CA3157785A1 - Polypeptides related to hmgb1 useful for promoting tissue regeneration, compositions comprising same, and uses thereof - Google Patents

Abstract

This invention provides a polypeptide represented by the following formula: H2N-A-X-B-A-X-B-HOOC wherein A represents consecutive amino acids, the sequence of which (1) includes a sequence identical to the sequence of amino acids 90 ? 93 of wildtype HMGB1, (2) has at its amino terminal end, between one and six consecutiveamino acids, for example, 1, 2, 3, 4, 5, or 6 amino acids, the sequence of which isidentical to the sequence of the corresponding one to six amino acids preceding aminoacid 90 in wild type HMGB1, and optionally (3) has a methionine at the aminoterminus;wherein X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 ? 162 of wild type HMGB1; and wherein B represents consecutive amino acids, the sequence of which (1) includes a sequence identical to the sequence of amino acids 163 ? 168 of wild type HMGB1 and (2) has at its carboxy terminal end, between one and six consecutive amino acids, for example, 1, 2, 3, 4, 5, or 6 amino acids, the sequence of which is identical to the sequence of the corresponding one to six amino acids following amino acid 168 in wild type HMGB1; and wherein each - represents a peptide bond between each of A and X, X and B, B and A, A and X, and X and B.

Description

REGENERATION. COMPOSITIONS COMPRISING SAME. AND USES THEREOF
[0001] This application claims the benefit of U.S. Provisional Application No.
62/934,299 filed November 12, 2019, the contents of which is hereby incorporated by reference.

[0002] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.
REFERENCE TO SEQUENCE LISTING

[0003] This application incorporates-by-reference nucleotide sequences which are present in the file named "201112 91203-A-PCT Sequence Listing AWG.txt", which is 78 kilobytes in size, and which was created on November 12, 2020 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed November 12, 2020 as part of this application.
TECHNICAL FIELD

[0004] This disclosure relates to engineered polypeptides related to HMGB1 that promote tissue regeneration without potential for deleterious inflammation and methods of treating an acute tissue injury by administering the engineered polypeptides to subjects in need thereof.
BACKGROUND OF THE INVENTION

[0005] Resident stem and progenitor cells play a key role in maintaining homeostasis and effecting repair of many tissues following injury [1]. However, most tissues in adults heal by scarring. Following the success of bone marrow transplantation [2] there has been considerable interest in exogenous stem cell therapies to promote regeneration of solid organs, with limited success in a few organs such as the eye [3] and skin [4]. The inflammatory environment following tissue injury is not conducive to stem cell engraftinent and the subsequent scarring disrupts the stem cell niche [5]. Therefore, focus has shifted to promoting tissue regeneration by stimulating endogenous repair mechanisms [6]. Development of a successful therapeutic would depend on identification of soluble mediators to promote these pathways.
We previously identified High Mobility Group Box 1 (HMGB1) as a key mediator of repair in multiple tissues, including bone, blood and skeletal muscle [7].

[0006] 1-1114GB1 is a prototypical alartnin [8,9] and under physiological conditions has an essential role in transcription [10,11]. On cell injury it is passively released from the damaged and necrotic cells into the extracellular space and the circulation to act on stem and progenitor cells to transition them to GAiert [7], a state intermediate between Go and GI
1121 On exposure to the appropriate activating factors, cells in GAlert are able to rapidly enter Gi and effect tissue repair. If not required, stem cells in GAlert revert back to Go after approximately 3 weeks [12], thereby ensuring that they are not exhausted and the niche is not depleted.

[0007] FEMBG1 comprises two L-shaped Box domains, A and B, each containing 3 a-helices (I - III) connected by flexible regions that are involved in LPS (N-terminus of Box A and adjacent C-terminal linker region) [13] or RAGE (C-terminus of Box B) binding [14]. The C-terminus of the protein is intrinsically disordered and contains a high proportion of carboxylic acid residues (Glu/Asp) comprising the acidic tail. This binds to the HMG
Boxes to regulate activities, including interactions with TLR-2 [10,15,16] and potentially also RAGE [15]
(Figures 1A-1B). The oxidation status of HMGB1 cysteine residues (Cys 22, Cys 44 in Box A
and Cys 105 in Box B) is a key determinant of the extracellular activities of HMGB1 and in turn is dependent on the mechanism of release. Three different redox forms have been described in vivo [17]. HMGB1 passively released from the nuclei following injury or cell necrosis is the fully-reduced form (FR-HMGB1). It binds to CXCL12 and the heterocomplex signals via the cell surface receptor CXCR4 to transition stem and progenitor cells to Gmen [7].
Partial oxidation in the local inflammatory environment results in the formation of disulfide (DS-HMGB1) [18,19], which has a disulfide bond between Cys 22 and Cys 44. This is also the form that is actively secreted by immune cells following acetylation [20] and N-glycosylation [21]. DS-HIVIGB1 signaling via the receptor for advanced glycation end products (RAGE) activates platelets and is a key mediator of thrombosis [22,23]. DS-HMGB1 also acts via TLR-4 and TLR-2, leading to release of proinflammatory cytokines, including TNF and IL-6 [24]. Intracellular signaling via all three receptors converges to induce NF-43 activity [25]
in a MyD88-dependent manner [26,27]. Oxidation of all three eysteine residues through the action of extracellular reactive oxygen species results in sulfonyl-HMGB1 (S03), which is biologically inactive [17,28].

[0008] The disulfide bridge in Box A of DS-H.MGB1 (Cys22-Cys44) is essential for TLR-4 signaling (Figures 1A-1B), initiating binding to TLR-4 but also has a relatively high dissociation rate. MD-2 then binds to Box B with low affinity but very low dissociation rates, stabilizing the interaction [29]; the Phe-Cys-Ser-Glu (FCSE, 104-107) peptide in Box B is essential for this interaction [30]. The capacity of DS-HMGB1 to signal via TLR-4 has been overcome by substituting cysteines at positions 22, 44 and 105 with senile, resulting in an engineered form described as 3S-FTMGB1 [17]. Whilst the authors claimed that has enhanced regenerative properties compared to FR-HMGB1 [31], we found that in bone, blood and skeletal muscle injuries it was equivalent to FR-HMGB1.
Interestingly, 3S-HMGB1 has been reported to be deleterious when administered locally following myocardial infraction whereas FR-HMGB1 resulted in a smaller infarct and enhanced cardiac function assessed over 4 weeks 132]. There are no published data on the effect of the 3S
substitutions on TLR-2 or RAGE signaling.

[0009] The sites for TLR-2 interaction are not clearly defined but glycyrrhizin is known to inhibit this interaction [33]. This suggests that at least one, and potentially both HMG Box domains and the acidic tail [101 are involved. The acidic tail negatively modulates HMGB1 and it has been reported that co-ligands [34] are necessary to displace the acidic tail of HMGB1 from the Box domains to permit signaling via TLR-2 [35]. However, several publications have reported TLR-2 dependent proinflammatory signaling with FIMGB1 alone [24,36]
and it is possible that the requirement for a co-ligand is cell type and context dependent. The role of the redox state of HMGB1 with regards to TLR-2 signaling remains unclear as both the disulfide form [22] and the fully-reduced form [34] have been proposed to signal through TLR-2. RAGE
interaction has been primarily mapped to a peptide in HMG Box B (residues 149-182) [14]
(Figures 1A-113 ), and peptides derived from this sequence can effectively inhibit 1-1MGB1-RAGE signaling [37]. In addition, there is also a second caspase-dependent site within Box A.
More recently a second RAGE binding site was identified on HMG Box A [38], accessible only after proteolysis by Caspase 11 [38]. However, the relative contribution of each site to RAGE signaling remains unknown. It is also recognized that prothrombotic signaling mediated by RAGE requires the disulfide form of HMGB1, implying that Box A is involved [22]. The acidic tail of FIMGB1 may negatively regulate RAGE signaling in a manner analogous to TLR-2 as it binds residues within the RAGE binding peptide [10,15,39] .

[0010] Successful translation of the regenerative activities of FR-HMGB1 to the clinic is dependent on the elimination of all potential deleterious signaling via RAGE, TLR-2 and TLR-4 whilst maintaining CXCL12-binding and signaling via CXCR4. Here the residues in HMGB1 that are critical for binding to CXCL12 are identified and an HMGB1 variant that retains regenerative activity whilst eliminating RAGE binding and TLR-2 and TLR-4 signaling is described.

SUMMARY OF THE INVENTION

[0011] This invention provides a polypeptide represented by the following formula:

wherein A represents consecutive amino acids, the sequence of which (1) includes a sequence identical to the sequence of amino acids 90- 93 of wild type HIAGB1, (2) has at its amino terminal end, between one and six consecutive amino acids, for example, 1, 2, 3, 4, 5, or 6 amino acids, the sequence of which is identical to the sequence of the corresponding one to six amino acids preceding amino acid 90 in wild type FIMGB I, and optionally (3) has a methionine at the amino terminus;
wherein X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94- 162 of wild type HMGB1; and wherein B represents consecutive amino acids, the sequence of which (1) includes a sequence identical to the sequence of amino acids 163 - 168 of wild type HMGB1 and (2) has at its carboxy terminal end, between one and six consecutive amino acids, for example, 1, 2, 3, 4, 5, or 6 amino acids, the sequence of which is identical to the sequence of the corresponding one to six amino acids following amino acid 168 in wild type 1-IMGB1; and wherein each - represents a peptide bond between each of A and X, X and B, B and A, A and X, and X and B.

[0012] This invention also provides a composition comprising the polypeptide in accordance with the invention and a carrier, and methods of treating a subject suffering from, or at risk for developing, a condition which would be alleviated by promoting regeneration of a tissue or cells that rely upon CXCR4 cells for repair which comprise administering to the subject a polypeptide of the invention in an amount effective to promote regeneration of the tissue or cells and a therapeutic or prophylactic dose of a pharmaceutical composition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figures 1A-1B show a schematic of the HMGB1 structure and locations of known immunogenic activities. Figure IA: Structure of HMGB1 (PDB 2YRQ, conformer 1, colored in PyMol according to known interactions with LPS, TLR-4 or RAGE. The acidic tail, which is involved in transcriptional modulation and bactericidal activities, is not shown in the structure. Figure 1B: Schematic representation of binding sites. Original figure coloring shows Box A - blue; Box B - green; Pink: residues involved in glycyrrhizin binding_ Red: flexible N-tenninal regions adjacent to Box A or Box B; Orange: cysteine residues; White:
linker region between FUVIG Boxes; Bright green and yellow: RAGE binding region (incomplete as it extends into the acidic tail, yellow).

[0014] Figures 2A-2F show conserved residues in each HMG Box domain are critical for CXCL12 binding and include the N-terminal D-P-X-X tetramer. Figure 2A: Peptide array (11x10) of FIMGB1 15-mers incubated with 1 !AM CXCL12-His6 and detected with anti-His5-HRP antibody. Intensity of spots corresponds to amount of CXCL12 bound to the peptides;
first two and last two spots in the array comprised 10-His positive controls.
Figure 2B:
Intensity quantification of spot intensity in (Figure 2A) (duplicate runs) normalized to 10-his control. Peptides used for alanine scanning experiments have been highlighted.
Peptides in the acidic tail were not included, as due to its high negative charge it would non-specifically bind cationic molecules such as CXCL12. Peptides in graphs are represented in SEQ
ID NOs: 8-104, left to right. Figure 2C: Peptide array of alanine mutagenesis at single positions within peptides identified in (Figures 2A-2B). First spot in each row corresponds to the positive control; second spot to the unmodified peptide. Peptides shown are represented in SEQ ID
NOs: 105-111. Figure 2D: Intensity quantification of the array in (Figure 2C, SEQ ID NOs:
105-111), normalized to the unmodified peptide. Residues which showed variation in CXCL12 signal greater than the variation observed seen in alanine residues (Ala ->
Ala; synonymous mutation, in grey) are show in red with original figure coloring. Figure 2E:
Michaelis-Menten saturation fits of biotinylated HIMGB1 constructs [full-length FR red/3S
black; minimal box A
8-78 (violet) and box B 94-162 (brown), extended box A 1-88 (pink) and box B
89-174 (gray)]
binding to CXCL12 with original figure coloring. Figure 2F: Summary of kinetic parameters derived from (Figure 2E). Affinity (Kd) constants from both fits follow the same relationship and are greatly decreased for FIMGB1 94-162; analyzed by 1-way Brown-Forsythe ANOVA
from the fitted data. Kd values were compared via a post-hoc 2-way ANOVA, averaging both values as no significant differences were found in the paired comparison (column factor). Raw interferograms can be found in Figure 9.

[0015] Figures 3A-3B show NMR validation of residues involved in CXCL12 binding.
Figure 3M Cumulative CSP of helical-only biotinylated Box B (94-162, HMGB1A-c028) or complete Box B (89-174, HMGB1A-c038) after titration with CXCL12 (0.42, 0.84 and 1.42 molar equivalents), calculated over several HSQC spectra including a parallel control with no CXCL12 measured after the last concentration point (CSP drift control).
Intensity of the green color in the graph indi ates relative CSP. The sequence of each HMGB1 construct has been overlaid with the residue number; an empty column (no number) represents residues which could not be mapped in the parallel 3D '1-1-'51\T HSQC/NOEfTOCSY experiments.
The sequence of the residues corresponding to each HMG Box is shown as follows with original figure coloring: Light blue, residues previously reported in the literature as involved in CXCL12 binding; red, residues weakly involved in the peptide array; purple, residues involved according to both published literature and peptide array. Grey, alanine residues within CXCL12 binding peptides (underlined) that could not be assessed in the peptide arrays. Sequence shown is represented in SEQ ID NO: 5. Figure 3B: Cumulative peak height change of (Figure 3A);
Heatmap of NMR changes. With original figure coloring, red indicates 1/10 change over 1 SEM
of all residues, blue decrease over -1 SEM. Sequence shown is represented in SEQ ID NO: 5.

[0016] Figures 4A-4C show design of the dBB12L construct. Figure 4A: Alignment of Box A + linkers (1-88) (SEQ ID NO: 3) with Box B + linkers (89-174) (SEQ ID NO:
4); values correspond to NMR nomenclature (excluding N-terminal methionine). Vertical lines designate strictly conserved positions, and double dots similar substitutions.
Underlined: peptide regions binding CXCL12 from the first peptide array. With original figure coloring, Red: residues flagged in the alanine scan as involved in CXCL12 binding which could not be verified by NMR; Orange: residues flagged in the alanine scan and also showing either CSP
or peak volume change by NMR; Cyan: residues not flagged in our NMR or peptide array experiments but described in the NMR literature as contributing to CXCL12 binding [44];
Purple: residues flagged peptide array experiments and confirmed by the published or our NMR
data; Green:
residues flagged only on NMR experiments, which can either directly bind CXCL12 or be affected by binding to nearby residues; Pink: residues flagged by both our NMR
experiments and the published data. Figure 4B: Structure of FR-HIVIGB1 1-166 (2YRQ), with the residues colored following the same color code as in (Figure 4A). Side chains of all colored residues have been shown. Dashed circles indicate the glycyrrhizin binding region in each HMG Box.

Figure 4C: Schematic of dBB I2L construct design. The initiation codon Met 1 is numbered as Met 0 herein, as it is partially lost in the cleaved peptide. Therefore, HIMGB1 Met 1 -Gly 2õ.Glu 215 becomes Met 0- Gly 1...Glu 214. Domain organization and sequences of FR-HMGB1 (top - SEQ ID NO: 1) and dBB12L construct (bottom - SEQ ID NO: 2). The dBB12L
construct is designed such that: 1. The acidic tail and part of the RAGE
binding domain (175-214) have been deleted; 2. Residues 1-88 (Box A) have been substituted by residues 89-174, resulting in two HMG Box B domains; and 3. Residues 163-174 C-terminal to Box B replace the native flexible linker (79-88) C-terminal to Box A in native H_MGB1.
CXCL12 binding peptides are shown as red letters. The repeat Box B units are separated in the diagram by a dashed black line.

[0017] Figures SA-SD show dBB12L has similar stability and surface charge conformation to FR-HMGB1 1-214/1-164. Figure 5A: Calculated Tmso values (in C) for full-length and 1-164 FR-111MGB1, and dBB12L. Shading of individual Tmso values indicate highest (green) and lowest (red) values within the global dataset for all constructs. N/A: curve not finable. Figure 5B: Native ESI/MS of HMGB1 constructs in either 50 mM or 02 M ammonium acetate, pH
6.5. All three HMGB1 constructs have similar native MIZ profiles, with dBB12L
closely resembling a reduced HIMGB1 construct with two HMG Boxes apart from each other.
Continuous line; compact monomer. Dashed line; extended monomer (HMG Boxes distal to each other). Removal of the acidic tail (FR FIMGB1 1-164, blue curves compared to FR-FIMGB1, red curves with original figure coloring) and higher ionic strength (Comparison of the spectra for the same construct in either 50 mM or 200 mM ammonium acetate) increases the prevalence of higher 1WZ states (partial unfolding). Figure SC: Solvent accessible surface area (SASA) calculations for the average folded HMGB1 monomer, the extended and compact monomer states, and the unfolded monomer from (Figure 5D). Figure 5D:
Denaturing ESI/MS
deconvolution, SDS-PAGE and SEC profiles of HMGB1 constructs after storage at room temperature for 180 days (DO-D180), in 0.2 M ammonium acetate, pH 6.5,

[0018] Figure 6A-6F show dBB12L has reduced binding to RAGE and does not signal through TLR-2 or TLR-4. Figure 6A: Michaelis-Menten saturation fits from hybrid ELISA
(n=4 per concentration, global fit) showing absence of RAGE binding by dBB12L
construct, regardless of oxidation status. DS-11MGB1 binds RAGE more avidly compared to FR-HIMGB1. Data normalized with respect to DS-HMGB1 control. Figure 6B: Michaelis-Menten saturation fits from biolayer interferometry (0-25 1iMI-IMGB1; n = 6 sensor runs). Association and dissociation rates were calculated from the raw interferogram data only.
Kinetics parameters and color legends (disulfide forms are indicated by dashed lines) are summarized in Figure 6C: Invalid fits represent R2 < 0.6 (poor binding). Data from ELISA
are proportionally more influenced by the dissociation constants than they are by association constants due to the nature of the experiment. For each kinetic parameter, green indicates the construct with the highest affinity, fastest association (km) or slowest dissociation (koff);
midpoint in yellow, lowest in red in the originally colored figure. Figures 6D-6E show DS-HIMGB I promoted NF-113 activity in reporter HEK-Dual cells expressing human TLR-2 and CD14 (Figure 6D) or murine TLR-4, MD-2 and CD14. (Figure 6E). DbB-HIVIGB1 did not promote NF-143 signaling, whereas FR-HMGB1 only induced minor NF-113 activation in both cell lines, potentially due to partial oxidation during the assay. Values are shown as mean + SEM fold change compared to control (media alone). Figure 6F: Disulfide HMGB1 (DS-H1VIGB1) increased TNF production in monocytes, which was further enhanced by the presence of suboptimal amounts of LTA, but not of LPS. Both FR-HIMGB1 and DbB-HMGB I
did not elicit TNF secretion, even when pre-incubated for 24h with LPS or LTA.
Response to LPS pre-incubated with these constructs was also significantly reduced n=3 donors, each with three (3) technical replicates.
Figures 7A-7J show the regenerative effects of optimal doses of dBB-HMGB1 and FR-FIMGB1 are identical to those of an activating injury. Figure 7A: Volcano plot showing differentially expressed genes in muscle stem cells by fold change following injury or HMGB1 induced auert. Integration demonstrates conserved up- (brown dots in figures with color) and down- (blue dots in figures with color) regulation of core genes in GAien induced by contralateral lower limb injury or intravenous (iv) I-IMGB1. Figure 7B:
Network map of gene ontology terms of differentially expressed cells during GAlert induction in muscle stem cells.
Figure 7C: Dose response of FR-FIMGB1 in a BaC12 skeletal muscle injury model, with regeneration quantified by fiber cross-sectional area. The optimal dose was 0.75 mg/kg (28.75 nmol/kg) and was used in subsequent assays. Figure 7D: Animals were dosed with FR-IIMGB1 (optimal dose) at the varying timepoints after injection of BaChto assess the interval where treatment with FR-FIMGB1 is effective post-injury. Values in (Figure 7C), (Figure 7D) shown as mean SEM in nested ANOVA with Holm-Sidak correction (values shown for post-hoc tests). Figure 7E: Pharmacokinetics of iv HMGB1 in mice, fitted by nonlinear least squares to a two-phase exponential decay curve (circulating HMGB1 after intravenous injection of the optimal dose). Figure 7F: Survival following myocardial infarction (MI) at 5 wk FR-FIMGB1 = 83 %, PBS = 52 %. Figure 7G: Ejection fraction. Dotted line indicates ejection fraction in normal/sham surgery mice. Figure 7H: Infarct size compared by 2-way ANOVA for treatment effect across times. Figure 71: Representative mid-ventricular short-axis cine-MRI images at end-diastolic and end-systolic phases of the cardiac cycle 1 and 5 wk after MI. Blood in the chambers appears bright. FR-HMGB1 group shows preservation of heart function and maintenance of wall thickness (yellow arrows in figures with color) with visible separation of right and left ventricles (red arrows in figures with color) during systole. In contrast, in the PBS treated group, there is significant left ventricle dilation (white arrows) and very limited contraction between diastole and systole. n = 10 per group. All MRI scans were performed and assessed by a blinded observer. Figure 7J: Plotted mean muscle cross-section area at given time points after BaC12 muscle injured animals treated with either PBS (black), 28.75 nM/kg of FR-HMGB1A-c001 (red in figures with color) or dBB12L (green in figures with color). N= 5 per group and timepoint, nested ANOVA (14ohn-Sidak post-hoc correction).
A representative image of each point is shown in Figure 12.

[0019] Figures 8A-8B show results of a peptide array of CXCL12 peptides interacting with IIIVIGB1. Figure 8A: Peptide array of fill-length CXCL12. "+" positions correspond to positive control 10-His peptides; the rest of the peptides comprise CXCL12 15-mers shifted two (2) residues in succession towards the C-terminus. The membrane was exposed to 1 uM
HMGB l(FR or 3S)-His6 (1-214), BoxA-His6 (8-78) and BoxB-His6 (94-162) for 24 hours.
Bound protein was detected by anti-His-HRP conjugate chemoluminescence. A
peptide of CXCL12 interacting with full length FINIGB1 cannot interact with either Box A
or Box B
alone, confirming the requirement of-the N-tenninal segment of each Box domain (particularly, D4 in box A/D90 in box B): intensity of the spots pertaining to the common CXCL12 peptide is also markedly decreased upon binding to the Box domains alone when compared to FL-HMGB1. Binding to 3S seems to be of higher intensity than that to FR; this is likely due to protein oxidation during the assay, although this was not quantified due to the low concentration of protein used being unsuitable for ESITTOF MS. BLI data, however, do suggest a lower off rate of CXCL12 from 35 than from FR-HMGB1. Figure 8B: CXCL12 dimer (PDB
2J7Z) with the regions binding IIMGB1 highlighted. In original figures with color, Red: shared binding region. Blue: non-shared binding region.

[0020] Figure 9 shows interferograms in BLI of CXCL12 binding to immobilized constructs. Biotinylated I-IMGB1 constructs were immobilized on streptavidin-coated Octet biosensors and dipped in rising concentration of CXCL12. Interferograms are colored according to CXCL12 concentration (key in top right). Each set of three replicates (cycles) for a given sensor is surrounded by a colored overlay according to construct FR FL-(c011), black: 3S-FL (c022), purple: FR-HIMGB1 Box A 8-78 (c027), brown: FR-HMGB1 Box B 94-162 (c028), pink: FR-HMGB1 Box A 1-88 (c037), grey: FR-FIMGB1 Box B 90-162 (c038).

[0021] Figures 10A-10D show NMR validation of residues involved in CXCL12 binding (continuation). Figure 10A: Cumulative CSP of F1MGB1 3S 1-184 (HMGB1A-c007) upon addition of 1:2 molar equivalents of CXCL12 in one step (1:1 HMG Box to CXCL12 ratio).
Box A and Box B residues have been considered separate molecules for the purposes of median CSP calculation. With the original figure coloring, intensity of the green color in the bar graph indicates higher relative CSP. The sequence of each HMGB1 construct has been overlaid with the residue number; an empty column (no number) represents residues which could not be mapped in the parallel 3D 1H-15N HSQC/NOEITOCSY experiments. The sequence of the residues corresponding to each box is in the middle of each condition, colored originally as per: Light blue, residues previously reported in the literature as involved in CXCL12 binding;
red, residues weakly involved in the peptide array; purple, residues involved according to both published literature and peptide array. The sequences shown are represented in SEQ ID NOs:
6 and 7. Figure 10B: 15N HSQC-HQMC peak spectra for (A) in 10 mM HEPES 150 mM
NaCl pH 75 buffer. Protein concentrations are indicated in the spectra overlay. Figures 10C -10D show 15N HSQC-HQMC peak spectra in 10 mM HEPES 150 mM NaC1 pH 75 buffer.
Protein concentrations are indicated in the spectra overlay. Figure 10C:
HMGB1A 94-162 (2-day experiment); Figure 10D: HMGB1A 89-174 (6-day experiment; minor degradation occurs after day 4).

[0022] Figure 11 shows Interferograrns in BLI of HMGB1 constructs binding to inunobilizekl Fe-RAGE. RAGE-Fc was immobilized in the surface of ABC sensors and dipped in rising concentration of different HIMGB1 constructs. Two experiments were run with different concentration ranges: the tee columns of graphs in the left, 0 to 22.22 RM HMGB1 over 9 steps; on the right, 0 to 25 !LIM over 7 steps. Both are originally color-coded by concentration (top). Colors indicate the specific construct concentration.
Each graph corresponds to a single sensor (replicate). hiterferograms surrounded by a red rectangle had data points excluded due to poor quality (e.g. drift).

[0023] Figure 12 shows histological images of regenerating muscle in response to FR-FIMGB1 (red) or dBB12L (green) compared to PBS control (black) in the originally colored figure.

[0024] Figures 13A-13C shows plasmid vector maps. Vector maps with features and restriction sites. TEV: Tobacco etch virus protease recognition site. 6-His:
10/6-his-tidine residue affinity epitope. FLAG: FLAG affinity epitope. StrepTag:
Streptactin3CT affinity epitope. SacB: Levansucrase precursor (negative selection in the presence of sucrose). pLIC:
Annealing sites for sequencing primers used in colony screening. All plasrnids contain kanamycin resistance (50 pg/mL).

[0025] Figure 14 shows mutagenesis of the FR-HMGB1 sequence to generate 3S-HMGB1.

DETAILED DESCRIPTION
Terms

[0026] In order to facilitate an understanding of the invention, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below.

[0027] As used herein, "engineered" means a non-naturally occurring compound that has been created based up changing a naturally occurring compound . An engineered compound, e.g. a polypeptide, may include portions of a naturally occurring compound that have been modified or rearranged. Such an engineered polypeptide may also be referred to as an "analogue" or "derivative" of the naturally occurring polypeptide.

[0028] As used herein, "stem cell" means any unspecialized cell that has the potential to develop into many different cell types in the body, including without limitation hemopoietic stem cells.

