HMGB1-related polypeptide useful for promoting tissue regeneration, composition containing same, and use thereof

Abstract

The present invention relates to a compound of the formula: H2N-AXBAXB-HOOC The present invention relates to HMBG1-based polypeptides represented by the formula:

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 190,429 (filed May 19, 2021), the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced, and each reference is incorporated herein by reference in its entirety.

[0003] Sequence Listing Reference This application also incorporates by reference each nucleotide sequence in the text file filed as part of this application, filed under the file name "220519_91203-B-PCT_Sequence_Listing_AWG.txt", having a size of 78 KB, created on May 18, 2022, in IBM-PC machine format, and operating system compatible with MS-Windows®.

[0004] Technical Field The present invention relates to HMGB1-related polypeptides that promote tissue regeneration without inducing harmful inflammation, and methods for treating acute or chronic conditions involving tissue damage by administering said polypeptides to a subject in need of treatment. [Background technology]

[0005] Resident stem and progenitor cells play a critical role in maintaining homeostasis and repairing many tissues after injury [1]. However, most adult tissues heal through scarring. Following the success of bone marrow transplantation [2], much interest focused on exogenous stem cell therapy to promote solid organ regeneration. However, success was limited to a few organs, such as the eye [3] and skin [4]. Inflammation after tissue injury does not lead 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]. Successful therapeutic development depends on identifying soluble mediators that promote these pathways [7]. We previously identified high-mobility group box 1 (HMGB1) as a key mediator of repair in multiple tissues, including bone, blood, and skeletal muscle [8].

[0006] HMGB1 is the prototype of alarmins [9, 10] and plays an important role in transcription under physiological conditions [11, 12]. Upon cell injury, it is passively released extracellularly and into the blood from damaged and necrotic cells and mediates the reversion of endogenous stem and progenitor cells to the G phase, an intermediate state between G0 and G1

[13] . Alert [8] When exposed to the appropriate activators, cells transition to G Alert It can rapidly transition from G1 to G2 and repair tissue. Alert Stem cells in the niche return to G0 after approximately three weeks

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

[0007] HMBG1 has two L-shaped box domains, A and B, each containing three α-helices (I–III) connected by a flexible region (Figure 1A). 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. The oxidation state of HMGB1 cysteine ​​residues (Cys22 and Cys44 in box A and Cys105 in box B) is a key determinant of HMGB1 extracellular activity, depending on the mechanism of release. Three different redox forms have been reported in vivo

[14] . HMGB1 passively released from the nucleus after injury or cell necrosis is the fully reduced form (FR-HMGB1). It binds to CXCL12, and the heterocomplex signals through the cell surface receptor CXCR4 to promote the GPCR of stem and progenitor cells. Alert [8]. Partial oxidation during local inflammation leads to the formation of disulfide HMGB1 (DS-HMGB1) linked at Cys22 and Cys44 [15, 16]. This is also the form actively secreted by immune cells after acetylation and N-glycosylation [17, 18]. TLR-4 signaling via DS-HMGB1 produces several pro-inflammatory cytokines, including TNF

[19] , whereas TLR-2 signaling has been shown to be detrimental in multiple processes, including thrombosis, reperfusion injury, and autoimmune diseases [20, 21]. DS-HMGB1 signaling via RAGE plays a key role in platelet activation and neutrophil-mediated NET formation, promoting thrombus formation [20, 22-24]. Intracellular signaling via all three receptors converges to induce NF-κB activity

[25] in a MyD88-dependent manner [26, 27]. Oxidation of all three cysteine ​​residues by the action of extracellular reactive oxygen species yields the biologically inactive sulfonyl-HMGB1(SO3) [14, 19].

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

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

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

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

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

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

[0009] The site of interaction with TLR2 is unclear, but glycyrrhizin is known to inhibit this interaction

[21] . It has been suggested that at least one, or possibly both, of the HMG box domains and the acidic tail are involved

[11] . It has been reported that the acidic tail negatively regulates binding, and that a co-ligand is required to displace the acidic tail of HMGB1 from the box domain, enabling signaling through TLR2 [32, 33]. However, several publications have reported TLR2-dependent pro-inflammatory signaling by HMGB1 alone [34, 35]. The role of the redox state of HMGB1 in TLR2 signaling remains unclear, as both the disulfide form

[36] and the fully reduced form

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

[37] (Figure 1B and 1C), and peptides derived from this sequence can effectively inhibit HMGB1-RAGE signaling

[38] . More recently, a second RAGE binding site has been identified in HMG box A

[39] , which has been shown to be active only after proteolysis by caspase-11

[39] . However, the relative contribution of each site to RAGE signaling remains unclear. Despite the current lack of understanding of the binding site, it is also recognized that RAGE-mediated prothrombotic signaling requires the disulfide form of HMGB1, implicating box A

[36] . The acidic tail of HMGB1 may negatively regulate RAGE signaling in a manner similar to TLR2, as it binds to residues within the RAGE-binding peptide [11, 40, 41].

[0010] Successfully translating the regenerative activity of FR-HMGB1 into medical applications depends on preserving CXCL12 binding and signaling through CXCR4 while eliminating all potential deleterious pro-inflammatory signaling. Here, we identify residues in HMGB1 that are important for binding to CXCL12, TLR4, TLR2, and RAGE, and describe HMGB1 variants that eliminate RAGE binding and TLR2 and TLR4 signaling while maintaining regenerative activity. Based on these data, we describe other HMGB1 constructs with these properties. Summary of the Invention

[0011] The present invention relates to a compound of the formula: H2N-AXBAXB-HOOC (In the formula, A is a sequence of consecutive amino acids, the sequence of which is (1) a sequence of four amino acids, (a) is identical to the sequence of amino acids 90 to 93 of wild-type human HMGB1 (SEQ ID NO: 1), or (b) differs from the sequence of (a) by one or more amino acids; and, (2) having 1 to 6 consecutive amino acids at its amino terminus, the sequence of which is (a) is identical to the corresponding sequence of 1 to 6 amino acids preceding amino acid 90 of wild-type human HMGB1, or (b) differs from the sequence of (a) by one or more amino acids; and, (3) optionally, the amino terminus is methionine; Each A may be the same or different; X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 to 162 of wild-type human HMGB1; B is a sequence of consecutive amino acids, the sequence of which is (1) a sequence of 5 or 6 amino acids, (a) It is identical to the sequence of amino acids 163–168 of wild-type human HMGB1; (b) identical to the sequence of amino acids 163–167 of wild-type human HMGB1; (c) in the sequence of (a), any one of amino acids 163, 167, or 168 is changed to another amino acid; (d) in the sequence of (b), any one of amino acids 163 and 167 is changed to another amino acid; (e) the sequence of (a) or (b), wherein amino acid 164 is changed from lysine to arginine; (f) in the sequence of (a) or (b), amino acid 165 is changed from glycine to alanine, serine, or threonine; (g) the sequence of (a) or (b), wherein amino acid 166 is changed from lysine to arginine; (h) the sequence of (a) or (b) is a combination of (e) and (f), (e) and (g), (f) and (g), or (e), (f) and (g); (i) the sequence of (a), (b), or (c) is a combination of one or more of the following modifications: (e), (f), and (g); or (j) the sequence of (d) is a combination of one or more of the modifications of (e), (f), and (g); and, (2) having 1 to 6 consecutive amino acids at its carboxy terminus, the sequence of which is (a) is identical to the corresponding sequence of 1 to 6 amino acids after amino acid 168 of wild-type human HMGB1; (b) is identical to the corresponding sequence of 1 to 6 amino acids after amino acid 167 of wild-type human HMGB1; (c) differs at one or more positions from the corresponding sequence of 1 to 6 amino acids after amino acid 168 of wild-type human HMGB1; or (d) differs at one or more positions from the corresponding sequence of 1 to 6 amino acids after amino acid 167 of wild-type human HMGB1; Each B may be the same or different; - represents a peptide bond between A and X, X and B, B and A, A and X, and X and B; wherein the number of amino acids in BA between two Xs must be at least 12; and in the carboxy-terminal B of the polypeptide, 1 to 6 consecutive amino acids in (2) may not be present. The present invention provides a polypeptide represented by the formula:

[0012] The present invention also provides compositions comprising a polypeptide of the invention and a carrier, and methods for treating a subject suffering from or at risk of developing a condition that can be alleviated by promoting regeneration of tissues or cells that depend on CXCR4+ cells for repair, comprising administering to the subject an amount of a polypeptide or composition of the invention effective to promote regeneration of the tissue or cells and achieve a therapeutic or prophylactic effect. [Brief explanation of the drawings]

[0013] [Figure 1A] 1A to 1C show an outline of the structure of HMGB1 and the locations of its known immunogenic activity. Figure 1A shows the structure of HMGB1 (PDB 2YRQ, conformer 1) showing the α-helices of each box domain. [Figure 1B] Figure 1B shows the structure from Figure 1A colored in PyMol according to its known interactions with LPS, TLR-4, or RAGE. The region involved in binding to TLR-2 is currently unknown. The acidic tail, which is involved in transcriptional regulation and bactericidal activity, is not shown. The structure of HMGB1 is shown, showing the α-helices of each box domain. The original colors are as follows: pink: residues involved in glycyrrhizin binding; red: flexible regions N-terminal to box A or box B; orange: cysteine ​​residues; white: linker regions between HMG boxes; and light green and yellow: RAGE-binding region (incomplete in Figure 1A because it extends into the yellow acidic tail). [Figure 1C] Figure 1C is a schematic diagram of the regions of HMGB1 known to regulate DAMP signaling, as described in the literature, with disulfide bonds shown. [Figure 2A]Figures 2A-2F show the conserved residues in each HMG box domain that are important for CXCL12 binding. Figure 2A shows a 15-mer peptide array (11 x 10) of HMGB1 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 and last two spots are 10-His positive controls. [Figure 2B] Figure 2B shows quantification of spot intensities from Figure 2A (from two experiments) normalized to the 10-His control. The peptides used for the alanine scanning experiment in Figure 2C are shown in the figure. Peptides within the acidic tail were not included because their high negative charge would nonspecifically bind to cationic molecules such as CXCL12. The peptides in the figure are represented by SEQ ID NOS: 8-104, from left to right. [Figure 2C] Figure 2C shows a peptide array of alanine point mutagenesis of the domains identified in Figures 2A and 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, from top to bottom. [Figure 2D] Figure 2D shows quantification of the intensities of the array of peptides (SEQ ID NOS: 105-111) from Figure 2C normalized to the unmodified peptide. Residues with increased CXCL12 binding compared to alanine residues (Ala->Ala, synonymous mutations, gray) are shown in red in the original figure and marked with an asterisk above the bar. [Figure 2E] Figure 2E shows Michaelis-Menten saturation fitting of biotinylated HMGB1 constructs [full-length FR 1–214 (circle), 3S 1–214 (square), minimal box A 8–78 (upward triangle) and box B 94–162 (downward triangle), extended box A 1–88 (diamond) and box B 89–174 (star)] binding to CXCL12. [Figure 2F]Figure 2F shows a summary of the kinetic parameters obtained from Figure 2E. The affinity constants (Kd) obtained from both fits followed the same relationship, with a significant decrease for HMGB1 94-162; fitted data were analyzed by one-way Brown-Forsythe ANOVA. When the AIC supported a model with multiple constants, Kd values ​​were compared by post-hoc two-way ANOVA. Since no significant differences were found in pairwise comparisons (column factors), the values ​​were averaged. The unprocessed interferograms are shown in Figure 12. Req: equilibrium response; kOff: dissociation constant (s-1); kOn: binding constant ((µM × s)-1); AIC: statistical comparison using the Aikaike information criterion (corrected). [Figure 3] Figure 3 shows the binding of HMGB1 to DAMP receptors analyzed by peptide array. These are intensity densitograms from CelluSpot® arrays using 15-mer peptides of HMGB1 baited against TLR-2 ("A", original orange), TLR-4 ("B", original red), or RAGE ("C", original green). Intensities were normalized from 0 (empty spot) to 100 (highest control value) and averaged across two membranes for each target. A schematic diagram of HMGB1 is shown to the left of the peptide sequence. The TLR-2 (A)-binding peptide in HMGB1 occupies similar positions in box A and box B, with a separate binding region immediately preceding the acidic tail. Unlike CXCL12, the binding patterns of TLR-4 (B) and RAGE (C) differ between the two boxes; TLR-4 binding peptides are primarily concentrated in box A and the linker, whereas RAGE binding peptide sequences are concentrated in the C-terminus of box B and a flexible region preceding the N-terminus of the acidic tail. [Figure 4]Figure 4 shows the DAMP receptor-binding peptide in box A and its conformational changes depending on the oxidation state of Cys22-Cys44. The surface of the bound peptide from Figure 3 is superimposed on the published Pymol NMR structure of HMGB1 (PDB 2YRQ). A top row: TLR-2; B middle row: TLR-4; C bottom row: RAGE. The original colors are as follows: cyan: cysteine ​​residues; pink (A only): glycyrrhizin-binding residues involved in the TLR-2 binding site; black (C only): caspase-1 site where HMGB1-induced RAGE-mediated immune tolerance has been reported. The peptides binding C-terminal to residue 164 of HMGB1 are not present in the available protein structure and are therefore not shown in any of the figures. In all three cases, the position of the peptide within box A is affected by oxidation. [Figure 5A]Figures 5A-5B show NMR validation of residues involved in CXCL12 binding. Figure 5A shows the 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 (CSP drift control) without CXCL12 measured after the final concentration point. The intensity of the shading in the bars indicates the relative CSP. The sequence of each HMGB1 construct is overlaid with residue numbers, and empty (unnumbered) columns represent residues that could not be mapped in parallel 3D 1H-15N HSQC / NOE / TOCSY experiments. The sequence of residues corresponding to each HMG box is shown in the original color as follows: light blue indicates residues reported in the literature to be involved in CXCL12 binding (specifically, K95, A100, I112, G114, L119, A136, and Y154); red indicates residues weakly involved in the peptide array (specifically, D90, P91, N92, R96, S99, F101, F104, C105, S106, E107, Y108, R109). , K113, G118, S120, K151, E152, K153, E155, Y161, R162, G165, K166, P167, and D168); purple indicates residues implicated in both the published literature and peptide arrays (specifically, F102, L103, E115, and D157); gray indicates alanine residues (A93, A159, and A160) within the CXCL12-binding peptide (underlined) that could not be assessed by peptide array. The sequence shown in the figure is represented as SEQ ID NO:5. [Figure 5B] Figure 5B shows a heat map of the cumulative peak height change; NMR change of Figure 5A. Red in the original color indicates an I / I change of more than 1 standard error for all residues, and blue in the original color indicates a decrease of more than -1 standard error. These bars are also labeled. The sequence shown in the figure is represented as SEQ ID NO:5. [Figure 6A]Figures 6A-6C show the design of the dBB12L construct, which ablates RAGE, TLR-2, and TLR-4 signaling. Figure 6A shows the alignment of the sequences of box A plus linker (1-88) (SEQ ID NO: 3) and box B plus linker (89-174) (SEQ ID NO: 4). The numbers correspond to the residue numbering in the NMR structure (excluding the N-terminal methionine). Vertical lines indicate strictly conserved positions, and double dots indicate similar amino acids. Underlined: CXCL12-binding peptide regions from the first peptide array. Red in original color: residues that could not be verified by NMR but were flagged as involved in CXCL12 binding in the alanine scan (specifically, in box A, D4, P8, M12, C22, R69, Y70, T76, and P80; in box B, P91, G118, and P167). Orange in original color: residues flagged in the alanine scan and showing either CSP or peak volume changes by NMR (specifically, in box A, K6, R9, G10, K11, S13, S14, H26, K28, K29, H30, K64, D66, E73, K75, Y77, and I78; in box B, D90, R96, S99, F101, F102, L103, F104, S106, R109, K113, K151, E152, I158, Y161, R162, G165, K166, and D168). Cyan in original color: residues that were not flagged in either NMR or peptide array experiments but have been implicated in the NMR literature

