involved in TLR-2 binding are currently unknown. The acidic tail, which is involved in transcriptional modulation and bactericidal activities is not shown in the structure.
  • Structure of HMGB1 showing the alpha helices of each Box domain is labeled, with the original coloring showing the following - Pink: residues involved in glycyrrhizin binding.
  • Figures 2A-2F show conserved residues in each HMG Box domain are critical for CXCL12 binding.
  • Figure 2A Peptide array (11x10) of HMGB1 15-mers incubated with 1 mM CXCL12-His6 and detected with anti-His5-HRP antibody. Intensity of spots corresponds to amount of CXCL12 bound to the peptides; first two and last two spots in the array comprised 10-His positive controls.
  • Figure 2B Intensity quantification of spot intensity in (Figure 2A) (duplicate runs) normalised to 10-his control. Peptides used for alanine scanning experiments in ( Figure 2C) are marked in the graph.
  • Peptides in the acidic tail were not included, as it would non-specifically bind cationic molecules such as CXCL12 due to the high negative charge.
  • Peptides in graphs are represented in SEQ ID NOs: 8-104, left to right.
  • Figure 2C Peptide arrays of alanine point mutagenesis of domains identified in Figure 2A and Figure 2B. First spot in each row corresponds to the positive control; second spot to the unmodified peptide. Peptides shown are represented in SEQ ID NOs: 105-111, top to bottom.
  • Figure 2D Intensity quantification of the array in ( Figure 2C, SEQ ID NOs: 105-111), normalized to the unmodified peptide.
  • Kd Affinity (Kd) constants from both fits follow the same relationship and are greatly decreased for HMGB194-162; analyzed by 1-way Brown-Forsythe ANOVA from the fitted data. Kd values were compared via a post-hoc 2-way ANOVA if AICc supported a model with multiple constants, averaging both values as no significant differences were found in the paired comparison (column factor). Raw interferograms can be found in Figure 12. Req: response at equilibrium, kOff: dissociation constant (s -1 ), kOn: association constant (( ⁇ M*s) -1 ), AICc: Statistical comparison by Aikaike ⁇ s Information Criterion (corrected).
  • FIG. 3 shows the binding of DAMP receptors to HMGB1 analyzed via peptide arrays.
  • TLR-2 A
  • TLR-4 B
  • RAGE red in original coloring
  • a schematic representation of HMGB1 is shown to the left of the peptide sequences.
  • TLR-2 (A) binding peptide sequences within HMGB1 occupy similar positions across Box A and Box B with another binding region just before the acidic tail.
  • FIG. 4 shows a representation of DAMP-receptor binding peptides and changes in their configuration dependent on the oxidation status of Cys22-Cys44 in Box A.
  • Figure 5A Cumulative CSP of helical-only biotinylated Box B (94-162, HMGB1A-c028) or complete Box B (89-174, HMGB1A-c038) after titration with CXCL12 (0.42, 0.84 and 1.42 molar equivalents), calculated over several HSQC spectra including a parallel control with no CXCL12 measured after the last concentration point (CSP drift control). Intensity of the darkness of the bars in the graph indicates relative CSP.
  • the sequence of each HMGB1 construct has been overlaid with the residue number; an empty column (no number) represents residues which could not be mapped in the parallel 3D 1 H- 15 N HSQC/NOE/TOCSY experiments.
  • Figures 6A-6C show design of dBB12L construct to eliminate RAGE, TLR-2 and TLR-4 signaling.
  • Figure 6A Sequence alignment of Box A + linkers (1-88) (SEQ ID NO: 3) with Box B + linkers (89-174) (SEQ ID NO: 4); numbers correspond to residue numbering from the NMR structure (excluding N-terminal methionine).
  • Vertical lines designate strictly conserved positions, and double dots similar amino acid types.
  • Underlined peptide regions binding CXCL12 from the first peptide array.
  • Red in original coloring residues flagged in the alanine scan as involved in CXCL12 binding which could not be verified by NMR (specifically Box A: D4, P8, M12, C22, R69, Y70, T76, and P80; and Box B: P91, G118, and P167.
  • Orange in original coloring residues flagged in the alanine scan and also showing either CSP or peak volume change by NMR (specifically Box A: K6, R9, G10, Kl l, S13, S14, H26, K28, K29, H30, K64, D66, E73, K75, Y77, and 178; and Box B: D90, R96, S99, F101, F102, L103, F104, SI 06, R109, K113, K151, E152, 1158, Y161, R162, G165, K166, and D168.
  • NMR specifically Box A: K6, R9, G10, Kl l, S13, S14, H26, K28, K29, H30, K64, D66, E73, K75, Y77, and 178; and Box B: D90, R96, S99, F101, F102, L103, F104, SI 06, R109, K113, K151, E152, 1158, Y161, R162,
  • Cyan in original coloring residues not flagged in our NMR or peptide array experiments but described in the NMR literature as contributing to CXCL12 binding [46] (specifically Box A: F37, S38, K49, and K56; and BoxB: A100, G114, LI 19, A136, and Y154. Purple in original coloring: residues flagged peptide array experiments and confirmed by the published or our NMR data (specifically Box A: V19 and R23; and Box B: El 15 and D 157.
  • FIG. 6C Schematic of dBB12L construct design.
  • the initiation codon Met 1 is numbered as Met 0 herein, as it is partially lost in the cleaved peptide. Therefore, HMGB1 Met 1 -Gly 2...Glu 215 becomes Met 0- Gly 1...Glu 214.
  • dBB12L construct is designed such that: 1. The acidic tail and part of the RAGE binding domain (175-214) deleted; 2. Residues 1-88 (Box A) substituted by residues 90-175, resulting in two HMG Box B domains; and 3. Residues 163- 174 C-terminal to Box B replace the native flexible linker (79-88) C-terminal to Box A in native HMGB1.
  • the repeat Box B units are separated in the diagram by a vertical dashed black line.
  • DAMP receptor binding peptides in Box A shown as dashed lines, those in Box B by solid lines.
  • the TLR-2 and RAGE peptides are truncated in DBB12L, and all Box A peptides are no longer present in the construct due to the substitution.
  • Figures 7A-7D show dBB12L has similar stability and surface charge conformation to FR-HMGB11-214/1-164.
  • Figure 7A Calculated Tm 50 values (in oC) for full-length and 1- 164 FR-HMGB1, and dBB12L under various buffer conditions, shown as a heat map of highest (green) and lowest (red) values within the global dataset for all constructs in original coloring. N/A: curve not fittable. Effects of pH and NaCl concentration have been summarized below the table (FR-HMGB1, circle; dBB12L, square; 1-164 FR-HMGB1, triangle).
  • Figure 7B Native ESI/MS of HMGB1 constructs in either 50 mM or 0.2 M ammonium acetate, pH 6.5.
  • HMGB1 constructs have similar native M/Z profiles, with dBB12L closely resembling a reduced HMGB1 construct with two HMG Boxes apart from each other. Continuous line; compact monomer. Dashed line; extended monomer (HMG Boxes distal to each other). Removal of the acidic tail (FR HMGB11-164, blue curves compared to FR- HMGB1, red curves) and higher ionic strength (Comparison of the spectra for the same construct in either 50 mM or 200 mM ammonium acetate) increases the prevalence of higher M/Z states (partial unfolding).
  • Figure 7C Solvent accessible surface area (SASA) calculations for the average folded HMGB1 monomer, the extended and compact monomer states, and the unfolded monomer from Figure 7D.
  • Figure 7D Denaturing ESI/MS deconvolution, SDS- PAGE and SEC profiles of HMGB1 constructs after storage at room temperature for 180 days (D0-D180), in 0.2 M ammonium acetate, pH 6.5.
  • Figure 8A-8F show dBB12L has reduced binding to RAGE and does not signal through TLR-2 or TLR-4.
  • DS-HMGB1 binds RAGE more avidly compared to FR- HMGB1.
  • FIGS. 8D-8E show DS-HMGB1 promoted NF- ⁇ activity in reporter HEK-Dual cells expressing human TLR-2 and CD14 ( Figure 8D) or murine TLR- 4, MD-2 and CD14. ( Figure 8E). dBB12L and FR-HMGB1 did not promote NF- ⁇ signaling in either cell line. Data shown as mean ⁇ SEM fold change compared to control (media alone).
  • FIG 8F Disulfide HMGB1 (DS-HMGB1) increased TNF production in monocytes, which was further enhanced by the presence of suboptimal amounts of LTA, but not of LPS.
  • FR- HMGB1 and dBB12L did not promote TNF expression, even when pre-incubated for 24h with LPS or LTA. Response to LPS pre-incubated with these constructs was also significantly reduced.
  • n 3 donors, each with three technical replicates.
  • Figure 9 shows the effects of modification of the linker on regenerative activity of FR-HMGB1.
  • the dBB12L sequence shown in the alignment is provided by residues 75-91 of SEQ ID NO: 2.
  • FR-HMGB1 sequence shown in the alignment (“FR”) is provided by residues 79-93 of SEQ ID NO: 1.
  • the “Sub(79-83)”, “Sub(84-88)”, and “Sub(89-93)” sequences shown in the alignment are provided by SEQ ID NO: 175.
  • Figures 10A-10J show the regenerative effects of optimal doses of dBB-HMGB1 and FR- HMGB1 are identical to those of an activating injury.
  • Figure 10A Volcano plot showing differentially expressed genes in muscle stem cells by fold change following injury or HMGB1 induced GAlert.
  • FIG. 10B Network map of gene ontology terms of differentially expressed cells during GAlert induction in muscle stem cells.
  • Figure 10C Dose response of FR-HMGB1 in a BaCl2 skeletal muscle injury model, with regeneration quantified by fiber cross-sectional area. The optimal dose was 0.75 mg/kg (28.75 nmol/kg) and was used in subsequent assays.
  • Figure 10D Animals were dosed with FR-HMGB1 (optimal dose) at the varying timepoints after injection of BaCl2 to assess the interval where treatment with FR- HMGB1 is effective post-injury. Values in Figure 10C and Figure 10D shown as mean ⁇ SEM in nested ANOVA with Holm-Sidak correction.
  • Figure 10E Pharmacokinetics of HMGB1 in the circulation following administration of 0.75mg/ml FR-HMGB1, fitted by nonlinear least squares to a two-phase exponential decay curve.
  • Figure 10G Ejection fraction over time calculated from serial MRI scans.
  • FR-HMGB1 Infarct size over time calculated from serial MRI scans.
  • Figure 10I Representative mid-ventricular short-axis cine-MRI images at end-diastolic and end-systolic phases of the cardiac cycle 1 and 5 wk after MI. Blood in the chambers appears bright.
  • FR- HMGB1 group shows preservation of heart function and maintenance of wall thickness (short, unlabeled arrows) with visible separation of right and left ventricles (arrows labeled “RV” or “LV”, respectively) during systole.
  • LV left ventricle dilation
  • n 10 per group. All MRI scans performed and assessed by a blinded observer.
  • Figure 10J Mean muscle cross-section area at given time points after BaCl2 muscle injured animals treated with PBS (black in original coloring, left bar at each day), 28.75 nM/kg of FR-HMGB1A-c001 (red in original coloring, middle bar at each day) or dBB12L (green in original coloring, right bar at each day).
  • N 5 per group and timepoint, nested ANOVA (Holm-Sidak post-hoc correction).
  • Figures 11A-11B show results of a peptide array of CXCL12 peptides interacting with HMGB1.
  • Figure 11A Peptide array of full-length CXCL12. “+” positions correspond to positive control 10-His peptides; the rest of the peptides comprise CXCL1215-mers shifted two (2) residues in succession towards the C-terminus.
  • the membrane was exposed to 1 uM HMGB1(FR or 3S)-His6 (1-214), BoxA-His6 (8-78) and BoxB-His6 (94-162) for 24 hours. Bound protein was detected by anti-His-HRP conjugate chemoluminescence.
  • a peptide of CXCL12 interacting with full length HMGB1 cannot interact with either Box A or Box B alone, confirming the requirement of the N-terminal segment of each Box domain (particularly, D4 in box A/D90 in box B): intensity of the spots pertaining to the common CXCL12 peptide is also markedly decreased upon binding to the Box domains alone when compared to FL- HMGB1. Binding to 3S seems to be of higher intensity than that to FR; this is likely due to protein oxidation during the assay, although this was not quantified due to the low concentration of protein used being unsuitable for ESI/TOF MS. BLI data, however, do suggest a lower off rate of CXCL12 from 3S than from FR-HMGB1.
  • Figure 11B CXCL12 dimer (PDB 2J7Z) with the regions binding HMGB1 highlighted. Red in original coloring: shared binding region. Blue in original coloring: non-shared binding region.
  • Figure 12 shows interferograms in BLI of CXCL12 binding to immobilized HMGB1 constructs. Biotinylated HMGB1 constructs were immobilized on streptavidin-coated Octet biosensors and dipped in rising concentration of CXCL12. Individual lines within each interferogram graph are in the same order of the key from top to bottom unless otherwise indicated. Interferograms are colored according to CXCL12 concentration (key in top right).
  • Each set of three replicates (cycles) for a given sensor is surrounded by a colored overlay according to construct.
  • Figures 13A-13D show NMR validation of residues involved in CXCL12 binding.
  • Figure 13A Cumulative CSP of HMGB13S 1-184 (HMGB1A-c007) upon addition of 1:2 molar equivalents of CXCL12 in one step (1:1 HMG Box to CXCL12 ratio). Box A and Box B residues have been considered separate molecules for the purposes of median CSP calculation. Intensity of the darkness of the bars in the bar graph indicates higher relative CSP.
  • the sequence of each HMGB1 construct has been overlaid with the residue number; an empty column (no number) represents residues which could not be mapped in the parallel 3D 1H-15N HSQC/NOE/TOCSY experiments.
  • Figure 13B 15N HSQC-HQMC peak spectra for (Figure 13A) in 10 mM HEPES 150 mM NaCl pH 7.5 buffer. Protein concentrations are indicated in the spectra overlay.
  • Figures 13C and 13D show 15N HSQC-HQMC peak spectra in 10 mM HEPES 150 mM NaCl pH 7.5 buffer. Protein concentrations are indicated in the spectra overlay.
  • Figure 13C HMGB1A 94-162 (2-day experiment);
  • Figure 13D HMGB1A 89-174 (6-day experiment; minor degradation occurs after day 4).
  • FIG 14 shows interferograms in BLI of HMGB1 constructs binding to immobilized Fc-RAGE.
  • RAGE-Fc was immobilized in the surface of AHC sensors and dipped in rising concentration of different HMGB1 constructs.
  • Two experiments were run with different concentration ranges: the three columns of graphs in the left, 0 to 22.22 ⁇ M HMGB1 over 9 steps; on the right, 0 to 25 ⁇ M over 7 steps. Both are color-coded by concentration (top). Colors indicate the specific construct concentration.
  • the lines in the graph from top to bottom correspond with the highest concentration to the lowest concentration in most cases.
  • Each graph corresponds to a single sensor (replicate).
  • Figure 15 shows histological images of regenerating muscle in response to FR- HMGB1 (red) or dBB12L (green) compared to PBS control (black), from Figure 10.
  • Figure 16 shows plasmid vector maps. Vector maps with features and restriction sites. TEV: Tobacco etch virus protease recognition site.6-His: 10/6-histidine residue affinity epitope. FLAG: FLAG affinity epitope. StrepTag: StreptactinXT affinity epitope. SacB: Levansucrase precursor (negative selection in the presence of sucrose). pLIC: Annealing sites for sequencing primers used in colony screening. All plasmids contain kanamycin resistance (50 ⁇ g/mL).
  • Figure 17 shows process to generate 3S-HMGB1.
  • This invention provides a polypeptide represented by the following formula: H2N-A-X-B-A-X-B-HOOC wherein each A represents consecutive amino acids, the sequence of which (1) is a sequence of four amino acids (a) identical to the sequence of amino acids 90 – 93 of wild type human HMGB1 (SEQ ID NO: 1), or (b) which differs from the sequence of (a) in respect to one or more amino acids; and (2) has at its amino terminal end, between one and six consecutive amino acids, the sequence of which (a) is identical to the sequence of the corresponding one to six amino acids preceding amino acid 90 in wild type human HMGB1, or (b) differs from the sequence of (a) in respect to one or more amino acids; and (3) optionally has a methionine at the amino terminus, wherein each A may be the same or different; wherein each X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 – 162 of
  • the methionine is present at the amino terminus of the polypeptide.
  • A has at its amino terminal end, the amino acid corresponding to amino acid 89 of wild type HMGB1 (SEQ ID NO: 1).
  • B has at its carboxy terminal end, six amino acids corresponding to amino acids 169-174 of wild type human HMGB1.
  • the number of amino acids in the B-A between the two Xs is at least 13. In such embodiments, the number of amino acids in the B-A between the two Xs is up to 22, more preferably up to 21, 20, 19, 18, 17, 16, 15, or 14.
  • the number of amino acids in the B-A between the two Xs is between 12 and 22 inclusive. [0035] In yet further embodiments, the number of amino acids in the B-A between the two Xs is between 13 and 22 inclusive. In some embodiments the number of amino acids in the B- A between the two Xs is between 13 and 21 inclusive, more preferably between 13 and 20 inclusive, more preferably between 13 and 19 inclusive, more preferably between 13 and 18 inclusive, more preferably between 13 and 17 inclusive, more preferably between 13 and 16 inclusive, more preferably between 13 and 15 inclusive, more preferably between 13 and 14 inclusive.
  • the sequence of (2)(c) or (2)(d) of either B or of both Bs differs at the one or more positions by the presence of a glycine, serine, proline, arginine, lysine, aspartic acid, glutamic acid, or histidine amino acid residue instead of the amino acid residue present at such position or positions in naturally occurring HMG1.
  • the sequence of (2)(c) or (2)(d) of either B or of both Bs is or comprises GSGSG (SEQ ID NO: 175).
  • the GSGSG (SEQ ID NO: 175) has been inserted into the region between the sequence of the B and the sequence of the A between the two Xs.
  • the order or number of the glycine and serine residues has been altered within the SEQ ID NO: 175 peptide sequence.
  • the sequence of (2)(c) or (2)(d) may be or comprise any of GSSSG (SEQ ID NO: 176), GSSGS (SEQ ID NO: 177), or GGSGG (SEQ ID NO: 178).
  • the amino acids of (2)(c) or (2)(d) of either B or of both Bs lack a defined secondary structure, or have a turn or random coil secondary structure.
  • either A or both As are five consecutive amino acids.
  • the five consecutive amino acids have a sequence identical to the sequence of amino acids 89 – 93 of wild type human HMGB1.
  • a) the A between the Xs is five consecutive amino acids and the B between the Xs is at least seven consecutive amino acids;
  • the A between the Xs is six consecutive amino acids and the B between the Xs is at least six consecutive amino acids;
  • c) the A between the Xs is seven consecutive amino acids and the B between the Xs is at least six consecutive amino acids;
  • the A between the Xs is eight consecutive amino acids and the B between the Xs is at least six consecutive amino acids;
  • e) the A between the Xs is nine consecutive amino acids and the B between the Xs is at least six consecutive amino acids; or f) the A between the Xs is ten consecutive amino acids and the B between the Xs is at least six consecutive amino acids.
  • a) the B between the Xs is six consecutive amino acids and the A between the Xs is at least six consecutive amino acids; b) the B between the Xs is seven consecutive amino acids and the A between the Xs is at least five consecutive amino acids; c) the B between the Xs is eight consecutive amino acids and the A between the Xs is at least five consecutive amino acids; d) the B between the Xs is nine consecutive amino acids and the A between the Xs is at least five consecutive amino acids; e) the B between the Xs is ten consecutive amino acids and the A between the Xs is at least five consecutive amino acids; f) the B between the Xs is eleven consecutive amino acids and the A between the Xs is at least five consecutive amino acids; or g) the B between the Xs is twelve consecutive amino acids and the A between the Xs is at least five consecutive amino acids.
  • This invention also provides a composition comprising any of the polypeptides of the invention and a carrier.
  • the polypeptide is present in a therapeutically or prophylactically effective amount and the carrier is a pharmaceutically acceptable carrier.
  • This invention also provides methods of treating a subject suffering from, or at risk for developing, a condition which would be alleviated by promoting regeneration of a tissue or cells that rely upon CXCR4 + cells for repair which comprise administering to the subject a polypeptide or composition of the invention in an amount or dose effective to promote regeneration of the tissue or cells, that is, achieve a therapeutic or prophylactic effective dose of the pharmaceutical composition of the invention in a subject in need thereof.
  • the condition is an acute injury.
  • the polypeptide is administered within 5 hours, preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours and most preferably within 1 hour of the acute injury.
  • the condition is a chronic condition.
  • the polypeptide is administered repeatedly at a daily, weekly, monthly, or yearly interval.
  • the acute injury is myocardial infarction.
  • the tissue is cardiac tissue or myocardium.
  • the acute injury is stroke, spinal cord injury, or peripheral nerve injury.
  • the polypeptide is administered within 5 hours of the myocardial infarction. In some embodiments, the polypeptide is administered within 5 hours, preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours and most preferably within 1 hour of the myocardial infarction. [0055] In a currently preferred embodiment, the polypeptide is administered within 5 hours of the stroke, spinal cord injury, or peripheral nerve injury. In some embodiments, the polypeptide is administered within 5 hours, preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours and most preferably within 1 hour of the stroke, spinal cord injury, or peripheral nerve injury.
  • the acute injury is a fracture, joint replacement or bone fusion.
  • the tissue is a bone.
  • the acute injury is a skeletal muscle injury, joint injury or ligament injury.
  • the condition involves liver damage.
  • the tissue is liver tissue.
  • the chronic condition is non-alcoholic steatohepatitis, liver cirrhosis or infective hepatitis.
  • the chronic condition involves damage to the brain or other parts of the central nervous system.
  • the chronic condition is Parkinson’s disease, dementia, multiple sclerosis, motor neuron disease or peripheral nerve injury.
  • the chronic condition involves a chronic joint injury.
  • the chronic joint injury is inflammatory arthritis or osteoarthritis.
  • the condition involves damage to the lung.
  • the acute injury is a viral infection of the lungs, bacterial infection of the lungs, fungal infection of the lungs, or mechanical injury to the lungs.
  • the viral infection of the lungs is an infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • the mechanical injury is a ventilator-induced injury.
  • the chronic condition is idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease, or emphysema.
  • the condition involves the gut.
  • the acute injury is a surgical injury to the gut.
  • the chronic injury is inflammatory bowel disease, Crohn’s disease, or ulcerative colitis.
  • the condition involves damage to the skin.
  • the acute injury is a burn or a surgical injury of the skin.
  • the chronic condition is a skin ulcer, diabetic ulcer, venous ulcer, arterial ulcer, or pressure ulcer.
  • the condition involves the pancreas and the cells are islet cells.
  • the condition is diabetes.
  • the condition is neutropenia following chemotherapy and the tissue is bone marrow.
  • the acute injury is chemotherapy, and optionally the polypeptide is administered before or within 5 hours of administration of the chemotherapy.
  • the polypeptide is administered before the acute injury.
  • the polypeptide is administered after the acute injury.
  • the condition is kidney failure and the tissue is kidney tissue.
  • the chronic condition is a disease that results in chronic renal failure.
  • the acute injury is elective surgery and the polypeptide is administered before, during or within 5 hours after the surgery.
  • the acute injury is a sport or military combat injury.
  • the chronic condition is a chronic skeletal muscle condition.
  • the chronic skeletal muscle condition is a muscular dystrophy such as Duchenne macular dystrophy, sarcopenia, or disuse atrophy.
  • engineered means a non-naturally occurring compound that has been created based upon changing a naturally occurring compound.
  • An engineered polypeptide may include amino acids corresponding to amino acids of a naturally occurring polypeptide and amino acids that vary in identity or location from those of a naturally occurring polypeptide. Such an engineered polypeptide may also be referred to as an “analogue” or “derivative” of the naturally occurring polypeptide.
  • stem cell means any unspecialized cell that has the potential to develop into many different cell types in the body, including without limitation hemopoietic stem cells.
  • an effective amount means an amount of a polypeptide of the invention that is capable of achieving a desired result, for example, alleviating a condition or one or more symptoms associated with a condition, for example, an acute or chronic tissue injury.
  • the specific amount or dose of a polypeptide administered according to this invention will, of course, be determined by the particular manner of treating or preventing the condition, for example, the route of administration, the physiological state of the subject, and the severity of the condition being treated.
  • an engineered HMGB1 polypeptide administered to a subject is preferably in the form of a composition comprising a therapeutically or prophylactically effective amount of the engineered HMGB1 polypeptide.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material.
  • suitable carries available and routinely used in pharmaceutical compositions are well within the knowledge of those skilled in the art. Accordingly, there is a wide variety of suitable carries available and routinely used in pharmaceutical compositions.
  • the first encompassed ⁇ -helix I and part of helix II of each HMG Box (peptides 1- 3 in Box A, peptides 5-6 in Box B), overlapping the glycyrrhizin binding site [43].
  • the second region (peptide 4 in Box A and 7 in B) was located at the C-terminal half of ⁇ -helix III.
  • the first CXCL12 binding peptide (helices I and II) had much greater intensity of the spots, indicating potentially higher affinity for CXCL12.
  • TLR-2 also bound to the linker region between the two Boxes, the peptide immediately adjacent to the acidic tail of HMGB1 and the C-terminal regions to each HMG Box, extending beyond the residues that bound CXCL12.
  • the binding interface for TLR-2 within Box A is within the alpha helices which twist upon oxidation [50] to adopt a different 3D configuration ( Figure 4, section A).
  • Peptides binding to TLR-4 ( Figure 3, section B) form a binding pocket in Box A that is only continuous when Box A is oxidized ( Figure 4, section B). These include the delipidated LPS binding segment and the region bordering the Lipid A binding region within Box A [51].
  • FR-HMGB194-162 and 89-174 to represent HMG Boxes with and without the flanking regions, as well as 3S HMGB11-184.
  • Non-oxidizable 3S-HMGB1 was used instead of native full-length FR-HMGB1 as the time required for obtaining a full set of 3D spectra at 750 MHz ( 15 N HSQC-TOCSY/NOESY and associated 15 N HSQC spectra) would result in oxidation of the latter.
  • CSP changes for residues not identified in the peptide arrays A147, M131, A169, K172, G173 in the construct with the flanking regions.
  • Other residues that have not been previously identified but were flagged as being potentially important in the peptide arrays did not display either CSP or volume changes upon addition of CXCL12 (C105, E107, Y108).
  • this engineered construct dBB12L ( Figure 6C) comprised the following segments of the native HMGB1 protein: flexible N-terminal region (from HMGB189-93), first Box B (from HMGB194-162), 12-residue linker C-terminal of native Box B (from HMGB1163- 174), and second Box B (from HMGB194-162).
  • the linker length of 12 residues in this dBB12L is similar to the 10 amino acids in the linker of native HMGB1 and included residues 172 and 173 which showed changes in CSP on CXCL12 binding.
  • DSF differential scanning fluorimetry
  • SASA solvent accessible surface area
  • dBB12L construct has greatly reduced affinity for RAGE and cannot signal through TLR-2 or TLR-4 [00107]
  • dBB12L had decreased TLR-2, TLR-4 signaling and RAGE binding, whilst preserving HMGB1-mediated regeneration. Due to the lack of an established signaling assay for RAGE, we assessed the binding of RAGE to HMGB1 using real-time kinetics (BLI) and an endpoint assay (ELISA). ELISA-based affinity measurements (Figure 8A) showed that 3S-, FR- and DS-HMGB1 at equilibrium bound similar amounts of RAGE, with DS-HMGB1 and 3S-HMGB1 having significantly higher affinity compared to FR-HMGB1.
  • dBB12L did not bind RAGE.
  • Three additional HMGB1 constructs were tested, DS-HMGB11-184, which has an intact RAGE binding peptide and oxidized Box A but no acidic tail and therefore has all the requisites for RAGE binding; DS-HMGB11-164, which lacks a significant portion of the RAGE binding peptide but retains an oxidized Box A; and DS-Box A alone.
  • DS-HMGB11-184 bound RAGE, but with reduced capacity and affinity compared to full-length DS-HMGB1.
  • DS-HMGB11-184 had much higher RAGE binding affinity than all other constructs, albeit with a slightly faster dissociation rate, compared to ELISA where it had lower affinity than DS-HMGB1.3S-HMGB1, whilst binding equivalent amounts of RAGE to DS- or FR- HMGB1, had similar affinity to FR-HMGB1 but much slower overall kinetic rates, whereas in ELISA it had affinity and binding capacity equivalent to DS- HMGB1.
  • the higher RAGE affinity of DS-HMGB1 compared to FR in both assays was due to a much faster association rate (k on ), whereas dissociation rates (k off ) were nearly identical for these two redox forms.
  • dBB12L which had an association rate closer to DS- HMGB1, exhibited very unstable binding due to a very high dissociation rate.
  • DS-HMGB11- 164 also had a faster RAGE binding equilibrium with overall lower binding affinity than full length DS-HMGB1, albeit with higher affinity than dBB12L-HMGB1.