[0029] As used herein, the term "effective amount" means an amount of a compound that is capable of achieving a desired result, for example, alleviating a condition or the symptoms associated therewith, for example, an acute tissue injury as described herein.
The specific dose of a compound administered according to this invention will, of course, be determined by the particular acts associated with the condition, for example, the route of administration, the physiological state of the subject, and the severity of the condition being treated. For example, an engineered HMGB1 protein administered to a subject is preferably in the form of a composition comprising a therapeutically effective amount of the engineered HIMGB1 protein.

[0030] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material. The choice of any specific pharmaceutically acceptable carriers is well within the knowledge of those skilled in the art..
Accordingly, there is a wide variety of suitable carries available and routinely used in phamiaceutical compositions.

[0031] It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components.

[0032] As used herein, all numerical ranges provided are intended to expressly include the endpoints and consistent with context all numbers that fall between the endpoints of range.
Embodiments of the Invention

[0033] This invention provides a polypeptide represented by the following formula:

wherein A represents consecutive amino acids, the sequence of which (1) includes a sequence identical to the sequence of amino acids 90 - 93 of wild type HA/16B1, (2) has at its amino terminal end, between one and six consecutive amino acids, the sequence of which is identical to the sequence of the corresponding one to six amino acids preceding amino acid 90 in wild type HMGB1, and optionally (3) has a methionine at the amino terminus;
wherein X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 - 162 of wild type HMGB1; and wherein B represents consecutive amino acids, the sequence of which (1) includes a sequence identical to the sequence of amino acids 163 - 168 of wild type HMGB1 and (2) has at its carboxy terminal end, between one and six consecutive amino acids, for example, 1, 2, 3, 4, 5, or 6 amino acids, the sequence of which is identical to the sequence of the corresponding one to six amino acids following amino acid 168 in wild type HMGB1; and wherein each - represents a peptide bond between each of A and X, X and 13, B and A, A and X, and X and B.

[0034] In some embodiments, the methionine is present at the amino terminus of the polypeptide.

[0035] In other embodiments, A has at its amino tenninal end, one amino acid corresponding to amino acid 89 of wild type 1-IMGB1.

[0036] In some embodiments, B has at its carboxy terminal end, six amino acids the sequence of which corresponds to the sequence of amino acids 169-174 of wild type I-11%4GBI.

[0037] This invention also provides a composition comprising the polypeptide of any one of the provided embodiments and a carrier.

[0038] In some embodiments, the polypeptide is present in a therapeutically or prophylactically effective amount and the carrier is a pharmaceutically acceptable carrier.

[0039] This invention also provides methods of treating a subject suffering from, or at risk for developing, a condition which would be alleviated by promoting regeneration of a tissue or cells that rely upon CXCR4+ cells for repair which comprise administering to the subject the polypeptide of any one of the provided embodiments in an amount effective to promote regeneration of the tissue or cells, that is, a therapeutically or prophylactically effective dose of the pharmaceutical composition of the invention.

[0040] In certain embodiments, the condition is myocardial infarction and the tissue is a cardiac tissue, particularly, myocardium.

[0041] In a currently preferred embodiment, the polypeptide is administered within 5 hours, preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours and most preferably within 1 hour of the myocardial infarction.

[0042] In some embodiments, the condition is a fracture and the tissue is a bone.

[0043] In other embodiments, the condition involves liver damage and the tissue is liver tissue.

[0044] In yet further embodiments, the condition involves damage to the brain or nervous system and includes stroke, Parkinson's disease and dementia

[0045] In some embodiments, the condition involves damage to the lung.

[0046] In yet further embodiments, the condition involves the gut and includes surgery and inflammatory bowel disease.

[0047] In some embodiments, the condition involves damage to the skin and includes surgical procedures, bums and ulcers.

[0048] In additional embodiments, the condition involves the pancreas including type 1 diabetes and the cells are islet cells.

[0049] In further embodiments, the condition is neutropenia, for example, neutropenia following chemotherapy and the tissue is bone marrow.

[0050] In some embodiments, the condition is kidney failure and the tissue is kidney tissue.

[0051] Further non-limiting details are described in the following Experimental Details section which is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the scope of the invention disclosed.

EXPERIMENTAL DETAILS
RESULTS
Identification of amino acids and motifs in HMGB1 involved in CXCL12 binding

[0052] Before considering which residues of FR-IIMGB1 can be mutated to eliminate proinflammatory signaling, it is essential to map the amino acids and motifs involved in binding to CXCL12. Peptide SPOT arrays were used, [40] where overlapping peptides covering the sequence of one of the target proteins (11MG131) are assessed by immunoblotting for their capacity to bind CXCL12 to identify key sequences involved in binding. Coarse peptide arrays with human HMGB1 peptides binding his-tagged CXCL12 highlighted a specific pattern of homologous sequences between Box A and Box B that bind CXCL12. Two main binding sites were identified by the clustering of CXCL12 binding peptides over the HMGB1 sequence (Figure 2A and Figure 2B). The first encompassed the initial one and a half a-helices of the HMG Box, overlapping the glycyrrhizin binding site [41]. The second was located at the C-terminal half of the third a-helix. In each HMG Box, the first CXCL12 binding peptide (helices I and II) appeared to be the most involved in CXCL12 binding as the intensity of CXCL12 binding to peptides from this segment was much higher. Binding of Box B
peptides to CXCL12 appeared to be slightly weaker compared to Box A based on the intensity of the immunoblots.

[0053] To further delineate the importance of individual residues, a second peptide array was generated in which each amino acid within the CXCL12 binding peptides were substituted to alanine and their effect on CXCL12 binding was assessed. The aim was to pinpoint amino acid(s) on the peptides that directly contribute to CXCL12 interaction. This confirmed that several residues are critical for CXCL12 binding as their substitution to alanine altered the intensity of bound CXCL12 (Figure 2C and Figure 2D) in otherwise homologous peptides.
Contrary to published data where the interaction with CXCL12 was shown to only involve the helical segments of the H:MG Boxes [42-44], we found that part of the flexible N-terminal flanking region to each box (D4-P-X-X-I), as well as C-terminal residues (Ile 78-Pro 80 for Box A and Ala 163-Asp168 for Box B), were involved in the interaction with CXCL12 (Figure 2E). This was confirmed with a reverse peptide array (Figure 9) of CXCL12 peptides. Full-length FR and 3S- HMGB1 bound to peptides containing sequences covering the whole of the I3-sheet of CXCL12, whereas HMGB1 Box constructs alone (8-78 Box A) or (94-162 Box B), without the flanking flexible region [45], only interacted with the peptides covering the N-tenninal strand of the I3-sheet.

[0054] We hypothesized that if these flexible regions adjacent to the HMG
Boxes were involved in CXCL12 binding, their absence should significantly alter the interaction of HMGB1 with CXCL12. Using biolayer interferometry (BLI), we assessed binding of to HMGB1 constructs comprising each of the HMG Boxes with (MI HMG Box constructs:
HMGBI 1-88 for Box A, HIMGB1 89-174 for Box B) [46] or without the flexible flanking residues (helical-only HMG Box constructs; FIMGB1 9-78, FINIGB1 94-162), full-length FR-HMOB1 and non-oxidizable (3S)-HIMGBI, which shares the CXCL12-binding properties of the wild-type protein [17]. As expected, binding of CXCL12 to IIMGB1 was greatly influenced by the presence of these flexible regions (Figure 2E and Figure 2F). Helical-only constructs had decreased CXCL12 affinity and binding capacity compared to full HMG Box constructs with intact flanking regions as evidenced by the increased dissociation rates (Iroft) of the helical-only constructs without the flanking regions. In contrast, affinities of CXCL12 for full-length HMGB1 (FR or 3S) and frill HMG Box constructs were comparable to each other and higher than for the helical-only constructs.

[0055] Our peptide arrays showed that the residues involved in binding CXCL12 were clustered in two peptides: one across a-helix I and the N-terminal half of II, and a second over the C-terminus of a-helix III. Both peptides included the flexible flanking regions at the N- and C-termini of each HMG Box. This pattern was mirrored across the HMG A and B
Boxes (Figure 2B, Figure 2D, Figure 2E). BLI kinetic data confirmed that the absence of the flanking regions in single HMG Box domains resulted in destabilization of the recruited CXCL12, which did not remain bound to the helical only constructs (Figure 2F).
CXCL12 binds to a concave pocket on the underside of each H:MG Box as shown by peptide array and NMR

[0056] Next NMR was used to confirm from a structural perspective the amino acid residues in HMGB1 involved in CXCL12 binding. Residues involved in CXCL12 binding would result in NMR signal changes upon formation of the complex. We used FR-HMGB1 94-162 and 89-174 to represent HMG Boxes with and without the flanking regions, as well as 184. Non-oxidizable 3S-HMGB1 was used as our full-length construct as the time required for obtaining a full set of 3D spectra at 750 MHz (15N HSQC-TOCSY/NOESY and associated 15N
HSQC spectra) would result in oxidation of FR-FIMGB1, altering both the peak resonances and the interaction with CXCL12.

[0057] CXCL12 titration of HMGB1 Box B 94-162 (Figure 3A) resulted in changes in NMR
signal, either cumulative chemical shift perturbation (CSP) or peak height changes (1no), of several residues at both the C-terminal and N-terminal binding regions identified in the peptide arrays. However, several residues (A100, 1112, L119, A136, Y154, D157, 1158) identified in our peptide arrays and also documented in the literature [43,44] showed no CSP
or volume changes. For the Box B construct that included the flanking regions, both the median CSP and mean volume change were significantly higher. Several residues (D90, (1165, K166) in these flanking regions were found to be involved in CXCL12 binding, in accordance with our peptide array data. Residues not affected by CXCL12 binding in the helical-only (94-162) construct, such as Y154, D157 and 1158, and which were identified as being involved in CXCL12 binding in the peptide arrays demonstrated significant CSP in the full HMG Box construct, indicative of improved binding when the flanking regions were present. In addition, we observed CSP
changes for residues not identified in the peptide arrays (A147, M131, A169, K172, (1173) in the construct with the flanking regions. Other residues that have not been previously identified but were flagged as being potentially important in the peptide arrays did not display either CSP
or volume changes upon addition of CXCL12 (C105, E107, Y108). This group of residues was, therefore, reclassified as being not critical for CXCL12 binding.

[0058] When the NMR experiment was repeated with 3S-HMGB1 1-184, CXCL12 addition resulted in CSP changes below the threshold for detection due to the signal to noise ratio.
Surprisingly, in contrast to published data [47], residues A100, 1112, L119 and A136 in Box B
of 3S-HMGB1 1-184 failed to produce significant CSP or volume changes, although residues (S99, K113) very close to some of these were affected. Within this construct, residues corresponding to HMG Box B showed weaker CSP changes for residues involved in interaction with CXCL12 compared to those in Box A. This could represent a more fluid equilibrium of CXCL12 binding, as suggested by higher association and dissociation rates for Box B (Figure 2G). This allowed us to also denote residues such as H30, D32 or S34 as false positives from the peptide array (Figure 11). Whilst K89 in Box B 89-174 showed high CSP, this is the third residue from the N-terminus (after the TEV cleavage leftover N-terminal residues ¨ Ser-Met ).
In the experiments with 3S H1vtGB1 1-184, where it is in the middle of the flexible linker, it did not demonstrate increased CSP changes. Therefore, the changes associated with this residue in Box B alone are likely related to its position at the N-terminus potentially permitting high conformational flexibility.

Design of a double Box B HMGB I construct that retains CXCL12 binding whilst eliminating proinflammatory signaling

[0059] We combined the data from the peptide arrays, alanine substitution, and NMR, and mapped them onto the NMR structure of HMGB1 (Figure 3A, PDB 2YRQ) to identify the residues involved in binding CXCL12. We found that the CXCL12 binding residues occupy the concave side of the HMG Box domains, forming a CXCL12 binding pocket on the underside of each HMG Box. These two pockets (one on each HMG box) also contain the described binding site for glycyrrhizin, a competitive inhibitor of HMGB1-CXCL12 binding [41], between Helix I and II on each Box. HMG Box A and B are each capable of binding one CXCL12 monomer with equivalent affinity. We also recognized that the distribution of peptides within each HMG Box that interact with CXCL12 is similar between both HMG
Boxes, and form almost identical binding pockets (Figure 4A).

[0060] Having identified the sequence determinants for binding CXCL12, we next designed HMGB1 constructs which preserved these interaction surfaces but altered sequences to attenuate proinflammatory signaling. Based on the fact that each HMG Box can bind to a CXCL12 monomer independently and the requirement of both Box A (oxidized) and Box B
for TLR-4 [48] and potentially for the prothrombotic RAGE [14,22] and TLR-2 [22,33]
signaling activities, we hypothesized that an HMGB1 construct where Box A is substituted by another Box B (i.e. 1-88 replaced by 89-174) would not signal through TLR-2, TLR4 or RAGE. In addition, we deleted part of the RAGE-binding sequence in Box B (175-184) to further decrease the affinity for this receptor. Replacing the Box A sequence for Box B also replaces the LPS glycan-binding peptide for a copy of the LPS lipid A binding peptide [13], which would further hamper the proinflanunatory activities of HIMGB I [49] and LPS transfer to TLR-4/MD-2 in a manner analogous to LBP. This engineered construct dBB12L
(Figure 4B) comprised of the following segments of the native HMGB1 protein: flexible N-terminal region (from HNIGB1 89-93), first Box B (from ITMGB1 94-162), 12-residue linker C-terminal of native Box B (from FIMGB1 163-174), and second Box B (from HMGB1 94-162).
The linker length of 12 residues in this dBB12L is similar to the 10 amino acids in the linker of native 1-IMGB1. This small increase in linker length was due to our preserving residues 172 and 173 which showed changes in CSP on CXCL12 binding.

[0061] We compared the thenuostability of dBB12L and wild-type HMGB1 using Dynamic Scanning Fluorimetry (DSF), and solvent accessible surface area (SASA) by both native mass spectrometry (native ESI/N1S) and size exclusion chromatography (SEC). dBB12L
had similar stability and surface charge profiles as FR-HIMGB1 1-164, which also contains two HMG Box domains and no C-terminal acid tail. Therrnostability trends were similar for all constructs (Figure 5A), with lower Tins() at pH nearing the isoelectric point (9.9 for tail-less constructs as dBB12L or 1-164, and 6 for FL-HMGB1) and they were all equally stable in conditions relevant to clinical practice (PBS, purification buffers, and saline solution), with Tm50 However, we observed different optimal ionic strength ranges for FR-HMGH1/FR-164 compared to dBB121a. In native ESUMS, all three HMGB1 constructs had similar charge state distributions, with a compact monomer as the main species and a higher SASA extended monomer (Figure 5B and Figure 5C).The extended monomer was more prevalent for tailless constructs or at higher ionic strength. Average monomer SASA values observed in native ES1/MS were in agreement with those derived from SEC or published NMR
structures (PDB
2YRQ; 1-TMGB1 1-164). Compact FL-HMGB1 monomer SASA matched computational models of FL-HMGB1 in water [44]. Storage up to 180 days did not affect SEC
profiles (always monodisperse at equal RV), degradation, or aggregation (Figure 5D).
This suggests that the conformations observed in ESUMS are representative or the native folding of FIMGB1 constructs, and that dBB12L is not significantly different from an HMGB1 construct with two HMG Boxes alone.
dBB12L construct has greatly reduced affinity for RAGE and cannot signal through TLR-2 or

[0062] We then evaluated whether dBB12L had decreased TLR-2, TLR-4 signaling and RAGE binding, whilst preserving HMGB1-mediated regeneration. Due to the lack of an established signaling assay for RAGE, we assessed the binding of RAGE to FIMGB1 using real-time kinetics (BLI) and an endpoint assay (ELISA). ELISA-based affinity measurements (Figure 6A) showed that 3S-, FR- and DS-HMGB1 at equilibrium bound similar amounts of RAGE, with DS-HMGB1 and 3S-HMGB1 having significantly higher apparent affinity than FR-HMGB1. In contrast, dBB12L did not to bind RAGE in this assay. Three additional HMGB1 constructs were tested, including DS-HMGB1 1-184, which has an intact RAGE
binding peptide and oxidized Box A but no acidic tail and therefore has all the requisites for RAGE binding; DS-HMGB1 1-164, which lacks a significant portion of the RAGE
binding peptide but retains an oxidized Box A; and DS-Box A alone. We found that DS-184 bound RAGE, but with reduced capacity and affinity compared to full-length DS-HMGB1.
By comparison, DS-HIvIGB1 1-164 had greatly diminished RAGE binding capacity compared to fill-length DS-HIMGB1 but still higher than dBB12L, whilst DS Box A 1-88 (hill HMG
Box construct with flanking regions) was unable to bind RAGE.

[0063] Kinetics analysis using BLI confirmed the ELISA results (Figure 6B), with two exceptions. In BLI (Figure 6C) DS-IIMGB1 1-184 had a much higher RAGE binding affinity than all other constructs, albeit with also slightly faster dissociation rate, compared to ELISA
where it had lower affinity than DS-H.MGBI. 3S-HMGB1, whilst binding equivalent amounts of RAGE to DS- or FR- HMGB1, had similar affinity to FR-HMGB1 but much slower overall kinetic rates, whereas in ELISA it had affinity and binding capacity for RAGE
equivalent to DS-HMGB1. Binding of all other HMGB1 constructs to RAGE in BLI reproduced the results from the ELISA. The higher RAGE affinity of DS-HMGB1 compared to FR in both assays was due to a much faster association rate (kon), whereas dissociation rates (Ls) were nearly identical for these two redox forms. In contrast, dBB12L, which had an association rate closer to DS-HMGB1, exhibited very unstable binding with a very high dissociation rate. DS-HIMGB1 1-164 also had a faster RAGE binding equilibrium with overall lower binding affinity than full length DS-FIMGB1, although with higher affinity than dBB12L-HMGB1, The deletion of both the final 10 residues in Box B (175-184) and the disulfide bridge in Box A (by substituting it with Box B) in dBB12L resulted in unstable binding of RAGE.

[0064] FIMGB1 binds TLR-2, TLR-4, and RAGE and signaling from all receptors converges to The NF-icfi pathway [25]. Consequently, it is difficult to attribute downstream proinflamrnatory cytokine production to a given receptor. Therefore, we first evaluated TLR-specific signaling using NF-113 reporter cell lines engineered to express either TLR-2 or TLR-4 and their co-receptors. Disulfide HMGB1 promoted NF-kB signaling via TLR-2 (Figure 6D) and TLR-4 (Figure 6E). In contrast, dBB12L failed to signal in either cell type. Next, we confirmed the effects of the various IIMGB1 constructs on primary human monocytes. It has been reported that DS-FIMGB1 synergizes with TLR-2 ligands such as LTA to promote proinflamrnatory signaling [341 We confirmed that DS-EINGB1 acted synergistically with LTA to promote higher TNF production than LTA or DS-HMGB1 alone. In contrast, dBB12L
or FR-HMGB1 did not exhibit this synergistic effect and alone failed to elicit TNF secretion greater than media alone (Figure 6F). No synergistic response has been described with DS-HMOB1 and the TLR-4 ligand LPS. When combined with LPS, DS-HMOB1 promoted TNF
expression by primary human monocytes to the same extent as LPS alone. In contrast, FR-FIMGB1 or dBB12L alone did not promote TNF production. However, when combined with LPS, FR-HMGB1 or dBB12L reduced TNF expression compared to LPS alone.

[0065] Taken together, these data show that dB1312L does not signal via TLR-2 or TLR-4, even in the presence of their cognate ligands and it has greatly decreased affinity for RAGE.
The dE3B12L construct has pro-regenerative activity comparable to that of FR-

[0066] Distant injury has been previously shown to transition stem cells to GAbmi [121 Therefore, we first compared the transcriptomic response of skeletal muscle stem cells of FR-HMGB1 to injury to the contralateral limb. The genes up- and down-regulated by or distant injury were remarkably similar (Figure 7A), with the major pathways upregulated being those associated with Gpaen [7,12], including mitochondrial metabolism, oxidative phosphoiylation and cell cycle (Figure 7B). Interestingly, CXCR4 was one of the most highly upregulated genes.

[0067] Next, we determined the optimal in vivo treatment dose for FR-HMGB1 using a validated murine model of skeletal muscle injury [7,12]. We found that 0.75 mg/kg (29 nmol/kg) resulted in the maximal response, with no further improvement in regenerative activity with higher doses (Figure 7C). We also assessed the optimal time for administration in vivo after injury. FR-HMGB1 was found to be effective in promoting repair when injected up to 5 hours post-injury (Figure 7D). Thereafter, there was no improvement. We then looked at the half-life of FR-FIMGB1 in the circulation following iv administration. We found that there was an initial rapid clearance (tu2 11 min) followed by subsequent slower clearance rate (tit2 120 min) (Figure 7E). This would be consistent with the half-life of 25 min in humans [50], with the protein being cleared by binding to haptoglobin [51,52],

[0068] We have previously shown that FR-HMGB1 accelerates regeneration of skeletal muscle, bone and blood following injury by promoting the transition of stem and progenitor cells to Gmert [7]. There are no significant stem cells in the mammalian heart and the majority of new cardiomyocytes following injury are derived from existing cardiomyocytes [53].
Therefore, we assessed whether adminisuation of FR-HMGB1 would promote cardiac regeneration. We found that iv injection at the time of myocardial infarction resulted in enhanced survival (83% in mice treated with FR-HMGB1 compared to 52% in PBS
controls) (Figure 7F). FR-HMGB1 resulted in approximately 60% reduction in infarct size as assessed by serial MRI scans over 5 weeks (Figure 7H) and 16% improvement in overall left ventricular ejection fraction (Figure 7G).

[0069] Finally, we assessed the efficacy of dBB12L compared to FR-FIMGB1 in promoting skeletal muscle regeneration in viva Mice injected with optimal doses (29 nM/kg) of FR-1-1114031 or dBB12L exhibited accelerated regeneration equally following injury (Figure 7J), as determined by an increase in the mean cross-sectional area of regenerating muscle fibers with centra nuclei [7,12]. This was most apparent at day 14, as previously described for FR-HMGB1 [7].
DISCUSSION
HMGB1 needs to be modified in order to be used as a tissue repair therapeutic

[0070] Therapies based on administration of exogenous stem cells to promote repair of solid organs have failed to deliver on the initial promise [6,54], and killed cells are just as effective by triggering an immune response [55]. An alternative, potentially more rewarding approach, would be to target endogenous regenerative repair processes, including resident stem and progenitor cells [56,57]. Inhibition of prostaglandin dehydrogenase is a promising approach [58], although progress through to clinical translation has been slow [59].
Administration of growth factors has also been described [60,61] but is limited by in vivo proteolysis [62].
Currently, there is no approved therapeutic for promoting regeneration and accelerating repair of multiple tissues.

[0071] We previously showed that exogenous administration of FR-FIMGB1 is effective in accelerating regeneration of bone, skeletal muscle and blood by transitioning resident stem and progenitor cells to GAiert, when they are able to readily respond to the appropriate activating factors released on tissue injury to effect repair [7]. Whilst most of the systemic DS-1-1MGB1 detected in patients after trauma is secreted in a second release event [50], there is accumulating evidence supporting the conversion of FR-FIMGB1 into DS-HIVIGB1 in vivo locally at the site of injury [18,19]. Therefore, the potential for deleterious proinflammatory signaling precludes the use of native FR-FINGB1 as a therapeutic.

[0072] Disulfide HMGB1 can signal through TLR-4 [24,30], TLR-2 [24,34,35] or RAGE
[14,63] to induce the expression of proinflammatory cytokines. TLR-4 signaling by DS-111V1GB1 results in production of several proinflammatory cytokines, including TNF [28], whilst TLR-2 signaling has been shown to be detrimental in multiple processes, including thrombosis and reperfusion injury [64], and autoimmune disorders [33]. DS-FIMGB1 signaling via RAGE plays a key role in platelet activation and NET formation by neutrophils to promote thrombus formation [23,64-66]. All three receptors ultimately converge on NF-113 [25,67], leading to synergistic expression of proinflanmiatory cytokines via all three receptors [68,69].

Therefore, development of HIVIGB1 as a therapeutic is crucially dependent on engineering the molecule to eliminate signaling via all three receptors.
Box A and Box B bind CXCL12 independently due to a shared peptide pattern

[0073] The regenerative activities of FR-HIMGB1 are critically dependent on the formation of a heterocomplex with CXCL12 and signaling via CXCR4. Whilst it is known that CXCL12 binds to HMG Boxes [42,70], the structural motifs involved remain unknown, with few residues proposed [47,71]. It is also unknown whether this signaling involves homodimers of CXCL12 via the CXCR4 axis, including enforcing quiescence of hemopoietic stem cells, or CXCL12 monomers, which promote chemotaxis [72-74].

[0074] Peptide arrays allowed us to identify the residues [75] involved in interaction. We identified a common pattern of two peptide binding regions for present in both Box A and Box B. The first peptide region extends from the N-terminal flexible segment into half of Helix II, overlapping with the glycyrrhizin binding site [41], and the second on the C-terminal portion of helix III in each HMG Box. We confirmed the importance of these residues using alanine mutation and also the importance of the flanking flexible regions by BLI, their removal resulting in a dramatic increase in the dissociation rate of CXCL12. BLI
also confirmed that each Box can bind a CXCL12 monomer independently with similar affinity, without cooperativity between the Boxes. Furthermore, our use of HEPES buffer prevented CXCL12 dimerization [76], allowing us to conclude that each Box is able to bind monomeric CXCL12, and dimerization of CXCL12 is not a requisite for complex formation.
This model is consistent with the proposed mechanism of signaling of the HMGB1-heterocomplex via CXCR4 [72].

[0075] We then used NMR to confirm the critical role of the peptide regions identified by the peptide arrays, including the flexible flanking regions whose absence resulted in a decrease in CXCL12-induced changes upon binding to either full or helical only HMG Box B constructs.
This is consistent with the faster dissociation rates observed in BLI for the helical only HMG
Box B construct compared to the one containing flanking regions. We also observed weaker signals (peak broadening) for Box B compared to Box A, supporting the faster exchange binding equilibrium observed in BLI for full-length Box B compared to Box A
and also the weaker signal for Box B CXCL12-binding sequences in the peptide arrays. Some residues demonstrated shift changes in NMR despite not being flagged in the peptide arrays. These may represent sequence-independent contribution to binding such as main chain interactions or relayed effects of other residues binding CXCL12 and affecting nearby positions in turn.

[0076] When we superimposed the residues involved in CXCL12 binding determined by both NMR and peptide arrays on the structure of HMGB1 1-164 (PDB 2YRQ), we identified pockets within each Box domain where the side chains of the residues all lined the center of the pocket. The residues identified both with alanine scan and NMR form a concave surface on Box B which also includes the glycyrrhizin binding site (Figure 3B, dotted circles). Conversely, several of the residues only showing changes in NMR have side chains pointing outwards of this pocket, suggesting a sequence-independent binding to CXCL12 (main-chain mediated) or that they are affected by indirect chemical environment changes in other parts of the HMG Box upon CXCL12 binding.

