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US-12624074-B2 - Polypeptides related to HMGB1 useful for promoting tissue regeneration, compositions comprising same, and uses thereof

US12624074B2US 12624074 B2US12624074 B2US 12624074B2US-12624074-B2

Abstract

This invention provides polypeptides represented by the following formula: H 2 N-A-X-B-A-X-B-HOOC which are based on HMGB1 as well as compositions comprising, and treatment methods using, such polypeptide.

Inventors

  • Jagdeep Nanchahal
  • Alvaro Vinals Guitart
  • Wyatt Yue
  • Nicola Burgess-Brown
  • Tzung Yuan Lee
  • Ana Isabel Espirito Santo

Assignees

  • OXFORD UNIVERSITY INNOVATION LIMITED

Dates

Publication Date
20260512
Application Date
20220512

Claims (20)

  1. 1 . A polypeptide represented by the following formula: H 2 N-A-X-B-A-X-B-HOOC wherein each A represents consecutive amino acids, the sequence of which (1) is a sequence of four amino acids (a) identical to the sequence of amino acids 90-93 of wild type human HMGB1 (SEQ ID NO: 1), or (b) which differs from the sequence of (a) in respect to one or more amino acids; and (2) has at its amino terminal end, between one and six consecutive amino acids, the sequence of which (a) is identical to the sequence of the corresponding one to six amino acids preceding amino acid 90 in wild type human HMGB1 (SEQ ID NO: 1), or (b) differs from the sequence of (a) in respect to one or more amino acids; and (3) optionally has a methionine at the amino terminus, wherein each A may be the same or different; wherein each X represents consecutive amino acids, the sequence of which is identical to the sequence of amino acids 94-162 of wild type human HMGB1 (SEQ ID NO: 1); wherein each B represents consecutive amino acids, the sequence of which (1) is a sequence of five or six amino acids (a) identical to the sequence of amino acids 163-168 of wild type human HMGB1 (SEQ ID NO: 1), (b) identical to the sequence of amino acids 163-167 of wild type human HMGB1 (SEQ ID NO: 1), (c) the sequence of (a) in which any one of amino acids 163, 167, or 168 is changed to any other amino acid; (d) the sequence of (b) in which any one of amino acids 163 or 167 is changed to any other amino acid; (e) the sequence of (a) or (b) in which amino acid 164 is changed from lysine to arginine; or (f) the sequence of (a) or (b) in which amino acid 165 is changed from glycine to alanine, serine or threonine; (g) the sequence of (a) or (b) in which amino acid 166 is changed from lysine to arginine; or (h) the sequence of (a) or (b) in combination with the changes in (e) and (f), (e) and (g), (f) and (g), or (e), (f) and (g); (i) the sequence of (a), (b), or (c) in combination with the changes in one or more of (e), (f), and (g); or (j) the sequence of (d) in combination with the changes in one or more of (e), (f), and (g); and (2) has at its carboxy terminal end, between one and six consecutive amino acids, the sequence of which (a) is identical to the sequence of the corresponding one to six amino acids following amino acid 168 in wild type human HMGB1 (SEQ ID NO: 1), (b) is identical to the sequence of the corresponding one to six amino acids following amino acid 167 in wild type human HMGB1 (SEQ ID NO: 1), (c) differs at one or more positions from the sequence of the corresponding one to six amino acids following amino acid 168 in wild type human HMGB1 (SEQ ID NO: 1), or (d) differs at one or more positions from the sequence of the corresponding one to six amino acids following amino acid 167 in wild type human HMGB1 (SEQ ID NO: 1), and wherein each B may be the same or different; and wherein each - represents a peptide bond between each of A and X, X and B, B and A, A and X, and X and B; with the proviso that in the B-A between the two Xs, the number of amino acids must be at least 12; and with the additional proviso that in the B at the carboxy terminal end of the polypeptide, the one to six consecutive amino acids of (2) may be absent.