  • the deletion of both the final 10 residues in Box B (175-184) and the disulfide bridge in Box A by substituting it with Box B in dBB12L resulted in unstable binding of RAGE.
  • HMGB1 binds TLR-2, TLR-4, and RAGE and signaling from all receptors converges to the NF- ⁇ pathway [25].
  • TLR-specific signaling using NF- ⁇ reporter cell lines engineered to express either TLR-2 or TLR- 4 and their co-receptors.
  • Disulfide HMGB1 promoted NF-kB signaling via TLR-2 ( Figure 8D) and TLR-4 ( Figure 8E).
  • dBB12L failed to signal in either cell type.
  • DS-HMGB1 synergizes with TLR-2 ligands such as lipoteichoic acid (LTA) to promote proinflammatory signaling [32].
  • FR-HMGB1 or dBB12L reduced TNF expression compared to LPS alone.
  • dBB12L does not signal via TLR-2 or TLR-4, even in the presence of their cognate ligands, has greatly decreased affinity for RAGE and reduces LPS-mediated pro-inflammatory signaling.
  • the dBB12L construct has pro-regenerative activity comparable to that of FR-HMGB1
  • Distant injury has previously been shown to transition stem cells to G Alert [13] . Therefore, we first compared the transcriptomic response of skeletal muscle stem cells to FR- HMGB1 or injury to the contralateral limb.
  • FR-HMGB1 was found to be effective in promoting repair when injected up to 5 h post-injury (Figure 10D).
  • Figure 10E We then assessed the half-life of FR-HMGB1 in the circulation following iv administration. We found that there was an initial rapid clearance (t1/2 ⁇ 11 min) followed by subsequent slower clearance rate (t1/2 ⁇ 120 min) (Figure 10E). This would be consistent with the half-life of 25 min in humans [54], with the protein being cleared by binding to haptoglobin [55,56].
  • FR-HMGB1 accelerates regeneration of skeletal muscle, bone and blood following injury by promoting the transition of stem and progenitor cells to G Alert [8].
  • DISCUSSION HMGB1 needs to be modified in order to be used as a tissue repair therapeutic [00116] Therapies based on administration of exogenous stem cells to promote repair of solid organs have failed to deliver on the initial promise [6,59], and killed cells are just as effective by triggering an immune response [60]. An alternative, potentially more effective approach, would be to target endogenous regenerative repair processes, including resident stem and progenitor cells [61,62].
  • FR-HMGB1 can signal through TLR-4 [29,35], TLR- 2 [32,33,35] or RAGE [37,67] to converge on NF- ⁇ [25,68], leading to synergistic expression of proinflammatory cytokines [69,70]. Therefore, development of HMGB1 as a therapeutic is crucially dependent on engineering the molecule to eliminate signaling via all three receptors.
  • dBB12L was as stable as 1-164 FR-HMGB1 or full-length FR-HMGB1, with no aggregation or degradation on storage for prolonged periods of time.
  • HMGB1 A similar motif is also present in other RAGE ligands such as S100 proteins, and homologous peptides to these sequences are effective antagonists of HMGB1-mediated RAGE signaling [38,52].
  • the acidic tail of HMGB1 shares residues with the RAGE binding peptide [40] and has been proposed as a regulator of RAGE interaction, analogous to its role in TLR-2 binding.
  • RAGE binding peptide in Box A [39] which was previously thought to require Caspase-1 processing for activity, is exposed in the intact HMGB1 when in solution and alters its conformation upon oxidation of the disulfide.
  • 3S- HMGB1 which has an affinity for RAGE similar to FR-HMGB1 in BLI, shows a much higher apparent affinity in ELISA similar to DS-HMGB1 due to a dissociation rate much lower than that of FR-HMGB1 or DS-HMGB1.
  • This increased RAGE binding may in part account for the increased fibrosis seen in mouse models of myocardial infarction compared to controls, whereas FR-HMGB1 promoted regeneration and improved function [31].
  • SPR others have also shown that binding of HMGB1 to RAGE likely requires two distinct binding sites, one of which varies according to the oxidation status [20].
  • FR-HMGB1 administered intravenously at the time of myocardial infarction resulted in improved survival, reduction in infarct size and improved left ventricular ejection fraction.
  • administration of dBB12L would also promote regeneration of tissues that rely on stem cells for repair such as bone, skeletal muscle and blood, as well as tissues where regeneration is predominantly reliant on mature cell populations such as cardiomyocytes in the heart.
  • dBB12L is likely to be effective if administered up to 5 h after injury. This is important as the median time for admission to hospital following MI in the USA is 3 h [81].
  • this region could be substituted with a linker of between 13 and 17 residues with any of the following modifications: [00129] a) Substitution of any, or all, residues equivalent to positions 168-174 (last 7 residues in the linker) in wild type human HMGB1 for any of the following: [00130] A random sequence of amino acids such that this region can adopt either no specific secondary structure, coil-turn structures, or alpha helical conformations, with the first two being preferred. [00131] Substitution of the amino acids (e.g., Lys ⁇ -> Arg, aliphatic to aliphatic substitutions). [00132] Flexible amino acid sequences [85] such as Gly-Ser, or turn-inducing residues such as Pro.
  • TLR-2 and RAGE binding and signaling is further impaired by the truncation of the binding sequences before the C-terminal acidic tail and the fact that the binding regions in oxidized Box A has been replaced by those equivalent in Box B.
  • Box B adopts a different surface arrangement than Box A when it is oxidized and therefore is unable to effectively bind to TLR4,TLR-2 or RAGE.
  • the double Box B construct maintains regenerative activity equivalent to FR-HMGB1. Taken together these data show that the double Box B constructs described above can be developed as clinical therapeutics.
  • dBB12L a construct that does not signal via TLR-2 or TLR-4 and fails to effectively bind RAGE was designed.
  • FR-HMGB1 transitions stem cells to GAlert in a manner similar to distant injury despite a short half-life and is effective when administered up to 5 hours after injury.
  • dBB12L promotes tissue regeneration in vivo as effectively as FR-HMGB1. Accordingly, dBB12L can be developed for clinical translation.
  • HMGB1 Reduced High Mobility Group Box 1
  • CXCL12 CXC Ligand 12
  • CXCR4 CXC Receptor 4
  • FR-HMGB1 CXC Ligand 12
  • DS-HMGB1 disulfide form
  • RAGE Receptor for Advanced Glycation End Products
  • Patent 9,623,078 refers to peptides limited to amino acids 1-44 (0-43 for our data) for cardiac regeneration.
  • U.S. Patent Application Publication US 2009/0202500 A1 discloses methods for tissue repair but only refers to full-length (1-215) wild-type HMGB1 (0-214 for our data).
  • the dBB12L construct presented herein has no RAGE binding or TLR-4/2 signalling, is 177 amino acids long, and includes amino acid substitutions that have not been previously described. Therefore, the constructs presented herein do not fall within the scope of the prior art.
  • CLINICAL APPLICATIONS [00142] This invention provides polypeptides and methods to harness endogenous regenerative processes to enhance tissue repair.
  • dBB12L a polypeptide of the invention
  • tissues include tissues where repair is primarily dependent on stem and progenitor cells, such as skeletal muscle and the haemopoietic system, as well tissues where repair is largely dependent on existing mature cells, e.g., cardiomyocytes in the adult mammalian heart.
  • the main target population are patients following MI, especially those at risk of developing heart failure (104).
  • a novel therapeutic that limits cardiac damage, promotes regeneration following MI and prevents the development of heart failure would dramatically reduce morbidity and mortality, and massively reduce healthcare burden.
  • Definitive data using an established (105-108) permanent ligation murine MI model that reliably leads to cardiomyocyte necrosis (109) show that a single iv dose of FR-HMGB1 at the time of injury leads to enhanced survival (83% for animals treated with FR-HMGB1 compared to 52% in the group treated with PBS placebo), and compared to controls, ⁇ 16% improvement of absolute cardiac ejection fraction and ⁇ 60% reduction in infarct size compared to PBS controls over 5 weeks (Figure 10F).
  • FR-HMGB1 While native FR-HMGB1 promotes functional recovery post MI ( Figures 10F-10I), local conversion to the disulfide form promotes thrombus formation and propagation via RAGE, TLR-2 and TLR-4 (110). Constructs reported by others such as 3S-HMGB1 that retain RAGE binding ( Figure 8B) result in excessive fibrosis and impairment of function following MI (111). FR-HMGB1 also binds RAGE, albeit to a lesser extent than DS-HMGB1 and, therefore, would not be suitable for clinical use.
  • HMGB1 signalling via TLR-2 plays a key role in ischaemia reperfusion injury following myocardial infarction (112) and thrombosis (110),
  • the inventors a have shown the key role of TLR-2 in human atherosclerosis (113).
  • TLR-4 signalling is also crucial in myocardial reperfusion injury (114).
  • the redox conditions in the ischemic and inflamed microcirculation of the damaged heart following myocardial infarction will promote conversion of FR-HMGB1 to the disulfide form (DS-HMGB1), which is a central mediator of thrombosis (110).
  • DS-HMGB1 disulfide form
  • adenoviral transduction and growth factors require intracardiac injection or topical patch application (FSTL1), manipulation of developmental pathways carries oncogenic risk (128) and viral transduction of miRNA199- a in pigs resulted in fatal arrhythmias (127).
  • An alternative strategy for stimulating cardiac regeneration by promoting clearance of immune cells requires repeated injection of VEGF-C (129).
  • Inhibition of MAP4K4 promotes myocardial survival and limits infarct size, but there was no regenerative effect (130). To date, none of these strategies have progressed to clinical trials.
  • This invention provides a unique solution which targets endogenous processes to promote cardiomyocyte survival and regeneration of multiple tissues.
  • FR-HMGB1 acts via the cell surface receptor CXCR4, it is not expected to have off target effects associated with targeting intracellular processes, e.g. by adenoviral transduction of transcription factors or miRNA.
  • HMGB1 inhibition increased infarct size following ischemia reperfusion injury (134) and whilst local upregulation (135, 136) or intramyocardial injection of FR-HMGB1 has been shown to be effective in both mice (111, 137, 138) and sheep (139), our data indicate iv administration is efficacious and more likely to reach all target cells.
  • the engineered double Box B construct of the invention which avoids deleterious proinflammatory signaling should be safe.
  • dBB12 can be used to promote healing following fracture or arthroplasty, thereby reducing the risk of potential complications such as loosening of components.
  • Brain and nervous system dBB12L may be used to improve patient outcomes following stroke. Other potential indications include Parkinson’s disease and dementia.
  • Lung. dBB12L is contemplated to improve outcomes following lung injury, for example, following Covid-19 or in patients with idiopathic pulmonary fibrosis.
  • dBB12L Liver.30% of people in the USA are estimated to suffer from non-alcoholic liver disease.60% of these go on to develop non-alcoholic steatohepatitis and 20% of those develop liver cirrhosis. Treatments are being developed to limit and prevent liver damage from tehse conditions. The inventors propose that dBB12L to be used in combination with these treatments to promote liver regeneration.
  • Gut. dBB12L may be used to promote healing of the gut, for example, following surgery or patients with inflammatory bowel disease such as ulceractive colitis in combination with treatments to control inflammation.
  • Kidney. dBB12L may be used to promote regeneration of the kideny, thereby potentially avoiding the need for dialysis or kidney transplantation.
  • dBB12L may be used to promote wound healing eg following surgery, burns or patients with ulcers eg diabetic ulders.
  • Pancreas. dBB12L may be used to improve outcomes in patients with type 1 diabetes mellitus by promoting regeneration of islet cells.
  • Bone marrow. dBB12L may promote regeneration of the haemopoetic system e.g. following chemotherapy, thereby preventing severe potentially life thereatening neutropenia.
  • the inventiors have previously shown that FR-HMGB1 is effective even if adminsitered up to 2 weeks before injury (142).
  • dBB12L is equally efficacious to FR- HMGB1 ( Figure 10J) it is contemplated that this polypeptide may be used prophylactically, for example, by the military or for sports injuries or before elective surgery or chemotherapy.
  • Mach-1 T1R cells Invitrogen, no antibiotic resistance or induction, BL21(DE3)-R3- pRARE2 (in-house BL21 derivative, chloramphenicol resistance 36 ⁇ g/mL , T7-polymerase lac induction [86]) and BL21(DE3)-R3-pRARE2-BirA (in vivo biotinylation derivative of the above, additional spectinomycin resistance 50 ⁇ g/mL) were sourced from chemically competent stocks made in-house.
  • Bacterial culture media 20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 0.1862g/L KCl were autoclaved and supplemented with 4.132 g/L MgCl2 and 20 mM glucose.
  • LB Lia Bertani
  • TB Terific Broth: 12 g/L tryptone, 24 g/L yeast extract, 4 g/L glycerol, 12.5 g/L K 2 HPO 4 , 2.35 g/L KH2PO 4 ., autoclave-sterilized.
  • TB supplement 1.6% w/v glycerol, 1% glucose, 25 mM (NH4)2SO 4 , 10 mM MgSO 4 , 10X trace metals, 0.22 ⁇ M sterile filtered.
  • Trace metal solution 50 mM FeCl 3 (13.5 g/L), 20 mM CaCl 2 (2.94 g/L), 10 mM MnCl2 (1.96 g/L), 10 mM ZnSO 4 (2.88 g/L), 2 mM CoCl 2 (0.48 g/L), 2 mM CuCl2 (0.34 g/L), and 2 mM NiCl 2 (0.48 g/L), in 0.1 M HCl, 0.22 ⁇ M sterile-filtered.
  • M9 minimal medium 16 g/L Na 2 HPO 4 , 4 g/L K 2 HPO 4 , 1 g/L NaCl, pH 7.2-7.3 and 2.5 g/L FeSO 4 , 0.25 mg/L ZnCl2, 0.05 mg/L CuSO 4 , 0.25 g/L EDTA, 1 mM MgSO 4 were autoclaved and supplemented with 4 g/L glucose, 1 g/L U-99% 15 NH 4 Cl (Cambridge Isotopes), 0.3 mM CaCl2,1.5 mg/L D-biotin and 1.5 mg/L Thiamine-HCL from sterile filtered stocks.
  • Plasmids were sourced from the SGC libraries [86]. All plasmids contain a 6xHis tag with a TEV-cleavage site; pNIC-Bio3 and pDsbC-HT-CBio also have C-terminal biotinylation epitopes (which can be removed with a stop codon). Plasmid DNA was linearized by restriction enzyme digestion: BfuA1 (3h, 60°C) for pNIC-CTHF or BsaI (2h, 37°C).
  • Cut vector DNA was purified with a PureLink PCR kit and treated with T4 DNA polymerase (NEB M0203) in the presence of 0.25 mM dGTP (pNIC-CTHF) or dCTP as per manufacturer protocols.
  • Reaction consisted of 5 ⁇ L Herculase II buffer, 1 ⁇ M of each primer, 6 ⁇ g/mL plasmid template, 1 ⁇ M dNTP mixture and 1 unit Herculase II polymerase (Agilent 600679; supplied with buffer and 100 ⁇ M dNTP stocks) in 25 ⁇ L final volume. PCR products were purified before further use (PureLink kit, ThermoFisher K310001). [00171] Amplified coding sequences (alleles) were cloned into the destination vector via ligation independent cloning (LIC).
  • LIC ligation independent cloning
  • the insert was treated with T4 DNA polymerase in the presence of a cognate nucleotide to that used for the vector (10 ⁇ L reaction volume), and 2 ⁇ L was mixed with 1 ⁇ L of treated vector and annealed for 30‘.40 ⁇ L ice-cold Mach-1 cells (for storage) or 20 ⁇ L BL21(DE3)-R3-pRARE2/ BL21(DE3)-R3-pRARE2-BirA cells (for expression) were added and heat-shocked for 45” at 42°C before chilling in ice. Recovery was performed for 2 h in SOC medium at 37°C prior to plating on selective media with 5% sucrose and antibiotics.
  • CXCL12 constructs were cloned with an in-frame SUMO protease site N-terminal to the mature protein to allow for periplasmic secretion with an N-terminal fusion protein in the pDsbC-HT-CBio vector (DsbC-SUMO-CXCL12) to avoid addition of N-terminal residues to the protein which could affect its activity [87,88] whilst obtaining folded, oxidized CXCL12 via the DsbC fusion protein system [89].
  • a detailed table of primers and vectors used for each construct, boundaries, expression strains, and base pairs/amino acid sequences can be found in the Supplemental Methods section. All mutants were verified by sequencing (SourceBioscience).
  • HMGB1-dBB The sequence for HMGB1-dBB was designed in silico by codon- optimizing a Box B 89-174 sequence according to E. coli BL21-DE3 genome (assembly ASM956v1) placed after the native HMGB1 Box B sequence, and synthetized in vitro by Twist Bioscience (San Francisco, USA) cloned in pNIC-CTHF.
  • Recombinant protein expression [00174] 20 mL of overnight culture of HMGB1-expression strain transformants, grown from a fresh agar plate streak, were inoculated into 1 L of TB (or M9) medium with supplement and allowed to grow up to OD 2.0 at 37°C with 0.45 RCF orbital shaking (OD 0.6 for M9 medium).
  • Precultures used for production of 15 N labelled HMGB1 were first spun down at 1000 RCF for 5 ‘and washed in M9 medium. Once the target OD was reached, were cooled to 18°C before addition of 0.5 mM or 0.25 mM IPTG (for HMGB1 and CXCL12 proteins respectively) and grown for 16 h before harvesting at 4000 RCF. For biotinylated proteins, 10 mM D-biotin in PBS was added before induction and again 1 h before cell harvesting.
  • HMGB1 purification Pellets of induced HMGB1-expressing cells were resuspended at 14 g/L in 1 M NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 10 mM Imidazole (Buffer A) supplemented with 1:1000 protease inhibitors (Calbiochem Set III, Merck 539134), 3 ⁇ g/mL Benzonase-MBP, 1 mM MgSO 4, 0.5 mg/L lysozyme (Sigma L6876) and 0.5% v/v Triton-X100 before freezing at - 80°C; from this point onwards all steps took place at 4°C.
  • Contaminants were washed with 15 CV of 0.5 M NaCl, 5% glycerol, 50 mM HEPES pH 7.5 (Buffer B) supplemented with 30 mM imidazole before elution directly into a PD-10 column (GE Healthcare; equilibrated in Buffer B + 20 mM imidazole) with 2.5 mL of Buffer B + 500 mM imidazole. Proteins were eluted from the column with 3.5 mL of Buffer B + 20 mM imidazole before tag removal with 1:20 OD TEV-GST protease over 16 hours.
  • Proteins were further purified by size exclusion chromatography (SEC) (Superdex S7510/300-0.35 mL/min or 16/600-1.2 mL/min flow rate) in either 10 mM HEPES pH 7.5 + 150 mM NaCl for biophysics work or cell-culture grade PBS for cell and animal work. Recombinant proteins were flash-frozen for storage, adding 1 mM TCEP in the case of reduced HMGB1 proteins. Recombinant CXCL12 purification [00177] Outer membranes of cells expressing DsbC-SUMO-CXCL12 were lysed by osmotic shock [90].
  • SEC size exclusion chromatography
  • Pellets were resuspended at 40 g/L in 1 M sucrose, 0.2 M Tris-HCl pH 8.0, 1 mM EDTA, 1 mg/mL lysozyme, 2X cOmplete protease inhibitor set (COEDTAF-RO, Roche), 50 mM Imidazole and 3 ⁇ g/mL benzonase. This was stirred for 45‘ at room temperature before adding 4 volumes of ice-cold 18.2 m ⁇ water and mixed for a further 10‘ before adding 1 mM MgSO 4 .
  • a 2% v/v of TX-114 was added to recombinant protein solutions, homogenized for 20 ⁇ with orbital shaking at 2000 RCF at 4°C, and separated for 5 ⁇ at 37°C before pelleting the detergent phase at 8000 RCF, 10 ⁇ , 25°C.
  • the supernatant was mixed with 5% w/v of SM-2 Biobeads (BioRad, 152-8920), cleaned with 2% TX-114 for 2 h and regenerated with 30 CV of methanol, 30 CV of endotoxin-free 18.2 m ⁇ water and 30 CV of endotoxin-free PBS.
  • TEV-GST protease (GST-fusion protein), Benzonase-MBP, and Ulp-1 protease were produced from transformants in storage at the SGC collection [86]; all had 200 ⁇ g/mL ampicillin resistance.
  • TEV and Ulp-1 were purified as per the protocols described for HMGB1 with only one IMAC step, whereas Benzonase-MBP was purified from outer membrane lysates obtained as with CXCL12 and isolated with use of amylose resin (NEB, E0821) as per manufacturer protocols. In both cases, the resulting proteins were concentrated to 10 mg/mL in 50 mM HEPES pH 7.5, 0.3 M NaCl, 10% glycerol.
  • the membranes were rehydrated at 20-25°C with 95% and 70% ethanol, equilibrated with PBST (PBS 1X + 0.05% Tween-20, 3x), and blocked with 10% BSA/PBST for 8 h.1 ⁇ M of the partner His-tagged protein construct was added (in PBS) and allowed to bind for 24 h at 4°C. Excess BSA and protein was removed with 3 washes in PBST; all washes lasted 1’ unless otherwise stated. To detect bound proteins, the membranes were treated with 1:3000 dilution of Qiagen anti-Penta His HRP conjugate (Qiagen 34460) and excess antibody removed with 3 washes in PBST for 20 ⁇ .
  • Qiagen anti-Penta His HRP conjugate Qiagen 34460
  • TLR-2, TLR-4 and RAGE peptide arrays were used instead (IntaVis, [94])
  • the membranes were baited with either CXCL12 or either of the three receptors in fusion with the CH domain of IgG (1530-TR, 9149-TR, or 1145-RG, BioTechne), as above, then probed with Anti-CXCL12 antibody (PA5-17238, Invitrogen).
  • Bound IgG whether by the Fc fusion proteins or anti CXCL12, was then detected with 0.25 ⁇ g/mL anti-human IgG CH2 domain antibody (NBP2-68464, custom conjugated to HRP).
  • a series of peptides covering human IgG CH2 domain were used as controls (Uniprot P01857, 111-223) were used as controls: the highest intensity spot was used as 100% signal threshold.
  • Bound antibody was in all cases via chemiluminescence (Pierce ECL substrate – 32109): the membrane was covered in substrate solution and placed between two clear plastic sheets before incremental imaging at 2 ⁇ intervals in a LAS-4000 camera. The intensity of the peptides in each membrane was measured in ImageJ and normalized to the controls and blank spots (100%-0%). Residues whose mutation to alanine resulted in higher intensity changes than those observed for alanine positions in the sequence were considered as significant contributors to CXCL12 binding.
  • NMR Nuclear magnetic resonance
  • a concentration range of RAGE-Fc chimera protein (BioTechne, 1145-RG; 0-640 nM in 1:4 dilutions) was added in 10% BSA/PBS and allowed to bind for 2 h, at 4°C.
  • Bound FC chimera was detected by incubation with Anti-Human IgG HRP (Agilent Dako P021402-2) diluted 1:10000 in 1% BSA/PBS for 2 h, at 20-25°C. Between each of these 3 steps, the plate was washed with 100 ⁇ L PBST, three times.
  • TMB substrate (ThermoFisher N301) was added to each well; the reaction was allowed to develop in the dark until the FL-DS-HMGB1 control developed a clear concentration-dependent color gradient before stopping the reaction with 25 ⁇ L of 0.5 M H2SO 4 .
  • OD 450 was measured as a readout (FluoStar OMEGA, BMG Labtech) and plotted as a saturation fit against 2x RAGE-Fc concentration (as the chimera is a RAGE dimer).
  • TLR-4 and TLR-2-mediated NF- ⁇ B signaling reporter assay [00189] HEK-Dual cells (Invivogen) expressing human TLR-2 and CD14 or murine TLR-4, MD-2 and CD14 were maintained in DMEM (Gibco), supplemented with 10 % FBS (Gibco), 1 % L-Glutamine (Gibco), and 1 % penicillin/streptomycin (Gibco), in standard tissue culture conditions (37oC; 5% CO2).
  • TLR-4 and TLR-2 HEK-Dual cells were plated in triplicate into wells of a 96 well plate and stimulated with 10 ⁇ g/mL l HMGB1 and (X concentration) FSL-1 for TLR-2 and 10 ng/mL LPS for TLR-4.24 hours after stimulation, NF- ⁇ activity was determined my measuring the induced levels of secreted embryonic alkaline phosphatase (SEAP).
  • SEAP embryonic alkaline phosphatase
  • Monocyte total NF- ⁇ B secretion assay Human monocytes (StemCell Technologies) were maintained in DMEM (Gibco), supplemented with 10% FBS (Gibco) in standard tissue culture conditions (37°C; 5% CO2). To determine if FR-HMGB1, DS-HMGB1 and dBB12L induces proinflammatory cytokine production, 10 5 human monocytes were plated in triplicate into wells of a 96-well plate and stimulated with 10 ⁇ g/mL HMGB1 and 50 ng/mL LPS or 10 ng/mL LTA. 24 h after stimulation, TNF levels were determined by Enzyme-linked immunosorbent assays (ELISA) (Abcam).
  • ELISA Enzyme-linked immunosorbent assays
  • mice were treated systemically with an i.v. injection of 30 ⁇ g FR-HMGB1 in 50 ⁇ L of PBS vehicle, or PBS only control.
  • Injury cell are from BaCl 2 injured mice as described below.
  • Alert cells are from the uninjured contralateral side of BaCl2 injured mice.
  • Murine muscle stem cells mMuSCs
  • Muscle cell suspensions were created by mincing thigh muscles and enzymatically digesting with collagenase 800 U/ml (Worthington-Biochem) and dispase 1 U/mL (Gibco).
  • mMuSC CD31-CD45-Sca-1- VCAM1 +
  • FACS fluorescence activated cell sorting
  • RNA extracted from freshly FACS isolated mMuSCs, was sent to RNA-seq analysis using Lexogen 3’kit library prep and sequenced using HiSeq400 (Illumina).
  • FASTQ files were assessed using FASTQC followed by the generation of TPM values with kallisto v0.42.4. TPM values were summed to obtain gene-level expression values using tximport and differential expression analysis was undertaken with DeSEQ2.
  • mice were anesthetized by aerosolized 2% isoflurane, given analgesia, transferred to a warming pad and the right lower hindlimb was disinfected with povidone iodine and the tail with 70% ethanol if intravenous injection was performed.50 ⁇ L of 1.2% BaCl2 (Sigma) was injected into and along the length of the tibialis anterior (TA) muscle to induce cell death. Mice were euthanized and lower limbs removed at the times indicated, fixed in 4% paraformaldehyde (Santa Cruz Biotechnology) for 24 h. The TA muscles were dissected and further fixed for 24 h before being embedded in paraffin and sectioned.
  • BaCl2 Sigma
  • Sections (5 ⁇ m) were stained with hematoxylin and eosin to identify fibers with central nuclei and imaged with an Olympus BX51 using a 10x ocular/ 40x objective lens.
  • the cross-sectional area (CSA) of the fibers from at least 4 images per mice was manually measured using the FIJI distribution of ImageJ2 software (NIH). Data were grouped per mice. Mice were injected with HMGB1 constructs (46 nM/kg, resuspended in PBS) or PBS vehicle control intramuscularly or intravenously at the time of injury or after injury for the optimal time administration of HMGB1 constructs after injury.
  • mice were subject to surgery between 10-14 weeks old, with body weight between 25-30 g. All mice had either an intravenous injection of FR-HMGB1 (46 nM/kg, resuspended in PBS) or vehicle control just before surgery.
  • Buprenorphine (buprenorphine hydrochloride; Vetergesic) was delivered as a 0.015 mg ml solution via intraperitoneal injection at 20 min before the procedure to provide analgesia. They were anaesthetized with 2.5% isoflurane and externally ventilated via an endotracheal tube.
  • Cardiac injury was induced by permanent ligation of the left anterior descending coronary artery (LAD) via a thoracotomy.
  • LAD left anterior descending coronary artery
  • Experimenters were blind to treatment groups for subsequent cardiac cine- MRI and analysis. Mice were housed and maintained in a controlled environment. All surgical and pharmacological procedures were performed in accordance with the Animals (Scientific Procedures) Act 1986, UK.
  • Cardiac cine-MRI and analysis [00195] Cardiac cine-MRI was performed post-LAD ligation at 7T using a Varian DDR system. Briefly, mice were anaesthetised with 2% isoflurane in O2, and positioned supine in a custom animal handling system with homeothermic control.
  • HMGB1-c038 (89-174, biotinylated) titration with CXCL12A-c021 at 0, 0.42, 0.82 and 1.42 molar equivalents, Figures 5A-5B [00198] Due to protein amount limitations, titration was performed by sequential addition of CXCL12 to HMGB1 samples, resulting in sample dilution. As the calculations in the chemical shift tracking module in CCPNMR are independent of peak, this does not alter the results; the median change has been indicated in the volume comparisons.
  • HMGB1 binds to activated platelets via platelet-expressed receptor for advanced glycation end products (RAGE) and is highly expressed in platelet rich coronary artery thrombi. Thromb Haemost 2015, 14:994–1003. 25. van Beijnum JR, Buurman WA, Griffioen AW: Convergence and amplification of toll- like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1). Angiogenesis 2008, 11:91–99. 26.