[0077] With this detailed understanding of CXCL12 binding, we were able to design a construct to eliminate proinflantinatory signaling via TLR-4, TLR-2 and RAGE.
This construct consisted of two 1-IMG Box B domains in tandem separated by a linker of similar length to that of wild-type HMGB1 (dBB12L), which is unable to oxidize as Box A has been replaced by Box B but still should bind CXCL12 as two HMG Boxes are present dBB12L was as stable as 1-164 FR-HMGB1 or full length FR-HMGB1, being equally thermostable in clinically relevant buffer conditions (PBS, saline solution), with no aggregation or degradation on storage for prolonged periods of time. The surface area and charge profile of dBB12L
also was like that of HMGB1 1-164, with monodisperse profiles in SEC and charge distribution in native ESI/MS. This similarity reflects a similar conformation in solution between dBB12L and a wild-type HMGB1 construct with two HMG Boxes and the linker region (FR-HMGB1 1-164) but without the acidic tail.
The engineered dBB12L construct does not signal via TLR-2 or TLR-4 or bind RAGE, whilst retaining full pro-regenerative properties

[0078] Binding of HMGB1 to TLR-4/MD-2 is well-described [291 Oxidized Box A
initiates the binding of DS-HMGB1 by interacting with TLR-4 and Box B stabilizes this interaction by binding MD-2; the FCSE motif within Box B has been shown to be essential for signaling [30].
Whilst DS-H1v1GB1 can signal on its own via TLR4/MD-2, it can also facilitate signaling via LPS by substituting for LPS-binding protein (LBP) which binds LPS and promotes transfer and recognition to TLR-4/IVID-2 [13]. Deletion of Box A in our dBB12L
construct effectively precluded signaling via TLR-4. Interestingly, we observed that both FR-HMGB1 and dBB12L

decrease INF expression by monocyte in response to LPS. This could be due to these proteins binding LPS but are unable to transfer it to TLR-4/MD2 due to the lack of oxidized Box A, unlike DS-HMGB1, which can effectively substitute for LPS binding protein (LBP) [13]
present in the serum of the culture medium [77].

[0079] The interaction between HMGB1 and TLR-2 has been less well described and there is some controversy as to whether HMGB1 can induce TLR-2 signaling on its own [24,36] or requires a co-ligand to induce activity, and also whether this response is dependent on the redox state of the protein 134,351 The published data where HMGB1 alone was unable to induce TLR-2 responses were performed either in absence of serum [35], or with low concentrations [34], suggesting that FIMGB1 alone retains some signaling capacity through TLR-2 but a co-ligand is required to induce higher levels of response. It is known that binding must involve at least one H:MG Box [33] and that the acidic tail negatively regulates binding to TLR-2 1351 We found that DS-HMGB1 alone was able to signal via TLR-2 and the effect was enhanced by the presence of LTA in serum-containing media. However, there was no response to FR-H:MGB1 or dBB12L, which did not synergize with LTA. This would suggest that, as with TLR-4, oxidized Box A with a disulfide bridge is necessary for TLR-2 mediated responses and that TLR-2 co-ligands synergize with DS-11MGBI, potentially by displacing the acidic tail to promote TLR-2 interaction.

[0080] The RAGE binding site within HIMGB1 (residues 150-183) has been previously described [14]. A similar motif is also present in other RAGE ligands such as S100 proteins, and homologous peptides to these sequences are effective antagonists of HMGB1-mediated RAGE signaling [37,78]. The acidic tail of IIMGB1 shares residues with the RAGE binding peptide [15] and has been proposed as a regulator of RAGE interaction, analogous to its role in TLR-2 binding. However, only the disulfide form of FIMGB1 has been linked specifically to prothrombotic activities via RAGE [22]. We found that constructs lacking the RAGE
binding peptide, including dBB12L, were unable to bind RAGE in the ELISA
assay. In contrast DS-HIMGB1, and interestingly also 3S-FIMGB1, were able to bind RAGE better than FR-HMGB1. When we analyzed the kinetics of the interaction using BLI we found that, in accordance with our ELISA data, dBB12L had lower affinity for RAGE compared to FR-HMOB1 and DS-H.MGB1. Whilst dBB12L had a three-fold higher association rate than FR-HIMGBI, potentially due to the presence of two partial RAGE binding domains in this construct, the dissociation rate was five-fold greater, indicative of overall unstable, weaker binding. The extensive washes associated with ELISA magnify the effect of the dissociation rates, leading to the different behavior of the HMGB1 constructs in each assay, and represent the binding status at equilibrium. For instance, 3S-HIMGBI, which has an affinity for RAGE
similar to FR-FIMGB1 in BIT, shows a much higher apparent affinity in ELISA
due to its dissociation rate being much lower than that of FR-HMGB1 or DS-1-1MGB 1, resulting in RAGE remaining bound to it. This increased RAGE binding may in part account for the increased fibrosis seen in mouse models of myocardial infarction compared to controls, whereas FR-HMGB1 promoted regeneration and improved function [32]. Using SPR
others have also shown that 3S-IIMGB1 likely binds RAGE with very high affinity, although comparison was not made with FR-HMGB1 1641 We found that the affinity for RAGE
for DS-HIMGB1 using BLI (Ka = 0.2-1.3 NI) was similar to that previously reported using SPR (0,1 [79] - 0.65 M [22]). Our BLI data also show that loss of the acidic tail increases the affinity of HMGB1 for RAGE, although it results in a less stable binding due to higher dissociation rates, whereas truncation of the RAGE binding peptide or reduction of Box A
greatly increases dissociation rates, resulting in unstable binding. Interestingly, oxidized Box A alone is incapable of binding RAGE, suggesting that the interaction is likely initiated by the RAGE
binding peptide. The absence of Box A together with truncation of the RAGE
binding peptide in dBB12L results in greatly reduced RAGE affinity, and reduced capacity to retain the RAGE
once bound, due to the faster dissociation rates when compared to either FR-or DS-HMGB1.

[0081] We found that the transcriptomic changes induced by FR-HMGB1 in skeletal muscle stem cells are very similar to those induced by distant injury and consistent with upregulation of pathways of mitochondria] metabolism, oxidative phosphorylation and cell cycle described Gideri [80,81]. Upregulation of CXCR4 expression by HMBG1 would potentially enhance its effects. Our data showing that FR-HNIGB1 is only effective if administered up to 5 hours post injury would be consistent with it acting by transitioning stem cells to Glum_ At later time points the stern cells will have been activated and hence cannot enter GAlert. Muscle stem cells have been shown to migrate to the site 5-6 hours after injury and start to actively divide at around 12 hours [82]. We confirmed that dBB12L retains regenerative activity in vivo equivalent to FR-11MGBI. Importantly, we found that FR-HMGB1 administered intravenously at the time of myocardial infarction resulted in improved survival, reduction in infarct size and improved left ventricular ejection fraction. Based on these data we would predict that administration of dBB12L would also promote regeneration of tissues that rely on stem cells for repair such as bone, skeletal muscle and blood, as well as tissues where regeneration is predominantly reliant on mature cell populations such as cardiomyocytes in the heart. We also predict that dBB12L

is likely to be effective if administered up to 5 hours after injury. This is important as the median time for admission to hospital following MI in the USA is 3 hours [83].
Approximately 800,000 people in the USA suffer from myocardial infarction every year [83]
and approximately 20-30% go on to develop cardiac failure. Despite US healthcare expenditure for heart failure of >$30 billion in 2012, projected to increase to $70Bn by 2030, 5-year survival is only ¨60%, which is worse than most cancers [83]. Based on our data we predict that administration of dB1312L within 5 hours of the event will enhance survival of patients experiencing a myocardial infarction, especially ST-elevation myocardial infarction, and through reduction in infarct size and preservation of ejection fraction, reduce the incidence and severity of cardiac failure. Others have shown in mouse [32,84,85] and sheep [86] models that direct injection of FR-H:MGB1 into the myocardium in the peri-infarct area 4 hours after infraction is effective in promoting cardiac repair. Our data demonstrating the efficacy of iv administration are important as this route is readily applicable for clinical use.

[0082] In conclusion, the sites of HIMGB1 critical for CXCL12 binding were mapped and a construct (dBB I2L) that does not signal via TLR-2 or TLR-4 and fails to effectively bind RAGE was designed. FR-HMGB1 transitions stem cells to Galen in a manner similar to distant injury despite a short half-life and is effective if administered up to 5 hours after injury.
Furthermore, dBB12L promotes tissue regeneration in vivo as effectively as FR-HMGBI.
Accordingly, dBB12L can be developed for clinical translation.
SUMMARY

[0083] Reduced High Mobility Group Box 1 (IIMGB1) protein binds to CXC Ligand (CXCL12) and signals through CXC Receptor 4 (CXCR4) to promote tissue and regeneration and accelerates repair by transitioning stem and progenitor cells to GAien.
However, local conversion of FR-HNIGB1 to the disulfide form (DS-HMGB1) may result in deleterious inflammation through signaling via Toll-Like Receptors 2 (TLR-2) and 4 (TLR-4), and the Receptor for Advanced Glycation End Products (RAGE). Therefore, before considering administration of HMGB1 to promote tissue regeneration in clinical practice, it is important to engineer the molecule to eliminate these potentially deleterious proinflammatory effects.

[0084] We have identified the residues involved in formation of the 1-IMGB1-heteroc,omplex using a combination of peptide arrays, biolayer interferometry and nuclear magnetic resonance. Combining these data with the available literature on the sites of interaction with the proinflammatory receptors, we designed a construct comprising two HMG

B Boxes in tandem (dB1312L) which has similar stability and conformation to that of a wild-type fully-reduced IlIMGB1 construct without the C-terminal acidic tail. As shown herein, dBB12L does not signal through TLR-2 or TLR-4, even in the presence of their co-ligands, and has greatly decreased RAGE binding. Furthermore, the dB1312L construct retains regenerative activity equivalent to FR-11MGBI in vivo.

[0085] A comprehensive review of the patent landscape and the scientific literature has identified U.S. Patent Application Publication US 2015/0203551 Al, which describes the substitution of cysteines with serines to prevent TLR-4 signalling; however, this construct has been shown to lead to excessive cardiac fibrosis following MI (17).
Furthermore, this construct has a slower dissociation for RAGE compared to FR-HMGB1, resulting in RAGE
remaining bound for longer after equilibrium, as shown herein in Figure 6B); therefore these substitutions were avoided in our constructs. U.S. Patent Application Publication US

stipulates that HIMGB1 constructs that promote stem cell migration and proliferation must include amino acids 1-187 (0-186 in our data, with 0 being the N-terminal Met) and U.S. Patent 9,623,078 refers to peptides limited to amino acids 1-44 (0-43 for our data) for cardiac regeneration. U.S. Patent Application Publication US 2009/0202500 Al discloses methods for tissue repair but only refers to full-length (1-215) wild-type HIMGB1 (0-214 for our data). The dBB12L construct presented herein has no RAGE binding or TLR-4/2 signalling, is 177 amino acids long, and includes amino acid substitutions that have not been previously described. Therefore, the constructs presented herein do not fall within the scope of the prior at CLINICAL APPLICATIONS

[0086] This invention provides polypeptides and methods to harness endogenous regenerative processes to enhance tissue repair. The polypeptides function similarly to fully reduced wild type HIMGB1 which promotes tissue regeneration by fanning a heterocomplex with two CXCL12 molecules, which in turn signals via CXCR4, likely two adjacent CXCR4 receptors on the cell surface.

[0087] Our data show that a polypeptide of the invention (dBB12L) acts in a similar way.
Therefore, it is contemplated that dBB12L will promote the regeneration of tissues that rely on CXCR4+ cells for repair. Such tissues include tissues where repair is primarily dependent on stem and progenitor cells, such as skeletal muscle and the haemopoietic system, as well tissues where repair is largely dependent on existing mature cells, e.g., cardiomyocytes in the adult mammalian heart.

[0088] Potential clinical indications:

[0089] Heart following myocardial infarction. This indication is ripe for a clinical trial.
Globally, ischemic heart disease affects 153 million people (101), with the loss of >105,000,000 Disability Adjusted Life Years in 2017 (102). Every year 205,000 people in the UK (103) and 805,000 in USA suffer from myocardial infarction (MI), 38% of them experiencing ST-elevation MI (STEMI) (101). Following MI, approximately 30-40%
of individuals develop heart failure, affecting 38 million worldwide. Despite US
healthcare expenditure for heart failure of >$30 billion in 2012, projected to increase to $70Bn by 2030, 5-year survival is only -60%, which is worse than most cancers (101). The main target population are patients following MI, especially those at risk of developing heart failure (104).
A novel therapeutic that limits cardiac damage, promotes regeneration following MI and prevents the development of heart failure would dramatically reduce morbidity and mortality, and massively reduce healthcare burden. Definitive data using an established (105-108) permanent ligation murine MI model that reliably leads to cardiomyocyte necrosis (109) show that a single iv dose of FR-HMGB1 at the time of injury leads to enhanced survival [83% for animals treated with FR-HMGB1 compared to 52% in the group treated with PBS
(placebo], and compared to controls, -16% improvement of absolute cardiac ejection fraction and -60%
reduction in infarct size compared to PBS controls over 5 weeks (Figure 7F).

[0090] In a skeletal injury model, the optimal dose of FR-FIMGB1 was 0.75 mg/kg (Figure 7C) and is effective even if administered iv up to 5 hours post injury (109) (Figure 7D), despite a very short half-life (Figure 7E). Following myoc.ardical infarction, reperfition of the ischemic cardiac muscle should be achieved as soon as possible. For example, following STENII patients should undergo percutaneous intervention. Data show that administration of HMGB1 as soon as possible and maximally up to 5 hours post-injury will preserve the damaged myocardium and promote regeneration.

[0091] While native FR-HNAGB1 promotes functional recovery post MI (Figures 7F-7I), local conversion to the disulfide form promotes thrombus formation and propagation via RAGE, TLR-2 and TLR-4 (110). Constructs reported by others such as 3S-HMGB1 that retain RAGE binding (Figure 6B) result in excessive fibrosis and impairment of function following Nil (111). FR-HMGB1 also binds RAGE, albeit to a lesser extent than DS-HMGB1 and, therefore, would not be suitable for clinical use. HIMGB1 signalling via TLR-2 plays a key role in ischaemia reperfusion injury following myocardial infarction (112) and Thrombosis (110), The inventors a have shown the key role of TLR-2 in human atherosclerosis (113). TLR-4 signalling is also crucial in myocardial reperfitsion injury (114). The redox conditions in the ischemic and inflamed microcirculation of the damaged heart following myocardial infarction will promote conversion of FR-H:MGB1 to the disulfide form (DS-H:MGB1), which is a central mediator ofthrombosis (110). There is no approved therapy for promoting cardiac regeneration following MI. Reports purporting to show regenerative effect of hematopoietic stem cells have been discredited (115), and killed cells are just as effective by triggering an immune response (116). Even with other cell types, including pluripotential stem cells, significant challenges remain, including arrhytlunogenesis, iinmunosuppression, scalability, batch variability, delivery, long-term viability and efficacy (115, 117). Large scale trials of cell-based temples showed no significant improvement in function, with arrhydunias reported in patients (118-120).

[0092] The absence of a significant stem population (121) and limited epicardial progenitor cells (122) in the adult heart, together with an understanding that the majority of new cardiomyocytes following injury are derived from existing cardiomyocytes, has shifted focus to promoting regeneration by manipulating endogenous pathways (121). This includes adenoviral transduction of multiple transcription factors(105, 108), manipulation of developmental pathways such as Hippo (106) or Meisl (123), addition of growth factors such as neuregulin (124), IGF/HGF (125) or FSTL1 (126), or manipulation of miRNAs (127). These approaches have significant shortcomings: adenoviml transduction and growth factors (IFGF1/HGF) require intracardiac injection or topical patch application (FSTL1), manipulation of developmental pathways carries oncogenic risk (128) and viral transduction of miRNA199-a in pigs resulted in fatal arrhythmias (127). An alternative strategy for stimulating cardiac regeneration by promoting clearance of immune cells requires repeated injection of VEGF-C
(129). Inhibition of MAP4K4 promotes myocardial survival and limits infarct size, but there was no regenerative effect (130). To date, none of these strategies have progressed to clinical trials.

[0093] This invention provides a unique solution which targets endogenous processes to promote cardiomyocyte survival and regeneration of multiple tissues. It overcomes the many hurdles associated with cell therapies, including anti-fibrotic CAR T cells (131), such as prohibitive expense (132, 133). Since FR-I-IMGB1 acts via the cell surface receptor CXCR4, it is not expected to have off target effects associated with targeting intracellular processes, e.g.
by alenoviral transduction of transcription factors or miRNA. IlMGB1 inhibition increased infarct size following ischemia reperfusion injury (134) and whilst local upregulation (135, 136) or intramyocardial injection of FR-HMGB1 has been shown to be effective in both mice (111, 137, 138) and sheep (139), our data indicate iv administration is efficacious and more likely to reach all target cells. The engineered double Box B construct of the invention which avoids deleterious proinflanunatory signaling should be safe.

[0094] A group in Milan described an FTMGB1 analogue (3S-HMGB1) where 3 cysteines are replaced with serines to negate TLR-4 signaling (140). Whilst they claimed that 3S-HIVIGB1 is superior to FR-HMGB1 in promoting tissue regeneration (141), the present inventors have not found this to be the case (142). Importantly, 3S-1-EMGB1 promoted fibrosis in a murine MI model, with deterioration in cardiac function, whereas FR-HMGB
I promoted tissue regeneration and improved left ventricular ejection fraction (111). The inventors have found that 3S-HMGB1 remains bound to RAGE for longer than either DS-HMGB1 or FR-HMGB1 (Figure 5A). Therefore, whilst FR-HMGB1, 3S-HMGB1 and DS-FINIGB1 bind equivalent amounts of RAGE at equilibrium, over time levels of RAGE bound by are comparable to the proinflaimnatory disulfide 11MGB1 (DS-I1MGB1) and higher than to FR-FIMGB1. The inventors double Box B construct eliminates undesirable proinflammatory signaling whilst retaining regenerative activity equivalent to FR-HMGB1.

[0095] Additional applications:

[0096] Fractures. Fractures occur following injury. However, one of the commonest skeletal 'injuries' is joint replacement or arthroplasty. The inventors propose that dBB12 can be used to promote healing following fracture or arthroplasty, thereby reducing the risk of potential complications such as loosening of components.

[0097] Brain and nervous system. dBB12L may be used to improve patient outcomes following stroke. Other potential indications include Parkinson's disease and dementia.

[0098] Lung. dB1312L is contemplated to improve outcomes following lung injury, for example, following Covid-19 or in patients with idiopathic pulmonary fibrosis.

[0099] Liver. 30% of people in the USA are estimated to suffer from non-alcoholic liver disease. 60% of these go on to develop non-alcoholic steatohepatitis and 20%
of those develop liver cirrhosis. Treatments are being developed to limit and prevent liver damage from tehse conditions. The inventors propose that dBB12L to be used in combination with these treatments to promote liver regeneration.

[00100] Gut dBB12L may be used to promote healing of the gut, for example, following surgery or patients with inflammatory bowel disease such as ulceractive colitis in combination with treatments to control inflammation,

[00101] Kidney. dBB12L may be used to promote regeneration of the kideny, thereby potentially avoiding the need for dialysis or kidney transplantation.

[00102] Skin. dBB12L may be used to promote wound healing eg following surgery, bums or patients with ulcers eg diabetic ulders.

[00103] Pancreas. dBB12L may be used to improve outcomes in patients with type 1 diabetes mellitus by promoting regeneration of islet cells.

[00104] Bone marrow, dBB12L may promote regeneration of the haemopoetic system e.g.
following chemotherapy, thereby preventing severe potentially life thereatening neutropenia.

[00105] The inventiors have previously shown that FR-HMGB1 is effective even if adminsitered up to 2 weeks before injury (142). Since dBB12L is equally efficacious to FR-HIMGB1 (Figure 7J) it is contemplated that this polypeptide may be used prophylactically, for example, by the military or for sports injuries or before elective surgery or chemotherapy.
MATERIALS AND METHODS
E. coil strains

[00106] Mach-I T IR cells (Invitrogen, no antibiotic resistance or induction, BL21(DE3)-R3-pRARE2 (in-house BL21 derivative, chloramphenicol resistance 36 pg/mL , T7-polymerase lac induction [87]) and BL21(DE3)-R3-pRARE2-BirA (in vivo biotinylation derivative of the above, additional spectinomycin resistance 50 pg/mL) were sourced from chemically competent stocks made in-house.
Bacterial culture media

[00107] SOC: 20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCI, 0.1862g/L KC1 were autoclaved and supplemented with 4.132 g/L MgCl2 and 20 mM glucose.

[00108] LB (Luria Bertani): 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCI, pH 7.2, autoclave sterilized. 2% w/v agar powder was added to make LB agar plates.

[00109] TB (Terrific Broth): 12 g/L tryptone, 24 g/L yeast extract, 4 g/L
glycerol, 12.5 g/L
K21-1PO4, 2.35 g/L KH2PO4., autoclave-sterilized

[00110] TB supplement 1.6% w/v glycerol, I% glucose, 25 mM (NH4)2SO4, 10 mM
MgSO4, 10X trace metals, 0.22 pM sterile filtered.

[00111] Trace metal solution: 50 mM FeCl3 (13.5 g/L), 20 mM CaC12 (2.94 g/L), 10 mM
MnCl2 (1.96 g/L), 10 mM ZnSO4 (2.88 g/L), 2 mM CoC12(0.48 g/L), 2 mM CuCl2 (0.34 g/L), and 2 mM NiC12 (0.48 gJL), in 0.1 M HC1, 0.22 p.M sterile-filtered.

[00112] M9 minimal medium: 16 g/L Na2HPO4, 4 g/L IC2HPO4, 1 g/L NaCl, pH 7.2-7.3 and 2.5 g/L FeSO4, 0.25 mg/L ZnC12, 0.05 mg/L CuSO4, 0.25 g/L EDTA, 1 mM MgSO4 were autoclaved and supplemented with 4 g/L glucose, 1 g/L U-99%1514H4C1 (Cambridge Isotopes), 0.3 mM CaC12,1.5 mg/L D-biotin and 1.5 mg/L Thiamine-HCL from sterile filtered stocks.
Plasmids

[00113] Plasmids were sourced from the SGC libraries [87]. All plasmids contain a 6xHis tag with a TEV-cleavage site; pNIC-Bio3 and pDsbC-HT-CBio also have C-terminal biotinylation epitopes (which can be removed with a stop codon). Plasmid DNA was linearized by restriction enzyme digestion: BfuAl (3h, 60 C) for pNIC-CTHF or Bsal (2h, 37 C). Cut vector DNA was purified with a PureLink PCR kit and treated with T4 DNA polymerase (NEB
M0203) in the presence of 0.25 mM dGTP (pNIC-CTHF) or dCTP as per manufacturer protocols.
Clcsaiin

[00114] HMGB1 constructs were sourced from the Mammalian Gene Collection (purified as plasmid from Machl cells grown overnight in LB medium with antibiotics).
Constructs were amplified via PCR: a program of 95 C /10', 25x (95 C/ 30", 52 C/1", 0.5-1.5 min at 68 C), 68 C/10`was used. Reaction consisted of 5 pt Herculase II buffer, 1 pM of each primer, 6 pg/mL plasmid template, 1 p.M dNTP mixture and 1 unit Herculase II polymerase (Agilent 600679; supplied with buffer and 100 pM dNTP stocks) in 25 L final volume.
PCR products were purified before further use (PureLink kit, ThermoFisher K310001).

[00115] Amplified coding sequences (alleles) were cloned into the destination vector via ligation independent cloning (LIC). The insert was treated with T4 DNA
polymerase in the presence of a cognate nucleotide to that used for the vector (10 pt reaction volume), and 2 pL
was mixed with 1 p.L of treated vector and annealed for 30`. 40 !IL ice-cold Mach-1 cells (for storage) or 20 pL BL21(DE3)-R3-pRARE2/ BL21(DE3)-R3-pRARE2-BirA cells (for expression) were added and heat-shocked for 45" at 42 C before chilling in ice. Recovery was performed for 2 hours in SOC medium at 37 C prior to plating on selective media with 5%
sucrose and antibiotics. After 24 h, positive colonies were picked and screened with MyTaq polymerase according to manufacturer protocols with specific sequencing primer pairs for bands of the correct molecular weight. Positive transformants were grown overnight in 1 inL
of 2X LB (double concentration of LB) with antibiotics and stocked with 12%
glycerol v/v at -80 C.

[00116] 3S-HMGB1 mutant sequence was generated in a similar manner. PCR was performed separately to generate a S23-S45 and a S106 fragment, which were annealed via PCR; 5 .1.1L of each purified PCR product substituted the primers and template in this reaction. The process is summarized in Figure 14.

[00117] CXCL12 constructs were cloned with an in-frame SUMO protease site N-terminal to the mature protein to allow for periplasmic secretion with an N-terminal fusion protein in the pDsbC-I-IT-CBio vector (DsbC-SUMO-CXCL12) to avoid addition of N-terminal residues to the protein which could affect its activity [88,89] whilst obtaining folded, oxidized CXCL12 via the DsbC fiision protein system 1901. All mutants were verified by sequencing (SourceBioscience). The sequence for FUVIGB1-dBB was designed in silico by codon-optimizing a Box B 89-174 sequence according to K coil BL21-DE3 genome (assembly ASM956v1) placed after the native IlIMGB1 Box B sequence, and synthetized in vitro by Twist Bioseience (San Francisco, USA) cloned in pNIC-CTHF.
Recombinant protein expression

[00118] 20 mL of overnight culture of HMGB1-expression strain transfonnants, grown from a fresh agar plate streak, were inoculated into 1 L of TB (or M9) medium with supplement and allowed to grow up to OD 2.0 at 37 C with 0.45 RCF orbital shaking (OD 0.6 for M9 medium).
Precultures used for production of 15N labelled HIVIGB1 were first spun down at 1000 RCF
for 5 'and washed in M9 medium. Once the target OD was reached, were cooled to 18 C before addition of 0.5 mM or 0.25 mM IPTG (for HMGBI and CXCL12 proteins respectively) and grown for 16 hours before harvesting at 4000 RCF. For biotinylated proteins, 10 mM D-biotin in PBS was added before induction and again 1 hour before cell harvesting.
Recombinant I-fMGB1 purification

[00119] Pellets of induced HMGB1-expressing cells were resuspended at 14 g/L
in 1 M NaCI, 5% glycerol, 50 mM HEPES pH 7.5, 10 mM Imidazole (Buffer A) supplemented with 1:1000 protease inhibitors (Calbiochem Set III, Merck 539134), 3 ps/mL Benzonase-MBP, 1 mM
MgSO4, 0.5 mg/L lysozyme (Sigma L6876) and 0.5% v/v Triton-X100 before freezing at -80 C; from this point onwards all steps took place at 4 C. Thawed pellets were spun down at 6780 RCF for 45' and the supematant was loaded into pre-equilibrated nickel-His GraviTrap (GE Healthcare) 1 na columns. After drip-through, columns were washed with 10 CV of 1 M
NaCI, 50 mM HEPES pH 7.5 and 1.5 CV of 0.4 M NaCI, 20 mM HEPES pH 7.5, 1 mM
MgSO4, and 3 gg/mL Benzonase-1VIBP solution to digest remaining DNA for 30`.
Contaminants were washed with 15 CV of 0.5 M NaCI, 5% glycerol, 50 mM HEPES pH
7.5 (Buffer B) supplemented with 30 mM imidazole before elution directly into a PD-10 column (GE Healthcare; equilibrated in Buffer B + 20 mM imidazole) with 15 mL of Buffer B + 500 mM imidazole. Proteins were eluted from the column with 3.5 mL of Buffer B +
20 mM
imidazole before tag removal with 1:20 OD TEV-GST protease over 16 h.

