Enhancement of wound healing by anti-ceramide antibodies

Abstract

The present disclosure provides compositions and methods for improving or accelerating wound healing. In various embodiments, the methods include the use of anti-ceramide antibodies and antibody fragments. In some embodiments, the wound is a chronic wound. In some embodiments, the wound is a diabetic wound.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 63 / 161,758, filed March 16, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] Description of sequence listing The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. A computer-readable copy of the sequence listing: Filename: CERA_020_00US_SeqList_ST25.txt, Recorded on: March 16, 2021, File size approximately 31.7 kilobytes.

[0003] The present disclosure relates to anti-ceramide compositions and methods of use thereof for treating or preventing wounds or enhancing wound healing, eg, diabetic wound healing. [Background technology]

[0004] Diabetes affects 340 million people worldwide, including 29.1 million in the U.S. A complication in diabetic patients is the inability of wounds to heal, which resulted in 73,000 lower-limb amputations in the U.S. in 2010.

[0005] In diabetic patients, hyperglycemia triggers chronic inflammation, poor circulation, and neuropathy over time. The combination of these factors slows or even stops the wound healing process. Despite extensive research in this field, the mechanisms underlying impaired healing of diabetic wounds are multifactorial and remain poorly understood.

[0006] Standard treatments for diabetic wounds include debridement of the wound, treatment of infection with antibiotics, and reduction or elimination of weight bearing from the lower extremities. However, there remains an unmet need for safe and effective treatments to accelerate wound healing, particularly for diabetic wounds. [Brief description of the drawings]

[0007] [Figure 1A] Representative photographs of wound healing over time are shown, with each row showing images of a particular lesion site in the indicated experimental group. [Figure 1B] Representative photographs of wound healing over time are shown, with each row showing images of a particular lesion site in the indicated experimental group. [Diagram 2] The average rate of wound healing in each experimental group over time is shown. The Y-axis shows wound size, normalized to the initial size of each wound as 1. The X-axis shows days. "D+A2A" is the diabetic+2A2 group and "C+A2A" is the control+2A2 group. [Figure 3A] 3A-3D are bar graphs showing the differences between treatments. Figure 3A is a bar graph showing wound size after 10 days, expressed as % normalized to initial wound size. [Figure 3B] Figure 3B shows a bar graph depicting the difference between treatments.Figure 3B shows a bar graph depicting the average number of days it took for mice to achieve 95% healing in each experimental group. [Figure 4] The average rate of wound healing in each experimental group over time is shown. The Y-axis shows wound size, normalized to the initial size of each wound as 1. The X-axis shows days. [Figure 5A] 1 is a bar graph showing the mean number of days it took for mice to achieve 25% healing for each experimental group, respectively. [Figure 5B] 1 is a bar graph showing the mean number of days it took for mice to achieve 50% healing for each experimental group, respectively. [Figure 5C] 1 is a bar graph showing the mean number of days it took for mice to achieve 75% healing for each experimental group, respectively. [Figure 5D] 1 is a bar graph showing the mean number of days it took for mice to achieve 90% healing for each experimental group, respectively. Summary of the Invention

[0008] In one aspect, the disclosure provides a method of treating or preventing a wound in a subject in need thereof, comprising administering to the subject an anti-ceramide antibody, or antigen-binding fragment thereof.

[0009] In one aspect, the disclosure provides a method of enhancing wound healing in a subject in need thereof, comprising administering to the subject an anti-ceramide antibody, or antigen-binding fragment thereof.

[0010] In some embodiments, the wound is a chronic wound or a diabetic wound. In some embodiments, the wound is a chronic wound. In some embodiments, the wound is a diabetic wound.

[0011] In some embodiments, the route of administration is selected from the group consisting of topical administration, intralesional administration, subcutaneous administration, transdermal administration, intramuscular administration, intravenous administration, and parenteral administration. In some embodiments, the route of administration is topical administration. In some embodiments, the route of administration is intravenous administration.

[0012] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses. In some embodiments, administration of successive doses is separated by at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 1 week. In some embodiments, the duration of administration is at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks.

[0013] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the inflammatory phase of wound healing. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative phase of wound healing. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the remodeling phase of wound healing.

[0014] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is an antibody. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single chain variable fragment (scFv).

[0015] In some embodiments, the method enhances wound healing by at least 10%, at least 20%, at least 30%, at least 50%, or at least 70% compared to a control wound. In some embodiments, enhanced wound healing is measured by mortality of the total surface area of ​​the wound at 10 days, 20 days, or 30 days after wounding. In some embodiments, enhanced wound healing is measured at 10 days after wounding. In some embodiments, enhanced wound healing is measured by mortality of the time it takes to achieve a 50%, 70%, 90%, 95%, or 100% reduction in the total surface area of ​​the wound. In some embodiments, enhanced wound healing is measured at a 95% reduction in the total surface area of ​​the wound.

[0016] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered prior to the onset of one or more symptoms of the wound, hi some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of the wound.

[0017] In some embodiments, the anti-ceramide antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1), an HCDR2 comprising the amino acid sequence of YNYPRDGSTKYNEKFKG (SEQ ID NO: 2), and an HCDR3 comprising the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3), L In some embodiments, V comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 comprising the amino acid sequence of SGSTLQS (SEQ ID NO: 5), and an LCDR3 comprising the amino acid sequence of QQHNEYPWT (SEQ ID NO: 6). H comprises the amino acid sequence of SEQ ID NO:7,L comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 6B5 antibody. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is 6B5scFv.