[46] as contributing to CXCL12 binding (specifically, in box A, F37, S38, K49, K56; in box B, A100, G114, L119, A136, Y154). Purple in original color: residues that were flagged in peptide array experiments and confirmed by published data or our NMR data (specifically, in box A, V19 and R23; in box B, E115 and D157). Green: residues that may bind CXCL12 directly, may be affected by binding to nearby residues, or were flagged only in NMR experiments (specifically, in box A, A33, K42, K43, S45, T50, S52, K58, D61, and A68; in box B, H116, G122, D123, V124, K126, K127, A147, A148, A159, A163, and K164).Pink in original color: residues flagged in both the NMR experiment and the published data (specifically, A16 in box A, K95 and I112 in box B). [Figure 6B] Figure 6B shows the structure of FR-HMGB1 1-166(2YRQ) with residues colored as shown in Figure 6A. The side chains of all colored residues are indicated. The dashed circles indicate the glycyrrhizin-binding region in each HMG box. The right panel shows a Pymol representation of the CXCL12-binding residues; when box A is reduced, residues flagged by NMR and peptide arrays form a similar binding pocket on the concave surface of each HMGB box. [Figure 6C] Figure 6C shows an overview of the design of the dBB12L construct. The initiation codon Met1 is numbered as Met0 in this specification because it is partially missing in the truncated peptide. Thus, HMGB1 Met1-Gly2···Glu215 becomes Met0-Gly1···Glu214. The domain organization and sequence of the FR-HMGB1 (top, SEQ ID NO: 1) and dBB12L constructs (bottom, SEQ ID NO: 2) are shown. The dBB12L construct was designed as follows: 1. The acidic tail and part of the RAGE-binding domain (175-214) are deleted; 2. Residues 1-88 (box A) are replaced with residues 90-175, resulting in two HMG box B domains; and 3. Residues 163-174 C-terminal to box B are replaced with the native flexible linker (79-88) C-terminal to box A in native HMGB1. CXCL12-binding peptides are shown in red text in their original color and with an asterisk above. Box B repeat units are separated in the diagram by black vertical dashed lines. Box A DAMP receptor-binding peptides are shown with dashed lines, and those in Box B are shown with solid lines. In DBB12L, the TLR-2 and RAGE peptides are truncated, and due to substitutions, not all Box A peptides are present in the construct. [Figure 7A]Figures 7A-7D show that dBB12L has similar stability and surface charge to FR-HMGB1 1-214 / 1-164. Figure 7A shows the calculated Tm50 (in °C) for full-length FR-HMGB1, 1-164 FR-HMGB1, and dBB12L in various buffers as a heat map of the highest (original green) and lowest (original red) values ​​within the global dataset for all constructs. N / A: curve not fittable. The effects of pH and NaCl concentration are summarized in the table below (FR-HMGB1: circles, dBB12L: squares, 1-164 FR-HMGB1: triangles). [Figure 7B] Figure 7B shows native ESI-MS of HMGB1 constructs in 50 mM or 0.2 M ammonium acetate (pH 6.5). The native M / Z profiles of all three HMGB1 constructs are similar, with dBB12L, in which the two HMG boxes are spaced apart, closely resembling 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 increasing ionic strength (compare spectra of the same constructs in 50 mM or 200 mM ammonium acetate) increase the high M / Z state (partial unfolding). [Figure 7C] FIG. 7C shows the calculated average solvent accessible surface area (SASA) of the folded HMGB1 monomer, the extended and compact monomer states, and the unfolded monomer obtained in FIG. 7D. [Figure 7D] FIG. 7D shows denaturing ESI / MS deconvolution, SDS-PAGE, and SEC profiles of HMGB1 constructs after storage in 0.2 M ammonium acetate (pH 6.5) at room temperature for 180 days (D0 to D180). [Figure 8A]Figures 8A-8F show that dBB12L reduces binding to RAGE and does not signal through TLR2 or TLR4. Figure 8A shows a Michaelis-Menten saturation fit of a hybrid ELISA (n=4 per concentration, global fit) showing that the dBB12L construct does not bind to RAGE regardless of oxidation state. DS-HMGB1 binds RAGE more strongly than FR-HMGB1. Data are normalized to the DS-HMGB1 control. [Figure 8B] Figure 8B shows the Michaelis-Menten saturation fitting of biolayer interferometry (0–25 μM HMGB1, six experiments shown in Figure 14). The binding and dissociation rates were calculated from the raw interferogram data alone. [Figure 8C] Figure 8C summarizes the kinetic parameters and color legends (disulfide forms are dashed lines) in Figures 8A and 8B. Invalid fitting indicates an R<0.6 (poor binding). ELISA is under steady-state conditions. For each kinetic parameter, green in the original color indicates the construct with the highest affinity, fastest on (k) or slowest off (k); yellow indicates the midpoint, and red indicates the lowest. [Figure 8D] Figure 8D shows that DS-HMGB1 promoted NF-κB activity in reporter HEK-Dual cells expressing human TLR2 and CD14. dBB12L and FR-HMGB1 did not promote NF-κB signaling. Data are presented as mean ± standard error fold change compared to control (medium alone). [Figure 8E] Figure 8E shows that DS-HMGB1 promoted NF-κB activity in reporter HEK-Dual cells expressing mouse TLR4, MD-2, and CD14. dBB12L and FR-HMGB1 did not promote NF-κB signaling. Data are presented as mean ± standard error fold change compared to control (medium alone). [Figure 8F]Figure 8F shows that disulfide HMGB1 (DS-HMGB1) increased TNF production in monocytes, which was further enhanced in the presence of suboptimal amounts of LTA, but not LPS. FR-HMGB1 and dBB12L did not promote TNF expression, even when preincubated with LPS or LTA for 24 hours. Preincubation with these constructs also significantly reduced the response to LPS. Three donors, three experiments each. [Figure 9] Figure 9 shows the effect of linker modification on the refolding activity of FR-HMGB1. The dBB12L sequence in the alignment is residues 75 to 91 of the sequence shown in SEQ ID NO: 2. The FR-HMGB1 sequence ("FR") in the alignment is residues 79 to 93 of the sequence shown in SEQ ID NO: 1. The sequences of "Sub(79-83)," "Sub(84-88)," and "Sub(89-93)" in the alignment are the sequences shown in SEQ ID NO: 175. [Figure 10A] Figures 10A-10J show that the regenerative effects of dBB-HMGB1 and FR-HMGB1 at optimal doses are identical to those of activated injury. Figure 10A is a volcano plot showing the fold change of differentially expressed genes in muscle stem cells after injury or HMGB1-induced GAlert. The integrals show the preservation of upregulation (brown dots with original color in brackets shown) and downregulation (blue dots with original color in brackets shown) of core genes in GAlert induced by contralateral leg injury or intravenous (iv) HMGB1. [Figure 10B] FIG. 10B shows a network map of gene ontology terms of differentially expressed genes during GAlert induction in muscle stem cells. [Figure 10C] Figure 10C shows the 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. [Figure 10D]Figure 10D shows the results of administering FR-HMGB1 (optimal dose) to animals at various time points after BaCl injection to assess the interval during which FR-HMGB1 treatment is effective after injury. Values ​​are shown as mean ± standard error in nested ANOVA with Holm-Sidak correction. [Figure 10E] FIG. 10E shows the pharmacokinetics of HMGB1 in the blood after administration of 0.75 mg / ml of FR-HMGB1, fitted by nonlinear least-squares to a biphasic exponential decay curve. [Figure 10F] Figure 10F shows a Kaplan-Meier survival plot: 5 weeks post-MI, FR-HMGB1 was 83% and PBS was 52%. [Figure 10G] Figure 10G shows ejection fraction over time calculated from serial MRI scans. The dashed line is the ejection fraction in normal / sham-operated mice. Squares: FR-HMGB1; Circles: PBS control. [Figure 10H] Figure 10H shows infarct size over time calculated from serial MRI scans. Squares: FR-HMGB1; circles: PBS control. [Figure 10I] Figure 10I shows representative mid-atrial short-axis cine MRI images at end-diastole and end-systole of the cardiac cycle at 1 week and 5 weeks after MI. Blood within the ventricle appears bright. The FR-HMGB1 group demonstrated preserved cardiac function and wall thickness (unlabeled short arrows), with separation of the right and left ventricles (arrows labeled RV and LV) during systole. In contrast, the PBS group had significant left ventricular dilation (arrow labeled LV), with very limited contraction between diastole and systole. 10 animals per group. All MRI scans were evaluated by a blinded observer. [Figure 10J] Figure 10J shows the mean muscle cross-sectional area at each time point after treatment of animals with BaCl2-induced muscle damage with PBS (black in original color, left bar), 28.75 nM / kg FR-HMGB1A-c001 (red in original color, middle bar), or dBB12L (green in original color, right bar). One group, five animals per day, nested ANOVA (Holm-Sidak post-hoc correction). Representative images from each time point are shown in Figure 15. [Figure 11A] Figures 11A-11B show the results of a peptide array of CXCL12 peptides that interact with HMGB1. Figure 11A shows a peptide array of full-length CXCL12. The "+" position corresponds to a positive control 10-His peptide, and the remaining peptides contain CXCL12 15-mers shifted toward the C-terminus by two consecutive residues. The membranes were exposed to 1 μM 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. The CXCL12 peptide interacting with full-length HMGB1 was 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 for box A and D90 for box B); the intensity of the spots associated with CXCL12 peptides was also significantly reduced when binding to the box domains alone compared to FL-HMGB1. Binding to 3S appeared to be stronger than binding to FR; this is likely due to protein oxidation during the assay, but was not quantified due to the low protein concentrations used being inappropriate for ESI / TOF MS. However, the BLI data suggest that the dissociation rate of CXCL12 from 3S is slower than that of FR-HMGB1. [Figure 11B] Figure 11B shows the CXCL12 dimer (PDB 2J7Z) with the HMGB1-binding regions highlighted. Red in original color: common binding regions. Blue in original color: non-common binding regions. [Figure 12]Figure 12 shows interferograms of BLI of CXCL12 binding to immobilized HMGB1 constructs. Biotinylated HMGB1 constructs were immobilized on streptavidin-coated Octet biosensors and immersed in increasing concentrations of CXCL12. The lines within each interferogram are in the same order as the key, from top to bottom, unless otherwise noted. Interferograms are colored according to CXCL12 concentration (legend in the upper right). Each set of three replicates (cycles) is surrounded by an overlay colored according to the construct. Line 1: FR FL-HMGB1 (shown as "Sensor information: c011" in the figure), Line 2: 3S-FL HMGB1 (shown as "Sensor information: c022" in the figure), Line 3: FR-HMGB1 Box A 8-78 (shown as "Sensor information: c027" in the figure), Line 4: FR-HMGB1 Box B 94-162 (shown as "Sensor information: c028" in the figure), Line 5: FR-HMGB1 Box A 1-88 (shown as "Sensor information: c037" in the figure), Line 6: FR-HMGB1 Box B 90-162 (shown as "Sensor information: c038" in the figure). [Figure 13A]Figures 13A-13D show NMR validation of residues involved in CXCL12 binding. Figure 13A shows the cumulative CSP (1:1 HMG box to CXCL12) of HMGB1 3S 1-184 (HMGB1A-c007) upon single-step addition of 1:2 molar equivalents of CXCL12. Residues in Box A and Box B are considered separate molecules for the purposes of calculating the median CSP. Darker bars in the bar graph indicate higher relative CSP. Residue numbers are overlaid on the sequence of each HMGB1 construct; empty columns (unnumbered) indicate residues that could not be mapped in parallel 3D 1H-15N HSQC / NOE / TOCSY experiments. The sequence of residues corresponding to each box is centered for each condition and is originally colored as follows: Light blue indicates residues reported to be involved in CXCL12 binding in the literature (specifically, in box B, K95, A100, I112, G114, L119, A136, and Y154; in box A, A16, F40, S41, K49, and K56); red indicates residues weakly involved in peptide arrays (specifically, in box B, D90, P91, N92, R96, S99, F101, F104, C105, S106, E107, Y108, R109, K113, G118, S120, K151, E152, K153, E155, Y161, R162, G165, and K166). , P167, and D168; in box A, D4, K6, K7, P8, R9, G10, K11, M12, S14, F17, F18, T21, E24, E25, H26, K27, K28, K29, H30, D32, S34, V35, N36, K64, D66, R69, Y70, E71, R72, E73, M74, K75, T76, Y77, H78, and P80; purple indicates residues implicated in both the published literature and peptide arrays (specifically, in box B, F102, L103, E115, and D157; in box A, S13, Y15, V19, and R23). The sequences shown are represented as SEQ ID NOs: 6 and 7. [Figure 13B] Figure 13B shows the 15N HSQC-HQMC peak spectrum in 10 mM HEPES, 150 mM NaCl (pH 7.5) buffer shown in Figure 13A. Protein concentration is shown in the spectral overlay. [Figure 13C] Figure 13C shows the N HSQC-HQMC peak spectrum of HMGB1A 94-162 (2-day experiment) in 10 mM HEPES, 150 mM NaCl (pH 7.5) buffer. Protein concentration is indicated by the spectral overlay. [Figure 13D] Figure 13D shows the N HSQC-HQMC peak spectrum of HMGB1A 89-174 (6-day experiment, slight degradation occurred after day 4) in 10 mM HEPES, 150 mM NaCl (pH 7.5) buffer. Protein concentration is shown by the spectral overlay. [Figure 14] Figure 14 shows interferograms of HMGB1 constructs bound to immobilized Fc-RAGE in BLI. RAGE-Fc was immobilized on the surface of an AHC sensor and soaked with various HMGB1 constructs at increasing concentrations. Two experiments were performed at different concentrations; the three columns on the left show 10 steps of HMGB1 from 0 to 22.22 μM, and the columns on the right show 9 steps from 0 to 25 μM. Each graph is color-coded by concentration (shown at the top). Colors indicate construct concentration. The lines from top to bottom in the graphs correspond, in most cases, to the highest to lowest concentrations. Each graph corresponds to a single sensor (replicate). Interferograms enclosed in black rectangles had data that was excluded due to poor quality (e.g., drift). [Figure 15] FIG. 15 shows histological images of regenerating muscles in response to FR-HMGB1 (red) or dBB12L (green) compared to the PBS control (black) shown in FIG. [Figure 16]Figure 16 shows the plasmid vector map. Vector map 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 primer used in colony screening. All plasmids contain kanamycin resistance (50 μg / mL). [Figure 17] FIG. 17 shows a method for producing 3S-HMGB1. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description of the Invention The present invention relates to a compound of the formula: H2N-AXBAXB-HOOC (In the formula, A is a sequence of consecutive amino acids, the sequence of which is (1) a sequence of four amino acids, (a) is identical to the sequence of amino acids 90 to 93 of wild-type human HMGB1 (SEQ ID NO: 1), or (b) differs from the sequence of (a) by one or more amino acids; and, (2) having 1 to 6 consecutive amino acids at its amino terminus, the sequence of which is (a) is identical to the corresponding sequence of 1 to 6 amino acids preceding amino acid 90 of wild-type human HMGB1, or (b) differs from the sequence of (a) by one or more amino acids; and, (3) optionally, the amino terminus is methionine; Each A may be the same or different; X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 to 162 of wild-type human HMGB1; B is a sequence of consecutive amino acids, the sequence of which is (1) a sequence of 5 or 6 amino acids, (a) It is identical to the sequence of amino acids 163–168 of wild-type human HMGB1; (b) identical to the sequence of amino acids 163–167 of wild-type human HMGB1; (c) in the sequence of (a), any one of amino acids 163, 167, or 168 is changed to another amino acid; (d) in the sequence of (b), any one of amino acids 163 and 167 is changed to another amino acid; (e) the sequence of (a) or (b), wherein amino acid 164 is changed from lysine to arginine; (f) in the sequence of (a) or (b), amino acid 165 is changed from glycine to alanine, serine, or threonine; (g) the sequence of (a) or (b), wherein amino acid 166 is changed from lysine to arginine; (h) the sequence of (a) or (b) is a combination of (e) and (f), (e) and (g), (f) and (g), or (e), (f) and (g); (i) the sequence of (a), (b), or (c) is a combination of one or more of the following modifications: (e), (f), and (g); or (j) the sequence of (d) is a combination of one or more of the modifications of (e), (f), and (g); and, (2) having 1 to 6 consecutive amino acids at its carboxy terminus, the sequence of which is (a) is identical to the corresponding sequence of 1 to 6 amino acids after amino acid 168 of wild-type human HMGB1; (b) is identical to the corresponding sequence of 1 to 6 amino acids after amino acid 167 of wild-type human HMGB1; (c) differs at one or more positions from the corresponding sequence of 1 to 6 amino acids after amino acid 168 of wild-type human HMGB1; or (d) differs at one or more positions from the corresponding sequence of 1 to 6 amino acids after amino acid 167 of wild-type human HMGB1; Each B may be the same or different; - represents a peptide bond between A and X, X and B, B and A, A and X, and X and B; wherein the number of amino acids in BA between two Xs must be at least 12; and in the carboxy-terminal B of the polypeptide, 1 to 6 consecutive amino acids in (2) may not be present. The present invention provides a polypeptide represented by the formula:

[0015] In some embodiments, there is a methionine at the amino terminus of the polypeptide.

[0016] In another embodiment, A has an amino acid on its amino terminal side that corresponds to amino acid 89 of wild-type HMGB1 (SEQ ID NO: 1).

[0017] In some embodiments, in B(2), B has 6 amino acids on its carboxy terminal side corresponding to amino acids 169 to 174 of wild-type human HMGB1.

[0018] In some embodiments, the number of amino acids in the BA between two X is at least 13. In such embodiments, the number of amino acids in the BA between two X is at most 22, more preferably at most 21, 20, 19, 18, 17, 16, 15, or 14.

[0019] In another embodiment, the number of amino acids in BA between two X's is 12 to 22.

[0020] In yet another embodiment, the number of amino acids in BA between two X's is 13 to 22. In some embodiments, the number of amino acids in BA between two X's is 13 to 21, more preferably 13 to 20, more preferably 13 to 19, more preferably 13 to 18, more preferably 13 to 17, more preferably 13 to 16, more preferably 13 to 15, or more preferably 13 to 14.

[0021] In some embodiments, the sequence of either or both of B, (2)(c) or (2)(d), has one or more positions that are substituted with glycine, serine, proline, arginine, lysine, aspartic acid, glutamic acid, or histidine in place of the amino acid residue present at that position in native HMG1.

[0022] In some embodiments, the sequence of (2)(c) or (2)(d) in either or both of B is or includes GSGSG (SEQ ID NO: 175).

[0023] In another embodiment, GSGSG (SEQ ID NO: 175) is present between sequence B and sequence A between two X's.

[0024] In yet another embodiment, the order or number of glycine and serine residues is altered within the peptide sequence set forth in SEQ ID NO: 175. By way of non-limiting example, the sequence of (2)(c) or (2)(d) may be or include any of GSSSG (SEQ ID NO: 176), GSSGS (SEQ ID NO: 177), or GGSGG (SEQ ID NO: 178).

[0025] In some embodiments, the amino acid sequence of either or both of B, (2)(c) or (2)(d), lacks any apparent secondary structure or has a turn or random coil secondary structure.

[0026] In some embodiments, either or both of A's are 5 consecutive amino acids.

[0027] In some embodiments, the 5 contiguous amino acids are identical to the sequence of amino acids 89 to 93 of wild-type human HMGB1.

[0028] In some embodiments, a) the A between the Xs is 5 consecutive amino acids and the B between the Xs is at least 7 consecutive amino acids; b) the A's between the X's are 6 consecutive amino acids and the B's between the X's are at least 6 consecutive amino acids; c) the A between the Xs is 7 consecutive amino acids and the B between the Xs is at least 6 consecutive amino acids; d) the A's between the X's are 8 consecutive amino acids and the B's between the X's are at least 6 consecutive amino acids; e) the A's between the X's are 9 consecutive amino acids and the B's between the X's are at least 6 consecutive amino acids; or f) The A's between the X's are 10 consecutive amino acids and the B's between the X's are at least 6 consecutive amino acids.