  • Non-oxidizable HMGB1 induces cardiac fibroblasts migration via CXCR4 in a CXCL12-independent manner and worsens tissue remodeling after myocardial infarction. Biochim Biophys Acta - Mol Basis Dis 2017, 1863:2693–2704. 32.
  • HMGB1 and thrombin mediate the blood-brain barrier dysfunction acting as biomarkers of neuroinflammation and progression to neurodegeneration in Alzheimer’s disease. J Neuroinflammation 2016, 13:1–12. 70. Hreggvidsdóttir HS, Lundberg AM, Aveberger A-C, Klevenvall L, Andersson U, Harris HE: High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor. Mol Med 2012, 18:224–30. 71. Livoti E: Experimentally validated computational docking to characterize protein- protein interactions. [date unknown]. 72.
  • Fassi EMA Sgrignani J, D’Agostino G, Cecchinato V, Garfalo M, Grazioso G, Uguccioni M, Cavalli A: Oxidation state dependent conformational changes of HMGB1 regulates the formation of the CXCL12/HMGB1 heterocomplex. bioRxiv Biochem 2019, doi:10.1101/555946. 73. Drury LJ, Ziarek JJ, Gravel S, Veldkamp CT, Takekoshi T, Hwang ST, Heveker N, Volkman BF, Dwinell MB: Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways.
  • Turchetto J Sequeira AF, Ramond L, Peysson F, Brás JLA, Saez NJ, Duhoo Y, Blémont M, Guerreiro CIPD, Quinton L, et al.: High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery. Microb Cell Fact 2017, 16:6. 90. Thein M, Sauer G, Paramasivam N, Grin I, Linke D: Efficient subfractionation of gram- negative bacteria for proteomics studies. J Proteome Res 2010, 9:6135–6147. 91.
  • Kaltashow IA, Mohimen A Electrospray ionization mass spectrometry can provide estimates of protein surface areas in solution.
  • Testa L Brocca S, Grandori R: Charge-surface correlation in electrospray ionization of folded and unfolded proteins.
  • Yu G Wang LG, Han Y, He QY: ClusterProfiler: An R package for comparing biological themes among gene clusters. Omi A J Integr Biol 2012, doi:10.1089/omi.2011.0118. 99.
  • Kubota, High-mobility group box 1 restores cardiac function after myocardial infarction in transgenic mice. Cardiovasc Res 80, 40-46 (2008). 136. Y. Nakamura, S. Suzuki, T. Shimizu, M. Miyata, T. Shishido, K. Ikeda, S. Saitoh, I. Kubota, Y. Takeishi, High Mobility Group Box 1 Promotes Angiogenesis from Bone Marrow-derived Endothelial Progenitor Cells after Myocardial Infarction. J Atheroscler Thromb 22, 570-581 (2015). 137. F. Limana, G. Esposito, D. D'Arcangelo, A. Di Carlo, S. Romani, G.

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Abstract

This invention provides polypeptides represented by the following formula: H2N-A-X-B-A-X-B-HOOC which are based on HMGB 1 as well as compositions comprising, and treatment methods using, such polypeptide.

Description

POLYPEPTIDES RELATED TO HMGB1 USEFUL FOR PROMOTING TISSUE REGENERATION, COMPOSITIONS COMPRISING SAME, AND USES THEREOF
[0001] This application claims the benefit of U.S. Provisional Application No. 63/190,429, filed May 19, 2021, the contents of which is hereby incorporated by reference.
[0002] Throughout this application, various publications are referenced, each such reference in its entirety is hereby incorporated by reference into this application.
REFERENCE TO SEQUENCE LISTING
[0003] This application also incorporates-by-reference each of the nucleotide sequences which are present in the text file named “220519_91203-B- PCT_Sequence_Listing_AWG.txt”, which is 78 kilobytes in size, and which was created on May 18, 2022 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is being filed as part of this application.
TECHNICAL FIELD
[0004] This invention concerns polypeptides related to HMGB1 that promote tissue regeneration without inducing deleterious inflammation and methods of treating acute and chronic conditions involving tissue injury by administering such a polypeptide to a subject in need of treatment for such a condition.
BACKGROUND OF THE INVENTION
[0005] Resident stem and progenitor cells play a key role in maintaining homeostasis and effecting repair of many tissues following injury [1] However, most tissues in adults heal by scarring. Following the success of bone marrow transplantation [2] there has been considerable interest in exogenous stem cell therapies to promote regeneration of solid organs. However, this has met with limited success, except in a few organs such as the eye [3] and skin [4] The inflammatory environment following tissue injury is not conducive to stem cell engraftment and the subsequent scarring disrupts the stem cell niche [5] Therefore, focus has shifted to promoting tissue regeneration by stimulating endogenous repair mechanisms [6] . Development of a successful therapeutic would depend on identification of soluble mediators to promote these pathways [7] We previously identified High Mobility Group Box 1 (HMGB1) as a key mediator of repair in multiple tissues, including bone, blood and skeletal muscle [8] .
[0006] HMGB1 is a prototypical alarmin [9,10] and under physiological conditions has an essential role in transcription [11,12] On cell injury it is passively released from the damaged and necrotic cells into the extracellular space and the circulation to act on endogenous stem and progenitor cells to transition them to GAiert [8], a state intermediate between Go and Gi [ 13] . On exposure to the appropriate activating factors, cells in GAiert can rapidly enter Gi and effect tissue repair. If not required, stem cells in GAiert revert back to Go after approximately 3 weeks [13], thereby ensuring that they are not exhausted and the niche is not depleted.
[0007] HMBG1 comprises two L-shaped Box domains, A and B, each containing three a- helices (I - III) connected by flexible regions (Figure 1A). The C-terminus of the protein is intrinsically disordered and contains a high proportion of carboxylic acid residues (Glu/Asp) comprising the acidic tail. The oxidation status of HMGB1 cysteine residues (Cys 22, Cys 44 in Box A and Cys 105 in Box B) is a key determinant of the extracellular activities of HMGB 1 and in turn is dependent on the mechanism of release. Three different redox forms have been described in vivo [14] HMGB1 passively released from the nuclei following injury or cell necrosis is the fully-reduced form (FR-HMGB1). It binds to CXCL12 and the heterocomplex signals via the cell surface receptor CXCR4 to transition stem and progenitor cells to GAiert [8] . Partial oxidation in the local inflammatory environment results in the formation of the disulfide HMGB1 (DS-HMGB1) [15,16], which has a disulfide bond between Cys 22 and Cys 44. This is also the form that is actively secreted by immune cells following acetylation [17] and N- glycosylation [18] TLR-4 signaling by DS-HMGB1 results in production of several proinflammatory cytokines, including TNF [19], whilst TLR-2 signaling has been shown to be detrimental in multiple processes, including thrombosis and reperfusion injury [20], and autoimmune disorders [21] DS-HMGB1 signaling via RAGE plays a key role in platelet activation and NET formation by neutrophils to promote thrombus formation [20,22-24] Intracellular signaling via all three receptors converges to induce NF-kb activity [25] in a MyD88-dependent manner [26,27] Oxidation of all three cysteine residues through the action of extracellular reactive oxygen species results in sulfonyl-HMGB 1 (SO3), which is biologically inactive [14,19]
[0008] The disulfide bridge in Box A of DS-HMGB1 (Cys22-Cys44) is essential for TLR-4 signaling (Figure IB and Figure 1C), initiating binding to TLR-4 but also has a relatively high dissociation rate. MD-2 then binds to Box B with low affinity but very low dissociation rates, stabilizing the interaction [28]; the Phe-Cys-Ser-Glu (FCSE, 104-107) peptide in Box B is essential for this interaction [29] The capacity of DS-HMGB1 to signal via TLR-4 has been overcome by substituting cysteines at positions 22, 44 and 105 with serine, resulting in an engineered form described as 3S-HMGB1 [14] Whilst the authors claimed that 3S-HMGB1 has enhanced regenerative properties compared to FR-HMGB1 [30], we found that in bone, blood and skeletal muscle injuries it was equivalent to FR-HMGB1. Interestingly, 3S-HMGB1 has been reported to be deleterious when administered locally following myocardial infraction whereas FR-HMGB 1 resulted in a smaller infarct and enhanced cardiac function assessed over 4 weeks [31] There are no published data on the effect of the 3S substitutions on TLR-2 or RAGE signaling.
[0009] The sites for TLR-2 interaction are not clearly defined but glycyrrhizin is known to inhibit this interaction [21] This suggests that at least one, and potentially both HMG Box domains and the acidic tail [11], are involved. The acidic tail negatively modulates the binding and it has been reported that co-ligands [32] are necessary to displace the acidic tail of HMGB 1 from the Box domains to permit signaling via TLR-2 [33] . However, several publications have reported TLR-2 dependent proinflammatory signaling with HMGB1 alone [34,35] The role of the redox state of HMGB1 with regards to TLR-2 signaling remains unclear as both the disulfide form [36] and the fully-reduced form [32] have been proposed to signal through TLR- 2. RAGE interaction has been primarily mapped to a peptide in HMG Box B (residues 149- 182) [37] (Figure IB and Figure 1C), and peptides derived from this sequence can effectively inhibit HMGB 1 -RAGE signaling [38] More recently a second RAGE binding site has identified on HMG Box A [39], although it is only been shown to be active after proteolysis by Caspase 11 [39] However, the relative contribution of each site to RAGE signaling remains unknown. It is also recognized that prothrombotic signaling mediated by RAGE requires the disulfide form of HMGB 1, implying that Box A may be involved [36], although the site of binding is currently not known. The acidic tail of HMGB 1 may negatively regulate RAGE signaling in a manner analogous to TLR-2 as it binds residues within the RAGE binding peptide [11,40,41]
[0010] Successful translation of the regenerative activities of FR-HMGB 1 to the clinic is dependent on the elimination of all potential deleterious proinflammatory signaling whilst maintaining CXCL 12-binding and signaling via CXCR4. Here we identify the residues in HMGB1 that are critical for binding to CXCL 12, TLR-4, TLR-2 and RAGE, and describe an HMGB 1 variant that retains regenerative activity whilst eliminating RAGE binding and TLR- 2 and TLR-4 signaling. Based on our data, other potential HMGB1 constructs that possess these properties are described. SUMMARY OF THE INVENTION
[0011] This invention provides a polypeptide represented by the following formula: H2N-A-X-B-A-X-B-HOOC wherein each A represents consecutive amino acids, the sequence of which
(1) is a sequence of four amino acids
(a) identical to the sequence of amino acids 90 - 93 of wild type human HMGB1 (SEQ ID NO: 1), or
(b) which differs from the sequence of (a) in respect to one or more amino acids; and
(2) has at its amino terminal end, between one and six consecutive amino acids, the sequence of which
(a) is identical to the sequence of the corresponding one to six amino acids preceding amino acid 90 in wild type human HMGB1, or
(b) differs from the sequence of (a) in respect to one or more amino acids; and
(3) optionally has a methionine at the amino terminus, wherein each A may be the same or different; wherein each X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 - 162 of wild type human HMGB 1; wherein each B represents consecutive amino acids, the sequence of which (1) is a sequence of five or six amino acids
(a) identical to the sequence of amino acids 163 - 168 of wild type human HMGB1,
(b) identical to the sequence of amino acids 163 - 167 of wild type human HMGB1, (c) the sequence of (a) in which any one of amino acids 163, 167, or 168 is changed to any other amino acid;
(d) the sequence of (b) in which any one of amino acids 163 or 167 is changed to any other amino acid;
(e) the sequence of (a) or (b) in which amino acid 164 is changed from lysine to arginine; or
(f) the sequence of (a) or (b) in which amino acid 165 is changed from glycine to alanine, serine or threonine;
(g) the sequence of (a) or (b) in which amino acid 166 is changed from lysine to arginine; or
(h) the sequence of (a) or (b) in combination with the changes in (e) and (f), (e) and (g), (f) and (g), or (e), (f) and (g);
(i) the sequence of (a), (b), or (c) in combination with the changes in one or more of (e), (f), and (g); or
(j) the sequence of (d) in combination with the changes in one or more of (e), (f), and (g); and
(2) has at its carboxy terminal end, between one and six consecutive amino acids, the sequence of which
(a) is identical to the sequence of the corresponding one to six amino acids following amino acid 168 in wild type human HMGB1,
(b) is identical to the sequence of the corresponding one to six amino acids following amino acid 167 in wild type human HMGB1,
(c) differs at one or more positions from the sequence of the corresponding one to six amino acids following amino acid 168 in wild type human HMGB 1 , or
(d) differs at one or more positions from the sequence of the corresponding one to six amino acids following amino acid 167 in wild type human HMGB 1 , and wherein each B may be the same or different; and wherein each - represents a peptide bond between each of A and X, X and B, B and A, A and X, and X and B; with the proviso that in the B-A between the two Xs, the number of amino acids must be at least 12; and with the additional proviso that in the B at the carboxy terminal end of the polypeptide, the one to six consecutive amino acids of (2) may be absent.
[0012] This invention also provides a composition comprising a polypeptide in accordance with the invention and a carrier, and methods of treating a subject suffering from, or at risk for developing, a condition which would be alleviated by promoting regeneration of a tissue or cells that rely upon CXCR4+ cells for repair which comprise administering to the subject a polypeptide or a composition of the invention in an amount effective to promote regeneration of the tissue or cells and to have a therapeutic or prophylactic effect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figures 1A-1C show a schematic of the HMGB1 structure and locations of known immunogenic activities. Figure 1A: Structure of HMGB1 (PDB 2YRQ, conformer 1) showing the alpha helices of each Box domain. Figure IB: Structure from (Figure 1A) coloured in PyMol according to known interactions with LPS, TLR-4 or RAGE. The regions involved in TLR-2 binding are currently unknown. The acidic tail, which is involved in transcriptional modulation and bactericidal activities is not shown in the structure. Structure of HMGB1 showing the alpha helices of each Box domain is labeled, with the original coloring showing the following - Pink: residues involved in glycyrrhizin binding. Red: flexible N-terminal regions adjacent to Box A or Box B. Orange: cysteine residues. White: linker region between HMG Boxes. Bright green and yellow: RAGE binding region (incomplete in Figure 1A, where it extends into the acidic tail, yellow). Figure 1C: Schematic representation of regions known to modulate DAMP signaling of HMGB1, as described in the literature. The disulfide bond has also been listed.
[0014] Figures 2A-2F show conserved residues in each HMG Box domain are critical for CXCL12 binding. Figure 2A: Peptide array (11x10) of HMGB1 15-mers incubated with 1 mM CXCL12-His6 and detected with anti-His5-HRP antibody. Intensity of spots corresponds to amount of CXCL12 bound to the peptides; first two and last two spots in the array comprised 10-His positive controls. Figure 2B: Intensity quantification of spot intensity in (Figure 2A) (duplicate runs) normalised to 10-his control. Peptides used for alanine scanning experiments in (Figure 2C) are marked in the graph. Peptides in the acidic tail were not included, as it would non-specifically bind cationic molecules such as CXCL12 due to the high negative charge. Peptides in graphs are represented in SEQ ID NOs: 8-104, left to right. Figure 2C: Peptide arrays of alanine point mutagenesis of domains identified in Figure 2A and Figure 2B. First spot in each row corresponds to the positive control; second spot to the unmodified peptide. Peptides shown are represented in SEQ ID NOs: 105-111, top to bottom. Figure 2D: Intensity quantification of the array in (Figure 2C, SEQ ID NOs: 105-111), normalized to the unmodified peptide. Residues which showed increase in CXCL12 binding compared to alanine residues (Ala -> Ala; synonymous mutation, in grey) are shown in red with the original figure coloring and have an asterisk above their bar. Figure 2E: Michaelis-Menten saturation fits of biotinylated HMGB1 constructs [full-length FR 1-214 (circle); 3S 1-214 (square); minimal Box A 8-78 (upright triangle) and Box B 94-162 (upsidedown triangle), extended box A 1-88 (diamond) and box B 89-174 (star)] binding to CXCL12. Figure 2F: Summary of kinetic parameters derived from (Figure 2E). Affinity (Kd) constants from both fits follow the same relationship and are greatly decreased for HMGB194-162; analyzed by 1-way Brown-Forsythe ANOVA from the fitted data. Kd values were compared via a post-hoc 2-way ANOVA if AICc supported a model with multiple constants, averaging both values as no significant differences were found in the paired comparison (column factor). Raw interferograms can be found in Figure 12. Req: response at equilibrium, kOff: dissociation constant (s-1), kOn: association constant ((µM*s)-1), AICc: Statistical comparison by Aikaike´s Information Criterion (corrected). [0015] Figure 3 shows the binding of DAMP receptors to HMGB1 analyzed via peptide arrays. Intensity densitogram of CelluSpot(TM) arrays with HMGB115-mer peptides baited with TLR-2 (“A”, orange in original coloring), TLR-4 (“B”, red in original coloring) or RAGE (“C”, green in original coloring). Intensity normalized from 0 (empty spots) to 100 (highest control) and averaged across two membranes for each target. A schematic representation of HMGB1 is shown to the left of the peptide sequences. TLR-2 (A) binding peptide sequences within HMGB1 occupy similar positions across Box A and Box B with another binding region just before the acidic tail. Unlike CXCL12, the binding pattern for TLR-4 (B) and RAGE (C) differs between the two Boxes, with TLR-4 binding peptides clustered mostly in Box A and the linker, and RAGE binding peptide sequences in the C-terminal end of Box B and the flexible region preceeding the acid tail. [0016] Figure 4 shows a representation of DAMP-receptor binding peptides and changes in their configuration dependent on the oxidation status of Cys22-Cys44 in Box A. Surface representation of the binding peptides described in Figure 3 overlaid on the published Pymol NMR structure of HMGB1 (PDB 2YRQ). A, top row) TLR-2, B, middle row) TLR-4, C, bottom row) RAGE. Original coloring shows the following: Cyan: cysteine residues. Pink (A only), glycyrrhizin binding residues contained in a TLR-2 binding site. Black (C only), Caspase-1 site reported to result in RAGE-mediated immune tolerance induced by HMGB1. Peptides binding after residue 164 in HMGB1 are not shown in any of the figures as they are absent in the available protein structures. In all three cases, the positioning of peptides within Box A is affected by oxidation. [0017] Figures 5A-5B show NMR validation of residues involved in CXCL12 binding. Figure 5A: Cumulative CSP of helical-only biotinylated Box B (94-162, HMGB1A-c028) or complete Box B (89-174, HMGB1A-c038) after titration with CXCL12 (0.42, 0.84 and 1.42 molar equivalents), calculated over several HSQC spectra including a parallel control with no CXCL12 measured after the last concentration point (CSP drift control). Intensity of the darkness of the bars in the graph indicates relative CSP. The sequence of each HMGB1 construct has been overlaid with the residue number; an empty column (no number) represents residues which could not be mapped in the parallel 3D 1H-15N HSQC/NOE/TOCSY experiments. The sequence of the residues corresponding to each HMG Box is shown with the original coloring as: Light blue, residues previously reported in the literature as involved in CXCL12 binding (specifically, K95, A100, 1112, G114, LI 19, A 136, and Y154); red, residues weakly involved in the peptide array (specifically, D90, P91, N92, R96, S99, LlOl, L104, C105, S106, E107, Y108, R109, K113, G118, S120, K151, E 152, K153, E155, Y161, R162, G165, K166, P167, and D168); purple, residues involved according to both published literature and peptide array (specifically, F102, L103, El 15, and D157). Grey, alanine residues (A93, A159, and A160) within CXCL12 binding peptides (underlined) that could not be assessed in the peptide arrays. Sequence shown is represented in SEQ ID NO: 5. Figure 5B: Cumulative peak height change of Figure 5A; Heatmap of NMR changes. Red in original coloring indicates I/IO change over 1 SEM of all residues, and blue in original coloring decrease over -1 SEM. These bars are also labeled. Sequence shown is represented in SEQ ID NO: 5.
[0018] Figures 6A-6C show design of dBB12L construct to eliminate RAGE, TLR-2 and TLR-4 signaling. Figure 6A: Sequence alignment of Box A + linkers (1-88) (SEQ ID NO: 3) with Box B + linkers (89-174) (SEQ ID NO: 4); numbers correspond to residue numbering from the NMR structure (excluding N-terminal methionine). Vertical lines designate strictly conserved positions, and double dots similar amino acid types. Underlined: peptide regions binding CXCL12 from the first peptide array. Red in original coloring: residues flagged in the alanine scan as involved in CXCL12 binding which could not be verified by NMR (specifically Box A: D4, P8, M12, C22, R69, Y70, T76, and P80; and Box B: P91, G118, and P167. Orange in original coloring: residues flagged in the alanine scan and also showing either CSP or peak volume change by NMR (specifically Box A: K6, R9, G10, Kl l, S13, S14, H26, K28, K29, H30, K64, D66, E73, K75, Y77, and 178; and Box B: D90, R96, S99, F101, F102, L103, F104, SI 06, R109, K113, K151, E152, 1158, Y161, R162, G165, K166, and D168. Cyan in original coloring: residues not flagged in our NMR or peptide array experiments but described in the NMR literature as contributing to CXCL12 binding [46] (specifically Box A: F37, S38, K49, and K56; and BoxB: A100, G114, LI 19, A136, and Y154. Purple in original coloring: residues flagged peptide array experiments and confirmed by the published or our NMR data (specifically Box A: V19 and R23; and Box B: El 15 and D 157. In green: residues flagged only on NMR experiments, which can either directly bind CXCL12 or be affected by binding to nearby residues (specifically Box A: A33, K42, K43, S45, T50, S52, K58, D61, and A68; and Box B: H116, G122, D123, V124, K126, K127, A147, A148, A159, A163, and K164. Pink in original coloring: residues flagged by both our NMR experiments and the published data (specifically Box A: A16; and Box B: K95, and I112. Figure 6B: Structure of FR-HMGB1 1-166 (2YRQ), with residues coloured as in Figure 6A. Side chains of all coloured residues shown. Dashed circles indicate the glycyrrhizin binding region in each HMG Box. On the right, Pymol surface representation of the CXCL12 binding residues; those residues flagged in NMR and peptide arrays form a similar binding pocket on the concave surface of each HMGB Box if Box A is reduced. Figure 6C: Schematic of dBB12L construct design. The initiation codon Met 1 is numbered as Met 0 herein, as it is partially lost in the cleaved peptide. Therefore, HMGB1 Met 1 -Gly 2…Glu 215 becomes Met 0- Gly 1…Glu 214. Comparison of the domain organization and sequences of FR-HMGB1 (top - SEQ ID NO: 1) and dBB12L construct (bottom - SEQ ID NO: 2) are shown. The dBB12L construct is designed such that: 1. The acidic tail and part of the RAGE binding domain (175-214) deleted; 2. Residues 1-88 (Box A) substituted by residues 90-175, resulting in two HMG Box B domains; and 3. Residues 163- 174 C-terminal to Box B replace the native flexible linker (79-88) C-terminal to Box A in native HMGB1. CXCL12 binding peptides shown as red letters as originally colored and also have an asterisk above them. The repeat Box B units are separated in the diagram by a vertical dashed black line. DAMP receptor binding peptides in Box A shown as dashed lines, those in Box B by solid lines. The TLR-2 and RAGE peptides are truncated in DBB12L, and all Box A peptides are no longer present in the construct due to the substitution. [0019] Figures 7A-7D show dBB12L has similar stability and surface charge conformation to FR-HMGB11-214/1-164. Figure 7A: Calculated Tm50 values (in ºC) for full-length and 1- 164 FR-HMGB1, and dBB12L under various buffer conditions, shown as a heat map of highest (green) and lowest (red) values within the global dataset for all constructs in original coloring. N/A: curve not fittable. Effects of pH and NaCl concentration have been summarized below the table (FR-HMGB1, circle; dBB12L, square; 1-164 FR-HMGB1, triangle). Figure 7B: Native ESI/MS of HMGB1 constructs in either 50 mM or 0.2 M ammonium acetate, pH 6.5. All three HMGB1 constructs have similar native M/Z profiles, with dBB12L closely resembling a reduced HMGB1 construct with two HMG Boxes apart from each other. Continuous line; compact monomer. Dashed line; extended monomer (HMG Boxes distal to each other). Removal of the acidic tail (FR HMGB11-164, blue curves compared to FR- HMGB1, red curves) and higher ionic strength (Comparison of the spectra for the same construct in either 50 mM or 200 mM ammonium acetate) increases the prevalence of higher M/Z states (partial unfolding). Figure 7C: Solvent accessible surface area (SASA) calculations for the average folded HMGB1 monomer, the extended and compact monomer states, and the unfolded monomer from Figure 7D. Figure 7D: Denaturing ESI/MS deconvolution, SDS- PAGE and SEC profiles of HMGB1 constructs after storage at room temperature for 180 days (D0-D180), in 0.2 M ammonium acetate, pH 6.5. [0020] Figure 8A-8F show dBB12L has reduced binding to RAGE and does not signal through TLR-2 or TLR-4. Figure 8A: Michaelis-Menten saturation fits from hybrid ELISA (n=4 per concentration, global fit) showing absence of RAGE binding by dBB12L construct, regardless of oxidation status. DS-HMGB1 binds RAGE more avidly compared to FR- HMGB1. Data normalized with respect to DS-HMGB1 control. Figure 8B: Michaelis-Menten saturation fits from biolayer interferometry (0-25 µM HMGB1; n = 6 sensor runs, shown in Figure 14). Association and dissociation rates were calculated from the raw interferogram data only. Kinetics parameters and color legends (disulfide forms are indicated by dashed lines) are summarized in Figure 8C: Invalid fits represent R2 < 0.6 (poor binding). ELISA data represent steady state conditions. For each kinetic parameter, in the original coloring green indicates the construct with the highest affinity, fastest association (kon) or slowest dissociation (kOff); midpoint in yellow, lowest in red. Figures 8D-8E show DS-HMGB1 promoted NF-κβ activity in reporter HEK-Dual cells expressing human TLR-2 and CD14 (Figure 8D) or murine TLR- 4, MD-2 and CD14. (Figure 8E). dBB12L and FR-HMGB1 did not promote NF-κβ signaling in either cell line. Data shown as mean ± SEM fold change compared to control (media alone). Figure 8F: Disulfide HMGB1 (DS-HMGB1) increased TNF production in monocytes, which was further enhanced by the presence of suboptimal amounts of LTA, but not of LPS. FR- HMGB1 and dBB12L did not promote TNF expression, even when pre-incubated for 24h with LPS or LTA. Response to LPS pre-incubated with these constructs was also significantly reduced. n = 3 donors, each with three technical replicates. [0021] Figure 9 shows the effects of modification of the linker on regenerative activity of FR-HMGB1. The dBB12L sequence shown in the alignment is provided by residues 75-91 of SEQ ID NO: 2. The FR-HMGB1 sequence shown in the alignment (“FR”) is provided by residues 79-93 of SEQ ID NO: 1. The “Sub(79-83)”, “Sub(84-88)”, and “Sub(89-93)” sequences shown in the alignment are provided by SEQ ID NO: 175. Figures 10A-10J show the regenerative effects of optimal doses of dBB-HMGB1 and FR- HMGB1 are identical to those of an activating injury. Figure 10A: Volcano plot showing differentially expressed genes in muscle stem cells by fold change following injury or HMGB1 induced GAlert. Integration demonstrates conserved up - (brown dots in original coloring, which fall within bracket as indicated) and down - (blue dots in original coloring, which fall within bracket as indicated) regulation of core genes in GAlert induced by contralateral lower limb injury or intravenous (iv) HMGB1. Figure 10B: Network map of gene ontology terms of differentially expressed cells during GAlert induction in muscle stem cells. Figure 10C: Dose response of FR-HMGB1 in a BaCl2 skeletal muscle injury model, with regeneration quantified by fiber cross-sectional area. The optimal dose was 0.75 mg/kg (28.75 nmol/kg) and was used in subsequent assays. Figure 10D: Animals were dosed with FR-HMGB1 (optimal dose) at the varying timepoints after injection of BaCl2 to assess the interval where treatment with FR- HMGB1 is effective post-injury. Values in Figure 10C and Figure 10D shown as mean ± SEM in nested ANOVA with Holm-Sidak correction. Figure 10E: Pharmacokinetics of HMGB1 in the circulation following administration of 0.75mg/ml FR-HMGB1, fitted by nonlinear least squares to a two-phase exponential decay curve. Figure 10F: Kaplan-Meier survival plot.5wk following MI FR-HMGB1 = 83%, PBS = 52%. Figure 10G: Ejection fraction over time calculated from serial MRI scans. Dotted line indicates ejection fraction in normal/sham surgery mice. Square: FR-HMGB1; Circle: PBS control. Figure 10H: Infarct size over time calculated from serial MRI scans. Square: FR-HMGB1; Circle: PBS control. Figure 10I: Representative mid-ventricular short-axis cine-MRI images at end-diastolic and end-systolic phases of the cardiac cycle 1 and 5 wk after MI. Blood in the chambers appears bright. FR- HMGB1 group shows preservation of heart function and maintenance of wall thickness (short, unlabeled arrows) with visible separation of right and left ventricles (arrows labeled “RV” or “LV”, respectively) during systole. In contrast, in the PBS group, there is significant left ventricle dilation ( “LV” arrows) and very limited contraction between diastole and systole. n=10 per group. All MRI scans performed and assessed by a blinded observer. Figure 10J: Mean muscle cross-section area at given time points after BaCl2 muscle injured animals treated with PBS (black in original coloring, left bar at each day), 28.75 nM/kg of FR-HMGB1A-c001 (red in original coloring, middle bar at each day) or dBB12L (green in original coloring, right bar at each day). N= 5 per group and timepoint, nested ANOVA (Holm-Sidak post-hoc correction). A representative image at each point is shown in Figure 15. [0022] Figures 11A-11B show results of a peptide array of CXCL12 peptides interacting with HMGB1. Figure 11A: Peptide array of full-length CXCL12. “+” positions correspond to positive control 10-His peptides; the rest of the peptides comprise CXCL1215-mers shifted two (2) residues in succession towards the C-terminus. The membrane was exposed to 1 uM HMGB1(FR or 3S)-His6 (1-214), BoxA-His6 (8-78) and BoxB-His6 (94-162) for 24 hours. Bound protein was detected by anti-His-HRP conjugate chemoluminescence. A peptide of CXCL12 interacting with full length HMGB1 cannot interact with either Box A or Box B alone, confirming the requirement of the N-terminal segment of each Box domain (particularly, D4 in box A/D90 in box B): intensity of the spots pertaining to the common CXCL12 peptide is also markedly decreased upon binding to the Box domains alone when compared to FL- HMGB1. Binding to 3S seems to be of higher intensity than that to FR; this is likely due to protein oxidation during the assay, although this was not quantified due to the low concentration of protein used being unsuitable for ESI/TOF MS. BLI data, however, do suggest a lower off rate of CXCL12 from 3S than from FR-HMGB1. Figure 11B: CXCL12 dimer (PDB 2J7Z) with the regions binding HMGB1 highlighted. Red in original coloring: shared binding region. Blue in original coloring: non-shared binding region. [0023] Figure 12 shows interferograms in BLI of CXCL12 binding to immobilized HMGB1 constructs. Biotinylated HMGB1 constructs were immobilized on streptavidin-coated Octet biosensors and dipped in rising concentration of CXCL12. Individual lines within each interferogram graph are in the same order of the key from top to bottom unless otherwise indicated. Interferograms are colored according to CXCL12 concentration (key in top right). Each set of three replicates (cycles) for a given sensor is surrounded by a colored overlay according to construct. First row: FR FL-HMGB1 (labeled as “Sensor Info: c011” above graph), second row: 3S-FL HMGB1 (labeled as “Sensor Info: c022” above graph), third row: FR-HMGB1 Box A 8-78 (labeled as “Sensor Info: c027” above graph), fourth row: FR- HMGB1 Box B 94-162 (labeled as “Sensor Info: c028” above graph), fifth row: FR-HMGB1 Box A 1-88 (labeled as “Sensor Info: c037” above graph), sixth row: FR-HMGB1 Box B 90- 162 (labeled as “Sensor Info: c038” above graph). [0024] Figures 13A-13D show NMR validation of residues involved in CXCL12 binding. Figure 13A: Cumulative CSP of HMGB13S 1-184 (HMGB1A-c007) upon addition of 1:2 molar equivalents of CXCL12 in one step (1:1 HMG Box to CXCL12 ratio). Box A and Box B residues have been considered separate molecules for the purposes of median CSP calculation. Intensity of the darkness of the bars in the bar graph indicates higher relative CSP. The sequence of each HMGB1 construct has been overlaid with the residue number; an empty column (no number) represents residues which could not be mapped in the parallel 3D 1H-15N HSQC/NOE/TOCSY experiments. The sequence of the residues corresponding to each box is in the middle of each condition, colored originally as: Light blue, residues previously reported in the literature as involved in CXCL12 binding (specifically Box B: K95, A100, I112, G114, L119, A136, and Y154; and Box A: A16, F40, S41, K49, and K56); red, residues weakly involved in the peptide array (specifically Box B: D90, P91, N92, R96, S99, F101, F104, C105, S106, E107, Y108, R109, K113, G118, S120, K151, E 152, K153, E155, Y161, R162, G165, K166, P167, and D168; Box A: D4, K6, K7, P8, R9, G10, K11, M12, S14, F17, F18, T21, E24, E25, H26, K27, K28, K29, H30, D32, S34, V35, N36, K64, D66, R69, Y70, E71, R72, E73, M74, K75, T76, Y77, H78, and P80); purple, residues involved according to both published literature and peptide array (specifically Box B: F102, L103, E115, and D157; Box A: S13, Y15, V19, and R23). The sequences shown are represented in SEQ ID NOs: 6 and 7. Figure 13B: 15N HSQC-HQMC peak spectra for (Figure 13A) in 10 mM HEPES 150 mM NaCl pH 7.5 buffer. Protein concentrations are indicated in the spectra overlay. Figures 13C and 13D show 15N HSQC-HQMC peak spectra in 10 mM HEPES 150 mM NaCl pH 7.5 buffer. Protein concentrations are indicated in the spectra overlay. Figure 13C: HMGB1A 94-162 (2-day experiment); Figure 13D: HMGB1A 89-174 (6-day experiment; minor degradation occurs after day 4). [0025] Figure 14 shows interferograms in BLI of HMGB1 constructs binding to immobilized Fc-RAGE. RAGE-Fc was immobilized in the surface of AHC sensors and dipped in rising concentration of different HMGB1 constructs. Two experiments were run with different concentration ranges: the three columns of graphs in the left, 0 to 22.22 µM HMGB1 over 9 steps; on the right, 0 to 25 µM over 7 steps. Both are color-coded by concentration (top). Colors indicate the specific construct concentration. The lines in the graph from top to bottom correspond with the highest concentration to the lowest concentration in most cases. Each graph corresponds to a single sensor (replicate). Interferograms surrounded by a black rectangle had data points excluded due to poor quality (e.g. drift). [0026] Figure 15 shows histological images of regenerating muscle in response to FR- HMGB1 (red) or dBB12L (green) compared to PBS control (black), from Figure 10. [0027] Figure 16 shows plasmid vector maps. Vector maps with features and restriction sites. TEV: Tobacco etch virus protease recognition site.6-His: 10/6-histidine residue affinity epitope. FLAG: FLAG affinity epitope. StrepTag: StreptactinXT affinity epitope. SacB: Levansucrase precursor (negative selection in the presence of sucrose). pLIC: Annealing sites for sequencing primers used in colony screening. All plasmids contain kanamycin resistance (50 µg/mL). [0028] Figure 17 shows process to generate 3S-HMGB1.