[0018] In some embodiments, the anti-ceramide antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of NYWMH (SEQ ID NO: 33), an HCDR2 comprising the amino acid sequence of AIYPGDSDTSYNQKFKG (SEQ ID NO: 34), and an HCDR3 comprising the amino acid sequence of LYYGYD (SEQ ID NO: 35); L In some embodiments, V comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of KSSQSLIDSDGKTFLN (SEQ ID NO: 36), an LCDR2 comprising the amino acid sequence of LVSKLDS (SEQ ID NO: 37), and an LCDR3 comprising the amino acid sequence of WQGTHFPYT (SEQ ID NO: 38). H comprises the amino acid sequence of SEQ ID NO: 39, L comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 2A2 antibody. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 2A2scFv.

[0019] In some embodiments, the anti-ceramide antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 and 43, an HCDR2 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 44 to 47, and an HCDR3 comprising or consisting of the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3), Lcomprises a light chain complementarity determining region 1 (LCDR1) that comprises or consists of the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 that comprises or consists of the amino acid sequence of SGSTLQS (SEQ ID NO: 5), and an LCDR3 that comprises or consists of the amino acid sequence of QQHNEYPWT (SEQ ID NO: 6). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYNEKFQG (SEQ ID NO: 44). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPREGSTKYNEKFQG (SEQ ID NO: 45). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDVSTKYNEKFQG (SEQ ID NO: 46). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYAEKFQG (SEQ ID NO: 47). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYNEKFQG (SEQ ID NO: 44). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPREGSTKYNEKFQG (SEQ ID NO: 45). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDVSTKYNEKFQG (SEQ ID NO: 46).In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYAEKFQG (SEQ ID NO: 47). In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48 and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48 and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:54.In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:55. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:53. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:54. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:55. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:53. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:54.In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:55. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:52, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:53. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:52, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:54. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:52, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:55.

[0020] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a humanized 6B5 (h6B5) antibody. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is h6B5scFv.

[0021] In some embodiments, the anti-ceramide antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 48, L comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the anti-ceramide antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (V H) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 48, L comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 49, L comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the anti-ceramide antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 49, L comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the anti-ceramide antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO:50, L comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the anti-ceramide antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO:50, L comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the anti-ceramide antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO:51, L comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the anti-ceramide antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO:52, Lcomprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a humanized antibody. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a humanized scFv. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] overview The present disclosure relates to compositions and methods for enhancing wound healing. In some embodiments, compositions of anti-ceramide antibodies and antigen-binding fragments thereof (e.g., scFvs) and methods of use in treating or preventing wounds are provided. In some embodiments, the wound is a chronic wound. In some embodiments, the wound is a diabetic wound. Such compositions and methods may be used to treat diabetic wounds in patients who have previously failed another treatment for the diabetic wound.

[0023] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0024] As used herein, the term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise specified.

[0025] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations of "comprises" or "comprising" will be understood to imply the inclusion of a stated element or integer, or group of elements or integers, but not the exclusion of any other element or integer, or group of elements or integers.

[0026] As used in this application, the terms "about" and "approximately" are used as equivalent terms. Any numbers used in this application, whether about / approximately or not, are meant to cover any normal variations understood by those of ordinary skill in the relevant art. In certain embodiments, the term "about" or "approximately" refers to a range of values ​​that falls within 10% or less in either direction of the stated reference value (greater or less), unless otherwise stated or clear from the context (except when such numbers are greater than 100% of the possible values ​​or less than 0% of the possible values).

[0027] The term "sample" refers to a biological composition (e.g., a portion of a cell or tissue) that is subjected to analysis and / or modification. In some embodiments, a sample is a "primary sample" in that it is obtained directly from a subject, and in some embodiments, a "sample" is the result of processing of a primary sample, e.g., to remove particular components and / or to isolate or purify a particular component of interest.

[0028] The term "subject" includes animals, such as, for example, mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, the subject is a livestock animal, such as a cattle, sheep, goat, cow, pig, etc., or a farm animal, such as a dog and a cat. In some embodiments (e.g., particularly in the context of research), the subject is a rodent (e.g., mouse, rat, hamster), rabbit, primate, or pig, such as an inbred pig. As used herein, the terms subject and patient are used interchangeably. In some embodiments, the subject may be a neonate, juvenile, or adult. Mammalian subjects are of particular interest. Mammalian species that may be treated with the present methods include dogs and cats, horses, cows, sheep, etc., as well as primates, particularly humans. Animal models, particularly small mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, etc.), may be used for experimental studies.

[0029] As used herein, the terms "treatment," "treating," or "palliating" refer to either therapeutic or preventative / prophylactic treatment. Treatment is therapeutic if at least one symptom of a disease in the individual receiving the treatment is improved, or the treatment can slow the progression of a progressive disease in the individual, or prevent the onset of additional related diseases.