[0029] In some embodiments, a) the B's between the X's are 6 consecutive amino acids and the A's between the X's are at least 6 consecutive amino acids; b) the B's between the X's are 7 consecutive amino acids and the A's between the X's are at least 5 consecutive amino acids; c) the B's between the X's are 8 consecutive amino acids and the A's between the X's are at least 5 consecutive amino acids; d) the B's between the X's are 9 consecutive amino acids and the A's between the X's are at least 5 consecutive amino acids; e) the B between the Xs is 10 consecutive amino acids and the A between the Xs is at least 5 consecutive amino acids; f) the B's between the X's are 11 consecutive amino acids and the A's between the X's are at least 5 consecutive amino acids; or g) The B's between the X's are 12 consecutive amino acids and the A's between the X's are at least 5 consecutive amino acids.

[0030] The present invention also provides a composition comprising a polypeptide of the invention and a carrier.

[0031] In some aspects, the polypeptide is present in a therapeutically or prophylactically effective amount and the carrier is a pharmaceutically acceptable carrier.

[0032] The present invention also provides a method for treating a subject suffering from or at risk of developing a condition that can be alleviated by promoting regeneration of tissues or cells that depend on CXCR4+ cells for repair, comprising administering to the subject an amount of a polypeptide or composition of the invention that promotes regeneration of the tissue or cells, i.e., achieving a therapeutically or prophylactically effective dose of the pharmaceutical composition of the invention in a subject in need thereof.

[0033] In some embodiments, the condition is an acute injury.

[0034] In currently preferred embodiments, the polypeptide is administered within 5 hours, preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours, and most preferably within 1 hour of acute injury.

[0035] In some embodiments, the condition is a chronic condition.

[0036] In some embodiments, the polypeptide is administered repeatedly daily, weekly, monthly, or yearly.

[0037] In some embodiments, the acute injury is a myocardial infarction. In some embodiments, the tissue is cardiac tissue or myocardium.

[0038] In some embodiments, the acute injury is a stroke, spinal cord injury, or peripheral nerve injury.

[0039] In currently preferred embodiments, the polypeptide is administered within 5 hours after myocardial infarction. In some embodiments, the polypeptide is administered within 5 hours, preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours, and most preferably within 1 hour of myocardial infarction.

[0040] In currently preferred embodiments, the polypeptide is administered within 5 hours after the stroke, spinal cord injury, or peripheral nerve injury. In some embodiments, the polypeptide is administered within 5 hours, preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours, and most preferably within 1 hour of the stroke, spinal cord injury, or peripheral nerve injury.

[0041] In some embodiments, the acute injury is a fracture, joint replacement, or bone fusion. In some embodiments, the tissue is bone.

[0042] In some embodiments, the acute injury is a skeletal muscle injury, a joint injury, or a ligament injury.

[0043] In some embodiments, the condition is associated with liver damage, hi some embodiments, the tissue is liver tissue.

[0044] In some embodiments, the chronic condition is non-alcoholic fatty liver disease, cirrhosis, or infectious hepatitis.

[0045] In some embodiments, the chronic condition is associated with damage to the brain or other parts of the central nervous system.

[0046] In some embodiments, the chronic condition is Parkinson's disease, dementia, multiple sclerosis, motor neuron disease, or peripheral nerve injury.

[0047] In some embodiments, the chronic condition is related to chronic joint damage.

[0048] In some aspects, the chronic joint damage is inflammatory arthritis or osteoarthritis.

[0049] In some embodiments, the condition is associated with lung damage.

[0050] In some embodiments, the acute injury is a viral infection of the lung, a bacterial infection of the lung, a fungal infection of the lung, or a mechanical injury of the lung.

[0051] In some embodiments, the pulmonary viral infection is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

[0052] In some embodiments, the mechanical injury is ventilator injury.

[0053] In some embodiments, the chronic condition is idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease, or emphysema.

[0054] In some embodiments, the condition involves the gut.

[0055] In some embodiments, the acute injury is a perioperative injury to the bowel.

[0056] In some embodiments, the chronic injury is inflammatory bowel disease, Crohn's disease or ulcerative colitis.

[0057] In some embodiments, the condition is associated with skin damage.

[0058] In some embodiments, the acute injury is a burn or a perioperative injury to the skin.

[0059] In some embodiments, the chronic condition is a skin ulcer, a diabetic ulcer, a venous ulcer, an arterial ulcer, or a pressure ulcer.

[0060] In some embodiments, the condition involves the pancreas and the cell is a pancreatic islet cell.

[0061] In some embodiments, the condition is diabetes.

[0062] In some embodiments, the condition is post-chemotherapy neutropenia and the tissue is bone marrow.

[0063] In some embodiments, the acute insult is chemotherapy, and optionally the polypeptide is administered within 5 hours before or after the administration of chemotherapy.

[0064] In some embodiments, the polypeptide is administered before the acute injury.

[0065] In some embodiments, the polypeptide is administered after an acute injury.

[0066] In some embodiments, the condition is renal failure and the tissue is renal tissue.

[0067] In some embodiments, the chronic condition is a disease that causes chronic renal failure.

[0068] In some embodiments, the acute injury is elective surgery and the polypeptide is administered before, during, or within 5 hours after surgery.

[0069] In some embodiments, the acute injury is a sports or combat injury.

[0070] In some embodiments, the chronic condition is a chronic muscular skeletal condition.

[0071] In some aspects, the chronic skeletal muscle condition is a muscular dystrophy, such as Duchenne muscular dystrophy, sarcopenia, or disuse syndrome.

[0072] term To facilitate understanding of the present invention, unless expressly stated otherwise in the specification, each of the following terms has the meaning set forth below.

[0073] As used herein, "engineered" refers to a non-naturally occurring compound produced by modifying a naturally occurring compound. Engineered polypeptides may contain amino acids that correspond to those in naturally occurring polypeptides, as well as amino acids that differ in identity or position from those in naturally occurring polypeptides. Such engineered polypeptides are also referred to as "analogs" or "derivatives" of naturally occurring polypeptides.

[0074] As used herein, "stem cells" refers to undifferentiated cells that have the potential to develop into many different cell types in the body, including but not limited to hematopoietic stem cells.

[0075] As used herein, the term "effective amount" refers to an amount of a polypeptide of the present invention that is capable of achieving a desired result, e.g., alleviating a condition or one or more symptoms associated therewith (e.g., acute or chronic tissue damage). The specific amount of a compound administered in accordance with the present invention will, of course, depend on the particular method of treating or preventing a 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 polypeptide administered to a subject is preferably in the form of a composition comprising a therapeutically or prophylactically effective amount of the engineered HMGB1 polypeptide.

[0076] The phrase "pharmaceutically acceptable" refers to a compound, material, composition, or dosage form that is, within the scope of sound medical judgment, suitable for use in humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable risk / benefit ratio. As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material. The selection of a particular pharmaceutically acceptable carrier is well within the knowledge of one of ordinary skill in the art. Accordingly, there are a wide variety of suitable carriers available and routinely used in pharmaceutical compositions.

[0077] As used herein, the term "a" or "an" should be understood to refer to "one or more" of the listed components.

[0078] In this specification, all numerical ranges recited are intended to expressly include the endpoints and, where consistent with the context, all numbers included between the endpoints of the range.

[0079] Further non-limiting details are provided in the "Experimental Details" section below, which are provided to aid in understanding the invention and are not intended, nor should they be construed, to limit the scope of the disclosed invention in any way. [Example]

[0080] Experiment details result Identification of amino acids and motifs within HMGB1 involved in CXCL12 binding Before investigating possible mutations or deletions of FR-HMGB1 residues to eliminate proinflammatory signaling, it is essential to map the amino acids and motifs involved in CXCL12 binding. Using peptide SPOT arrays

[42] , overlapping peptides covering the sequence of one of the target proteins (HMGB1) were assessed for their ability to bind to CXCL12 by immunoblotting. Two major binding sites were identified (Figures 2A and 2B). The first encompassed α-helix I and part of helix II of each HMG box (peptides 1–3 in box A and peptides 5–6 in box B) and overlapped with the glycyrrhizin-binding site

[43] . The second region (peptide 4 in box A and peptide 7 in box B) was located in the C-terminal half of α-helix III. Within each HMG box, the first CXCL12-binding peptides (helices I and II) showed much greater spot intensity, potentially indicating higher affinity for CXCL12.

[0081] Next, to identify amino acids that directly contribute to CXCL12 interaction, we generated a second peptide array in which each amino acid in the CXCL12-binding peptide was replaced with alanine. This confirmed the important role of several residues (Figures 2C and 2D). In contrast to published data showing that CXCL12 interacts only with the helical segments of the HMG boxes [44-46], we found that CXCL12 interacts with a portion of the flexible N-terminal flanking region of each box (D -4 -PXX -1The subscripted numbers indicate the relative position of the first residue in each HMG box (Pro9 in box A, Pro94 in box B) and residues at the C-terminus (Ile78-Pro80 in box A, Ala163-Asp168 in box B) were also found to be involved (Figure 2E). This was confirmed by reverse peptide arrays (Figure S11) in which CXCL12 peptides were probed for binding to HMGB1. 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))

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

[0082] To further confirm the role of these flexible regions, we used biolayer interferometry (BLI) to assess the binding of HMGB1 constructs with different boundaries to CXCL12. We created either complete HMG box constructs containing the entire HMG box with flanking regions (HMGB1 1-88 for box A and HMGB1 89-174 for box B)

[48] , or helix-only HMG box constructs containing only the core HMG box and no flexible flanking residues (HMGB1 9-78 and HMGB1 94-162). These were compared with full-length HMGB1 constructs (1-214), FR-HMGB1, and nonoxidized (3S)-HMGB1. The latter retains the CXCL12-binding properties of the wild-type protein

[14] . As expected, the helix-only constructs exhibited lower affinity and binding capacity for CXCL12 compared with the full HMG box constructs with intact flanking regions due to their higher dissociation rates. In contrast, the affinities of CXCL12 for full-length FR-HMGB1, full-length 3S-HMGB1, and the full HMG box constructs were comparable to each other and higher than those for the helix-only constructs (Figures 2E and 2F).

[0083] Identification of amino acids and motifs in HMGB1 involved in TLR-2, TLR-4, and RAGE binding Next, we probed the HMGB1 peptide array for proinflammatory receptors (TLR-2, TLR-4, and RAGE). To account for nonspecific binding, the normalized signal of the unbaited array was subtracted from that of the baited array. TLR-2 was found to bind to peptides in box A and box B of HMGB1, which correspond to the glycyrrhizin-binding site in the HMG box (Figure 1B)

[49] (Figure 3, Section A). Furthermore, TLR-2 also bound to the linker region between the two boxes, peptides directly adjacent to the acidic tail of HMGB1, and the C-terminal region of each HMG box, extending beyond the residues that bind CXCL12. Interestingly, the binding interface for TLR-2 within box A lies within an α-helix (Figure 4, Section A) that twists upon oxidation

[50] and adopts a different 3D configuration.

[0084] Peptides that bind to TLR-4 (Figure 3, section B) form a contiguous binding pocket (Figure 4, section A) within box A only when box A is oxidized. This includes the delipidated LPS-binding segment and the region adjacent to the lipid A-binding region within box A

[51] .

[0085] The spot array data for RAGE (Figure 3, section C) confirmed the role of peptides previously shown to regulate RAGE activity [39, 52] (Figure 4, section C), namely, the Rage antagonist peptide (RAP) between positions 149-182 and the caspase-1 activation site in helix 2 of box A. However, in contrast to a previous study in which activity was only seen after caspase-1 cleavage of HMGB1

[39] , we observed that in full-length box A, the box A site is solvent accessible and its position is affected by bending of helix 2 in box A upon oxidation.

[0086] Collectively, these data indicate that distinct regions within each HMG box are involved in the binding of HMGB1 to the three proinflammatory receptors, and that the spatial arrangement of box A is oxidation-dependent. In addition, the linker sequences between the boxes and the N-terminal sequence of the acidic tail have high affinity in all three proinflammatory receptors. Therefore, substitution or deletion of these sequences effectively reduces binding to all these proinflammatory receptors.

[0087] CXCL12 binds to a concave pocket beneath each HMG box, as shown by peptide arrays and NMR (578). Next, NMR was used to identify the amino acid residues within HMGB1 involved in CXCL12 binding from a structural point of view. FR-HMGB1 94-162 and 89-174, and 3S-HMGB1 1-184 were used to represent the HMG box with or without adjacent regions. A complete set of 3D spectra ( 15 N HSQC-TOCSY / NOESY and related 15 Non-oxidized 3S-HMGB1 was used instead of native full-length FR-HMGB1 because the time required to acquire N HSQC spectra would result in oxidation of the latter.

[0088] CXCL12 titration of HMGB1 box B 94-162 (Figure 5A) resulted in changes in the cumulative chemical shift perturbation (CSP) or peak height (I / I0) of several residues in the C- and N-terminal binding regions identified in the peptide array. For box B constructs containing flanking regions, both the median CSP and mean volume change were significantly higher, confirming that several residues in these flanking regions (D90, G165, and K166) are involved in CXCL12 binding. Residues identified in the peptide array as involved in CXCL12 binding, such as Y154, D157, and I158, that were unaffected by CXCL12 binding in the helix-only (94-162) construct, showed significant CSP in the full HMG box construct (89-174), indicating improved binding when the flanking regions were present. Additionally, in constructs with adjacent regions, we observed CSP changes at residues not identified in the peptide array (A147, M131, A169, K172, G173). Other previously unidentified residues flagged as potentially important in the peptide array did not show CSP or volume changes upon addition of CXCL12 (C105, E107, Y108). Therefore, these groups of residues were reclassified as not important for CXCL12. Surprisingly, in contrast to published data

[53] , residues A100, I112, L119, and A136 in the box B construct failed to produce significant CSP changes, although residues very close to some of these (S99, K113) were affected.

[0089] When we repeated the NMR experiment with 3S-HMGB1 1-184, the CSP changes upon CXCL12 addition were below the detection threshold for most residues due to the low signal-to-noise ratio. However, binding above the median CSP change was still observed for some residues. Residues corresponding to HMG box B showed generally weaker CSP changes compared to those in box A. This may represent a more fluid equilibrium, consistent with the larger binding and dissociation rates of box B (Figure 2F). This also allowed us to identify residues in box A identified by peptide array, such as H30, D32, or S34, as false positives (Figure 11). K89 in box B 89-174 showed a high CSP, but this is the third residue from the N-terminus (the remaining N-terminal residue after TEV cleavage, Ser-Met). In experiments with 3S-HMGB1 1-184, the CSP did not increase when it was located in the center of the flexible linker. Therefore, changes involving this residue in box B alone may be related to its N-terminal position, which may allow for high conformational flexibility.

[0090] Design of a double box B HMGB1 construct that retains CXCL12 binding while abrogating pro-inflammatory signaling Combining peptide array, alanine substitution, and NMR data, we mapped the residues involved in binding to CXCL12 onto the NMR structure of HMGB1 (Figures 6A and 6B, PDB 2YRQ) and identified the residues involved in CXCL12 binding. These were found to form a concave pocket beneath each HMG box. These two pockets also contain the binding site for glycyrrhizin, which inhibits HMGB1-CXCL12 binding

[43] . Surprisingly, the distribution of CXCL12-interacting peptides within each HMG box was similar between the two HMG boxes, forming nearly identical binding pockets (Figure 6B). Furthermore, HMG box A and box B can bind a single CXCL12 monomer with comparable affinity, and binding of the CXCL12 monomer does not require cooperation between the two box domains. In contrast, binding to pro-inflammatory receptors requires cooperation between the two box domains. Therefore, we hypothesized that an engineered HMGB1 construct in which box A was replaced with a different box B (i.e., replacing 1-88 with 89-174) would effectively bind two CXCL12 monomers and present CXCR4 membrane dimers, but would be unable to signal through TLR2, TLR4, or RAGE. Deleting the RAGE-binding sequence in box B (175-184) further reduced its propensity to bind RAGE. Replacing the box A sequence with box B also replaces the LPS glycan-binding peptide with an LPS lipid A-binding peptide

[51] . Thus, the engineered construct dBB12L (Figure 6C) consisted of the following segments of the native HMGB1 protein: the 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 and includes residues 172 and 173, which are altered in CSP upon binding to CXCL12.

[0091] We performed differential scanning fluorescence (DSF) and solvent-accessible surface area (SASA) measurements, and compared the thermal stability of dBB12L and wild-type HMGB1 under various buffer conditions using native mass spectrometry (native ESI / MS) and size-exclusion chromatography (SEC). dBB12L, which contains two HMG box domains but lacks the C-terminal acidic tail, had similar stability and surface charge profiles to FR-HMGB1 1-164. The thermal stability trends in various buffers were similar for all constructs (Figure 7A), with Tm 1.0 and Tm 2.0 in buffers with pH values ​​near their isoelectric points (9.9 for dBB12L or the tailless construct 1-164, and 6 for FL-HMGB1). 50 All constructs were equally stable in PBS, purification buffer, and saline solution, with Tm 50 The optimum ionic strength and pH were approximately 50 °C. However, differences in the optimum ionic strength and pH were observed for FR-HMGB1 / FR-HMGB1 1–164 compared to dBB12L. Native ESI-MS analysis revealed that all three HMGB1 constructs had similar charge state distributions, with compact monomers as the predominant species and extended monomers with higher SASA (Figures 7B and 7C). Extended monomers were more prevalent in tailless constructs or at higher ionic strengths. The average monomer SASA values ​​observed by native ESI-MS (Figure 7C) were consistent with those derived from SEC or the published NMR structure (PDB 2YRQ, HMGB1 1–164). The SASA of the compact FL-HMGB1 monomers was consistent with the computational model of FL-HMGB1 in water

[46] . Storage for up to 180 days did not affect the SEC profiles (always monodisperse with equal RVs), degradation, or aggregation (Figure 7D). Taken together, these data indicate that dBB12L has similar folding and stability to native FR-HMGB1 in clinically relevant solutions.