[00120] Proteases and further contaminants were removed by recirculating the protein solutions over the same GraviTrap column used to purify initially (equilibrated in Buffer 8+20 mM imidazole). For biotinylated proteins, streptavidin-XT resin was used instead to select biotinylated molecules only: after 30 min of incubation in the resin, sample was allowed to drip through, washed with 30 CV of buffer A and 1 CV of buffer B + 100 mM D-biotin, and eluted by incubation in 3 CV of the same buffer for 2 h. Proteins were further purified by size exclusion chromatography (SEC) (Superdex 575 10/300-0.35 mL/min or 16/600-1,2 mL/min flow rate) in either 10 mM HEPES pH 7.5 + 150 mM NaCI for biophysics work or cell-culture grade PBS for cell and animal work. Recombinant proteins were flash-frozen for storage, adding 1 mM TCEP in the case of reduced HMGB1 proteins.
Recombinant CXCL12 purification

[00121] Outer membranes of cells expressing DsbC-SUMO-CXCL12 were lysed by osmotic shock 1911 Pellets were resuspended at 40 g/L in 1 M sucrose, 0.2 M Tris-HCI
pH 8.0, 1 mM
EDTA, 1 mg/mL lysozyme, 2X cOmplete protease inhibitor set (COEDTAF-RO, Roche), 50 mM Imidazole and 3 gg/rnL benzonase. This was stirred for 45 min at room temperature before adding 4 volumes of ice-cold 18.2 m12 water and mixed for a further 10 min before adding 1 mM MgSO4. This was centrifuged for 1 hour at 16000 RCF, 4 C, and the supernatant loaded at 10 mL/min into Ni-NTA Superflow columns (Qiagen, 30761) on an Akta Xpress FPLC
system; 1 column was used for every 6 L of cells. Proteins were eluted via an imidazole gradient (10-25 mM over 10 CV, and 25-500 mM over 8 CV) in Buffer B, and 1:10 OD of Ulp-protease were added before dialysis in 100 volumes of 0.2 M NaCl, 20 mM HEPES
pH 8.0 (Buffer Ac) overnight. On the next day, the protein was loaded into CaptoS
columns (CaptoS
hripAct, GE 17-3717-47) at 2.3 mL/min and eluted in a gradient of 0.2-1.5 M
NaC1 in 20 mM
HEPES pH 8.0 to separate cut CXCL12 from DsbC and Ulp-1. Proteins were further purified via SEC in the same way as HIMGB1 and flash-frozen for storage.
Removal of endotoxins

[00122] Endotoxin was removed in all cases before size-exclusion chromatography via phase separation with Triton Tx-114 [92]. A 2% v/v of TX-114 was added to recombinant protein solutions, homogenized for 20' with orbital shaking at 2000 RCF at 4 C, and separated for 5' at 37 C before pelleting the detergent phase at 8000 RCF, 10', 2.5 C. The supernatant was mixed with 5% w/v of SM-2 Biobeads (BioRad, 152-8920), cleaned with 2% TX-114 for 2 hours and regenerated with 30 CV of methanol, 30 CV of endotoxin-free 18.2 mil water and 30 CV of endotoxin-free PBS. This was incubated for 4 hours at room temperature to adsorb remaining Triton and PEG [93] before injection onto a sanitized SEC system (with 0.5 M
NaOH contact over 12 h, followed by 0.2 M acetic acid/20% ethanol contact over 6 hours and equilibration in cell-culture grade PBS) to fully remove leftover polymer contaminants whilst performing size exclusion. The absence of Triton and PEG was verified by lack of their respective charge state species in ESI/QTOF-MS mass spectrometry [94]. LPS
content of the recombinant proteins was assessed via the LAL method (GenScript ToxinSensor L000350).
Samples were approved for cell and animal use when they contained <4 EU LPS/mg protein.
Enzyme production

[00123] TEV-GST protease (GST-fusion protein), Benzonase-MBP, and Ulp-1 protease were produced from transformants in storage at the SGC collection [87]; all had 200 1.1g/mL
ampicillin resistance. TEV and U1p-1 were purified as per the protocols described for HMGB1 with only one IMAC step, whereas Benzonase-MBP was purified from outer membrane lysates obtained as with CXCL12 and isolated with use of amylose resin (NEB, E0821) as per manufacturer protocols. In both cases, the resulting proteins were concentrated to 10 mg/mL
in 50 mM HEPES pH 7.5, 0.3 M NaC1, 10% glycerol. GST-TEV protease and Ulp-1 were flash-frozen with liquid nitrogen and supplemented with 0.5 mM TCEP during purification;
Benzonase-MBP was supplemented with 50% glycerol and 2 mM MgCl2 and stored at -20 C.
Peptide arrays

[00124] Membranes with FMOC-coupled 15-mer peptides of human IIMGB1 (Uniprot P09429) or CXCL12 (Uniprot P48061, excluding secretion signal) were printed by Dr. Sarah Picaud at the SGC upon request following published protocols [401. The membranes were rehydrated at 20-25 C with 95% and 70% ethanol, equilibrated with PBST (PBS IX
+0.05%
Tween-20, 3x), and blocked with 10% BSA/PBST for 8 h. 1 u.M of the partner His-tagged protein construct was added (in PBS) and allowed to bind for 24 hours at 4 C.
Excess BSA and protein was removed with 3 washes in PBST; all washes lasted 1 min unless otherwise stated.
To detect bound proteins, the membranes were treated with 1:3000 dilution of Qiagen anti-Penta His HRP conjugate (Qiagen 34460) and excess antibody removed with 3 washes in PBST
for 20'. Bound antibody was then quantified via chemiluminescence (Pierce ECL
substrate -32109): the membrane was covered in substrate solution and placed between two clear plastic sheets before incremental imaging at 2' intervals in a LAS-4000 camera (chemiltuninescence settings). The intensity of the peptides in each membrane was measured in ImageJ and normalized to the 10-His control. Alanine mutagenesis scans were performed in the same manner, with the printed peptides consisting of those identified during the initial peptide array.
Residues whose mutation to alanine resulted in higher intensity changes than those observed for alanine positions in the sequence were considered as significant contributors to CXCL12 binding.
Biolayer interferometry (BLI)

[00125] Pre-hydrated streptavidin Octet Biosensors (ForteBio 18-5019) were coated with 4 pM solutions of biotinylated 1-IMGB1 proteins in 10 mM HEPES, pH 7.5, 150 mM
NaC1 (Base buffer-BB) plus, 0.5 mM TCEP (60 sec baseline, 60 sec binding). Nonspecific binding was minimized by incubation for 3' in BB+ 1% BSA + 0.05% Tween-20 (Kinetics Buffer, KB) prior to kinetics assays. Interaction with CXCL12 was measured by performing incremental immersion of the sensors in solutions with increasing CXCL12 concentration (0-150 uM, in 1:2 dilutions) in KB (60 sec baseline, 500 sec association, 420 sec dissociation, 180 sec reduction in BB + 0.5 mM TCEP). An OctetRed 384 instrument was used for these experiments. Kinetics data were extracted with DataAnalysis 9.0 (ForteBio).
Response at equilibrium RE4 was plotted against concentration in a Michaelis-Menten saturation plot to calculate kD/Benox (saturation plot of concentration against rEq). Kinetic rates (association rate;
/con and dissociation rate; kW were derived from the direct measurements of each parameter from all the association and dissociation steps in the interferogram, and fitted to a horizontal line (mean) across all measurements. Data from each replicate run were pooled in the same manner to calculate the overall mean. For measuring RAGE binding kinetics to HMGB1, 15 pg/mL RAGE-Fe in PBS+ 0.1% BSA + 0.02% Tween-20 was immobilized on the surface of anti-IgG biosensors (AHC, 18-5060) over 30 sec and dipped in serial concentrations of each HIVIGB1 construct (60 sec baseline, 200 sec association/dissociation) and fitted in the same manner to derive kinetic parameters.
Nuclear magnetic resonance (NMR)

[00126] 'N-labelled recombinant HMG131 constructs in 10 mM HEPES 150 mM NaCl pH
7.5 (identical buffering and ionic strength to BLI experiments) were supplemented with 5%
v/v 920, and pipetted with a glass Pasteur pipette into a 5 mm Shigeimi tube, sealed with paraffin. Final volumes were > 330 L. CXCL12 was added in the same buffer and final volumes were adjusted to avoid modification of the referencing. Signal locking, tube shimming, and nuclei tuning were manually performed via Bruker TopSpin software. Water signal was suppressed by acquiring a III spectra with power level 1 (P1) =
estimated pulse calibration (pulsecal). When a single peak was observed, a P1 value of 4 times the initial was used as a baseline and adjusted until a symmetric peak could be observed in the 111 spectrum.
NMR experiments were performed after these calibration steps CH-NMR, 'N-HSQC, "N-NOESY-HSQC, "N-TOCSY-HSQC Peaks in "N-HSQC spectra were assigned based on published NMR tables for HMGB1 1-184 (BMRB 15418) and our own NOESY/TOCSY data for each construct analyzed. To measure CXCL12 binding, the chemical shift position and volume of identified peaks was tracked across different molar equivalences of CXCL12 (these are listed in the relevant image) via CCPNMR 3.0 Analyze's Chemical Shift Tracking module.
For peak intensity changes, the median intensity change in each set was considered as a baseline. Full chemical shift tables and experiment parameters are detained at the end of this section.
Mass spectrometry

[00127] For protein identification via MS/MS and tryptic digest, bands from SDS-PAGE gels were excised and submitted to the SGC open access MS platform and analysed by Dr. Rod Chalk, Dr. Tiago Moreira and Oktawia Borkowska, as published 94,95]. Data analysis (peptide mapping) to annotate protein identity was performed with the MASCOT
search engine, against the Uniprot (reference protein sequences) and SGC (construct sequence) databases. Native ESI/MS experiments were performed by manual injection in volatile buffer (50 or 200 mM ammonium acetate, 6.5) into an ESI/QTOF instrument (Agilent Q-TOF
6545) at 360 tit/h. Signal was acquired for at least 10 counts (30 sec) once a steady ionic stream was observed in the total ion chromatogram. For denaturing experiments, samples were diluted to 1 mg/mL in 0.2% formic acid and injected via HPLC (Agilent 1100 HPLC) and eluted in a mobile phase of formic acid/methanol, as described [94]. Each continuous distribution of charge states was considered a distinct conformation; charge states (Z) were assigned according to the formula where mW= (mW/Z-proton mass )* Z. Surface areas were derived from the formulas proposed in the literature [96,97] which resulted in the formula In(SASA)= ln(M/Z)*0.6897-4.063 for native MS and In(SASA)= ln(M/Z)*0.9024-5.9013 for denatured samples. At least three independent injections were performed for all MS samples.
All solutions in these experiments were made with HPLC water (electrochemical grade) and solvents.
SEC surface area quantitation

[00128] To correlate SEC chromatograms with surface area, standard sets with known structures (BioRad 1511901) were run through a Superdex 75 pg, 10/300 column used in these experiments. SASA for the proteins contained in these and the GE-supplied calibration curve standards were derived from published PDB structures (BSA, 3V03,0valbumin, 1JTI;Myoglobin, 2V1I;RNAseA, 1A5P;Aprotinin, 1NAG;Vitamin B12, 3 BUL) and correlated with retention volume by nonlinear least squares fitting (SASA= 331.2*R12-1.19e4*RV+1.08e5). All experiments compared between LIMOS 1 samples were performed in the same buffer as native MS (200 mM ammonium acetate, pH 65); injections were performed at 1 mg/mL to avoid saturation of signal and all samples were eluted at 0.4 mL/min.
RAGE binding ELISA assay

[00129] 384-well protein-binding ELISA plates (Santa Cruz Biotechnology, se-206072) were coated with 50 pt of 40 nM solutions in PBS (+ 0.5 inM TCEP for FR-HIMGB1 constructs) of 14M681 constructs for 24 h_ at 4 C, including FL/DS HMGB1 full-length controls and blank, with 4 replicates of each. Nonspecific binding was blocked by incubation with 10% BSA in PBS for 2 h, at 20-25 C. A concentration range of RAGE-Fe chimera protein (BioTectme, 1145-RG; 0-640 nM in 1:4 dilutions) was added in 10% BSA/PBS and allowed to bind for 2 h, at 4 C. Bound FC chimera was detected by incubation with Anti-Human IgG
FIRP (Agilent DaIco P021402-2) diluted 1:10000 in 1% BSA/PBS for 2 h, at 20-25 C. Between each of these 3 steps, the plate was washed with 100 pt PBST, 3 times.

[00130] To detect bound antibody, 25 pL of TMB substrate (ThermoFisher N301) was added to each well; the reaction was allowed to develop in the dark until the FL-DS-FIMGB1 control developed a clear concentration-dependent color gradient before stopping the reaction with 25 pL of 0.5 M H2504. OD 450 was measured as a readout (FluoStar OMEGA, BMG
Labtech) and plotted as a saturation fit against 2x RAGE-Fe concentration (as the chimera is a RAGE
dimer) TLR-4 and TLR-2-mediated NF-icB signaling reporter assay

[00131] HEK-Dual cells (Invivogen) expressing human TLR-2 and CD14 or murine TLR-4, MD-2 and CD14 were maintained in DMEM (Gibco), supplemented with 10 % FBS
(Gibco), 1 % L-Glutamine (Gibco), and 1 % penicillin/stieptomycin (Gibco), in standard tissue culture conditions (37oC; 5% CO2). To determine if FR-FIMGB1, DS-FIMGB1 and dBB12L
induces activation of TLR-4 and TLR-2 signaling, 104 TLR-4 and TLR-2 1-113K-Dual cells were plated in triplicate into wells of a 96 well plate and stimulated with 10 pg/mL 1 HIMGB1 and (X
concentration) FSL-1 for TLR-2 and 10 ng/mL LPS for TLR-4. 24 hours after stimulation, NF-KO activity was determined my measuring the induced levels of secreted embryonic alkaline phosphatase (SEAP).
Monocyte total NF-KB secretion assay

[00132] Human monocytes (StemCell Technologies) were maintained in DMEM
(Gibco), supplemented with 10% FBS (Gibco) in standard tissue culture conditions (37 C;
5% CO2).
To determine if FR-H1MGB1, DS-HMGB1 and dBB12L induces proinflammatory cytokine production, 105 human monocytes were plated in triplicate into wells of a 96-well plate and stimulated with 10 pg/mL HMGB1 and 50 ng/mL LPS or 10 ng/mL LTA. 24 hours after stimulation, TNF levels were determined by Enzyme-linked immunosorbent assays (ELISA) (Abeam).
Transcriptomic analysis

[00133] Mice were treated systemically with an i.v. injection of 30 pg FR-HMGB1 in 50 pL
of PBS vehicle, or PBS only control. Injury cell are from BaC12 injured mice as described below. Alert cells are from the uninjured contralateral side of BaC12 injured mice. Murine muscle stem cells (mMuSCs) were defined and freshly isolated according to previously reported protocols. Muscle cell suspensions were created by mincing thigh muscles and enzymatically digesting with collagenase 800 U/ml (Worthington-Biochem) and dispase 1 U/mL (Gibco). Thereafter, all suspensions were strained through 70 pm and 40 pm filters (Greiner Rio-One) and stained with respective antibodies. mMuSC, CD31-CD4.5-Sca-1-VCAM1+, were isolated by fluorescence activated cell sorting (FACS) using BD
FACSAria III machine. RNA, extracted from freshly FACS isolated mMuSCs, was sent to RNA-seq analysis using Lexogen 3'kit library prep and sequenced using HiSeq400 (Illumina). FASTQ
files were assessed using FASTQC followed by the generation of TPM values with kallisto v0.42.4. TPM values were summed to obtain gene-level expression values using ix/import and differential expression analysis was undertaken with DeSEQ2. GO enrichment of differentially expressed genes was performed using the R package 'clusterProfiler'[98] with a Benjamini-Hochberg multiple testing adjustment and a false-discovery rate cut-off of 0.1.
Visualization was performed using the R packages cggplotT and igraph' In vivo mouse muscle injury model

[00134] C578L/6 inbred mouse strain, females of 11-12 weeks of age were purchased from Charles River UK and housed in the local Biological Safety Unit (BSU) at the Kennedy Institute. Acclimatization period was 1-2 weeks. All protocols performed on live animals have been approved by the UK Home Office (PPL 30/3330 and PPL P12F5C2AF) as well as the local animal facility named persons and are registered under the appropriate project and personal licenses under ASPA regulations. All consumables were surgically certified;
recombinant proteins were endotoxin-free. Surgeries were done in a clean environment separate from culling facilities. All animals were monitored for 6 hours post-operatively, and daily for the following 3 days; monitoring was then transferred to the NVS/NACWO.

[00135] Surgeries were performed as described previously ([7,12]). Animals were anesthetized by aerosolized 2% isoflurane, given analgesia, transferred to a warming pad and the right lower hindlimb was disinfected with povidone iodine and the tail with 70% ethanol if intravenous injection was performed. 50 EIL of 1.2% BaCl2 (Sigma) was injected into and along the length of the tibialis anterior (TA) muscle to induce cell death. Mice were euthanized and lower limbs removed at the times indicated, fixed in 4% paraformaldehyde (Santa Cruz Biotechnology) for 24 h. The TA muscles were dissected and further fixed for 24 hours before being embedded in paraffin and sectioned. Sections (5 pm) were stained with hematoxylin and eosin to identify fibers with central nuclei and imaged with an Olympus BX51 using a 10x ocular/ 40x objective lens. The cross-sectional area (CSA) of the fibers from at least 4 images per mice was manually measured using the FIR distribution of ImageJ2 software (N111). Data were grouped per mice. Mice were injected with HMGB1 constructs (46 nM/kg, resuspended in PBS) or PBS vehicle control intramuscularly or intravenously at the time of injury or after injury for the optimal time administration of FUVIGB1 constructs after injury.
In vivo mouse cardiac injury model

[00136] C57BL/6 female mice were subject to surgery between 10-14 weeks old, with body weight between 25-30 g, All mice had either an intravenous injection of FR-FUVIGB1 (46 n/vI/kg, resuspended in PBS) or vehicle control just before surgery.
Buprenorphine (buprenorphine hydrochloride; Vetergesic) was delivered as a 0.015 mg ml solution via intraperitoneal injection at 20 min before the procedure to provide analgesia.
They were anaesthetized with 2.5% isoflurane and externally ventilated via an endotracheal tube. Cardiac injury was induced by permanent ligation of the left anterior descending coronary artery (LAD) via a thoracotomy. Experimenters were blind to treatment groups for subsequent cardiac eine-MRI and analysis. Mice were housed and maintained in a controlled environment.
All surgical and pharmacological procedures were performed in accordance with the Animals (Scientific Procedures) Act 1986, UK.
Cardiac cine-MRI and analysis

[00137] Cardiac cine-MRI was performed post-LAD ligation at 7T using a Varian DDR
system. Briefly, mice were anaesthetised with 2% isoflurane in 02, and positioned supine in a custom animal handling system with homeothermic control. Prospectively gated proton cardiac images were acquired with a partial Fourier accelerated spoiled gradient echo CINE sequence (TR 5.9 ms, TE 2.2 ms, 30 kHz bandwidth, 30 FA, approximately 20-30 frames gated to the R wave with a 4 ms postlabel delay; 20% partial acquisition; 4 averages) with a 72 mm volume transmit/4 channel surface receive coil (Rapid Biomedical (3mbH) in order to acquire two and four chamber long-axis views and a short axis stack for finictional quantification (128x128 matrix; 25.6 nunA2 FOV; 0.2 min resolution in-plane). Non-acquired partial Fourier data was reconstructed via the method of projection onto convex sets prior to a simple, cartesian, DFT.
Blinded image analysis was performed using ImageJ (NH-1). Left ventricular mass, volumes and ejection fraction were calculated as previously described'. The relative infarct size was calculated from the average of the endocardial and epicardial circumferential lengths of the thinned, akinetic region of all slices, measured at diastole, and expressed as a percentage of the total myocardial surface [99].
Statistical analysis

[00138] All calculations were performed with GraphPad Prism (v. 8.41). For kinetics experiments (BLURAGE ELISA), all fits were performed via nonlinear least squares. For the RAGE ELISA, as each concentration of RAGE was independent from the rest of the wells, all data were considered as one kinetics fit; in BLI however each sensor was considered an independent fit for the purposes of calculation. Comparisons between parameters were performed via the AUC method. Mouse muscle injury model dam were analyzed as a nested ANOVA, where each sub-column comprises all the muscle CSA values for a given animal, and each group contains all the animals in order to separate biological variation from treatment effect. If the equal variance assumption could not be met in either case data were analyzed via a Kruskal-Wallis test; for nested ANOVA equal numbers of data from each animal would be randomly selected to avoid skewing. Any other data were analyzed via one-way ANOVA if the heteroskedasticity plot and Q/Q plot supported the equal variance assumption [100]; these were also verified by Spearman's Test. For multivariate experiments (e.g.
cardiac experiments) a two-factor ANOVA was used under the same assumptions (no dataset violated heteroskedasticity in this case). Post-hoc comparisons were weighted by Holms-Sidak correction (ANOVA family tests) or Dunn's method (Kruskal-Wallis). The test selected in each case is noted under each figure legend. Significance legends: n.s; not significant, *; p <0.033, **; p< 0,002, ***; p< 0.0002, ****; p< 0,0001.
NMR chemical shift tables HMGB1-c028 (94462, biotinylated) titration with CXCL12A-c021 at 0, 0.42, 0.82 and 1.42 molar equivalents, Figures 3A-3B.

[00139] Due to protein amount limitations, titration was performed by sequential addition of CXCL12 to H]%'IGB1 samples, resulting in sample dilution. As the calculations in the chemical shift tracking module in CCPNMR are independent of peak, this does not alter the results; the median change has been indicated in the volume comparisons.
BROKER AVM HD 500,5 MM CPTCI 1H-13C/15N/D
Z-GRD PROBE
1H frequency (Hz) 500.01233645 1H sweep width (ppm) 11.9044681870088 Itisweep width (Hz) 5952.38095238095 1FIN sweep width (ppm) 10.9286921061064 1.514 frequency (Hz) 50.6715211382702 '5N sweep width (ppm) 32.0372585410812 15N sweep width (Hz) 1623.37662337662 Water suppression gradient pulse width (Hz) 2336.45 Buffer 10 mM HEPES, pH 7.5, 150 mM Had Baseline 1 (Day 1) Experiment type 2D