[0030] As used herein, the term "effective amount" refers to the amount of an agent or composition required to produce a particular physiological effect. The effective amount of a particular agent can be expressed in a variety of ways based on the nature of the agent, such as mass / volume, number of cells / volume, particles / volume, (mass of agent) / (mass of subject), number of cells / (mass of subject), or particles / (mass of subject). The effective amount of a particular agent is also referred to as the median effective concentration (EC 50 ), which refers to the concentration of an agent that produces a particular physiological response magnitude that is midway between a reference level and a maximum response level.

[0031] The term "antibody" refers to an immunoglobulin (Ig) molecule capable of binding to a specific target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one epitope recognition site located in the variable region of the Ig molecule. As used herein, the term encompasses intact polyclonal or monoclonal antibodies, and antigen-binding fragments thereof. For example, a natural immunoglobulin molecule is composed of two heavy chain polypeptides and two light chain polypeptides. Each of the heavy chain polypeptides associates with a light chain polypeptide through an interchain disulfide bond between the heavy and light chain polypeptides to form two heterodimeric proteins or polypeptides (i.e., a protein composed of two heterologous polypeptide chains). The two heterodimeric proteins then associate through an additional interchain disulfide bond between the heavy chain polypeptides to form an immunoglobulin protein or polypeptide.

[0032] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment that contains at least one complementarity determining region (CDR) of an immunoglobulin heavy and / or light chain that binds to at least one epitope of an antigen of interest. In this regard, an antigen-binding fragment of an antibody described herein may contain one, two, three, four, five, or all six CDRs of the variable heavy (VH) and variable light (VL) chain sequences from an antibody that specifically binds to ceramide. Antigen-binding fragments include proteins that generally contain the antigen-binding or variable regions thereof, such as portions of a full-length antibody, Fab, F(ab')2, Fab', Fv fragments, minibodies, diabodies, single domain antibodies (dAbs), single chain variable fragments (scFv), multispecific antibodies formed from antibody fragments, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site or fragment of the required specificity. In certain embodiments of the present disclosure, antigen-binding fragments are used rather than intact antibodies to increase tissue or tumor penetration. In other embodiments, the antigen-binding fragments are further modified to increase serum half-life.

[0033] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from a portion of an antibody that can bind to Fc receptors on cells and / or the C1q component of complement, thereby mediating the antibody's effector functions. Fc stands for "crystallizable fragment," a fragment of an antibody that readily forms protein crystals. The different protein fragments, first described by proteolytic digestion, can define the overall general structure of immunoglobulin proteins. As originally defined in the literature, the Fc region is a homodimeric protein comprising two polypeptides associated by disulfide bonds, each comprising a hinge region, a CH2 domain, and a CH3 domain. However, more recently, the term has been applied to a single-chain monomeric component consisting of a CH3, a CH2, and at least a portion of a hinge sufficient to form a disulfide-linked dimer with a second such chain. Thus, depending on the context, the use of the term "Fc region" or "Fc domain" herein refers to either the dimeric form or the individual monomers that associate to form the dimeric protein. For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140, and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, the term Fc domain includes variants of naturally occurring sequences.

[0034] The term "immunoglobulin constant region" or "constant region" refers to a peptide or polypeptide sequence that corresponds to or is derived from part or all of one or more constant domains of an immunoglobulin (e.g., CH1, CH2, CH3). In certain embodiments, the constant region does not include a CH1 domain. In certain embodiments, the constant domains that make up the constant region are human.

[0035] The terms "light chain variable region" (also referred to as "light chain variable domain" or "VL") and "heavy chain variable region" (also referred to as "heavy chain variable domain" or "VH") refer to the variable binding regions from antibody light and heavy chains, respectively. The variable binding regions are composed of distinct and clearly defined subregions known as "complementarity determining regions" (CDRs) and "framework regions" (FRs).

[0036] The term "immunoglobulin light chain constant region" (also referred to as "light chain constant region" or "CL") is the constant region from an antibody light chain.

[0037] The term "immunoglobulin heavy chain constant region" (also referred to as "heavy chain constant region" or "CH") refers to the constant region from an antibody heavy chain. The CH can be further divided depending on the antibody isotype into CH1, CH2, and CH3 (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4 domains (IgE, IgM).

[0038] The term "F(ab)" refers to two of the protein fragments resulting from proteolytic cleavage of an IgG molecule by the enzyme papain. Each F(ab) contains a covalent heterodimer of a VH and a VL chain and contains an intact antigen-binding site. Each F(ab) is a monovalent antigen-binding fragment. The term "Fab" refers to a fragment derived from F(ab')2 and may contain a small portion of Fc. Each Fab' fragment is a monovalent antigen-binding fragment.

[0039] The term "F(ab')2" refers to the protein fragment of IgG generated by proteolytic cleavage with the enzyme pepsin. Each F(ab')2 fragment contains two F(ab') fragments and is therefore a bivalent antigen-binding fragment.

[0040] An "Fd fragment" contains the VH and CH1 domains.

[0041] "Fv fragment" refers to a non-covalent VH::VL heterodimer containing an antigen-binding site that retains much of the antigen recognition and binding ability of a native antibody molecule, but lacks the CH1 and CL domains contained within the Fab. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662, Hochman et al. (1976) Biochem 15:2706-2710, and Ehrlich et al. (1980) Biochem 19:4091-4096. In some embodiments, Fv fragments may be produced by preferential proteolytic cleavage of IgM, and rarely IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art.