[0092] The dBB12L construct has significantly reduced affinity for RAGE and is unable to signal through TLR2 or TLR4 We next assessed whether dBB12L reduced TLR2 and TLR4 signaling and RAGE binding while maintaining HMGB1-mediated regeneration. Because no RAGE signaling assays have been established, we assessed the binding of HMGB1 to RAGE using real-time kinetics (BLI) and endpoint assays (ELISA). Affinity measurements by ELISA (Figure 8A) showed that at equilibrium, 3S-, FR-, and DS-HMGB1 bound to the same amount of RAGE, and the affinities of DS-HMGB1 and 3S-HMGB1 were significantly higher than those of FR-HMGB1. In contrast, dBB12L did not bind to RAGE. Three other 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 most of the RAGE-binding peptide but retains oxidized box A; and DS-box A alone. DS-HMGB1 1-184 bound to RAGE, but with reduced potency and affinity compared to full-length DS-HMGB1. The RAGE-binding ability of DS-HMGB1 1-164 was significantly reduced compared to full-length DS-HMGB1 but was still higher than that of dBB12L, whereas DS-box A 1-88 (a complete HMG box construct with adjacent regions) failed to bind RAGE.

[0093] Kinetic analysis using BLI was consistent with the ELISA results, with two exceptions (Figure 8B). In BLI (Figure 8C), DS-HMGB1 1-184 had much higher RAGE binding affinity than all other constructs, despite 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 much slower overall kinetics, whereas in ELISA, its affinity and binding capacity were comparable to DS-HMGB1. The higher affinity of DS-HMGB1 for RAGE compared to FR in both assays was due to a faster binding rate (kon ), while the dissociation rate (k off ) were nearly identical in these two redox forms. In contrast, dBB12L, whose binding rate is similar to that of DS-HMGB1, exhibited very unstable binding due to a very fast dissociation rate. DS-HMGB1 1–164 also exhibited a higher binding affinity than dBB12L-HMGB1, but had an overall lower affinity than full-length DS-HMGB1 and a faster RAGE binding equilibrium. In dBB12L, the deletion of the last 10 residues (175–184) of box B and the disulfide bridge in box A, resulting in substitution with box B, destabilized RAGE binding.

[0094] 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 each receptor. Therefore, we first evaluated TLR-specific signaling using NF-κB reporter cell lines engineered to express either TLR2 or TLR4 and their co-receptors. Disulfide HMGB1 promoted NF-κB signaling through TLR2 (Figure 8D) and TLR4 (Figure 8E). In contrast, dBB12L failed to signal in either cell type. Next, we examined the effects of various HMGB1 constructs on primary human monocytes. DS-HMGB1 has been reported to synergize with TLR2 ligands, such as lipoteichoic acid (LTA), to promote pro-inflammatory signaling

[32] . 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 failed to induce greater TNF secretion than medium alone (Figure 8F). The synergistic response between 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 with LPS alone.

[0095] Taken together, these data indicate that dBB12L does not signal through TLR2 or TLR4, even in the presence of its cognate ligand, significantly reduces its affinity for RAGE, and reduces LPS-mediated pro-inflammatory signaling.

[0096] The dBB12L construct exhibits pro-regenerative activity comparable to that of FR-HMGB1 Distal injury induces stem cells to G AlertIt has previously been shown that FR-HMGB1 induces a transition to the G pathway

[13] . Therefore, we first compared the transcriptomic response of skeletal muscle stem cells to FR-HMGB1 or contralateral limb injury. The genes up- and down-regulated by FR-HMGB1 or distal injury were highly similar (Figure 10A), and the major pathways up-regulated were G pathways, including mitochondrial metabolism, oxidative phosphorylation, and cell cycle. Alert (Figure 10B) [8, 13]. Interestingly, CXCR4 was one of the most highly upregulated genes.

[0097] Next, we determined the optimal in vivo therapeutic dose of FR-HMGB1 using a validated mouse model of skeletal muscle injury [8, 13]. We found that the maximal response was achieved at 0.75 mg / kg (29 nmol / kg), and that higher doses did not further improve regenerative activity (Figure 7C). We also evaluated the optimal timing of in vivo administration after injury. FR-HMGB1 was found to be effective in promoting repair when injected up to 5 hours after injury (Figure 10D). Next, we examined the half-life of FR-HMGB1 in the blood after intravenous administration. The initial rapid clearance (t 1 / 2 11 minutes), followed by a slow clearance (t 1 / 2 The half-life of the protein was found to be approximately 120 min (Figure 10E), consistent with a half-life of 25 min in humans

[54] , and the protein is cleared by binding to haptoglobin [55, 56].

[0098] FR-HMGB1 promotes the G-regulation 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 stem cells [8]. The mammalian cardiac progenitor cell population is small

[57] , and the majority of new cardiomyocytes after injury are derived from pre-existing cardiomyocytes

[58] . To confirm the efficacy of FR-HMGB1 in tissues that do not rely on resident stem and progenitor cells for regeneration, we evaluated whether administration of FR-HMGB1 has an effect on cardiac regeneration. Intravenous infusion of FR-HMGB1 during myocardial infarction improved survival (83% in FR-HMGB1-treated mice compared with 52% in PBS controls) (Figure 10F), reduced infarct size by approximately 60% as assessed by serial MRI scans over a 5-week period (Figure 7H), and improved overall left ventricular ejection fraction by 16% (Figure 7G).

[0099] Next, we evaluated the efficacy of dBB12L compared with FR-HMGB1 in promoting skeletal muscle regeneration in vivo. Mice injected with the optimal dose (29 nM / kg) of FR-HMGB1 or dBB12L showed similar accelerated regeneration after injury, as measured by an increase in the average cross-sectional area of ​​regenerating muscle fibers with central nuclei (Figure 10J) [8, 13]. This was most pronounced on day 14, as previously described for FR-HMGB1 [8].

[0100] Finally, we evaluated the role of the flexible region between the HMG boxes in the refolding process. First, we tested whether changes in this linker region of wild-type human FR-HMGB1 affected protein stability. By reducing the number of amino acid residues in the linker to 13 or less, we were able to increase the Tm 50We found that the linker length decreased expression, indicating that linker length plays an important role in protein folding and stability. Furthermore, deletion of the first two prolines in the linker region completely abolished expression, indicating that linker length likely governs protein folding and stability as well. We then tested the in vivo regenerative activity of constructs with various linkers (Figure). Deletion of the entire linker (Δ79-88) resulted in a loss of regenerative activity, while deletion of the first four linker residues (Δ79-82) resulted in some loss of regenerative activity. Deletion of the central region (Δ84-88) completely abolished tissue regeneration, but surprisingly, replacement of residues 84-88 with a sequence of common flexible amino acid residues fully restored regenerative activity. Substitution of a DPNA peptide before the second box B domain also had a negative effect on regenerative activity.

[0101] Consideration HMGB1 needs to be modified for use as a tissue repair therapeutic Therapies using exogenous stem cells to promote solid organ repair have not met their initial promise [6, 59], and dead cells are also effective in eliciting immune responses

[60] . An alternative, potentially more effective approach is to target endogenous regenerative repair processes involving resident stem and progenitor cells [61, 62]. Prostaglandin dehydrogenase inhibition is a promising approach [7] but has been slow to translate into clinical practice

[63] . Growth factor administration has also been used [64, 65], but is limited by in vivo proteolysis

[66] . Currently, no therapeutic agents have been approved to promote regeneration and accelerate repair in multiple tissues.

[0102] We hypothesize that if resident stem and progenitor cells can readily respond to appropriate activating factors released upon tissue injury and repair, exogenous administration of FR-HMGB1 may activate these cells to G AlertWe have previously shown that HMGB1 is effective in accelerating bone, skeletal muscle, and blood regeneration by shifting it to DS-HMGB1 [8]. Meanwhile, accumulating in vivo evidence supports localized conversion of FR-HMGB1 to DS-HMGB1 at injury sites [18, 19]. Therefore, native FR-HMGB1 cannot be used as a therapeutic agent due to the potential for harmful pro-inflammatory signaling. DS-HMGB1 signals through TLR4 [29, 35], TLR2 [32, 33, 35], or RAGE [37, 67], concentrating on NF-κB [25, 68] and resulting in the synergistic expression of pro-inflammatory cytokines [69, 70]. Therefore, the development of HMGB1 as a therapeutic agent will depend heavily on engineering the molecule to eliminate signaling through all three receptors.

[0103] Box A and Box B bind independently to CXCL12 The regenerative activity of FR-HMGB1 is highly dependent on the formation of a heterocomplex with CXCL12 and signaling through CXCR4. While CXCL12 is known to bind to the HMG box [44, 71], the structural motif involved remains unknown [53, 72]. Whether this signaling involves homodimerization of CXCL12 monomers, which promotes chemotaxis, is also unknown [73-75]. By combining data from forward peptide arrays (Figures 2A and 2B) and reverse peptide arrays (Figure 11), alanine substitution arrays (Figures 2C and 2D), and NMR experiments (Figures 5A and 5B), we were able to identify residues involved in the HMGB1-CXCL12 interaction

[76] . We identified a common pattern in HMGB1 peptides that bind to CXCL12, occupying the concave pockets below box A and box B. Using BLI (Figures 2E and 2F), we identified the role of the adjacent flexible region by BLI in CXCL12 binding. Furthermore, because HEPES buffer was used in BLI and NMR experiments to prevent CXCL12 dimerization

[77] , we concluded that each box can bind to CXCL12 monomers without the need for cooperation between the box domains, and that CXCL12 dimerization is not a prerequisite for complex formation.

[0104] Design of engineered dBB12L constructs that do not signal through TLR-2 or TLR-4 or bind RAGE while fully retaining their pro-regenerative properties We found a similar reflection of the HMG box in the region of HMGB1 involved in TLR-2 binding (Figure 3, Section A and Figure 4, Section A), but found that the disulfide form of HMGB1 exhibited enhanced ability to signal through TLR-2 (Figures 8D and 8F). This indicates that, unlike CXCL12 (which has two identical HMG boxes in the reduced molecule), TLR-2 signaling requires two distinct HMG boxes (reduced and oxidized conformations). TLR-4 has long been known to preferentially bind to oxidized HMGB1 box A. We identified a distinct binding pocket for TLR-4 on HMGB1 that overlaps with the LPS-binding segment (1–15, 80–96) and is continuous only when box A is oxidized. RAGE binding requires intact sequences in addition to boxes A and B before oxidation of the acidic tail and box A enhances RAGE binding. Interestingly, in all cases, the linker region of HMGB1 appears to be involved in binding to pro-inflammatory receptors.

[0105] This detailed understanding enabled the design of a construct that maintained CXCL12 binding while abrogating pro-inflammatory signaling via TLR4, TLR2, and RAGE. This construct consisted of two tandem HMG box B domains separated by a linker of similar length to that of wild-type HMGB1 (dBB12L). The absence of box A in dBB12L prevents oxidation and thus prevents the rearrangement of the binding interface observed with TLR-2 (helix 3 rotation), RAGE (helix 2 bending), and TLR-4 (continuous binding surface), while allowing binding to two CXCL12 monomers via the two box B domains. We found that dBB12L was as stable as either 1-164 FR-HMGB1 or full-length FR-HMGB1, neither aggregating nor degrading upon long-term storage.

[0106] DS-HMGB1 alone can bind and signal through TLR4 / MD-2

[28] , but replacing it with LPS-binding protein (LBP), which binds to LPS and promotes its transmission and recognition to TLR4 / MD-2, can enhance LPS-mediated signaling

[51] . Deletion of box A in our dBB12L construct effectively eliminated TLR4-mediated signaling. Interestingly, we observed that FR-HMGB1 and dBB12L reduced TNF expression in monocytes in response to LPS. Unlike DS-HMGB1

[51] , which can effectively replace residual LPS present in serum in the culture medium

[78] , this may be due to the fact that these proteins bind LPS but are unable to transmit LPS to TLR4 / MD-2 due to the lack of oxidized box A.

[0107] There is controversy as to whether HMGB1 can induce TLR2 signaling by itself [34, 35] or whether it requires a co-ligand to induce activity, and whether this response depends on the redox state of the protein [32, 33]. Available data [32, 33] suggest that HMGB1 alone can signal through TLR-2 but requires a co-ligand to induce a high response. We found that DS-HMGB1 alone can signal through TLR2, and its effect was enhanced in the presence of LTA in serum-containing medium. However, there was no response to FR-HMGB1 or dBB12L, which did not synergize with LTA. This suggests that, like TLR4, TLR2-mediated responses require the oxidized box A with a disulfide bridge, and that TLR2 co-ligands synergize with DS-HMGB1, possibly by displacing the acidic tail to facilitate TLR2 interaction.

[0108] The RAGE-binding peptide (residues 149–182) within HMGB1 has been previously described

[37] . Similar motifs are present in other RAGE ligands, such as S100 proteins, and peptides homologous to these sequences are effective antagonists of HMGB1-mediated RAGE signaling [38, 52]. The acidic tail of HMGB1 shares residues with the RAGE-binding peptide

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

[36] . We observed that the RAGE-binding peptide in box A, which was thought to require caspase-1 processing for activation

[39] , is exposed in intact HMGB1 in solution and undergoes disulfide oxidation, changing its conformation. We found that constructs lacking the complete RAGE-binding peptide 149-182, including dBB12L, were unable to bind to RAGE in ELISA assays. Interestingly, in contrast to DS-HMGB1, 3S-HMGB1 also bound to RAGE better than FR-HMGB1. Our BLI data indicated that the affinity for RAGE was lower for dBB12L compared with FR-HMGB1 and DS-HMGB1. dBB12L exhibited a threefold higher binding rate and a fivefold higher dissociation rate than FR-HMGB1, likely due to the presence of two partial RAGE-binding domains in this construct. ELISA data represent equilibrium binding and reflect the balance between association and dissociation rates. For example, 3S-HMGB1, which has similar affinity for RAGE as FR-HMGB1 in BLI, exhibits a much higher apparent affinity in ELISA, similar to DS-HMGB1, due to its much slower dissociation rate than either FR-HMGB1 or DS-HMGB1. This increased RAGE binding may partially explain the increased fibrosis seen in a mouse model of myocardial infarction compared to controls, whereas FR-HMGB1 promoted regeneration and improved function

[31] .Other researchers have also used SPR to show that two distinct binding sites are likely required for HMGB1-RAGE binding, one of which changes depending on the oxidation state

[20] . We used BLI to measure the affinity (K) of DS-HMGB1 for RAGE. d We found that the β-hMGB1 affinity (β = 0.2–1.3 μM) was similar to that previously reported using SPR (0.1

[79] –0.65 μM

[36] ), which also reported high affinity for 3S-HMGB1. The BLI data indicate that loss of the acidic tail significantly increased the affinity of HMGB1 for RAGE due to a significant increase in the binding rate, while truncation of the RAGE-binding peptide or reduction of box A significantly increased the dissociation rate. Interestingly, oxidized box A alone was unable to bind to RAGE, suggesting that the interaction was fully stabilized by the RAGE-binding peptide, and that the acidic tail negatively regulated this binding by competing with residues within the RAGE-binding peptide

[40] . The absence of box A in dBB12L and truncation of the RAGE-binding peptide significantly reduced RAGE affinity.

[0109] The transcriptome changes induced by FR-HMGB1 in skeletal muscle stem cells closely resemble those induced by distal injury, and upregulation of CXCR4 expression by HMBG1 may potentiate its effects. Our data showing that FR-HMGB1 is effective only when administered within 5 hours of injury suggests that it promotes stem cell growth. AlertThis is consistent with the drug acting by promoting the transition of stem cells to the endothelial cell membrane. At this later time point, stem cells are fully activated

[80] . We confirmed that dBB12L retains regenerative activity in vivo comparable to that of FR-HMGB1. Importantly, we found that intravenous administration of FR-HMGB1 at the time of myocardial infarction 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 (e.g., bone, skeletal muscle, blood) and tissues that primarily rely on mature cell populations, such as cardiomyocytes, for regeneration. We predict that dBB12L is likely to be effective when administered within 5 hours after injury. This is important because the median time to hospitalization after an MI in the United States is 3 hours

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

[81] , and approximately 20% progress to heart failure. Despite the medical costs of heart failure in the United States 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

[81] . Based on our data, we predict that administration of dBB12L within 5 hours of onset of symptoms will improve survival in patients with myocardial infarction and reduce the incidence and severity of heart failure by reducing infarct size and preserving ejection fraction. Studies have shown in mouse [31, 82, 83] and sheep

[84] models that direct injection of FR-HMGB1 into the peri-infarct myocardium 4 hours after infarction is effective in promoting cardiac repair. Our data demonstrating its efficacy are important because intravenous administration is easily adaptable for clinical use.

[0110] Based on our data using constructs with various linker lengths and linker substitutions, additional constructs for dBB12L with similar activity profiles can be designed. These may include the following variations:

[0111] 1) Residues corresponding to positions 169-174 at the C-terminus of the second B box in the double box B construct of the present invention (i.e., residues 167-172 of the sequence set forth in SEQ ID NO:2, "AAKKGV") are either completely deleted or substituted as described in 3) below. We hypothesize that substituting the equivalent residues at the C-terminus of the first B box domain in the double box B construct will further help eliminate potential binding to TLR-2.

[0112] 2) The current dBB12L construct of the present invention contains 12 amino acid residues at the C-terminus of the first box B domain, corresponding to positions 163-174 of the native HMGB1 sequence. An additional five amino acids at the N-terminus of the second B box, which were found to be important for CXCL12 binding, result in a total linker length of 17 amino acids between the boxes. Constructs with linker lengths of less than 13 amino acid residues exhibit poor renaturation activity and thermal stability, resulting in unstable molecular folding. Therefore, the optimal linker length is predicted to be 13-20 amino acids. The loss of renaturation activity of constructs with shorter linkers may be related to an inability to adopt the proper conformation to present two CXCL12 monomers to a CXCR4 dimer.