DETAILED DESCRIPTION OF THE INVENTION [0029] This invention provides a polypeptide represented by the following formula: H2N-A-X-B-A-X-B-HOOC wherein each A represents consecutive amino acids, the sequence of which (1) is a sequence of four amino acids (a) identical to the sequence of amino acids 90 – 93 of wild type human HMGB1 (SEQ ID NO: 1), or (b) which differs from the sequence of (a) in respect to one or more amino acids; and (2) has at its amino terminal end, between one and six consecutive amino acids, the sequence of which (a) is identical to the sequence of the corresponding one to six amino acids preceding amino acid 90 in wild type human HMGB1, or (b) differs from the sequence of (a) in respect to one or more amino acids; and (3) optionally has a methionine at the amino terminus, wherein each A may be the same or different; wherein each X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 – 162 of wild type human HMGB1; wherein each B represents consecutive amino acids, the sequence of which (1) is a sequence of five or six amino acids (a) identical to the sequence of amino acids 163 – 168 of wild type human HMGB1, (b) identical to the sequence of amino acids 163 – 167 of wild type human HMGB1, (c) the sequence of (a) in which any one of amino acids 163, 167, or 168 is changed to any other amino acid; (d) the sequence of (b) in which any one of amino acids 163 or 167 is changed to any other amino acid; (e) the sequence of (a) or (b) in which amino acid 164 is changed from lysine to arginine; or (f) the sequence of (a) or (b) in which amino acid 165 is changed from glycine to alanine, serine or threonine; (g) the sequence of (a) or (b) in which amino acid 166 is changed from lysine to arginine; or (h) the sequence of (a) or (b) in combination with the changes in (e) and (f), (e) and (g), (f) and (g), or (e), (f) and (g); (i) the sequence of (a), (b), or (c) in combination with the changes in one or more of (e), (f), and (g); or (j) the sequence of (d) in combination with the changes in one or more of (e), (f), and (g); and (2) has at its carboxy terminal end, between one and six consecutive amino acids, the sequence of which (a) is identical to the sequence of the corresponding one to six amino acids following amino acid 168 in wild type human HMGB1, (b) is identical to the sequence of the corresponding one to six amino acids following amino acid 167 in wild type human HMGB1, (c) differs at one or more positions from the sequence of the corresponding one to six amino acids following amino acid 168 in wild type human HMGB1, or (d) differs at one or more positions from the sequence of the corresponding one to six amino acids following amino acid 167 in wild type human HMGB1, and wherein each B may be the same or different; and wherein each - represents a peptide bond between each of A and X, X and B, B and A, A and X, and X and B; with the proviso that in the B-A between the two Xs, the number of amino acids must be at least 12; and with the additional proviso that in the B at the carboxy terminal end of the polypeptide, the one to six consecutive amino acids of (2) may be absent. [0030] In some embodiments, the methionine is present at the amino terminus of the polypeptide. [0031] In other embodiments, A has at its amino terminal end, the amino acid corresponding to amino acid 89 of wild type HMGB1 (SEQ ID NO: 1). [0032] In some embodiments, in B(2), B has at its carboxy terminal end, six amino acids corresponding to amino acids 169-174 of wild type human HMGB1. [0033] In some embodiments, the number of amino acids in the B-A between the two Xs is at least 13. In such embodiments, the number of amino acids in the B-A between the two Xs is up to 22, more preferably up to 21, 20, 19, 18, 17, 16, 15, or 14. [0034] In other embodiments, the number of amino acids in the B-A between the two Xs is between 12 and 22 inclusive. [0035] In yet further embodiments, the number of amino acids in the B-A between the two Xs is between 13 and 22 inclusive. In some embodiments the number of amino acids in the B- A between the two Xs is between 13 and 21 inclusive, more preferably between 13 and 20 inclusive, more preferably between 13 and 19 inclusive, more preferably between 13 and 18 inclusive, more preferably between 13 and 17 inclusive, more preferably between 13 and 16 inclusive, more preferably between 13 and 15 inclusive, more preferably between 13 and 14 inclusive. [0036] In some embodiments, the sequence of (2)(c) or (2)(d) of either B or of both Bs differs at the one or more positions by the presence of a glycine, serine, proline, arginine, lysine, aspartic acid, glutamic acid, or histidine amino acid residue instead of the amino acid residue present at such position or positions in naturally occurring HMG1. [0037] In some embodiments, the sequence of (2)(c) or (2)(d) of either B or of both Bs is or comprises GSGSG (SEQ ID NO: 175). [0038] In other embodiments, the GSGSG (SEQ ID NO: 175) has been inserted into the region between the sequence of the B and the sequence of the A between the two Xs. [0039] In yet further embodiments, the order or number of the glycine and serine residues has been altered within the SEQ ID NO: 175 peptide sequence. As non-limiting examples, the sequence of (2)(c) or (2)(d) may be or comprise any of GSSSG (SEQ ID NO: 176), GSSGS (SEQ ID NO: 177), or GGSGG (SEQ ID NO: 178). [0040] In some embodiments, the amino acids of (2)(c) or (2)(d) of either B or of both Bs lack a defined secondary structure, or have a turn or random coil secondary structure. [0041] In some embodiments, either A or both As are five consecutive amino acids. [0042] In some embodiments, the five consecutive amino acids have a sequence identical to the sequence of amino acids 89 – 93 of wild type human HMGB1. [0043] In some embodiments, a) the A between the Xs is five consecutive amino acids and the B between the Xs is at least seven consecutive amino acids; b) the A between the Xs is six consecutive amino acids and the B between the Xs is at least six consecutive amino acids; c) the A between the Xs is seven consecutive amino acids and the B between the Xs is at least six consecutive amino acids; d) the A between the Xs is eight consecutive amino acids and the B between the Xs is at least six consecutive amino acids; e) the A between the Xs is nine consecutive amino acids and the B between the Xs is at least six consecutive amino acids; or f) the A between the Xs is ten consecutive amino acids and the B between the Xs is at least six consecutive amino acids. [0044] In some embodiments, a) the B between the Xs is six consecutive amino acids and the A between the Xs is at least six consecutive amino acids; b) the B between the Xs is seven consecutive amino acids and the A between the Xs is at least five consecutive amino acids; c) the B between the Xs is eight consecutive amino acids and the A between the Xs is at least five consecutive amino acids; d) the B between the Xs is nine consecutive amino acids and the A between the Xs is at least five consecutive amino acids; e) the B between the Xs is ten consecutive amino acids and the A between the Xs is at least five consecutive amino acids; f) the B between the Xs is eleven consecutive amino acids and the A between the Xs is at least five consecutive amino acids; or g) the B between the Xs is twelve consecutive amino acids and the A between the Xs is at least five consecutive amino acids. [0045] This invention also provides a composition comprising any of the polypeptides of the invention and a carrier. [0046] In some embodiments, the polypeptide is present in a therapeutically or prophylactically effective amount and the carrier is a pharmaceutically acceptable carrier. [0047] This invention also provides methods of treating a subject suffering from, or at risk for developing, a condition which would be alleviated by promoting regeneration of a tissue or cells that rely upon CXCR4+ cells for repair which comprise administering to the subject a polypeptide or composition of the invention in an amount or dose effective to promote regeneration of the tissue or cells, that is, achieve a therapeutic or prophylactic effective dose of the pharmaceutical composition of the invention in a subject in need thereof. [0048] In some embodiments, the condition is an acute injury. [0049] In a currently preferred embodiment, the polypeptide is administered within 5 hours, preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours and most preferably within 1 hour of the acute injury. [0050] In some embodiments, the condition is a chronic condition. [0051] In some embodiments, the polypeptide is administered repeatedly at a daily, weekly, monthly, or yearly interval. [0052] In some embodiments, the acute injury is myocardial infarction. In some embodiments, the tissue is cardiac tissue or myocardium. [0053] In some embodiments, the acute injury is stroke, spinal cord injury, or peripheral nerve injury. [0054] In a currently preferred embodiment, the polypeptide is administered within 5 hours of the myocardial infarction. In some embodiments, the polypeptide is administered within 5 hours, preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours and most preferably within 1 hour of the myocardial infarction. [0055] In a currently preferred embodiment, the polypeptide is administered within 5 hours of the stroke, spinal cord injury, or peripheral nerve injury. In some embodiments, the polypeptide is administered within 5 hours, preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours and most preferably within 1 hour of the stroke, spinal cord injury, or peripheral nerve injury. [0056] In some embodiments, the acute injury is a fracture, joint replacement or bone fusion. In some embodiments, the tissue is a bone. [0057] In some embodiments, the acute injury is a skeletal muscle injury, joint injury or ligament injury. [0058] In some embodiments, the condition involves liver damage. In some embodiments the tissue is liver tissue. [0059] In some embodiments, the chronic condition is non-alcoholic steatohepatitis, liver cirrhosis or infective hepatitis. [0060] In some embodiments, the chronic condition involves damage to the brain or other parts of the central nervous system. [0061] In some embodiments, the chronic condition is Parkinson’s disease, dementia, multiple sclerosis, motor neuron disease or peripheral nerve injury. [0062] In some embodiments, the chronic condition involves a chronic joint injury. [0063] In some embodiments, the chronic joint injury is inflammatory arthritis or osteoarthritis. [0064] In some embodiments, the condition involves damage to the lung. [0065] In some embodiments, the acute injury is a viral infection of the lungs, bacterial infection of the lungs, fungal infection of the lungs, or mechanical injury to the lungs. [0066] In some embodiments, the viral infection of the lungs is an infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). [0067] In some embodiments, the mechanical injury is a ventilator-induced injury. [0068] In some embodiments, the chronic condition is idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease, or emphysema. [0069] In some embodiments, the condition involves the gut. [0070] In some embodiments, the acute injury is a surgical injury to the gut. [0071] In some embodiments, the chronic injury is inflammatory bowel disease, Crohn’s disease, or ulcerative colitis. [0072] In some embodiments, the condition involves damage to the skin. [0073] In some embodiments, the acute injury is a burn or a surgical injury of the skin. [0074] In some embodiments, the chronic condition is a skin ulcer, diabetic ulcer, venous ulcer, arterial ulcer, or pressure ulcer. [0075] In some embodiments, the condition involves the pancreas and the cells are islet cells. [0076] In some embodiments, the condition is diabetes. [0077] In some embodiments, the condition is neutropenia following chemotherapy and the tissue is bone marrow. [0078] In some embodiments, the acute injury is chemotherapy, and optionally the polypeptide is administered before or within 5 hours of administration of the chemotherapy. [0079] In some embodiments, the polypeptide is administered before the acute injury. [0080] In some embodiments, the polypeptide is administered after the acute injury. [0081] In some embodiments, the condition is kidney failure and the tissue is kidney tissue. [0082] In some embodiments, the chronic condition is a disease that results in chronic renal failure. [0083] In some embodiments, the acute injury is elective surgery and the polypeptide is administered before, during or within 5 hours after the surgery. [0084] In some embodiments, the acute injury is a sport or military combat injury. [0085] In some embodiments, the chronic condition is a chronic skeletal muscle condition. [0086] In some embodiments, the chronic skeletal muscle condition is a muscular dystrophy such as Duchenne macular dystrophy, sarcopenia, or disuse atrophy. Terms [0087] In order to facilitate an understanding of the invention, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. [0088] As used herein, “engineered” means a non-naturally occurring compound that has been created based upon changing a naturally occurring compound. An engineered polypeptide may include amino acids corresponding to amino acids of a naturally occurring polypeptide and amino acids that vary in identity or location from those of a naturally occurring polypeptide. Such an engineered polypeptide may also be referred to as an “analogue” or “derivative” of the naturally occurring polypeptide. [0089] As used herein, “stem cell” means any unspecialized cell that has the potential to develop into many different cell types in the body, including without limitation hemopoietic stem cells. [0090] As used herein, the term "effective amount" means an amount of a polypeptide of the invention that is capable of achieving a desired result, for example, alleviating a condition or one or more symptoms associated with a condition, for example, an acute or chronic tissue injury. The specific amount or dose of a polypeptide administered according to this invention will, of course, be determined by the particular manner of treating or preventing the condition, for example, the route of administration, the physiological state of the subject, and the severity of the condition being treated. For example, an engineered HMGB1 polypeptide administered to a subject is preferably in the form of a composition comprising a therapeutically or prophylactically effective amount of the engineered HMGB1 polypeptide. [0091] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material. The choice of any specific pharmaceutically acceptable carriers is well within the knowledge of those skilled in the art. Accordingly, there is a wide variety of suitable carries available and routinely used in pharmaceutical compositions. [0092] It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. [0093] As used herein, all numerical ranges provided are intended to expressly include the endpoints and consistent with context all numbers that fall between the endpoints of range. [0094] Further non-limiting details are described in the following Experimental Details section which is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the scope of the invention disclosed. EXPERIMENTAL DETAILS RESULTS Identification of amino acids and motifs in HMGB1 involved in CXCL12 binding [0095] Before considering which residues of FR-HMGB1 can be mutated or deleted to eliminate proinflammatory signaling, it is essential to map the amino acids and motifs involved in binding to CXCL12. We used peptide SPOT arrays, [42] where overlapping peptides covering the sequence of one of the target proteins (HMGB1) are assessed by immunoblotting for their capacity to bind CXCL12. Two main binding sites were identified (Figure 2A and Figure 2B). The first encompassed α-helix I and part of helix II of each HMG Box (peptides 1- 3 in Box A, peptides 5-6 in Box B), overlapping the glycyrrhizin binding site [43]. The second region (peptide 4 in Box A and 7 in B) was located at the C-terminal half of α-helix III. In each HMG Box, the first CXCL12 binding peptide (helices I and II) had much greater intensity of the spots, indicating potentially higher affinity for CXCL12. [0096] Next, to identify the amino acids that directly contribute to CXCL12 interaction, we generated a second peptide array where each amino acid within the CXCL12 binding peptides was substituted with alanine, This confirmed the critical role of several residues (Figure 2C and Figure 2D). Contrary to published data where the CXCL12 has only been shown to interact with the helical segments of the HMG Boxes [44–46], we found that part of the flexible N- terminal flanking region of each box (D-4-P-3-X-2-X-1,, where the subscript number indicates relative position to the first residue in each HMG Box – Pro 9 in A, and Pro 94 in B), as well as C-terminal residues (Ile 78-Pro 80 for Box A and Ala 163-Asp168 for Box B), are also involved (Figure 2E). This was confirmed with a reverse peptide array (Figure 11) in which CXCL12 peptides were probed for binding to HMGB1. Full-length FR- and 3S-HMGB1 bound to peptides containing sequences covering the whole β-sheet of CXCL12, whereas HMGB1 Box constructs alone (8-78 Box A) or (94-162 Box B), without the flanking flexible region [47], only interacted with the peptides covering the N-terminal strand of the β-sheet. [0097] To further confirm the role of these flexible regions, we used biolayer interferometry (BLI) to assess binding of CXCL12 to HMGB1 constructs with different boundaries. We generated either full HMG Box constructs comprising the entire HMG Box with the flanking regions (HMGB11-88 for Box A, HMGB189-174 for Box B) [48] and helical-only HMG Box constructs comprising the core HMG Box alone, without the flexible flanking residues (HMGB19-78, HMGB194-162). These were compared to full-length HMGB1 constructs (1- 214), both FR-HMGB1 and non-oxidizable (3S)-HMGB1, the latter sharing the CXCL12- binding properties of the wild-type protein [14]. As expected, the helical-only constructs had lower CXCL12 affinity and binding capacity compared to full HMG Box constructs with intact flanking regions due to much faster dissociation rates. In contrast, affinities of CXCL12 for full-length FR- or 3S-HMGB1 and full HMG Box constructs were comparable to each other and greater than for the helical-only constructs (Figure 2E and Figure 2F). Identification of amino acids and motifs in HMGB1 involved in TLR-2, TLR-4 and RAGE binding [0098] Next, we probed the HMGB1 peptide arrays against each of the proinflammatory receptors (TLR-2, TLR-4 and RAGE) To account for nonspecific binding, the normalized signal from unbaited arrays was subtracted from the baited arrays We found that TLR-2 bound to a peptide in HMGB1 in Box A and Box B (Figure 3, section A) that coincides with the glycyrrhizin binding site in both HMG Boxes (Figure 1B) [49]. In addition, TLR-2 also bound to the linker region between the two Boxes, the peptide immediately adjacent to the acidic tail of HMGB1 and the C-terminal regions to each HMG Box, extending beyond the residues that bound CXCL12. Interestingly, the binding interface for TLR-2 within Box A is within the alpha helices which twist upon oxidation [50] to adopt a different 3D configuration (Figure 4, section A). [0099] Peptides binding to TLR-4 (Figure 3, section B) form a binding pocket in Box A that is only continuous when Box A is oxidized (Figure 4, section B). These include the delipidated LPS binding segment and the region bordering the Lipid A binding region within Box A [51]. [00100] Our spot array data for RAGE (Figure 3, section C) confirmed the role of peptides previously shown to modulate RAGE activity [39] [52] (Figure 4, section C), namely Rage Antagonistic Peptide (RAP) between positions 149-182, and the Caspase-1 activated site in Helix 2 of Box A. However, in contrast to previous studies where activity was only seen following Caspase-1 cleavage of HMGB1 [39], we observed that the Box A site is solvent- accessible in a full-length Box A and that its positioning is affected by the bending of Helix 2 in Box A on oxidation. [00101] Taken together, these data show that binding of HMGB1 to all three proinflammatory receptors involves distinct regions in each of the HMG Boxes, and the spatial arrangement within Box A is dependent on oxidation. In addition, the sequences in the inter-Box linker and those preceding the acidic tail have high affinity for all three proinflammatory receptors. Therefore, substitution or elimination of these sequences would effectively reduce binding to all these proinflammatory receptors. CXCL12 binds to a concave pocket on the underside of each HMG Box as shown by peptide array and NMR (578) [00102] We next used NMR to confirm, from a structural perspective, the amino acid residues in HMGB1 involved in CXCL12 binding. We used FR-HMGB194-162 and 89-174 to represent HMG Boxes with and without the flanking regions, as well as 3S HMGB11-184. Non-oxidizable 3S-HMGB1 was used instead of native full-length FR-HMGB1 as the time required for obtaining a full set of 3D spectra at 750 MHz (15N HSQC-TOCSY/NOESY and associated 15N HSQC spectra) would result in oxidation of the latter. [00103] CXCL12 titration of HMGB1 Box B 94-162 (Figure 5A) resulted in changes in cumulative chemical shift perturbation (CSP) or peak height changes (I/I0), of several residues at both the C-terminal and N-terminal binding regions identified in the peptide arrays. For the Box B construct that included the flanking regions, both the median CSP and mean volume change were significantly higher, confirming that several residues (D90, G165, K166) in these flanking regions are involved in CXCL12 binding. Residues not affected by CXCL12 binding in the helical-only (94-162) construct, such as Y154, D157 and I158, and which were identified as being involved in CXCL12 binding in the peptide arrays, demonstrated significant CSP in the full HMG Box construct (89-174), indicative of improved binding when the flanking regions were present. In addition, we observed CSP changes for residues not identified in the peptide arrays (A147, M131, A169, K172, G173) in the construct with the flanking regions. Other residues that have not been previously identified but were flagged as being potentially important in the peptide arrays did not display either CSP or volume changes upon addition of CXCL12 (C105, E107, Y108). Consequently, this group of residues was reclassified as being not critical for CXCL12 binding. Surprisingly, in contrast to published data [53], residues A100, I112, L119 and A136 in either Box B construct failed to produce significant CSP changes, although residues (S99, K113) that are very close to some of these were affected. [00104] When the NMR experiment was repeated with 3S-HMGB11-184, CSP changes upon CXCL12 addition were below the threshold for detection for most residues due to a lower signal to noise ratio. However, we were able to still observe binding to some residues over the median CSP change. Residues corresponding to HMG Box B showed overall weaker CSP changes compared to those in Box A. This could represent a more fluid equilibrium, consistent with the faster association and dissociation rates for Box B (Figure 2F). This allowed us to also denote residues such as H30, D32 or S34 in Box A identified in the peptide array as false positives (Figure 11). Whilst K89 in Box B 89-174 showed high CSP, this is the third residue from the N-terminus (after the TEV cleavage leftover N-terminal residues – Ser-Met -). In the experiments with 3S HMGB11-184, where it is in the middle of the flexible linker, it did not demonstrate increased CSP changes. Therefore, the changes associated with this residue in Box B alone are likely related to its position at the N-terminus potentially permitting high conformational flexibility. Design of a double Box B HMGB1 construct that retains CXCL12 binding whilst eliminating proinflammatory signaling [00105] We combined the data from the peptide arrays, alanine substitution and NMR, and mapped them onto the NMR structure of HMGB1 (Figure 6A and Figure 6B, PDB 2YRQ), to identify the residues involved in binding CXCL12. We found they form concave pocket on the underside of each HMG Box. These two pockets also contain the described binding site for glycyrrhizin, which inhibits HMGB1-CXCL12 binding [43]. Surprisingly, the distribution of peptides within each HMG Box that interact with CXCL12 is similar between both HMG Boxes and form almost identical binding pockets (Figure 6B). Furthermore, the HMG Box A and B are each capable of binding one CXCL12 monomer with equivalent affinity and binding of a monomer of CXCL12 does not require cooperation between the two Box domains. In contrast, binding to the proinflammatory receptors requires cooperation between the two Box domains. Therefore, we hypothesized that an engineered HMGB1 construct where Box A is substituted by another Box B (i.e. 1-88 replaced by 89-174) would effectively bind two CXCL12 monomers to present to a CXCR4 membrane dimer but would be unable to signal through TLR-2, TLR-4 or RAGE. We further reduced the propensity for binding to RAGE by deleting the RAGE-binding sequence in Box B (175-184). Replacing the Box A sequence for Box B also replaces the LPS glycan-binding peptide for the LPS lipid A binding peptide [51]. Therefore, this engineered construct dBB12L (Figure 6C) comprised the following segments of the native HMGB1 protein: flexible N-terminal region (from HMGB189-93), first Box B (from HMGB194-162), 12-residue linker C-terminal of native Box B (from HMGB1163- 174), and second Box B (from HMGB194-162). The linker length of 12 residues in this dBB12L is similar to the 10 amino acids in the linker of native HMGB1 and included residues 172 and 173 which showed changes in CSP on CXCL12 binding. [00106] We compared the thermostability of dBB12L and wild-type HMGB1 across a variety of buffer conditions using differential scanning fluorimetry (DSF) and solvent accessible surface area (SASA) determination by both native mass spectrometry (native ESI/MS) and size exclusion chromatography (SEC). dBB12L had similar stability and surface charge profiles to FR-HMGB11-164, which also contains two HMG Box domains and no C-terminal acid tail. Thermostability trends across different buffer conditions were similar for all constructs (Figure 7A), with lower Tm50 for buffers with pH close to the isoelectric point (9.9 for tail-less constructs as dBB12L or 1-164, and 6 for FL-HMGB1). All constructs were equally stable in PBS, purification buffers, and saline solution, with Tm50 ≈50°C. However, we observed different optimal ionic strength and pH ranges for FR-HMGB1/FR-HMGB11-164 compared to dBB12L. In native ESI/MS, all three HMGB1 constructs had similar charge state distributions, with a compact monomer as the main species and a second species with higher SASA representing an extended monomer conformation (Figure 7B and Figure 7C). The extended monomer was more prevalent for tail-less constructs or at higher ionic strength. Average monomer SASA values observed in native ESI/MS (Figure 7C) agreed with those derived from SEC or published NMR structures (PDB 2YRQ; HMGB11-164). Compact FL- HMGB1 monomer SASA matched computational models of FL-HMGB1 in water [46]. Storage up to 180 days did not affect SEC profiles (always monodisperse at equal RV), degradation, or aggregation (Figure 7D). Taken together, these data show that dBB12L has a similar folding and stability to native FR-HMGB1 in clinically relevant solutions. dBB12L construct has greatly reduced affinity for RAGE and cannot signal through TLR-2 or TLR-4 [00107] Next, we evaluated whether dBB12L had decreased TLR-2, TLR-4 signaling and RAGE binding, whilst preserving HMGB1-mediated regeneration. Due to the lack of an established signaling assay for RAGE, we assessed the binding of RAGE to HMGB1 using real-time kinetics (BLI) and an endpoint assay (ELISA). ELISA-based affinity measurements (Figure 8A) showed that 3S-, FR- and DS-HMGB1 at equilibrium bound similar amounts of RAGE, with DS-HMGB1 and 3S-HMGB1 having significantly higher affinity compared to FR-HMGB1. In contrast, dBB12L did not bind RAGE. Three additional HMGB1 constructs were tested, DS-HMGB11-184, which has an intact RAGE binding peptide and oxidized Box A but no acidic tail and therefore has all the requisites for RAGE binding; DS-HMGB11-164, which lacks a significant portion of the RAGE binding peptide but retains an oxidized Box A; and DS-Box A alone. We found that DS-HMGB11-184 bound RAGE, but with reduced capacity and affinity compared to full-length DS-HMGB1. By comparison, DS-HMGB11-164 had greatly diminished RAGE binding capacity compared to full-length DS-HMGB1 but still higher than dBB12L, whilst DS Box A 1-88 (full HMG Box construct with flanking regions) was unable to bind RAGE. [00108] Kinetics analysis using BLI were consistent with the ELISA results (Figure 8B), with two exceptions. In BLI (Figure 8C) DS-HMGB11-184 had much higher RAGE binding affinity than all other constructs, albeit with a slightly faster dissociation rate, compared to ELISA where it had lower affinity than DS-HMGB1.3S-HMGB1, whilst binding equivalent amounts of RAGE to DS- or FR- HMGB1, had similar affinity to FR-HMGB1 but much slower overall kinetic rates, whereas in ELISA it had affinity and binding capacity equivalent to DS- HMGB1. The higher RAGE affinity of DS-HMGB1 compared to FR in both assays was due to a much faster association rate (kon), whereas dissociation rates (koff) were nearly identical for these two redox forms. In contrast, dBB12L, which had an association rate closer to DS- HMGB1, exhibited very unstable binding due to a very high dissociation rate. DS-HMGB11- 164 also had a faster RAGE binding equilibrium with overall lower binding affinity than full length DS-HMGB1, albeit with higher affinity than dBB12L-HMGB1. The deletion of both the final 10 residues in Box B (175-184) and the disulfide bridge in Box A by substituting it with Box B in dBB12L resulted in unstable binding of RAGE. [00109] HMGB1 binds TLR-2, TLR-4, and RAGE and signaling from all receptors converges to the NF-κβ pathway [25]. Consequently, it is difficult to attribute downstream proinflammatory cytokine production to each receptor. Therefore, we first evaluated TLR- specific signaling using NF-κβ reporter cell lines engineered to express either TLR-2 or TLR- 4 and their co-receptors. Disulfide HMGB1 promoted NF-kB signaling via TLR-2 (Figure 8D) and TLR-4 (Figure 8E). In contrast, dBB12L failed to signal in either cell type. Next, we confirmed the effects of the various HMGB1 constructs on primary human monocytes. It has been reported that DS-HMGB1 synergizes with TLR-2 ligands such as lipoteichoic acid (LTA) to promote proinflammatory signaling [32]. We confirmed that DS-HMGB1 acted synergistically with LTA to promote higher TNF production than LTA or DS-HMGB1 alone. In contrast, dBB12L or FR-HMGB1 did not exhibit this synergistic effect and failed to elicit TNF secretion greater than media alone (Figure 8F). No synergistic response has been described with DS-HMGB1 and the TLR-4 ligand LPS. When combined with LPS, DS- HMGB1 promoted TNF expression by primary human monocytes to the same extent as LPS alone. In contrast, FR-HMGB1 or dBB12L alone did not promote TNF production. However, when combined with LPS, FR-HMGB1 or dBB12L reduced TNF expression compared to LPS alone. [00110] Taken together, these data show that dBB12L does not signal via TLR-2 or TLR-4, even in the