[0042] A "dAb fragment" (Ward et al., Nature 341:544 546, 1989) contains a VH domain.

[0043] A "single-chain antibody" or "scFv" is a fusion protein of an immunoglobulin heavy chain variable region (VH) and a light chain variable region (VL), linked by a short linker peptide of 10 to about 25 amino acids. The linker may be rich in glycine for flexibility, serine or threonine for solubility, and may connect the N-terminus of VH to the C-terminus of VL, or vice versa. scFvs retain the specificity of the original immunoglobulin despite the removal of the constant regions and introduction of the linker. In this disclosure, any reference to an antibody or antibody fragment or uses thereof is intended to include scFv molecules and uses thereof.

[0044] The term "minibody" refers to a fusion protein comprising an scFv linked to a CH3 domain, and is included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, for example, Ward, ES et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0045] The term "diabody" refers to bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain using a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing these domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and Poljak et al., Structure 2:1121-23 (1994)).

[0046] The term "nanobody" or "single domain antibody" refers to an antigen-binding fragment consisting of a single monomeric variable antibody domain. The Nanoclone method is a method for generating nanobodies against a desired target based on automated high-throughput selection of B cells. (See WO2006 / 079372)

[0047] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0048] The term "chimeric antibody" as used herein refers to monoclonal antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0049] The term "single chain variable fragment" or "scFv" refers to a fusion protein of immunoglobulin heavy chain variable region (VH) and light chain variable region (VL), connected by a short linker peptide of 10 to about 25 amino acids. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. The linker may connect the N-terminus of VH to the C-terminus of VL, or vice versa. Many methods have been described for identifying chemical structures for converting naturally aggregated, but chemically separated, light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of the antigen binding site. See, for example, U.S. Pat. Nos. 5,091,513 and 5,132,405 to Huston et al., and U.S. Pat. No. 4,946,778 to Ladner et al.

[0050] As used herein, the term "CDR" refers to the "complementarity determining region" of an immunoglobulin (antibody) molecule. A CDR is that part of the variable domain in an antibody that binds to its specific antigen. There are three CDRs per variable domain (i.e., CDR1, CDR2, and CDR3 in the variable domain of the light chain, and CDR1, CDR2, and CDR3 in the variable domain of the heavy chain). Within the variable domain, CDR1 and CDR2 are found in the variable (V) region of the polypeptide chain, and CDR3 shows the greatest variability since it is encoded by VJ in the case of the light chain region and VDJ in the case of the heavy chain region.

[0051] An "isolated antibody" is an antibody that is: (1) not associated with naturally associated components, including other naturally associated antibodies, that accompany it in its natural state; (2) free of other proteins from the same species; (3) expressed by cells from a different species; or (4) not naturally occurring.

[0052] The term "human antibody" includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In preferred embodiments, all of the variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies). These antibodies can be prepared in a variety of ways, as described below.

[0053] As used herein, the term "humanized" refers to an antibody or antigen-binding fragment thereof derived from a non-human species that retains the antigen-binding properties of the original non-human antibody. In some embodiments, the binding fragments of the antibody (e.g., light and heavy chain variable regions, Fab, scFv) are humanized. Non-human antigen-binding fragments can be synthesized using a technique known as CDR grafting (Jones et al., Nature 321:522 (1986)), as well as "reshaping" (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio / Technol 1991 9:266-271) and "hyperchimerization" (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J. Immunol. 2002; 2003; 2004). Immunol 148:1149-1154), and variations thereof including "veneering" (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994:291-312). Other regions of the antibody, such as the hinge region and constant region domains, can also be humanized if derived from a non-human source.

[0054] As used herein, the term "pharmacologically acceptable" refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions that are approved by a federal or state government regulatory agency or are listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans, are considered to be "pharmacologically acceptable."

[0055] The terms "prevent," "prevention," and "prophylactically" refer to administration of a compound, e.g., an anti-ceramide antibody or antigen-binding fragment thereof, prior to the onset of a disease (e.g., prior to the onset of a particular symptom of a disease). Preventing a disease can include reducing the likelihood of a disease occurring, delaying the onset of a disease, alleviating long-term symptoms, or delaying the ultimate progression of a disease.

[0056] As used herein, the term "specifically binds" refers to a binding that is at least 10 times stronger than that of an antigen, while not significantly binding to other components or antigens present in a mixture. 5 M -1 It refers to the ability of an antibody or antigen-binding fragment thereof to bind to a target antigen with a binding affinity (Ka) of at least about 1. Reference herein to an anti-ceramide antibody refers to an antibody or antigen-binding fragment thereof that specifically binds to ceramide.

