HMGB1-related polypeptides useful for promoting tissue regeneration, compositions containing the same, and uses thereof

Abstract

The present invention relates to a compound of the formula: H2N-AXBAXB-HOOC (wherein A is a sequence of consecutive amino acids, the sequence of which (1) contains the same sequence as amino acids 90 to 93 of wild-type HMGB1, (2) has 1 to 6 consecutive amino acids, e.g., 1, 2, 3, 4, 5, or 6 amino acids, at its amino terminal side, whose sequence is the same as the sequence of the corresponding 1 to 6 amino acids preceding amino acid 90 of wild-type HMGB1, and optionally (3) the amino terminus is methionine, and X is a sequence of consecutive amino acids, the sequence of which is the same as amino acid 94 of wild-type HMGB1. (1) a sequence identical to the sequence of amino acids 163 to 168 of wild-type HMGB1; and (2) a sequence identical to the sequence of 1 to 6 consecutive amino acids, e.g., 1, 2, 3, 4, 5, or 6 amino acids, at its carboxy terminus, whose sequence is identical to the sequence of 1 to 6 corresponding amino acids after amino acid 168 of wild-type HMGB1, and each - represents a peptide bond between A and X, X and B, B and A, A and X, and X and B, respectively. The present invention provides a polypeptide represented by the formula:

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 934,299, filed November 12, 2019, the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced, including those referenced within parentheses. The disclosures of all publications mentioned in this application are incorporated in their entirety into this application to provide a description of the art to which this invention pertains and features in the art that can be used in accordance with the present invention.

[0003] Sequence Listing Reference This application incorporates the nucleotide sequence filed "201112_91203-A-PCT_Sequence_Listing_AWG.txt", which is 78 kilobytes in size, created on November 12, 2020 in IBM-PC machine format, and operating system compatible with MS-Windows®, and is contained in a text file submitted as part of this application on November 12, 2020.

[0004] Technical Field The present disclosure relates to engineered polypeptides related to HMGB1 that promote tissue regeneration without harmful inflammation, and methods of treating acute tissue injury by administering the engineered polypeptides to a subject in need thereof. [Background technology]

[0005] Resident stem and progenitor cells play a critical role in maintaining homeostasis and repairing numerous tissues after injury [1]. However, most tissues in adults heal through scarring. Following the success of bone marrow transplantation [2], there has been considerable interest in exogenous stem cell therapy to promote solid organ regeneration, but success has been limited to certain organs, such as the eye [3] and skin [4]. The inflammatory environment following tissue injury is not conducive to stem cell engraftment, and subsequent scarring destroys the stem cell niche [5]. Therefore, the focus has shifted to promoting tissue regeneration by stimulating endogenous repair mechanisms [6]. The development of successful therapeutics depends on identifying soluble mediators that promote these pathways. We previously identified high-mobility group box 1 (HMGB1) as a key mediator of repair in multiple tissues, including bone, blood, and skeletal muscle [7].

[0006] HMGB1 is a prototypic alarmin [8, 9] that, under physiological conditions, plays an important role in transcription [10, 11]. It is passively released extracellularly and into the circulation from damaged and necrotic cells upon cell injury, and mediates the transition of stem and progenitor cells to the G phase, an intermediate state between G0 and G1

[12] . Alert [7]. Upon exposure to the appropriate activator, Alert Cells in G1 rapidly enter G1 and can carry out tissue repair. Alert Stem cells in the nucleus return to G0 after approximately three weeks

[12] , thereby ensuring that they do not become exhausted and their niche is not depleted.

[0007] HMBG1 contains two L-shaped box domains, A and B, each containing three α-helices (I-III) connected by a flexible region involved in binding LPS (the N-terminus of box A and the adjacent C-terminal linker region)

[13] or RAGE (the C-terminus of box B)

[14] . The C-terminus of the protein is intrinsically disordered and contains a high proportion of carboxylic acid residues (Glu / Asp) that constitute the acidic tail. This binds to the HMG box and regulates its activity, including interactions with TLR2 [10, 15, 16] and potentially RAGE

[15] (Figures 1A-1B). The oxidation state of HMGB1 cysteine ​​residues (Cys22 and Cys44 in box A and Cys105 in box B) is a key determinant of its extracellular activity, which depends on the mechanism of release. Three different redox forms have been described in vivo

[17] . HMGB1, which is passively released from the nucleus after injury or cell necrosis, is in the fully reduced form (FR-HMGB1), which binds to CXCL12 and the heterocomplex mediates the G pathway of stem and progenitor cells via the cell surface receptor CXCR4. Alert It signals to promote the transition to endothelial cells [7]. Partial oxidation in the local inflammatory environment leads to the formation of disulfide-type HMGB1 (DS-HMGB1) [18, 19], which has a disulfide bond between Cys22 and Cys44. This is also the form actively secreted by immune cells after acetylation

[20] and N-glycosylation

[21] . DS-HMGB1 signaling via the receptor for advanced glycation end products (RAGE) activates platelets and is a key mediator of thrombosis [22, 23]. DS-HMGB1 also acts via TLR4 and TLR2, resulting in the release of pro-inflammatory cytokines, including TNF and IL-6

[24] . Intracellular signaling via all three receptors converges to induce NF-κB activity

[25] in a MyD88-dependent manner [26, 27]. Oxidation of the three cysteine ​​residues by the action of extracellular reactive oxygen species leads to sulfonyl-HMGB1(SO3), which is biologically inactive [17, 28].

[0008] The disulfide bridge (Cys22-Cys44) in box A of DS-HMGB1 is essential for TLR4 signaling (Figures 1A-1B), initiating binding to TLR4 but with a relatively high dissociation rate. MD-2 then binds to box B with low affinity but a very slow dissociation rate, stabilizing the interaction

[29] , and the Phe-Cys-Ser-Glu (FCSE, 104-107) peptide in box B is essential for this interaction

[30] . The ability of DS-HMGB1 to signal through TLR4 was attenuated by substituting serine for the cysteines at positions 22, 44, and 105, resulting in an engineered form described as 3S-HMGB1

[17] . Although Tirone M et al. claimed that 3S-HMGB1 has enhanced regenerative properties compared to FR-HMGB1

[31] , we found that it was equivalent to FR-HMGB1 in bone, blood, and skeletal muscle injury. Interestingly, 3S-HMGB1 was harmful when administered locally after myocardial infarction, whereas FR-HMGB1 reduced infarct size and enhanced cardiac function over a 4-week period

[32] . There are no published data regarding the effects of 3S substitutions on TLR2 or RAGE signaling.

[0009] Although the site of TLR2 interaction has not been clearly defined, glycyrrhizin is known to inhibit this interaction

[33] , suggesting that at least one, and possibly both, of the HMG box domain and the acidic tail are involved

[10] . It has been reported that the acidic tail negatively regulates HMGB1, and that a co-ligand

[34] is required to displace the acidic tail of HMGB1 from the box domain and enable signaling through TLR2

[35] . However, several publications have reported TLR2-dependent pro-inflammatory signaling by HMGB1 alone [24, 36], and the requirement for a co-ligand may be cell type- and context-dependent. The role of the redox state of HMGB1 in TLR2 signaling remains unclear, as both the disulfide form

[22] and the fully reduced form

[34] have been proposed to signal through TLR2. RAGE interaction has been primarily mapped to peptides within HMG box B (residues 149–182)

[14] (Figures 1A–1B), and peptides derived from this sequence can effectively inhibit HMGB1-RAGE signaling

[37] . In addition, a second caspase-dependent site exists within box A. More recently, a second RAGE-binding site has been identified in HMG box A

[38] , accessible only after proteolysis by caspase-11

[38] . However, the relative contribution of each site to RAGE signaling remains unclear. It has also been recognized that RAGE-mediated prothrombotic signaling requires the disulfide form of HMGB1, implicating box A

[22] . The acidic tail of HMGB1 binds to residues within the RAGE-binding peptide and may negatively regulate RAGE signaling in a manner similar to TLR2 [10, 15, 39].

[0010] Successful translation of FR-HMGB1's regenerative activity into a pharmaceutical product depends on abolishing all potentially deleterious signaling via RAGE, TLR2, and TLR4 while maintaining CXCL12 binding and signaling via CXCR4. Here, we identify residues within HMGB1 that are important for CXCL12 binding and describe HMGB1 variants that abolish RAGE binding and TLR2 and TLR4 signaling while maintaining regenerative activity. DISCLOSURE OF THE INVENTION

[0011] The present invention relates to a compound of the formula: H2N-AXBAXB-HOOC (wherein A represents consecutive amino acids, the sequence of which (1) comprises a sequence identical to the sequence of amino acids 90 to 93 of wild-type HMGB1, (2) has 1 to 6 consecutive amino acids, for example, 1, 2, 3, 4, 5, or 6 amino acids, at its amino terminal side, whose sequence is identical to the sequence of the corresponding 1 to 6 amino acids preceding amino acid 90 of wild-type HMGB1, and optionally (3) the amino terminus is methionine, X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 to 162 of wild-type HMGB1; B is a sequence of consecutive amino acids, the sequence of which (1) contains a sequence identical to the sequence of amino acids 163 to 168 of wild-type HMGB1, and (2) has 1 to 6 consecutive amino acids, for example, 1, 2, 3, 4, 5, or 6 amino acids, at its carboxy terminus, whose sequence is identical to the sequence of the corresponding 1 to 6 amino acids following amino acid 168 of wild-type HMGB1; Each "-" represents a peptide bond between A and X, X and B, B and A, A and X, and X and B, respectively. The present invention provides a polypeptide represented by the formula:

[0012] The present invention also relates to a composition comprising a polypeptide according to the present invention and a carrier, as well as a method for repairing CXCR4. +Provided is a method of treating a subject suffering from or at risk of developing a condition that is alleviated by promoting the regeneration of cell-dependent tissues or cells, comprising administering to the subject a therapeutic or prophylactic dose of a polypeptide of the invention, or a pharmaceutical composition of the invention, in an amount effective to promote tissue or cell regeneration. [Brief explanation of the drawings]

[0013] [Figure 1A] Figures 1A-1B show a schematic representation of the structure of HMGB1 and the location of its known immunogenic activity. Figure 1A: Structure of HMGB1 (PDB 2YRQ, conformer 1). Colored in PyMol according to known interactions with LPS, TLR4, or RAGE. The acidic tail, which is involved in transcriptional modulation and bactericidal activity, is not shown. [Figure 1B] Figure 1B: Schematic representation of the binding site. Box A is blue, box B is green, pink indicates residues involved in glycyrrhizin binding. Red indicates flexible regions on the N-terminal side adjacent to box A or box B. Orange indicates cysteine ​​residues. White indicates linker regions between HMG boxes. Light yellow-green indicates RAGE binding region (incomplete as it extends into the yellow acidic tail). [Figure 2A] Figures 2A-2F show the conserved residues within each HMG box domain, including the N-terminal DPXX tetramer, that are important for CXCL12 binding. Figure 2A: HMGB1 15-mer peptide array (11 x 10) incubated with 1 μM CXCL12-His6 and detected with anti-His5-HRP antibody. Spot intensity corresponds to the amount of CXCL12 bound to the peptide. The first two and last two spots on the array contained the 10-His positive control. [Figure 2B]Figure 2B: Quantification of spot intensities in Figure 2A normalized to the 10-his control (two experiments). Peptides used in the alanine scanning experiments are highlighted. Peptides in the acidic tail were not included because their high negative charge may lead to nonspecific binding to cationic molecules such as CXCL12. Peptides in the graph are represented by SEQ ID NOs: 8-104, from left to right. [Figure 2C] Figure 2C: Peptide array of alanine mutagenesis at single positions within the peptides identified in Figures 2A-2B. The first spot in each row corresponds to the positive control, and the second spot corresponds to the unmodified peptide. The peptides shown are represented by SEQ ID NOS: 105-111. [Figure 2D] Figure 2D: Quantification of the intensity of the array of peptides from Figure 2C (SEQ ID NOs: 105-111) normalized to the unmodified peptide. Residues that showed higher variation in CXCL12 signal than the observed variation seen in alanine residues (Ala->Ala, synonymous mutation, grey) are shown in red. [Figure 2E] Figure 2E: Michaelis-Menten saturation fitting of biotinylated HMGB1 constructs binding to CXCL12 [full-length FR red / 3S black, minimal box A 8–78 (purple) and box B 94–162 (brown), extended box A 1–88 (pink) and box B 89–174 (gray)]. [Figure 2F] Figure 2F: Summary of kinetic parameters derived from Figure 2E. The affinity (Kd) constants obtained from both fits followed the same relationship, with a significant decrease for HMGB1 94-162, and were analyzed by one-way Brown-Forsythe ANOVA from the fitted data. Since no significant differences were found in pairwise comparisons (column factor), Kd values ​​were compared by post hoc two-way ANOVA and averaged across both values. The raw interferograms are shown in Figure 9. [Figure 3A]Figures 3A-3B show NMR validation of residues involved in CXCL12 binding. Figure 3A: Cumulative CSP of helix-only biotinylated box B (94-162, HMGB1A-c028) or complete box B (89-174, HMGB1A-c038) calculated across multiple HSQC spectra after titration with CXCL12 (0.42, 0.84, and 1.42 molar equivalents), including a parallel control without CXCL12 (CSP drift control) measured after the final concentration point. Green intensity in the graph indicates relative CSP. Sequences of each HMGB1 construct are overlaid with residue numbers; empty columns (unnumbered) represent residues that could not be mapped in parallel 3D 1H-15N HSQC / NOE / TOCSY experiments. The sequences of residues corresponding to each HMG box are shown as follows: light blue, residues previously reported in the literature to be involved in CXCL12 binding; red, residues weakly involved in peptide arrays; purple, residues involved in both the published literature and peptide arrays. Grey, alanine residues within CXCL12-binding peptides (underlined) that could not be assessed by peptide arrays. The sequences shown are represented by SEQ ID NO:5. [Figure 3B] Figure 3B: Heat map of cumulative peak height change, NMR change, of Figure 3A. Red indicates I / I change of more than 1 standard error for all residues, and blue indicates a decrease of more than -1 standard error. The sequence shown is represented by SEQ ID NO: 5. [Figure 4A]Figures 4A-4C show the design of the dBB12L construct. Figure 4A: Alignment of box A plus linker (1-88) (SEQ ID NO: 3) with box B plus linker (89-174) (SEQ ID NO: 4). Values ​​correspond to the NMR nomenclature (excluding the N-terminal methionine). Vertical lines indicate strictly conserved positions, and double dots indicate analogous substitutions. Underlined: CXCL12-binding peptide regions from the first peptide array. Red: Residues flagged in the alanine scan as involved in CXCL12 binding that could not be verified by NMR. Orange: Residues flagged in the alanine scan that show either CSP or NMR peak volume changes. Cyan: Residues not flagged in our NMR or peptide array experiments but described in the NMR literature