HMGB1 construct and concentration 0.45 mM, HMGB1-c028 (Box B 94-162), C-terminal Avi tag C.XCL12 construct and concentration 0 mM
Temperature 25 Volume Sample pH (after adjusting) 7.7 Number of scans 8 Assign Fl position F2 position LW Fl (Hz) i LW F2 (Hz) Height BoxBFL95.LYS.N 8.47816 i 122.87351 16.67468 12.22962 108864312 i , BoxBFL.96.4FtG.N
8.30965 i 123.9316 14.96291 11.62421 i 144139104 ;. , BoxBFL99.SER.N
7.95885 ' 114.22726 22.5074 15.03176 1 16802856 BoxBFL.100.ALA.N 9.0643 122.83852 12.15917 12.63149 138071536 BoxBFL1.01.PHEN
8.44324 116.14361 13.00283 12.61897 1 92877416 BoxBFL102.PHEM
8.26304 122.13933 14.85477 12.7452 ' 88662424 BoxBFL.103.LEU.N
8.27748 121.1555 13.77712 12.44091 112430712 BoxBFL.104.PHE.N
8.15422 122.51526 17.30965 12.77095 208687344 BoxBFL.105.CYS.P1 8.49978 118.38542 12.79441 13.0002 114087696 BoxBFL1DESER.N
8.12921 115.05247 11.81293 12.01762 185743392 BoxBFL.107.GLU.N
7.15414 119.18761 15.14408 11.95417 174642688 BoxBFL.108.7YR.N
7.944 114.61662 17.02596 12.46204 BoxBFL109.ARG.N
9.23012 123.69626 13.5621 12.34021 130691488 BoxBFL111.LYS.N
6.80053 117.70067 15.27665 12.39016 143073536 BoxBFL112.1LEN
8.1617 119.74645 14.25943 12.83013 126874280 BoxBFL113.LYSeN
8.24755 118.47981 17.01044 13.8.802 71528184 BoxBFL.114.GLY.N 7.6363 103.71903 15.16014 12.02905 120973232 BoxBFL.115.GLU.N
7.50091 119.58688 15.18257 12.11395 146458640 BoxBFL.116.111S.N
7.8198 114.78302 14.39534 11.92497 1175052%
BoxBFL.119.LEU.N
7.36285 121.19842 12.72385 11.91713 222764784 BoxBFL120SER.N
9.31614 121.05943 13.74707 12.14133 126779632 BoxBFL121.1LEN
8.7484 120.66613 14.38154 12.69317 61496764 BoxBFL122.GLY.N
8.70938 109.44017 17.19009 12.98604 15080434 BoxBFL.123.ASP.N
7.96042 124.28299 14.86623 12.46038 123154200 BoxBFL124.VALN
8.63541 124.07082 13.35656 11.92144 172075232 BoxBFL.12S.ALA.N
7.92766 121.34983 12.19988 11.82723 225860864 BoxBFL.126.LYS.N
8.01853 119.68738 12.11349 12.34419 232838720 BoxBFL.127.LYS.N
8.03865 121.30632 12.54305 12.01855 257706416 BoxBFL.128.LEU.N 8.6442 120.16728 12.9965 12.28793 158055184 BoxBFL.129.GLY.N
8.26246 106.03693 14.92945 11.88241 144422592 BoxBFL130.GLU.N
8.07143 123.2476 13.07349 12.04715 202424016 BoxBFL131.MET.N
8.77019 118.9603 12.65128 12.17389 190716976 BoxBFL132.7RP.N
8.7155 122.68159 13.66438 12.0197 BoxBFL.133.ASN.N
8.17909 117.36655 11.90513 11.83855 191488080 BoxBFL.134.ASN.N
7.58752 115.82865 19.62284 12.10783 166835600 BoxBFL.135.71IR.N
7.30723 119.95451 15.53231 12.28047 175465168 BoxBFL.136.ALA.N
9.24925 131.44171 12.05279 11.8376 183664944 BoxBFL138.ASP.N
8.99204 115.70793 13.82959 13.53115 i 66696728 BoxBFL139.4SP.N
7.33843 117.87656 17.35284 12.00825 ! 172632800 BoxBFL140.LYS.N 7.8113 119.11469 15.11934 12.32708 i 144059264 BoxEIFL.141.G.N.N
7.42681 118.19987 12.23114 12.40879 203757632 BoxBFL143.7YR.N
7.21691 115.66054 15.9391 12.2 135669520 BoxBFL144.GLU.N
8.18987 120.68771 12.95833 12.4239 148173872 BoxBFL.14S.LYS.N
9.18301 121.23245 13.19779 12.25029 171717696 BoxBFL.146.LYS.N, Box0FL.156.1.11S.N
7.69692 120.57372 12.71031 12.47383 196874368 BoxBFL147.ALAM
8.48249 121.09351 12.35114 12.39023 191332288 BoxBFL148.ALALN
8.36965 122.00069 12.19025 12.15233 220547328 BoxBFL149.LYS_N
7.97224 120.72971 13.19222 12.42346 213554816 BoxBFL15O.LEU.N
8.28652 120.07973 14.56355 13.14455 145004448 BoxBFL.151.LYS.N
8.42411 123.51065 12.54219 12.56125 167598160 BoxBFL.152ØU.N
8.08663 119.9904 12.53193 12.7395 BoxBFL.153.LYS.N
7.75259 119.74852 13.195 12.42426 BoxBFL154.7YR.N
8.13041 120.47623 13.12379 12.69255 159451584 BoxBFL1SS.GLU.N
8.52484 117.50521 13.60921 12.71924 154408976 BoxBFL1S1.ASP.N
8.85459 123.10409 12.45843 12.23066 174923680 BoxBFL158.ILE.N
9.0667 122.28071 13.73538 12.53844 157092272 BoxBFL.159.ALA.N
7.45046 124.74939 12.51372 12.04599 i 200120928 BoxBFL.160.ALAM
7.74386 120.563 14.38809 12.29212 1 198915568 BoxBFL.161.7YR.N
8.20147 120.05331 14.39746 13.18223 ! 167227632 BoxBFL.162.ARG.N
8.26383 118.79358 13.15573 12.41703 ; 173894048 3D experiments (Day 1) These spectra are not shown due to their 3D nature; data available if requested. Sample is that of Baseline 1.
Experiment type 3D, 15N
EDITED ItI-1H TOCSY-HSQC, THROUGH-SPACE
INTERACTION
Temperature I 25 QC
Volume 300 piL
Number of scans 12 Experiment type 3D, 15N
EDITED 111-1H NOEST-HSQC, THROUGH-BOND
------------------------------------------------------------------ , INTERACTION
Temperature 25 QC
Volume 300 LILL
Number of scans I 16 Assi n F1 sition i F2 osition LW F1 Hz LW F2 Hz Hei ht I
Boxl3FL.95.LYS.N
8.47806 122.8889 15.74549 1239893 1.04E+08 BoxBFL.96.ARG.N
8.31955 123.9958 15.32754 11.79906 1.23E-F08 I3oxBFL.99.SER.N 7.95975 114.213 18.91249 16.00748 19314842 BoxBFL.100.ALA.N 9.06482 122.8403 12.12244 12.51287 1.23E-108 BoxBFL.101.PHE.N
8.44328 116.1449 14.02058 12.94259 83270120 BoxBFL.102.PHE.N
8.2626 122.1026 14.57979 12.61002 83484184 BoxBFL.103.LEU.N
8.27876 121.1543 13.21545 12.41884 98850448 BoxBFL.104.PHE.N
8.1723 122.6037 17.85543 16.70344 1.63E+08 BoxBFL.105.CYS.N 8.49894 118.3888 12.67018 13.00159 1.03E+08 BoxBFL.106.SER.N 8.12904 115.0558 11.922 12.17556 1.66E+08 EIoxBFL.107.GLU.N 7.15246 119.1841 15.05754 12.02976 1.54E+08 BoxBFL.108.TYR.N 7.94272 114.6176 17.11915 12.55614 1.04E+08 BoxBFL.109.ARG.N 9.23036 123.7013 13.06226 12.53714 1.15E+08 BoxBFL.111.LYS.N 6.79969 117.703 15.74346 12.62776 1.25E+08 BoxBFL.112.I1E.N
8.1614 119.7481 15.19044 12.89975 1.06E+08 BoxBFL.113.LYS.N
8.25273 118.4894 16.03035 15.20607 71142304 BoxBFL.114.GLY.N 7.63597 103.7241 14.57231 12.02882 1.1E+08 BoxBFL.115.GLU.N 7.49968 119.5819 15.16195 12.5197 1.35E+08 BoxBFL.116.HIS.N 7.82235 114.787 15.19416 11.98251 1.02E+08 BoxBFL.119.LEU.N
7.3624 121.2015 12.53706 12.04396 2.03E+08 BoxBFL.120SER.N 9.31525 121.061 13.81227 12.2652 1.13E+08 BoxBFL.121.ILE.N 8.74925 120.6769 12.5753 12.7098 61919540 BoxBFL.122.GLY.N
8.71207 109.4584 25.89949 13.91125 16954762 BoxBFL.123.ASP.N 7.95784 124.2831 14.47967 12.59113 1.13E+08 EloxBFL.124.VALN 8.63489 124.0688 13.34502 12.07557 1.51E+08 BoxBFL.125.ALA.N 7.92846 121.3424 11.96993 12.06949 1.99E+08 BoxBFL.126.LYS.N
8.01973 119.6945 12.24395 12.62986 2.08E+08 EloxBFL.127.LYS.N
8.03911 121.3059 12.17418 12.02556 2.24E+08 BoxBFL.128.LEU.N 8.64346 120.1657 12.73252 12.33711 1.36E+08 BoxBFL.129.GLY.N 8.26155 106.0419 14.87585 12.01202 1.26E+08 BoxBFL.130.GLU.N 8.07094 123.248 12.8194 12.1305 1.81E+08 EloxBFL.131.MET.N 8.77033 118.9628 12.47681 12.18055 1.65E+08 BoxBFL.132.TRP.N i 8.71521 122.6821 13.43252 i 12.15419 1.09E+08 BoxBFL.133.ASN.N 8.17879 117.3654 11.81466 11.94108 1.72E+08 BoxBFL.134.ASN.N 7.58738 115.8377 20.34615 11.95454 1.47E+08 BoxBFL.135.THR.N 7.30659 119.9511 15.61869 12.3645 1.57E+08 BoxBFL.136.ALA.N 9.2484 131.4382 11.79903 12.0221 1.7E+08 BoxBFL.138.ASP.N
8.99068 115.7088 13.61787 13.15666 66921064 BoxBFL.139.ASP.N 7.33797 117.8781 18.54773 12.14095 1.52E+08 BoxBFL.140.LYS.N 7.81083 119.1164 14.67427 12.45762 1.24E+08 BoxBFL.141.GLN.N 7.42677 118.2031 12.06407 12.71254 1.77E+08 BoxBFL.143.TYR.N 7.21631 115.6619 15.45741 12.35014 1.19E+08 BoxBFL.144.GLU.N 8.18784 120.6799 13.13889 12.30444 1.27E+08 BoxBFL.145.LYS.N
9.183 121.238 13.17983 12.43273 1.47E+08 BoxBFL.146.LYS.N, BoxBFL.156.LYS.N 7.69709 120.5754 12.94948 12.65531 1.73E-W8 BoxBFL.147.ALA.N 8.48271 121.0951 12.06422 12.53668 1.63E+08 BoxBFL.148.ALA.N 8.37014 122.0096 12.01385 12.60817 1.9E+08 BoxBFL.149.LYS.N 7.97339 120.7383 13.42094 12.64648 1.82E+08 BoxBFL.150.LEU.N 8.28676 120.0845 14.4103 13.05275 1.22E-W8 BoxBFL.151.LYS.N 8.42457 123.5222 12.66033 12.96606 1.42E+08 BoxBFL.152.GLU.N 8.08704 119.9998 12.5224 13.21315 1.98E+08 BoxBFL.153.LYS.N 7.75272 119.7489 13.27981 12.63584 1.63E-1-08 BoxBFL.154.TYR.N 8.12892 120.4783 12.99281 12.73019 1.34E+08 BoxBFL.155.GLU.N 8.52395 117.5111 13.25587 12.99665 1.34E-W8 BoxBFL.157.ASP.N 8.85469 123.1047 12.50397 12.25568 1.51E+08 BoxBFL.158.ILE.N
9.06751 122.2825 13.25933 12.71521 1.38E+08 BoxBFL.159.ALA.N 7.44862 124.7591 12.4575 12.38733 1.75E+08 BoxBFL.160.ALA.N
7.7427 120.5687 14.46225 12.64065 1.71E+08 13oxBFL.161.TYR.N 8.19946 120.0547 15.35591 ! 13.74534 1.39E+08 BoxBFL.162.ARG.N 8.2623 118.7997 12.52352 ! 12.69996 1.54E+08 0.84 molar ratio CXCL12/1-17MG Box (Day 2) Experiment type 2D 1H-15N HSQC
HMGB1 construct and concentration 0.337 mM, U-15N HMGB1A-c028 (Box B 94-162), C-terminal biotinylation tag (300 p.L of 0.45 mM stock) CXCL12 construct and concentration 0.285 mM, CXCL12A-c021, wt 1-67, no tag (100 1.1L of 1.14 mM stock) Volume 350 pi Sample pH (after adjusting) 7.73 Temperature 25 QC
Number of scans 8 Fl assign F1 F2 LW Fl LW F2 Height position position (Hz) (Hz) BoxBFL.95.LYS.N 8.47806 122.8889 15.74549 12.59893 1.04E+08 BoxBFL.96.ARG.N 8.31955 123.9958 15.32754 11.79906 1.23E+08 BoxBFL.99.SER.N 7.95975 114.213 18.91249 16.00748 19314842 BoxBFL.100ALA.N 9.06482 122.8403 12.12244 12.51287 1.23E+08 BoxBFL.101.PHE.N 8.44328 116.1449 14.02058 12.94259 83270120 BoxBFL.102.PHE.N 8.2626 122.1026 1437979 12.61002 83484184 BoxBFL.103.LEU.N 8.27876 121.1543 13.21545 12.41884 98850448 BoxBFL.104.PHE.N 8.1723 122.6037 17.85543 16.70344 1.63E+08 EIoxBFL.105.CYS.N 8.49894 118.3888 12.67018 13.00159 1.03E+08 BoxBFL.106.SER.N 8.12904 115.0558 11.922 12.17556 1.66E+08 BoxBFL.107.GLU.N 7.15246 119.1841 15.05754 12.02976 1.54E+08 BoxBFL.108.TYR.N 7.94272 114.6176 17.11915 12.55614 1.04E+08 BoxBFL.109.ARG.N 9.23036 123.7013 13.06226 12.53714 1.15E+08 BoxBFL.111.LYS.N 6.79969 117.703 15.74346 12.62776 1.25E+08 BoxBFL.112.ILE.N
8.1614 119.7481 15.19044 12.89975 1.06E+08 BoxBFL.113.LYS.N 8.25273 118.4894 16.03035 15.20607 71142304 BoxBFL.114.GLY.N 7.63597 103.7241 ! 14.57231 12.02882 1.1E+08 BoxBFL.115.GLU.N 7.49968 1193819 15.16195 12.5197 1.35E+08 BoxBFL.116.111S.N 7.82235 114.787 15.19416 11.98251 1.02E+08 BoxBFL.119.LEU.N 7.3624 121.2015 12.53706 12.04396 2.03E+08 BoxBFL.120SER.N 9.31525 121.061 13.81227 12.2652 1.13E+08 BoxBFL.121.ILE.N 8.74925 120.6769 12.5753 12.7098 61919540 BoxBFL.122.GLY.N 8.71207 109.4584 25.89949 13.91125 16954762 BoxBFL.123.ASP.N 7.95784 124.2831 14.47967 12.59113 1.13E+08 BoxBFL.124.VALN 8.63489 124.0688 13.34502 12.07557 1.51E+08 BoxBFL.125.ALA.N 7.92846 121.3424 11.96993 12.06949 1.99E+08 BoxBFL.126.LYS.N 8.01973 119.6945 12.24395 12.62986 2.08E+08 BoxBFL.127.LYS.N 8.03911 121.3059 12.17418 12.02556 2.24E+08 BoxBFL.128.LEU.N 8.64346 120.1657 12.73252 12.33711 1.36E+08 BoxBFL.129.GLY.N 8.26155 106.0419 14.87585 12.01202 1.26E+08 4.
BoxBFL.130.GLU.N 8.07094 123.248 122194 12.1305 121E+08 BoxBFL.131.MET.N 8.77033 118.9628 12.47681 12.18055 1.65E+08 BoxBFL.132.TRP.N 8.71521 122.6821 13.43252 12.15419 1.09E+08 BoxBFL.133.A.SN.N 8.17879 117.3654 11.81466 11.94108 1.72E+08 BoxBFL.134.ASN.N 7.58738 115.8377 20.34615 11.95454 1.47E+08 BoxBFL.135.THR.N 7.30659 119.9511 15.61869 12.3645 1.57E+08 BoxBFL.136.ALA.N 9.2484 131.4382 11.79903 12.0221 1.7E+08 EIoxBFL.138.ASP.N 8.99068 115.7088 13.61787 13.15666 66921064 BoxBFL.139.ASP.N 7.33797õ117.8781 ! 18.54773 1 12.14095 1.52E+08 BoxBFL.140.LYS.N 7.81083 119.1164 14.67427 12.45762 1.24E+08 BoxBFL.141.GLN.N 7.42677 118.2031 12.06407 12.71254 1.77E+08 BoxBFL.143.TYR.N 7.21631 115.6619 15.45741 12.35014 1.19E+08 BoxBFL.144.GLU.N 8.18784 120.6799 13.13889 12.30444 1.27E+08 BoxBFL.145.LYS.N
9.183 121.238 13.17983 12.43273 1.47E+08 BoxBFL.146.LYS.N, BoxBFL.156.LYS.N 7.69709 120.5754 12.94948 12.65531 1.73E+08 13oxl3FL.147.ALA.N 8.48271 121.0951 12.06422 12.53668 1.63E+08 , BoxBFL.148.ALA.N_ 8.37014 122:0096_12_01385_ 12:60817 , 1.9E+08 iiiiiiiiiiiiiiiiiiiiii 7.97339 120.7383 13.42094 12.64648 1.82E+08 BoxBFL.150.LEU.N 8.28676 120.0845 14.4103 13.05275 1.22E+08 13oxl3FL.151.LYS.N 8.42457 123.5222 12.66033 12.96606 1.42E+08 BoxBFL.152.GLU.N 8.08704 119.9998 i 12.5224 13.21315 1.98E+08 EIoxBFL.153.LYS.N 7.75272 119.7489 13.27981 12.63584 1.63E+08 BoxBFL.154.TYR.N 8.12892 120.4783 12.99281 12.73019 1.34E+08 BoxBFL.155.GLU.N 8.52395 117.5111 13.25587 12.99665 1.34E+08 BoxBFL.157.ASP.N 8.85469 123.1047 12_50397 12.25568 1.51E+08 BoxBFL.158.ILE.N 9.06751 122.2825 13.25933 12.71521 1.38E+08 BoxBFL.159.ALA.N 7.44862 124.7591 12.4575 12.38733 1.75E+08 BoxBFL.160.ALA.N 7.7427 120.5687 14.46225 12.64065 1.71E+08 BoxBFL.161.TYR.N 8.19946 120.0547 15.35591 13.74534 1.39E+08 BoxBFL.162.ARG.N 8.2623 118.7997 12.52352 12.69996 1.54E+08 1.43 molar ratio CXCL12/1-11VIG Box (Day 2) Experiment type 2D 1H-15N HSQC
HMGB1 construct and concentration 0.28 mM, U-15N
HMGB1A-c028 (Box B 94-162), C-terminal biotinylation tag, 300 p.L of 0.45 mM stock CXCL12 construct and concentration 0.412 mM, CXCL12A-c021, wt 1-67, no tag, 170 pl 01 1.14 mM stock Temperature 25 QC
Volume 350 pth Sample pH (after adjusting) 7.69 Number of scans 8 F 1 assign Fl F2 LW Fl LW F2 Height position position (Hz) (Hz) BoxBFL.95.LYS.N 8.47778 i 122.9103 15.37565 !
12.39804 80050272 BoxBFL.96.ARG.N 8.33007 124.0602 14.92746 12.00118 82355536 !
13oxBFL.99.SER.N 7.96392 = 114.2034 N/A

BoxBFL.100.ALA.N 9.06578 122.8423 12.95809 12.6583 87116544 BoxBFL.101.PHE.N 8.44289 116.1454 13.47969 12.85253 61494512 ------------------------BoxBFL.102.PHE.N 8.26155 122.0515 14.49526 12.56936 63868476 BoxBFL.103.LEU.N 8.28051 121.1503 14.84782 12.62126 65927464 BoxBFL.104.PHE.N 8.18982 122.6828 20.78995 15.31712 1E+08 BoxBFL.105.CYS.N 8.49843 118.3925 13.1053 13.13926 70645200 BoxBFL.106.SER.N 8.1287 115.0622 12.0922 12.5294 1.18E+08 !
BoxBFL.107.GLU.N 7.15039 119.185 14.99568 ! 11.952 1.12E+08 BoxBFL.108.TYR.N
7.9412 114.6199 17.85822 12.78123 73545448 BoxBFL.109ARG.N 9.23053 123.7071 13.66025 13.12308 78615272 EkncBFL.111.LYS.N 6.79981 117.7058 16.96135 !
12.39598 86118672 BoxBFL.112.ILE.N 8.16151 119.7459 14.73583 13.27134 71542432 !
BoxBFL.113.LYS.N 8.25631 118.4871 16.07722 14.62748 55390008 BoxBFL.114.GLY.N 7.63682 103.7319 15.93962 12.34204 83576064 BoxBFL.115.GLU.N 7.49983 119.5699 15.3896 12.37429 96911488 !
BoxBFL.116.HIS.N 7.82619 114.7906 14.24248 i 12.35403 74137880 !
BoxBFL.119.LEU.N 7.36266 121.2025 12.44261 !
12.21601 1.49E+08 BoxBFL.120SER.N 9.31408 121.062 12.93199 12.44319 84698824 BoxBFL.121.ILE.N 8.75038 120.6813 14.48357 12.89635 BoxBFL.122.GLY.N 8.71118 109.4804 16.4655 14.14866 16957850 BoxBFL.123.ASP.N 7.95741 124.2905 15.0023 12.39813 80795464 !
BoxBFL.124.1/ALN 8.63479 124.0656 13.47634 12.10198 1.07E+08 BoxBFL.125.ALA.N 7.92779 121.3329 13.01599 12.5379 1.33E+08 BoxBFL.126.LYS.N 8.02119 119.7027 12.30924 12.8741 1.48E+08 ------------------------I3oxBFL.127.LYS.N 8.03986 121.3062 12.4847 ! 12.16844 1.55E-i-08 BoxBFL.128.LEU.N 8.64351 120.1664 12.98093 12.34936 95774424 BoxBFL.129.GLY.N 8.26062 106.0395 14.57273 1.
12.29455 i 88870768 BoxBFL.130.GLU.N i 8.07098 123.2514 12.85049 12.30451 1.28E+08 BoxBFL.131.MET.N 8.77103 118.9657 12.95376 12.1418 1.15E+08 BoxBFL.132.TRP.N 8.71439 122.6801 14.10342 12.19979 76116600 BoxBFL.133.ASN.N 8.17827 117.365 1233999 12.19573 1.2E+08 BoxBFL.134.ASN.N 7.58849 115.8472 19.64464 11.72657 1.07E+08 BoxBFL.135.THR.N 7.30642 119.9458 15.21751 12.24418 1.12E+08 BoxBFL.136.ALA.N 9.24753 131.4348 12.65711 12.04662 1.19E+08 BoxBFL.138.ASP.N 8.99115 115.7039 12.95325 13.45469 55523416 BoxBFL.139.ASP.N 7.33717 117.8755 1736433 12.18072 1.09E+08 BoxBFL.140.LYS.N 7.80982 119.1179 15.67278 12.39083 82706752 BoxlIFL.141-GLN-N 7.42724 118.2084 12.20378 12.60333 1.22E+08 I
BoxBFL.143.TYR.N 7.21658 115.6631 17.78092 12.5108 80885808 z BoxBFL.144.GLUM 8.18558 120.6672 12.65054 12.60763 91853544 BoxBFL.145.LYS.N 9.1835 121.244 13.52547 12.49804 1.01E+08 Boxl3FL.146-LYS-N, 13oxBFL.156.LYS.N 7.69757 120.5767 13.43106 13.08878 1.22E+08 BoxBFL.147.ALA_N 8.48332 121.097 12.54408 12.70391 1.12E+08 BoxBFL.148.ALA.N 8.37158 122.0199 12.14356 12.99704 1.29E+08 BoxBFL.149.LYS.N 7.97496 120.7468 12.94812 12.72338 1.28E+08 BoxBFL.150.LEU.N 8.28799 120.0826 13.26184 13.21101 83975808 BoxBFL.151-LYS-N 8.42544 123.5327 13.10847 12.87192 96388256 BoxBFL.152.GLU.N 8.08805 120.0097 13.34088 13.96487 1.37E-i-08 BoxBFL.153.LYS.N 7.75361 119.7537 13.4631 12.86615 1.13E+08 BoxBFL.154.TYR.N 8.12773 120.4857 13.65716 12.83688 92183904 BoxBFL.155.GLU.N 8.522 117.5173 12.9949 13.07435 92028096 BoxBFL.157.ASP.N 8.85612 123.1054 13.64785 12.54087 99610256 BoxBFL.158.ILE.N 9.06839 122.2825 13.74015 12.91936 I3oxBFL.159.ALA.N 7.44725 124.7733 12.59718 12.9055 1.24E+08 BoxBFL.160.ALA.N 7.74231 120.5797 15.01396 13.34523 1.18E+08 BoxBFL.161.TYR.N 8.19948 120.0505 15.71875 13.97336 94945976 BoxBFL.162.ARG.Ni 8.26146 118.804 12.47976 12.94011 1.12E+08 Baseline 2 (Day 2) Experiment type 2D '11-'5N HSQC
HMGB1 construct and concentration 0.45 mM,I-IMGB1-c028 (Box B 94-162), C-terminal bioti nyl anon tag CXCL12 construct and concentration 0 mM
Temperature 25 QC
Volume 300 !IL
Sample pH (after adjusting) 7.7 Number of scans 8 Fl assign Fl F2 LW Fl (Hz) LW F2 (Hz) Height position position __________ BoxBFL.95.121S.N 8.47836 122.8704 i 15.45555 12.31895 1.15E+08 BoxBFL.96.ARG.N 8.30905 123.928 14.69757 11.67602 1.52E+08 BoxBFL.99.SER.N 7.95323 114.2133 15.92668 15.37737 19244536 13oxBFL.100.ALA.N 9.06443 122.8375 12.04384 12.58362 1.45E+08 BoxBFL.101.PHEN 8.44327 116.1467 13.37094 12.57032 99456376 BoxBFL.102.PHE.N
8.26291 122.1401 13.91643 12.69516 i 93582568 BoxBFL.103.LEU.N
8.27739 121.1559 13.54853 12.32854 1.2E+08 BoxBFL.104.PHE.N
8.15312 122.5067 16.98582 12.61768 2.23E+08 BoxBFL.105.CYS.N
8.49958 118.3879 12.45479 13.04816 1.23E+08 BoxBFL.106SER.N
8.12962 115.053 11.71118 11.95992 2.01E+08 BoxBFL.107.GLU.N
7.15443 119.1857 15.15976 11.875 1.85E+08 BoxBFL.108.TYR.N
7.94312 114.6181 16.58447 ^ 12.32968 1.25E+08 BoxBFL.109.ARG.N
9.2303 123.6965 13.1266 12.39272 1.36E+08 BoxBFL.111.LYS.N
6.80044 117.7026 15.51854 12.47282 1.51E+08 BoxBFL.112.ILE.N
8.16214 119.7445 14.38896 12.72125 1.33E+08 BoxBFL.113.LYS.N
8.24788 118.4867 17.04567 13.89265 75341960 BoxBFL.114.GLY.N
7.63545 103.7167 16.12903 11.9801 1.23E+08 BoxBFL.115.GLU.N
7.50084 119.5887 15.33825 12.18563 1.53E+08 BoxBFL.116.HIS.N
7.81992 114.7797 14.07903 12.00191 1.27E+08 BoxBFL.119.LEU.N
7.36245 121.1993 12.49408 11.92292 2.35E+08 BoxBFL.1203ER.N
9.31637 121.0591 13.96408 12.31166 1.32E+08 BoxBFL.121.ILE.N
8.74889 120.6692 14.04081 13.00014 64885424 13oxBFL.122.GLY.N
8.71247 109.452 14.17681 13.60233 17286296 BoxBFL.123.ASP.N
7.95965 124.2811 13.89649 12.49605 1.34E+08 BoxBFL.124.VALN
8.63524 124.0725 13.34482 12.04966 1.82E+08 BoxBFL.125.ALA.N
7.92792 121.3503 11.86774 11.79008 2.42E+08 BoxBFL.126.LYS.N
8.01841 119.6864 11.86371 12.33853 245E+08 BoxBFL.127.LYS.N
8.03849 121.3063 111092 12.01079 2.71E+08 BoxBFL.128.LEU.N
8.64384 120.1687 12.82931 12.23453 1.65E+08 BoxBFL.129.GLY.N
8.26299 106.0359 14.80106 = 11.90484 1.55E+08 BoxBFL.130.GLU.N
8.07168 123.2465 12.61599 12.00749 2.15E+08 BoxBFL.131.MET.N 8.76967 118.9613 12.58221 12.16772 2E+08 BoxBFL.132.TRP.N
8.71566 122.6842 13.26303 12.06045 1.35E+08 BoxBFL.133.ASN.N
8.17935 117.3676 11.78966 1 11.94741 2.04E+08 BoxBFL.134.ASN.N
7.58744 115.8288 20.03676 12.02406 1.76E+08 BoxBFL.135.THR.N
7.30721 119.9545 14.90053 12.28766 1.9E+08 EIoxBFL.136.ALA.N
9.2491 131.4409 11.88622 11.94243 1.93E+08 BoxBFL.138.ASP.N
8.99179 115.703 14.27261 13.33337 68493488 BoxBFL.139.ASP.N
7.33887 117.8767 18.17146 11.95364 1.82E+08 BoxBFL.140.LYS.N
7.81148 119.1162 15.20448 12.14857 1.5E+08 BoxBFL.141.GLN.N
7.4268 118.1975 11.86842 12.43174 2.19E+08 BoxBFL.143.TYR.N
7.21681 115.6633 16.06949 12.06739 1.45E+08 BoxBFL.144.GLU.N
8.19039 120.6913 12.43979 12.40533 1.6E+08 BoxBFL.145.LYS.N
9.18288 121.2343 13.02849 12.2664 1.81E+08 BoxBFL.146.LYS.N, 13oxBFL.156.LYS.N 7.69649 120.5726 12.4096 = 12.47784 2.08E+08 BoxBFL.147.ALA.N
8.48223 121.0936 11.71767 ^ 12.54903 2.05E+08 BoxBFL.148.ALA.N
8.36946 122.0011 12.08468 12.21854 2.33E+08 BoxBFL.149.LYS.N
7.9718 120.7293 12.63602 12.4563 2.26E+08 13oxBFL.150.LEU.N
8.28613 120.0804 13.87273 13.26832 1.52E+08 BoxBFL.151.LYS.N
8.42463 123.5087 12.67228 12.49152 1.76E+08 EIoxBFL.152.GLU.N
8.08647 119.9901 12.2303 12.58471 2.36E+08 BoxBFL.153.LYS.N
7.75247 119.748 13.27241 12.43002 1.97E+08 BoxBFL.154.TYR.N
8.13017 120.4741 12.9694 12.56059 1.68E+08 BoxBFL.155.GLU.N
8.52488 117.5048 13.26926 12.59952 1.65E+08 BoxBFL.157.ASP.N i 8.85488 123.1048 12.53023 12.19821 1.85E+08 BoxBFL.158.I1E.N 9.06773 122.2845 12.97983 12.55507 1.7E+08 BoxBFL.159.ALA.N 7.45038 124.7516 12.10878 12.01767 2.17E+08 BoxBFL.160ALA.N 7.74351 120.5631 14.03576 12.41792 2.12E+08 BoxBFL.161.TYR.N 8.20134 120.056 14.08572 13.06065 1.75E+08 BoxBFL.162ARG.N 8.26373 118.7947 12.78627 12.3573 1.81E+08 1-11VIGB1-c038 (89-174, biotinylated) titration with CXCL12A-c021 at 0, 0.42, 0.82 and 1.42 molar equivalents, Figures 3A-3B