presence of their cognate ligands, has greatly decreased affinity for RAGE and reduces LPS-mediated pro-inflammatory signaling. The dBB12L construct has pro-regenerative activity comparable to that of FR-HMGB1 [00111] Distant injury has previously been shown to transition stem cells to GAlert [13]. Therefore, we first compared the transcriptomic response of skeletal muscle stem cells to FR- HMGB1 or injury to the contralateral limb. The genes up- and down-regulated by FR-HMGB1 or distant injury were remarkably similar (Figure 10A), with the major pathways upregulated being those associated with GAlert [8,13], including mitochondrial metabolism, oxidative phosphorylation and cell cycle (Figure 10B). Interestingly, CXCR4 was one of the most highly upregulated genes. [00112] Next, we determined the optimal in vivo treatment dose for FR-HMGB1 using a validated murine model of skeletal muscle injury [8,13]. We found that 0.75 mg/kg (29 nmol/kg) resulted in the maximal response, with no further improvement in regenerative activity with higher doses (Figure 10C). We also assessed the optimal time for administration in vivo after injury. FR-HMGB1 was found to be effective in promoting repair when injected up to 5 h post-injury (Figure 10D). We then assessed the half-life of FR-HMGB1 in the circulation following iv administration. We found that there was an initial rapid clearance (t1/2 ≈11 min) followed by subsequent slower clearance rate (t1/2 ≈ 120 min) (Figure 10E). This would be consistent with the half-life of 25 min in humans [54], with the protein being cleared by binding to haptoglobin [55,56]. [00113] We have previously shown that FR-HMGB1 accelerates regeneration of skeletal muscle, bone and blood following injury by promoting the transition of stem and progenitor cells to GAlert [8]. There is a small population of progenitor cells in the mammalian heart [57] and the majority of new cardiomyocytes following injury are derived from existing cardiomyocytes [58]. To ascertain the efficacy of FR-HMGB1 on tissues that do not rely on resident stem and progenitor cells for regeneration, we assessed whether administration of FR- HMGB1 on cardiac regeneration. We found that iv injection at the time of myocardial infarction resulted in enhanced survival (83% in mice treated with FR-HMGB1 compared to 52% in PBS controls) (Figure 10F) and approximately 60% reduction in infarct size as assessed by serial MRI scans over 5 weeks (Figure 10H) and 16% improvement in overall left ventricular ejection fraction (Figure 10G). [00114] Next, we assessed the efficacy of dBB12L compared to FR-HMGB1 in promoting skeletal muscle regeneration in vivo. Mice injected with optimal doses (29 nM/kg) of FR- HMGB1 or dBB12L exhibited accelerated regeneration equally following injury (Figure 10J), as determined by an increase in the mean cross-sectional area of regenerating muscle fibers with central nuclei [8,13]. This was most apparent at day 14, as previously described for FR- HMGB1 [8]. [00115] Finally, we assessed the role of the flexible region between HMG Boxes in the regenerative process. We first tested whether alterations in this linker region of wild type human FR-HMGB1 affected protein stability. We found that reducing the linker length to 13 amino acid residues or less led to a decrease in Tm50 indicating that the linker length plays a key role in protein folding and stability. Furthermore, deletion of the first two prolines in the linker region completely abrogated expression. This indicates that the linker length likely also governs protein folding and stability. We then tested in vivo regenerative activity of the constructs with variations in their linkers (Figure). We found that deletion of the entire linker (Δ79-88) resulted in loss of regenerative activity and deletion of the first four linker residues (Δ79-82) led to some loss of regenerative activity. Whilst deletion of the central region (Δ84- 88) resulted in complete loss of tissue regeneration, surprisingly substitution of residues 84-88 with a sequence of generic flexible amino acid residues restored full regenerative activity. Substitution of the DPNA peptide before the second Box B domain also adversely affected regenerative activity. DISCUSSION HMGB1 needs to be modified in order to be used as a tissue repair therapeutic [00116] Therapies based on administration of exogenous stem cells to promote repair of solid organs have failed to deliver on the initial promise [6,59], and killed cells are just as effective by triggering an immune response [60]. An alternative, potentially more effective approach, would be to target endogenous regenerative repair processes, including resident stem and progenitor cells [61,62]. Inhibition of prostaglandin dehydrogenase is a promising approach [7], although progress through to clinical translation has been slow [63]. Administration of growth factors has also been described [64,65] but is limited by in vivo proteolysis [66]. Currently, there is no approved therapeutic for promoting regeneration and accelerating repair of multiple tissues. [00117] We previously showed that exogenous administration of FR-HMGB1 is effective in accelerating regeneration of bone, skeletal muscle and blood by transitioning resident stem and progenitor cells to GAlert, when they are able to readily respond to the appropriate activating factors released on tissue injury to effect repair [8]. However, there is accumulating evidence supporting the conversion of FR-HMGB1 into DS-HMGB1 in vivo locally at the site of injury [15,16]. Therefore, the potential for deleterious proinflammatory signaling precludes the use of native FR-HMGB1 as a therapeutic. DS-HMGB1 can signal through TLR-4 [29,35], TLR- 2 [32,33,35] or RAGE [37,67] to converge on NF-κβ [25,68], leading to synergistic expression of proinflammatory cytokines [69,70]. Therefore, development of HMGB1 as a therapeutic is crucially dependent on engineering the molecule to eliminate signaling via all three receptors. Box A and Box B bind CXCL12 independently [00118] The regenerative activities of FR-HMGB1 are crucially dependent on the formation of a heterocomplex with CXCL12 and signaling via CXCR4. Whilst it is known that CXCL12 binds to HMG Boxes [44,71], the precise structural motifs involved remain unknown [53,72]. It is also unclear whether this signaling involves homodimers of CXCL12 monomers, which promote chemotaxis [73–75]. By combining the data from our forward (Figure 2A and Figure 2B) and reverse peptide arrays (Figure 11), alanine substitution arrays (Figure 2C and Figure 2D), and NMR experiments (Figure 5A and Figure 5B), we were able to identify the residues [76] involved in HMGB1-CXCL12 interaction. We identified a common pattern of HMGB1 peptides binding CXCL12 that occupy concave pockets on the underside of Box A and Box B. Using BLI (Figure 2E and Figure 2F) we identified the role of the flanking flexible regions by BLI in CXCL12 binding. Furthermore, as we used HEPES buffer during BLI and NMR experiments to prevent CXCL12 dimerization [77], we concluded that each Box is able to bind monomeric CXCL12 without the requisite for cooperation between the Box domains and that dimerization of CXCL12 is not a requisite for complex formation. Design of the engineered dBB12L construct that does not signal via TLR-2 or TLR-4 or bind RAGE, whilst retaining full pro-regenerative properties [00119] We observed a similar mirroring across HMG Boxes of the regions in HMGB1 involved in TLR-2 binding (Figure 3, section A and Figure 4, section A), but found that the disulfide form of HMGB1 is more capable of signaling through TLR-2 (Figure 8D and Figure 8F) This indicates that, unlike with CXCL12 (two identical HMG Boxes in the reduced molecule), TLR-2 signaling requires two different HMG Boxes (one in the reduced and another in the oxidized conformation). TLR-4 has long been known to preferentially bind oxidized HMGB1 Box A. We identified a clear binding pocket for TLR-4 on HMGB1 that overlaps with the LPS binding segments (1-15, 80-96) and is only continuous when Box A is oxidized. In addition to both Box A and B, RAGE binding requires an intact sequence before the acidic tail and oxidation of Box A enhances RAGE binding. Interestingly, in all cases the linker region of HMGB1 appears to be involved in binding to the proinflammatory receptors. [00120] With this detailed understanding, we were able to design a construct to eliminate proinflammatory signaling via TLR-4 and TLR-2 and RAGE whilst preserving CXCL12 binding. This construct consisted of two HMG Box B domains in tandem separated by a linker of similar length to that of wild-type HMGB1 (dBB12L). The absence of Box A in dBB12L precludes oxidation and hence the rearrangement of the observed binding interfaces for TLR- 2 (rotation of Helix 3), RAGE (bending of Helix 2) and TLR-4 (continuous binding surface), whilst permitting binding to two CXCL12 monomers via the two Box B domains. We found that dBB12L was as stable as 1-164 FR-HMGB1 or full-length FR-HMGB1, with no aggregation or degradation on storage for prolonged periods of time. [00121] Whilst DS-HMGB1 alone can bind and signal via TLR-4/MD-2 [28], it can also facilitate signaling via LPS by substituting for LPS-binding protein (LBP), which binds LPS and promotes transfer and recognition to TLR-4/MD-2 [51]. Deletion of Box A in our dBB12L construct effectively precluded signaling via TLR-4. Interestingly, we observed that both FR- HMGB1 and dBB12L decrease TNF expression by monocytes on exposure to LPS. This could be due to these proteins binding LPS but are unable to transfer it to TLR-4/MD2 due to the lack of oxidized Box A, unlike DS-HMGB1, which can effectively substitute for residual LBP [51] present in the serum of the culture medium [78]. [00122] There is controversy as to whether HMGB1 can induce TLR-2 signaling on its own [34,35] or requires a co-ligand to induce activity, and also whether this response is dependent on the redox state of the protein [32,33]. Available data [32][33] suggest that that HMGB1 alone can signal through TLR-2 but a co-ligand is required to induce higher levels of response. We found that DS-HMGB1 alone was able to signal via TLR-2 and the effect was enhanced in the presence of LTA in serum-containing media. However, there was no response to FR- HMGB1 or dBB12L, which did not synergize with LTA. This would suggest that, as with TLR-4, oxidized Box A with a disulfide bridge is necessary for TLR-2 mediated responses and that TLR-2 co-ligands synergize with DS-HMGB1, potentially by displacing the acidic tail to promote TLR-2 interaction. [00123] A RAGE binding peptide within HMGB1 (residues 149-182) has been previously described [37]. A similar motif is also present in other RAGE ligands such as S100 proteins, and homologous peptides to these sequences are effective antagonists of HMGB1-mediated RAGE signaling [38,52]. The acidic tail of HMGB1 shares residues with the RAGE binding peptide [40] and has been proposed as a regulator of RAGE interaction, analogous to its role in TLR-2 binding. However, only the disulfide form of HMGB1 has been linked to prothrombotic activities via RAGE [36]. We observed that the RAGE binding peptide in Box A [39], which was previously thought to require Caspase-1 processing for activity, is exposed in the intact HMGB1 when in solution and alters its conformation upon oxidation of the disulfide. We found that constructs lacking a complete RAGE binding peptide 149-182, including dBB12L, were unable to bind RAGE in the ELISA assay. In contrast DS-HMGB1, and interestingly also 3S-HMGB1, were able to bind RAGE better than FR-HMGB1. Our BLI data showed that dBB12L has lower affinity for RAGE compared to FR-HMGB1 and DS- HMGB1. Whilst dBB12L had a three-fold higher association rate than FR-HMGB1, potentially due to the presence of two partial RAGE binding domains in this construct, the dissociation rate was five-fold greater. The ELISA data represent the binding status at equilibrium and, therefore, reflects the balance between the association and dissociation rates. For instance, 3S- HMGB1, which has an affinity for RAGE similar to FR-HMGB1 in BLI, shows a much higher apparent affinity in ELISA similar to DS-HMGB1 due to a dissociation rate much lower than that of FR-HMGB1 or DS-HMGB1. This increased RAGE binding may in part account for the increased fibrosis seen in mouse models of myocardial infarction compared to controls, whereas FR-HMGB1 promoted regeneration and improved function [31]. Using SPR others have also shown that binding of HMGB1 to RAGE likely requires two distinct binding sites, one of which varies according to the oxidation status [20]. We found that the affinity for RAGE for DS-HMGB1 using BLI (Kd = 0.2-1.3 µM) was similar to that previously reported using SPR (0.1 [79] - 0.65 µM [36]), these studies also reporting high affinity for 3S-HMGB1. Our BLI data also show that loss of the acidic tail increases the affinity of HMGB1 for RAGE by greatly increasing the association rate, whereas truncation of the RAGE binding peptide or reduction of Box A greatly increased dissociation rates. Interestingly, oxidized Box A alone is incapable of binding RAGE, suggesting that the interaction is fully stabilized by the RAGE binding peptide, and the acidic tail negatively regulates this binding by competing with residues within the RAGE binding peptide [40]. The absence of Box A together with truncation of the RAGE binding peptide in dBB12L results in greatly reduced RAGE affinity. [00124] The transcriptomic changes induced by FR-HMGB1 in skeletal muscle stem cells are very similar to those induced by distant injury and upregulation of CXCR4 expression by HMBG1 would potentially enhance its effects. Our data showing that FR-HMGB1 is only effective if administered up to 5 h post injury would be consistent with it acting by transitioning stem cells to GAlert. At later time points the stem cells will have been fully activated [80]. We confirmed that dBB12L retains regenerative activity in vivo equivalent to FR-HMGB1. Importantly, we found that FR-HMGB1 administered intravenously at the time of myocardial infarction resulted in improved survival, reduction in infarct size and improved left ventricular ejection fraction. Based on these data we would predict that administration of dBB12L would also promote regeneration of tissues that rely on stem cells for repair such as bone, skeletal muscle and blood, as well as tissues where regeneration is predominantly reliant on mature cell populations such as cardiomyocytes in the heart. We also predict that dBB12L is likely to be effective if administered up to 5 h after injury. This is important as the median time for admission to hospital following MI in the USA is 3 h [81]. Approximately 800,000 people in the USA suffer from myocardial infarction every year [81] and approximately 20% go on to develop cardiac failure. Despite US healthcare expenditure for heart failure of >$30 billion in 2012, projected to increase to $70Bn by 2030, 5-year survival is only ~60%, which is worse than most cancers [81]. Based on our data we predict that administration of dBB12L within 5 h of the event will enhance survival of patients experiencing a myocardial infarction, and through reduction in infarct size and preservation of ejection fraction, reduce the incidence and severity of cardiac failure. Others have shown in mouse [31,82,83] and sheep [84] models that direct injection of FR-HMGB1 into the myocardium in the peri-infarct area 4 h after infraction is effective in promoting cardiac repair. Our data demonstrating the efficacy of iv administration are important as this route is readily applicable for clinical use. [00125] Based on our data using constructs with different linker lengths and linker substitutions, alternative constructs to dBB12L with similar activity profiles can also be designed. These could include the following variations: [00126] 1) Residues equivalent to positions 169-174 (i.e. “AAKKGV,” shown as residues 167-172 of SEQ ID NO: 2) at the C terminus of the second B Box in our double Box B construct could be eliminated altogether or substituted as described below (3). We would also envisage that substituting the equivalent residues at the C terminus of the first B Box domain in the double Box B construct would further help eliminate any potential binding to TLR-2. [00127] 2) Our current dBB12L construct contains 12 amino acid residues corresponding to positions 163-174 of the native HMGB1 sequence at the C terminus of the first Box B domain. With the additional 5 amino acids at the N terminus of the second B Box that we have found to be critical for CXCL12 binding, this gives an overall inter Box linker length of 17 amino acids. Constructs with linker length less than 13 amino acid residues have lower regenerative activity and thermostability indicative of unstable molecular folding. Therefore, we would predict that the optimal linker length would be 13-20 amino acids. The loss of regenerative activity of constructs with shorter linkers may also be related to an inability to adopt the appropriate conformation to present two CXCL12 monomers to a CXCR4 dimer. [00128] 3) Deletion of the linker affects the regenerative effect differently depending on the deleted positions: deletion of residues 79-82 (11 residues including the beginning of Box B) resulted in a molecule with decreased regenerative effect whilst deletion of residues 84-88 led to complete loss of regenerative activity. We also found that substitution of amino acids 84-88 with a sequence of generic flexible amino acids (GSGSG (SEQ ID NO: 175)) in the inter Box linker after the C terminus of Box A did not adversely affect in vivo regenerative activity. This region was not involved in CXCL12 binding in in our peptide arrays or NMR. Therefore, in our next generation molecules this region could be substituted with a linker of between 13 and 17 residues with any of the following modifications: [00129] a) Substitution of any, or all, residues equivalent to positions 168-174 (last 7 residues in the linker) in wild type human HMGB1 for any of the following: [00130] A random sequence of amino acids such that this region can adopt either no specific secondary structure, coil-turn structures, or alpha helical conformations, with the first two being preferred. [00131] Substitution of the amino acids (e.g., Lys <-> Arg, aliphatic to aliphatic substitutions). [00132] Flexible amino acid sequences [85] such as Gly-Ser, or turn-inducing residues such as Pro. [00133] Charged residues to improve solubility. [00134] b) Substitution of any, or all residues equivalent to positions 163-167 by any of the options in (3a) or point deletion of individual residues. However, we anticipate that substitution of K164, G165 or K166 is likely to affect CXCL12 binding capacity, as substitution or deletion of residues equivalent to this region on wild type HMGB1 led to reduction in regenerative activity. Therefore, those would likely need to be substituted with more chemically similar residues. [00135] c) Substitution of any, or all, residues in the immediate 5 amino acids before Box B, including the D-P-X-X motif. Whilst this resulted in loss of some regenerative activity, it is possible that this region is only required as a flexible extension of the HMG Box as it is positioned parallel to the third alpha helix. [00136] The above amino acid sequences will be designed with the intention of reducing proinflammatory activities (by removal of specific epitopes to e.g., further reduce the affinity for RAGE, LPS, and other DAMP/PAMPs), improve protein stability and folding, or introduction of chemical functionalization (e.g. Click chemistry or unnatural amino acids), with the overriding objective of preserving CXCL12 binding capacity to the modified HMG Box domains including the altered linkers. Whilst more profound modifications to the HMG Box core domain are also possible, these would be more likely to disrupt the structure of the HMG Box domains and therefore the CXCL12 binding pockets. [00137] Our data show that whilst the peptides involved in binding CXCL12 are mirrored in Box A and Box B, this is not the case with the proinflammatory receptors, as each Box and the adjacent domains are involved in the binding to TLR-2, TLR-4 or RAGE. The loss of Box A and its C-terminal linker in dBB12L accounts for the absence of signaling via all three proinflammatory receptors. TLR-2 and RAGE binding and signaling is further impaired by the truncation of the binding sequences before the C-terminal acidic tail and the fact that the binding regions in oxidized Box A has been replaced by those equivalent in Box B. Box B adopts a different surface arrangement than Box A when it is oxidized and therefore is unable to effectively bind to TLR4,TLR-2 or RAGE. Importantly, the double Box B construct maintains regenerative activity equivalent to FR-HMGB1. Taken together these data show that the double Box B constructs described above can be developed as clinical therapeutics. [00138] In conclusion, the sites of HMGB1 critical for CXCL12 binding were mapped and a construct (dBB12L) that does not signal via TLR-2 or TLR-4 and fails to effectively bind RAGE was designed. FR-HMGB1 transitions stem cells to GAlert in a manner similar to distant injury despite a short half-life and is effective when administered up to 5 hours after injury. Furthermore, dBB12L promotes tissue regeneration in vivo as effectively as FR-HMGB1. Accordingly, dBB12L can be developed for clinical translation. SUMMARY [00139] Reduced High Mobility Group Box 1 (HMGB1) protein binds to CXC Ligand 12 (CXCL12) and signals through CXC Receptor 4 (CXCR4) to promote tissue regeneration and accelerates repair by transitioning stem and progenitor cells to GAlert. However, local conversion of FR-HMGB1 to the disulfide form (DS-HMGB1) may result in deleterious inflammation through signalling via Toll-Like Receptors 2 and 4, and the Receptor for Advanced Glycation End Products (RAGE). Therefore, it is important to engineer the molecule to eliminate these potentially deleterious proinflammatory effects when considering HMGB1 for use in clinical practice. [00140] We have identified the residues involved in formation of the HMGB1-CXCL12 heterocomplex using a combination of peptide arrays, biolayer interferometry and nuclear magnetic resonance. In addition, we used peptide arrays to define the peptide sequences required for the binding of TLR-2, RAGE, TLR-4. Based on these data we designed a construct comprising two HMG B Boxes in tandem (dBB12L). This has similar regenerative capacity, stability and conformation to wild-type fully-reduced HMGB1, does not signal through TLR-2 or TLR-4, even in the presence of their co- ligands, and has greatly decreased RAGE binding. We also describe a series of other double Box B constructs that would have similar attributes. [00141] A comprehensive review of the patent landscape and the scientific literature has identified U.S. Patent Application Publication US 2015/0203551 A1, which describes the substitution of cysteines with serines to prevent TLR-4 signalling; however, this construct has been shown to lead to excessive cardiac fibrosis following MI [14]. Furthermore, this construct has a slower dissociation for RAGE compared to FR-HMGB1, resulting in RAGE remaining bound for longer after equilibrium, as shown herein in Figure 8B); therefore these substitutions were avoided in our constructs. U.S. Patent Application Publication US 2009/0069227 A9 stipulates that HMGB1 constructs that promote stem cell migration and proliferation must include amino acids 1-187 (0-186 in our data, with 0 being the N-terminal Met) and U.S. Patent 9,623,078 refers to peptides limited to amino acids 1-44 (0-43 for our data) for cardiac regeneration. U.S. Patent Application Publication US 2009/0202500 A1 discloses methods for tissue repair but only refers to full-length (1-215) wild-type HMGB1 (0-214 for our data). The dBB12L construct presented herein has no RAGE binding or TLR-4/2 signalling, is 177 amino acids long, and includes amino acid substitutions that have not been previously described. Therefore, the constructs presented herein do not fall within the scope of the prior art. CLINICAL APPLICATIONS [00142] This invention provides polypeptides and methods to harness endogenous regenerative processes to enhance tissue repair. The polypeptides function similarly to fully reduced wild type HMGB1 which promotes tissue regeneration by forming a heterocomplex with two CXCL12 molecules, which in turn signals via CXCR4, likely two adjacent CXCR4 receptors on the cell surface. [00143] Our data show that a polypeptide of the invention (dBB12L) acts in a similar way. Therefore, it is contemplated that dBB12L will promote the regeneration of tissues that rely on CXCR4+ cells for repair. Such tissues include tissues where repair is primarily dependent on stem and progenitor cells, such as skeletal muscle and the haemopoietic system, as well tissues where repair is largely dependent on existing mature cells, e.g., cardiomyocytes in the adult mammalian heart. [00144] Potential clinical indications: [00145] Heart following myocardial infarction. This indication is ripe for a clinical trial. Globally, ischemic heart disease affects 153 million people (101), with the loss of >105,000,000 Disability Adjusted Life Years in 2017 (102). Every year 205,000 people in the UK (103) and 805,000 in USA suffer from myocardial infarction (MI), 38% of them experiencing ST-elevation MI (STEMI) (101). Following MI, approximately 30-40% of individuals develop heart failure, affecting 38 million worldwide. Despite US healthcare expenditure for heart failure of >$30 billion in 2012, projected to increase to $70Bn by 2030, 5-year survival is only ~60%, which is worse than most cancers (101). The main target population are patients following MI, especially those at risk of developing heart failure (104). A novel therapeutic that limits cardiac damage, promotes regeneration following MI and prevents the development of heart failure would dramatically reduce morbidity and mortality, and massively reduce healthcare burden. Definitive data using an established (105-108) permanent ligation murine MI model that reliably leads to cardiomyocyte necrosis (109) show that a single iv dose of FR-HMGB1 at the time of injury leads to enhanced survival (83% for animals treated with FR-HMGB1 compared to 52% in the group treated with PBS placebo), and compared to controls, ~16% improvement of absolute cardiac ejection fraction and ~60% reduction in infarct size compared to PBS controls over 5 weeks (Figure 10F). [00146] In a skeletal injury model, the optimal dose of FR-HMGB1 was 0.75 mg/kg (Figure 10C) and is effective even if administered iv up to 5 hours post injury (109) (Figure 10D), despite a very short half-life (Figure 10E). Following myocardical infarction, reperfuion of the ischemic cardiac muscle should be achieved as soon as possible. For example, following STEMI patients should undergo percutaneous intervention. Data show that administration of HMGB1 as soon as possible and maximally up to 5 hours post-injury will preserve the damaged myocardium and promote regeneration. [00147] While native FR-HMGB1 promotes functional recovery post MI (Figures 10F-10I), local conversion to the disulfide form promotes thrombus formation and propagation via RAGE, TLR-2 and TLR-4 (110). Constructs reported by others such as 3S-HMGB1 that retain RAGE binding (Figure 8B) result in excessive fibrosis and impairment of function following MI (111). FR-HMGB1 also binds RAGE, albeit to a lesser extent than DS-HMGB1 and, therefore, would not be suitable for clinical use. HMGB1 signalling via TLR-2 plays a key role in ischaemia reperfusion injury following myocardial infarction (112) and thrombosis (110), The inventors a have shown the key role of TLR-2 in human atherosclerosis (113). TLR-4 signalling is also crucial in myocardial reperfusion injury (114). The redox conditions in the ischemic and inflamed microcirculation of the damaged heart following myocardial infarction will promote conversion of FR-HMGB1 to the disulfide form (DS-HMGB1), which is a central mediator of thrombosis (110). There is no approved therapy for promoting cardiac regeneration following MI. Reports purporting to show regenerative effect of hematopoietic stem cells have been discredited (115), and killed cells are just as effective by triggering an immune response (116). Even with other cell types, including pluripotential stem cells, significant challenges remain, including arrhythmogenesis, immunosuppression, scalability, batch variability, delivery, long-term viability and efficacy (115, 117). Large scale trials of cell-based therapies showed no significant improvement in function, with arrhythmias reported in patients (118- 120). [00148] The absence of a significant stem population (121) and limited epicardial progenitor cells (122) in the adult heart, together with an understanding that the majority of new cardiomyocytes following injury are derived from existing cardiomyocytes, has shifted focus to promoting regeneration by manipulating endogenous pathways (121). This includes adenoviral transduction of multiple transcription factors(105, 108), manipulation of developmental pathways such as Hippo (106) or Meis1 (123), addition of growth factors such as neuregulin (124), IGF/HGF (125) or FSTL1 (126), or manipulation of miRNAs (127). These approaches have significant shortcomings: adenoviral transduction and growth factors (IFGF1/HGF) require intracardiac injection or topical patch application (FSTL1), manipulation of developmental pathways carries oncogenic risk (128) and viral transduction of miRNA199- a in pigs resulted in fatal arrhythmias (127). An alternative strategy for stimulating cardiac regeneration by promoting clearance of immune cells requires repeated injection of VEGF-C (129). Inhibition of MAP4K4 promotes myocardial survival and limits infarct size, but there was no regenerative effect (130). To date, none of these strategies have progressed to clinical trials. [00149] This invention provides a unique solution which targets endogenous processes to promote cardiomyocyte survival and regeneration of multiple tissues. It overcomes the many hurdles associated with cell therapies, including anti-fibrotic CAR T cells (131), such as prohibitive expense (132, 133). Since FR-HMGB1 acts via the cell surface receptor CXCR4, it is not expected to have off target effects associated with targeting intracellular processes, e.g. by adenoviral transduction of transcription factors or miRNA. HMGB1 inhibition increased infarct size following ischemia reperfusion injury (134) and whilst local upregulation (135, 136) or intramyocardial injection of FR-HMGB1 has been shown to be effective in both mice (111, 137, 138) and sheep (139), our data indicate iv administration is efficacious and more likely to reach all target cells. The engineered double Box B construct of the invention which avoids deleterious proinflammatory signaling should be safe. [00150] A group in Milan described an HMGB1 analogue (3S-HMGB1) where 3 cysteines are replaced with serines to negate TLR-4 signaling (140). Whilst they claimed that 3S- HMGB1 is superior to FR-HMGB1 in promoting tissue regeneration (141), the present inventors have not found this to be the case (142). Importantly, 3S-HMGB1 promoted fibrosis in a murine MI model, with deterioration in cardiac function, whereas FR-HMGB1 promoted tissue regeneration and improved left ventricular ejection fraction (111). The inventors have found that 3S-HMGB1 remains bound to RAGE for longer