[44] as contributing to CXCL12 binding. Purple: Residues flagged in the peptide array experiment that have been published or confirmed by our NMR data. Green: residues flagged only in NMR experiments that may either bind directly to CXCL12 or be affected by binding to nearby residues. Pink: residues flagged by both our NMR experiments and published data. [Figure 4B] Figure 4B: Structure of FR-HMGB1 1-166(2YRQ). Residues are colored according to the same colors as in Figure 4A. The side chains of all colored residues are shown. The dashed circles indicate the glycyrrhizin-binding regions in each HMG box. [Figure 4C]Figure 4C: Overview of the dBB12L construct design. The initiation codon Met1 is numbered as Met0 here because it is partially missing in the truncated peptide. Thus, HMGB1 Met1-Gly2...Glu215 becomes Met0-Gly1...Glu214. Domain composition and sequence of the FR-HMGB1 (top, SEQ ID NO: 1) and dBB12L construct (bottom, SEQ ID NO: 2). The dBB12L construct was designed as follows: 1. The acidic tail and part of the RAGE-binding domain (175-214) were deleted; 2. Residues 1-88 (box A) were replaced with residues 89-174, resulting in two HMG box B domains; and 3. Residues 163-174 C-terminal to box B were replaced with the native flexible linker (79-88) C-terminal to box A in native HMGB1. The CXCL12-binding peptide is shown in red. The repeat units of Box B are separated in the diagram by dashed black lines. [Figure 5A] Figures 5A-5D show that dBB12L has similar stability and surface charge structure to FR-HMGB1 1-214 / 1-164. Figure 5A: Calculated Tm50 values ​​(in °C) for full-length and 1-164 FR-HMGB1, and dBB12L. Shading of individual Tm50 values ​​indicates the highest (green) and lowest (red) values ​​within the global dataset of all constructs. N / A: No curve fit. [Figure 5B]Figure 5B: Native ESI-MS of HMGB1 constructs at 50 mM or 0.2 M ammonium acetate, pH 6.5. All three HMGB1 constructs have similar native M / Z profiles, with dBB12L, in which the two HMG boxes are spaced apart, approximating the reduced HMGB1 construct. Solid line: compact monomer. Dashed line: extended monomer (HMG boxes are distal to each other). Removal of the acidic tail (compare FR HMGB1 1-164 in the blue curve with FR-HMGB1 in the red curve) and higher ionic strength (compare spectra of the same constructs at 50 mM or 200 mM ammonium acetate) increase the abundance of higher M / Z states (partially unfolded). [Figure 5C] FIG. 5C: Calculation of the solvent accessible surface area (SASA) of the average folded HMGB1 monomer, the extended and compact monomer states, and the unfolded monomer from FIG. 5D. [Figure 5D] Figure 5D: 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 6A] Figures 6A-6F show that dBB12L has reduced binding to RAGE and does not signal through TLR2 or TLR4. Figure 6A: Michaelis-Menten saturation fit obtained by hybrid ELISA (n = 4 per concentration, global fit) showing the absence of RAGE binding by dBB12L constructs regardless of oxidation state. DS-HMGB1 binds RAGE more strongly compared to FR-HMGB1. Data are normalized to the DS-HMGB1 control. [Figure 6B]Figure 6B: Michaelis-Menten saturation fits obtained by biolayer interferometry (0–25 μM HMGB1, six experiments). Association and dissociation rates were calculated from raw interferogram data only. Kinetic parameters and color legends are summarized in Figure 6C, with invalid fits indicating R<0.6 (poor binding). Due to the nature of the experiment, data obtained from ELISA are proportionally more influenced by the dissociation constant than by the association constant. For each kinetic parameter, green indicates the construct with the highest affinity, fastest association (k), or slowest dissociation (kOff), while yellow indicates the midpoint and red indicates the lowest. [Figure 6C] FIG. 6C: Summary of kinetic parameters and color legend from FIG. 6B (disulfide forms are indicated by dashed lines). [Figure 6D] Figure 6D: DS-HMGB1 promoted NF-κβ activity in reporter HEK-Dual cells expressing human TLR2 and CD14. DbB-HMGB1 did not promote NF-κβ signaling, whereas FR-HMGB1 only induced slight NF-κβ activity in both cell lines, which may be due to partial oxidation during the assay. Values ​​are shown as mean ± standard error fold change compared to control (medium alone). [Figure 6E] Figure 6E: DS-HMGB1 promoted NF-κβ activity in reporter HEK-Dual cells expressing mouse TLR4, MD-2, and CD14. DbB-HMGB1 did not promote NF-κβ signaling, whereas FR-HMGB1 only induced slight NF-κβ activity in both cell lines, which may be due to partial oxidation during the assay. Values ​​are shown as mean ± standard error fold change compared to control (medium alone). [Figure 6F](Figure 6F) Disulfide HMGB1 (DS-HMGB1) increased TNF production in monocytes, which was further enhanced by the presence of suboptimal amounts of LTA, but not LPS. Neither FR-HMGB1 nor DbB-HMGB1 induced TNF secretion, even when preincubated with LPS or LTA for 24 hours. The response to LPS preincubated with these constructs was also significantly reduced in n = 3 donors, each with three technical replicates. [Figure 7A] Figures 7A-7J show that the regenerative effects of optimal doses of dBB-HMGB1 and FR-HMGB1 are identical to those of activated injury. Figure 7A: Volcano plot showing fold change of differentially expressed genes in muscle stem cells after injury or HMGB1-induced GAlert. The integrals show the preservation of up- (brown dots) and down- (blue dots) regulation of core genes in GAlert induced by contralateral leg injury or intravenous (iv) HMGB1. [Figure 7B] FIG. 7B: Network map of gene ontology terms of differentially expressed genes during GAlert induction in muscle stem cells. [Figure 7C] Figure 7C: Dose response of FR-HMGB1 in a BaCl2 skeletal muscle injury model, where regeneration was quantified by fiber cross-sectional area. The optimal dose was 0.75 mg / kg (28.75 nmol / kg), which was used in subsequent assays. Values ​​are shown as mean ± standard error in nested ANOVA with Holm-Sidak correction (values ​​for post-hoc tests are shown). [Figure 7D] (Figure 7D) Animals were dosed with FR-HMGB1 (optimal dose) at various time points after BaCl injection to assess the interval during which FR-HMGB1 treatment was effective after injury. Values ​​are shown as mean ± standard error in nested ANOVA with Holm-Sidak correction (values ​​for post-hoc tests are shown). [Figure 7E]FIG. 7E: Pharmacokinetics of intravenous HMGB1 in mice (circulating HMGB1 after intravenous infusion of an optimal dose) fitted by nonlinear least-squares to a biphasic exponential decay curve. [Figure 7F] Figure 7F: Survival rate after myocardial infarction (MI) at week 5. FR-HMGB1 = 83%, PBS = 52%. [Figure 7G] Figure 7G: Ejection fraction. Dashed line indicates ejection fraction in normal / sham-operated mice. [Figure 7H] FIG. 7H: Infarct size compared by two-way ANOVA for the effect of treatment over time. [Figure 7I] Figure 7I: Representative mid-atrial short-axis cineMRI images at end-diastole and end-systole of the cardiac cycle at 1 and 5 weeks post-MI. Blood within the ventricle appears bright. The FR-HMGB1 group demonstrates preserved cardiac function and wall thickness (yellow arrows), with visible separation of the right and left ventricles during systole (red arrows). In contrast, in the PBS-treated group, there is significant left ventricular dilation (white arrow), with very limited contraction between diastole and systole. 10 animals per group. All MRI scans were performed and evaluated by a blinded observer. [Figure 7J] Figure 7J: Mean muscle cross-sectional area plotted at given time points after treatment of BaCl2-injured animals with PBS (black), 28.75 nM / kg FR-HMGB1A-c001 (red), or dBB12L (green). Five animals per group and time point. Nested ANOVA (Holm-Sidak post-hoc correction). Representative images from each time point are shown in Figure 12. [Figure 8A]Figures 8A-8B show the results of peptide arrays of CXCL12 peptides that interact with HMGB1. Figure 8A: Peptide array of full-length CXCL12. The "+" position corresponds to a positive control 10-His peptide, and the remaining peptides contain a 15-mer of CXCL12 shifted toward the C-terminus by two consecutive residues. The membrane was exposed to 1 μM of HMGB1 (FR or 3S)-His6(1-214), box A-His6(8-78), and box B-His6(94-162) for 24 hours. Bound proteins were detected by chemiluminescence of an anti-His-HRP conjugate. CXCL12 peptides interacting with full-length HMGB1 were unable to interact with either box A or box B alone, confirming the necessity of the N-terminal segments of each box domain (particularly D4 in box A and D90 in box B). The intensity of spots associated with common CXCL12 peptides was also significantly reduced compared to FL-HMGB1 when binding to the box domains alone. Binding to 3S was observed to be more intense than that to FR, likely due to protein oxidation during the assay; however, this was not quantified due to the low protein concentration used being unsuitable for ESI / TOF MS. BLI data, however, indicate a lower dissociation rate of CXCL12 from 3S than from FR-HMGB1. [Figure 8B] Figure 8B: CXCL12 dimer (PDB 2J7Z) with HMGB1-binding regions highlighted. Red: common binding regions. Blue: non-common binding regions. [Figure 9]Figure 9 shows interferograms of CXCL12 binding to immobilized HMGB1 constructs in BLI. Biotinylated HMGB1 constructs were immobilized on streptavidin-coated Octet biosensors and immersed in increasing concentrations of CXCL12. The interferograms are colored according to the CXCL12 concentration (legend shown in the upper right). Each set of three replicates (cycles) for a given sensor is surrounded by an overlay colored according to the construct. FR FL-HMGB1 (c011), black: 3S-FL HMGB1 (c022), purple: FR-HMGB1 Box A 8-78 (c027), brown: FR-HMGB1 Box B 94-162 (c028), pink: FR-HMGB1 Box A 1-88 (c037), gray: FR-HMGB1 Box B 90-162 (c038). [Figure 10A] Figures 10A-10D show NMR validation of residues involved in CXCL12 binding (continuation of Figure 3). Figure 10A: Cumulative CSP of HMGB1 3S 1-184 (HMGB1A-c007) upon single-step addition of 1:2 molar equivalents of CXCL12 (1:1 HMG box to CXCL12 ratio). Box A and box B residues are considered separate molecules for the purposes of median CSP calculation. Green intensity in the bar graph indicates higher relative CSP. Residue numbers are overlaid on the sequence of each HMGB1 construct; empty columns (unnumbered) represent residues that could not be mapped in parallel 3D 1H-15N HSQC / NOE / TOCSY experiments. The sequence of the residues corresponding to each box is shown in the middle of each condition, colored according to the following: light blue, residues previously reported to be involved in CXCL12 binding in the literature; red, residues weakly involved in peptide arrays; purple, residues involved in both the published literature and peptide arrays. The sequences shown are represented by SEQ ID NOs: 6 and 7. [Figure 10B] Figure 10B: (A) 15N HSQC-HQMC peak spectrum in 10 mM HEPES, 150 mM NaCl pH 7.5 buffer. Protein concentration is shown by spectral overlay. [Figure 10C] Figure 10C: HMGB1A 94-162 (2-day experiment). 15N HSQC-HQMC peak spectrum in 10 mM HEPES, 150 mM NaCl pH 7.5 buffer. Protein concentration is indicated by the spectral overlay. [Figure 10D] Figure 10D: HMGB1A 89-174 (6-day experiment, slight degradation occurs after day 4). 15N HSQC-HQMC peak spectrum in 10 mM HEPES, 150 mM NaCl pH 7.5 buffer is shown. Protein concentration is indicated by the spectral overlay. [Figure 11] Figure 11 shows interferograms of HMGB1 constructs bound to immobilized Fc-RAGE in BLI. RAGE-Fc was immobilized on the surface of an AHC sensor and immersed in increasing concentrations of different HMGB1 constructs. Two experiments were performed with different concentration ranges: the left column of three graphs shows 0–22.22 μM HMGB1 over nine steps, and the right column shows 0–25 μM HMGB1 over seven steps. All graphs are color-coded by concentration (top). Colors indicate specific construct concentrations. Each graph corresponds to a single sensor (replicate). Interferograms surrounded by red rectangles had data points excluded due to poor quality (e.g., drift). [Figure 12] FIG. 12 shows histological images of regenerating muscles in response to FR-HMGB1 (red) or dBB12L (green) compared to PBS control (black). [Figure 13]Figures 13A-13C show 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: Strepactin XT affinity epitope. SacB: Levansucrase precursor (negative selection in the presence of sucrose). pLIC: annealing site for sequencing primers used in colony screening. All plasmids contain kanamycin resistance (50 μg / mL). [Figure 14] FIG. 14 shows the mutagenesis of the FR-HMGB1 sequence to generate 3S-HMGB1. BEST MODE FOR CARRYING OUT THE INVENTION

[0014] term To facilitate understanding of the present invention, unless otherwise expressly provided herein, each of the following terms has the meaning set forth below.