[00140] Due to protein amount limitations, titration was performed by sequential addition of CXCL12 to 1-1MGB1 samples, resulting in sample dilution. As the calculations in the chemical shift tracking module in CCPNMR are independent of peak, this does not alter the results; the median change has been indicated in the volume comparisons.
BRUKER AVM HD 500,5 MM CPTCI 11-1-13C/15N/D
Z-GRD PROBE
1H frequency (Hz) 500.01233645 1H sweep width (ppm) 11.9044681870088 11-Isweep width (Hz) 5952.38095238095 1HN sweep width (ppm) 10.9286921061064 "N frequency (Hz) 50.6715211382702 15N sweep width (ppm) 32.0372585410812 15N sweep width (Hz) 1623.37662337662 Water suppression gradient pulse width (Hz) 2336.45 Buffer 10 mM HEPES, pH 7.5, 150 mM NaCI
Baseline 1 (Day 1) Experiment type 2D

HMGB1 construct and concentration 0.81 mM, HMGB1-c038 (Box B 89-174), C-terminal biotinylation tag CXCL12 construct and concentration 0 mM
Temperature 25 QC
Volume 300 pi Sample pH (after adjusting) 7.8 Number of scans 8 Assign Fl position F2 position LW Fl (Hz) LW F2 (Hz) Height BoxBFL.119.LYS.H 8.22877 123.15 13.43706 11.76465 2.84E+08 BoxBFL.90.ASP.H 8.06507 119.1836 17.47528 13.0415 71477080 BomBFL92.ASN.H 8.38837 116.1932 26.72516 13.63039 40775376 BoxBFL.93.ALA.H 7.31759 123.8361 12.03289 11.75968 2.88E+08 BoxBFL95.1.1S.H 8.54394 123.6499 15.56655 12.66447 1.57E+08 BoxBFL96.ARG.H 8.31244 123.9103 13.30976 11.8029 3.19E+08 BoxBFL.99.SERM 7.89322 114.8385 1234573 13.41732 83696192 4.
BoxBFL100.ALA.H 9.06159 122.8259 12.5231 12.3534 1.5E+08 BoxBFL.101.PHE.H 8.44257 116.1455 14.2976 12.88207 1.18E+08 BoxBFL.102.PHE.H 8.26477 122.1713 14.09453 12.29629 2.95E+08 BoxBFL.103.LEU.H 8.27759 121.1528 15.09128 13.06243 1.22E+08 BoxBFL.104.PHE.H 8.13387 122.4785 13.88173 12.22486 2.22E+08 , BoxBFL105.CYS.H
8.50131 i 118.3952 14119029 116882 i 1.26E+08 ;. , BoxBFL106SER.H 8.1324 , 115.0572 11.86819 12.2455 2.18E+08 , BoxBFL107.GLU.H
7.15821 i 119.2011 15.07209 12.23544 1.97E+08 BoxBFL108.1YR.H
7.94721 114.6268 17.12946 12.56173 j 1.29E+08 BoxBFL109.ARG.H
9.22527 123.6917 14.35796 12.40158 ' 1.33E+08 BoxBFL111.LYS.H
6.80367 117.7105 15.83919 12.77867 1.48E+08 BoxBFL112.ILE.H
8.16585 119.7589 15.93766 14.10622 1.3E+08 BoxBFL11.3.LYS.H
8.24667 118.3979 14.18155 12.282 1.95E+08 BoxBFL114.GLY.H 7.6419 103.7578 15.9097 12.86625 1.49E+08 BoxBFL115.011.11 7.50312 119.5592 15.3641 12.25047 1.76E+08 BoxBFL116.HIS.H
7.82141 114.7108 15.60333 12.79576 1.45E+08 BoxBFL119.LEU.H
7.36645 121.1961 12.25315 11.84566 2.79E+08 BoxBFL1205ER.H
9.31869 121.0561 13.67984 12.45399 1.5E+08 BoxBFL121.ILE.H 8.7507 120.6719 13.37699 12.68743 93999208 BoxBFL122.GLY.H
8.71122 109.4492 18.2158 13.7384 40704368 BoxBFL123.ASP.H
7.96237 124.2817 14.31821 13.03276 1.64E+08 BoxBFL124.VALH 8.6379 124.0546 13.5712 12.12567 1.86E+08 BoxBFL125.ALA.H
7.93334 121.344 12.65998 12.7035 2.76E+08 ..
BoxBFL126.LYS.H
8.02273 119.6775 12.14567 12.20318 2.67E+08 .,.
BoxBFL127.LYS.H
8.04282 121.3008 13.53438 16.44289 2.72E+08 BoxBFL128.LEU.H
8.64819 120.1742 13.66523 12.49721 1.57E+08 BoxBFL129.GLY.H
8.26351 106.0328 15.35471 12.18689 1.69E+08 BoxBFL130.G.U.H
8.07435 123.2474 13.07314 12.31509 2.26E4-08 Boxl3FL131.ME1.H 837512 118.9617 13.39615 12.51007 2.05E+08 Boxl3FL.132.1RP.H
8.71654 122.6792 14.56383 12.46287 1.3E+08 BoxBFL.133.ASN.H
8.17988 117.3613 12.44756 12.16788 2.1E+08 BoxBFL134.A5N.H
7.59333 115.8407 17.61059 12.39088 1.9E+08 Boxl3FL.135.111R.H
7.31004 119.9467 15.60722 12.12025 2.02E+08 BoxBFL.136.ALA.H
9.25013 131.437 12.17023 12.15813 2.27E+08 BoxBFL137.ALA.H
8.85817 124.999 15.52392 16.42924 11136171 BoxlIFL138.A.SP.H
8.99358 115.7103 13.01277 13.43146 1.17E+08 BoxEIFL.139.ASP.H
7.34269 117.8836 17.65566 12.28682 1.93E+08 BoxBFL140.LYS.H
7.81346 119.1053 15.13034 12.58181 1.53E4-08 BoxBFL141.GLN.H
7.43127 118.216 12.22585 12.9068 2.03E+08 BoxBFL143.111R.H
7.21936 115.67 15.42101 12.81604 1.43E+08 .4 BoxBFL.144.GLU.H 8.1673 120.3573 19.36893 15.09454 2.23E+08 BoxBFL145.LYS.H
9.19147 121.2642 13.95073 13.10497 1.73E+08 BoxBFL146.LYS.H, BoxBFL156.LYS.H
7.70091 1203702 14.84964 12.91125 3.78E+08 BoxBFL.147.41A.H
8.47542 120.5044 16.38659 13.35601 I 61059660 BoxBEL.148.ALA.H
8.38049 122.0622 13.43968 12.61324 2.27E+08 BoxBFL149.LYS.H
7.98075 120.7812 13.45082 13.37624 2.21E+08 BoxBFL150.LEU.H
8.29642 120.0874 14.65693 13.57267 1.39E+08 BoxBFL.151.LYS.H 8.4373 123.66 13.67398 13.22858 1.71E+08 BoxBFL152.GLU.H 8.0907 120.0978 14.82538 13.76241 2.37E+08 BoxBFL153.LYS.H
7.76448 119.7662 14.67797 13.05371 1.95E+08 BoxBFL.154.1YR.H
8.14526 120.4576 #VALUE! #VALUE! 1.83E+08 Boxl3FL155.GLU.H
8.52465 117.6406 14.85626 13.30374 1.65E+08 BoxBFL157.A5P.H
8.90183 123.2173 13.33878 13.30286 1.79E+08 BoxBFL158.ILE.H
9.20351 122.5498 13.98115 12.71351 1.81E+08 BoxBFL.159.ALA.H
7.36561 124.8259 12.81959 12.53986 2.31E+08 BoxBFL160.ALA.H
7.78333 120.4019 13.47788 13.29924 2.25E+08 Boxl3FL.161.1YR.H
8.19889 120.0636 16.81491 18.13778 2.19E+08 BoxBFL.162.ARG.H
8.26173 118.8178 12.98114 12.65685 1.88E+138 Boxl3FL163.ALAH
7.63926 121.3704 13.43373 12.39034 1.48E+08 BoxBFL164.LYS.H
7.65612 118.3003 14.35415 12.33745 1.22E+08 BoxBFL165.GLY.H
8.05505 108.6412 27.92431 13.8698 20239174 BoxBFL166.LYS.H
8.04195 121.5844 15.77089 13.22022 j 4.24E+08 Boxl3FL.168.ASP.H 8.4885 121.098 12.61027 13.02291 1.96E+08 BoxBFL.169.ALA.H
8.27056 124.8752 14.6408 13.13492 53273208 BoxBFL170.ALA.H
8.13312 121.3951 14.00858 12.18198 1.5E+08 , BoxBFL.172.LYS.H
8.4277 i 124.6737 7.48588 9.20329 i 3518457 1- , i BoaFL.173.GLY.H
8.57505 1 111.1121 15.89317 13.54035 25440228 t.
3D experiments (Day 1-5) These spectra are not shown due to their 3D nature; data available if requested. Sample is that of Baseline 1.
Experiment type 3D, IsN
EDITED 41-1H TOC.SY-FISQC, THROUGH-SPACE
INTERACTION
Temperature 1 25 9C
Volume 1 300 pi Number of scans i 32 Experiment type 3D, 15N
EDITED 111-1H NOESY-HSQC, THROUGH-BOND
INTERACTION
Temperature i 25 2C
Volume .
:.., 300 IA
Number of scans ;! 32 0.42 molar ratio CXCL12/HNIG Box (Day 5) Experiment type 2D 1H-15N HSQC
.......................................................... , ........................................................
HMGB1 construct and concentration 0.7 mM, HMGB1-c038 (Box B 94-162), C-terminal hiotinylation tag (285 pi of 0.8 mM stock) CXCL12 construct and concentration 0.29 mM, CXCL12A-c021, wt 1-67, no tag (41 a of 1.8 mM
stock) Volume 252C
Sample pH (after adjusting) 320 1.
Temperature 7.8 Number of scans 32 Assign Fl position F2 position i LW Fl (Hz) LW F2 (Hz) I Height E
BogBFL.89.LYS.H
8.23317 123.0882 , 15.99121 13.36991 1 8.54E+138 BoxBFL.90.A.SP.H
8.07425 119.195 17.03548 12.94404 3.32E+08 litexBFL.92.ASN.H
8.39252 116.22 20.52278 13.76841 1.57E408 BoxBFL.93.ALA.H
7.32309 123.8461 13.23603 12.04781 8.1E+08 BoxBFL95.LYS.H 835152 123.6861 15.235 12.47899 5.7E+08 BoxBFL96.ARG.H
8.33348 124.0077 14.64892 11.53036 6.66E+08 , BoxBFL.99.SER.H
7.89458 114.8324 17.06332 13.29929 3.9E+08 BoxBFL.100.ALA.H
9.07237 122.8303 12.45091 12.65528 6.1E4-08 BoxBFL101.PHE.H
8.45267 116.1672 12.95977 E 12.54238 5.08E+08 BoxBFL.102.PHE.H 8.2712 122.1831 13,20017 13.34009 8.34E+08 BoxBFL.103.LEU.H
8.28642 121.127 15.05638 12.72235 4.44E+08 , BoxBFL104.PHE.H 8.154 _,...... 122.5034 15.1555812.55092õ 4.93E+08 _ ,,,,,,,,,,,,,,,,,,,,,,,,,, 175-0321 113.34e iigdiii iaTiiaei iiii;Cia BoxBFL.106SER.H
8.14042 115.0895 12.31066 12.82245 7.92E+08 BoxBFL.107.GLU.H 7.1606 119.2044 15.04631 12.06946 7.79E4-08 BoxBFL108.1YR.H
7.95517 114.6331 17.1485 12.51247 5.1E+08 BomEtFL109.ARG.H
9.24196 123.7109 14.77907 12.44116 5.14E+08 BoxBFL.111-LYS.H
6.80383 117.7113 16.35257 12.50023 5.75E+08 BoxBFL112.1LEH
8.17228 119.7666 15.12234 13.68265 5.3E+08 BoxBFL.113.LYS.H
8.26058 118.4342 14.6.3114 12.6851 5.11E+08 ., BoxBFL.114.GLY.H
7.64965 103.7836 14.15269 12.03673 6.32E+08 :-BoxBFL115.GLU.H
7.50893 i 119.5449 15.10834 i 12.14579 7E+08 , BoxBFL.116.HIS.H
7.83669 i 114.7278 4VALUE! itVALUE! i 3.31E+08 ;. , BoxBFL119.LEU.H
7.37631 ' 121.2095 11.82599 11.95517 1.11E+09 BoxBFL.120SER.H
9.32759 121.0738 13.32814 12.27617 : 6.4E+08 BoxBFL121.1LE.H
8.75541 120.6669 13.45308 12.74538 I 4.21E+08 BoxBFL122.GLY.H
8.72192 109.4783 18.02792 13.29589 , 1.9E+08 BoxBFL.123.4SP.H
7.97614 124.3289 15.78848 12.93298 5.54E+08 BoxBFL.124.VALH 8.6467 124.0535 13.39795 11.91108 7.39E+08 BoxBFL.125.ALA.H
7.93311 121.3373 20.94036 12.50751 7.5E+08 BoxBFL126.LYS.H
8.03459 119.7401 12.11644 12.969 9.82E+08 BoxBFL.127.LYS.H
8.05395 121.3131 13.08531 12.0213 1.04E+09 BoxBFL.128.LEU.H
8.65647 120.1666 12.77726 12.56328 6.48E+08 BoxBFL129.GLY.H
8.26041 106.0026 16.56723 12.47707 5.83E+08 BoxBFL.130.GLU.H 8.0809 123.2669 12.42406 12.29425 8.83E+08 BoxBFL131.ME1.H
8.78921 118.9705 13.20277 12.33316 7.89E+08 BoxBFL132.7RP.H
8.71893 122.6622 13.85963 12.75907 4.98E+08 BoxBFL.133.ASN.H
8.18222 117.3634 13.90003 12.0061 7.53E+033 t BoxBFL.134.ASN.H 7.6005 115.8484 17.9579 11.96318 7.22E+08 BoxBFL.135.710tH
7.31604 119.9459 15.68761 11 .89589 7.84E+08 , BoxBFL.136.ALA.H
9.25887 131.4427 12.04897 11.90835 9.4E+08 BoxBFL137.ALAH
8.86544 124.9937 18.19465 14 .33638 71698952 BoxBFL138.45P.H
9.00067 115.712 12.28957 13.24047 5.28E+08 BoxBFL139.ASP.H
7.34953 117.8877 17.87632 12.18195 7.56E+08 BoxBFL.140.LYS.H
7.82254 119.1251 15.16611 12.34065 6.15E+08 BoxBFL141.GLN.H 7A4009 118.2284 11.84562 12.39199 8.33E+08 BoxBFL.143.7YR.H
7.22876 115.6684 16.28895 12.57946 5.48E+08 BoxBFL.144.GLU.H
8.18291 120.313 24.30189 16.31598 7.26E+013 BoxBFL145.LYS.H
9.20235 121.2804 13.87277 13.31669 6.47E+08 BoxBFL.146.LYS.H, BoxlIFL156.LYS.H
7.70895 120.5874 14.10502 13.01551 1.38E+09 BoxBFL.147.ALA.H
8.48426 120.552 16.0703 14.31819 2.91E+08 BoxBFL148.ALA.H
8.39134 122.0914 13.96231 13.19139 7.81E+08 BoxBFL149.LYS.H
7.99377 120.7978 13.72404 13.22412 7.88E+08 BoxBFL150.LEU.H
8.30338 120.106 14.84471 13.15063 5.18E-N38 BoxBFL.151.LYS.H
8.44582 123.6944 13.55023 13.11479 6.01E4-08 t BoxBFL.152.GLU.H
8.10037 120.1197 14.86852 13.77938 8.15E+08 BoxBFL.153.LYS.H
7.77409 119.7673 13.03363 13.46475 7.36E+08 .*
BoxBFL.154.7YR.H
8.15602 120.4585 24.22866 16.12356 6.94E+08 BoxBFL155.GLU.H
8.53056 117.6515 13.90664 13.6262 5.87E4-08 BoxBFL157.ASP.H
8.91261 123.2303 14.37862 13.03578 5.44E+08 BoxBFL158.H.E.H
9.21552 122.5667 15.94888 12.94384 I 4.83E+08 BoxEIFL.159.AL&H
7.36871 124.8542 14.00455 12.99954 6.93E+08 BoxBFL.160.ALA.H
7.79079 120.4098 13.48341 13.45411 7.53E+08 BoxBFL161.7YR.H
8.20906 120.0582 17.84468 15.5777 8.17E+08 BoxBFL.162.ARG.H
8.26922 118.8094 13.23997 12.86921 7.66E+033 BoxBFL.163.ALA.H
7.64365 121.401 13.62331 13.15062 4.69E+08 BoxBFL164.LYS.H
7.66395 118.3578 14.08222 14 .60296 3.61E+08 BoxBFL.165.GLY.H
8.06949 108.6489 23.00117 14 .26944 84792952 BoxBFL166.LYS.H
8.06804 121.6875 16.22206 12.67618 1.02E+09 BoxBFL.168.ASP.11 8.49698 121.1145 12.93091 12.80312 7.46E+08 BoxBFL.169.ALA.H
8.27748 124.8788 14.08935 13.77469 2.91E+033 BoxBFL.170.ALA.H
8.13889 121.4079 15.50116 12.51434 5.16E+08 BoxBFL.172.LYS.H
8.44213 124.8148 14.07011 12.03688 79426568 . .
BoxBFL173.431.Y.F1 8.58133 111.1267 : 16.71041 13.95112 : .. 1.23E+08 ,.
0.84 molar ratio CXCL12/1-17MG Box (Day 5) Experiment type 2D 11-HMGB1 construct and concentration 0.58 mM, HMGB1-c038 (Box B 94-162), C-terminal biotinylation tag (285 ptl_ of 0.8 mM stock) CXCL12 construct and concentration 0.482 mM, CXCL12A-c021, wt 1-67, no tag (104 1AL of 1.8 mM stock) Volume 252C
Sample pH (after adjusting) 320 LEL
Temperature 7.8 Number of scans 32 I Assign i Fl position i F2 position LW Fl (Hz) i LW F2 (Hz) Height -:
BoxBFL.89.LY5.H
8.22679 12238562 19.92847 15.30362 6.04E+08 BoxBFL90.ASP.H
8.07636 119.19941 17.45582 13.22061 2.24E+08 BoxBFL.92.A.SN.H 839239 116.22614 23.07716 14 .06705 1.06E+08 BoxBFL.93.ALA.H
7.32259 123.8468 13.46498 12.02831 6.25E+08 BoxBFL95.LYS.H
8.55168 123.69223 15.31781 12.58495 4.41E+08 BoxBFL.96.AFtG.H
8.34299 124.0494 14.88799 11.9875 4.06E+08 BoxBFL99.5ER.H
7.89423 114.84247 #VALUE! #VALU E! 3.07E+08 C.
BoxBFL.100.ALA.H
9.07369 122.83003 11.94043 12.61474 5.02E+08 BoxBFLILY1.PHE.H
8.45401 116.1693 13.37439 12.53442 3.98E+08 BoxEIFL.102.PHE.H
8.27231 122.15451 13.98629 14.5081 5.11E+08 BoxBFL.103.LEU.H
8.28889 121.12078 14.37061 12.72043 3.73E+08 BoxBFL104.PHE.H
8.16438 122.51969 15.78414 13.39462 2.93E+08 BoxBFL.105.CYS.H
8.50866 118.40024 13.19461 13.85193 4.4E+ 08 BoxBFL.106.SER.H 8.1423 115.09842 11.9209 12.43735 6.58E4-08 BoxBFL107.GLU.H
7.16005 119.20473 14.95609 12.03505 6.36E+08 BoxBFL108.1YR.H
7.95609 114.63347 16.3564 12.6025 4.15E+08 BoxBFL109.ARG.H
9.24419 123.71548 14.94347 12.70613 4.15E+08 BoxBFL.111.LYS.H
6.80375 117.70835 16.02119 12.42776 4.77E+08 BoxBFL1123LE.H
8.17303 119.76733 15.02702 13.64763 4.37E+08 BoxBFL113.LYS.H
8.26665 118.45194 14.62975 13.06685 3.35E+08 BoxBFL.114.GLY.H
7.64978 103.7787 14.53777 12.13962 5.15E+08 BoxBFL115.GLU.H
7.50983 119.55375 15.22445 11.99963 5.72E+08 BoxBFL.116.H15.H
7.84327 114.73761 #VALUE! #VALU E! 2.63E+08 BoxBFL.119.LEU.H
7.37738 121.21246 11.7834 12.0448 9.01E4-08 BoxBFL.120-SER.H
9.32827 121.07659 13.31064 12.27752 5.09E+08 BoxBFL.121.1LE.H 8.7561 120.66594 13.60291 12.71327 3.22E+08 BoxEIFL122.GLY.H
8.72507 109.47718 17.30426 13.44831 1.43E+08 BoxBFL.123.ASP.H
7.97787 124.33699 16.31857 12.97771 4.33E+08 BoxBFL.124.VALH 8.6484 124 .05721 12.86612 11.89578 6.24E+08 BoxBFL125.ALA.H
7.92439 121.32801 19.13773 11.92173 6.11E+08 BoxBFL.126.LYS.H 8.0362 119.75212 12.45829 12.85125 7.81E4-08 BoxBFL127.LYS_H
8.05575 121.31674 12.43276 12.04567 8.57E+08 BoxBFL128.LEU.H
8.65767 120.16374 13.11087 12.67279 5.26E+08 BoxBFL129.GLY.H
8.25962 105.99904 16.21275 12.32688 4.86E+08 BoxBFL.130.6LU.H
8.08214 123.27002 12.67708 12.37817 7.09E+08 BoxBFL.131.MET.H
8.79146 118.97292 13.56391 12.24356 6.42E+08 BoxBFL.132.TRP.H
8.71828 122.65646 13.66393 12.75142 4.11E+08 BoxBFL133.A.SN.H 8.1825 117.36324 13.94004 11.96683 6.19E+08 BoxBFL134.A.SN.H
7.60102 115.8515 19.86751 11.87762 5.95E+08 BoxBFL135.711R.H
7.31691 119.94345 15.34212 11.78759 6.45E+08 BoxBFL.136.ALA.H
9.25983 131.44194 11.68396 11.88563 7.59E4-08 BoxBFL137.ALA.H
8.86664 124 .98894 17.53135 14 .89353 51062092 BoxEIFL.138.ASP.H 9.0022 115.71168 12.51951 13.18965 4.13E+08 BoxBFL.139.ASP.H 7.3505 117.8869 19.06678 12.10936 6.11E+08 BoxBFL140.LYS_H
7.82332 119.12697 14.98048 12.40774 4.99E+08 BoxBFL.141.GLN.H
7.44172 118.2299 11.81727 12.36063 6.81E+ 08 BoxBFL.143.1YR.H
7.23052 115.66761 15.72802 12.52565 4.53E4-08 BoxBFL.144.431.U.H
8.17729 120.35761 20.14083 15.18319 5.71E+08 BoxBFL145.LYS_H
9.20417 1212843 13.91087 13.13152 5.12E+08 BoxBFL146.LYS.H, BoxBFL156.LYS.H
7.70982 120.59018 14.12825 13.12581 1.11E+09 BoxBFL.147.ALA.H
8.48613 1203644 16.41386 14 .34424 2.09E+08 BoxBFL148.ALA.H
8.39403 122.09686 i 13.11396 13.17476 i 6.52E+08 ' BoxBFL.149.LYSII
7.99631 i 120.80367 1332788 13.03509 i 6.35E+08 , BomEIFL150.LEU.H
8.30472 , 120.10823 15.05319 13.15084 4.31E+08 t.
BoxBFL.151.LYS.H
8.44699 i 123.70209 13.71114 13.30569 4.8E+013 BoxBFL152.GLU.H
8.10211 120.12205 13.45462 13.68813 I 6.67E+08 BoxBFL153.LYS.H
7.77558 119.76647 13.7091 13.64753 ' 5.96E+08 BoxBFL.154.11fR.H
8.15632 120.46388 21.61287 16.30266 5.62E+08 , BoxEWL.155.G.U.H
8.53123 117.6528 13.66215 13.47564 4.65E+08 BoxBFL157.ASP.H
8.91586 123.23537 143729 12.90289 4.42E+08 +
BoxBFL158.ILE.H
9.21936 122.58191 16.30721 13.2986 3.82E+08 BoxBFL.159.ALAM
7.36846 124.86417 13.94085 12.75957 5.54E+08 r BoxBFL160.ALA-H
7.79128 120.4108 13.753 13.44963 5.87E+08 BoxBFL161.1YR.H
8.21265 120.05605 16.82215 14.29685 6.46E+08 +
BoxBR.162.ARG.H
8.27145 118.80874 12.38965 12.75367 6.47E+08 BoxBFL163.ALAM
7.64337 121.40884 14.1601 13.35172 3.57E+08 BoxBFL164.LYS.H
7.66408 118.38396 14.21213 14.54701 2.69E+08 BoxBFL.165.GLY.H
8.07305 108.67299 20.01365 13.50363 59059740 +
BoxBFL.166.LYS.H
8.07769 121.72575 15.87162 13.20124 6.24E+08 BoxBFL.168.ASP.H 8.4982 121.11929 12.80238 12.73258 6.09E+08 , BoxBFL.169.ALAH
8.28032 124.89477 1536254 1533651 1.81E+08 BoxBFL170.ALAH
8.14155 121.40211 15.22818 12.63552 i 3.56E+08 BoxBFL172.LYS.11 8.44239 124.80508 14.17767 12.23471 67949384 ..-BoxBFL173.GLY.H
8.58353 111.11154 15.74456 14.03704 i 74405800 1.43 molar ratio CXCL12/1-11VIG Box (Day 5) Experiment type 2D 1H-15N HSQC
HMGB1 construct and concentration 1 0.48 mM, HMGB1-c038 (Box B 94-162), C-terminal i biotinylation tag (285 iti of 0.8 mM stock) CXCL12 construct and concentration i 0.69 rnhl, CXCL12A-c021, wt 1-67, no tag (171 pt of 1.8 mM stock) Temperature i 25 st Volume i 320 1.1th Sample pH (after adjusting) 1 7.8 Number of scans 32 Assign Fl position F2 position LW Fl (Hz) ; LW F2 (Hz) Height BoxBFL89.LYS.H
8.21895 122.9077 18.92121 17.02404 5.39E+08 BoxBFL90.ASP.H
8.07743 119.2044 17.38461 13.70778 1.57E+08 BoxBFL92-ASN.H
8.39578 116.2228 22.65251 14.06046 78748568 BoxBFL.93.AIA.H 7.3206 123.844 13.85399 11.97782 5.01E+08 BoxBFL95.LYS.H
8.55161 123.6949 15.60312 12.56883 3.5E+08 BoxBFL.96.AlitH
8.34894 124.0718 15.43549 12.28544 2.6E+08 BoxBFL.99.SER.H
7.89399 114.8337 #VALUE! WALUEI 2.52E+08 BoxBFL100.ALA.H
9.07401 122.83 12.0362 12.69173 4.12E+08 BoxBFL101.PHE.H
8.45444 116.1709 13.37824 E 12.49155 3.25E+08 BoxBFL.102.PHE.H
8.27168 122.1244 14.24496 16.08647 3.25E+08 BoxBFL.103.LEU.H
8.28943 121.1158 14.34292 12.71141 3.12E+08 BoxBFL104.PHEH
8.17112 1223365 17.78199 16.29353 1.94E+08 BoxBFL105.CYS.H
8.50875 118.4054 12.72396 13.22091 3.64E-N38 BoxBFL.106-SER.H
8.14279 115.1046 11.79716 12.39792 5.45E+08 BoxBFL107.GLU.H
7.15923 119.2044 15.5058 12.08644 5.2E+08 BoxBFL108.TYR.H
7.95617 114.6327 17.84136 12.54391 3.41E+08 C.
BoxBFL.109.ARG.H
9.24629 123.7188 14.08981 12.70662 3.49E+013 BoxBFL111.LYSII 6.8034 117.7047 16.29311 12.28933 4E+08 BoxBFL112.ILE.H
8.17337 119.7674 14.76928 13.50054 3.6E+08 BoxBFL113.LYS.H
8.27134 118.4462 INALUE! #VALUE1 2.17E+08 BoxBFL.114.431.Y.H
7.64931 103.7749 14.43105 12.23031 4.15E+08 BoxBFL115.GLUM 7.50986 i 119.5617 14.96744 i 11.95792 4.69E+08 , BoxBFL116.HIS.H
7.87127 i 114.7661 19.92977 12.94964 i 2.92E+08 ;. , BoxBFL119.LEU.H
7.37742 ' 121.2158 1132553 12.11307 7.45E+08 BoxBFL.120SER.H
9.32858 121.0789 13.18459 12.45593 4.18E+013 BoxBFL121.ILE.H 8.7553 120.6649 13.54667 12.77952 I 2.56E+08 BoxBFL122.GLY.H
8.72559 109.4867 18.08209 13.61802 ' 1.03E+08 BoxBFL.123.ASP.H
7.98069 124.3565 15.26493 12.68456 3.59E4-08 BoxBFL124.VALH 8.6488 124.0599 12.70186 11.8932 5.19E+08 BoxBFL.125.ALA.H
7.92234 121.3274 19.67901 12.01544 5.27E+08 BoxBFL126.LYS.H
8.03667 119.7712 12.36815 13.1321 6.59E+08 BoxBFL.127.LYS.H
8.05631 121.3212 12.09821 12.05342 7.14E+08 BoxBFL.128.LEU.H
8.65776 120.1602 13.10106 12.67615 4.37E+08 BoxBFL129.GLY.H
8.25895 105.9973 16.02096 12.32461 4.1E+08 BoxBR.130.GLU.H
8.08217 123.2734 12.61882 12.35737 5.86E+08 BoxBFL131.ME1.H
8.79223 118.9771 12.92803 12.14025 5.33E+08 BoxBFL132.7RP.H
8.71806 122.6543 13.59301 12.72152 3.45E+08 BoxBFL.133.ASPI.H
8.18174 117.3624 14.1966 12.05599 5.03E+033 BoxBFL.134.ASPIH
7.60142 115.8532 19.14308 11.90938 4.93E+08 BoxBFL.135.710tH
7.31694 119.9423 15.52944 11.84047 5.33E+08 ..
BoxBFL.136.ALA.H
9.25989 131.4404 11.56485 11.90864 6.26E+013 BoxBFL137.ALAH
8.86475 125.0108 16.06168 15.67273 35378044 BoxBFL138.ASP.H
9.00211 115.711 12.86091 13.34747 3.22E+08 BoxBFL139.ASP.H
7.35096 117.8866 19.0693 12.15302 5.06E+08 BoxBFL140.LYS.H
7.82374 119.1295 14.6706 12.41802 4.18E4-08 Boxl3FL141.GLN.H
7.44219 118.232 11.85067 12.27391 5.63E+08 BoxBFL.143.7YR.H
7.23072 115.6698 15.74818 12.51927 3.75E+08 BoxBFL.144.GLU.H
8.17663 120.3647 19.90498 14.8137 4.57E+013 BoxBFL.145.LYS.H
9.20511 121.2878 14.30353 13.0189 4.26E+08 BoxBFL.146.LYS.H, BoxlIFL156.LYS.11 7.71006 120.5924 14.14949 13.23139 9.14E+08 BoxBFL.147.ALA.H
8.48712 120.5785 16.9957 14.34865 1.49E+08 BoxBFL148.ALA.H
8.39484 122.1015 12.93976 12.98248 5.41E+08 BoxBFL149.LYS_H
7.99731 120.8071 13.2779 13.16866 5.32E+08 BoxBFL150.LEU.H 8.3049 120.1088 14.94034 13.2186 3.55E-N38 BoxBFL.151.LYS.H 8.4477 123.7082 13.98336 13.38566 3.93E4-08 BoxBFL.152.GLU.H
8.10253 120.1254 13.30973 13.69574 5.51E+08 BoxBFL.153.LYS.H
7.77618 119.767 13.49419 13.56752 4.88E+08 .4 BoxBFL.154.7YR.H
8.15722 120.4673 22.68082 16.71144 4.54E+013 BoxBFL155.GLU.H
8.53108 117.6557 13.40247 13.49932 3.86E4-08 BoxBFL157.ASP.H
8.91702 123.2376 13.94689 12.93051 3.63E+08 BoxBFL158.ILE.H
9.22334 122.5976 15.59077 13.35733 I 3.16E+08 BoxEIFL.159.AL&H
7.36744 124.8739 13.98725 12.61412 4.56E+08 BoxBFL.160.ALA.H
7.79157 120.411 13.64762 13.47195 4.83E+08 BoxBFL161.7YR.H
8.21447 120.0592 16.95219 14.11511 5.1E+08 BoxBFL.162.ARG.H
8.27207 118.8079 12.06017 12.68513 5.39E+033 BoxBFL.163.ALA.H
7.64246 121.4127 13.9688 13.29345 2.85E+08 BoxBFL164.LYS.H
7.66442 118.4079 14.86267 15.95431 2.05E+08 BoxBR.165.GLY.H
8.07387 108.6809 24.49469 14.82814 37366064 BoxBFL166.LYS_H
8.08076 121.7464 18.19369 13.05761 3.83E+08 BoxBFL162-ASP.11 8.49868 121.1201 12.66135 12.73158 5.01E+08 BoxBFL.169.ALA.H
8.28128 124.9183 14.6585 15.5819 1.24E+033 BoxBFL.170.ALA.H
8.14173 121.3982 16.83083 12.7598 2.45E+08 BoxBFL.172.LYS.H
8.44324 124.8133 14.79008 12.58927 50445744 . .
BoxBFL173.431.Y.F1 8.57949 111.0939 : 20.28256 15.57476 : 46002192 ,.
Baseline 2 (Day 5) Experiment type HMGB1 construct and concentration OS mM, HMGB1-c038 (Box B 94-162), C-terminal biotinylation tag CXCL12 construct and concentration 0 mM