[0057] As used herein, the term "sequence identity" refers to the relationship between two or more polynucleotide sequences or two or more polypeptide sequences. If a position in one sequence is occupied by the same nucleic acid base or amino acid residue at the corresponding position of the comparison sequence, the sequences are said to be "identical" at that position. The percentage of sequence identity is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of identical positions. The number of identical positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage of sequence identity. The percentage of sequence identity is determined by comparing two optimally aligned sequences over the comparison window. A comparison window for polynucleotide sequences can be, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more nucleic acids in length. A comparison window for polypeptide sequences can be, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more nucleic acids in length. To optimally align sequences for comparison, portions of the polynucleotide or polypeptide sequences in the comparison window may contain additions or deletions, referred to as gaps, while the reference sequence is held constant. An optimal alignment is one that produces the maximum number of "identical" positions possible between the reference and comparison sequences, even with gaps.The percentage of "sequence identity" between two sequences can be determined using the version of the program "BLAST2Sequences" available from the National Center for Biotechnology Information as of September 1, 2004, which incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison) and is based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When using "BLAST2Sequences", the parameters that were the default parameters as of September 1, 2004 can be used for any other required parameters, including, but not limited to, word size (3), open gap penalty (11), extension gap penalty (1), gap dropoff (50), expectation value (10), and matrix options. Two nucleotide or amino acid sequences are considered to have "substantially similar sequence identity" or "substantial sequence identity" if the two nucleotide or amino acid sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to each other.

[0058] "Angiogenesis" refers to the formation of new blood vessels.

[0059] As used herein, the term "wound" refers to injury to tissue, including, but not limited to, acute, subacute, delayed or difficult to heal wounds, and chronic wounds. Injuries may be due to trauma, violence, accidents, surgery, disease. Wounds occur due to tears or breaks in membranes (such as the skin), which can usually cause damage to the underlying tissue. Wounds may be caused by pressure injuries from extended bed rest. Chronic wounds may be caused by diseases, including but not limited to diabetes, diseases of internal organs, including but not limited to liver, kidney, or lung disease, cancer, or any other condition that slows the healing process. In some embodiments, wounds may be caused by a combination of factors described in this paragraph. Wounds may occur in a localized location or internally. Wounds may be open or closed wounds. Wounds include, for example, diabetic wounds or ulcers, burns, incisions, excisions, lacerations, abrasions, puncture or penetration wounds, surgical wounds, contusions, hematomas, crush injuries, and ulcers.

[0060] The term "diabetic wound" refers to a wound that occurs in a diabetic subject that is at least partially caused by a diabetic condition, e.g., type I or type II diabetes. Diabetic wounds often occur on the lower extremities (e.g., diabetic foot wounds).

[0061] The terms "enhanced wound healing" or "accelerated wound healing" are used interchangeably and refer to an improvement in the wound healing process in a treatment group compared to a control group.

[0062] Anti-ceramide antibodies, antibody fragments, and derivatives The present disclosure relates to anti-ceramide antibodies and antigen-binding fragments thereof for enhancing wound healing or for treating or preventing wounds. In some embodiments, the wound is a chronic wound. In some embodiments, the wound is a diabetic wound.

[0063] Ceramides are a family of waxy lipid molecules. They are composed of sphingosine and fatty acids. Ceramides are found in high concentrations in the plasma membrane of eukaryotic cells, as they are constituent lipids that make up sphingomyelin, one of the major lipids in the lipid bilayer. Ceramides are involved in a variety of cell signaling pathways, including regulating cell differentiation, proliferation, and programmed cell death (PCD). As a bioactive lipid, ceramides are involved in diverse physiological functions, including apoptosis, cell growth arrest, differentiation, cell senescence, cell migration, and adhesion. Roles of ceramides and their downstream metabolites have also been suggested in several pathological conditions, including cancer, neurodegeneration, diabetes, microbial pathologies, obesity, and inflammation.

[0064] Exemplary anti-ceramide antibody sequences and properties are also disclosed in U.S. Patent Publication Nos. 2010 / 0239572 and 2017 / 0335014, each of which is incorporated herein by reference. Exemplary anti-ceramide antibody sequences are provided in Table 1. However, any anti-ceramide antibody or antigen-binding fragment thereof may be used in accordance with the disclosed methods and uses. [Table 1] TIFF2024511079000003.tif233170 TIFF2024511079000004.tif192170

[0065] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is humanized 6B5 (h6B5). In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In some embodiments, the sequence of the h6B5 antibody is provided in U.S. Provisional Application No. 62 / 991,232, filed March 18, 2020, the contents of which are incorporated herein by reference in their entirety for all purposes. The sequences of each CDR of the h6B5 antibody or antigen-binding fragment thereof are disclosed throughout the present specification and summarized in Table 2 below. [Table 2]

[0066] In some embodiments, the anti-ceramide antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a recombinant antibody, or a synthetic antibody. In some embodiments, the anti-ceramide antigen-binding antibody fragment is an antigen-binding fragment of any one of the foregoing. In some embodiments, the anti-ceramide antigen-binding antibody fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, an Fd fragment, a dAb fragment, a diabody, an scFv, or the like. In some embodiments, the anti-ceramide antibody and antigen-binding fragments thereof are produced using recombinant DNA technology. Procedures for expression and purification of recombinant proteins are well established in the art.

[0067] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single chain variable fragment (scFv). In some embodiments, the scFv comprises the CDR sequences and / or variable chain sequences of the 2A2, h2A2, 6C8, 7B10, 9H10, h6B5, or 6B5 antibody.