[0015] As used herein, "engineered" refers to a non-naturally occurring compound that is created by altering a naturally occurring compound. An engineered compound, e.g., a polypeptide, may contain portions of a naturally occurring compound that have been modified or rearranged. Such engineered polypeptides are also referred to as "analogs" or "derivatives" of naturally occurring polypeptides.

[0016] As used herein, "stem cell" means any unspecialized cell that has the potential to develop into many different cell types in the body, including but not limited to hematopoietic stem cells.

[0017] As used herein, the term "effective amount" refers to an amount of a compound that is capable of achieving a desired result, e.g., alleviating a condition or symptoms associated therewith, such as acute tissue injury as described herein. The specific dose of a compound administered in accordance with the present invention will, of course, be determined by the particular effects associated with the condition, e.g., the route of administration, the physiological condition of the subject, and the severity of the condition being treated. For example, the engineered HMGB1 protein administered to a subject is preferably in the form of a composition comprising a therapeutically effective amount of the engineered HMGB1 protein.

[0018] The phrase "pharmaceutically acceptable" refers to a compound, material, composition, or dosage form that is suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable risk / benefit ratio. As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material. The selection of any particular pharmaceutically acceptable carrier is well within the knowledge of one of ordinary skill in the art. Accordingly, a wide variety of suitable carriers are available and routinely used in pharmaceutical compositions.

[0019] As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the listed components.

[0020] All numerical ranges provided herein are intended to expressly include the endpoints thereof and, where consistent with context, all numbers subsumed between the endpoints of the range.

[0021] Aspects of the invention The present invention relates to a compound of the formula: H2N-AXBAXB-HOOC (wherein A is a sequence of consecutive amino acids, the sequence of which (1) contains a sequence identical to the sequence of amino acids 90 to 93 of wild-type HMGB1, (2) has 1 to 6 consecutive amino acids on the amino terminal side thereof, the sequence of which is identical to the sequence of the corresponding 1 to 6 amino acids preceding amino acid 90 of wild-type HMGB1, and optionally (3) the amino terminus is methionine; X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 to 162 of wild-type HMGB1; B is a sequence of consecutive amino acids, the sequence of which (1) contains a sequence identical to the sequence of amino acids 163 to 168 of wild-type HMGB1, and (2) has 1 to 6 consecutive amino acids, for example, 1, 2, 3, 4, 5, or 6 amino acids, at its carboxy terminus, whose sequence is identical to the sequence of the corresponding 1 to 6 amino acids following amino acid 168 of wild-type HMGB1; Each "-" represents a peptide bond between A and X, X and B, B and A, A and X, and X and B, respectively. The present invention provides a polypeptide represented by the formula:

[0022] In some embodiments, the methionine is present at the amino terminus of the polypeptide.

[0023] In other embodiments, A has one amino acid at its amino terminus that corresponds to amino acid 89 of wild-type HMGB1.

[0024] In some embodiments, B has 6 amino acids at its carboxy terminus whose sequence corresponds to amino acids 169 to 174 of wild-type HMGB1.

[0025] The invention also provides a composition comprising a polypeptide according to any one of the aspects provided and a carrier.

[0026] In some embodiments, the polypeptide is present in a therapeutically or prophylactically effective amount and the carrier is a pharmaceutically acceptable carrier.

[0027] The present invention also relates to the repair of CXCR4+ Also provided is a method of treating a subject suffering from or at risk of developing a condition that is alleviated by promoting cell-dependent tissue or cell regeneration, comprising administering to the subject an amount of a polypeptide according to any one of the provided aspects effective to promote tissue or cell regeneration, i.e., a therapeutically or prophylactically effective dose of a pharmaceutical composition of the invention.

[0028] In certain embodiments, the condition is myocardial infarction and the tissue is cardiac tissue, particularly myocardium.

[0029] 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 myocardial infarction.

[0030] In some embodiments, the condition is a fracture and the tissue is bone.

[0031] In other embodiments, the condition is associated with liver injury and the tissue is liver tissue.

[0032] In still a further aspect, the condition is related to damage to the brain or nervous system, including stroke, Parkinson's disease, and dementia.

[0033] In some embodiments, the condition is associated with damage to the lungs.

[0034] In still a further aspect, the condition involves the digestive tract and includes surgery and inflammatory bowel disease.

[0035] In some embodiments, the condition is related to injury to the skin, including surgical procedures, burns, and ulcers.

[0036] In additional embodiments, the condition involves the pancreas, including type 1 diabetes, and the cell is a pancreatic islet cell.

[0037] In a further embodiment, the condition is neutropenia, eg, post-chemotherapy neutropenia, and the tissue is bone marrow.

[0038] In some embodiments, the condition is renal failure and the tissue is kidney tissue.

[0039] Further non-limiting details are provided in the Experimental Details section below, which are set forth to aid in the understanding of the invention, but are not intended, nor are they to be construed, in any way to limit the scope of the invention as disclosed. [Example]

[0040] result Identification of amino acids and motifs within HMGB1 involved in CXCL12 binding Before considering which residues in FR-HMGB1 can be mutated to eliminate proinflammatory signaling, it is essential to map the amino acids and motifs involved in binding to CXCL12. Using peptide SPOT arrays

[40] , overlapping peptides covering the sequence of one of the target proteins (HMGB1) were assessed for their ability to bind CXCL12 by immunoblotting to identify key sequences involved in binding. A coarse-grained peptide array using human HMGB1 peptides that bind to his-tagged CXCL12 highlighted a specific pattern of homologous sequences binding to CXCL12 between box A and box B. Clustering of CXCL12-binding peptides against the HMGB1 sequence identified two major binding sites (Figures 2A and 2B). The first encompassed the first 1.5 α-helices of the HMG box, overlapping with the glycyrrhizin-binding site

[41] . The second was located in the C-terminal half of the third α-helix. Within each HMG box, the first CXCL12-binding peptide (helices I and II) appeared to be most involved in CXCL12 binding, as the binding intensity of peptides derived from this segment to CXCL12 was much higher. Binding of peptides from box B to CXCL12 appeared slightly weaker compared to box A, based on immunoblot intensity.

[0041] To further clarify the importance of individual residues, we generated a second peptide array in which each amino acid in the CXCL12-binding peptide was substituted with alanine and assessed their effect on CXCL12 binding. The goal was to identify amino acids on the peptide that directly contribute to CXCL12 interaction. This confirmed that these residues are important for CXCL12 binding, as substitution of several residues with alanine altered the strength of binding to CXCL12 from an otherwise homologous peptide (Figures 2C and 2D). In contrast to published data indicating that interaction with CXCL12 is only mediated by the helical segment of the HMG box [42-44], we focused on the flexible N-terminal flanking region of each box (D). -4 -PXX -1 We found that residues at the C-terminus (Ile78–Pro80 for box A and Ala163–Asp168 for box B) are involved in the interaction with CXCL12 (Fig. 2E). This was confirmed by a reverse peptide array of CXCL12 peptides (Fig. 9). Full-length FR and 3S-HMGB1 bound to peptides containing sequences covering the entire β-sheet of CXCL12, whereas HMGB1 box constructs alone (8–78 box A) or (94–162 box B)

[45] , which did not contain the adjacent flexible regions, interacted only with peptides covering the N-terminal strand of the β-sheet.

[0042] We hypothesized that if these flexible regions adjacent to the HMG box are involved in CXCL12 binding, their absence would significantly alter the interaction between HMGB1 and CXCL12. Using biolayer interferometry (BLI), we assessed the binding of CXCL12 to HMGB1 constructs containing each HMG box with or without flexible flanking residues (full HMG box constructs, HMGB1 1–88 for box A and HMGB1 89–174 for box B)

[46] or without (helix-only HMG box constructs, HMGB1 9–78 and HMGB1 94–162), full-length FR-HMGB1, and nonoxidizable (3S)-HMGB1, which share the CXCL12-binding properties of the wild-type protein

[17] . As expected, the binding of HMGB1 to CXCL12 was significantly affected by the presence of these flexible regions (Figures 2E and 2F). The dissociation rate (k off The helix-only constructs had reduced CXCL12 affinity and binding capacity compared to the full HMG box constructs with intact flanking regions, as evidenced by increased CXCL12 affinity. In contrast, the CXCL12 affinity for full-length HMGB1 (FR or 3S) and the full HMG box constructs was similar to each other and higher than that of the helix-only constructs.

[0043] Our peptide arrays showed that residues involved in CXCL12 binding were clustered in two peptides: one spanning the N-terminal halves of α-helices I and II, and the second spanning the C-terminus of α-helix III. Both peptides contained flexible flanking regions at the N- and C-termini of the respective HMG boxes. These patterns were mirrored across the HMG A and B boxes (Figures 2B, 2D, and 2E). BLI kinetic data confirmed that the absence of flanking regions in the single HMG box domain destabilized recruited CXCL12, which did not remain bound to the helix-only construct (Figure 2F).

[0044] CXCL12 binds to a concave pocket beneath each HMG box, as shown by peptide array and NMR Next, we used NMR to identify the amino acid residues in HMGB1 involved in CXCL12 binding from a structural perspective. Residues involved in CXCL12 binding result in changes in NMR signals upon complex formation. We used FR-HMGB1 94-162 and 89-174, which represent the HMG box with and without flanking regions, and 3S-HMGB1 1-184. A complete set of 3D spectra ( 15 N HSQC-TOCSY / NOESY and related 15 Because the time required to acquire the N HSQC spectrum would result in oxidation of FR-HMGB1, altering its peak resonance and interaction with CXCL12, non-oxidizable 3S-HMGB1 was used as our full-length construct.

[0045] CXCL12 titration of HMGB1 box B 94-162 (Figure 3A) resulted in changes in the NMR signals of several residues in the C- and N-terminal binding regions identified in the peptide array, either cumulative chemical shift perturbations (CSPs) or peak height changes (I / I). However, several residues (A100, I112, L119, A136, Y154, D157, and I158) identified in our peptide array and documented in the literature [43, 44] showed no changes in CSP or volume. For box B constructs containing adjacent regions, both the median CSP and mean volume changes were significantly higher. Several residues in these adjacent regions (D90, G165, and K166) had been found to be involved in CXCL12 binding according to our peptide array data. Residues unaffected by CXCL12 binding in the helix-only (94-162) construct, such as Y154, D157, and I158, and those identified in the peptide array as involved in CXCL12 binding, showed significant CSP in the full HMG box construct and improved binding when the flanking regions were present. In addition, we observed CSP changes for residues not identified in the peptide array in the construct with the flanking regions (A147, M131, A169, K172, G173). Other residues previously unidentified but flagged as potentially important in the peptide array did not show CSP or volume changes upon addition of CXCL12 (C105, E107, Y108). These groups of residues were therefore reclassified as not important for CXCL12.

[0046] When the NMR experiment was repeated using 3S-HMGB1 1-184, CXCL12 addition resulted in CSP changes below the threshold of detection due to the signal-to-noise ratio. Surprisingly, in contrast to published data

[47] , residues A100, I112, L119, and A136 in box B of 3S-HMGB1 1-184 failed to induce significant CSP or volume changes, although residues very close to some of these (S99, K113) were affected. Within this construct, residues corresponding to HMG box B showed weaker CSP changes compared to those in box A, which are involved in the interaction with CXCL12. This may represent a more fluid equilibrium for CXCL12 binding, as suggested by the higher association and dissociation rates of box B (Figure 2G). This also allowed us to identify residues H30, D32, or S34 as false positives from the peptide array (Figure 11). K89 in box B 89-174 showed high CSP, but this residue is the third residue from the N-terminus (the remaining N-terminal residue after TEV cleavage, Ser-Met). Experiments with 3S HMGB1 1-184 showed no increase in CSP when it was located in the middle of the flexible linker. Therefore, changes associated with this residue in box B alone are likely related to its N-terminal position, which may allow for high conformational flexibility.

[0047] Design of a double-box B HMGB1 construct that retains CXCL12 binding while abrogating pro-inflammatory signaling We combined data from peptide arrays, alanine substitution, and NMR and mapped them to the NMR structure of HMGB1 (Figure 3A, PDB 2YRQ) to identify residues involved in CXCL12 binding. We found that CXCL12-binding residues occupy the concave side of the HMG box domain, forming a CXCL12-binding pocket beneath each HMG box. These two pockets (one for each HMG box) also contain the binding site for glycyrrhizin, a competitive inhibitor of HMGB1-CXCL12 binding, between helices I and II of each box

[41] . HMG boxes A and B can each bind one CXCL12 monomer with comparable affinity. We also found that the distribution of CXCL12-interacting peptides within each HMG box is similar between the two HMG boxes, forming nearly identical binding pockets (Figure 4A).