[0113] 3) Linker deletions altered the regenerating activity depending on the location of the deletion: deletion of residues 79-82 (11 residues including the beginning of box B) reduced the regenerating activity of the molecule, whereas deletion of residues 84-88 completely abolished regenerating activity. We also found that substitution of amino acids 84-88 in the interbox linker following the C-terminus of box A with a common flexible amino acid sequence (GSGSG (SEQ ID NO: 175)) did not adversely affect regenerating activity in vivo. Our peptide array and NMR studies did not reveal that this region was involved in CXCL12 binding. Therefore, in the next-generation molecules of the present invention, this region can be replaced with a 13-17 residue linker with the following modifications:

[0114] a) Substitution of any or all of residues corresponding to positions 168-174 of wild-type human HMGB1 (the last seven residues in the linker) with any of the following: A random sequence of amino acids that allows this region to adopt a non-specific secondary structure, either a coil-turn structure or an α-helical conformation (the first two are preferred); Amino acid substitutions (e.g., Lys<->Arg, aliphatic to aliphatic); Flexible amino acid sequences such as Gly-Ser

[85] or turn-inducing residues such as Pro; Charged residues that improve solubility.

[0115] b) Substitution of any or all of the residues corresponding to positions 163 to 167 with any of the options in a), or point deletion of individual residues. However, because substitution or deletion of residues corresponding to this region of wild-type HMGB1 reduced refolding activity, it is predicted that substitution of K164, G165, or K166 may affect CXCL12 binding ability. Therefore, it is thought that these should be replaced with chemically more similar residues.

[0116] c) Substitution of any or all of the five amino acids immediately preceding box B, which comprise the DPXX motif, resulted in the loss of some renaturing activity, but this region, which lies parallel to the third α-helix, may only be required as a flexible extension of the HMG box.

[0117] The amino acid sequences described above are designed with the primary goal of preserving the binding ability of CXCL12 to the modified HMG box domain, including linker modifications, to reduce pro-inflammatory activity (e.g., by removing specific epitopes to further reduce affinity for RAGE, LPS, and other DAMPs / PAMPs), improve protein stability and folding, or introduce chemical functionalization (e.g., click chemistry or unnatural amino acids). Significant modifications of the HMG box core domain are also possible, but are likely to disrupt the structure of the HMG box domain and therefore the CXCL12-binding pocket.

[0118] Our data indicate that while the peptide responsible for binding to CXCL12 is reflected in box A and box B, this is not the case for proinflammatory receptors, as each box and adjacent domains are involved in binding to TLR-2, TLR-4, or RAGE. The deletion of box A and its C-terminal linker in dBB12L explains the absence of signaling through all three proinflammatory receptors. TLR-2 and RAGE binding and signaling are further impaired by the truncation of the binding sequence before the C-terminal acidic tail and the fact that the binding region in oxidized box A is replaced by its equivalent in box B. Because the surface configuration of box B differs from that of box A upon oxidation, it cannot effectively bind to TLR4, TLR-2, or RAGE. Importantly, the dual box B construct maintains regenerative activity equivalent to that of FR-HMGB1. Collectively, these data indicate that the dual box B construct described so far can be developed as a clinical therapeutic agent.

[0119] 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 This is effective when administered within 5 hours after injury. Furthermore, dBB12L promotes tissue regeneration in vivo with efficacy comparable to that of FR-HMGB1. Therefore, dBB12L is clinically viable.

[0120] summary Reduced high mobility group box 1 (HMGB1) protein binds to CXC ligand 12 (CXCL12) and signals through CXC receptor 4 (CXCR4), promoting stem and progenitor cells to G AlertThis promotes tissue regeneration and accelerates repair by transitioning HMGB1 to its disulfide form (DS-HMGB1). However, local conversion of FR-HMGB1 to its disulfide form (DS-HMGB1) can lead to harmful inflammation through signaling via Toll-like receptors 2 and 4 and the receptor for advanced glycation end products (RAGE). Therefore, when considering clinical applications of HMGB1, it is important to manipulate the molecule to eliminate these potentially harmful pro-inflammatory effects.

[0121] We used a combination of peptide arrays, biolayer interferometry, and nuclear magnetic resonance spectroscopy to identify residues involved in the formation of the HMGB1-CXCL12 heterocomplex. Furthermore, we used peptide arrays to define the peptide sequences required for TLR-2, RAGE, and TLR-4 binding. Based on these data, we designed a construct (dBB12L) containing two HMG B boxes in tandem. This construct has similar renaturation ability, stability, and conformation to wild-type, fully reduced HMGB1, does not signal through TLR-2 or TLR-4, even in the presence of their co-ligands, and exhibits significantly reduced RAGE binding. We also describe a series of other double box B structures with similar attributes.

[0122] A comprehensive review of patents and literature identified U.S. Patent Application Publication No. 2015 / 0203551, which describes the substitution of cysteine ​​with serine to prevent TLR4 signaling; however, this construct has been shown to induce excessive cardiac fibrosis after MI

[14] . Furthermore, this construct slowed the dissociation of RAGE compared to FR-HMGB1, resulting in RAGE remaining bound for longer after equilibration, as shown in Figure 8B. Therefore, these substitutions were avoided in the constructs of the present invention. 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 (referred to herein as 0-186, with position 0 being the N-terminal Met), and U.S. Patent No. 9,623,078 cites peptides limited to amino acids 1-44 (referred to herein as 0-43) for cardiac regeneration. U.S. Patent Application Publication No. 2009 / 0202500 discloses methods for tissue repair, but only refers to full-length (1-215) wild-type HMGB1 (herein, 0-214). The dBB12L construct presented herein lacks RAGE binding and TLR4 / 2 signaling, is 117 amino acids long, and contains previously undescribed amino acid substitutions. Therefore, the construct presented herein does not fall within the scope of the prior art.

[0123] Clinical applications The present invention provides polypeptides and methods for harnessing endogenous regenerative processes to enhance tissue repair. 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.

[0124] 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.

[0125] Potential clinical applications: Heart after myocardial infarction This indication is suitable for clinical trials. Globally, ischemic heart disease affects 153 million people

[0101] , with over 105 million disability-adjusted age losses in 2017

[0102] . Each year, 205,000 people in the UK

[0103] and 805,000 people in the US suffer a myocardial infarction (MI), of which 38% experience ST-segment elevation MI (STEMI)

[0101] . 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 that for most cancers

[0101] . The primary target population is post-MI patients, particularly those at risk of developing heart failure

[0104] . Novel therapeutic agents that limit cardiac injury, promote post-MI regeneration, and prevent the development of heart failure would dramatically reduce morbidity and mortality, significantly lowering healthcare costs. Using a well-established permanent ligation murine MI model [105-108] that reliably results in cardiomyocyte necrosis, definitive data show that a single intravenous administration of FR-HMGB1 at the time of injury enhanced survival (83% in FR-HMGB1-treated animals compared with 52% in PBS placebo-treated animals), improved absolute cardiac ejection fraction by approximately 16% compared with controls, and reduced infarct size by approximately 60% compared with PBS controls at 5 weeks (Figure 10F).

[0126] The optimal dose of FR-HMGB1 in the skeletal injury model was 0.75 mg / kg (Figure 10C), and despite its very short half-life (Figure 10E), it was effective even when administered intravenously up to 5 hours after injury (Figure 10D). 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.

[0127] Native FR-HMGB1 promotes functional recovery after MI (Figures 10F–10I), whereas local conversion to the disulfide form promotes thrombus formation and signaling via RAGE, TLR2, and TLR4.

[0110] Constructs reported by others, such as 3S-HMGB1 that retains RAGE binding (Figure 8B), result in excessive fibrosis and dysfunction after MI.

[0111] FR-HMGB1 also binds to RAGE, although 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 and thrombosis after myocardial infarction.

[0112] We have demonstrated an important role for TLR2 in human atherosclerosis.

[0113] TLR4 signaling is also important in myocardial reperfusion injury.

[0114] 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.

[0110] There are no approved therapies to promote cardiac regeneration after MI. Reports claiming to demonstrate the regenerative properties of hematopoietic stem cells have been contradicted.

[0115] Even dead cells can be effective in eliciting an immune response.

[0116] Even with the use of other cell types, including pluripotent stem cells, considerable challenges remain, including arrhythmias, 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]

[0128] The absence of a significant stem cell population in the adult heart

[0121] and the low number of epicardial progenitor cells

[0122] , which suggests that the majority of new cardiomyocytes after injury are derived from pre-existing cardiomyocytes, have shifted the focus to promoting regeneration through manipulation of endogenous pathways

[0121] . These include adenoviral transduction of multiple transcription factors [105,108], manipulation of developmental pathways such as Hippo

[0106] or Meis1

[0123] , addition of growth factors such as neuregulin

[0124] , IGF / HGF

[0125] , or FSTL1

[0126] , or manipulation by miRNA

[0127] . These approaches have significant drawbacks: adenoviral transduction and growth factors (IFGF1 / HGF) require intracardiac injection or application by local patch (FSTL1), manipulation of developmental pathways carries the risk of oncogenicity

[0128] , and viral transduction of miRNA199-a in pigs resulted in fatal arrhythmias

[0127] . An alternative strategy, stimulating cardiac regeneration by promoting immune cell clearance, requires repeated injections of VEGF-C.

[0129] Inhibition of MAP4K4 promoted myocardial survival and limited infarct size but had no regenerative effects.

[0130] To date, none of these strategies have progressed into clinical trials.

[0129] The present invention provides a unique solution that targets endogenous processes to promote cardiomyocyte survival and regeneration of multiple tissues. It utilizes anti-fibrotic CAR-T cells.

[0131] This overcomes many of the obstacles associated with cell therapy, including FR-HMGB1, 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. While HMGB1 inhibition increases infarct size after ischemia-reperfusion injury

[0134] , and local upregulation of FR-HMGB1 [135, 136] or intramyocardial injection has been shown to be effective in both mice [111, 137, 138] and sheep

[0139] , our data indicate that intravenous administration is more likely to be effective and reach all target cells. Our engineered double box B construct, which avoids harmful pro-inflammatory signaling, is safe.

[0130] The Milan group described an HMGB1 analog (3S-HMGB1) in which three cysteines were replaced with serine to abolish TLR4 signaling.

[0140] They claimed that 3S-HMGB1 was superior to FR-HMGB1 in promoting tissue regeneration.

[0141] However, we did not find this to be the case.

[0142] 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.

[0111] We found that 3S-HMGB1 remained bound to RAGE longer than DS-HMGB1 or FR-HMGB1 (Figure 7A). Thus, FR-HMGB1, 3S-HMGB1, and DS-HMGB1 bind comparable amounts of RAGE at equilibrium, but over time, 3S-HMGB1 binds RAGE at levels comparable to pro-inflammatory disulfide HMGB1 (DS-HMGB1) and higher than FR-HMGB1. The dual box B construct of the present invention retains regenerative activity comparable to FR-HMGB1 while eliminating undesirable pro-inflammatory signaling.

[0131] Other applications: fracture Fractures occur after injury. However, one of the most common skeletal "injuries" is joint replacement or arthroplasty. We propose that dBB12 can be used to promote healing after fractures or arthroplasty, thereby reducing potential complications, such as the risk of component loosening.

[0132] Brain and Nervous System dBB12L may be used to improve the prognosis of patients after stroke. Other indications include Parkinson's disease and dementia.

[0133] lung dBB12L is contemplated to improve prognosis after lung injury, for example, after COVID-19 infection or in patients with idiopathic pulmonary fibrosis.

[0134] liver An estimated 30% of the US population suffers from nonalcoholic liver disease. Of these, 60% develop nonalcoholic steatohepatitis, and 20% of these develop cirrhosis. Methods are being developed to limit and prevent liver damage from these conditions. We propose that dBB12L be used in combination with these methods to promote liver regeneration.

[0135] intestines dBB12L may also be used in combination with treatments to control inflammation, for example to promote intestinal healing after surgery or in patients with inflammatory bowel disease (eg ulcerative colitis).

[0136] kidney dBB12L may be used to promote kidney regeneration, thereby potentially avoiding the need for dialysis or kidney transplantation.

[0137] skin dBB12L may be used, for example, to promote wound healing in patients after surgery, with burns, or with ulcers (eg, diabetic ulcers).

[0138] pancreas dBB12L may be used to improve prognosis in patients with type 1 diabetes by promoting regeneration of pancreatic islet cells.

[0139] bone marrow dBB12L may promote regeneration of the hematopoietic system, for example, after chemotherapy, thereby preventing severe, potentially life-threatening neutropenia.

[0140] We have previously shown that FR-HMGB1 is effective even when administered up to 2 weeks before injury. Because dBB12L is as effective as FR-HMGB1 (Figure 10J), this polypeptide may be used prophylactically, for example, for combat or sports injuries, or before elective surgery or chemotherapy.

[0141] Materials and Methods E. coli strains Mach-1 T1R cells (Invitrogen, no antibiotic resistance or induction), BL21(DE3)-R3-pRARE2 (an in-house BL21 derivative, chloramphenicol resistant at 36 μg / mL, T7-polymerase lac induction

[86] ), and BL21(DE3)-R3-pRARE2-BirA (an in vivo biotinylated derivative of the above BL21(DE3)-R3-pRARE2, additionally harboring spectinomycin resistance at 50 μg / mL) were obtained from chemically compatible stocks prepared in-house.

[0142] 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 MgCl2 and 20 mM glucose. 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. TB (Terrific Broth): 12 g / L tryptone, 24 g / L yeast extract, 4 g / L glycerin, 12.5 g / L K2HPO4, 2.35 g / L KH2PO4, autoclave sterilization. TB supplement: 1.6 w / v% glycerol, 1% glucose, 25 mM (NH4)2SO4, 10 mM MgSO4, 10x trace metals, 0.22 μM, sterile filtered. 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 to 0.22 μM. 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 Supplemented with NH4Cl (Cambridge Isotopes), 0.3 mM CaCl2, 1.5 mg / L D-biotin, and 1.5 mg / L thiamine-HCl from sterile filtered stocks.

[0143] Plasmid Plasmids were obtained from the SGC library

[86] . 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 using 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.

[0144] Cloning HMGB1 constructs (Mach1 cells) obtained from the Mammalian Gene Collection were amplified by PCR using a program of 95°C / 10 min, 25 cycles of [95°C / 30 s, 52°C / 1 min, 68°C / 0.5–1.5 min], followed by 68°C / 10 min. Reactions 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 dNTP stock) in a final volume of 25 μL. PCR products were purified (PureLink kit, ThermoFisher K310001) before further use.

[0145] 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 for 2 hours at 37°C before plating on selective medium with 5% sucrose and antibiotics. Positive clones were picked after 24 hours 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 in 12% v / v glycerol at -80°C.

[0146] The 3S-HMGB1 mutant sequence was generated in a similar manner. Separate PCRs were performed to generate the S23-S45 and S106 fragments, which were then annealed by PCR, and 5 μL of each purified PCR product was used as a substitute for primers and template in the reaction. This method is summarized in Figure 17.

[0147] CXCL12 constructs were cloned using an in-frame SUMO protease site N-terminal to the mature protein, allowing 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 [87, 88]. Oxidized CXCL12 was folded via the DsbC fusion protein system

[89] . A detailed table of the primers and vectors used for each construct, their boundaries, expression strains, and base pair / amino acid sequences is provided in the Supplemental Methods section. 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 at Twist Bioscience (San Francisco, USA) and cloned into pNIC-CTHF.

[0148] Recombinant protein expression A 20 mL overnight culture of a transformant of an HMGB1-expressing strain grown from a fresh agar plate streak 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). 15Precultures used to produce N-labeled HMGB1 were first spun down at 1000 RCF for 5 minutes and washed with M9 medium. They were grown to the target OD and cooled to 18°C. Then, 0.5 mM or 0.25 mM IPTG (for HMGB1 and CXCL12 proteins, respectively) was added and grown for 16 hours before harvesting at 4000 RCF. For biotinylated proteins, induction was performed after the addition of 10 mM D-biotin in PBS, followed by cell harvesting after an additional hour.

[0149] 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 pellet was 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 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.

[0150] The protein solution was removed by recirculating through the column (equilibrated in Buffer B + 20 mM imidazole). For biotinylated proteins, streptavidin-XT resin was used instead to select only biotinylated molecules. After 30 minutes of incubation with 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 incubation in 3 CV of the same buffer for 2 hours. Proteins were further purified by size-exclusion chromatography (SEC) (Superdex S75 10 / 300, flow rate 0.35 mL / min; 16 / 600, flow rate 1.2 mL / min) using 10 mM HEPES pH 7.5 + 150 mM NaCl for biophysical studies and cell culture-grade PBS for cell and animal studies. Recombinant proteins were flash-frozen for storage with the addition of 1 mM TCEP in the case of reduced HMGB1 protein.

[0151] Purification of recombinant CXCL12 The outer membranes of cells expressing DsbC-SUMO-CXCL12 were lysed by osmotic shock

[90] . 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 another 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 0.2-1.5 M NaCl gradient 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.

[0152] Endotoxin removal Endotoxin was removed in all cases by phase separation using Triton® Tx-114 prior to size-exclusion chromatography

[91] . 2% v / v TX-114 was added to the recombinant protein solution, homogenized for 20 min with rotary shaking at 2000 RCF at 4°C, and separated for 5 min at 37°C. The detergent phase was then pelleted for 10 min 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 h, 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

[92] , 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, completely removing polymer contaminants. The absence of Triton® and PEG was verified by the absence of these charge-state species using ESI / QTOF-MS mass spectrometry

[93] . 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.