[0068] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a 2A2 antibody or antigen-binding fragment thereof, as described in U.S. Patent Publication No. 2010 / 0239572. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a 6B5 antibody or antigen-binding fragment thereof, as described in U.S. Patent Publication No. 2017 / 0335014. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is an h6B5 antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is an scFv. In some embodiments, the scFV comprises the CDR sequences of any of the antibodies disclosed in Table 1. In some embodiments, the scFv comprises the CDR sequences of 2A2. In some embodiments, the scFv comprises the CDR sequences of 6B5. In some embodiments, the scFv comprises the CDR sequences of h6B5. In some embodiments, the scFv comprises the variable heavy and light chain sequences of h2A2. In some embodiments, the scFv comprises the variable heavy and light chain sequences of 6B5. In some embodiments, the scFv comprises the variable heavy and light chain sequences of h6B5.

[0069] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof has any immunoglobulin isotype. Immunoglobulins may be from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. The IgG isotype is divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises one or more modifications in the Fc region. Certain modifications may provide desired effector functions or serum half-life. In some embodiments, with the appropriate Fc region, naked antibodies bound to cell surfaces can induce cytotoxicity, for example, via antibody-dependent cellular cytotoxicity (ADCC), or by recruiting complement in complement-dependent cytotoxicity (CDC), or by recruiting non-specific cytotoxic cells expressing one or more effector ligands that recognize the bound antibody on the target cell and subsequently cause phagocytosis of the target cell in antibody-dependent cell-mediated phagocytosis (ADCP), or some other mechanism. Certain other Fc regions may be used when it is desirable to eliminate or reduce effector functions to minimize side effects or therapeutic complications. The Fc region of the antibody can be modified to increase binding affinity to FcRn and thus increase serum half-life. Alternatively, the Fc region can be conjugated to PEG or albumin to increase serum half-life, or some other conjugation that produces the desired effect.

[0070] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a detectable label or tag. Exemplary detectable labels include fluorescent tags, affinity tags, radioisotopes, luminescent markers, particle labels, chromophores, phosphate markers, and enzyme labels. Exemplary fluorescent labels include GFP, RFP, and YFP. Exemplary enzyme labels include horseradish peroxidase and alkaline phosphatase. Exemplary peptide tags include His tags, MBP, and streptavidin.

[0071] The means of detection will be determined by the label chosen. Appearance of the label or its reaction products can be accomplished using the naked eye, if the label is particulate and accumulates at appropriate levels, or using instruments such as spectrophotometers, luminometers, fluorometers, or by ELISA or Western blot.

[0072] wound healing In one aspect, the present disclosure provides methods and compositions for enhancing wound healing.In one aspect, the present disclosure provides methods and compositions for treating or preventing wounds.

[0073] Natural wound healing occurs in well-defined stages. Acute skin wounds can heal in 1-3 weeks in a biological process that restores the integrity and function of the skin and underlying tissue. Such wounds can be the result of scrapes, abrasions, cuts, abrasions, incisions, lacerations, or bruises to the skin.

[0074] Wounds can be classified into one of four grades depending on the depth of the wound: i) Grade I: wounds limited to the epithelium, ii) Grade II: wounds extending into the dermis, iii) Grade III: wounds extending into the subcutaneous tissue, and iv) Grade IV (or full thickness wounds): wounds that expose bone (e.g., bony pressure points such as the greater trochanter or sacrum). The term "partial thickness wound" refers to wounds that encompass grades I-III, and examples of partial thickness wounds include pressure wounds, venous ulcers, and diabetic wounds or ulcers. The term "deep wound" is meant to include both grade III and grade IV wounds.

[0075] Natural wound healing occurs primarily according to three major time sequences. Each of these sequences is characterized by specific cellular activities and controlled by numerous regulatory signals (both positive and negative) that collectively govern and support the progression of the repair process. The following are therefore distinguished: (a) Inflammatory phase; (b) the proliferative phase (including the granulation and epithelialization phases), and (c) Remodeling period.

[0076] The first phase, the inflammatory phase, begins as soon as the blood vessel ruptures, an event that leads to the formation of a thrombus (blood clot), composed mainly of fibrin and fibronectin, constituting a provisional matrix. This matrix partially fills the lesion and allows the migration of inflammatory cells within the damaged area that are recruited to ensure the clearance of the wound. Platelets also release factors (e.g. cytokines and / or growth factors) that allow the recruitment of cells involved in the healing process. This period is characterized by the infiltration and activation of a large number of inflammatory cells (polymorphonuclear cells, macrophages) at the site of the lesion, which protect the organism from any foreign microorganisms and also cleanse or wipe the wound.

[0077] The second phase corresponds to the development of granulation tissue. First, the colonization of the wound by migration and proliferation of fibroblasts is observed. Then, migration of endothelial cells from healthy blood vessels allows angiogenesis, or neovascularization, of the damaged tissue. In the granulation tissue, fibroblasts are activated and differentiate into myofibroblasts with remarkable contractile properties provided by actin microfilaments, thus allowing wound contraction. The microfilaments are expressed through the protein, alpha smooth muscle actin. These myofibroblasts play a key role in the formation and contraction of granulation tissue, which leads to the healing of the lesion. Then, keratinocytes migrate from the wound edge and then differentiate, leading to the reconstruction of the epidermis. This developmental phase of granulation tissue is initiated following a prior decrease in the general state of inflammation in the lesion, the gradual disappearance of polymorphonuclear neutrophils, and the appearance of macrophages, including "repair" macrophages. This transition from the inflammatory phase to the proliferation / repair phase is known as the resolution phase of inflammation.