than either DS-HMGB1 or FR- HMGB1 (Figure 7A). Therefore, whilst FR-HMGB1, 3S-HMGB1 and DS-HMGB1 bind equivalent amounts of RAGE at equilibrium, over time levels of RAGE bound by 3S-HMGB1 are comparable to the proinflammatory disulfide HMGB1 (DS-HMGB1) and higher than to FR-HMGB1. The inventors double Box B construct eliminates undesirable proinflammatory signaling whilst retaining regenerative activity equivalent to FR-HMGB1. [00151] Additional applications: [00152] Fractures. Fractures occur following injury. However, one of the commonest skeletal ‘injuries’ is joint replacement or arthroplasty. The inventors propose that dBB12 can be used to promote healing following fracture or arthroplasty, thereby reducing the risk of potential complications such as loosening of components. [00153] Brain and nervous system. dBB12L may be used to improve patient outcomes following stroke. Other potential indications include Parkinson’s disease and dementia. [00154] Lung. dBB12L is contemplated to improve outcomes following lung injury, for example, following Covid-19 or in patients with idiopathic pulmonary fibrosis. [00155] Liver.30% of people in the USA are estimated to suffer from non-alcoholic liver disease.60% of these go on to develop non-alcoholic steatohepatitis and 20% of those develop liver cirrhosis. Treatments are being developed to limit and prevent liver damage from tehse conditions. The inventors propose that dBB12L to be used in combination with these treatments to promote liver regeneration. [00156] Gut. dBB12L may be used to promote healing of the gut, for example, following surgery or patients with inflammatory bowel disease such as ulceractive colitis in combination with treatments to control inflammation. [00157] Kidney. dBB12L may be used to promote regeneration of the kideny, thereby potentially avoiding the need for dialysis or kidney transplantation. [00158] Skin. dBB12L may be used to promote wound healing eg following surgery, burns or patients with ulcers eg diabetic ulders. [00159] Pancreas. dBB12L may be used to improve outcomes in patients with type 1 diabetes mellitus by promoting regeneration of islet cells. [00160] Bone marrow. dBB12L may promote regeneration of the haemopoetic system e.g. following chemotherapy, thereby preventing severe potentially life thereatening neutropenia. [00161] The inventiors have previously shown that FR-HMGB1 is effective even if adminsitered up to 2 weeks before injury (142). Since dBB12L is equally efficacious to FR- HMGB1 (Figure 10J) it is contemplated that this polypeptide may be used prophylactically, for example, by the military or for sports injuries or before elective surgery or chemotherapy. MATERIALS AND METHODS E. coli strains [00162] Mach-1 T1R cells (Invitrogen, no antibiotic resistance or induction, BL21(DE3)-R3- pRARE2 (in-house BL21 derivative, chloramphenicol resistance 36 µg/mL , T7-polymerase lac induction [86]) and BL21(DE3)-R3-pRARE2-BirA (in vivo biotinylation derivative of the above, additional spectinomycin resistance 50 µg/mL) were sourced from chemically competent stocks made in-house. Bacterial culture media [00163] SOC: 20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 0.1862g/L KCl were autoclaved and supplemented with 4.132 g/L MgCl2 and 20 mM glucose. [00164] LB (Luria Bertani): 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.2, autoclave sterilized.2% w/v agar powder was added to make LB agar plates. [00165] TB (Terrific Broth): 12 g/L tryptone, 24 g/L yeast extract, 4 g/L glycerol, 12.5 g/L K2HPO4, 2.35 g/L KH2PO4., autoclave-sterilized. [00166] TB supplement: 1.6% w/v glycerol, 1% glucose, 25 mM (NH4)2SO4, 10 mM MgSO4, 10X trace metals, 0.22 µM sterile filtered. [00167] Trace metal solution: 50 mM FeCl3 (13.5 g/L), 20 mM CaCl2 (2.94 g/L), 10 mM MnCl2 (1.96 g/L), 10 mM ZnSO4 (2.88 g/L), 2 mM CoCl2 (0.48 g/L), 2 mM CuCl2 (0.34 g/L), and 2 mM NiCl2 (0.48 g/L), in 0.1 M HCl, 0.22 µM sterile-filtered. [00168] M9 minimal medium: 16 g/L Na2HPO4, 4 g/L K2HPO4, 1 g/L NaCl, pH 7.2-7.3 and 2.5 g/L FeSO4, 0.25 mg/L ZnCl2, 0.05 mg/L CuSO4, 0.25 g/L EDTA, 1 mM MgSO4 were autoclaved and supplemented with 4 g/L glucose, 1 g/L U-99% 15NH4Cl (Cambridge Isotopes), 0.3 mM CaCl2,1.5 mg/L D-biotin and 1.5 mg/L Thiamine-HCL from sterile filtered stocks. Plasmids [00169] Plasmids were sourced from the SGC libraries [86]. All plasmids contain a 6xHis tag with a TEV-cleavage site; pNIC-Bio3 and pDsbC-HT-CBio also have C-terminal biotinylation epitopes (which can be removed with a stop codon). Plasmid DNA was linearized by restriction enzyme digestion: BfuA1 (3h, 60°C) for pNIC-CTHF or BsaI (2h, 37°C). Cut vector DNA was purified with a PureLink PCR kit and treated with T4 DNA polymerase (NEB M0203) in the presence of 0.25 mM dGTP (pNIC-CTHF) or dCTP as per manufacturer protocols. Cloning [00170] HMGB1 constructs sourced from the Mammalian Gene Collection (Mach1 cells) were amplified via PCR: a program of 95°C /10´, 25x (95ºC/ 30”, 52ºC/1´, 0.5-1.5’ at 68ºC), 68ºC/10´was used. Reaction consisted of 5 µL Herculase II buffer, 1 µM of each primer, 6 µg/mL plasmid template, 1 µM dNTP mixture and 1 unit Herculase II polymerase (Agilent 600679; supplied with buffer and 100 µM dNTP stocks) in 25 µL final volume. PCR products were purified before further use (PureLink kit, ThermoFisher K310001). [00171] Amplified coding sequences (alleles) were cloned into the destination vector via ligation independent cloning (LIC). The insert was treated with T4 DNA polymerase in the presence of a cognate nucleotide to that used for the vector (10 µL reaction volume), and 2 µL was mixed with 1 µL of treated vector and annealed for 30‘.40 µL ice-cold Mach-1 cells (for storage) or 20 µL BL21(DE3)-R3-pRARE2/ BL21(DE3)-R3-pRARE2-BirA cells (for expression) were added and heat-shocked for 45” at 42°C before chilling in ice. Recovery was performed for 2 h in SOC medium at 37°C prior to plating on selective media with 5% sucrose and antibiotics. After 24 h, positive colonies were picked and screened with MyTaq polymerase according to manufacturer protocols with specific sequencing primer pairs for bands of the correct molecular weight. Positive transformants were grown overnight in 1 mL of 2X LB (double concentration of LB) with antibiotics and stocked with 12% glycerol v/v at -80°C. [00172] 3S-HMGB1 mutant sequence was generated in a similar manner. PCR was performed separately to generate a S23-S45 and a S106 fragment, which were annealed via PCR; 5 µL of each purified PCR product substituted the primers and template in this reaction. The process is summarized in Figure 17. [00173] CXCL12 constructs were cloned with an in-frame SUMO protease site N-terminal to the mature protein to allow for periplasmic secretion with an N-terminal fusion protein in the pDsbC-HT-CBio vector (DsbC-SUMO-CXCL12) to avoid addition of N-terminal residues to the protein which could affect its activity [87,88] whilst obtaining folded, oxidized CXCL12 via the DsbC fusion protein system [89]. A detailed table of primers and vectors used for each construct, boundaries, expression strains, and base pairs/amino acid sequences can be found in the Supplemental Methods section. All mutants were verified by sequencing (SourceBioscience). The sequence for HMGB1-dBB was designed in silico by codon- optimizing a Box B 89-174 sequence according to E. coli BL21-DE3 genome (assembly ASM956v1) placed after the native HMGB1 Box B sequence, and synthetized in vitro by Twist Bioscience (San Francisco, USA) cloned in pNIC-CTHF. Recombinant protein expression [00174] 20 mL of overnight culture of HMGB1-expression strain transformants, grown from a fresh agar plate streak, were inoculated into 1 L of TB (or M9) medium with supplement and allowed to grow up to OD 2.0 at 37°C with 0.45 RCF orbital shaking (OD 0.6 for M9 medium). Precultures used for production of 15N labelled HMGB1 were first spun down at 1000 RCF for 5 ‘and washed in M9 medium. Once the target OD was reached, were cooled to 18°C before addition of 0.5 mM or 0.25 mM IPTG (for HMGB1 and CXCL12 proteins respectively) and grown for 16 h before harvesting at 4000 RCF. For biotinylated proteins, 10 mM D-biotin in PBS was added before induction and again 1 h before cell harvesting. Recombinant HMGB1 purification [00175] Pellets of induced HMGB1-expressing cells were resuspended at 14 g/L in 1 M NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 10 mM Imidazole (Buffer A) supplemented with 1:1000 protease inhibitors (Calbiochem Set III, Merck 539134), 3 µg/mL Benzonase-MBP, 1 mM MgSO4, 0.5 mg/L lysozyme (Sigma L6876) and 0.5% v/v Triton-X100 before freezing at - 80°C; from this point onwards all steps took place at 4°C. Thawed pellets were spun down at 6780 RCF for 45´ and the supernatant was loaded into pre-equilibrated nickel-His GraviTrap (GE Healthcare) 1 mL columns. After drip-through, columns were washed with 10 CV of 1 M NaCl, 50 mM HEPES pH 7.5 and 1.5 CV of 0.4 M NaCl, 20 mM HEPES pH 7.5, 1 mM MgSO4, and 3 µg/mL Benzonase-MBP solution to digest remaining DNA for 30’. Contaminants were washed with 15 CV of 0.5 M NaCl, 5% glycerol, 50 mM HEPES pH 7.5 (Buffer B) supplemented with 30 mM imidazole before elution directly into a PD-10 column (GE Healthcare; equilibrated in Buffer B + 20 mM imidazole) with 2.5 mL of Buffer B + 500 mM imidazole. Proteins were eluted from the column with 3.5 mL of Buffer B + 20 mM imidazole before tag removal with 1:20 OD TEV-GST protease over 16 hours. [00176] Proteases and further contaminants were removed by recirculating the protein solutions over the same GraviTrap column used to purify initially (equilibrated in Buffer B+20 mM imidazole). For biotinylated proteins, streptavidin-XT resin was used instead to select biotinylated molecules only: after 30‘ of incubation in the resin, sample was allowed to drip through, washed with 30 CV of buffer A and 1 CV of buffer B + 100 mM D-biotin, and eluted by incubation in 3 CV of the same buffer for 2 h. Proteins were further purified by size exclusion chromatography (SEC) (Superdex S7510/300-0.35 mL/min or 16/600-1.2 mL/min flow rate) in either 10 mM HEPES pH 7.5 + 150 mM NaCl for biophysics work or cell-culture grade PBS for cell and animal work. Recombinant proteins were flash-frozen for storage, adding 1 mM TCEP in the case of reduced HMGB1 proteins. Recombinant CXCL12 purification [00177] Outer membranes of cells expressing DsbC-SUMO-CXCL12 were lysed by osmotic shock [90]. Pellets were resuspended at 40 g/L in 1 M sucrose, 0.2 M Tris-HCl pH 8.0, 1 mM EDTA, 1 mg/mL lysozyme, 2X cOmplete protease inhibitor set (COEDTAF-RO, Roche), 50 mM Imidazole and 3 µg/mL benzonase. This was stirred for 45‘ at room temperature before adding 4 volumes of ice-cold 18.2 mΩ water and mixed for a further 10‘ before adding 1 mM MgSO4. This was centrifuged for 1 h at 16000 RCF, 4°C, and the supernatant loaded at 10 mL/min into Ni-NTA Superflow columns (Qiagen, 30761) on an Äkta Xpress FPLC system; 1 column was used for every 6 L of cells. Proteins were eluted via an imidazole gradient (10- 25 mM over 10 CV, and 25-500 mM over 8 CV) in Buffer B, and 1:10 OD of Ulp-1 protease were added before dialysis in 100 volumes of 0.2 M NaCl, 20 mM HEPES pH 8.0 (Buffer Ac) overnight. On the next day, the protein was loaded into CaptoS columns (CaptoS ImpAct, GE 17-3717-47) at 2.3 mL/min and eluted in a gradient of 0.2-1.5 M NaCl in 20 mM HEPES pH 8.0 to separate cut CXCL12 from DsbC and Ulp-1. Proteins were further purified via SEC in the same way as HMGB1 and flash-frozen for storage. Removal of endotoxins [00178] Endotoxin was removed in all cases before size-exclusion chromatography via phase separation with Triton Tx-114 [91]. A 2% v/v of TX-114 was added to recombinant protein solutions, homogenized for 20´ with orbital shaking at 2000 RCF at 4°C, and separated for 5´ at 37°C before pelleting the detergent phase at 8000 RCF, 10´, 25°C. The supernatant was mixed with 5% w/v of SM-2 Biobeads (BioRad, 152-8920), cleaned with 2% TX-114 for 2 h and regenerated with 30 CV of methanol, 30 CV of endotoxin-free 18.2 mΩ water and 30 CV of endotoxin-free PBS. This was incubated for 4 h at room temperature to adsorb remaining Triton and PEG [92] before injection onto a sanitized SEC system (with 0.5 M NaOH contact over 12 h, followed by 0.2 M acetic acid/20% ethanol contact over 6 h and equilibration in cell- culture grade PBS) to fully remove leftover polymer contaminants whilst performing size exclusion. The absence of Triton and PEG was verified by lack of their respective charge state species in ESI/QTOF-MS mass spectrometry [93]. LPS content of the recombinant proteins was assessed via the LAL method (GenScript ToxinSensor L000350). Samples were approved for cell and animal use when they contained < 4 EU LPS/mg protein. Enzyme production [00179] TEV-GST protease (GST-fusion protein), Benzonase-MBP, and Ulp-1 protease were produced from transformants in storage at the SGC collection [86]; all had 200 µg/mL ampicillin resistance. TEV and Ulp-1 were purified as per the protocols described for HMGB1 with only one IMAC step, whereas Benzonase-MBP was purified from outer membrane lysates obtained as with CXCL12 and isolated with use of amylose resin (NEB, E0821) as per manufacturer protocols. In both cases, the resulting proteins were concentrated to 10 mg/mL in 50 mM HEPES pH 7.5, 0.3 M NaCl, 10% glycerol. GST-TEV protease and Ulp-1 were flash-frozen with liquid nitrogen and supplemented with 0.5 mM TCEP during purification; Benzonase-MBP was supplemented with 50% glycerol and 2 mM MgCl2 and stored at -20ºC. Peptide arrays [00180] For alanine scanning and the initial HMGB1 and CXCL12 arrays, membranes with the identified peptides in the original membrane, the full HMGB1 sequence, or CXCL12 (Uniprot P48061, excluding secretion signal) were printed by Dr. Sarah Picaud at the SGC upon request following published protocols [42]. The membranes were rehydrated at 20-25°C with 95% and 70% ethanol, equilibrated with PBST (PBS 1X + 0.05% Tween-20, 3x), and blocked with 10% BSA/PBST for 8 h.1 µM of the partner His-tagged protein construct was added (in PBS) and allowed to bind for 24 h at 4°C. Excess BSA and protein was removed with 3 washes in PBST; all washes lasted 1’ unless otherwise stated. To detect bound proteins, the membranes were treated with 1:3000 dilution of Qiagen anti-Penta His HRP conjugate (Qiagen 34460) and excess antibody removed with 3 washes in PBST for 20´. [00181] For the TLR-2, TLR-4 and RAGE peptide arrays, CelluSpot arrays were used instead (IntaVis, [94]) The membranes were baited with either CXCL12 or either of the three receptors in fusion with the CH domain of IgG (1530-TR, 9149-TR, or 1145-RG, BioTechne), as above, then probed with Anti-CXCL12 antibody (PA5-17238, Invitrogen). Bound IgG, whether by the Fc fusion proteins or anti CXCL12, was then detected with 0.25 µg/mL anti-human IgG CH2 domain antibody (NBP2-68464, custom conjugated to HRP). A series of peptides covering human IgG CH2 domain were used as controls (Uniprot P01857, 111-223) were used as controls: the highest intensity spot was used as 100% signal threshold. [00182] Bound antibody was in all cases via chemiluminescence (Pierce ECL substrate – 32109): the membrane was covered in substrate solution and placed between two clear plastic sheets before incremental imaging at 2´ intervals in a LAS-4000 camera. The intensity of the peptides in each membrane was measured in ImageJ and normalized to the controls and blank spots (100%-0%). Residues whose mutation to alanine resulted in higher intensity changes than those observed for alanine positions in the sequence were considered as significant contributors to CXCL12 binding. Biolayer interferometry (BLI) [00183] Pre-hydrated streptavidin Octet Biosensors (FortéBio 18-5019) were coated with 4 µM solutions of biotinylated HMGB1 proteins in 10 mM HEPES, pH 7.5, 150 mM NaCl (Base buffer-BB) plus, 0.5 mM TCEP (60´´ baseline, 60´´ binding). Nonspecific binding was minimized by incubation for 3´ in BB+ 1% BSA + 0.05% Tween-20 (Kinetics Buffer, KB) prior to kinetics assays. Interaction with CXCL12 was measured by performing incremental immersion of the sensors in solutions with increasing CXCL12 concentration (0-150 µM, in 1:2 dilutions) in KB (60´´ baseline, 500´´ association, 420´´ dissociation, 180´´ reduction in BB + 0.5 mM TCEP). An OctetRed 384 instrument was used for these experiments. Kinetics data were extracted with DataAnalysis 9.0 (FortéBio). Response at equilibrium REq was plotted against concentration in a Michaelis-Menten saturation plot to calculate kD/Bmax (saturation plot of concentration against rEq). Kinetic rates (association rate; kon and dissociation rate; koff) were derived from the direct measurements of each parameter from all the association and dissociation steps in the interferogram, and fitted to a horizontal line (mean) across all measurements. Data from each replicate run were pooled in the same manner to calculate the overall mean. For measuring RAGE binding kinetics to HMGB1, 15 µg/mL RAGE-Fc in PBS+ 0.1% BSA + 0.02% Tween-20 was immobilized on the surface of anti-IgG biosensors (AHC, 18-5060) over 30” and dipped in serial concentrations of each HMGB1 construct (60” baseline, 200” association/dissociation) and fitted in the same manner to derive kinetic parameters. Nuclear magnetic resonance (NMR) [00184] 15N-labelled recombinant HMGB1 constructs in 10 mM HEPES 150 mM NaCl pH 7.5 (identical buffering and ionic strength to BLI experiments) were supplemented with 5% v/v D2O, and pipetted with a glass Pasteur pipette into a 5 mm Shigeimi tube, sealed with paraffin. Final volumes were > 330 µL. CXCL12 was added in the same buffer and final volumes were adjusted to avoid modification of the referencing. Signal locking, tube shimming, and nuclei tuning were manually performed via Bruker TopSpin software. Water signal was suppressed by acquiring a 1H spectra with power level 1 (P1) = estimated pulse calibration (pulsecal). When a single peak was observed, a P1 value of 4 times the initial was used as a baseline and adjusted until a symmetric peak could be observed in the 1H spectrum. NMR experiments were performed after these calibration steps (1H-NMR, 15N-HSQC, 15N- NOESY-HSQC, 15N-TOCSY-HSQC Peaks in 15N-HSQC spectra were assigned based on published NMR tables for HMGB11-184 (BMRB 15418) and our own NOESY/TOCSY data for each construct analyzed. To measure CXCL12 binding, the chemical shift position and volume of identified peaks was tracked across different molar equivalences of CXCL12 (these are listed in the relevant image) via CCPNMR 3.0 Analyze´s Chemical Shift Tracking module. For peak intensity changes, the median intensity change in each set was considered as a baseline. Full chemical shift tables and experiment parameters are detained at the end of this section. Mass spectrometry [00185] For protein identification via MS/MS and tryptic digest, bands from SDS-PAGE gels were excised and submitted to the SGC open access MS platform and analysed by Dr. Rod Chalk, Dr. Tiago Moreira and Oktawia Borkowska, as published [93,95]. Data analysis (peptide mapping) to annotate protein identity was performed with the MASCOT search engine, against the Uniprot (reference protein sequences) and SGC (construct sequence) databases. Native ESI/MS experiments were performed by manual injection in volatile buffer (50 or 200 mM ammonium acetate, pH 6.5) into an ESI/QTOF instrument (Agilent Q-TOF 6545) at 360 µL/h. Signal was acquired for at least 10 counts (30 sec) once a steady ionic stream was observed in the total ion chromatogram. For denaturing experiments, samples were diluted to 1 mg/mL in 0.2% formic acid and injected via HPLC (Agilent 1100 HPLC) and eluted in a mobile phase of formic acid/methanol, as described [93]. Each continuous distribution of charge states was considered a distinct conformation; charge states (Z) were assigned according to the formula where mW= (mW/Z-proton mass )* Z. Surface areas were derived from the formulas proposed in the literature [96,97] which resulted in the formula ln(SASA)= ln(M/Z)*0.6897-4.063 for native MS and ln(SASA)= ln(M/Z)*0.9024-5.9013 for denatured samples. At least three independent injections were performed for all MS samples. All solutions in these experiments were made with HPLC water (electrochemical grade) and solvents. SEC surface area quantitation [00186] To correlate SEC chromatograms with surface area, standard sets with known structures (BioRad 1511901) were run through a Superdex 75 pg, 10/300 column used in these experiments. SASA for the proteins contained in these and the GE-supplied calibration curve standards were derived from published PDB structures (BSA, 3V03;Ovalbumin, 1JTI;Myoglobin, 2V1I;RNAseA, 1A5P;Aprotinin, 1NAG;Vitamin B12, 3BUL) and correlated with retention volume by nonlinear least squares fitting (SASA= 331.2*RV2- 1.19e4*RV+1.08e5). All experiments compared between HMGB1 samples were performed in the same buffer as native MS (200 mM ammonium acetate, pH 6.5); injections were performed at 1 mg/mL to avoid saturation of signal and all samples were eluted at 0.4 mL/min. RAGE binding ELISA assay [00187] 384-well protein-binding ELISA plates (Santa Cruz Biotechnology, sc-206072) were coated with 50 µL of 40 nM solutions in PBS (+ 0.5 mM TCEP for FR-HMGB1 constructs) of HMGB1 constructs for 24 h, at 4°C, including FL/DS HMGB1 full-length controls and blank, with 4 replicates of each. Nonspecific binding was blocked by incubation with 10% BSA in PBS for 2 h, at 20-25°C. A concentration range of RAGE-Fc chimera protein (BioTechne, 1145-RG; 0-640 nM in 1:4 dilutions) was added in 10% BSA/PBS and allowed to bind for 2 h, at 4°C. Bound FC chimera was detected by incubation with Anti-Human IgG HRP (Agilent Dako P021402-2) diluted 1:10000 in 1% BSA/PBS for 2 h, at 20-25°C. Between each of these 3 steps, the plate was washed with 100 µL PBST, three times. [00188] To detect bound antibody, 25 µL of TMB substrate (ThermoFisher N301) was added to each well; the reaction was allowed to develop in the dark until the FL-DS-HMGB1 control developed a clear concentration-dependent color gradient before stopping the reaction with 25 µL of 0.5 M H2SO4. OD 450 was measured as a readout (FluoStar OMEGA, BMG Labtech) and plotted as a saturation fit against 2x RAGE-Fc concentration (as the chimera is a RAGE dimer). TLR-4 and TLR-2-mediated NF-κB signaling reporter assay [00189] HEK-Dual cells (Invivogen) expressing human TLR-2 and CD14 or murine TLR-4, MD-2 and CD14 were maintained in DMEM (Gibco), supplemented with 10 % FBS (Gibco), 1 % L-Glutamine (Gibco), and 1 % penicillin/streptomycin (Gibco), in standard tissue culture conditions (37oC; 5% CO2). To determine if FR-HMGB1, DS-HMGB1 and dBB12L induces activation of TLR-4 and TLR-2 signaling, 104 TLR-4 and TLR-2 HEK-Dual cells were plated in triplicate into wells of a 96 well plate and stimulated with 10 µg/mL l HMGB1 and (X concentration) FSL-1 for TLR-2 and 10 ng/mL LPS for TLR-4.24 hours after stimulation, NF- κβ activity was determined my measuring the induced levels of secreted embryonic alkaline phosphatase (SEAP). Monocyte total NF-κB secretion assay [00190] Human monocytes (StemCell Technologies) were maintained in DMEM (Gibco), supplemented with 10% FBS (Gibco) in standard tissue culture conditions (37°C; 5% CO2). To determine if FR-HMGB1, DS-HMGB1 and dBB12L induces proinflammatory cytokine production, 105 human monocytes were plated in triplicate into wells of a 96-well plate and stimulated with 10 µg/mL HMGB1 and 50 ng/mL LPS or 10 ng/mL LTA. 24 h after stimulation, TNF levels were determined by Enzyme-linked immunosorbent assays (ELISA) (Abcam). Transcriptomic analysis [00191] Mice were treated systemically with an i.v. injection of 30 μg FR-HMGB1 in 50 µL of PBS vehicle, or PBS only control. Injury cell are from BaCl2 injured mice as described below. Alert cells are from the uninjured contralateral side of BaCl2 injured mice. Murine muscle stem cells (mMuSCs) were defined and freshly isolated according to previously reported protocols. Muscle cell suspensions were created by mincing thigh muscles and enzymatically digesting with collagenase 800 U/ml (Worthington-Biochem) and dispase 1 U/mL (Gibco). Thereafter, all suspensions were strained through 70 μm and 40 μm filters (Greiner Bio-One) and stained with respective antibodies. mMuSC, CD31-CD45-Sca-1- VCAM1+, were isolated by fluorescence activated cell sorting (FACS) using BD FACSAria III machine. RNA, extracted from freshly FACS isolated mMuSCs, was sent to RNA-seq analysis using Lexogen 3’kit library prep and sequenced using HiSeq400 (Illumina). FASTQ files were assessed using FASTQC followed by the generation of TPM values with kallisto v0.42.4. TPM values were summed to obtain gene-level expression values using tximport and differential expression analysis was undertaken with DeSEQ2. GO enrichment of differentially expressed genes was performed using the R package ‘clusterProfiler’[98] with a Benjamini-Hochberg multiple testing adjustment and a false-discovery rate cut-off of 0.1. Visualization was performed using the R packages ‘ggplot2’ and ‘igraph’. In vivo mouse muscle injury model [00192] C57BL/6 inbred mouse strain, females of 11-12 weeks of age were purchased from Charles River UK and housed in the local Biological Safety Unit (BSU) at the Kennedy Institute. Acclimatization period was 1-2 weeks. All protocols performed on live animals have been approved by the UK Home Office (PPL 30/3330 and PPL P12F5C2AF) as well as the local animal facility named persons and are registered under the appropriate project and personal licenses under ASPA regulations. All consumables were surgically certified; recombinant proteins were endotoxin-free. Surgeries were done in a clean environment separate from culling facilities. All animals were monitored for 6 h post-operatively, and daily for the following 3 days; monitoring was then transferred to the NVS/NACWO. [00193] Surgeries were performed as described previously ([8,13]). Animals were anesthetized by aerosolized 2% isoflurane, given analgesia, transferred to a warming pad and the right lower hindlimb was disinfected with povidone iodine and the tail with 70% ethanol if intravenous injection was performed.50 μL of 1.2% BaCl2 (Sigma) was injected into and along the length of the tibialis anterior (TA) muscle to induce cell death. Mice were euthanized and lower limbs removed at the times indicated, fixed in 4% paraformaldehyde (Santa Cruz Biotechnology) for 24 h. The TA muscles were dissected and further fixed for 24 h before being embedded in paraffin and sectioned. Sections (5 µm) were stained with hematoxylin and eosin to identify fibers with central nuclei and imaged with an Olympus BX51 using a 10x ocular/ 40x objective lens. The cross-sectional area (CSA) of the fibers from at least 4 images per mice was manually measured using the FIJI distribution of ImageJ2 software (NIH). Data were grouped per mice. Mice were injected with HMGB1 constructs (46 nM/kg, resuspended in PBS) or PBS vehicle control intramuscularly or intravenously at the time of injury or after injury for the optimal time administration of HMGB1 constructs after injury. In vivo mouse cardiac injury model [00194] C57BL/6 female mice were subject to surgery between 10-14 weeks old, with body weight between 25-30 g. All mice had either an intravenous injection of FR-HMGB1 (46 nM/kg, resuspended in PBS) or vehicle control just before surgery. Buprenorphine (buprenorphine hydrochloride; Vetergesic) was delivered as a 0.015 mg ml solution via intraperitoneal injection at 20 min before the procedure to provide analgesia. They were anaesthetized with 2.5% isoflurane and externally ventilated via an endotracheal tube. Cardiac injury was induced by permanent ligation of the left anterior descending coronary artery (LAD) via a thoracotomy. Experimenters were blind to treatment groups for subsequent cardiac cine- MRI and analysis. Mice were housed and maintained in a controlled environment. All surgical and pharmacological procedures were performed in accordance with the Animals (Scientific Procedures) Act 1986, UK. Cardiac cine-MRI and analysis [00195] Cardiac cine-MRI was performed post-LAD ligation at 7T using a Varian DDR system. Briefly, mice were anaesthetised with 2% isoflurane in O2, and positioned supine in a custom animal handling system with homeothermic control. Prospectively gated proton cardiac images were acquired with a partial Fourier accelerated spoiled gradient echo CINE sequence (TR 5.9 ms, TE 2.2 ms, 30 kHz bandwidth, 30º FA, approximately 20-30 frames gated to the R wave with a 4 ms postlabel delay; 20% partial acquisition; 4 averages) with a 72 mm volume transmit/4 channel surface receive coil (Rapid Biomedical GmbH) in order to acquire two and four chamber long-axis views and a short axis stack for functional quantification (128x128 matrix; 25.6 mm^2 FOV; 0.2 mm resolution in-plane). Non-acquired partial Fourier data was reconstructed via the method of projection onto convex sets prior to a simple, cartesian, DFT. Blinded image analysis was performed using ImageJ (NIH). Left ventricular mass, volumes and ejection fraction were calculated as previously described1. The relative infarct size was calculated from the average of the endocardial and epicardial circumferential lengths of the thinned, akinetic region of all slices, measured at diastole, and expressed as a percentage of the total myocardial surface [99]. Statistical analysis [00196] All calculations were performed with GraphPad Prism (v. 8.41). For kinetics experiments (BLI/RAGE ELISA), all fits were performed via nonlinear least squares. For the RAGE ELISA, as each concentration of RAGE was independent from the rest of the wells, all data were considered as one kinetics fit; in BLI however each sensor was considered an independent fit for the purposes of calculation. Comparisons between parameters were performed via the AUC method. Mouse muscle injury model data were analyzed as a nested ANOVA, where each sub-column comprises all the muscle CSA values for a given animal, and each group contains all the animals in order to separate biological variation from treatment effect. If the equal variance assumption could not be met in either case data were analyzed via a Kruskal-Wallis test; for nested ANOVA equal numbers of data from each animal would be randomly selected to avoid skewing. Any other data were analyzed via one-way ANOVA if the heteroskedasticity plot and Q/Q plot supported the equal variance assumption [100]; these were also verified by Spearman´s Test. For multivariate experiments (e.g. cardiac experiments) a two-factor ANOVA was used under the same assumptions (no dataset violated heteroskedasticity in this case). Post-hoc comparisons were weighted by Holms-Sidak correction (ANOVA family tests) or Dunn´s method (Kruskal-Wallis). The test selected in each case is noted under each figure legend. Significance legends: n.s; not significant, *; p < 0.033, **; p< 0.002, ***; p< 0.0002, ****; p< 0.0001. NMR chemical shift tables HMGB1-c028 (94-162, biotinylated) titration with CXCL12A-c021 at 0, 0.42, 0.82 and 1.42 molar equivalents, Figures 5A-5B. [00197] Due to protein amount limitations, titration was performed by sequential addition of CXCL12 to HMGB1 samples, resulting in sample dilution. As the calculations in the chemical shift tracking module in CCPNMR are independent of peak, this does not alter the results; the median change has been indicated in the volume comparisons. B
3D experiments (Day 1) These spectra are not shown due to their 3D nature; data available if requested. Sample is that of Baseline 1.