[0153] Enzyme production TEV-GST protease (GST fusion protein), Benzonase-MBP, and Ulp-1 protease were produced from transformants stored in the SGC collection

[86] ; all were ampicillin-resistant at 200 μg / mL. TEV and Ulp-1 were purified once with an IMAC step according to the protocol described for HMGB1. Benzonase-MBP was purified from outer membrane lysates obtained similarly to 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.

[0154] Peptide array For alanine scanning and initial HMGB1 and CXCL12 arrays, membranes of peptides identified on the original membrane, membranes of the complete HMGB1 sequence, or membranes with CXCL12 (Uniprot P48061, minus the secretion signal) were printed by Dr. Sarah Picaud at SGC according to published protocols, as appropriate. Membranes were rehydrated with 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 hours. 1 μM partner His-tagged protein constructs were added (in PBS) and allowed to bind for 24 hours at 4°C. Excess BSA and protein were removed by washing three times with PBST; all washes were for 1 minute unless otherwise noted. 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 min in PBST.

[0155] For TLR-2, TLR-4, and RAGE peptide arrays, CelluSpot arrays were used instead (IntaVis

[94] ). Membranes were baited with one of three receptors fused to the CH2 domain of CXCL12 or IgG (530-TR, 9149-TR, or 1145-RG, BioTechne) as described above and then probed with an anti-CXCL12 antibody (PA5-17238, Invitrogen). Binding of IgG by the Fc fusion protein or anti-CXCL12 was then detected with 0.25 μg / mL of an anti-human IgG CH2 domain antibody (NBP2-68464, custom-conjugated to HRP). A series of peptides covering the CH2 domain of human IgG (Uniprot P01857, 111–223) was used as a control; the highest intensity spot was used as the 100% signal threshold.

[0156] In all cases, bound antibodies were visualized by chemiluminescence (Pierce ECL substrate - 32109): membranes were covered with substrate solution, placed between two clear plastic sheets, and then incrementally imaged at 2-minute intervals using an LAS-4000 camera. The intensity (100%-0%) of each peptide on each membrane was measured using ImageJ and normalized to control and blank spots. Residues whose mutation to alanine resulted in a greater change in intensity than that observed for the alanine position in the sequence were considered to be important contributors to CXCL12 binding.

[0157] 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 (basal 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 a 1:2 dilution) 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 obtained from all binding and dissociation steps in the interferogram and fitted to a horizontal line (mean) for all measurements. Data from each replicate was pooled in the same manner to calculate the overall mean. To measure the kinetics of RAGE binding to HMGB1, 15 μg / mL 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 kinetic parameters were derived by fitting in the same manner.

[0158] 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 In the 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 were analyzed. To measure CXCL12 binding, the chemical shift positions and volumes of identified peaks were tracked at different molar equivalents of CXCL12 (these are listed in the associated images) using the chemical shift tracking module in CCPNMR 3.0 Analyze. For peak intensity shifts, 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.

[0159] mass spectrometry For protein identification by MS / MS and trypsin digestion, bands obtained from SDS-PAGE gels were excised and submitted to the SGC open-access MS platform and analyzed as published by Dr. Rod Chalk, Dr. Tiago Moreira, and Oktawia Borkowska [93, 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 (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

[93] . Each continuous distribution of charge states was considered a distinct conformation, and the charge state (Z) was defined as: Formula: mW = (mW / Z - proton mass) × Z The surface area was derived from the formula proposed in the literature [96, 97], which gives, for native MS, Formula ln(SASA)=ln(M / Z)×0.6897-4.063 and for denatured samples, Formula ln(SASA)=ln(M / Z)×0.9024-5.9013 At least three independent injections were performed for all MS samples. All solutions in these experiments were prepared using HPLC water (electrochemical grade) and solvents.

[0160] 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 of the proteins included in these and the 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.

[0161] 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. Various concentrations of RAGE-Fc chimeric protein (BioTechne, 1145-RG, 0-640 nM at a 1:4 dilution) were 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.

[0162] To detect bound antibody, 25 μL of TMB substrate (ThermoFisher N301) was added to each well and developed in the dark until the FL-DS-HMGB1 control developed a clear concentration-dependent color gradient. The reaction was then 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).

[0163] 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).

[0164] 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).

[0165] Transcriptome analysis Mice were treated systemically 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 identified 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). All suspensions were then passed 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. 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 Benjamini-Hochberg multiple testing adjustment and a false discovery rate cutoff of 0.1. Visualization was performed using the R packages "ggplot2" and "igraph."

[0166] 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 Kennedy Institute's Biological Safety Unit (BSU). 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 designated personnel at the local animal facility, and are registered under the appropriate project and personal licenses under ASPA regulations. All consumables were certified for surgical use, and recombinant proteins were endotoxin-free. Surgeries were performed in a clean environment separate from the euthanasia facility. All animals were monitored for 6 hours postoperatively and daily for three days thereafter; monitoring was then transferred to NVS / NACWO.

[0167] Surgery was performed as previously described [8, 13]. Animals were anesthetized with 2% aerosolized isoflurane and transferred to a heating pad. The right hind limb was disinfected with povidone-iodine, and the tail was disinfected with 70% ethanol for intravenous injections. 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 the hind limbs were removed at specified times 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 of ImageJ2 software (NIH). Data were grouped by mouse. Mice were injected intramuscularly or intravenously with HMGB1 constructs (46 nM / kg, suspended in PBS) or PBS vehicle control at the time of injury or at the optimal time point for the HMGB1 constructs after injury.

[0168] In vivo mouse cardiac injury model C57BL / 6 female mice weighing 25–30 g and aged 10–14 weeks were operated on. All mice were intravenously injected with either FR-HMGB1 (46 nM / kg, suspended in PBS) or vehicle control immediately before surgery. Buprenorphine (buprenorphine hydrochloride; Vetergesic) was delivered as a 0.015 mg / ml solution via intraperitoneal injection 20 minutes before surgery to provide analgesia. Mice were anesthetized with 2.5% isoflurane and ventilated externally 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 cine MRI and analysis. Mice were housed and maintained in a controlled environment. All surgical and pharmacological procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986.

[0169] Cardiac cine MRI and analysis Cardiac cine MRI was performed at 7T using a Varian DDR system after LAD ligation. Briefly, mice were anesthetized with 2% isoflurane in O and placed supine on a custom-built animal handling system with thermoregulation. Prospectively gated proton cardiac images were scanned (128 × 128 matrix, 25.6 mm) to obtain two- and four-atrial long-axis views and short-axis stacks for functional quantification. 2 The partial Fourier accelerated, spoiled gradient echo-cine sequence (FOV, 0.2 mm planar resolution) was acquired with a 72 mm volume-transmit / four-channel surface-receive coil (Rapid Biomedical GmbH) using a 5.9 ms TR, 2.2 ms TE, 30 kHz bandwidth, 30° FA, approximately 20-30 frames gating on the R wave with a 4 ms post-labeling delay; 20% partial acquisition; averaging of four). Unacquired partial Fourier data were reconstructed by the projection onto a convex set method before simple Cartesian DFT. Blinded image analysis was performed using ImageJ (NIH). Left ventricular mass, volume, and ejection fraction were calculated as previously described [1]. Relative infarct size was calculated from the average endocardial and epicardial perimeter of the thinned akinetic area in all slices measured during diastole and expressed as a percentage of the total myocardial surface

[99] .

[0170] statistical analysis All calculations were performed using GraphPad Prism (v. 8.41). For kinetic experiments (BLI / RAGE ELISA), all fitting was performed using nonlinear least-squares. For RAGE ELISA, all data were considered as a single kinetic fit because each RAGE concentration was independent of the remaining wells; however, for BLI, each sensor was considered as an independent fit for calculation purposes. Comparisons between parameters were performed using the AUC method. Data from the mouse muscle injury model 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 was not met in either case, data were analyzed using the Kruskal-Wallis test; for nested ANOVA, an equal number of data from each animal was randomly selected to avoid skew. If unequal variance plots and Q / Q plots supported the equal variance assumption, other data were analyzed with one-way ANOVA; these were also verified by Spearman's test. For multivariate experiments (e.g., cardiac experiments), two-way ANOVA was used with the same assumption (in this case, there were no data sets that violated the unequal variance). Post-hoc comparisons were weighted by the Holm-Sidak correction (ANOVA family test) or the Dunns method (Kruskal-Wallis). The test selected in each case is stated below the respective figure legend. Significance legend: ns; not significant; * ;p<0.033, ** ;p<0.002, *** ;p<0.0002, **** ;p<0.0001.

[0171] 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 5A and 5B

[0172] 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.

[0173] [Table 1]

[0174] [Table 2]

[0175] [Table 3-1]

[0176] [Table 3-2]

[0177] 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.

[0178] [Table 4]

[0179] [Table 4-1]

[0180] [Table 5-1]

[0181] [Table 5-2]

[0182] [Table 6]

[0183] [Table 7-1]

[0184] [Table 7-2]

[0185] [Table 8]

[0186] [Table 9-1]

[0187] [Table 9-2]

[0188] [Table 10]

[0189] [Table 11-1]

[0190] [Table 11-2]

[0191] HMGB1-c038 (89–174, biotinylated) titrated with 0, 0.42, 0.82, and 1.42 molar equivalents of CXCL12A-c021, Figures 5A and 5B

[0192] Due to protein limitations, 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 for volume comparisons.

[0193] [Table 12]

[0194] [Table 13]

[0195] [Table 14-1]

[0196] [Table 14-2]

[0197] 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.

[0198] [Table 15]

[0199] [Table 16]

[0200] [Table 17-1]

[0201] Table 17-2

[0202] Table 18

[0203] Table 19-1

[0204] Table 19-2

[0205] Table 20

[0206] Table 21-1

[0207] Table 21-2

[0208] Table 22

[0209] Table 23-1

[0210] Table 23-2

[0211] 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, Figures 13A–13D

[0212] [Table 24]

[0213] [Table 25]

[0214] [Table 26-1]

[0215] [Table 26-2]

[0216] [Table 26-3]

[0217] 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.

[0218] [Table 27]

[0219] [Table 28]

[0220] [Table 29-1]