  2. 2 . The polypeptide of claim 1 , wherein each A represents consecutive amino acids, the sequence of which (1) is a sequence of four amino acids (a) identical to the sequence of amino acids 90-93 of wild type human HMGB1 (SEQ ID NO: 1); (2) has at its amino terminal end, between one and six consecutive amino acids; and (3) optionally has a methionine at the amino terminus.
  3. 3 . The polypeptide of claim 1 , wherein each B represents consecutive amino acids, the sequence of which (1) is a sequence of six amino acids identical to the sequence of amino acids 163-168 of wild type human HMGB1 (SEQ ID NO: 1), and (2) has at its carboxy terminal end, between one and six consecutive amino acids.
  4. 4 . The polypeptide of claim 1 , 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 HMGB1 (SEQ ID NO: 1), (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 (SEQ ID NO: 1), and optionally (3) has a methionine at the amino terminus; and 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 (SEQ ID NO: 1) 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 HMGB1 (SEQ ID NO: 1).
  5. 5 . The polypeptide of claim 1 , wherein a methionine is present at the amino terminus of the polypeptide.
  6. 6 . The polypeptide of claim 1 , wherein A has at its amino terminal end one amino acid corresponding to amino acid 89 of wild type human HMGB1 (SEQ ID NO: 1).
  7. 7 . The polypeptide of claim 1 , wherein B has at its carboxy terminal end six amino acids corresponding to amino acids 169-174 of wild type human HMGB1 (SEQ ID NO: 1).
  8. 8 . The polypeptide of claim 1 , wherein the number of amino acids in the B-A between the two Xs is between 12-22 inclusive.
  9. 9 . The polypeptide of claim 1 , wherein the sequence of (2) (c) or (2) (d) of either B or of both Bs differs at the one or more positions by the presence of a glycine, serine, proline, arginine, lysine, aspartic acid, glutamic acid, or histidine amino acid residue.
  10. 10 . The polypeptide of claim 1 , wherein either A or both As are five consecutive amino acids.
  11. 11 . The polypeptide of claim 1 , wherein the A between the Xs is five consecutive amino acids and the B between the Xs is at least seven consecutive amino acids.
  12. 12 . The polypeptide of claim 1 , wherein the B between the Xs is twelve consecutive amino acids and the A between the Xs is at least five consecutive amino acids.
  13. 13 . The polypeptide of claim 1 , wherein the one to six consecutive amino acids of the B at the carboxy terminal end of the polypeptide are absent.
  14. 14 . The polypeptide of claim 1 , wherein each A represents consecutive amino acids, the sequence of which is KDPNA.
  15. 15 . The polypeptide of claim 1 , wherein each B represents consecutive amino acids, the sequence of which is AKGKPDAAKKGV.
  16. 16 . The polypeptide of claim 1 , the sequence of which is SEQ ID NO: 2.
  17. 17 . The polypeptide of claim 1 , the sequence of which is a methionine followed by SEQ ID NO: 2.
  18. 18 . A method of treating a subject suffering from, or at risk for developing, a condition which would be alleviated by promoting regeneration of a tissue or cells that rely upon CXCR4+ cells for repair which comprises administering to the subject the polypeptide of claim 16 in an amount effective to promote regeneration of the tissue or cells.
  19. 19 . The method of claim 18 , wherein the condition is a myocardial infarction.
  20. 20 . The method of claim 19 , wherein the polypeptide is administered within 5 hours following the myocardial infarction.