0.84 molar ratio CXCL12/HMG Box (Day 2)
1.43 molar ratio CXCL12/HMG Box (Day 2) E i t t 2D 1H15N HSQC
Baseline 2 (Day 2)
HMGB1-c038 (89-174, biotinylated) titration with CXCL12A-c021 at 0, 0.42, 0.82 and 1.42 molar equivalents, Figures 5A-5B [00198] Due to protein amount limitations, titration was performed by sequential addition of CXCL12 to HMGB1 samples, resulting in sample dilution. As the calculations in the chemical shift tracking module in CCPNMR are independent of peak, this does not alter the results; the median change has been indicated in the volume comparisons. BRUKER AVIII HD 500 5 MM CPTCI 1H13C/15N/D
3D experiments (Day 1-5) These spectra are not shown due to their 3D nature; data available if requested. Sample is that of Baseline 1. E i t t 3D 15N EDITED 1H1H TOCSYHSQC THROUGHSPACE
TL95LYSH 85094 1237507 195433 186325 167E07
REFERENCES 1. Weissman IL: Stem cells: Units of development, units of regeneration, and units in evolution. Cell 2000, 100:157–168. 2. Gratwohl A, Pasquini MC, Aljurf M, Atsuta Y, Baldomero H, Foeken L, Gratwohl M, Bouzas LF, Confer D, Frauendorfer K, et al.: One million haemopoietic stem-cell transplants: a retrospective observational study. Lancet Haematol 2015, 2:e91–e100. 3. Sharma R, Khristov V, Rising A, Jha BS, Dejene R, Hotaling N, Li Y, Stoddard J, Stankewicz C, Wan Q, et al.: Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs. Sci Transl Med 2019, 11:1–14. 4. Ojeh N, Pastar I, Tomic-Canic M, Stojadinovic O: Stem cells in skin regeneration, wound healing, and their clinical applications. Int J Mol Sci 2015, 16:25476–25501. 5. Domb AJ, Nudelman R: In vivo and in vitro elimination of aliphatic polyanhydrides. Biomaterials 1995, 16:319–23. 6. Trainor N, Pietak A, Smith T: Rethinking clinical delivery of adult stem cell therapies. Nat Biotechnol 2014, 32:729–735. 7. Zhang Y, Desai A, Yang SY, Bae KB, Antczak MI, Fink SP, Tiwari S, Willis JE, Williams NS, Dawson DM, et al.: Inhibition of the prostaglandin-degrading enzyme 15- PGDH potentiates tissue regeneration. Science 2015, 348. 8. Lee G*, Espirito Santo, A.I.*, Zwingenberger Cai L, Vogl T, Feldmann M, Horwood N, J.K-K C, Nanchahal J: HMGB1 accelerates the regeneration of multiple tissues by transitioning stem cells to G(Alert). Proc Natl Acad Sci U S A (*authors Contrib Equal to this Proj 2018, doi:10.1073/pnas.1802893115. 9. Oppenheim JJ, Yang D: Alarmins: Chemotactic activators of immune responses. Curr Opin Immunol 2005, 17:359–365. 10. Yang D, Rosa G De, Tewary P, Oppenheim JJ: Alarmins Link Neutrophils and Dendritic Cells. Trends Inmunol 2009, 30:531–537. 11. Belgrano FS, De Abreu Da Silva IC, Bastos De Oliveira FM, Fantappié MR, Mohana- Borges R: Role of the acidic tail of high mobility group protein B1 (HMGB1) in protein stability and DNA bending. PLoS One 2013, 8:1–12. 12. Joshi SR, Sarpong YC, Peterson RC, Scovell WM: Nucleosome dynamics: HMGB1 relaxes canonical nucleosome structure to facilitate estrogen receptor binding. Nucleic Acids Res 2012, 40:10161–10171. 13. Rodgers JT, King KY, Brett JO, Cromie MJ, Charville GW, Maguire KK, Brunson C, Mastey N, Liu L, Tsai C-R, et al.: mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert). Nature 2014, 509:393–6. 14. Venereau E, Casalgrandi M, Schiraldi M, Antoine DJ, Cattaneo A, De Marchis F, Liu J, Antonelli A, Preti A, Raeli L, et al.: Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release. J Exp Med 2012, 209:1519–28. 15. Cecchinato V, D’Agostino G, Raeli L, Nerviani A, Schiraldi M, Danelon G, Manzo A, Thelen M, Ciurea A, Bianchi ME, et al.: Redox-Mediated Mechanisms Fuel Monocyte Responses to CXCL12/HMGB1 in Active Rheumatoid Arthritis. Front Immunol 2018, 9:1–12. 16. Ferrara M, Chialli G, Ferreira LM, Ruggieri E, Careccia G, Preti A, Piccirillo R, Bianchi ME, Sitia G, Venereau E: Oxidation of HMGB1 Is a Dynamically Regulated Process in Physiological and Pathological Conditions. Front Immunol 2020, 11:1–13. 17. Bonaldi T, Talamo F, Scaffidi P, Ferrera D, Porto A, Bachi A, Rubartelli A, Agresti A, Bianchi ME: Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. EMBO J 2003, 22:5551–5560. 18. Kim YH, Kwak MS, Park JB, Lee SA, Choi JE, Cho HS, Shin JS: N-linked glycosylation plays a crucial role in the secretion of HMGB1. J Cell Sci 2016, 129:29–38. 19. Venereau E, Schiraldi M, Uguccioni M, Bianchi ME: HMGB1 and leukocyte migration during trauma and sterile inflammation. Mol Immunol 2013, 55:76–82. 20. Stark K, Philippi V, Stockhausen S, Busse J, Antonelli A, Miller M, Schubert I, Hoseinpour P, Chandraratne S, Von Bruhl ML, et al.: Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice - SM. Blood 2016, 128:2435–2449. 21. Teng W, He X, Shan L, Fan C, Peng S, Wang X, Shan Z, Wang W, Liu X, Liu S, et al.: Glycyrrhizin, a Direct HMGB1 Antagonist, Ameliorates Inflammatory Infiltration in a Model of Autoimmune Thyroiditis via Inhibition of TLR2-HMGB1 Signaling. Thyroid 2017, 27:722–731. 22. Vogel S, Bodenstein R, Chen Q, Feil S, Feil R, Rheinlaender J, Schäffer TE, Bohn E, Frick JS, Borst O, et al.: Platelet-derived HMGB1 is a critical mediator of thrombosis. J Clin Invest 2015, 125:4638–4654. 23. Maugeri N, Campana L, Gavina M, Covino C, De Metrio M, Panciroli C, Maiuri L, Maseri A, D’Angelo A, Bianchi ME, et al.: Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps. J Thromb Haemost 2014, 12:2074–2088. 24. Ahrens I, Agrotis A, Topcic D, Bassler N, Chen YC, Bobik A, Bode C, Peter K: HMGB1 binds to activated platelets via platelet-expressed receptor for advanced glycation end products (RAGE) and is highly expressed in platelet rich coronary artery thrombi. Thromb Haemost 2015, 14:994–1003. 25. van Beijnum JR, Buurman WA, Griffioen AW: Convergence and amplification of toll- like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1). Angiogenesis 2008, 11:91–99. 26. Lotze MT, Tracey KJ: High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 2005, 5:331–42. 27. Sakaguchi M, Murata H, Yamamoto K, Ono T, Sakaguchi Y, Motoyama A, Hibino T, Kataoka K, Huh N: TIRAP, an adaptor protein for TLR2/4, transduces a signal from RAGE phosphorylated upon ligand binding. PLoS One 2011, 6:e23132. 28. He M, Bianchi ME, Coleman TR, Tracey KJ, Al-abed Y, Unit CD, Biology C: Exploring the biological functional mechanism of the HMGB1 / TLR4 / MD-2 complex by surface plasmon resonance. [date unknown]. 29. Yang H, Hreggvidsdottir HS, Palmblad K, Wang H, Ochani M, Li J, Lu B, Chavan S, Rosas-Ballina M, Al-Abed Y, et al.: A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release. Proc Natl Acad Sci U S A 2010, 107:11942–7. 30. Tirone M, Tran NL, Ceriotti C, Gorzanelli A, Canepari M, Bottinelli R, Raucci A, Maggio S Di, Santiago C, Mellado M, et al.: High mobility group box 1 orchestrates tissue regeneration via CXCR4. J Exp Med 2018, doi:10.1084/jem.20160217. 31. Di Maggio S, Milano G, De Marchis F, D’Ambrosio A, Bertolotti M, Palacios BS, Badi I, Sommariva E, Pompilio G, Capogrossi MC, et al.: Non-oxidizable HMGB1 induces cardiac fibroblasts migration via CXCR4 in a CXCL12-independent manner and worsens tissue remodeling after myocardial infarction. Biochim Biophys Acta - Mol Basis Dis 2017, 1863:2693–2704. 32. Kwak MS, Lim M, Lee YJ, Lee HS, Kim YH, Ho Youn J, Choi JE, Shin J-S: HMGB1 Binds to Lipoteichoic Acid and Enhances TNF-α and IL-6 Production through HMGB1- Mediated Transfer of Lipoteichoic Acid to CD14 and TLR2. J Innate Immun 2015, 7:405–416. 33. Aucott H, Sowinska A, Harris HE, Lundback P: Ligation of free HMGB1 to TLR2 in the absence of ligand is negatively regulated by the C-terminal tail domain. Mol Med 2018, 24:19. 34. Das N, Dewan V, Grace PM, Gunn RJ, Tamura R, Tzarum N, Watkins LR, Wilson IA, Yin H: HMGB1 Activates Proinflammatory Signaling via TLR5 Leading to Allodynia. Cell Rep 2016, 17:1128–1140. 35. Yu M, Wang H, Ding A, Golenbock DT, Latz E, Czura CJ, Fenton MJ, Tracey KJ, Yang H: HMGB1 signals through toll-like receptor (TLR) 4 and TLR2. Shock 2006, 26:174– 179. 36. Stark K, Philippi V, Stockhausen S, Busse J, Antonelli A, Miller M, Schubert I, Hoseinpour P, Chandraratne S, Von Bruhl ML, et al.: Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice. Blood 2016, 128:2435–2449. 37. Huttunen HJ, Fages C, Kuja-Panula J, Ridley AJ, Rauvala H: Receptor for advanced glycation end products-binding COOH-terminal motif of amphoterin inhibits invasive migration and metastasis. Cancer Res 2002, 62:4805–4811. 38. Arumugam T, Ramachandran V, Gomez SB, Schmidt AM, Logsdon CD: S100P-derived RAGE antagonistic peptide reduces tumor growth and metastasis. Clin Cancer Res 2012, 18:4356–4364. 39. LeBlanc PM, Doggett TA, Choi J, Hancock MA, Durocher Y, Frank F, Nagar B, Ferguson TA, Saleh M: An immunogenic peptide in the A-box of HMGB1 protein reverses apoptosis-induced tolerance through RAGE receptor. J Biol Chem 2014, 289:7777–7786. 40. Anggayasti WL, Ogino K, Yamamoto E, Helmerhorst E, Yasuoka K, Mancera RL: The acidic tail of HMGB1 regulates its secondary structure and conformational flexibility: A circular dichroism and molecular dynamics simulation study. Comput Struct Biotechnol J 2020, 18:1160–1172. 41. Rauvala H, Rouhiainen A: Physiological and pathophysiological outcomes of the interactions of HMGB1 with cell surface receptors. Biochim Biophys Acta - Gene Regul Mech 2010, 1799:164–170. 42. Nady N, Min J, Kareta MS, Chédin F, Arrowsmith CH: A SPOT on the chromatin landscape? Histone peptide arrays as a tool for epigenetic research. Trends Biochem Sci 2008, 33:305–313. 43. Mollica L, De Marchis F, Spitaleri A, Dallacosta C, Pennacchini D, Zamai M, Agresti A, Trisciuoglio L, Musco G, Bianchi ME: Glycyrrhizin binds to high-mobility group box 1 protein and inhibits its cytokine activities. Chem Biol 2007, 14:431–41. 44. Schiraldi M, Raucci A, Muñoz LM, Livoti E, Celona B, Venereau E, Apuzzo T, De Marchis F, Pedotti M, Bachi A, et al.: {HMGB1} promotes recruitment of inflammatory cells to damaged tissues by forming a complex with {CXCL12} and signaling via {CXCR4.}. J Exp Med 2012, 209:551–563. 45. De Leo F, Quilici G, Tirone M, Mannella V, De Marchis F, Preti A, Gori A, Casalgrandi M, Mezzapelle R, Bianchi M, et al.: Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment. bioRxiv 2019, doi:10.1101/563890. 46. Fassi EMA, Sgrignani J, D’Agostino G, Cecchinato V, Garofalo M, Grazioso G, Uguccioni M, Cavalli A: Oxidation State Dependent Conformational Changes of HMGB1 Regulate the Formation of the CXCL12/HMGB1 Heterocomplex. Comput Struct Biotechnol J 2019, 17:886–894. 47. Bianchi ME, Falciola L, Ferrari S, Lilley DM: The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins. Embo J 1992, 11:1055–1063. 48. Li J, Wang H, Mason JM, Levine J, Yu M, Ulloa L, Czura CJ, Tracey KJ, Yang H: Recombinant HMGB1 with cytokine-stimulating activity. J Immunol Methods 2004, 289:211–223. 49. Girard JP: A Direct Inhibitor of HMGB1 Cytokine. Chem Biol 2007, 14:345–347. 50. Wang J, Tochio N, Takeuchi A, Uewaki J ichi, Kobayashi N, Tate S ichi: Redox- sensitive structural change in the A-domain of HMGB1 and its implication for the binding to cisplatin modified DNA. Biochem Biophys Res Commun 2013, 441:701–706. 51. Youn JH, Kwak MS, Wu J, Kim ES, Ji Y, Min HJ, Yoo JH, Choi JE, Cho HS, Shin JS: Identification of lipopolysaccharide-binding peptide regions within HMGB1 and their effects on subclinical endotoxemia in a mouse model. Eur J Immunol 2011, 41:2753– 2762. 52. Lee S, Piao C, Kim G, Kim JY, Choi E, Lee M: Production and application of HMGB1 derived recombinant RAGE-antagonist peptide for anti-inflammatory therapy in acute lung injury. Eur J Pharm Sci 2018, 114:275–284. 53. De Leo F, Quilici G, Tirone M, Mannella V, De Marchis F, Preti A, Gori A, Casalgrandi M, Mezzapelle R, Bianchi M, et al.: Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment - SM. bioRxiv 2019, doi:10.1101/563890. 54. Ottestad W, Rognes IN, Pischke SE, Mollnes TE, Andersson U, Eken T: Biphasic Release of the Alarmin High Mobility Group Box 1 Protein Early After Trauma Predicts Poor Clinical Outcome. Crit Care Med 2019, 47:e614–e622. 55. Yang H, Wang H, Wang Y, Addorisio M, Li J, Postiglione MJ, Chavan SS, Al-Abed Y, Antoine DJ, Andersson U, et al.: The haptoglobin beta subunit sequesters HMGB1 toxicity in sterile and infectious inflammation. J Intern Med 2017, 282:76–93. 56. Yang H, Wang H, Levine YA, Gunasekaran MK, Wang Y, Addorisio M, Zhu S, Li W, Li J, de Kleijn DP V, et al.: Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes. JCI insight 2016, 1:1–14. 57. Smart N, Bollini S, Dubé KN, Vieira JM, Zhou B, Davidson S, Yellon D, Riegler J, Price AN, Lythgoe MF, et al.: De novo cardiomyocytes from within the activated adult heart after injury. Nature 2011, 474:640–644. 58. Sadek H, Olson EN: Toward the Goal of Human Heart Regeneration. Cell Stem Cell 2020, 26:7–16. 59. Brooks M: Stem cell research: time for a dose of realism. BMJ 2017, 356. 60. Vagnozzi RJ, Maillet M, Sargent MA, Khalil H, Johansen AKZ, Schwanekamp JA, York AJ, Huang V, Nahrendorf M, Sadayappan S, et al.: An acute immune response underlies the benefit of cardiac stem cell therapy. Nature 2020, 577:405–409. 61. Forbes SJ, Rosenthal N: Preparing the ground for tissue regeneration: From mechanism to therapy. Nat Med 2014, 20:857–869. 62. Lane SW, Williams DA, Watt FM: Modulating the stem cell niche for tissue regeneration. Nat Biotechnol 2014, 32:795–803. 63. Antczak MI, Zhang Y, Wang C, Doran J, Naidoo J, Voruganti S, Williams NS, Markowitz SD, Ready JM: Inhibitors of 15-Prostaglandin Dehydrogenase To Potentiate Tissue Repair. J Med Chem 2017, 60:3979–4001. 64. Ren X, Zhao M, Lash B, Martino MM, Julier Z: Growth Factor Engineering Strategies for Regenerative Medicine Applications. Front Bioeng Biotechnol 2020, 7:1–9. 65. Yamakawa S, Hayashida K: Advances in surgical applications of growth factors for wound healing. Burn Trauma 2019, 7:1–13. 66. Mitchell AC, Briquez PS, Hubbell JA, Cochran JR: Engineering growth factors for regenerative medicine applications. Acta Biomater 2016, 30:1–12. 67. Hori O, Brett J, Slattery T, Cao R, Zhang J, Chen JX, Stern D, Schmidt AM: The Receptor for Advanced Glycation End Products (RAGE) Is a Cellular Binding Site for Amphoterin. J Biol Chem 1995, 270:25752–25761. 68. Ibrahim ZA, Armour CL, Phipps S, Sukkar MB: RAGE and TLRs: Relatives, friends or neighbours? Mol Immunol 2013, 56:739–744. 69. Festoff BW, Sajja RK, van Dreden P, Cucullo L: HMGB1 and thrombin mediate the blood-brain barrier dysfunction acting as biomarkers of neuroinflammation and progression to neurodegeneration in Alzheimer’s disease. J Neuroinflammation 2016, 13:1–12. 70. Hreggvidsdóttir HS, Lundberg AM, Aveberger A-C, Klevenvall L, Andersson U, Harris HE: High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor. Mol Med 2012, 18:224–30. 71. Livoti E: Experimentally validated computational docking to characterize protein- protein interactions. [date unknown]. 72. Fassi EMA, Sgrignani J, D’Agostino G, Cecchinato V, Garfalo M, Grazioso G, Uguccioni M, Cavalli A: Oxidation state dependent conformational changes of HMGB1 regulates the formation of the CXCL12/HMGB1 heterocomplex. bioRxiv Biochem 2019, doi:10.1101/555946. 73. Drury LJ, Ziarek JJ, Gravel S, Veldkamp CT, Takekoshi T, Hwang ST, Heveker N, Volkman BF, Dwinell MB: Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways. Proc Natl Acad Sci U S A 2011, 108:17655–17660. 74. Chang S, Li Y, Yuan F, Qu M, Song Y, Zhang Z, Yang GY, Wang Y: Monomeric CXCL12 outperforms its dimeric and wild type variants in the promotion of human endothelial progenitor cells’ function. Biochem Biophys Res Commun 2017, 488:303– 310. 75. Ziarek JJ, Kleist AB, London N, Raveh B, Montpas N, Bonneterre J, St-onge G, Dicosmo-ponticello CJ, Koplinski CA, Roy I, et al.: Structural basis for chemokine recognition by a G protein – coupled receptor and implications for receptor activation. Sci Signal 2017, 5756. 76. Nady N, Min J, Kareta MS, Chédin F, Arrowsmith CH: Supplementary Materials: A SPOT on the chromatin landscape? Histone peptide arrays as a tool for epigenetic research. Trends Biochem Sci 2008, 33:305–313. 77. Veldkamp CT, Peterson FC, Pelzek AJ, Volkman BF: The monomer – dimer equilibrium of stromal cell-derived factor-1 (CXCL12) is altered by pH, phosphate, sulfate, and heparin. Protein Sci 2005, 1:1071–1081. 78. Akira S: Toll-like Receptors and Innate Immunity. In Edited by Dixon FJBT-A in I. Academic Press; 2001:1–56. 79. Xu D, Young J, Song D, Esko JD: Heparan sulfate is essential for high mobility group protein 1 (HMGB1) signaling by the receptor for advanced glycation end products (RAGE). J Biol Chem 2011, 286:41736–41744. 80. Gurevich DB, Nguyen PD, Siegel AL, Ehrlich O V., Sonntag C, Phan JMN, Berger S, Ratnayake D, Hersey L, Berger J, et al.: Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo. Science 2016, 353. 81. Benjamin EJ, Muntner P, Alonso A, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Chang AR, Cheng S, Das SR, et al.: Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association.2019. 82. Limana F, Esposito G, D’Arcangelo D, Di Carlo A, Romani S, Melillo G, Mangoni A, Bertolami C, Pompilio G, Germani A, et al.: HMGB1 attenuates cardiac remodelling in the failing heart via enhanced cardiac regeneration and miR-206-mediated inhibition of TIMP-3. PLoS One 2011, 6:1–11. 83. Foglio E, Puddighinu G, Germani A, Russo MA, Limana F: HMGB1 Inhibits Apoptosis Following MI and Induces Autophagy via mTORC1 Inhibition. J Cell Physiol 2017, 232:1135–1143. 84. Bauzá M del R, Giménez CS, Locatelli P, De Lorenzi A, Hnatiuk A, Capogrossi MC, Crottogini A, Cuniberti L, Olea FD: High-dose intramyocardial HMGB1 induces long- term cardioprotection in sheep with myocardial infarction. Drug Deliv Transl Res 2019, 9:935–944. 85. Chen X, Zaro JL, Shen WC: Fusion protein linkers: Property, design and functionality. Adv Drug Deliv Rev 2013, 65:1357–1369. 86. Savitsky P, Bray J, Cooper CDO, Marsden BD, Mahajan P, Burgess-Brown NA, Gileadi O: High-throughput production of human proteins for crystallization: The SGC experience. J Struct Biol 2010, 172:3–13. 87. Yang OO, Swanberg SL, Lu Z, Dziejman M, McCoy J, Luster AD, Walker BD, Herrmann SH: Enhanced inhibition of human immunodeficiency virus type 1 by Met- stromal-derived factor 1beta correlates with down-modulation of CXCR4. J Virol 1999, 73:4582–4589. 88. Crump MP, Gong JH, Loetscher P, Rajarathnam K, Amara A, Arenzana-Seisdedos F, Virelizier JL, Baggiolini M, Sykes BD, Clark-Lewis I: Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J 1997, 16:6996–7007. 89. Turchetto J, Sequeira AF, Ramond L, Peysson F, Brás JLA, Saez NJ, Duhoo Y, Blémont M, Guerreiro CIPD, Quinton L, et al.: High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery. Microb Cell Fact 2017, 16:6. 90. Thein M, Sauer G, Paramasivam N, Grin I, Linke D: Efficient subfractionation of gram- negative bacteria for proteomics studies. J Proteome Res 2010, 9:6135–6147. 91. Aida Y, Pabst MJ: Removal of endotoxin from protein solutions by phase separation using triton X-114. J Immunol Methods 1990, 132:191–195. 92. Teodorowicz M, Perdijk O, Verhoek I, Govers C, Savelkoul HFJ, Tang Y, Wichers H, Broersen K: Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins. PLoS One 2017, 12:e0173778. 93. Chalk R: Mass Spectrometric Analysis of Proteins. Humana Press, New York, NY; 2017:373–395. 94. Wu C, Li SS-C: CelluSpotsTM: A Reproducible Means of Making Peptide Arrays for the Determination of SH2 Domain Binding Specificity BT - Peptide Microarrays: Methods and Protocols. In Edited by Cretich M, Chiari M. Humana Press; 2009:197–202. 95. Chalk R, Berridge G, Shrestha L, Strain-Damerell C, Mahajan P, Yue W, Gileadi O, Burgess-Brown N: High-Throughput Mass Spectrometry Applied to Structural Genomics. Chromatography 2014, 1:159–175. 96. Kaltashow IA, Mohimen A: Electrospray ionization mass spectrometry can provide estimates of protein surface areas in solution. Anal Chem 2005, 77:5370–5379. 97. Testa L, Brocca S, Grandori R: Charge-surface correlation in electrospray ionization of folded and unfolded proteins. Anal Chem 2011, 83:6459–6463. 98. Yu G, Wang LG, Han Y, He QY: ClusterProfiler: An R package for comparing biological themes among gene clusters. Omi A J Integr Biol 2012, doi:10.1089/omi.2011.0118. 99. Carr CA, Stuckey DJ, Tatton L, Tyler DJ, Hale SJM, Sweeney D, Schneider JE, Martin- Rendon E, Radda GK, Harding SE, et al.: Bone marrow-derived stromal cells home to and remain in the infarcted rat heart but fail to improve function: An in vivo cine-MRI study. Am J Physiol - Hear Circ Physiol 2008, 295. 100. Lee S, Lee DK: Multiple Comparison Test and Its Imitations What is the proper way to apply the multiple comparison test? KJA. Korean J Anesth 2018, doi:10.4097/kja.d.18.00242. 101. E. J. Benjamin, P. Muntner, A. Alonso, M. S. Bittencourt, C. W. Callaway, A. P. Carson, A. M. Chamberlain, A. R. Chang, S. Cheng, S. R. Das, F. N. Delling, L. Djousse, M. S. V. Elkind, J. F. Ferguson, M. Fornage, L. C. Jordan, S. S. Khan, B. M. Kissela, K. L. Knutson, T. W. Kwan, D. T. Lackland, T. T. Lewis, J. H. Lichtman, C. T. Longenecker, M. S. Loop, P. L. Lutsey, S. S. Martin, K. Matsushita, A. E. Moran, M. E. Mussolino, M. O'Flaherty, A. Pandey, A. M. Perak, W. D. Rosamond, G. A. Roth, U. K. A. Sampson, G. M. Satou, E. B. Schroeder, S. H. Shah, N. L. Spartano, A. Stokes, D. L. Tirschwell, C. W. Tsao, M. P. Turakhia, L. B. VanWagner, J. T. Wilkins, S. S. Wong, S. S. Virani, E. American Heart Association Council on, C. Prevention Statistics, S. Stroke Statistics, Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. Circulation 139, e56-e528 (2019). 102. G. B. D. DALYs, H. Collaborators, Global, regional, and national disability-adjusted life-years (DALYs) for 359 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 392, 1859-1922 (2018). 103. BritishHeartFoundation, bhf.org.uk/what-we-do/our-research/heart-statistics/heart- statistics-publications/cardiovascular-disease-statistics-2019, (2020). 104. T. J. Cahill, R. K. Kharbanda, Heart failure after myocardial infarction in the era of primary percutaneous coronary intervention: Mechanisms, incidence and identification of patients at risk. World J Cardiol 9, 407-415 (2017). 105. L. Qian, Y. Huang, C. I. Spencer, A. Foley, V. Vedantham, L. Liu, S. J. Conway, J. D. Fu, D. Srivastava, In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593-598 (2012). 