Temperature 25 eC
Volume 285 RI_ Sample pH (after adjusting) 7.8 Number of scans 32 , ...............................................................................
............................................
I Assign i Fl position i F2 position ; LW Fl (Hz) i LW F2 (1.14 ; Height I
BoxBFL8tLYS.H E 8.23102 1 123.1415 14.76968 12.01671 1.13E+09 i a EtoxBFL.90.ASP.H 8.06917 119.1829 17.02515 12.22975 3.96E+08 BoxBFL92.ASN.H 8.38929 116.2163 20.63226 13.77677 2.02E+08 r BoxBFL.93.ALA.H 7.32084 123.8384 14.33885 11.63902 8.9E+08 BoxBFL95-LYS.F1 8.5478 123.6697 15.96042 12.62552 6.07E+08 c=
BoxElF1-96.ARG.H 8.3189 123.9301 14.50868 11.3829 1.03E+09 BoxBFL.993ER.H 7.89207 114.8246 15.31161 12.80624 3.66E+08 BoxBFL100.ALA.11 9.06739 122.8207 14.07546 12.19279 5.84E+08 BoxBFL.101.PHE.H 8.44676 116.1525 14.74364 12.91541 4.61E-N38 BoxBFL102.PHE.H 8.26698 122.1883 14.22121 12.44991 1.11E+09 BoxBFL103.LEU.H 8.28133 121.1339 16.10868 12.48103 4.42E+08 BoxBFL.104.PHE.H 1 8.13782 ._ 122.4713 14.37141 12.44314 7.5E-N38 BoxBFL10S.CYS.H 8.50419 118.3867 15.14844 14.2131 4.87E+08 BoxBFL.106.SER.H 8.13524 , ^
115.0683 13.16398 E 12.31969 7.42E+08 BoxBFL107.GLU.H 7.15834 119.1995 15.9825 11.95677 I .. 7.25E+08 BoxBFL.108.1YR.H 7.9497 114.6278 17.47581 12.33864 4.82E+08 , BoxBFL.109.ARG.H 9.23234 123.6965 16.59866 12.29412 4.81E+08 BoxBFL111.LYS_H 6.80327 117.711 16.90439 12.46518 5.6E+08 a BoxBFL.112.1LE.H 8.16856 119.7594 16.15624 13.91137 4.88E+08 BoxBFL113.LYS.H 8.24926 118.3993 14.3358 12.1708 6.95E+08 BoxBFL114.43111.11 7.64664 103.781 15.76333 12.14132 5.87E+08 BoxBFL.115.GLU.H 7.50528 1195289 15.83641 12.53165 6.74E+08 -:-BoxEIFL116.HIS.H 7.82554 114.7129 17.52591 12.91339 4.1E+08 BoxBFL.119.LEU.H 7.37163 121.1972 13.24197 11 .65462 1.03E+09 BoxBFL.120SER.H 9.32283 121.0625 15.14154 12.10113 5.93E+08 BoxBFL121.ILE.H 8.7522 120.6657 14.87619 12.56095 4.04E+08 , BoxBFL122.GLY.H 8.71514 109.4591 18.21003 13.47553 2E4-03 BoxBFL123.ASP.F1 7.96996 124.3035 16.15828 12.66772 5.78E+08 BoxBFL124.VALH 8.64104 124.0413 14.34107 12.30741 7.01E+08 f BoxBFL125.AULH 7.93386 121.3372 15.70871 12.46088 I 8.51E4-08 BoxBFL.126.LYS.H 8.02815 ;
119.7021 13.46969 12.7483 9.18E+08 ..
BoxBFL.127.LYS.H 8.0478 121.2943 14.12423 15.42969 9.72E+08 BoxBFL.128.LEU.H 8.65186 120.1671 14.50135 12.59408 5.79E+08 BoxBFL129.431.Y.H 8.26077 106.0107 16.79447 12.54485 5.75E+08 BoxBFL130.GLU.H 8.07653 +
123.2546 14.00315 12.1069 8.25E+08 BoxBFL131.MET.H 8.7821 118.9601 14.7908 12.21398 7.26E+138 BoxBFL.132.TRP.H 8.71726 122.6675 15.33509 12.4674 4.67E+03 BoxBFL133.ASH.H 8.17966 117.3578 14.21937 12.09116 7.35E+08 BoxElF1-134.ASH.H 7.59622 115.839 17.11067 12.24948 7.11E+08 -:-BoxBFL135.7HR.H 7.31205 119.9435 16.01333 11.84476 7.59E+08 BoxBFL136.ALAH 9.25411 131.4374 13.61094 12.00251 8.78E+08 EtoxBFL.137.ALA.H 8.86096 124.9828 16.96746 13.9175 82783192 BoxBFL138.ASP.H 8.99667 ., 115.7095 14.2258 12.81374 5.22E+08 BoxBFL139.ASP.H 7.34551 117.8838 17.77517 12.06753 7.26E+08 BoxBFL140.LYS.F1 7.81795 119.1111 16.26883 12.4296 5.8E+08 _ BoxBFL141.GLN.H _ _ _ _ 7.4356 118.2188 _ _ 13744592 12.84284 _ _ _ _ 7.56E408 _ BoxBFL.143.1YR.H 7.22342 115.6648 17.19563 12.6802 5.23E+08 BoxBFL144.GLU.H 8.17886 ;
120.3107 24.6535 15.3662 j 8.3E+08 f EtexBFL.145.LYS.H 9.19676 121.2667 15.33294 12.93118 6.33E408 BoxBFL.146.LYS.H, 8ox8FL.156.LYS.H 7.70415 120.5771 15.33781 12.75619 1.39E+09 _ BoxBFL147.ALA.11 8.47838 120.5221 16.72412 13.05465 3.2E+08 ____________________________ 8.38608 122.0761 15.33089 12.69376 7.72ia-BoxBFL149.LYS.H 7.98664 120.7873 15.37662 13.186 7.8E+08 , BoxBFL.150.LEU.H
8.29876 i 120.0933 15.78188 13.31339 i 5.08E+08 ;. , BoxBFL151.LYS.H
8.44117 ' 123.678 14.86959 12279 6.28E+08 BoxBFL152.GLU.H
8.09634 120.1123 17.64454 13.74299 8.61E+08 BoxBFL153.LYS_H
7.76844 119.7644 14.87574 12.75453 I 7.39E+08 BoxBFL154.1YR.H
8.14945 120.4238 #VALU E! #VALUE! ' 7.23E+08 BoxBFL155.GLU.H
8.52687 117.6413 15.0031 13.04017 6.17E4-08 BoxBFL.157.ASP.H 8.906 123.2206 15.30218 13.15 6.04E+08 BoxBFL158.ILE.H
9.20733 122.5499 17.72024 12.87546 5.48E+08 BoxBFL159.A1A.H
7.36719 124.835 14.8669 12.75735 7.55E+08 BoxBFL.160.ALA.H
7.78634 120.3982 14.86213 13.04298 7.84E+08 BoxBFL.161.1YR.H
8.20162 120.0455 17.64211 17.63803 8.72E+08 BoxBFL162.ARG.H
8.26363 118.8125 15.04827 12.83358 7.06E+08 BoxBFL163.ALA.H
7.64059 121.3835 14.5465 12.45633 5.34E+08 BoxBFL164.LYS_H
7.65996 118.3228 15.81194 12.97592 4.19E+08 BoxBFL165.GLY.H 8.0589 108.6345 195729 13.76712 1.13E+08 BoxBFL166.LYS.H
8.04566 1215924 15.74549 13.70187 1.31E+09 -*
BoxBFL.168.ASP.H
8.49244 121.104 14.18502 12.88319 7.2E+08 13oxBFL.169.ALA-H 8.2712 124.8749 14.52477 12.28691 3.58E+08 ..
BoxBFL170.AL&H
8.13452 121.4021 14.64616 11.71461 6.15E+08 BoxBFL172.LYS_H
8.42733 124.7554 12.80903 10 .72519 i 46624344 BoxBFL173.GLY.H
8.57669 111.124 19.67674 13.22967 1.55E4-08 HIMGB1A-c007 (3S, 1-184) with 1:2 molar ratio CXCL12 (1:1 molar ratio CXCL12 to 111VIG Box), in 10 mM HEPES pH 7.5 150 mM NaCI, Figure 12 SPECTROMETER WM SMM TCI CRYOPROBE
1H frequency (Hz) N
749.91352 1H sweep width (ppm) 12.122609 1H sweep width (Hz) 9090.9090 1HN sweep width (ppm) 12.122609 15N frequency (Hz) 75.99661 15N sweep width (ppm) 32.01577 15N sweep width (Hz) 2433.09002 Water suppression gradient pulse width (Hz) 3527.31 Baseline 1 (Day 1) Experiment type 2D 1I-1-HMGB1 construct and concentration 0.30 mM, HMG B1-c007 (35 1-184), TEV-cleaved (200 ra 0.45 m141 stock + 185 pl. buffer) CXCL12 construct and concentration 0 mM
Temperature 25 QC
Volume 350 pt Sample pH (after adjusting) 7.66 Number of scans 16 Buffer 10 mM
HEPES, pH 73 F1 assign : Fl position I F2 position i LW Fl (Hz) LW F2 (Hz) i Height .=' TL3.GLY.H, TL.10.GLY.H 8.6061 111.0604 ] 24.6728 23.8470 i 4.24E4-06 i TL6.LYS.H 8.3808 120.7275 19.3276 19.0515 1.26E+07 TI-7-LYS.H 7.9672 123.9738 18.9452 18.4814 1.22E+07 TL9.ARG.H 8.9422 124.5857 3o. 5 1 as 22.5922 2.68E+06 --------------------------- ------------------- -------------- --- ------------------- ------------------ ------------- --------- -------------- --------:.---------- --------TL11.LYS.H 7.6533 119.5407 18.7001 20.1359 %, 3.98E+06 TL.1.3.SER.H,TL.26.HIS.H 8.1990 119.2086 26.4193 23.6946 6.96E+06 ,.

...............................................................................
............................ , i TL14.SER.H 93037 116.5728 17.7398 203154 1.52E+06 TL1S.TYR.H 7.9749 122.3508 213872 20.0836 / 9.77E+06 t TL16.ALA.H 7.8898 122.0710 18.6785 21.9854 i 1.16E+07 71_17.PHE.H 8.3224 118.1346 25.4892 18.2495 5.66E+06 t 711-18.PHE.H 8.1246 125.6475 19.4733 26.9249 6.38E-106 TL21.THR.H 8.1820 116.6683 21.3497 19.6855 7.47E+06 TL23.ARG.H 8.9337 123.5827 19.6637 19.5765 1.34E+07 TL25.GLU.H 8.3297 119.6119 17.9559 20.8464 4.41E+06 TL27.LYS.H 7.9199 118.2035 17.4027 24.5903 %' 1.32E+07 TL28.LYS.H 7.4656 117.6101 21.4259 22.0467 2.19E+06 TL29.LYS.H 7.5008 116.9197 26.2391 24A263 i 3.84E+06 TL30.HIS.H 7.9045 116.8771 21.2366 19.0908 i 3.22E-107 TL32.ASP.H 8.5014 116.2147 21.6349 20.6257 : 1.15E+07 , TL33.ALA.H 7.6405 123.1947 27.1908 18.4877 i 6.05E+06 TL36.ASN.H 8.5631 124.5616 21.3601 19.8298 t 1.05E+07 =..
TL38.SER.H 8.5266 116.5707 20.6963 19.7437 1.75E+07 =..
TL39.GLU.H 7.8017 121.11763 24 .3844 20.7391 ,= 5.60E+06 t TL41.SER.H 8.4328 114.6754 21.9986 19.2519 7.61E+06 TL43.LYS.H 7.9047 120.0881 24.5054 21.5027 8.26E+06 1.
TL48.TRP.H 8.6119 121.3719 27.7321 19.6527 6.67E+06 TL49.LYS.H 7.5392 114.9177 22.4466 26.2994 ,= 6.95E+06 t TL50.THR.H 7.4184 106.6545 19.9202 26.9454 3.55E-106 TL51.14ET.H 7.1286 124.1028 34.8660 19.9547 3.59E+06 -.
TL52.SER.H 9.0214 120.2824 25.7658 22.2592 3.14E+06 TL54.LYS.H 8.2612 118.6673 21.4006 20.8276 1.72E+07 TLSS.GLU.H, TL146.LYS. H, TL-156.LYS.H 7.6743 =
120.5743 39.0944 20.46491 4.08E+07 TL57.GLY.H 7.8481 107.2718 24.8060 21.1106 4.92E+06 :
TL58.LYS.H 7.9240 118.8658 23.1257 30.2328 : 535E+06 , TL60.GLU.H 8.4408 121.0589 18.5170 21.8991 i 2.43E+07 TL61.ASP.H 8.5793 121.9550 25.9461 21.0215 z 2.41E+06 , TL63.ALA.H 8.0272 123.2985 18.8092 20.8840 3.01E+07 TL64.LYS.H 8.5925 122.4879 24.6164 26.1814 3.30E+06 TL65.ALA.1-1 7.9334 123.0276 19.4945 20.6248 1.77E+07 TL66.ASP.H 8.3.119 120.0786 22.2268 23.C/110 ,= 5.95E+06 t TL67.LYS.H 8.2124 120.8922 19.2067 21.7276 134E+07 TL68.ALA.H 7.4653 120.9539 26.2159 21.1243 4.51E+06 t 71_71.GLU.H 8.4294 117.1838 20.4156 25.0309 7.16E+06 711-72.ARG.H 8.0622 120.1074 18.6953 20.2934 i 2.46E-107 t 711...73.GLU.H 8.4749 120.0201 22.5415 19.5028 9.11E+06 TL74.14ET.H 8.4409 117.6522 #VALU E! #VALUE! 6.40E+06 -:
TL75.LYS.H 7.5100 119.0899 22.5073 22.2834 3.08E+06 TL77.7YR.H 7.5024 123.9352 30.9003 17.6211 ,= 3.53E+06 71.78.1LE.H 7.8034 129.2718 20.0866 19.3005 %% 1.72E+07 , TL88.PHE.H 8.3596 122.1156 17.9211 19.8933 i 2.66E+07 TL89.LYS.H 8.2181 123.3632 21.8312 19.6249 1.19E-107 z TL90.ASP.H 8.0110 119.1287 20.5283 23.7061 9.96E+06 TL93.ALA.H 7.3090 123.8485 18.6517 18.5426 : 2.41E+07 TL95.LYS.H 8.5094 123.7507 19.5433 18.6325 1.67E+07 TL99.SER.H 7.8684 114.9049 20.2451 19.8589 i 2.20E+07 TL100.ALA.H 9.0706 122.9058 17.9269 19.6686 1.43E+07 TL102.PHE.H 8.2289 122.4276 19.9106 19.5888 ,=% 137E+07 t TL103.LEU.H 8.2529 120.0969 21.6806 25.4949 1.99E+07 TL104.PHE.H 8.1450 122.5447 19.7785 17.8570 i 3.42E+06 t TL106.SER.H 7.9866 116.8141 16.6297 18.4050 2.77E+07 711-107.GLU.H 7.1139 119.4856 18.6763 18.2751 i 2.77E-107 t TL111.LYS.H 6.8070 117.8973 20.9968 19.4882 i 1.97E+07 TL112.1LE.H 8.1471 119.7094 19.6956 20.7896 = 2.21E+07 +
TL113.LYS.H 8.2277 118.6620 18.9931 20.0020 2.62E+07 TL114.GLY.H 7.6276 103.8053 19.2245 18.7965 2.17E+07 -:..
TL115.GLU.H 7.4519 119.5050 , 19.0651 18.6530 2.50E+07 ...............................................................................
.............................. , i TL116.1115.11 7.8557 114.5584 21.3730 26.3315 1.95E+07 TL119.LEU.H 7.3441 121.1282 16.9517 18.0939 4.11E+07 t Tt.120.SER.H 9.2783 120.9530 19.8514 19.2891 i 1.57E+07 TL121.ILE.H 8.7093 120.6277 19.2620 19.2230 1.13E+07 t TL122.GLY.H 8.6846 109.4587 26.8288 22.3716 431E-106 Tt.123.ASP.H 7.9382 124.3219 18.3663 18.7325 2.13E+07 TL124.VALH 8.6040 124.0412 18.2886 18.7654 2.25E+07 TL125.ALA.H 7.8785 121.3328 16.5530 19.1095 3.63E+07 TL126.LYS.H 7.9989 119.7206 19.3006 19.3155 %' 7.93E+07 TL127.1.YS.H 8.0145 121.3551 17.2034 17.8224 7.22E+07 TL17.8.LEU.11 8.6505 120.2524 18.6803 18.7237 i 2.09E+07 , TI.129.GLY.H 8.2032 106.2602 19.1920 18.8485 i 2.46E-107 C.
11.131.MET.H 8.7231 118.8760 18.0856 18.4060 : 2.38E+07 , TL132.TRP.H 8.7095 122.5759 19.0186 19.0721 i 1.59E+07 T11..133.ASN.H 8.1328 117.2422 19.0104 18.8528 , 3.15E+07 Tt.134.ASN.H 7.5431 115.7695 19.1824 18.8449 2.98E+07 Tl...135.THR.H 7.2603 119.9046 19.374-2 19.5603 ,% 2.60E+07 t TL136.ALA.H 9.2045 131.3561 17.2667 18.5735 2.23E+07 , Tt.137.AIA.H 8.8342 124.9935 33.3526 21.2936 1.19E+06 t Tt_138.ASP.H 8.9528 115.6245 18.8620 19.6655 1.34E+07 Tt_139.ASP.H 7.2986 117.8616 19.8829 18.6225 ,z 2.53E+07 t TI.140.A1A.H 7.7883 119.0980 20.0357 19.4836 1.79E-107 TL141.GLN.H 7.4069 118.1124 18.1893 19.2718 2.30E+07 , TI_143.TYR.H 7.1940 115.5652 20.7215 19.7599 1.68E+07 TL145.LYS.H 9.1712 121.2666 18.7285 20.6529 1.58E+07 Tt.147.A1A.H 8.3287 120.5992 22.2428 21.8046 %% 1.24E+07 TL149.LYS.H 7.9709 120.8132 19.5777 23.5142 2.79E+07 :
TL150.LEU.11 8.2579 119.8355 19.6870 24.4644 : 1.95E+07 , Tt.151.LYS.H 8.3943 123.7275 19.8464 22.6956 i 2.13E+07 TL1.52.GLU.H 8.0005 120.0908 18.5048 18.9795 z 4.56E+07 , TL1.53.LYS.H 7.7378 119.7316 20.1171 19.9428 2.47E+07 TI.1.54.TYR.H 8.1211 120.2560 27.4207 25.0214 4.56E+07 TL155.GLU.H ................................................... 8.4779 117.6292 20.1839 20.3442 1.81E+07 ,. -0 Tt.157.ASP.H 8.8596 123.1691 18.1489 19.7980 ,% 1.95E+07 t T1-158.1LE.11 ................................................. 9.1467 122.4891 19.2484 19.6793 1.60E+07 ,. 0 Tt.1.59.A1A.H 7.3324 124.8211 18.1448 19.5515 2.61E+07 t TL160.A1A.H 7.7523 120.3814 18.8194 20.3354 2.87E+07 T11...1.62.AFIG.H 8.2660 119.1276 20.2383 21.2173 i 2.28E-107 t Tl...163.ALA.H 7.5998 121.5022 18.4569 19.0784 1.90E+07 TL1.64.LYS.H 7.6444 118.6458 18.5535 19.1662 1.93E+07 TL166.LYS.H 8.0107 121.8105 21.0399 22.8760 1.20E+07 TL168.ASP.H 8.4217 120.2067 20.8680 20.3203 ,% 1.18E+07 Tt.169.A1A.H 8.2713 124.9694 23.2824 19.9815 %% 1.63E+07 c.
Tt.170.ALA.H 8.1977 121.6039 19.5714 19.2612 i 1.48E+07 TL174.VALH 7.9243 119.5174 23.1927 24.7751 6.62E-106 z 11.175.VALH 8.3105 125.5123 16.0756 16.4540 6.65E+07 C.
T1._176.LYS.H 8.4655 126.6128 28.7022 20.5965 : 3.74E+06 TL177.ALA.H 8.3773 125.8179 31.4688 20.8340 3.36E+06 Tt.178.GLU.H 8.4450 120.6799 IIVALUEI #VALUEI i 9.04E+06 Tl...179.LYS.H 8.4425 122.3029 19.9586 24.6041 1.34E+07 TL185.GLU.H 8.2782 122.8421 19.8975 i 21.0209 1.81E+07 , 3D experiments (Day 1) These spectra are not shown due to their 3D nature; data available if requested. Sample is that of Baseline 1.
Experiment type 3D, IsN
EDITED 1H-'IITOCSY-HSQC, THROUGH-SPACE INTERACTION (1 MAR 19) Temperature 25 2C
Volume Number of scans 8 Experiment type 3D, 15N
EDITED 41-11-1 NOESY-HSQC, THROUGH-BOND INTERACTION (28 FEB 19) Temperature 25 at Volume 300 tiLL
Number of scans 16 Baseline 2 (Day 3) Experiment type 2D 1H-NIMB1 construct and concentration 0.30 mM, H MG B1-c007 (35 1-184), TEV-cleaved (200 pi_ 0.45 mM stock + 185 p.1_ buffer) CXCL12 construct and concentration 0 mM
Temperature 25 QC
Sample pH (after adjusting) 7.66 Volume 350 pLL
Number of scans 16 Buffer 10 mM
HEPES, pH 7.5 FL assign FL F2 position LW Fl LW F2 Height position (Hz) (Hz) TL3.GLY.H, TL.10.GLY.H 8.6065 111.0499 23.1613 23.4948 E 4.43E+06 ;