[0078] The third phase of the process is the remodeling stage, which aims to reconstruct functional tissue so that the newly formed tissue takes on the initial characteristics and properties of the original tissue. Part of the extracellular matrix is ​​digested by proteases (essentially matrix metalloproteases and elastases), and a reorganization of the extracellular matrix is ​​observed. Type III collagen, which predominates in the granulation tissue, is gradually replaced by type I collagen, the main matrix component of the dermis. At the end of the maturation phase, fibroblasts, myofibroblasts, and vascular cells experience a decrease in proliferation and / or activity. Excess cells then die by apoptosis, with a concomitant remodeling of the extracellular matrix.

[0079] If the wound does not heal within a normal period of time, it is considered to be a "chronic wound".For example, a chronic wound may take longer than 2 weeks, longer than 3 weeks, longer than 4 weeks, longer than 5 weeks, longer than 6 weeks, longer than 6 weeks, longer than 7 weeks, longer than 8 weeks, longer than 9 weeks, longer than 10 weeks, longer than 11 weeks, longer than 12 weeks, longer than 3 months, longer than 4 months, longer than 5 months, or longer than 6 months to heal.In some embodiments, a chronic wound does not start to heal after 3, 4, 5, 6, 7, 8, 9, or 10 weeks starting from the appearance of the wound.

[0080] In the case of chronic wounds, the wound may be weakened in one of the stages of healing or may not progress through the normal stages of healing. Chronic wounds may include, for example, wounds that are at least partially characterized by one or more of the following: 1) a prolonged inflammatory phase; 2) delayed or defective extracellular matrix (ECM); and 3) stagnant or reduced epithelialization rate. Chronic wounds may exist for a relatively short period of time, such as one month, or may exist for several years. The compositions and methods described herein can initiate and enhance the healing of chronic wounds.

[0081] Chronic skin wounds include, but are not limited to, skin ulcers, pressure sores, diabetic wounds (e.g., diabetic ulcers and pressure sores), and other skin disorders. In some embodiments, the chronic wound is selected from diabetic wounds, venous ulcers, arterial ulcers, pressure ulcers, and vasculitic ulcers. In some embodiments, the chronic wound is a diabetic wound.

[0082] Chronic skin wounds may be of any size, shape, or depth and may appear discolored compared to normal healthy skin pigmentation. Chronic skin wounds may bleed, swell, permeate with purulent secretions or other fluids, cause pain or make movement of the affected area difficult or painful. Chronic skin wounds may become infected, develop an elevated temperature, and produce milky, yellow, green, or brown pus or secretions. The secretions may be odorless or have a pungent odor. When infected, chronic skin wounds may be red, tender, or warm to the touch.

[0083] Chronic skin wounds may be caused by conditions that reduce blood flow due to diabetes, poor blood supply, low blood oxygen, low blood pressure, or conditions characterized by blockage, blockage, or narrowing of blood vessels. Poor oxygen supply may be caused by certain blood, heart, and lung diseases, and / or smoking. Chronic skin wounds may also be the result of repeated trauma to the skin, such as increased swelling or pressure in the tissue, or constant pressure on the wound area. Chronic skin wounds may also be caused by a weakened or damaged immune system. A weakened or damaged immune system may be caused by increasing age, radiation, malnutrition, and / or drug therapy, such as anti-cancer drugs or steroids. Chronic skin wounds may also be caused by bacterial, viral, or fungal infection, or the presence of a foreign body.

[0084] Impaired wound healing after injury in diabetic subjects represents a major clinical problem, resulting in prolonged hospitalization and significant medical costs. Two-thirds of all non-traumatic amputations are preceded by diabetic wounds. Impaired healing of diabetic wounds is multifactorial and characterized by reduced production of chemokines, reduced angiogenesis, and abnormal inflammatory responses.

[0085] Without being bound to any particular theory, it is believed that the absence of healing of these diabetic wounds is at least partially associated with the increased bioavailability of glucose. This leads to a number of physiological and metabolic changes, such as skin thickening, or significant oxidative stress leading to neuropathy and arteriopathy. Arteriopathy and neuropathy are the two main risk factors for chronicity and therefore delayed healing of diabetic wounds, more specifically diabetic foot wounds. The most well-known types of chronic non-diabetic wounds, such as pressure ulcers, venous ulcers, arterial ulcers or mixed ulcers, do not originate from the same pathology. For example, venous insufficiency is responsible for the formation, chronicity and therefore delayed healing of venous ulcers. Pressure ulcers, as far as they are concerned, are wounds that arise after cycles of ischemia and reperfusion following excessive and prolonged pressure and friction on skin tissue.

[0086] Several major problems disrupt the correct wound healing sequence in diabetic subjects. The first delay in healing arises from the inflammatory phase, where the transition from the inflammatory phase to the proliferative phase is interrupted. The inflammatory phase is a period essential for healing, but it must be temporary. The resolution of inflammation is the key point that conditions the start of the other periods of healing. This dynamic event is accompanied by the disappearance of inflammatory cells (polymorphonuclear neutrophils) and the appearance of macrophages. Chemotactic and angiogenic anti-inflammatory mediators are then produced, which allow in particular the migration and differentiation of fibroblasts, the key cells of the granulation phase. As in the case of the aforementioned subjects, the interruption of this inflammatory phase causes an abnormal prolongation of the inflammatory phase, leading to chronicity of the wound, thereby delaying all subsequent stages of healing. Finally, the period of wound closure is delayed, especially during epithelialization, or even does not occur in most cases.