[0048] After identifying the CXCL12-binding sequences, we next designed HMGB1 constructs that contained these interaction surfaces but with altered sequences to reduce pro-inflammatory signaling. Based on the fact that each HMG box can independently bind to CXCL12 monomers and the requirement of both box A (oxidized) and box B for TLR4

[48] and potentially prothrombotic RAGE [14, 22] and TLR2 [22, 33] signaling activity, we hypothesized that an HMGB1 construct in which box A was replaced with a different box B (i.e., replacing 1-88 with 89-174) would not signal through TLR2, TLR4, or RAGE. In addition, we deleted a portion of the RAGE-binding sequence in box B (175-184) to further reduce affinity for this receptor. Replacing the box A sequence with box B also replaces the LPS glycan-binding peptide with a copy of the LPS lipid A-binding peptide

[13] , which may further disrupt the proinflammatory activity of HMGB1

[49] and LPS transduction to TLR4 / MD-2 in a manner similar to LBP. The engineered construct, dBB12L (Figure 4B), consisted of the following segments of the native HMGB1 protein: a flexible N-terminal region (derived from HMGB1 89-93), the first box B (derived from HMGB1 94-162), a 12-residue linker C-terminal to the native box B (derived from HMGB1 163-174), and the second box B (derived from HMGB1 94-162). The 12-residue linker in dBB12L is similar to the 10 amino acids in the linker of native HMGB1. This slight increase in linker length results from the preservation of residues 172 and 173, which we showed altered CSP in CXCL12 binding.

[0049] We compared the thermal stability of dBB12L and wild-type HMGB1 by native mass spectrometry (ESI / MS) and size-exclusion chromatography (SEC) using dynamic scanning fluorimetry (DSF) and solvent-accessible surface area (SASA). dBB12L had a similar stability and surface charge profile to FR-HMGB1 1-164, which also contains two HMG box domains but lacks the C-terminal acidic tail. The thermal stability trends were similar for all constructs (Figure 5A), with Tm values ​​near the isoelectric points (9.9 for dBB12L or the tailless construct of 1-164, and 6 for FL-HMGB1). 50 and are all equally stable in conditions relevant to clinical practice (PBS, purification buffer, and saline solution), with Tm 50 The optimum ionic strength range for FR-HMGB1 / FR-HMGB1 1–164 was approximately 50°C. However, we observed a different optimal ionic strength range for FR-HMGB1 / FR-HMGB1 1–164 compared to dBB12L. In native ESI / MS, all three HMGB1 constructs had similar charge state distributions, with compact monomers as the major species and extended monomers with higher SASA (Figures 5B and 5C). Extended monomers were more abundant in the tailless construct or at higher ionic strengths. The average monomer SASA values ​​observed in native ESI / MS were consistent with those derived from SEC or the published NMR structure (PDB 2YRQ, HMGB1 1–164). The compact FL-HMGB1 monomer SASA was consistent with the computational model of FL-HMGB1 in water

[44] . Storage for up to 180 days did not affect the SEC profile (always monodisperse at equal RVs), degradation, or aggregation (Figure 5D). This suggests that the conformation observed in ESI / MS represents the HMGB1 construct or its native fold, and that dBB12L is not significantly different from the HMGB1 construct containing two HMG boxes alone.

[0050] The dBB12L construct has significantly reduced affinity for RAGE and is unable to signal through TLR2 or TLR4 We next evaluated whether dBB12L reduced TLR2 and TLR4 signaling and RAGE binding while preserving HMGB1-mediated regeneration. Due to the lack of established signaling assays for RAGE, we assessed RAGE binding to HMGB1 using real-time kinetics (BLI) and endpoint assays (ELISA). ELISA-based affinity measurements (Figure 6A) showed that 3S-, FR-, and DS-HMGB1 bound similar amounts of RAGE at equilibrium, with DS-HMGB1 and 3S-HMGB1 having significantly higher apparent affinities than FR-HMGB1. In contrast, dBB12L did not bind to RAGE in this assay. Three additional HMGB1 constructs were tested: DS-HMGB1 1-184, which contains the intact RAGE-binding peptide and oxidized box A but lacks the acidic tail and thus possesses all the requirements for RAGE binding; DS-HMGB1 1-164, which lacks a significant portion of the RAGE-binding peptide but retains oxidized box A; and DS-box A alone. We found that DS-HMGB1 1-184 bound to RAGE but with reduced potency and affinity compared to full-length DS-HMGB1. By comparison, DS-HMGB1 1-164 exhibited significantly reduced RAGE-binding potency compared to full-length DS-HMGB1 but still greater than dBB12L, whereas DS-box A 1-88 (a complete HMG box construct with adjacent regions) failed to bind RAGE.

[0051] Kinetic analysis using BLI confirmed the ELISA results (Figure 6B), with two exceptions. In BLI (Figure 6C), DS-HMGB1 1-184 had a much higher RAGE binding affinity than all other constructs, although it also had a slightly faster dissociation rate compared to ELISA, where it had lower affinity than DS-HMGB1. 3S-HMGB1 bound to RAGE in comparable amounts to DS- or FR-HMGB1, with affinity similar to FR-HMGB1 but a much slower overall kinetic rate, whereas in ELISA it had affinity and binding capacity for RAGE comparable to DS-HMGB1. The binding of all other HMGB1 constructs to RAGE in BLI reproduced the results obtained by ELISA. The higher affinity of DS-HMGB1 for RAGE compared to FR in both assays was due to its faster binding rate (k on ), while the dissociation rate (k off ) were nearly identical in these two redox forms. In contrast, dBB12L, which had a binding rate close to that of DS-HMGB1, exhibited very unstable binding and a very high dissociation rate. DS-HMGB1 1-164 also had a faster RAGE binding equilibrium with an overall lower binding affinity than full-length DS-HMGB1 but higher affinity than dBB12L-HMGB1. The deletion of the last 10 residues (175-184) within box B and the disulfide bridge within box A (by replacing it with box B) in dBB12L resulted in unstable binding of RAGE.

[0052] HMGB1 binds to TLR2, TLR4, and RAGE, and signaling from all receptors converges on the NF-κB pathway

[25] . As a result, it is difficult to attribute downstream pro-inflammatory cytokine production to a given receptor. Therefore, we first evaluated TLR-specific signaling using NF-κB reporter cell lines engineered to express TLR2 or TLR4 and their co-receptors. Disulfide HMGB1 promoted NF-κB signaling through TLR2 (Figure 6D) and TLR4 (Figure 6E). In contrast, dBB12L failed to signal in either cell type. Next, we confirmed the effects of various HMGB1 constructs on primary human monocytes. DS-HMGB1 has been reported to synergize with TLR2 ligands, such as LTA, to promote pro-inflammatory signaling

[34] . We confirmed that DS-HMGB1 synergized with LTA to promote greater TNF production than LTA or DS-HMGB1 alone. In contrast, dBB12L or FR-HMGB1 did not exhibit this synergistic effect and were unable to induce greater TNF secretion than medium alone (Figure 6F). The synergistic response using DS-HMGB1 and the TLR4 ligand LPS has not been explained. 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.

[0053] Taken together, these data demonstrate that dBB12L does not signal through TLR2 or TLR4, even in the presence of their cognate ligands, and has a greatly reduced affinity for RAGE.

[0054] The dBB12L construct has pro-regenerative activity comparable to that of FR-HMGB1 Distal injury induces stem cells to G AlertIt has previously been shown that HMGB1 promotes the transition to a skeletal muscle cell-like state

[12] . Therefore, we first compared the transcriptome responses of FR-HMGB1 and skeletal muscle stem cells to injury to the contralateral limb. Genes up- and down-regulated by FR-HMGB1 or distal injury were highly similar (Figure 7A), and the major pathways up-regulated were G pathways, including mitochondrial metabolism, oxidative phosphorylation, and cell cycle (Figure 7B). Alert [7, 12]. Interestingly, CXCR4 was one of the most highly regulated genes.

[0055] Next, we used a validated mouse model of skeletal muscle injury to determine the optimal in vivo therapeutic dose of FR-HMGB1 [7, 12]. We found that 0.75 mg / kg (29 nmol / kg) resulted in the maximal response, with no further improvement in regenerative activity at higher doses (Figure 7C). We also evaluated the optimal time of in vivo administration after injury. FR-HMGB1 was found to be effective in promoting repair when infused up to 5 hours after injury (Figure 7D). There was no improvement thereafter. We then investigated the half-life of FR-HMGB1 in the circulation after intravenous administration. We observed an initial rapid clearance (t 1 / 2 11 min), followed by a subsequent slower clearance rate (t 1 / 2 We found that the half-life of α-glucan was approximately 120 min (Figure 7E), consistent with a half-life of 25 min in humans

[50] , and that the protein is cleared by binding to haptoglobin [51, 52].

[0056] The present inventors have demonstrated that FR-HMGB1 promotes the GTPase activity of stem and progenitor cells after injury. AlertWe have previously shown that FR-HMGB1 accelerates the regeneration of skeletal muscle, bone, and blood by promoting the transition to endothelial cells [7]. There are no significant stem cells in the mammalian heart, and the majority of new cardiomyocytes after injury are derived from pre-existing cardiomyocytes

[53] . Therefore, we evaluated whether administration of FR-HMGB1 promotes cardiac regeneration. We found that intravenous infusion during myocardial infarction resulted in enhanced survival (83% in FR-HMGB1-treated mice compared with 52% in PBS controls) (Figure 7F). FR-HMGB1 resulted in an approximately 60% reduction in infarct size as assessed by serial MRI scans over a 5-week period (Figure 7H) and a 16% improvement in overall left ventricular ejection fraction (Figure 7G).

[0057] Finally, we evaluated the efficacy of dBB12L compared with FR-HMGB1 in promoting skeletal muscle regeneration in vivo. Mice injected with optimal doses (29 nM / kg) of FR-HMGB1 or dBB12L showed comparable accelerated regeneration after injury, as determined by an increase in the average cross-sectional area of ​​regenerating muscle fibers with central nuclei (Figure 7J) [7, 12]. This was most pronounced on day 14, as previously described for FR-HMGB1 [7].

[0058] Consideration HMGB1 needs to be modified for use as a tissue repair therapeutic Therapies based on the administration of exogenous stem cells to promote solid organ repair have failed to fulfill their initial promise [6, 54], and cell killing is similarly effective by triggering an immune response

[55] . An alternative, and potentially more meaningful, approach is to target endogenous regenerative repair processes involving resident stem and progenitor cells [56, 57]. Prostaglandin dehydrogenase inhibition is a promising approach

[58] , but progress to clinical trials has been slow

[59] . Administration of growth factors has also been described [60, 61], but is limited by in vivo proteolysis

[62] . Currently, there are no approved therapeutic agents to promote regeneration and accelerate repair in multiple tissues.

[0059] The present inventors have previously demonstrated that exogenous administration of FR-HMGB1 activates resident stem and progenitor cells into G cells that are readily responsive to appropriate activators released to carry out repair upon tissue injury. Alert Studies have shown that FR-HMGB1 is effective in accelerating bone, skeletal muscle, and blood regeneration by shifting it to DS-HMGB1 [7]. Although the majority of systemic DS-HMGB1 detected in patients after trauma is secreted in a secondary release event

[50] , accumulating evidence supports local conversion of FR-HMGB1 to DS-HMGB1 at the site of injury in vivo [18, 19]. Therefore, the potential for deleterious pro-inflammatory signaling precludes the use of native FR-HMGB1 as a therapeutic agent.

[0060] Disulfide HMGB1 can signal through TLR4 [24, 30], TLR2 [24, 34, 35], or RAGE [14, 63] to induce the expression of proinflammatory cytokines. TLR4 signaling via DS-HMGB1 leads to the production of several proinflammatory cytokines, including TNF

[28] , whereas TLR2 signaling has been shown to be detrimental in multiple processes, including thrombosis and reperfusion injury

[64] and autoimmune disorders

[33] . DS-HMGB1 signaling via RAGE plays a critical role in neutrophil-mediated NET formation, which promotes platelet activation and thrombus formation [23, 64-66]. All three receptors ultimately converge on NF-κB [25, 67], leading to the synergistic expression of proinflammatory cytokines via all three receptors [68, 69]. Therefore, the development of HMGB1 as a therapeutic agent relies heavily on engineering the molecule to eliminate signaling through all three receptors.

[0061] Box A and Box B bind independently to CXCL12 due to a common peptide pattern The regenerative activity of FR-HMGB1 is highly dependent on the formation of a heterocomplex with CXCL12 and signaling through CXCR4. CXCL12 is known to bind to the HMG box [42, 70], but the structural motif involved remains unclear, with few residues proposed [47, 71]. It is also unclear whether this signaling involves CXCL12 homodimers via the CXCR4 axis, including enforcing hematopoietic stem cell quiescence, or CXCL12 monomers promoting chemotaxis [72-74].

[0062] Peptide arrays allowed us to identify residues involved in the HMGB1-CXCL12 interaction

[75] . We identified a common pattern of two peptide-binding regions for CXCL12, located in box A and box B. In each HMG box, the first peptide region extends from the N-terminal flexible segment to half of helix II and overlaps with the glycyrrhizin-binding site

[41] , while the second is located in the C-terminal portion of helix III. We confirmed the importance of these residues using alanine mutations and confirmed the importance of the adjacent flexible regions by BLI, confirming that their removal dramatically increases the dissociation rate of CXCL12. We also confirmed by BLI that each box can bind to CXCL12 monomers independently with similar affinity, without cooperativity between the boxes. Furthermore, the use of HEPES buffer prevented CXCL12 dimerization

[76] , allowing us to conclude that each box can bind to monomeric CXCL12 and that CXCL12 dimerization is not a prerequisite for complex formation. This model is consistent with the proposed signaling mechanism of the HMGB1-CXCL12 heterocomplex via CXCR4

[72] .