Description

This application is (a) a continuation-in-part of PCT International Application No. PCT/IB2020/060674, filed Nov. 12, 2020, which claims the benefit of U.S. Provisional Application No. 62/934,299, filed Nov. 12, 2019; and (b) claims the benefit of U.S. Provisional Application No. 63/190,429, filed May 19, 2021, the entire contents of each of which are hereby incorporated by reference into the subject application. Throughout this application, various publications are referenced, each such reference in its entirety is hereby incorporated by reference into this application. REFERENCE TO SEQUENCE LISTING This application also incorporates-by-reference each of the nucleotide sequences which are present in the text file named “220512_91203-B_Sequence_Listing_AWG.txt”, which is 78 kilobytes in size, and which was created on May 11, 2022 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is being filed as part of this application. TECHNICAL FIELD This invention concerns polypeptides related to HMGB1 that promote tissue regeneration without inducing deleterious inflammation and methods of treating acute and chronic conditions involving tissue injury by administering such a polypeptide to a subject in need of treatment for such a condition. BACKGROUND OF THE INVENTION Resident stem and progenitor cells play a key role in maintaining homeostasis and effecting repair of many tissues following injury [1]. However, most tissues in adults heal by scarring. Following the success of bone marrow transplantation [2] there has been considerable interest in exogenous stem cell therapies to promote regeneration of solid organs. However, this has met with limited success, except in a few organs such as the eye [3] and skin [4]. The inflammatory environment following tissue injury is not conducive to stem cell engraftment and the subsequent scarring disrupts the stem cell niche [5]. Therefore, focus has shifted to promoting tissue regeneration by stimulating endogenous repair mechanisms [6]. Development of a successful therapeutic would depend on identification of soluble mediators to promote these pathways [7]. We previously identified High Mobility Group Box 1 (HMGB1) as a key mediator of repair in multiple tissues, including bone, blood and skeletal muscle [8]. HMGB1 is a prototypical alarmin [9,10] and under physiological conditions has an essential role in transcription [11,12]. On cell injury it is passively released from the damaged and necrotic cells into the extracellular space and the circulation to act on endogenous stem and progenitor cells to transition them to Gaien [8], a state intermediate between G0 and G1 [13]. On exposure to the appropriate activating factors, cells in GAlert can rapidly enter G1 and effect tissue repair. If not required, stem cells in GAlert revert back to G0 after approximately 3 weeks [13], thereby ensuring that they are not exhausted and the niche is not depleted. HMBG1 comprises two L-shaped Box domains, A and B, each containing three α-helices (I-III) connected by flexible regions (FIG. 1A). The C-terminus of the protein is intrinsically disordered and contains a high proportion of carboxylic acid residues (Glu/Asp) comprising the acidic tail. The oxidation status of HMGB1 cysteine residues (Cys 22, Cys 44 in Box A and Cys 105 in Box B) is a key determinant of the extracellular activities of HMGB1 and in turn is dependent on the mechanism of release. Three different redox forms have been described in vivo [14]. HMGB1 passively released from the nuclei following injury or cell necrosis is the fully-reduced form (FR-HMGB1). It binds to CXCL12 and the heterocomplex signals via the cell surface receptor CXCR4 to transition stem and progenitor cells to Gaien [8]. Partial oxidation in the local inflammatory environment results in the formation of the disulfide HMGB1 (DS-HMGB1) [15,16], which has a disulfide bond between Cys 22 and Cys 44. This is also the form that is actively secreted by immune cells following acetylation [17] and N-glycosylation [18]. TLR-4 signaling by DS-HMGB1 results in production of several proinflammatory cytokines, including TNF [19], whilst TLR-2 signaling has been shown to be detrimental in multiple processes, including thrombosis and reperfusion injury [20], and autoimmune disorders [21]. DS-HMGB1 signaling via RAGE plays a key role in platelet activation and NET formation by neutrophils to promote thrombus formation [20,22-24]. Intracellular signaling via all three receptors converges to induce NF-κβ activity [25] in a MyD88-dependent manner [26,27]. Oxidation of all three cysteine residues through the action of extracellular reactive oxygen species results in sulfonyl-HMGB1 (SO3), which is biologically inactive [14,19]. The disulfide bridge in Box A of DS-HMGB1 (Cys22-Cys44) is essential for TLR-4 signaling (FIG. 1B and FIG. 1C), initiating binding to TLR-4 but also has a relatively high dissociation rate. MD-2 then binds t