106. M. Xin, Y. Kim, L. B. Sutherland, M. Murakami, X. Qi, J. McAnally, E. R. Porrello, A. I. Mahmoud, W. Tan, J. M. Shelton, J. A. Richardson, H. A. Sadek, R. Bassel-Duby, E. N. Olson, Hippo pathway effector Yap promotes cardiac regeneration. Proc Natl Acad Sci U S A 110, 13839-13844 (2013). 107. J. P. Leach, T. Heallen, M. Zhang, M. Rahmani, Y. Morikawa, M. C. Hill, A. Segura, J. T. Willerson, J. F. Martin, Hippo pathway deficiency reverses systolic heart failure after infarction. Nature 550, 260-264 (2017). 108. T. M. A. Mohamed, Y. S. Ang, E. Radzinsky, P. Zhou, Y. Huang, A. Elfenbein, A. Foley, S. Magnitsky, D. Srivastava, Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration. Cell 173, 104-116 e112 (2018). 109. S. Hashmi, S. Al-Salam, Acute myocardial infarction and myocardial ischemia- reperfusion injury: a comparison. Int J Clin Exp Pathol 8, 8786-8796 (2015). 110. K. Stark, V. Philippi, S. Stockhausen, J. Busse, A. Antonelli, M. Miller, I. Schubert, P. Hoseinpour, S. Chandraratne, M. L. von Bruhl, F. Gaertner, M. Lorenz, A. Agresti, R. Coletti, D. J. Antoine, R. Heermann, K. Jung, S. Reese, I. Laitinen, M. Schwaiger, A. Walch, M. Sperandio, P. P. Nawroth, C. Reinhardt, S. Jackel, M. E. Bianchi, S. Massberg, Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice. Blood 128, 2435-2449 (2016). 111. S. Di Maggio, G. Milano, F. De Marchis, A. D'Ambrosio, M. Bertolotti, B. S. Palacios, I. Badi, E. Sommariva, G. Pompilio, M. C. Capogrossi, A. Raucci, Non-oxidizable HMGB1 induces cardiac fibroblasts migration via CXCR4 in a CXCL12-independent manner and worsens tissue remodeling after myocardial infarction. Biochim Biophys Acta 1863, 2693-2704 (2017). 112. J. Mersmann, F. Iskandar, K. Latsch, K. Habeck, V. Sprunck, R. Zimmermann, R. R. Schumann, K. Zacharowski, A. Koch, Attenuation of myocardial injury by HMGB1 blockade during ischemia/reperfusion is toll-like receptor 2-dependent. Mediators Inflamm 2013, 174168 (2013). 113. C. Monaco, S. M. Gregan, T. J. Navin, B. M. Foxwell, A. H. Davies, M. Feldmann, Toll- like receptor-2 mediates inflammation and matrix degradation in human atherosclerosis. Circulation 120, 2462-2469 (2009). 114. J. Oyama, C. Blais, Jr., X. Liu, M. Pu, L. Kobzik, R. A. Kelly, T. Bourcier, Reduced myocardial ischemia-reperfusion injury in toll-like receptor 4-deficient mice. Circulation 109, 784-789 (2004). 115. K. R. Chien, J. Frisen, R. Fritsche-Danielson, D. A. Melton, C. E. Murry, I. L. Weissman, Regenerating the field of cardiovascular cell therapy. Nat Biotechnol 37, 232-237 (2019). 116. R. J. Vagnozzi, M. Maillet, M. A. Sargent, H. Khalil, A. K. Z. Johansen, J. A. Schwanekamp, A. J. York, V. Huang, M. Nahrendorf, S. Sadayappan, J. D. Molkentin, An acute immune response underlies the benefit of cardiac stem cell therapy. Nature 577, 405-409 (2020). 117. C. E. Murry, W. R. MacLellan, Stem cells and the heart-the road ahead. Science 367, 854-855 (2020). 118. S. Bollini, N. Smart, P. R. Riley, Resident cardiac progenitor cells: at the heart of regeneration. J Mol Cell Cardiol 50, 296-303 (2011). 119. P. K. Nguyen, E. Neofytou, J. W. Rhee, J. C. Wu, Potential Strategies to Address the Major Clinical Barriers Facing Stem Cell Regenerative Therapy for Cardiovascular Disease: A Review. JAMA Cardiol 1, 953-962 (2016). 120. T. J. Cahill, R. P. Choudhury, P. R. Riley, Heart regeneration and repair after myocardial infarction: translational opportunities for novel therapeutics. Nat Rev Drug Discov 16, 699-717 (2017). 121. H. Sadek, E. N. Olson, Toward the Goal of Human Heart Regeneration. Cell Stem Cell 26, 7-16 (2020). 122. N. Smart, S. Bollini, K. N. Dube, J. M. Vieira, B. Zhou, S. Davidson, D. Yellon, J. Riegler, A. N. Price, M. F. Lythgoe, W. T. Pu, P. R. Riley, De novo cardiomyocytes from within the activated adult heart after injury. Nature 474, 640-644 (2011). 123. A. I. Mahmoud, F. Kocabas, S. A. Muralidhar, W. Kimura, A. S. Koura, S. Thet, E. R. Porrello, H. A. Sadek, Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature 497, 249-253 (2013). 124. K. Bersell, S. Arab, B. Haring, B. Kuhn, Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury. Cell 138, 257-270 (2009). 125. S. Koudstaal, M. M. Bastings, D. A. Feyen, C. D. Waring, F. J. van Slochteren, P. Y. Dankers, D. Torella, J. P. Sluijter, B. Nadal-Ginard, P. A. Doevendans, G. M. Ellison, S. A. Chamuleau, Sustained delivery of insulin-like growth factor-1/hepatocyte growth factor stimulates endogenous cardiac repair in the chronic infarcted pig heart. J Cardiovasc Transl Res 7, 232-241 (2014). 126. K. Wei, V. Serpooshan, C. Hurtado, M. Diez-Cunado, M. Zhao, S. Maruyama, W. Zhu, G. Fajardo, M. Noseda, K. Nakamura, X. Tian, Q. Liu, A. Wang, Y. Matsuura, P. Bushway, W. Cai, A. Savchenko, M. Mahmoudi, M. D. Schneider, M. J. van den Hoff, M. J. Butte, P. C. Yang, K. Walsh, B. Zhou, D. Bernstein, M. Mercola, P. Ruiz-Lozano, Epicardial FSTL1 reconstitution regenerates the adult mammalian heart. Nature 525, 479-485 (2015). 127. K. Gabisonia, G. Prosdocimo, G. D. Aquaro, L. Carlucci, L. Zentilin, I. Secco, H. Ali, L. Braga, N. Gorgodze, F. Bernini, S. Burchielli, C. Collesi, L. Zandona, G. Sinagra, M. Piacenti, S. Zacchigna, R. Bussani, F. A. Recchia, M. Giacca, MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs. Nature 569, 418-422 (2019). 128. A. Vujic, N. Natarajan, R. T. Lee, Molecular mechanisms of heart regeneration. Semin Cell Dev Biol, (2019). 129. J. M. Vieira, S. Norman, C. Villa Del Campo, T. J. Cahill, D. N. Barnette, M. Gunadasa- Rohling, L. A. Johnson, D. R. Greaves, C. A. Carr, D. G. Jackson, P. R. Riley, The cardiac lymphatic system stimulates resolution of inflammation following myocardial infarction. J Clin Invest 128, 3402-3412 (2018). 130. L. R. Fiedler, K. Chapman, M. Xie, E. Maifoshie, M. Jenkins, P. A. Golforoush, M. Bellahcene, M. Noseda, D. Faust, A. Jarvis, G. Newton, M. A. Paiva, M. Harada, D. J. Stuckey, W. Song, J. Habib, P. Narasimhan, R. Aqil, D. Sanmugalingam, R. Yan, L. Pavanello, M. Sano, S. C. Wang, R. D. Sampson, S. Kanayaganam, G. E. Taffet, L. H. Michael, M. L. Entman, T. H. Tan, S. E. Harding, C. M. R. Low, C. Tralau-Stewart, T. Perrior, M. D. Schneider, MAP4K4 Inhibition Promotes Survival of Human Stem Cell- Derived Cardiomyocytes and Reduces Infarct Size In Vivo. Cell Stem Cell 24, 579-591 e512 (2019). 131. H. Aghajanian, T. Kimura, J. G. Rurik, A. S. Hancock, M. S. Leibowitz, L. Li, J. Scholler, J. Monslow, A. Lo, W. Han, T. Wang, K. Bedi, M. P. Morley, R. A. Linares Saldana, N. A. Bolar, K. McDaid, C. A. Assenmacher, C. L. Smith, D. Wirth, C. H. June, K. B. Margulies, R. Jain, E. Pure, S. M. Albelda, J. A. Epstein, Targeting cardiac fibrosis with engineered T cells. Nature 573, 430-433 (2019). 132. N. Trainor, A. Pietak, T. Smith, Rethinking clinical delivery of adult stem cell therapies. Nat Biotechnol 32, 729-735 (2014). 133. M. J. Foglia, K. D. Poss, Building and re-building the heart by cardiomyocyte proliferation. Development 143, 729-740 (2016). 134. S. Oozawa, S. Mori, T. Kanke, H. Takahashi, K. Liu, Y. Tomono, M. Asanuma, I. Miyazaki, M. Nishibori, S. Sano, Effects of HMGB1 on ischemia-reperfusion injury in the rat heart. Circ J 72, 1178-1184 (2008). 135. T. Kitahara, Y. Takeishi, M. Harada, T. Niizeki, S. Suzuki, T. Sasaki, M. Ishino, O. Bilim, O. Nakajima, I. Kubota, High-mobility group box 1 restores cardiac function after myocardial infarction in transgenic mice. Cardiovasc Res 80, 40-46 (2008). 136. Y. Nakamura, S. Suzuki, T. Shimizu, M. Miyata, T. Shishido, K. Ikeda, S. Saitoh, I. Kubota, Y. Takeishi, High Mobility Group Box 1 Promotes Angiogenesis from Bone Marrow-derived Endothelial Progenitor Cells after Myocardial Infarction. J Atheroscler Thromb 22, 570-581 (2015). 137. F. Limana, G. Esposito, D. D'Arcangelo, A. Di Carlo, S. Romani, G. Melillo, A. Mangoni, C. Bertolami, G. Pompilio, A. Germani, M. C. Capogrossi, HMGB1 attenuates cardiac remodelling in the failing heart via enhanced cardiac regeneration and miR-206-mediated inhibition of TIMP-3. PLoS One 6, e19845 (2011). 138. E. Foglio, G. Puddighinu, A. Germani, M. A. Russo, F. Limana, HMGB1 Inhibits Apoptosis Following MI and Induces Autophagy via mTORC1 Inhibition. J Cell Physiol 232, 1135-1143 (2017). 139. M. D. R. Bauza, C. S. Gimenez, P. Locatelli, A. De Lorenzi, A. Hnatiuk, M. C. Capogrossi, A. Crottogini, L. Cuniberti, F. D. Olea, High-dose intramyocardial HMGB1 induces long-term cardioprotection in sheep with myocardial infarction. Drug Deliv Transl Res 9, 935-944 (2019). 140. M. E. Bianchi, Casalgrandi, M., Venereau, E. J., Brunelli, S., HMGB1 variants and uses thereof. European Patent Office - patentimages.storage.googleapis.com/cf/3c/6c/3b9d7ca70f194a/EP2877248B1.pdf (2012). 141. M. Tirone, N. L. Tran, C. Ceriotti, A. Gorzanelli, M. Canepari, R. Bottinelli, A. Raucci, S. Di Maggio, C. Santiago, M. Mellado, M. Saclier, S. Francois, G. Careccia, M. He, F. De Marchis, V. Conti, S. Ben Larbi, S. Cuvellier, M. Casalgrandi, A. Preti, B. Chazaud, Y. Al-Abed, G. Messina, G. Sitia, S. Brunelli, M. E. Bianchi, E. Venereau, High mobility group box 1 orchestrates tissue regeneration via CXCR4. J Exp Med 215, 303- 318 (2018). 142. G. Lee, A. I. Espirito Santo, S. Zwingenberger, L. Cai, T. Vogl, M. Feldmann, N. J. Horwood, J. K. Chan, J. Nanchahal, Fully reduced HMGB1 accelerates the regeneration of multiple tissues by transitioning stem cells to GAlert. Proc Natl Acad Sci U S A 115, E4463-E4472 (2018).

Claims

CLAIMS What is claimed is: 1. A polypeptide represented by the following formula: H2N-A-X-B-A-X-B-HOOC wherein each A represents consecutive amino acids, the sequence of which (1) is a sequence of four amino acids (a) identical to the sequence of amino acids 90 – 93 of wild type human HMGB1 (SEQ ID NO: 1), or (b) which differs from the sequence of (a) in respect to one or more amino acids; and (2) has at its amino terminal end, between one and six consecutive amino acids, the sequence of which (a) is identical to the sequence of the corresponding one to six amino acids preceding amino acid 90 in wild type human HMGB1, or (b) differs from the sequence of (a) in respect to one or more amino acids; and (3) optionally has a methionine at the amino terminus, wherein each A may be the same or different; wherein each X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 – 162 of wild type human HMGB1; wherein each B represents consecutive amino acids, the sequence of which (1) is a sequence of five or six amino acids (a) identical to the sequence of amino acids 163 – 168 of wild type human HMGB1, (b) identical to the sequence of amino acids 163 – 167 of wild type human HMGB1, (c) the sequence of (a) in which any one of amino acids 163, 167, or 168 is changed to any other amino acid; (d) the sequence of (b) in which any one of amino acids 163 or 167 is changed to any other amino acid; (e) the sequence of (a) or (b) in which amino acid 164 is changed from lysine to arginine; or (f) the sequence of (a) or (b) in which amino acid 165 is changed from glycine to alanine, serine or threonine; (g) the sequence of (a) or (b) in which amino acid 166 is changed from lysine to arginine; or (h) the sequence of (a) or (b) in combination with the changes in (e) and (f), (e) and (g), (f) and (g), or (e), (f) and (g); (i) the sequence of (a), (b), or (c) in combination with the changes in one or more of (e), (f), and (g); or (j) the sequence of (d) in combination with the changes in one or more of (e), (f), and (g); and (2) has at its carboxy terminal end, between one and six consecutive amino acids, the sequence of which (a) is identical to the sequence of the corresponding one to six amino acids following amino acid 168 in wild type human HMGB1, (b) is identical to the sequence of the corresponding one to six amino acids following amino acid 167 in wild type human HMGB1, (c) differs at one or more positions from the sequence of the corresponding one to six amino acids following amino acid 168 in wild type human HMGB1, or (d) differs at one or more positions from the sequence of the corresponding one to six amino acids following amino acid 167 in wild type human HMGB1, and wherein each B may be the same or different; and wherein each - represents a peptide bond between each of A and X, X and B, B and A, A and X, and X and B; with the proviso that in the B-A between the two Xs, the number of amino acids must be at least 12; and with the additional proviso that in the B at the carboxy terminal end of the polypeptide, the one to six consecutive amino acids of (2) may be absent.
2. The polypeptide of claim 1, wherein the methionine is present at the amino terminus.
3. The polypeptide of claim 2, wherein 2(a) of A has at its amino terminal end, one amino acid corresponding to amino acid 89 of wild type human HMGB1.
4. The polypeptide of any one of claims 1–3, wherein in B(2), B has at its carboxy terminal end, six amino acids corresponding to amino acids 169-174 of wild type human HMGB1.
5. The polypeptide of any one of claims 1–4, wherein the number of amino acids in the B-A between the two Xs is at least 13.
6. The polypeptide of any one of claims 1–4, wherein the number of amino acids in the B-A between the two Xs is between 12-22 inclusive.
7. The polypeptide of any one of claims 1–4, wherein the number of amino acids in the B-A between the two Xs is between 13-22 inclusive.
8. The polypeptide of any one of claims 1–7, wherein the sequence of (2)(c) or (2)(d) of either B or of both Bs differs at the one or more positions by the presence of a glycine, serine, proline, arginine, lysine, aspartic acid, glutamic acid, or histidine amino acid residue.
9. The polypeptide of any one of claims 1–7, wherein the sequence of (2)(c) or (2)(d) of either B or of both Bs is or comprises GSGSG (SEQ ID NO: 175).
10. The polypeptide of claim 9, wherein the GSGSG (SEQ ID NO: 175) has been inserted into the region between the B and A between the two Xs.
11. The polypeptide of claim 9, wherein the order or number of the glycine and serine residues has been altered within the SEQ ID NO: 175 peptide sequence.
12. The polypeptide of any one of claims 1–11, wherein the amino acids of (2)(c) or (2)(d) of either B or of both Bs lack a defined secondary structure, or have a turn or random coil secondary structure.
13. A polypeptide of any one of claims 1-12, wherein either A or both As are five consecutive amino acids.
14. The polypeptide of claim 13, wherein the five consecutive amino acids have a sequence identical to the sequence of amino acids 89 – 93 of wild type human HMGB1.
15. The polypeptide of any one of claims 1-14, wherein (a) the A between the Xs is five consecutive amino acids and the B between the Xs is at least seven consecutive amino acids; (b) the A between the Xs is six consecutive amino acids and the B between the Xs is at least six consecutive amino acids; (c) the A between the Xs is seven consecutive amino acids and the B between the Xs is at least six consecutive amino acids; (d) the A between the Xs is eight consecutive amino acids and the B between the Xs is at least six consecutive amino acids; (e) the A between the Xs is nine consecutive amino acids and the B between the Xs is at least six consecutive amino acids; or (f) the A between the Xs is ten consecutive amino acids and the B between the Xs is at least six consecutive amino acids.
16. The polypeptide of any one of claims 1-14, wherein (a) the B between the Xs is six consecutive amino acids and the A between the Xs is at least six consecutive amino acids; (b) the B between the Xs is seven consecutive amino acids and the A between the Xs is at least five consecutive amino acids; (c) the B between the Xs is eight consecutive amino acids and the A between the Xs is at least five consecutive amino acids; (d) the B between the Xs is nine consecutive amino acids and the A between the Xs is at least five consecutive amino acids; (e) the B between the Xs is ten consecutive amino acids and the A between the Xs is at least five consecutive amino acids; (f) the B between the Xs is eleven consecutive amino acids and the A between the Xs is at least five consecutive amino acids; or (g) the B between the Xs is twelve consecutive amino acids and the A between the Xs is at least five consecutive amino acids.
17. A composition comprising the polypeptide of any one of claims 1-16 and a carrier.
18. A pharmaceutical composition of claim 17, wherein the polypeptide is present in a therapeutically or prophylactically effective amount and the carrier is a pharmaceutically acceptable carrier.
19. A method of treating a subject suffering from, or at risk for developing, a condition which would be alleviated by promoting regeneration of a tissue or cells that rely upon CXCR4+ cells for repair which comprise administering to the subject the polypeptide of any one of claims 1-16 in an amount effective to promote regeneration of the tissue or cells or a therapeutic or prophylactic dose of the pharmaceutical composition of claim 18.
20. The method of claim 19, wherein the condition is an acute injury.
21. The method of claim 20, wherein the polypeptide is administered within 5 hours following the acute injury.
22. The method of claim 19, wherein the condition is a chronic condition.
23. The method of claim 22, wherein the polypeptide is administered repeatedly at a daily, weekly, monthly, or yearly interval.
24. The method of claim 20, wherein the acute injury is myocardial infarction.
25. The method of claim 20, wherein the acute injury is stroke, spinal cord injury, or peripheral nerve injury.
26. The method of claim 24, wherein the polypeptide is administered within 5 hours of the myocardial infarction.
27. The method of claim 25, wherein the polypeptide is administered within 5 hours of the stroke, spinal cord injury, or peripheral nerve injury.
28. The method of claim 20, wherein the acute injury is a fracture, joint replacement or bone fusion.
29. The method of claim 20, wherein the acute injury is a skeletal muscle injury, joint injury or ligament injury.
30. The method of claim 19, wherein the condition involves liver damage.
31. The method of claim 22, wherein the chronic condition is non-alcoholic steatohepatitis, liver cirrhosis or infective hepatitis.
32. The method of claim 22, wherein the chronic condition involves damage to the brain or other parts of the central nervous system.
33. The method of claim 22, wherein the chronic condition is Parkinson’s disease, dementia, multiple sclerosis, motor neuron disease or peripheral nerve injury.
34. The method of claim 22, wherein the chronic condition involves a chronic joint injury.
35. The method of claim 34, wherein the chronic joint injury is inflammatory arthritis or osteoarthritis.
36. The method of claim 19, wherein the condition involves damage to the lung.
37. The method of claim 20, wherein the acute injury is a viral infection of the lungs, bacterial infection of the lungs, fungal infection of the lungs, or mechanical injury to the lungs.
38. The method of claim 37, wherein the viral infection of the lungs is an infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
39. The method of claim 37, wherein the mechanical injury is a ventilator-induced injury.
40. The method of claim 22, wherein the chronic condition is idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease, or emphysema.
41. The method of claim 19, wherein the condition involves the gut.
42. The method of claim 20, wherein the acute injury is a surgical injury to the gut.
43. The method of claim 22, wherein the chronic injury is inflammatory bowel disease, Crohn’s disease, or ulcerative colitis.
44. The method of claim 19, wherein the condition involves damage to the skin.
45. The method of claim 20, wherein the acute injury is a burn or a surgical injury of the skin.
46. The method of claim 22, wherein the chronic condition is a skin ulcer, diabetic ulcer, venous ulcer, arterial ulcer, or pressure ulcer.
47. The method of claim 19, wherein the condition involves the pancreas and the cells are islet cells.
48. The method of claim 47, wherein the condition is diabetes.
49. The method of claim 19, wherein the condition is neutropenia following chemotherapy and the tissue is bone marrow.
50. The method of claim 20, wherein the acute injury is chemotherapy, and optionally the polypeptide is administered before or within 5 hours of administration of the chemotherapy.
51. The method of claim 50, wherein the polypeptide is administered before the acute injury.
52. The method of claim 50, wherein the polypeptide is administered after the acute injury.
53. The method of claim 19, wherein the condition is kidney failure and the tissue is kidney tissue.
54. The method of claim 22, wherein the chronic condition is a disease that results in chronic renal failure.
55. The method of claim 20, wherein the acute injury is elective surgery and the polypeptide is administered before, during or within 5 hours after the surgery.
56. The method of claim 20, wherein the acute injury is a sport or military combat injury.
57. The method of claim 22, wherein the chronic condition is a chronic skeletal muscle condition.
58. The method of claim 57, wherein the chronic skeletal muscle condition is a muscular dystrophy such as Duchenne macular dystrophy, sarcopenia, or disuse atrophy.
EP22728676.2A 2021-05-19 2022-05-19 Polypeptides related to hmgb1 useful for promoting tissue regeneration, compositions comprising same, and uses thereof Pending EP4341280A1 (en)

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AU2003228099A1 (en) * 2002-07-03 2004-01-23 Centro Cardiologico Monzino S.P.A.-Irccs Use of hmgb1 in the treatment of tissue damage and/or to promote tissue repair
US20060121047A1 (en) * 2002-11-20 2006-06-08 Tracey Kevin J Use of hmgb polypetides for increasing immune responses
US20090069227A9 (en) 2003-04-29 2009-03-12 Capogrossi Maurizio C Use of HMGB1 to promote stem cell migration and/or proliferation
US20080305120A1 (en) * 2004-06-17 2008-12-11 Medimmune, Inc. Immunogenic Compositions Comprising Hmgb 1 Polypeptides
ES2629086T3 (en) 2006-10-30 2017-08-07 Genomix Co., Ltd. Pharmaceutical substance to promote functional regeneration of damaged tissue
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ES2661500T3 (en) 2012-07-26 2018-04-02 Ospedale San Raffaele S.R.L. HMGB1 variants and their uses
ES2660420T3 (en) 2012-10-25 2018-03-22 Genomix Co., Ltd. Novel method to treat heart attack using HMGB1 fragment
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