[0221] Table 29-2

[0222] Table 29-3

[0223] Table 30

[0224] Table 31-1

[0225] Table 31-2

[0226] Table 31-3

[0227] References 1. Weissman IL: Stem cells: Units of development, units of regeneration, and units in evolution. Cell 2000, 100:157-168. 2. Gratwohl A, Pasquini MC, Aljurf M, Atsuta Y, Baldomero H, Foeken L, Gratwohl M, Bouzas LF, Confer D, Frauendorfer K, et al.: One million haemopoietic stem-cell transplants: a retrospective observational study. Lancet Haematol 2015, 2:e91-e100. 3. Sharma R, Khristov V, Rising A, Jha BS, Dejene R, Hotaling N, Li Y, Stoddard J, Stankewicz C, Wan Q, et al.: Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs. Sci Transl Med 2019, 11:1-14. 4. Ojeh N, Pastar I, Tomic-Canic M, Stojadinovic O: Stem cells in skin regeneration, wound healing, and their clinical applications. Int J Mol Sci 2015, 16:25476-25501. 5. Domb AJ, Nudelman R: In vivo and in vitro elimination of aliphatic polyanhydrides. Biomaterials 1995, 16:319-23. 6. Trainor N, Pietak A, Smith T: Rethinking clinical delivery of adult stem cell therapies. Nat Biotechnol 2014, 32:729-735. 7. Lee G*, Espirito Santo, A.I.*, Zwingenberger Cai L, Vogl T, Feldmann M, Horwood N, J.K-K C, Nanchahal J: HMGB1 accelerates the regeneration of multiple tissues by transitioning stem cells to G(Alert). Proc Natl Acad Sci U S A (*authors Contrib Equal to this Proj 2018, doi:10.1073 / pnas.1802893115. 8. Oppenheim JJ, Yang D: Alarmins: Chemotactic activators of immune responses. Curr Opin Immunol 2005, 17:359-365. 9. Yang D, Rosa G De, Tewary P, Oppenheim JJ: Alarmins Link Neutrophils and Dendritic Cells. Trends Inmunol 2009, 30:531-537. 10. Belgrano FS, De Abreu Da Silva IC, Bastos De Oliveira FM, Fantappie MR, Mohana-Borges R: Role of the acidic tail of high mobility group protein B1 (HMGB1) in protein stability and DNA bending. PLoS One 2013, 8:1-12. 11. Joshi SR, Sarpong YC, Peterson RC, Scovell WM: Nucleosome dynamics: HMGB1 relaxes canonical nucleosome structure to facilitate estrogen receptor binding. Nucleic Acids Res 2012, 40:10161-10171. 12. Rodgers JT, King KY, Brett JO, Cromie MJ, Charville GW, Maguire KK, Brunson C, Mastey N, Liu L, Tsai C-R, et al.: mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert). Nature 2014, 509:393-6. 13. Youn JH, Kwak MS, Wu J, Kim ES, Ji Y, Min HJ, Yoo JH, Choi JE, Cho HS, Shin JS: Identification of lipopolysaccharide-binding peptide regions within HMGB1 and their effects on subclinical endotoxemia in a mouse model. Eur J Immunol 2011, 41:2753-2762. 14. Huttunen HJ, Fages C, Kuja-Panula J, Ridley AJ, Rauvala H: Receptor for advanced glycation end products-binding COOH-terminal motif of amphoterin inhibits invasive migration and metastasis. Cancer Res 2002, 62:4805-4811. 15. Anggayasti WL, Ogino K, Yamamoto E, Helmerhorst E, Yasuoka K, Mancera RL: The acidic tail of HMGB1 regulates its secondary structure and conformational flexibility: A circular dichroism and molecular dynamics simulation study. Comput Struct Biotechnol J 2020, 18:1160-1172. 16. Watson M, Stott K, Thomas JO: Mapping Intramolecular Interactions between Domains in HMGB1 using a Tail-truncation Approach. J Mol Biol 2007, 374:1286-1297. 17. Venereau E, Casalgrandi M, Schiraldi M, Antoine DJ, Cattaneo A, De Marchis F, Liu J, Antonelli A, Preti A, Raeli L, et al.: Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release. J Exp Med 2012, 209:1519-28. 18. Cecchinato V, D’Agostino G, Raeli L, Nerviani A, Schiraldi M, Danelon G, Manzo A, Thelen M, Ciurea A, Bianchi ME, et al.: Redox-Mediated Mechanisms Fuel Monocyte Responses to CXCL12 / HMGB1 in Active Rheumatoid Arthritis. Front Immunol 2018, 9:1-12. 19. Ferrara M, Chialli G, Ferreira LM, Ruggieri E, Careccia G, Preti A, Piccirillo R, Bianchi ME, Sitia G, Venereau E: Oxidation of HMGB1 Is a Dynamically Regulated Process in Physiological and Pathological Conditions. Front Immunol 2020, 11:1-13. 20. Bonaldi T, Talamo F, Scaffidi P, Ferrera D, Porto A, Bachi A, Rubartelli A, Agresti A, Bianchi ME: Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. EMBO J 2003, 22:5551-5560. 21. Kim YH, Kwak MS, Park JB, Lee SA, Choi JE, Cho HS, Shin JS: N-linked glycosylation plays a crucial role in the secretion of HMGB1. J Cell Sci 2016, 129:29-38. 22. Stark K, Philippi V, Stockhausen S, Busse J, Antonelli A, Miller M, Schubert I, Hoseinpour P, Chandraratne S, Von Bruhl ML, et al.: Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice. Blood 2016, 128:2435-2449. 23. Ahrens I, Agrotis A, Topcic D, Bassler N, Chen YC, Bobik A, Bode C, Peter K: HMGB1 binds to activated platelets via platelet-expressed receptor for advanced glycation end products (RAGE) and is highly expressed in platelet rich coronary artery thrombi. Thromb Haemost 2015, 14:994-1003. 24. Yu M, Wang H, Ding A, Golenbock DT, Latz E, Czura CJ, Fenton MJ, Tracey KJ, Yang H: HMGB1 signals through toll-like receptor (TLR) 4 and TLR2. Shock 2006, 26:174-179. 25. van Beijnum JR, Buurman WA, Griffioen AW: Convergence and amplification of toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1). Angiogenesis 2008, 11:91-99. 26. Lotze MT, Tracey KJ: High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 2005, 5:331-42. 27. Sakaguchi M, Murata H, Yamamoto K, Ono T, Sakaguchi Y, Motoyama A, Hibino T, Kataoka K, Huh N: TIRAP, an adaptor protein for TLR2 / 4, transduces a signal from RAGE phosphorylated upon ligand binding. PLoS One 2011, 6:e23132. 28. Venereau E, Schiraldi M, Uguccioni M, Bianchi ME: HMGB1 and leukocyte migration during trauma and sterile inflammation. Mol Immunol 2013, 55:76-82. 29. He M, Bianchi ME, Coleman TR, Tracey KJ, Al-abed Y, Unit CD, Biology C: Exploring the biological functional mechanism of the HMGB1 / TLR4 / MD-2 complex by surface plasmon resonance. [date unknown], 30. Yang H, Hreggvidsdottir HS, Palmblad K, Wang H, Ochani M, Li J, Lu B, Chavan S, Rosas-Ballina M, Al-Abed Y, et al.: A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release. Proc Natl Acad Sci U S A 2010, 107:11942-7. 31. Tirone M, Tran NL, Ceriotti C, Gorzanelli A, Canepari M, Bottinelli R, Raucci A, Maggio S Di, Santiago C, Mellado M, et al.: High mobility group box 1 orchestrates tissue regeneration via CXCR4. J Exp Med 2018, doi:10.1084 / jem.20160217. 32. Di Maggio S, Milano G, De Marchis F, D'Ambrosio A, Bertolotti M, Palacios BS, Badi I, Sommariva E, Pompilio G, Capogrossi MC, et al.: Non-oxidizable HMGB1 induces cardiac fibroblast migration via CXCR4 in a CXCL12-independent manner and worsens tissue remodeling after myocardial infarction. Biochim Biophys Acta - Mol Basis Dis 2017, 1863:2693–2704. 33. Teng W, He X, Shan L, Fan C, Peng S, Wang X, Shan Z, Wang W, Liu Thyroid 2017, 27:722-731. 34. Kwak MS, Lim M, Lee YJ, Lee HS, Kim YH, Ho Youn J, Choi JE, Shin J-S: HMGB1 Binds to Lipoteichoic Acid and Enhances TNF-α and IL-6 Production through HMGB1-Mediated Transfer of Lipoteichoic Acid to CD14 and TLR2. J Innate Immun 2015, 7:405-416. 35. Aucott H, Sowinska A, Harris HE, Lundback P: Ligation of free HMGB1 to TLR2 in the absence of ligand is negatively regulated by the C-terminal tail domain. Mol Med 2018, 24:19. 36. Das N, Dewan V, Grace PM, Gunn RJ, Tamura R, Tzarum N, Watkins LR, Wilson IA, Yin H: HMGB1 Activates Proinflammatory Signaling via TLR5 Leading to Allodynia. Cell Rep 2016, 17:1128-1140. 37. Arumugam T, Ramachandran V, Gomez SB, Schmidt AM, Logsdon CD: S100P-derived RAGE antagonistic peptide reduces tumor growth and metastasis. Clin Cancer Res 2012, 18:4356-4364. 38. LeBlanc PM, Doggett TA, Choi J, Hancock MA, Durocher Y, Frank F, Nagar B, Ferguson TA, Saleh M: An immunogenic peptide in the A-box of HMGB1 protein reverses apoptosis-induced tolerance through RAGE receptor. J Biol Chem 2014, 289:7777-7786. 39. Rauvala H, Rouhiainen A: Physiological and pathophysiological outcomes of the interactions of HMGB1 with cell surface receptors. Biochim Biophys Acta - Gene Regul Mech 2010, 1799:164-170. 40. Nady N, Min J, Kareta MS, Chedin F, Arrowsmith CH: A SPOT on the chromatin landscape? Histone peptide arrays as a tool for epigenetic research. Trends Biochem Sci 2008, 33:305-313. 41. Mollica L, De Marchis F, Spitaleri A, Dallacosta C, Pennacchini D, Zamai M, Agresti A, Trisciuoglio L, Musco G, Bianchi ME: Glycyrrhizin binds to high-mobility group box 1 protein and inhibits its cytokine activities. Chem Biol 2007, 14:431-41. 42. Schiraldi M, Raucci A, Munoz LM, Livoti E, Celona B, Venereau E, Apuzzo T, De Marchis F, Pedotti M, Bachi A, et al.: {HMGB1} promotes recruitment of inflammatory cells to damaged tissues by forming a complex with {CXCL12} and {XCR} signaling via {4}. J Exp Med 2012, 209:551-563. 43. De Leo F, Quilici G, Tirone M, Mannella V, De Marchis F, Preti A, Gori A, Casalgrandi M, Mezzapelle R, Bianchi M, et al.: Diflunisal targets the HMGB1 / CXCL12 heterocomplex and blocks immune cell recruitment. bioRxiv 2019, doi:10.1101 / 563890. 44. Fassi EMA, Sgrignani J, D'Agostino G, Cecchinato V, Garofalo M, Grazioso G, Uguccioni M, Cavalli A: Oxidation State Dependent Conformational Changes of HMGB1 Regulate the Formation of the CXCL12 / HMGB1 Heterocomplex. Comput Struct Biotechnol J 2019, 17:886-894. 45. Bianchi ME, Falciola L, Ferrari S, Lilley DM: The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins. Embo J 1992, 11:1055-1063. 46. Li J, Wang H, Mason JM, Levine J, Yu M, Ulloa L, Czura CJ, Tracey KJ, Yang H: Recombinant HMGB1 with cytokine-stimulating activity. J Immunol Methods 2004, 289:211-223. 47. De Leo F, Quilici G, Tirone M, Mannella V, De Marchis F, Preti A, Gori A, Casalgrandi M, Mezzapelle R, Bianchi M, et al.: Diflunisal targets the HMGB1 / CXCL12 heterocomplex and blocks immune cell recruitment - SM. bioRxiv 2019, doi:10.1101 / 563890. 48. He M, Bianchi ME, Coleman TR, Tracey KJ, Al-abed Y: Exploring the biological functional mechanism of the HMGB1 / TLR4 / MD-2 complex by surface plasmon resonance. Mol Med 2018, 24:1-9. 49. Deng M, Tang Y, Li W, Wang X, Zhang R, Zhang X, Zhao X, Liu J, Tang C, Liu Z, et al.: The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis. Immunity 2018, 49:740-753.e7. 50. Ottestad W, Rognes IN, Pischke SE, Mollnes TE, Andersson U, Eken T: Biphasic Release of the Alarmin High Mobility Group Box 1 Protein Early After Trauma Predicts Poor Clinical Outcome. Crit Care Med 2019, 47:e614-e622. 51. Yang H, Wang H, Wang Y, Addorisio M, Li J, Postiglione MJ, Chavan SS, Al-Abed Y, Antoine DJ, Andersson U, et al.: The haptoglobin beta subunit sequesters HMGB1 toxicity in sterile and infectious inflammation. J Intern Med 2017, 282:76-93. 52. Yang H, Wang H, Levine YA, Gunasekaran MK, Wang Y, Addorisio M, Zhu S, Li W, Li J, de Kleijn DP V, et al.: Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes. JCI insight 2016, 1:1-14. 53. Sadek H, Olson EN: Toward the Goal of Human Heart Regeneration. Cell Stem Cell 2020, 26:7-16. 54. Brooks M: Stem cell research: time for a dose of realism. BMJ 2017, 356. 55. Vagnozzi RJ, Maillet M, Sargent MA, Khalil H, Johansen AKZ, Schwanekamp JA, York AJ, Huang V, Nahrendorf M, Sadayappan S, et al.: An acute immune response underlies the benefit of cardiac stem cell therapy. Nature 2020, 577:405-409. 56. Forbes SJ, Rosenthal N: Preparing the ground for tissue regeneration: From mechanism to therapy. Nat Med 2014, 20:857-869. 57. Lane SW, Williams DA, Watt FM: Modulating the stem cell niche for tissue regeneration. Nat Biotechnol 2014, 32:795-803. 58. Zhang Y, Desai A, Yang SY, Bae KB, Antczak MI, Fink SP, Tiwari S, Willis JE, Williams NS, Dawson DM, et al.: Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration. Science (80- ) 2015, 348. 59. Antczak MI, Zhang Y, Wang C, Doran J, Naidoo J, Voruganti S, Williams NS, Markowitz SD, Ready JM: Inhibitors of 15-Prostaglandin Dehydrogenase To Potentiate Tissue Repair. J Med Chem 2017, 60:3979-4001. 60. Ren X, Zhao M, Lash B, Martino MM, Julier Z: Growth Factor Engineering Strategies for Regenerative Medicine Applications. Front Bioeng Biotechnol 2020, 7:1-9. 61. Yamakawa S, Hayashida K: Advances in surgical applications of growth factors for wound healing. Burn Trauma 2019, 7:1-13. 62. Mitchell AC, Briquez PS, Hubbell JA, Cochran JR: Engineering growth factors for regenerative medicine applications. Acta Biomater 2016, 30:1-12. 63. Hori O, Brett J, Slattery T, Cao R, Zhang J, Chen JX, Stern D, Schmidt AM: The Receptor for Advanced Glycation End Products (RAGE) Is a Cellular Binding Site for Amphoterin. J Biol Chem 1995, 270:25752-25761. 64. Stark K, Philippi V, Stockhausen S, Busse J, Antonelli A, Miller M, Schubert I, Hoseinpour P, Chandraratne S, Von Bruhl ML, et al.: Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice - SM. Blood 2016, 128:2435-2449. 65. Vogel S, Bodenstein R, Chen Q, Feil S, Feil R, Rheinlaender J, Schaeffer TE, Bohn E, Frick JS, Borst O, et al.: Platelet-derived HMGB1 is a critical mediator of thrombosis. J Clin Invest 2015, 125:4638-4654. 66. Maugeri N, Campana L, Gavina M, Covino C, De Metrio M, Panciroli C, Maiuri L, Maseri A, D’Angelo A, Bianchi ME, et al.: Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps. J Thromb Haemost 2014, 12:2074-2088. 67. Ibrahim ZA, Armour CL, Phipps S, Sukkar MB: RAGE and TLRs: Relatives, friends or neighbours? Mol Immunol 2013, 56:739-744. 68. Festoff BW, Sajja RK, van Dreden P, Cucullo L: HMGB1 and thrombin mediate the blood-brain barrier dysfunction acting as biomarkers of neuroinflammation and progression to neurodegeneration in Alzheimer’s disease. J Neuroinflammation 2016, 13:1-12. 69. Hreggvidsdottir HS, Lundberg AM, Aveberger A-C, Klevenvall L, Andersson U, Harris HE: High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor. Mol Med 2012, 18:224-30. 70. Livoti E: Experimentally validated computational docking to characterize protein-protein interactions. [date unknown], 71. Fassi EMA, Sgrignani J, D’Agostino G, Cecchinato V, Garfalo M, Grazioso G, Uguccioni M, Cavalli A: Oxidation state dependent conformational changes of HMGB1 regulates the formation of the CXCL12 / HMGB1 heterocomplex. bioRxiv Biochem 2019, doi:10.1101 / 555946. 72. Drury LJ, Ziarek JJ, Gravel S, Veldkamp CT, Takekoshi T, Hwang ST, Heveker N, Volkman BF, Dwinell MB: Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways. Proc Natl Acad Sci U S A 2011, 108:17655-17660. 73. Chang S, Li Y, Yuan F, Qu M, Song Y, Zhang Z, Yang GY, Wang Y: Monomeric CXCL12 outperforms its dimeric and wild type variants in the promotion of human endothelial progenitor cells min function. Biochem Biophys Res Commun 2017, 488:303-310. 74. Ziarek JJ, Kleist AB, London N, Raveh B, Montpas N, Bonneterre J, St-onge G, Dicosmo-ponticello CJ, Koplinski CA, Roy I, et al.: Structural basis for chemokine recognition by a G protein - coupled receptor and implications for receptor activation. Sci Signal 2017, 5756. 75. Nady N, Min J, Kareta MS, Chedin F, Arrowsmith CH: Supplementary Materials: A SPOT on the chromatin landscape? Histone peptide arrays as a tool for epigenetic research. Trends Biochem Sci 2008, 33:305-313. 76. Veldkamp CT, Peterson FC, Pelzek AJ, Volkman BF: The monomer - dimer equilibrium of stromal cell-derived factor-1 (CXCL12) is altered by pH, phosphate, sulfate, and heparin. Protein Sci 2005, 1:1071-1081. 77. Akira S: Toll-like Receptors and Innate Immunity. In Edited by Dixon FJBT-A in I. Academic Press; 2001:1-56. 78. Lee S, Piao C, Kim G, Kim JY, Choi E, Lee M: Production and application of HMGB1 derived recombinant RAGE-antagonist peptide for anti-inflammatory therapy in acute lung injury. Eur J Pharm Sci 2018, 114:275-284. 79. Xu D, Young J, Song D, Esko JD: Heparan sulfate is essential for high mobility group protein 1 (HMGB1) signaling by the receptor for advanced glycation end products (RAGE). J Biol Chem 2011, 286:41736-41744. 80. Schlessinger K, Hall A, Tolwinski N: Wnt signaling pathways meet Rho GTPases. Genes Dev 2009, 23:265-77. 81. Bryja V, Cervenka I, Cajanek L: The connections of Wnt pathway components with cell cycle and centrosome: side effects or a hidden logic? Crit Rev Biochem Mol Biol 2017, 52:614-637. 82. Gurevich DB, Nguyen PD, Siegel AL, Ehrlich O V., Sonntag C, Phan JMN, Berger S, Ratnayake D, Hersey L, Berger J, et al.: Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo. Science (80- ) 2016, 353. 83. Benjamin EJ, Muntner P, Alonso A, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Chang AR, Cheng S, Das SR, et al.: Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. 2019. 84. Limana F, Esposito G, D’Arcangelo D, Di Carlo A, Romani S, Melillo G, Mangoni A, Bertolami C, Pompilio G, Germani A, et al.: HMGB1 attenuates cardiac remodelling in the failing heart via enhanced cardiac regeneration and miR-206-mediated inhibition of TIMP-3. PLoS One 2011, 6:1-11. 85. Foglio E, Puddighinu G, Germani A, Russo MA, Limana F: HMGB1 Inhibits Apoptosis Following MI and Induces Autophagy via mTORC1 Inhibition. J Cell Physiol 2017, 232:1135-1143. 86. Bauza M del R, Gimenez CS, Locatelli P, De Lorenzi A, Hnatiuk A, Capogrossi MC, Crottogini A, Cuniberti L, Olea FD: High-dose intramyocardial HMGB1 induces long-term cardioprotection in sheep with myocardial infarction. Drug Deliv Transl Res 2019, 9:935-944. 87. Savitsky P, Bray J, Cooper CDO, Marsden BD, Mahajan P, Burgess-Brown NA, Gileadi O: High-throughput production of human proteins for crystallization: The SGC experience. J Struct Biol 2010, 172:3-13. 88. Yang OO, Swanberg SL, Lu Z, Dziejman M, McCoy J, Luster AD, Walker BD, Herrmann SH: Enhanced inhibition of human immunodeficiency virus type 1 by Met-stromal-derived factor 1beta correlates with down-modulation of CXCR4. J Virol 1999, 73:4582-4589. 89. Crump MP, Gong JH, Loetscher P, Rajarathnam K, Amara A, Arenzana-Seisdedos F, Virelizier JL, Baggiolini M, Sykes BD, Clark-Lewis I: Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J 1997, 16:6996-7007. 90. Turchetto J, Sequeira AF, Ramond L, Peysson F, Bras JLA, Saez NJ, Duhoo Y, Blemont M, Guerreiro CIPD, Quinton L, et al.: High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery. Microb Cell Fact 2017, 16:6. 91. Thein M, Sauer G, Paramasivam N, Grin I, Linke D: Efficient subfractionation of gram-negative bacteria for proteomics studies. J Proteome Res 2010, 9:6135-6147. 92. Aida Y, Pabst MJ: Removal of endotoxin from protein solutions by phase separation using triton X-114. J Immunol Methods 1990, 132:191-195. 93. Teodorowicz M, Perdijk O, Verhoek I, Govers C, Savelkoul HFJ, Tang Y, Wichers H, Broersen K: Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins. PLoS One 2017, 12:e0173778. 94. Chalk R: Mass Spectrometric Analysis of Proteins. Humana Press, New York, NY; 2017:373-395. 95. Chalk R, Berridge G, Shrestha L, Strain-Damerell C, Mahajan P, Yue W, Gileadi O, Burgess-Brown N: High-Throughput Mass Spectrometry Applied to Structural Genomics. Chromatography 2014, 1:159-175. 96. Kaltashow IA, Mohimen A: Electrospray ionization mass spectrometry can provide estimates of protein surface areas in solution. Anal Chem 2005, 77:5370-5379. 97. Testa L, Brocca S, Grandori R: Charge-surface correlation in electrospray ionization of folded and unfolded proteins. Anal Chem 2011, 83:6459-6463. 98. Yu G, Wang LG, Han Y, He QY: ClusterProfiler: An R package for comparing biological themes among gene clusters. Omi A J Integr Biol 2012, doi:10.1089 / omi.2011.0118. 99. Carr CA, Stuckey DJ, Tatton L, Tyler DJ, Hale SJM, Sweeney D, Schneider JE, Martin-Rendon E, Radda GK, Harding SE, et al.: Bone marrow-derived stromal cells home to and remain in the infarcted rat heart but fail to improve function: An in vivo cine-MRI study. Am J Physiol - Hear Circ Physiol 2008, 295. 100. Lee S, Lee DK: Multiple Comparison Test and Its Imitations What is the proper way to apply the multiple comparison test? KJA. Korean J Anesth 2018, doi:10.4097 / kja.d.18.00242. 101. E. J. Benjamin, P. Muntner, A. Alonso, M. S. Bittencourt, C. W. Callaway, A. P. Carson, A. M. Chamberlain, A. R. Chang, S. Cheng, S. R. Das, F. N. Delling, L. Djousse, M. S. V. Elkind, J. F. Ferguson, M. Fornage, L. C. Jordan, S. S. Khan, B. M. Kissela, K. L. Knutson, T. W. Kwan, D. T. Lackland, T. T. Lewis, J. H. Lichtman, C. T. Longenecker, M. S. Loop, P. L. Lutsey, S. S. Martin, K. Matsushita, A. E. Moran, M. E. Mussolino, M. O'Flaherty, A. Pandey, A. M. Perak, W. D. Rosamond, G. A. Roth, U. K. A. Sampson, G. M. Satou, E. B. Schroeder, S. H. Shah, N. L. Spartano, A. Stokes, D. L. Tirschwell, C. W. Tsao, M. P. Turakhia, L. B. VanWagner, J. T. Wilkins, S. S. Wong, S. S. Virani, E. American Heart Association Council on, C. Prevention Statistics, S. Stroke Statistics, Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. Circulation 139, e56-e528 (2019). 102. G. B. D. DALYs, H. Collaborators, Global, regional, and national disability-adjusted life-years (DALYs) for 359 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 392, 1859-1922 (2018). 103. BritishHeartFoundation, bhf.org.uk / what-we-do / our-research / heart-statistics / heart-statistics-publications / cardiovascular-disease-statistics-2019, (2020). 104. T. J. Cahill, R. K. Kharbanda, Heart failure after myocardial infarction in the era of primary percutaneous coronary intervention: Mechanisms, incidence and identification of patients at risk. World J Cardiol 9, 407-415 (2017). 105. L. Qian, Y. Huang, C. I. Spencer, A. Foley, V. Vedantham, L. Liu, S. J. Conway, J. D. Fu, D. Srivastava, In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593-598 (2012). 106. M. Xin, Y. Kim, L. B. Sutherland, M. Murakami, X. Qi, J. McAnally, E. R. Porrello, A. I. Mahmoud, W. Tan, J. M. Shelton, J. A. Richardson, H. A. Sadek, R. Bassel-Duby, E. N. Olson, Hippo pathway effector Yap promotes cardiac regeneration. Proc Natl Acad Sci U S A 110, 13839-13844 (2013). 107. J. P. Leach, T. Heallen, M. Zhang, M. Rahmani, Y. Morikawa, M. C. Hill, A. Segura, J. T. Willerson, J. F. Martin, Hippo pathway deficiency reverses systolic heart failure after infarction. Nature 550, 260-264 (2017). 108. T. M. A. Mohamed, Y. S. Ang, E. Radzinsky, P. Zhou, Y. Huang, A. Elfenbein, A. Foley, S. Magnitsky, D. Srivastava, Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration. Cell 173, 104-116 e112 (2018). 109. S. Hashmi, S. Al-Salam, Acute myocardial infarction and myocardial ischemia-reperfusion injury: a comparison. Int J Clin Exp Pathol 8, 8786-8796 (2015). 110. K. Stark, V. Philippi, S. Stockhausen, J. Busse, A. Antonelli, M. Miller, I. Schubert, P. Hoseinpour, S. Chandraratne, ML von Bruhl, F. Gaertner, M. Lorenz, A. Agresti, R. Coletti, DJ Antoine, R. Heermann, K. Jung, S. Reese, I. Laitinen, M. Schwaiger, A. Walch, M. Sperandio, PP Nawroth, C. Reinhardt, S. Jackel, ME Bianchi, S. Massberg, Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice. Blood 128, 2435-2449 (2016). 111. S. Di Maggio, G. Milano, F. De Marchis, A. D'Ambrosio, M. Bertolotti, BS Palacios, I. Badi, E. Sommariva, G. Pompilio, MC Capogrossi, A. Raucci, Non-oxidizable HMGB1 induces cardiac fibroblast migration via CXCR4 in a CXCL12-independent manner and worsens tissue remodeling after myocardial infarction. Biochim Biophys Acta 1863, 2693–2704 (2017). 112. J. Mersmann, F. Iskandar, K. Latsch, K. Habeck, V. Sprunck, R. Zimmermann, R. R. Schumann, K. Zacharowski, A. Koch, Attenuation of myocardial injury by HMGB1 blockade during ischemia / reperfusion is toll-like receptor 2-dependent. Mediators Inflamm 2013, 174168 (2013). 113. C. Monaco, S. M. Gregan, T. J. Navin, B. M. Foxwell, A. H. Davies, M. Feldmann, Toll-like receptor-2 mediates inflammation and matrix degradation in human atherosclerosis. Circulation 120, 2462-2469 (2009). 114. J. Oyama, C. Blais, Jr., X. Liu, M. Pu, L. Kobzik, R. A. Kelly, T. Bourcier, Reduced myocardial ischemia-reperfusion injury in toll-like receptor 4-deficient mice. Circulation 109, 784-789 (2004). 115. K. R. Chien, J. Frisen, R. Fritsche-Danielson, D. A. Melton, C. E. Murry, I. L. Weissman, Regenerating the field of cardiovascular cell therapy. Nat Biotechnol 37, 232-237 (2019). 116. R. J. Vagnozzi, M. Maillet, M. A. Sargent, H. Khalil, A. K. Z. Johansen, J. A. Schwanekamp, A. J. York, V. Huang, M. Nahrendorf, S. Sadayappan, J. D. Molkentin, An acute immune response underlies the benefit of cardiac stem cell therapy. Nature 577, 405-409 (2020). 117. C. E. Murry, W. R. MacLellan, Stem cells and the heart-the road ahead. Science 367, 854-855 (2020). 118. S. Bollini, N. Smart, P. R. Riley, Resident cardiac progenitor cells: at the heart of regeneration. J Mol Cell Cardiol 50, 296-303 (2011). 119. P. K. Nguyen, E. Neofytou, J. W. Rhee, J. C. Wu, Potential Strategies to Address the Major Clinical Barriers Facing Stem Cell Regenerative Therapy for Cardiovascular Disease: A Review. JAMA Cardiol 1, 953-962 (2016). 120. T. J. Cahill, R. P. Choudhury, P. R. Riley, Heart regeneration and repair after myocardial infarction: translational opportunities for novel therapeutics. Nat Rev Drug Discov 16, 699-717 (2017). 121. H. Sadek, E. N. Olson, Toward the Goal of Human Heart Regeneration. Cell Stem Cell 26, 7-16 (2020). 122. N. Smart, S. Bollini, K. N. Dube, J. M. Vieira, B. Zhou, S. Davidson, D. Yellon, J. Riegler, A. N. Price, M. F. Lythgoe, W. T. Pu, P. R. Riley, De novo cardiomyocytes from within the activated adult heart after injury. Nature 474, 640-644 (2011). 123. A. I. Mahmoud, F. Kocabas, S. A. Muralidhar, W. Kimura, A. S. Koura, S. Thet, E. R. Porrello, H. A. Sadek, Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature 497, 249-253 (2013). 124. K. Bersell, S. Arab, B. Haring, B. Kuhn, Neuregulin1 / ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury. Cell 138, 257-270 (2009). 125. S. Koudstaal, MM Bastings, DA Feyen, CD Waring, FJ van Slochteren, PY Dankers, D. Torella, JP Sluijter, B. Nadal-Ginard, PA Doevendans, GM Ellison, SA Chamuleau, Sustained delivery of insulin-like growth factor-1 / hepatocyte growth factor stimulates endogenous cardiac repair in the chronic infarcted pig heart. J Cardiovasc Transl Res 7, 232-241 (2014). 126. K. Wei, V. Serpooshan, C. Hurtado, M. Diez-Cunado, M. Zhao, S. Maruyama, W. Zhu, G. Fajardo, M. Noseda, K. Nakamura, Butte, PC Yang, K. Walsh, B. Zhou, D. Bernstein, M. Mercola, P. Ruiz-Lozano, Epicardial FSTL1 reconstitution regenerates the adult mammalian heart. Nature 525, 479-485 (2015). 127. K. Gabisonia, G. Prosdocimo, G. D. Aquaro, L. Carlucci, L. Zentilin, I. Secco, H. Ali, L. Braga, N. Gorgodze, F. Bernini, S. Burchielli, C. Collesi, L. Zandona, G. Sinagra, M. Piacenti, S. Zacchigna, R. Bussani, FA Recchia, M. Giacca, MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs. Nature 569, 418–422 (2019). 128. A. Vujic, N. Natarajan, RT Lee, Molecular mechanisms of heart regeneration. Semin Cell Dev Biol, (2019). 129. JM Vieira, S. Norman, C. Villa Del Campo, TJ Cahill, DN Barnette, M. Gunadasa-Rohling, LA Johnson, DR Greaves, CA Carr, DG Jackson, PR Riley, The cardiac lymphatic system stimulates resolution of inflammation following myocardial infarction. J Clin Invest 128, 3402-3412 (2018). 130. L. R. Fiedler, K. Chapman, M. Xie, E. Maifoshie, M. Jenkins, P. A. Golforoush, M. Bellahcene, M. Noseda, D. Faust, A. Jarvis, G. Newton, M. A. Paiva, M. Harada, D. J. Stuckey, W. Song, J. Habib, P. Narasimhan, R. Aqil, D. Sanmugalingam, R. Yan, L. Pavanello, M. Sano, S. C. Wang, R. D. Sampson, S. Kanayaganam, G. E. Taffet, L. H. Michael, M. L. Entman, T. H. Tan, S. E. Harding, C. M. R. Low, C. Tralau-Stewart, T. Perrior, M. D. Schneider, MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo. Cell Stem Cell 24, 579-591 e512 (2019). 131. H. Aghajanian, T. Kimura, J. G. Rurik, A. S. Hancock, M. S. Leibowitz, L. Li, J. Scholler, J. Monslow, A. Lo, W. Han, T. Wang, K. Bedi, M. P. Morley, R. A. Linares Saldana, N. A. Bolar, K. McDaid, C. A. Assenmacher, C. L. Smith, D. Wirth, C. H. June, K. B. Margulies, R. Jain, E. Pure, S. M. Albelda, J. A. Epstein, Targeting cardiac fibrosis with engineered T cells. Nature 573, 430-433 (2019). 132. N. Trainor, A. Pietak, T. Smith, Rethinking clinical delivery of adult stem cell therapies. Nat Biotechnol 32, 729-735 (2014). 133. M. J. Foglia, K. D. Poss, Building and re-building the heart by cardiomyocyte proliferation. Development 143, 729-740 (2016). 134. S. Oozawa, S. Mori, T. Kanke, H. Takahashi, K. Liu, Y. Tomono, M. Asanuma, I. Miyazaki, M. Nishibori, S. Sano, Effects of HMGB1 on ischemia-reperfusion injury in the rat heart. Circ J 72, 1178-1184 (2008). 135. T. Kitahara, Y. Takeishi, M. Harada, T. Niizeki, S. Suzuki, T. Sasaki, M. Ishino, O. Bilim, O. Nakajima, I. Kubota, High-mobility group box 1 restores cardiac function after myocardial infarction in transgenic mice. Cardiovasc Res 80, 40-46 (2008). 136. Y. Nakamura, S. Suzuki, T. Shimizu, M. Miyata, T. Shishido, K. Ikeda, S. Saitoh, I. Kubota, Y. Takeishi, High Mobility Group Box 1 Promotes Angiogenesis from Bone Marrow-derived Endothelial Progenitor Cells after Myocardial Infarction. J Atheroscler Thromb 22, 570-581 (2015). 137. F. Limana, G. Esposito, D. D'Arcangelo, A. Di Carlo, S. Romani, G. Melillo, A. Mangoni, C. Bertolami, G. Pompilio, A. Germani, M. C. Capogrossi, HMGB1 attenuates cardiac remodelling in the failing heart via enhanced cardiac regeneration and miR-206-mediated inhibition of TIMP-3. PLoS One 6, e19845 (2011). 138. E. Foglio, G. Puddighinu, A. Germani, M. A. Russo, F. Limana, HMGB1 Inhibits Apoptosis Following MI and Induces Autophagy via mTORC1 Inhibition. J Cell Physiol 232, 1135-1143 (2017). 139. M. D. R. Bauza, C. S. Gimenez, P. Locatelli, A. De Lorenzi, A. Hnatiuk, M. C. Capogrossi, A. Crottogini, L. Cuniberti, F. D. Olea, High-dose intramyocardial HMGB1 induces long-term cardioprotection in sheep with myocardial infarction. Drug Deliv Transl Res 9, 935-944 (2019). 140. M. E. Bianchi, Casalgrandi, M., Venereau, E. J., Brunelli, S., HMGB1 variants and uses thereof. European Patent Office - patentimages.storage.googleapis.com / cf / 3c / 6c / 3b9d7ca70f194a / EP2877248B1.pdf (2012). 141. M. Tirone, NL Tran, C. Ceriotti, A. Gorzanelli, M. Canepari, R. Bottinelli, A. Raucci, S. Di Maggio, C. Santiago, M. Mellado, M. Saclier, S. Francois, G. Careccia, M. He, F. De Marchis, V. Conti, S. Ben Larbi, S. Cuvellier, M. Casalgrandi, A. Preti, B. Chazaud, Y. Al-Abed, G. Messina, G. Sitia, S. Brunelli, ME Bianchi, E. Venereau, High mobility group box 1 orchestrates tissue regeneration via CXCR4. J Exp Med 215, 303–318 (2018). 142. G. Lee, AI Espirito Santo, S. Zwingenberger, L. Cai, T. Vogl, M. Feldmann, NJ Horwood, JK Chan, J. Nanchahal, Fully reduced HMGB1 accelerates the regeneration of multiple tissues by transitioning stem cells to GAlert. Proc Natl Acad Sci USA 115, E4463-E4472 (2018).