[0063] We then used NMR to confirm the important role of the peptide regions identified by peptide arrays, including the flexible flanking regions, whose absence resulted in reduced CXCL12-induced changes upon binding to either full-length or helix-only HMG Box B constructs. This is consistent with the faster dissociation rates observed in BLI for the helix-only HMG Box B constructs compared to those containing the flanking regions. We also observed a weaker signal (peak broadening) for Box B compared to Box A, corroborating the faster exchange binding equilibrium observed for full-length Box B compared to Box A in BLI and further supporting the weaker signal for the CXCL12-binding sequence of Box B in peptide arrays. Some residues exhibited shift changes in NMR despite not being flagged in the peptide arrays. These may represent sequence-independent contributions to binding, such as backbone interactions or the relaying effect of other residues that bind to CXCL12 and affect nearby positions.

[0064] When we superimposed the residues involved in CXCL12 binding determined by NMR and peptide arrays onto the structure of HMGB1 1–164 (PDB 2YRQ), we identified pockets within each box domain, with the side chains of all residues aligned to the center of the pocket. The residues identified by alanine scanning and NMR form a concave surface in box B, which also contains the glycyrrhizin-binding site (Figure 3B, dashed circle). In contrast, several of the residues that showed only changes in NMR had side chains pointing outward from this pocket, suggesting that they either bind to CXCL12 independently (backbone-mediated) or are affected by indirect changes in the chemical environment in other parts of the HMG box upon CXCL12 binding.

[0065] This detailed understanding of CXCL12 binding allowed us to design a construct that abrogates pro-inflammatory signaling via TLR4, TLR2, and RAGE. This construct (dBB12L) consists of two tandem HMG box B domains separated by a linker of similar length to that of wild-type HMGB1. Because box A is replaced by box B, it is not oxidized, but the presence of two HMG boxes should still allow it to bind CXCL12. dBB12L is as stable as 1-164 FR-HMGB1 or full-length FR-HMGB1, is equally thermostable in clinically relevant buffer conditions (PBS, saline solution), and exhibits no aggregation or degradation upon prolonged storage. The surface area and charge profile of dBB12L were also similar to those of HMGB1 1-164, with a monodisperse profile by SEC and a distributed charge profile by native ESI-MS. This similarity reflects the similar conformation in solution between dBB12L and the wild-type HMGB1 construct, which has two HMG boxes and a linker region (FR-HMGB1 1-164) but no acidic tail.

[0066] The engineered dBB12L construct does not signal through TLR2 or TLR4 or bind RAGE, yet retains full pro-regenerative properties The binding of HMGB1 to TLR4 / MD-2 has been well described

[29] . Oxidized box A initiates the binding of DS-HMGB1 by interacting with TLR4, box B stabilizes this interaction by binding to MD-2, and the FCSE motif within box B has been shown to be essential for signal transduction

[30] . DS-HMGB1 can signal via TLR4 / MD-2 on its own, but it can also promote LPS-mediated signaling by replacing it with LPS-binding protein (LBP), which binds to LPS and promotes its transmission and recognition to TLR4 / MD-2

[13] . Deletion of box A in our dBB12L construct effectively eliminates TLR4-mediated signaling. Interestingly, we observed that FR-HMGB1 and dBB12L reduced TNF expression by monocytes in response to LPS. This may be due to the fact that these proteins bind to LPS but are unable to communicate it to TLR4 / MD2 due to the lack of oxidized box A, unlike DS-HMGB1, which can effectively replace LPS-binding protein (LBP) present in serum in the culture medium

[77]

[13] .

[0067] The interaction between HMGB1 and TLR2 has not been well described, and there is some debate regarding whether HMGB1 can induce TLR2 signaling by itself [24, 36] or whether it requires a co-ligand to induce activity, and whether this response depends on the redox state of the protein [34, 35]. Published data demonstrating that HMGB1 alone was unable to induce TLR2 responses were conducted either in the absence of serum

[35] or at low HMGB1 concentrations

[34] , suggesting that HMGB1 alone retains some ability to signal through TLR2 but requires a co-ligand to induce a higher level of response. It is known that binding must involve at least one HMG box

[33] and that the acidic tail negatively regulates binding to TLR2

[35] . We found that DS-HMGB1 alone could signal through TLR2, and that this effect was enhanced by the presence of LTA in serum-containing medium. However, there was no response to FR-HMGB1 or dBB12L, which do not synergize with LTA, suggesting that, like TLR4, TLR2-mediated responses require the disulfide-bridged oxidized box A and that TLR2 coligands synergize with DS-HMGB1, possibly by displacing the acidic tail to facilitate TLR2 interaction.

[0068] The RAGE-binding site within HMGB1 (residues 150–183) has been previously described

[14] . Similar motifs exist in other RAGE ligands, e.g., S100 proteins, and peptides homologous to these sequences are effective antagonists of HMGB1-mediated RAGE signaling [37, 78]. The acidic tail of HMGB1 shares residues with the RAGE-binding peptide

[15] and has been proposed as a regulator of RAGE interaction, similar to its role in TLR2 binding. However, only the disulfide form of HMGB1 has been specifically associated with RAGE-mediated prothrombotic activity

[22] . We found that constructs lacking the RAGE-binding peptide, including dBB12L, were unable to bind RAGE in ELISA assays. In contrast to DS-HMGB1, and interestingly, 3S-HMGB1 also bound RAGE better than FR-HMGB1. When we analyzed the interaction kinetics using BLI, we found that dBB12L had lower affinity for RAGE compared to FR-HMGB1 and DS-HMGB1, consistent with our ELISA data. dBB12L had a threefold higher association rate than FR-HMGB1, likely due to the presence of two partial RAGE-binding domains in this construct, but the dissociation rate was fivefold higher, indicating an overall unstable and weak binding. The extensive washing involved in ELISA emphasizes the effect of dissociation rates, which result in different HMGB1 construct behavior in each assay and represent the binding state at equilibrium. For example, 3S-HMGB1, which has similar affinity for RAGE to FR-HMGB1 in BLI, shows a much higher apparent affinity in ELISA due to its dissociation rate being much slower than that of FR-HMGB1 or DS-HMGB1, resulting in RAGE remaining bound. This increased RAGE binding may partially explain the increased fibrosis seen in mouse models of myocardial infarction compared to controls, whereas FR-HMGB1 promoted regeneration and improved function

[32] .Using SPR, it has also been shown that 3S-HMGB1 likely binds to RAGE with very high affinity, although no comparison with FR-HMGB1 has been made

[64] . We used BLI to measure the affinity (K ) of DS-HMGB1 for RAGE. d We found that the oxidized HMGB1 binding affinity (0.2–1.3 μM) was similar to that previously reported using SPR (0.1

[79] –0.65 μM

[22] ). Our BLI data also show that loss of the acidic tail increases the affinity of HMGB1 for RAGE but results in a less stable binding due to a higher dissociation rate, whereas truncation of the RAGE-binding peptide or reduction of box A significantly increases the dissociation rate, resulting in an unstable binding. Interestingly, oxidized box A alone is unable to bind to RAGE, suggesting that the interaction is likely initiated by the RAGE-binding peptide. The absence of box A, along with truncation of the RAGE-binding peptide in dBB12L, results in a significant reduction in RAGE affinity and a reduced ability to retain RAGE once bound due to a faster dissociation rate compared to either FR- or DS-HMGB1.

[0069] We found that the transcriptome changes induced by FR-HMGB1 in skeletal muscle stem cells closely resemble those induced by distal injury, affecting mitochondrial metabolism, oxidative phosphorylation, and cell cycle G Alert We found that this is consistent with upregulation of the G pathway [80, 81]. Upregulation of CXCR4 expression by HMBG1 may enhance its effects. Our data showing that FR-HMGB1 is effective only when administered within 5 hours of injury suggests that it may promote stem cells to G pathway. Alert At this later time point, stem cells are activated and therefore transition to G AlertMuscle stem cells have been shown to migrate to the site of injury 5–6 hours after injury and begin actively differentiating at approximately 12 hours

[82] . We confirmed that dBB12L retains regenerative activity in vivo comparable to that of FR-HMGB1. Importantly, we found that intravenously administered FR-HMGB1 at the time of myocardial infarction resulted in improved survival, reduced infarct size, and improved left ventricular ejection fraction. Based on these data, we predict that administration of dBB12L will also promote the regeneration of tissues that rely on stem cells for repair, such as bone, skeletal muscle, and blood, as well as tissues whose regeneration relies primarily on mature cell populations, such as cardiomyocytes in the heart. We also predict that dBB12L is likely to be effective if administered within 5 hours after injury. This is important because the median time to hospital admission after MI in the United States is 3 hours

[83] . Approximately 800,000 people in the United States suffer myocardial infarction each year

[83] , and approximately 20–30% will develop heart failure. Despite the fact that the medical costs of heart failure in the United States exceeded $30 billion in 2012 and are expected to increase to $70 billion by 2030, the 5-year survival rate is only approximately 60%, worse than that of most cancers

[83] . Based on our data, we predict that administration of dBB12L within 5 hours of the event will improve survival in patients experiencing myocardial infarction, particularly ST-segment elevation myocardial infarction, and reduce the incidence and severity of heart failure through reduced infarct size and preservation of ejection fraction. In mouse [32, 84, 85] and sheep

[86] models, direct injection of FR-HMGB1 into the myocardium in the peri-infarct region 4 hours after infarction has been shown to be effective in promoting cardiac repair. Our data demonstrating the efficacy of intravenous administration is important because this route is readily adaptable for clinical use.

[0070] In conclusion, we mapped the site of HMGB1 important for CXCL12 binding and designed a construct (dBB12L) that does not signal through TLR2 or TLR4 and cannot effectively bind RAGE. Despite its short half-life, FR-HMGB1 inhibits stem cells in a manner similar to distal injury. Alert and is effective when administered within 5 hours after injury. Furthermore, dBB12L promotes tissue regeneration in vivo as effectively as FR-HMGB1. Therefore, dBB12L can be developed for clinical translation.

[0071] overview Reduced high mobility group box 1 (HMGB1) protein binds to CXC ligand 12 (CXCL12) and signals through CXC receptor 4 (CXCR4) to promote tissue and regeneration, promoting stem and progenitor cell proliferation. Alert However, local conversion of FR-HMGB1 to its disulfide form (DS-HMGB1) can lead to harmful inflammation through signaling via Toll-like receptors 2 (TLR2) and 4 (TLR4) and the receptor for advanced glycation end products (RAGE). Therefore, before considering administering HMGB1 to promote tissue regeneration in clinical practice, it is important to engineer the molecule to eliminate these potentially harmful pro-inflammatory effects.

[0072] The present inventors used a combination of peptide arrays, biolayer interferometry, and nuclear magnetic resonance to identify residues involved in the formation of the HMGB1-CXCL12 heterocomplex. Combining these data with available literature on the sites of interaction with proinflammatory receptors, the present inventors designed a construct containing two HMG B boxes in tandem (dBB12L), which has stability and conformation similar to that of the wild-type, fully reduced HMGB1 construct lacking the C-terminal acidic tail. As shown herein, dBB12L does not signal through TLR2 or TLR4, even in the presence of their co-ligands, and has significantly reduced RAGE binding. Furthermore, the dBB12L construct retains regenerative activity comparable to that of FR-HMGB1 in vivo.

[0073] A comprehensive review of the patent landscape and scientific literature identified U.S. Patent Application Publication No. 2015 / 0203551, which describes replacing cysteine ​​with serine to prevent TLR4 signaling; however, this construct has been shown to result in excessive cardiac fibrosis after myocardial infarction. (17) Furthermore, this construct has a slower dissociation of RAGE compared to FR-HMGB1, resulting in RAGE remaining bound longer after equilibration, as shown here in Figure 6B; therefore, these substitutions were avoided in our constructs. U.S. Patent Application Publication No. 2009 / 0069227 (A9) specifies that HMGB1 constructs promoting stem cell migration and proliferation must contain amino acids 1-187 (0-186 in our data, where 0 is the N-terminal Met), and U.S. Patent No. 9,623,078 cites peptides restricted to amino acids 1-44 (0-43 in our data) for cardiac regeneration. U.S. Patent Application Publication No. 2009 / 0202500 discloses methods for tissue repair but refers only to full-length (1-215) wild-type HMGB1 (0-214 in our data). The dBB12L construct presented herein lacks RAGE binding or TLR4 / 2 signaling, is 117 amino acids long, and contains previously undescribed amino acid substitutions. Therefore, the constructs presented herein do not fall within the scope of the prior art.

[0074] clinical application The present invention provides polypeptides and methods for enhancing tissue repair by utilizing endogenous regenerative processes. The polypeptides function similarly to fully reduced wild-type HMGB1, forming a heterocomplex with two CXCL12 molecules, which promotes tissue regeneration by signaling through CXCR4, presumably through two adjacent CXCR4 receptors on the cell surface.

[0075] Our data indicate that the polypeptide of the present invention (dBB12L) acts in a similar manner. Therefore, it is expected that dBB12L will promote the regeneration of tissues that depend on CXCR4+ cells for repair. Such tissues include tissues whose repair depends primarily on stem and progenitor cells, such as skeletal muscle and the hematopoietic system, and tissues whose repair depends largely on pre-existing mature cells, such as cardiomyocytes in the adult mammalian heart.