T1.6.LYS.H 8.3807 120.7320 19.6417 18.6975 1.24E+07 TL-7.LYS.H 7.9677 123.9750 18.5170 18.1121 1.19E+07 11_9_ARG.H 8.9453 124.5715 24.2572 23.7356 2.61E+06 T1.11.LYS.H 7.6528 119.5498 17.4576 20.3878 3.89E+ 06 71-13.5ER.H, T1.26.HIS.H 8.1991 119.1995 26.6500 22.3922 6.54E+06 TL14.5ER.H
9.2956 116.5826 16.8521 20.2197 1.33E+06 TL15.7YR.H 7.9757 122.3477 21.7938 19.9370 E 9.68E+06 71-16.ALA.H
7.8896 122.0725 19.8069 21.8309 1.12E+07 TL17.PHE.H
8.3224 118.1373 25.3998 18.6893 5.67E+06 TL18.PHE.H
8.1241 125.6449 20.5389 27.4613 6.00E+06 T1.21.THR.H
8.1825 116.6667 21.1937 19.7947 7.42E+06 TL23.ARG.H
8.9329 123.5826 19.1345 19.5535 1.33E+07 TL25.GLU.H
8.3294 119.6141 20.8289 20.5729 4.29E+06 Tl...27.LYS.H 7.9196 118.2075 17.7875 24.4847 1.31E+07 71.28.LYS.H
7.4656 1173781 22.7406 23.1308 2.32E+06 TL29.LYS.H
7.4989 116.9374 23.0209 25.0045 4.05E+06 TL.30.HIS.H 7.9042 116.8739 21.0887 18.9026 3.21E+07 TL.32.ASP.H 8.5004 116.2125 20.0730 20.5524 1.17E+07 TL33.ALA.H
7.6424 123.1937 25.8311 18.9977 6.03E+06 TL36.45N.H
8.5629 124.5621 20.2634 20.1827 1.01E+07 Tl...38.SER.H 8.5266 116.5762 20.3927 20.2102 1.75E+07 TL39.GLU.H
7.8015 121.4813 25.4307 21.5986 5.49E+06 TL41.SER.H
8.4315 114.6769 24.0654 18.8439 7.20E+06 TL43.LYS_H 7.9025 120.0835 25.2682 22.0179 E 8.40E+06 TL.48.TRP.H 8.6129 121.3660 22.6050 19.2575 ; 6.76E+06 TL49.LYS.H 7.5396 114.9173 20.9942 25.1553 E 6.76E+06 T1.50.THR.H
7.4186 106.6388 24.1325 25.7242 3.44E+06 11.51.MET.H
7.1310 124.1048 22.9068 19.6438 3.75E+06 t.
TL52.SER.H
9.0209 120.2880 28.9274 22.9000 2.95E+06 TL54.LYS.H
: 8.2621 118.6676 20.9063 21.0484 1.74E+07 TL55.GLU.H, 11.146.LYS.H, TL156.LYS.H
7.6741 1203737 35.8117 20.3103 4.07E+07 TL57.GLY.H
7.8487 107.2598 28.3292 20.2673 4.70E4-06 TL58.LYS.H
7.9229 118.8497 24.2289 27.7675 5.26E+06 TL60.GLU.H
8.4408 121.0595 18.5310 21.9295 2.41E+07 T1-61.ASP.H 8.5746 121.9512 28.9797 22.9634 2.66E+06 TL.63.AULH
8.0269 123.2962 18.7324 21.1710 2.95E407 T1.64.LYS.H 8.5909 122.4962 24.9341 24.0694 2.94E+ 06 1L65.ALAII
7.9332 123.0304 20.3519 20.5074 1.74E4-07 T1-66.ASP.H 8.3409 120.0857 22.2493 23.7054 5.80E+06 TL67.LYS.H
8.2130 120.8955 19.6156 22.3669 1.54E+07 TL.68.ALA.H 7.4616 120.9561 37.9570 19.8115 ; 4.30E+06 TL71.GLU.H 8.4286 117.1801 21.4128 25.4454 E 6.76E+06 TL72.ARG.H
8.0621 120.1083 18.7908 20.0752 2.45E407 TL73.GLU.H
8.4738 120.0142 23.7008 19.2906 8.71E+06 TL74.MET.H 8.4409 117.6522 #VALUE I INALUE I 6.44E+06 TL75.LYS.H
7.5068 119.0858 26.7860 22.3687 3.23E+06 TL77.TYR.H 7.5032 123.9308 38.9311 17.2302 3.49E+06 TL78.ILE.H 7.8036 129.2703 19.9513 19.4233 1.70E+07 TL88.PHE.H
8.3595 122.1143 17.6506 19.7390 2.67E+07 TL89.LYS.H
8.2182 123.3647 19.6893 20.1167 1.23E+07 TL.90.ASP.H
8.0126 119.1425 20.7630 25.2387 9.55E+06 TL93.ALA.H
7.3090 123.8482 18.8016 18.5673 2.40E+07 T1.95.LYS.H 8.5090 123.7493 18.6872 18.3947 1.70E4 07 TL99.SER.H
7.8687 114.9053 20.2014 20.0612 2.21E+07 TL100.AIA.H
9.0705 122.9049 17.6281 19.4111 1.43E+07 TL102.PHE.H
8.2287 122.4261 20.3823 19.7821 1.38E+07 TL103.LEU.H
8.2533 120.0963 22.3290 25.7323 1.99E+07 TL104.PHE.H 7.9865 116.8146 16.6032 18.4916 ; 2.71E+07 -t-TL106.5ER.H 7.1139 119.4847 18.9087 18.2576 ; 2.71E+07 TL107.GLU.H 6.8074 117.8959 20.3505 19.3071 E 1.99E4 07 S.
TL111.LYS.H
8.1473 119.7047 19.6728 20.5823 2.19E+07 TI-112.1LE.H 8.2277 118.6631 19.2041 19.9527 2.58E+07 TL113.LYS.H 7.6278 t 103.8060 20.2430 18.4257 2.16E+07 TL114.GLY.H
7.4522 1193041 18.8464 18.7187 2.51E+07 TL115.GLU.H
7.8558 114.5590 21.3428 26.4292 1.94E+07 TL116.HIS.H
7.3444 121.1280 17.0541 18.0097 4.07E+07 TL119.LEU.H
9.2779 120.9556 19.1260 18.9896 1.56E+07 TL120.SER.H
8.7096 120.6282 19.3699 19.3593 1.16E+07 TL121.ILE.H 8.6834 109.4592 23.3770 21.1555 4.56E+06 TL122.GLY.H 7.9382 124.3228 18.0611 18.8783 2.14E4 07 1L123.ASP.H 8.6038 = 124.0408 18.3007 18.9123 2.25E+07 TL124.VAL.H
7.8787 121.3329 16.2690 19.0869 3.68E+07 TL125.ALA.H
7.9987 119.7210 19.0408 19.1946 7.83E+07 TL.126.LYS.H 8.0145 121.3562 17.2124 17.7746 7.14E+07 TL127.LY5.H
8.6507 120.2533 18.8465 18.5636 2.06E+07 -t-TL.17J3.LEU.H 8.2034 106.2583 19.1542 18.5945 2.46E407 T1.129.GLY.H 7.9939 122.7099 31.0113 24.8633 2.83E4 06 -t-TL131.MET.H
8.7234 118.8753 17.8826 18.2976 2.41E+07 TL132.TRP.H 8.7097 z 122.5757 19.4286 19.1503 1.60E+07 TL133.ASN.H
8.1327 117.2411 19.1545 18.7949 3.10E+07 TL.134.ASN.H
7.5430 115.7691 19.2434 18.7462 2.97E+07 TL135.THR.H
7.2604 119.9055 18.9668 19.2131 2.62E+07 TL136.AIA.H
9.2048 131.3556 16.6770 18.6275 2.27E+07 TL.137.AIA.H
8.8215 124.9797 28.9631 18.8998 9.53E+05 T1-138.ASP.H
8.9534 115.6244 19.3079 19.6390 1.34E+07 1L139.ASP.H
7.2990 117.8613 19.5011 18.4882 2.54E+07 T1.1413.ALA.H 7.7881 119.0997 19.6218 19.4625 1.80E+07 TL141.GLN.H
7.4070 118.1139 18.2290 19.3873 2.28E+07 TL143.TYR.H
7.1941 115.5640 20.6936 19.5627 1.75E+07 TL145.LYS.H 9.1714 121.2694 18.9207 20.5145 1.55E+07 TL147.ALA.H 83294 120.5969 21.2897 22.3662 1.25E+07 TL149.LYS.H 7.9711 120.8080 19.0205 22.9038 2.77E+07 TL1SO.LEILH 8.2586 119.8352 19.6218 24.8315 1.93E+07 T1.151.LYS.H 8.3946 123.7260 20.5952 23.0334 2.12E407 TL1S2.GLU.H 8.0004 120.0915 18.6445 18.7793 4.53E4-07 TL1S3.LYS.H 7.7381 119.7331. 20.2180 19.8851 2.46E407 TL1S4.TYR.H 8.1209 120.2560 28.0283 25.0515 4.45E+07 TL1SS.GLU.H 8.4777 117.6292 19.6687 20.1632 1.81E+07 TL1.57.4SP.H 8.8597 123.1714 18.3048 19.6916 1.96E4-07 TL1S8.ILE.H 9.1469 122.4919 19.3081 19.5812 1.61E+07 T1.159.41A.H 7.3325 124.8205 17.9034 19.2404 2.59E407 11.160.41A.H 7.7526 120.3794 18.6070 20.3336 2.91E+07 TL162.ARG.H 8.2667 119.1253 20.5620 21.2604 2.28E-s-07 TL163.41A.H 7.5996 121.4996 18.4419 19.1701 1.89E+07 TL164.LYS.H 7.6446 118.6434 18.3150 18.9990 1.94E+07 TL166.LYS.H 8.0103 121.8118 20.8605 22.0326 1.21E4-07 TL168.4SP.H 8.4199 120.2046 21.4934 19.9990 1.18E+07 TL169.41A.H 8.2714 124.9725 23.9158 19.9230 1.64E+07 T1_170.41A.H 8.1980 121.6044 19.8101 19.2270 1.50E+07 T1_174.VALH 7.9239 119.5328 23.8954 29.0599 E 6.20E+06 T1.175.VALH 8.3108 125.5127 16.0825 16.4752 6.74E407 S.
TL176.LYS.H 8.4651 126.6236 22.4539 20.5401 4.14E4-06 T1_177.ALA.H 83781 125.8126 25.5412 22.8124 3.71E+06 TL178.GLU.H 8.4450 120.6799 #VALUE1 #VALUE1 9.25E+06 TL179.LYS.H 8.4424 122.3058 19.6823 24.3392 1.27E+07 TL185.GLU.H 8.2779 122.8405 19.1833 22.8993 1.91E+07 1:1 molar ratio CXCL12/H1VIG Box (1:2 ratio HIVIGB1A-c007/CXCL12A-c021, Day 4) Experiment type 2D 1H-HMGB1 construct and concentration 0.30 mM, MG 81-c007 (35 1-184), TEV-cleaved (200 pi 0.45 mitil stock) CXCL12 construct and concentration 0.6 mM, CXCL12A-c021, wt 1-67, no tag (185 (IL
1.14 mM stock) Temperature 25 QC
Volume 350 pi Sample pH (after adjusting) 7.65 Number of scans 16 Buffer 10 mhol HEPES, pH 7.5 Fl assign F1 F2 position LW Fl LW F2 Height position (Hz) (Hz) TL3.GLY.H, TL10.GLY.H 8.6044 111.0338 27.0394 23.1335 5.94E+06 TL&LYS.H 8.3809 120.6948 22.5007 18.0287 1.34E4-07 TL7.LYS.H 7.9643 123.9632 20.3742 18.4134 134E407 TL9.ARG.H 8.9503 124.5797 31.7140 24.0875 3.87E+06 T1_11.LYSII 7.6557 119.5615 16.0398 21.6463 5.42E+06 TLIS.SER.H, TL.26.HIS.H 8.2004 119.2105 23.4564 24.3179 1.04E+07 TL14.SER.H 9.3238 116.5556 14.7611 15.5346 1.67E+06 TL15.7YR.H 7.9778 122.3535 21.1121 19.3913 1.51E+07 TL16.414.H 7.8925 122.0870 193520 22.0446 1.84E+07 S.
TL17.111E.H 8.3240 118.1495 273926 18.0447 9.03E-106 TL.18.PHE.H 8.1278 125.6553 19.8250 26.5265 1.13E+07 TL21.THR.H 8.1835 116.6724 213629 19.5787 9.24E+06 11..23.ARG.H 8.9348 123.5796 18.8791 19.0832 240E+07 ! TL.25.GLUM j 8.3321 119.6265 22.0156 18.9298 6.69E+06 i TL27.LYS.H 7.9208 118.2122 E 17.2389 24.5277 2.00E+07 TL28.LYS.H t 7.4693 117.6188 E
20.5149 21.7352 3.16E+06 TL29.LYS.H 7.5019 116.9291 ;
20.0353 22.0649 5.99E+06 TL30.HIS.H 7.9083 116.8927 i 20.9767 19.1705 5.23E+07 TL32.ASP.H z = 8.5041 116.2183 ;
21.3681 20.8293 1.80E+07 TL33.AL.A.H t 7.6486 123.1983 i 33.3099 19.1418 7.80E406 TL36.ASN.H
i 8.5655 124.5629 18.7902 19.8770 1.75E+07 TL38.SER.H
8.5296 116.5760 19.5825 19.3443 3.10E+07 TL39.GLU.H
7.8000 121.4911 27.7099 20.3638 8.06E+06 TL41.SER .11 8.4349 114.6887 23.1352 19.2946 1.18E+07 TL43.LYS.H
1 7.9049 120.0830 25.4006 223369 1.19E+07 TL48.TRP.H
t 8.6160 121.3765 22.4001 19.4978 1.10E+07 TL49.LYS.H ,t = 7.5429 13.4.9206 21.7660 26.6768 1.04E407 11.50.THR.H
7.4225 106.6598 25.9188 26.8533 5.09E+06 TL51..NIET.H
t 7.1282 124.0983 25.1134 21.1386 5.30E+06 TL52.SER.H 9.0270 120.2729 23.7729 19.7149 4 .97E+06 TL54.LYS.H
8.2637 118.6728 213251 20.6048 2.61E+07 TL55.(A.U.H, 1L146.LYS.H, TL1.56.LYS.H 7.6767 120.5832 30.2398 20.3845 6.71E+07 TL57.GLY.H
7.8522 107.2682 26.6428 20.8923 8.03E+06 TL58. LYS.H 7.9244 118.8701 22.7163 29.6656 7.94E+06 TL60.GLU.H
8.4437 121.0610 18.2434 20.3448 4.09E+07 TL61.ASP.H 8.5815 121.9597 33.1388 21.6707 3.39E+06 TL63.ALA.H
8.0294 123.2984 18.2389 19.6294 5.05E+07 8.5980 122.5146 26.0412 25.2214 4 .71E+06 TL65.ALA.H
7.9368 123.0339 18.6025 19.9725 2.82E+07 TL66.ASP.H
8.3457 120.0879 22.5058 23.2892 7.23E+06 TL67.LYS.H t 8.2154 120.8999 18.9826 22.1852 2.52E-8-07 TL68.ALAM
7.4697 120.9568 23.2933 19.0137 6.76E+06 TL71.GLU.H 8.4315 117.1750 21.7295 ; 26.3586 1.06E+07 TL72.ARG.H = 8.0645 120.1128 18.0569 ; 19.4262 4.23E-8-07 TL73.GLU.H
t 8.4787 120.0270 23.1364 19.3270 1.51E407 TL74.101ET.H 8.4409 117.6522 INALU E I #VALU E I 9.83E+06 TL75.1.YS.14 7.5049 119.1098 31.7802 21.8213 4.59E+06 TL77.7YR.H
7.5105 123.9396 38.1218 16.9483 4.14E+06 TL78.ILE.H
7.8084 129.2854 193578 19.9301 2.60E+07 TL88.PHE.H
= 8.3628 122.1204 16.9053 19.1178 4.49E+07 TI-89.LYS.H t 8.2210 123.3706 19.7502 19.2731 2.10E-8-07 TL90.ASP.H
= 8.0147 119.1518 21.2737 24.1312 1.43E+07 TL93.AL.A.H
7.3126 123.8531 193345 18.4537 3.57E+07 TL95.1.YS.14 8.5127 123.7578 18.2433 18.4107 2.55E+07 7.8719 114.9159 19.1639 18.9030 3.80E+07 1L100.AIA.H
9.0737 122.9134 17.9203 18.9328 2.47E+07 TL102.PHE.II
8.2316 1224356 224797 20.9918 1.28E+07 TL103.LEU.H
tµ 8.2553 120.1006 213513 23.9579 3.34E+07 TL104.PHE.H
8.1434 122.5532 26.6494 18.4699 3.85E+06 TL106.SER.H
7.9897 116.8195 16.1819 18.1182 4.66E+07 TL1.07.GLU.H
= 7.1171 119.4906 17.8505 18.0469 4.73E-i-07 TL111.LYS.H 6.8097 117.9049 19.8483 19.1076 3.40E-807 TL112.ILE.H 8.1508 119.7121 19.4877 20.4021 3.72E+07 TL113.LYS.H
8.2307 118.6666 18.3996 19.2564 437E+07 TL114.GLY.H
7.6300 103.8047 18.7943 18.4306 3.60E+07 TI-11S.GLU.H t 7.4552 119.5222 19.2213 18.9640 4 .04E+07 11.1.16.HIS-H 7.8582 114.5580 20.7604 23.4804 3.16E+07 TL119.LEU.H 7.3458 121.1340 16..1812 17..8123 6.86E+07 TL120.SER.H
9.2812 120.9606 18.7787 19.3743 2.64E+07 TL121.ILE.H 8.7114 120.6306 20.5564 19.1568 1.71E+07 TL122.GLY.H
.t 8.6882 109.4736 24.3584 20.5405 5.40E+06 TL123.ASP.H 7.9414 124.3316 18.9583 18.7555 3.43E-8-07 TL124.VALH
8.6075 124.0518 17.5597 18.0450 4.01E+07 TL125.ALA.H
7.8803 121.3377 163135 18.8634 6.13E+07 TL126.LYS.H 8.0024 119.7348 E
20.5705 18.3671 1.04E+08 TL127.LYS.H 8.0190 121.3711 17.2894 17.8534 1.01E+08 11-128.LEU.H t 8.6532 120.2570 i 18.0062 18.2445 3.66E+07 TL129.G1Y.H
z 8.2053 106.2627 18.0361 18.4504 4.24E+07 TL131.MET.H I 8.7256 118.8819 i 16.6866 17.9953 4.34E407 TL132.TRP.H
i 8.7119 122.5814 18.4799 18.7626 2.80E+07 TL133.ASN.H
8.1348 117.2495 17.4409 18.4657 5.40E+07 TL134.ASN.H
7.5461 115.7741 193366 18.8391 4.91E-8-07 TL135.THR.H
I 7.2627 119.9122 18.4851 18.5540 4.44E+07 TL136.ALA.H
1 9.2071 131.3622 17.0411 18.2227 3.66E+07 TL137.ALA.H
8.8274 124.9859 14.4547 22.1480 1.45E+06 71.138.45P.H
t 8.9571 115.6309 18.9345 19.4487 1.95E407 11.139.ASP.H
7.3010 117.8635 19.8679 18.2453 4.22E+07 11_140.ALA.H
I 7.7905 119.1034 19.3181 18.7588 3.13E-807 71-141.GLN.H
7.4097 118.1173 17.1880 18.8418 4.01E+07 71.143.TYR.H
z 7.1967 115.5677 19.8319 19.1363 2.97E+07 71.145.LYS.H 9.1741 121.2738 17.8887 20.0482 2.77E+07 TL147.ALA.H
tµ 8.3318 120.6021 21.9785 20.7642 1.98E+07 =..
TL149.LYS.H
7.9737 120.8159 17.7823 22.3931 4.89E+07 11_150.LEILH 8.2612 119.8328 19.4856 23.9293 2.86E+07 TL151.LYS.H
t 8.3980 123.7315 19.0907 22.3943 3.45E+07 71.152.GLU.H
8.0036 120.1009 183759 18.4348 5.46E407 TL153.LYS.H
7.7406 119.7378 19.3471 19.4416 4.18E+07 TLI54.TYR.H
I 8.1247 120.2565 24.0128 22.4221 7.15E+07 TL155.GLU.H
8.4809 117.6318 183609 19.8254 3.19E+07 71-157.ASP.H
I 8.8614 123.1717 17.8349 19.3205 3.35E+07 TL158.ILE.H
9.1480 122A894 19.2904 19.2156 2.73E+07 71-159.ALA.11 7.3355 124.8233 173550 19.1822 4.38E+07 TL160.AIA.H
7.7549 120.3869 17.8564 19.8791 4.70E+07 TL162.ARG.H
8.2685 119.1365 19.6143 21.0415 3.36E+07 TL163.ALA.H
I 7.6030 121.5109 18.4123 19.0334 2.70E+07 11.164.LYS.H
7.6483 118.6508 183044 19.7095 2.57E+07 TL166.LYS.H 8.0157 121.7960 20.9936 22.5167 1.29E+07 11-168.ASP.H 8.4254 120.2086 22.2976 21.9565 1.05E+07 TL169.41A.H
z 8.2750 124.9759 19.4096 19.6958 2.71E+07 k.
TL170.AIA.H
8.2009 121.6082 19.1281 18.5205 2.58E+07 71_174.VALH
7.9258 119.5189 26.7477 26.1396 8.34E+06 71.175.VALH
8.3137 125.5231 16.9953 16.9413 7.98E+07 71...176.LYS.H I 8.4697 126.6299 26.1100 20.5983 2.90E+06 TL177.AIA.H
t 8.3806 125.7978 28.0424 18.8813 2.71E+06 TL178.GLU.H I 8.4483 120.7051 WALUE I #VALUEI 9.69E+06 TL179.LYS.H 8.4452 122.3144 19.8139 22.4131 2.10E+07 71-185.GLU.H
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Claims (18)

What is claimed is:

1. A polypeptide represented by the following formula:

wherein A represents consecutive amino acids, the sequence of which (1) includes a sequence identical to the sequence of amino acids 90 ¨ 93 of wild type FUVIGB1, (2) has at its amino terminal end, between one and six consecutive amino acids, the sequence of which is identical to the sequence of the corresponding one to six amino acids preceding amino acid 90 in wild type HMGB1, and optionally (3) has a methionine at the amino terminus;
wherein X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94 ¨ 162 of wild type HMGB1; and wherein B represents consecutive amino acids, the sequence of which (1) includes a sequence identical to the sequence of amino acids 163 ¨ 168 of wild type HMGB1 and (2) has at its carboxy terminal end, between one and six consecutive amino acids, the sequence of which is identical to the sequence of the corresponding one to six amino acids following amino acid 168 in wild type FIMGB1; and wherein each - represents a peptide bond between each of A and X, X and B, B and A, A and X, and X and B.

2. A polypeptide of claim 1, wherein the methionine is present at the amino terminus.

3. A polypeptide of claim 1 or 2, wherein A has at its amino terminal end, one amino acid corresponding to amino acid 89 of wild type FIMGB1.

4. A polypeptide of any one of claims 1-3, wherein B has at its carboxy terminal end, six amino acids corresponding to amino acids 169-174 of wild type HMGB1.

5. A composition comprising the polypeptide of any one of claims 1-4 and a carrier.

6. A pharmaceutical composition of claim 5, wherein the polypeptide is present in a therapeufically or prophylactically effective amount and the canier is a phannaceutically acceptable carrier.

7. A method of treating a subject suffering from, or at risk for developing, a condition which would be alleviated by promoting regeneration of a tissue or cells that rely upon occRe cells for repair which comprise administering to the subject the polypeptide of any one of claims 1-4 in an amount effective to promote regeneration of the tissue or a therapeutic or prophylactic dose of the pharmaceutical composition of Claim 6.

8. A method of claim 7, wherein the condition is myocardial infarction and the tissue is a cardiac tissue / myocardium.

9. The method of claim 8, wherein the polypeptide is administered within 5 hours of the myocardial infarction.

10. A method of claim 7, wherein the condition is a fracture and the tissue is a bone.

11. A method of claim 7, wherein the condition involves liver damage and the tissue is liver tissue.

12. A method of claim 7, wherein the condition involves damage to the brain or nervous system and includes stroke, Parkinson's disease and dementia.

13. A method of claim 7, wherein the condition involves damage to the lung.

14. A method of claim 7, wherein the condition involves the gut and includes surgery and inflammatory bowel disease.

15. A method of claim 7, wherein the condition involves damage to the skin and includes surgical procedures, bums and ulcers.

16. A method of claim 7, wherein the condition involves the pancreas including type 1 diabetes and the cells are islet cells.

17. A method of claim 7, wherein the condition is neutropenia for example following chemotherapy and the tissue is bone marrow.

18. A method of claim 7, wherein the condition is kidney failure and the tissue is kidney tissue.