Claims

1. The following formula: H ２ N−A−X−B−A−X−B−COOH (wherein A is a consecutive amino acid, and its sequence is (1) a sequence of 4 amino acids, (a) the same as the sequence of amino acids 90 to 93 of wild-type human HMGB1 (SEQ ID NO: 1), or (b) different from the sequence in (a) by one or more amino acids; and (2) having 1 to 6 consecutive amino acids on its amino-terminal side, and its sequence is (a) the same as the sequence of the corresponding 1 to 6 amino acids before amino acid 90 of wild-type human HMGB1, or (b) different from the sequence in (a) by one or more amino acids; and (3) optionally, the amino terminus is methionine, each A may be the same or different; X is a consecutive amino acid, and its sequence is the same as the sequence of amino acids 94 to 162 of wild-type human HMGB1; B is a consecutive amino acid, and its sequence is (1) a sequence of 5 or 6 amino acids, (a) the same as the sequence of amino acids 163 to 168 of wild-type human HMGB1; (b) the same as the sequence of amino acids 163 to 167 of wild-type human HMGB1; (c) in the sequence of (a), any one of amino acids 163, 167 or 168 is changed to another amino acid; (d) in the sequence of (b), any one of amino acids 163 or 167 is changed to another amino acid; (e) in the sequence of (a) or (b), amino acid 164 is changed from lysine to arginine; (f) in the sequence of (a) or (b), amino acid 165 is changed from glycine to alanine, serine or threonine; (g) in the sequence of (a) or (b), amino acid 166 is changed from lysine to arginine; (h) the sequence of (a) or (b) is a combination of (e) and (f), (e) and (g), (f) and (g) or (e), (f) and (g); (i) the sequence of (a), (b) or (c) is a combination of one or more changes of (e), (f) and (g); or (j) the sequence of (d) is a combination of one or more changes of (e), (f) and (g); and (2) having 1 to 6 consecutive amino acids on its carboxy-terminal side, and its sequence is (a) the same as the sequence of the corresponding 1 to 6 amino acids after amino acid 168 of wild-type human HMGB1, (b) identical to the sequence of the corresponding 1 to 6 amino acids after amino acid 167 of wild-type human HMGB1, (c) differing from the sequence of the corresponding 1 to 6 amino acids after amino acid 168 of wild-type human HMGB1 at one or more positions, or (d) differing from the sequence of the corresponding 1 to 6 amino acids after amino acid 167 of wild-type human HMGB1 at one or more positions, each B may be the same or different; - represents a peptide bond between A and X, X and B, B and A, A and X, and X and B; wherein the number of amino acids of B - A between the two Xs must be at least 12; and at B at the carboxy terminus of the polypeptide, the consecutive 1 to 6 amino acids of (2) may not be present) a polypeptide represented by. **Claim 2** The polypeptide according to claim 1, wherein the amino terminus is methionine. **Claim 3** The polypeptide according to claim 2, wherein (2)(a) of said A is 1 amino acid corresponding to amino acid 89 of wild-type human HMGB1. **Claim 4** The polypeptide according to claim 1, wherein in (2) of said B, B has 6 amino acids corresponding to amino acids 169 - 174 of wild-type human HMGB1 on its carboxy terminus side. **Claim 5** The polypeptide according to claim 1, wherein the sequence of (2)(c) or (2)(d) of either or both of B is GSGSG (SEQ ID NO: 175) or contains GSGSG (SEQ ID NO: 175). **Claim 6** The polypeptide according to claim 5, wherein said GSGSG (SEQ ID NO: 175) is present between B and A between the two Xs. **Claim 7** The polypeptide according to claim 5, wherein the order or number of glycine residues and serine residues is changed within the peptide sequence shown by SEQ ID NO:

175. **Claim 8** The polypeptide according to claim 1, wherein either or both of A are 5 consecutive amino acids, and said 5 consecutive amino acids have the same sequence as the sequence of amino acids 89 - 93 of wild-type human HMGB1. **Claim 9** A composition comprising the polypeptide according to any one of claims 1 - 8 and a carrier. **Claim 10** The pharmaceutical composition according to claim 9, wherein the polypeptide is present in a therapeutically or prophylactically effective amount and the carrier is a pharmaceutically acceptable carrier. **Claim 11** A composition comprising the polypeptide according to any one of claims 1 to 8 for use in a method of treating a subject suffering from a condition alleviated by promoting the regeneration of a tissue or cell that depends on CXCR4+ cells or a subject at risk of developing said condition, the composition comprising administering to the subject an effective amount of the polypeptide according to any one of claims 1 to 8 to promote the regeneration of said tissue or cell.

12. The composition according to claim 11, wherein the condition is an acute injury selected from myocardial infarction; stroke, brain injury, spinal cord injury or peripheral nerve injury; fractures, joint replacement or bone union, skeletal muscle injury, joint injury or ligament injury including sports injuries; lung injury, viral lung infection, bacterial lung infection, fungal lung infection, or mechanical lung injury such as ventilator-induced injury; skin injury such as burns or surgical skin injury including surgery; acute renal failure; bone marrow injury such as chemotherapy; waiting or emergency surgery; and liver injury.

13. The composition according to claim 12, wherein the polypeptide is administered within 5 hours after the acute injury.

14. The composition according to claim 11, wherein the condition is a chronic condition selected from heart failure; chronic conditions related to injury to the brain or other parts of the central nervous system, Parkinson's disease, dementia, multiple sclerosis, motor neuron disease or peripheral nerve injury; age-related macular degeneration; chronic joint injury, inflammatory arthritis or osteoarthritis; idiopathic pulmonary fibrosis (IPF); chronic obstructive pulmonary disease or emphysema; skin ulcers, diabetic ulcers, venous ulcers, arterial ulcers or pressure ulcers; diseases causing chronic renal failure; non-alcoholic fatty liver disease, cirrhosis or infectious hepatitis; inflammatory bowel disease, Crohn's disease or ulcerative colitis; chronic skeletal muscle conditions such as muscular dystrophy such as Duchenne muscular dystrophy, sarcopenia or disuse syndrome; pancreatic islet cell diseases; and diabetes.

15. The composition according to claim 14, wherein the polypeptide is administered repeatedly daily, weekly, monthly or annually.

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