[0076] Potential clinical indications: Heart after myocardial infarction This indication is suitable for clinical trials. Globally, ischemic heart disease affects 153 million people (101), resulting in over 105,000,000 disability-adjusted age losses in 2017 (102). Each year, 205,000 people in the UK (103) and 805,000 people in the US suffer a myocardial infarction (MI), of which 38% experience an ST-segment elevation MI (STEMI) (101). After an MI, approximately 30-40% of individuals develop heart failure, affecting 38 million people worldwide. Despite medical costs for heart failure in the US exceeding $30 billion in 2012 and projected to increase to $70 billion by 2030, the 5-year survival rate is only approximately 60%, worse than most cancers (101). A key target population is post-MI patients, particularly those at risk for developing heart failure (104). Novel therapeutic agents that limit cardiac damage, promote post-MI regeneration, and prevent the onset of heart failure would dramatically reduce morbidity and mortality, significantly lowering healthcare costs. Using a well-established (105-108) permanent ligation mouse MI model (109), which reliably results in cardiomyocyte necrosis, conclusive data show that a single intravenous administration of FR-HMGB1 at the time of injury resulted in enhanced survival [83% in FR-HMGB1-treated animals compared with 52% in PBS (placebo-treated) animals], as well as an approximately 16% improvement in absolute cardiac ejection fraction compared to controls and an approximately 60% reduction in infarct size compared to PBS controls over 5 weeks (Figure 7F).

[0077] In a skeletal injury model, the optimal dose of FR-HMGB1 was 0.75 mg / kg (Figure 7C), and despite its very short half-life (Figure 7E), it was effective even when administered intravenously up to 5 hours after injury (109) (Figure 7D). After myocardial infarction, reperfusion of the ischemic myocardium should be achieved as soon as possible. For example, patients after STEMI should undergo percutaneous intervention. The data indicate that administration of HMGB1 as early as possible, and up to 5 hours after injury, preserves damaged myocardium and promotes regeneration.

[0078] Native FR-HMGB1 promotes functional recovery after MI (Figures 7F-7I), whereas local conversion to the disulfide form promotes thrombus formation and signaling via RAGE, TLR2, and TLR4 (110). Constructs reported by others, such as 3S-HMGB1 that retains RAGE binding (Figure 6B), result in excessive fibrosis and dysfunction after MI (111). FR-HMGB1 also binds to RAGE, albeit to a lesser extent than DS-HMGB1, and is therefore not suitable for clinical use. HMGB1 signaling via TLR2 plays an important role in ischemia-reperfusion injury (112) and thrombosis (110) after myocardial infarction, and we have demonstrated a critical role for TLR2 in human atherosclerosis (113). TLR4 signaling is also important in myocardial reperfusion injury (114). Redox conditions in the ischemic and inflamed microcirculation of the injured heart after myocardial infarction promote the conversion of FR-HMGB1 to its disulfide form (DS-HMGB1), which is a central mediator of thrombosis (110). There are no approved therapies to promote cardiac regeneration after MI. Reports claiming to demonstrate the regenerative properties of hematopoietic stem cells have been refuted (115), and cytotoxic cells are equally effective at triggering an immune response (116). Even with the use of other cell types, including pluripotent stem cells, considerable challenges remain, including arrhythmogenicity, immunosuppression, scalability, batch variability, delivery, long-term feasibility, and efficacy (115, 117). Large-scale clinical studies of cell-based therapies have not shown significant improvement in function, and arrhythmias have been reported in patients (118-120).

[0079] The absence of a significant stem cell population in the adult heart (121) and limited epicardial progenitor cells (122), together with the understanding that the majority of new cardiomyocytes after injury are derived from pre-existing cardiomyocytes, has shifted the 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 drawbacks: adenoviral transduction and growth factors (IFGF1 / HGF) require intracardiac injection or local patch application (FSTL1), manipulation of developmental pathways carries the risk of oncogenicity (128), and viral transduction of miRNA199-a in pigs resulted in fatal arrhythmias (127). An alternative strategy to stimulate cardiac regeneration by promoting immune cell clearance requires repeated injections of VEGF-C (129). Inhibition of MAP4K4 promoted myocardial survival and limited infarct size but had no regenerative effects (130). To date, none of these strategies have progressed into clinical trials.

[0080] The present invention offers a unique solution for targeting endogenous processes to promote cardiomyocyte survival and regeneration of multiple tissues. It overcomes many of the obstacles associated with cell therapies, including antifibrotic CAR-T cells (131), such as prohibitive cost (132, 133). Because FR-HMGB1 acts through the cell surface receptor CXCR4, it is not expected to have off-target effects associated with targeting intracellular processes, for example, by adenoviral transduction of transcription factors or miRNAs. HMGB1 inhibition increases infarct size after ischemia-reperfusion injury (134), while local upregulation of FR-HMGB1 (135, 136) or intramyocardial injection has been shown to be effective in both mice (111, 137, 138) and sheep (139). However, our data indicate that intravenous administration is more likely to be effective and reach all target cells. The engineered double box B constructs of the present invention that avoid harmful pro-inflammatory signaling should be safe.

[0081] The Milan group described an HMGB1 analog (3S-HMGB1) in which three cysteines were replaced with serine to abolish TLR4 signaling (140). They claimed that 3S-HMGB1 was superior to FR-HMGB1 in promoting tissue regeneration (141), but we did not find this to be the case (142). Importantly, 3S-HMGB1 promoted fibrosis in a mouse MI model, accompanied by deterioration of cardiac function, whereas FR-HMGB1 promoted tissue regeneration and improved left ventricular ejection fraction (111). We found that 3S-HMGB1 remained bound to RAGE longer than either DS-HMGB1 or FR-HMGB1 (Figure 5A). Thus, FR-HMGB1, 3S-HMGB1, and DS-HMGB1 bind comparable amounts of RAGE at equilibrium, but over time, the level of RAGE bound by 3S-HMGB1 becomes comparable to that of pro-inflammatory disulfide HMGB1 (DS-HMGB1) and higher than that of FR-HMGB1. Our double box B construct retains regenerative activity comparable to that of FR-HMGB1 while eliminating undesirable pro-inflammatory signaling.

[0082] Additional applications: fracture Fractures occur after injury. However, one of the most common skeletal "injuries" is joint replacement or arthroplasty. The inventors propose that dBB12 can be used to promote healing after fractures or arthroplasty, thereby reducing the risk of potential complications, such as component loosening.

[0083] Brain and Nervous System dBB12L may be used to improve the prognosis of patients after stroke. Other potential indications include Parkinson's disease and dementia.

[0084] lung dBB12L is contemplated to improve prognosis after lung injury, for example, after COVID-19 infection, or in patients with idiopathic pulmonary fibrosis.

[0085] liver It is estimated that 30% of the population in the United States suffers from non-alcoholic liver disease.60% of these will develop non-alcoholic steatohepatitis, and 20% of these will develop cirrhosis.Treatments have been developed to limit and prevent liver damage caused by these conditions.The present inventors propose that dBB12L can be used in combination with these treatments to promote liver regeneration.

[0086] digestive tract dBB12L may also be used in combination with treatments to control inflammation in the gastrointestinal tract, for example, after surgery or to promote healing in patients with inflammatory bowel disease, such as ulcerative colitis.

[0087] kidney dBB12L may be used to promote kidney regeneration, thereby potentially avoiding the need for dialysis or kidney transplantation.

[0088] skin dBB12L may be used to promote wound healing, for example, after surgery, in patients with burns, or ulcers, eg, diabetic ulcers.

[0089] pancreas dBB12L may be used to improve prognosis in patients with type 1 diabetes mellitus by promoting pancreatic islet cell regeneration.

[0090] bone marrow dBB12L may promote regeneration of the hematopoietic system, for example, after chemotherapy, thereby preventing severe, potentially life-threatening neutropenia.

[0091] We have previously shown that FR-HMGB1 is effective even when administered up to 2 weeks before injury. (142) Because dBB12L is as effective as FR-HMGB1 (Figure 7J), this polypeptide may be used prophylactically, for example, by the military or for athletic injuries, or before elective surgery or chemotherapy.

[0092] Materials and Methods E. coli strains 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

[87] ), and BL21(DE3)-R3-pRARE2-BirA (in vivo biotinylated derivative as above, additional spectinomycin resistance, 50 μg / mL) were obtained from chemically compatible stocks made in-house.

[0093] Bacterial culture medium SOC: 20 g / L tryptone, 5 g / L yeast extract, 0.5 g / L NaCl, 0.1862 g / L KCl, autoclaved and supplemented with 4.132 g / L MgCl and 20 mM glucose.

[0094] LB (Luria Bertani): 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.2, autoclave sterilization. LB agar plates were prepared by adding 2 w / v% agar powder.

[0095] 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 sterilization.

[0096] TB supplement: 1.6% w / v glycerol, 1% glucose, 25 mM (NH4)2SO4, 10 mM MgSO4, 10X trace metals, 0.22 μM, sterile filtered.

[0097] Trace metals 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, sterile filtered at 0.22 μM.

[0098] 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, autoclaved, 4 g / L glucose, 1 g / L U-99% 15 NH4Cl (Cambridge Isotopes), 0.3 mM CaCl2, 1.5 mg / L D-biotin, and 1.5 mg / L thiamine-HCl were supplemented from sterile filtered stocks.

[0099] Plasmid Plasmids were obtained from the SGC library

[87] . All plasmids contain a 6xHis tag with a TEV cleavage site, and pNIC-Bio3 and pDsbC-HT-CBio also contain a C-terminal biotinylation epitope (removable with a stop codon). Plasmid DNA was linearized by restriction enzyme digestion: BfuA1 (3 h, 60°C) for pNIC-CTHF or BsaI (2 h, 37°C) for pNIC-CTHF. The cleaved 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 according to the manufacturer's protocol.

[0100] Cloning The HMGB1 construct was obtained from the mammalian gene collection (purified as a plasmid from Mach1 cells grown overnight in LB medium with antibiotics). The construct was amplified by PCR using a program of 95°C / 10 min, 25 cycles (95°C / 30 s, 52°C / 1 min, 68°C for 0.5–1.5 min), 68°C / 10 min). The reaction consisted of 5 μL of Hercules II buffer, 1 μM of each primer, 6 μg / mL of plasmid template, 1 μM of dNTP mix, and 1 unit of Hercules II polymerase (Agilent 600679, supplied with buffer and 100 μM of dNTP stock) in a final volume of 25 μL. The PCR product was purified (PureLink kit, ThermoFisher K310001) before further use.

[0101] The amplified coding sequences (alleles) were cloned into the desired vector by ligation-independent cloning (LIC). The insert was treated with T4 DNA polymerase in the presence of the same nucleotides as used in the vector (10 μL reaction volume), and 2 μL was mixed with 1 μL of the treated vector and annealed for 30 minutes. 40 μL of ice-cold Mach-1 cells (for storage) or 20 μL of BL21(DE3)-R3-pRARE2 / BL21(DE3)-R3-pRARE2-BirA cells (for expression) were added, heat-shocked at 42°C for 45 seconds, and then cooled on ice. Recovery was allowed to occur in SOC medium at 37°C for 2 hours before plating on selective medium with 5% sucrose and antibiotics. After 24 hours, positive clones were picked and screened with MyTaq polymerase using a sequencing primer pair specific for the correct molecular weight band according to the manufacturer's protocol. Positive transformants were grown overnight in 1 mL of 2x LB (double concentrated LB) with antibiotics and stocked at -80°C in 12% v / v glycerol.

[0102] The 3S-HMGB1 mutant sequence was generated in a similar manner. PCR was performed separately to generate the S23-S45 and S106 fragments, which were annealed by PCR, and 5 μL of each purified PCR product was used as a substitute for primers and template in the reaction. This process is summarized in Figure 14.

[0103] The CXCL12 construct was cloned using an in-frame SUMO protease site at the N-terminus of the mature protein to enable periplasmic secretion of the N-terminal fusion protein in the pDsbC-HT-CBio vector (DsbC-SUMO-CXCL12), avoiding the addition of N-terminal residues to the protein that could affect its activity [88, 89]. Oxidized CXCL12 was obtained via the DsbC fusion protein system

[90] . All mutants were verified by sequencing (SourceBioscience). The sequence of HMGB1-dBB was designed in silico by codon-optimizing the sequence of box B 89–174 according to the E. coli BL21-DE3 genome (assembly ASM956v1) and placing it after the native HMGB1 box B sequence. It was synthesized in vitro by Twist Bioscience (San Francisco, USA) and cloned into pNIC-CTHF.

[0104] Recombinant protein expression A 20 mL overnight culture of a transformant of an HMGB1-expressing strain grown from a streak on a fresh agar plate was inoculated into 1 L of TB (or M9) medium with supplements and grown at 37°C with 0.45 RCF rotary shaking to a maximum OD of 2.0 (OD of 0.6 for M9 medium). The preliminary culture used for the production of 15N-labeled HMGB1 was first spun down at 1000 RCF for 5 minutes and washed in M9 medium. This was grown to the target OD, cooled to 18°C, and then 0.5 mM or 0.25 mM IPTG (for HMGB1 and CXCL12 proteins, respectively) was added. After 16 hours of growth, the culture was harvested at 4000 RCF. For biotinylated proteins, induction was performed by adding 10 mM D-biotin in PBS, followed by cell harvesting after an additional hour.

[0105] Purification of recombinant HMGB1 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 inhibitor (Calbiochem Set III, Merck 539134), 3 μg / mL benzonase-MBP, 1 mM MgSO, 0.5 mg / L lysozyme (Sigma L6876), and 0.5% (v / v) Triton® X100, then frozen at −80°C; all subsequent steps were performed at 4°C. The thawed pellets were spun down at 6780 RCF for 45 minutes, and the supernatant was loaded onto a 1 mL pre-equilibrated nickel-His GraviTrap (GE Healthcare) column. After loading, the column was 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 the remaining DNA for 30 minutes. Contaminants were washed with 15 CV of 0.5 M NaCl, 5% glycerol, 50 mM HEPES pH 7.5 supplemented with 30 mM imidazole (Buffer B), and then eluted directly onto a PD-10 column (GE Healthcare, equilibrated in Buffer B + 20 mM imidazole) with 2.5 mL of Buffer B + 500 mM imidazole. Protein was eluted from the column with 3.5 mL of Buffer B + 20 mM imidazole, followed by tag removal with 1:20 OD of TEV-GST protease for 16 hours.

[0106] Proteases and further contaminants were removed by recirculating the protein solution through the same GraviTrap column (equilibrated in Buffer B + 20 mM imidazole) used for the initial purification. For biotinylated proteins, streptavidin-XT resin was instead used to select only biotinylated molecules. After a 30-minute incubation on the resin, the sample was applied dropwise, washed with 30 CV of Buffer A and 1 CV of Buffer B + 100 mM D-biotin, and eluted by a 2-hour incubation in 3 CV of the same buffer. Proteins were further purified by size-exclusion chromatography (SEC) (Superdex S75, flow rate 10 / 300-0.35 mL / min or 16 / 600-1.2 mL / min) in 10 mM HEPES pH 7.5 + 150 mM NaCl for biophysical studies or cell culture-grade PBS for cell and animal studies. The recombinant proteins were flash-frozen for storage with the addition of 1 mM TCEP in the case of reduced HMGB1 protein.

[0107] Purification of recombinant CXCL12 The outer membranes of cells expressing DsbC-SUMO-CXCL12 were lysed by osmotic shock

[91] . The pellet was 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 kit (COEDTAF-RO, Roche), 50 mM imidazole, and 3 μg / mL benzonase. After stirring for 45 minutes at room temperature, 4 volumes of ice-cold 18.2 mΩ water were added. After mixing for an additional 10 minutes, 1 mM MgSO4 was added. The mixture was centrifuged at 16,000 RCF for 1 hour at 4°C, and the supernatant was loaded onto a Ni-NTA Superflow column (Qiagen, 30761) on an Aekta Xpress FPLC system at 10 mL / min, using one column for every 6 L of cells. The protein was eluted with an imidazole gradient (10-25 mM over 10 CV and 25-500 mM over 8 CV) in Buffer B and dialyzed overnight in 100 volumes of 0.2 M NaCl, 20 mM HEPES pH 8.0 (Buffer Ac) after addition of an OD of 1:10. The next day, the protein was loaded onto a CaptoS column (CaptoS ImpAct, GE 17-3717-47) at 2.3 mL / min and eluted with a gradient of 0.2-1.5 M NaCl in 20 mM HEPES pH 8.0 to separate cleaved CXCL12 from DsbC and Ulp-1. The protein was further purified by SEC in the same manner as HMGB1 and flash-frozen for storage.

[0108] Endotoxin removal Endotoxin was removed in all cases by phase separation using Triton® Tx-114 prior to size-exclusion chromatography

[92] . 2% v / v TX-114 was added to the recombinant protein solution, homogenized for 20 minutes with rotary shaking at 2000 RCF at 4°C, and separated for 5 minutes at 37°C. The detergent phase was then pelleted for 10 minutes at 8000 RCF at 25°C. The supernatant was mixed with 5% w / v SM-2 biobeads (BioRad, 152-8920), clarified with 2% TX-114 for 2 hours, and reconstituted with 30 CV of methanol, 30 CV of endotoxin-free 18.2 mΩ water, and 30 CV of endotoxin-free PBS. This was incubated at room temperature for 4 hours to adsorb the remaining Triton® and PEG

[93] , and then injected into a sterile SEC system (contact with 0.5 M NaOH for 12 hours, followed by 0.2 M acetic acid / 20% ethanol for 6 hours, and equilibrated in cell culture-grade PBS) for size exclusion while completely removing residual polymer contaminants. The absence of Triton® and PEG was verified by the absence of their respective charge-state species in ESI / QTOF-MS mass spectrometry

[94] . The LPS content of the recombinant protein was assessed by the LAL method (GenScript ToxinSensor L000350). Samples were approved for cell and animal use if they contained less than 4 EU of LPS per mg of protein.

[0109] Enzyme production TEV-GST protease (GST fusion protein), Benzonase-MBP, and Ulp-1 protease were produced from transformants stored in the SGC collection

[87] ; all were ampicillin-resistant at 200 μg / mL. TEV and Ulp-1 were purified in a single IMAC step according to the protocol described for HMGB1, while Benzonase-MBP was purified from outer membrane lysates obtained with CXCL12 and isolated using amylose resin (NEB, E0821) according to the manufacturer's protocol. In both cases, the resulting proteins were concentrated to 10 mg / mL in 50 mM HEPES pH 7.5, 0.3 M NaCl, and 10% glycerol. GST-TEV protease and Ulp-1 were flash frozen in liquid nitrogen and supplemented with 0.5 mM TCEP during purification, while Benzonase-MBP was supplemented with 50% glycerol and 2 mM MgCl2 and stored at -20°C.

[0110] Peptide array Membranes bearing FMOC-linked 15-mer peptides of human HMGB1 (Uniprot P09429) or CXCL12 (Uniprot P48061, secretion signal omitted) were printed at SGC by Dr. Sarah Picaud according to published protocols upon request

[40] . Membranes were rehydrated in 95% and 70% ethanol at 20–25°C, equilibrated in PBST (PBS 1× + 0.05% Tween® 20, 3×), and blocked with 10% BSA / PBST for 8 h. 1 μM partner His-tagged protein constructs were added (in PBS) and allowed to bind for 24 h at 4°C. Excess BSA and protein were removed by three washes in PBST, all lasting 1 min unless otherwise indicated. To detect bound proteins, membranes were treated with a 1:3000 dilution of Qiagen anti-pentaHis HRP conjugate (Qiagen 34460), and excess antibody was removed by washing three times for 20 minutes in PBST. Bound antibody was then quantified by chemiluminescence (Pierce ECL Substrate-32109). The membrane was covered with substrate solution, placed between two clear plastic sheets, and then imaged stepwise at 2-minute intervals on an LAS-4000 camera (chemiluminescence setting). The intensity of the peptides on each membrane was measured in ImageJ and normalized to the 10-His control. Alanine mutagenesis scans were performed in the same manner using printed peptides from those identified during the initial peptide array. Residues whose mutation to alanine resulted in a higher intensity change than that observed at alanine positions within the sequence were considered significant contributors to CXCL12 binding.

[0111] Biolayer Interferometry (BLI) Prehydrated streptavidin Octet biosensors (ForteBio 18-5019) were coated with a 4 μM solution of biotinylated HMGB1 protein in 10 mM HEPES, pH 7.5, 150 mM NaCl (basic buffer - BB) plus 0.5 mM TCEP (60 s baseline, 60 s binding). Nonspecific binding was minimized by incubating in BB + 1% BSA + 0.05% Tween® 20 (kinetic buffer, KB) for 3 min prior to kinetic assays. Interaction with CXCL12 was measured by stepwise immersion of the sensor in solutions of increasing CXCL12 concentrations (0-150 μM in 1:2 dilutions) in KB (60 s baseline, 500 s binding, 420 s dissociation, 180 s reduction in BB + 0.5 mM TCEP). An OctetRed 384 instrument was used for these experiments. Kinetic data were extracted using DataAnalysis 9.0 (ForteBio). Response at equilibrium, R Eq is plotted against concentration in a Michaelis-Menten saturation plot to obtain kD / B max The kinetic rate (binding rate k on and dissociation rate k off ) were derived from direct measurements of each parameter from all binding and dissociation steps in the interferogram and fitted to a horizontal line (mean) across all measurements. Data from each replicate were pooled in the same manner to calculate the overall mean. To measure the kinetics of RAGE binding to HMGB1, 15 μg / mL of RAGE-Fc in PBS + 0.1% BSA + 0.02% Tween® 20 was immobilized on the surface of an anti-IgG biosensor (AHC, 18-5060) for 30 seconds and immersed in serial concentrations of each HMGB1 construct (60 seconds baseline, 200 seconds binding / dissociation), and fitted in the same manner to derive kinetic parameters.

[0112] nuclear magnetic resonance (NMR) in 10 mM HEPES, 150 mM NaCl pH 7.5 (same buffer and ionic strength as in the BLI experiments)15 N-labeled recombinant HMGB1 constructs were supplemented with 5% v / v D2O, pipetted into 5 mm Shigeimi tubes with a glass Pasteur pipette, and sealed with paraffin. The final volume was more than 330 μL. CXCL12 was added in the same buffer, and the final volume was adjusted to avoid altering the reference. Signal locking, tube shimming, and nucleus adjustment were performed manually using Bruker TopSpin software. The water signal was 1 H spectra were suppressed by acquiring them at power level 1 (P1) = estimated pulse calibration (pulsecal). If a single peak was observed, a P1 value four times higher than the initial value was used as the baseline. 1 The H spectrum was adjusted until a symmetric peak was observed. NMR experiments were performed after these calibration steps ( 1 H-NMR, 15 N-HSQC, 15 N-NOESY-HSQC, 15 N-TOCSY-HSQC peaks are 15 For N-HSQC spectra, assignments were based on the published NMR table for HMGB1 1-184 (BMRB 15418), and our own NOESY / TOCSY data for each construct was analyzed. To measure CXCL12 binding, the chemical shift positions and volumes of identified peaks were tracked across different molar equivalents of CXCL12 (listed in the associated images) using the chemical shift tracking module in CCPNMR 3.0 Analyze. For peak intensity changes, the median intensity change in each set was considered the baseline. The complete chemical shift table and experimental parameters are provided at the end of this section.

[0113] mass spectrometry For protein identification by MS / MS and trypsin digestion, bands from SDS-PAGE gels were excised and submitted to the SGC open-access MS platform, where they were analyzed by Dr. Rod Chalk, Dr. Tiago Moreira, and Oktawia Borkowska as published [94, 95]. Data analysis (peptide mapping) to annotate protein identity was performed using the MASCOT search engine against the Uniprot (reference protein sequence) and SGC (construct sequence) databases. Native ESI / MS experiments were performed by manual injection into an ESI / QTOF instrument (Agilent Q-TOF 6545) in volatile buffer (50 or 200 mM ammonium acetate, pH 6.5) at 360 μL / h. After a stable ion flow was observed in the total ion chromatogram, signals were acquired for at least 10 counts (30 s). For denaturation experiments, samples were diluted to 1 mg / mL in 0.2% formic acid, injected by HPLC (Agilent 1100 HPLC), and eluted in a formic acid / methanol mobile phase as described

[94] . Each continuous distribution of charge states was considered a distinct conformation, and charge states (Z) were assigned according to the formula mW = (mW / Z - proton mass) * Z. Surface areas were derived from formulas proposed in the literature [96, 97], yielding 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 using HPLC water (electrochemical grade) and solvents.

[0114] SEC surface area quantification To correlate SEC chromatograms with surface area, a set of standards with known structure (BioRad 1511901) was run on the Superdex 75 pg, 10 / 300 column used in this experiment. SASAs for these proteins and calibration curve standards supplied by GE were derived from public PDB structures (BSA, 3V03; ovalbumin, 1JTI; myoglobin, 2V1I; RNAse A, 1A5P; aprotinin, 1NAG; vitamin B12, 3BUL) and correlated to retention volume by nonlinear least-squares fitting (SASA = 331.2 × RV). 2 -1.19e4 × RV + 1.08e5). All experiments comparing 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 signal saturation, and all samples were eluted at 0.4 mL / min.

[0115] RAGE binding ELISA assay A 384-well protein-binding ELISA plate (Santa Cruz Biotechnology, sc-206072) was coated with 50 μL of 40 nM HMGB1 constructs in PBS (+0.5 mM TCEP for FR-HMGB1 constructs), including FL / DS HMGB1 full-length controls and blanks, in replicates of four, for 24 hours at 4°C. Nonspecific binding was blocked by incubation with 10% BSA in PBS for 2 hours at 20-25°C. A range of concentrations of RAGE-Fc chimeric protein (BioTechne, 1145-RG, 0-640 nM at a 1:4 dilution) was added in 10% BSA / PBS and allowed to bind for 2 hours at 4°C. Bound FC chimeras were detected by incubation with anti-human IgG HRP (Agilent Dako P021402-2) diluted 1:10,000 in 1% BSA / PBS for 2 hours at 20-25°C. Between each of these three steps, the plate was washed three times with 100 μL of PBST.

[0116] To detect bound antibody, 25 μL of TMB substrate (ThermoFisher N301) was added to each well, and the reaction was developed in the dark until the FL-DS-HMGB1 control developed a clear concentration-dependent color gradient, after which the reaction was stopped with 25 μL of 0.5 M H2SO4. OD450 was measured as the readout (FluoStar OMEGA, BMG Labtech) and plotted as a saturation fit against 2× RAGE-Fc concentration (because the chimera is a RAGE dimer).

[0117] TLR4- and TLR2-mediated NF-κB signaling reporter assay HEK-Dual cells (Invivogen) expressing human TLR2 and CD14 or mouse TLR4, MD-2, and CD14 were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco), 1% L-glutamine (Gibco), and 1% penicillin / streptomycin (Gibco) under standard tissue culture conditions (37°C, 5% CO). To determine whether FR-HMGB1, DS-HMGB1, and dBB12L induce activation of TLR4 and TLR2 signaling, 10 TLR4 and TLR2 HEK dual cells were seeded in wells of a 96-well plate (triplicate experiments) and stimulated with 10 μg / mL HMGB1 and (X concentration) FSL-1 for TLR2 and 10 ng / mL LPS for TLR4. Twenty-four hours after stimulation, NF-κβ activity was determined by measuring the levels of induced secreted embryonic alkaline phosphatase (SEAP).

[0118] Monocyte total NF-κB secretion assay Human monocytes (StemCell Technologies) were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) under standard tissue culture conditions (37°C, 5% CO). To determine whether FR-HMGB1, DS-HMGB1, and dBB12L induce pro-inflammatory cytokine production, 10 5Human monocytes were seeded in wells of a 96-well plate (triplicate experiments) and stimulated with 10 μg / mL HMGB1 and 50 ng / mL LPS or 10 ng / mL LTA. Twenty-four hours after stimulation, TNF levels were determined by enzyme-linked immunosorbent assay (ELISA) (Abcam).

[0119] Transcriptome analysis Mice were systemically treated with an intravenous injection of 30 μg of FR-HMGB1 in 50 μL of PBS vehicle or PBS-only control. Injured cells were derived from BaCl2-injured mice as described below. Alert cells were derived from the uninjured contralateral side of BaCl2-injured mice. Mouse muscle stem cells (mMuSCs) were defined and freshly isolated according to a previously reported protocol. Muscle cell suspensions were prepared by mincing thigh muscles and enzymatically digesting them with collagenase 800 U / ml (Worthington-Biochem) and dispase 1 U / mL (Gibco). Subsequently, all suspensions were filtered through 70 μm and 40 μm filters (Greiner Bio-One) and stained with the respective antibodies. mMuSCs, CD31 - CD45 - Sca-1 - VCAM1 +mMuSCs were isolated by fluorescence-activated cell sorting (FACS) using a BD FACSAria III instrument. RNA extracted from freshly FACS-isolated mMuSCs was sent for RNA-seq analysis using the Lexogen 3' kit library prep and sequenced using a HiSeq400 (Illumina). FASTQ files were evaluated using FASTQC, followed by generation of TPM values ​​using kallisto v0.42.4. TPM values ​​were summed to obtain gene-level expression values ​​using tximport, and differential expression analysis was performed 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 cutoff of 0.1. Visualization was performed using the R packages "ggplot2" and "igraph."

[0120] In vivo mouse muscle injury model Female C57BL / 6 inbred mice, 11–12 weeks old, were purchased from Charles River UK and housed in the local Biological Safety Unit (BSU) at the Kennedy Institute. The acclimation period lasted 1–2 weeks. All protocols performed on live animals were approved by the UK Home Office (PPL 30 / 3330 and PPL P12F5C2AF) and by a designated individual from the local animal facility, and are registered under ASPA regulations under the appropriate project and person licenses. All consumables were certified for surgical use, and recombinant proteins were endotoxin-free. Surgeries were performed in a separate, controlled environment from the selection facility. All animals were monitored for 6 hours postoperatively and daily for the following 3 days, after which monitoring was transitioned to NVS / NACWO.

[0121] Surgery was performed as previously described [7, 12]. Animals were aerosol anesthetized with 2% isoflurane, anesthetized, and transferred to a heating pad. The right lower hind limb was sterilized with povidone-iodine and the tail with 70% ethanol if intravenous infusion was being performed. 50 μL of 1.2% BaCl2 (Sigma) was injected along the length of the tibialis anterior (TA) muscle to induce cell death. Mice were euthanized, and at the indicated time points, the hind limbs were removed and fixed in 4% paraformaldehyde (Santa Cruz Biotechnology) for 24 hours. The TA muscle was dissected and fixed for an additional 24 hours before being embedded in paraffin and sectioned. Sections (5 μm) were stained with hematoxylin and eosin to identify fibers with central nuclei and imaged on an Olympus BX51 using a 10x eyepiece / 40x objective. Fiber cross-sectional area (CSA) from at least four images per mouse was measured manually using the FIJI distribution in ImageJ2 software (NIH). Data were grouped by mouse. Mice were intramuscularly or intravenously injected with HMGB1 constructs (46 nM / kg, resuspended in PBS) or PBS vehicle control, either simultaneously with injury or after injury for optimal administration of HMGB1 constructs after injury.

[0122] In vivo mouse cardiac injury model Female C57BL / 6 mice, 10-14 weeks old and weighing 25-30 g, were subjected to surgery. All mice received an intravenous infusion of either FR-HMGB1 (46 nM / kg, resuspended in PBS) or vehicle control immediately prior to surgery. Buprenorphine (buprenorphine hydrochloride, Vetergesic) was delivered by intraperitoneal injection as a 0.015 mg / ml solution 20 minutes before the procedure to provide analgesia. Mice were anesthetized 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 thoracotomy. Experimenters were blinded to treatment groups for subsequent cardiac cineMRI 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.

[0123] Cardiac cineMRI and analysis Cardiac cineMRI was performed at 7T using a Varian DDR system after LAD ligation. Briefly, mice were anesthetized with 2% isoflurane in oxygen gas and placed supine in a custom animal handling system with thermoregulation. Prospectively gated proton cardiac images were acquired with a 72mm volume transmit / four-channel surface receive coil (Rapid Biomedical GmbH) using a partial Fourier accelerated spoiled gradient echo CINE procedure (TR 5.9 ms, TE 2.2 ms, 30 kHz bandwidth, 30° FA, approximately 20-30 frames gated on the R wave with a 4 ms post-labeling delay; 20% partial acquisition; averaging of four) (128x128 matrix, 25.6 mm^2 FOV, 0.2 mm planar resolution) to obtain two and four atrial long-axis views and short-axis stacks for functional quantification. Unacquired partial Fourier transform data were reconstructed by the convex projection method prior to simple Cartesian DFT. Blinded image analysis was performed using ImageJ (NIH). Left ventricular mass, volume, and ejection fraction were calculated as previously described. 1Relative infarct size was calculated from the average endocardial and epicardial circumference of the thin akinetic area of ​​all sections measured in diastole and expressed as a percentage of the total myocardial surface

[99] .

[0124] statistical analysis All calculations were performed using GraphPad Prism (v.8.41). For kinetic experiments (BLI / RAGE ELISA), all fits were performed using nonlinear least-squares regression. For RAGE ELISA, each RAGE concentration was independent of the rest of the wells, and all data were considered as a single kinetic fit; however, in BLI, each sensor was considered as an independent fit for calculation purposes. Comparisons between parameters were performed using the AUC method. Mouse muscle injury model data were analyzed using nested ANOVA, where each column contained all muscle CSA values ​​for a given animal, and each group contained all animals to separate biological variation from treatment effects. If the equal variance assumption could not be met in any case, data were analyzed using the Kruskal-Wallis test. For nested ANOVA, an equal number of data points from each animal were randomly selected to avoid skewness. Any other data were analyzed by one-way ANOVA if the heteroscedasticity plot and Q / Q plot supported the equal variance assumption; these were also verified by Spearman's test. For multivariate experiments (e.g., cardiac experiments), two-way ANOVA was used under the same assumption (no data sets violated heteroscedasticity in this case). Post-hoc comparisons were weighted by the Holm-Sidak correction (ANOVA family test) or the Dunns method (Kruskal-Wallis). The test chosen in each case is stated below the respective figure legend. Legend for significance: ns; not significant; * ;p<0.033, ** ;p<0.002, *** ;p<0.0002, **** ;p<0.0001.

[0125] NMR Chemical Shift Table HMGB1-c028 (94–162, biotinylated) titrated with 0, 0.42, 0.82, and 1.42 molar equivalents of CXCL12A-c021, Figures 3A and 3B Due to the limited amount of protein, the HMGB1 sample was titrated with CXCL12 and diluted sequentially. This does not change the results, as the calculations in the CCPNMR chemical shift tracking module are peak-independent. Median changes are shown by comparing volumes.

[0126] [Table 1]

[0127] [Table 2]

[0128] [Table 3-1]

[0129] [Table 3-2]

[0130] 3D Experiment (Day 1) Due to the 3D nature of these spectra are not shown, but the data is available if required. The sample is from Baseline 1.

[0131] [Table 4]

[0132] [Table 5-1]

[0133] [Table 5-2]

[0134] Table 6

[0135] Table 7-1

[0136] Table 7-2

[0137] Table 8

[0138] Table 9-1

[0139] Table 9-2

[0140] Table 10

[0141] Table 11-1

[0142] Table 11-2

[0143] HMGB1-c038 (89–174, biotinylated) titrated with 0, 0.42, 0.82, and 1.42 molar equivalents of CXCL12A-c021, Figures 3A and 3B Due to the limited amount of protein, the HMGB1 sample was titrated with CXCL12 and diluted sequentially. This does not change the results, as the calculations in the CCPNMR chemical shift tracking module are peak-independent. Median changes are shown by comparing volumes.

[0144] [Table 12]

[0145] [Table 13]

[0146] [Table 14-1]

[0147] [Table 14-2]

[0148] 3D Experiments (Days 1-5) Due to the 3D nature of these spectra are not shown, but the data is available if required. The sample is from Baseline 1.

[0149] [Table 15]

[0150] [Table 16]

[0151] [Table 17-1]

[0152] Table 17-2

[0153] Table 18

[0154] Table 19-1

[0155] Table 19-2

[0156] Table 20

[0157] Table 21-1

[0158] Table 21-2

[0159] Table 22

[0160] Table 23-1

[0161] Table 23-2

[0162] HMGB1A-c007(3S, 1–184) and CXCL12 at a molar ratio of 1:2 (1:1 molar ratio CXCL12 / HMG box) in 10 mM HEPES pH 7.5, 150 mM NaCl, Figure 12

[0163] [Table 24]

[0164] [Table 25]

[0165] [Table 26-1]

[0166] [Table 26-2]

[0167] [Table 26-3]

[0168] 3D Experiment (Day 1) Due to the 3D nature of these spectra are not shown, but the data is available if required. The sample is from Baseline 1.

[0169] [Table 27]

[0170] [Table 28]

[0171] [Table 29-1]

[0172] Table 29-2

[0173] Table 29-3

[0174] Table 30

[0175] Table 31-1

[0176] Table 31-2

[0177] Table 31-3

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Claims

1. The following formula: H ２ N-A-X-B-A-X-B-HOOC (wherein each A is a consecutive amino acid, the sequence of which is (1) DPNA, and (2) the amino terminal side of which is either K, FK, KFK, KKFK, KKKFK, or TKKKFK; X is consecutive amino acids, the sequence of which is PKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYR; each B is a sequence of consecutive amino acids, the sequence of which is (1) AKGKPD, and (2) the carboxy terminal side of which is either A, AA, AAK, AAKK, AAKKG, or AAKKGV; - represents a peptide bond between A and X, X and B, B and A, A and X, and X and B; optionally containing a methionine at the amino terminus of the formula H 2 NAXBAXB-HOOC) A polypeptide represented by the formula:

2. The polypeptide of claim 1, wherein the amino terminus is methionine.

3. 3. The polypeptide of claim 1, wherein each A has a K on its amino terminal side.

4. The polypeptide according to any one of claims 1 to 3, wherein each B has the amino acid sequence AAKKGV at its carboxy terminal side.

5. 5. The polypeptide of claim 1, wherein each A is the amino acid sequence KDPNA and each B is the amino acid sequence AKGKPDAAKKGV.

6. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 5 and a carrier.

7. 7. The pharmaceutical composition of claim 6, wherein the polypeptide is present in a therapeutically or prophylactically effective amount and the carrier is a pharmaceutically acceptable carrier.

8. Repair CXCR4 ＋ 10. A polypeptide according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 7, for use in a method of treating a subject suffering from or at risk of developing a condition which is alleviated by promoting the regeneration of a cell-dependent tissue or cell, the method comprising administering to the subject a therapeutic or prophylactic dose of a polypeptide according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 7, in an amount effective to promote regeneration of the tissue.

9. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in the method according to claim 8, wherein the condition is myocardial infarction and the tissue is cardiac tissue / myocardium.

10. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in the method according to claim 9, wherein the polypeptide is administered within 5 hours of the myocardial infarction.

11. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in the method according to claim 8, wherein the condition is a fracture and the tissue is bone.

12. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in a method according to claim 8, wherein the condition is associated with liver damage and the tissue is liver tissue.

13. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in a method according to claim 8, wherein the condition is related to damage to the brain or nervous system and includes stroke, Parkinson's disease and dementia.

14. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in a method according to claim 8, wherein the condition is related to lung damage.

15. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in a method according to claim 8, wherein the condition is related to the gastrointestinal tract and includes surgery and inflammatory bowel disease.

16. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in a method according to claim 8, wherein the condition is related to skin damage and includes surgical procedures, burns and ulcers.

17. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in a method according to claim 8, wherein the condition is related to the pancreas, including type 1 diabetes, and the cells are pancreatic islet cells.

18. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in the method according to claim 8, wherein the condition is post-chemotherapy neutropenia and the tissue is bone marrow.

19. A polypeptide according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 7 for use in the method according to claim 8, wherein the condition is renal failure and the tissue is kidney tissue.

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