[0087] The role of ceramide and ASM in wound healing Without being bound to any particular theory, it is contemplated that the formation of ceramide-rich platforms (CRPs) underlies microvascular endothelial pathology that contributes to wound healing difficulties, e.g., in the case of chronic or diabetic wounds.

[0088] Sphingolipids represent major constituents of membrane microdomains, and ceramide-enriched microdomains appear to be a prerequisite for inflammatory cytokine signaling. Acid sphingomyelinase (ASM) and neutral sphingomyelinase (NSM) are key regulatory enzymes of sphingolipid metabolism, promoting sphingomyelin hydrolysis towards proinflammatory ceramide. ASM is a key early responder in inflammatory cytokine signaling.

[0089] Endothelial tissue injury causes membrane damage and acid sphingomyelinase-dependent ceramide formation in the outer cell leaflet of the plasma membrane. During the development of tissue damage after injury, CRP-dependent apoptosis likely represents a feed-forward process. In immune-mediated tissue injury, cytolytic T cells also induce CRP on target cells, which is necessary for efficient cell killing. Disruption of this process prevents further evolution of immune-mediated tissue injury and leads to the initiation of a robust and durable tissue repair process.

[0090] Administration of anti-ceramide antibodies or antigen-binding fragments thereof restores vascular endothelial homeostasis, regulating signaling between blood and tissues, transporting hematopoietic cells, maintaining non-thrombotic blood flow, promoting immune and inflammatory responses, and leading to homeostatic repair and regeneration without inducing fibrosis after injury. Furthermore, because the ceramide-rich platform is primarily present in damaged cells, systematic delivery of anti-ceramide antibodies or antigen-binding fragments thereof can act locally in damaged tissues without affecting normal tissues, potentially offering a wide therapeutic index.

[0091] Pharmaceutical compositions, routes of administration, dosages, and dosing schedules In some embodiments, the disclosure provides pharmaceutical compositions comprising an anti-ceramide antibody or antigen-binding fragment thereof for enhancing wound healing or treating or preventing wounds.

[0092] For administration, the antibodies or fragments (e.g., anti-ceramide antibodies and antigen-binding fragments thereof) of the present disclosure may be formulated as pharmaceutical compositions. The pharmaceutical compositions may include (i) an anti-ceramide antibody or antigen-binding fragment thereof, and (ii) a pharma- ceutically acceptable carrier, diluent, or excipient. Pharmaceutical compositions including anti-ceramide antibodies or antigen-binding fragments thereof, and / or scFvs may be formulated according to known methods for preparing pharma- ceutically useful compositions, whereby the therapeutic molecule is combined in a mixture with a pharma- ceutically acceptable carrier, diluent, or excipient. Suitable carriers, diluents, or excipients are well known to those skilled in the art. (See, e.g., Gennaro (Ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995).) The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, and the like.

[0093] In some embodiments, the pharmaceutical composition may be formulated in one of the following dosage forms: oral unit dosage form, intravenous unit dosage form, intranasal unit dosage form, suppository unit dosage form, intradermal unit dosage form, intramuscular unit dosage form, intraperitoneal unit dosage form, subcutaneous unit dosage form, or topical dosage form. In some embodiments, the pharmaceutical composition may be formulated in a topical dosage form. In some embodiments, the pharmaceutical composition may be formulated in an intravenous dosage form.

[0094] In some embodiments, the pharmaceutical composition is formulated for topical administration. In some embodiments, the topical pharmaceutical composition of the anti-ceramide antibody or antigen-binding fragment thereof may be formulated in combination with a pharma- ceutically acceptable carrier. Non-limiting examples of dosage forms for topical compositions include powders, sprays, foams, jellies, ointments, pastes, creams, lotions, gels, solutions, patches, suppositories, and liposomal preparations. The dosage form may be formulated with a mucoadhesive polymer for sustained release of the active ingredient. The active compound may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and any preservatives, buffers, or propellants that may be required. Topical formulations can be prepared by combining the active ingredient with conventional pharmaceutical diluents and carriers commonly used in topical dry, liquid, cream, and aerosol formulations. Ointments and creams may be formulated, for example, with an aqueous or oily base with the addition of a suitable thickening and / or gelling agent. Such a base may include water and / or an oil, such as liquid paraffin, or a vegetable oil, such as peanut oil or castor oil. Thickening agents that may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycol, wool fat, hydrogenated lanolin, beeswax, etc. Lotions may be formulated with an aqueous or oily base and will generally also include one or more of the following: stabilizers, emulsifiers, dispersing agents, suspending agents, thickening agents, coloring agents, fragrances, etc. Powders may be formed with any suitable powder base, for example, talc, lactose, starch, etc. Drops may be formulated with an aqueous or non-aqueous base also including one or more dispersing agents, suspending agents, solubilizing agents, etc.

[0095] In some embodiments, ointments, pastes, creams and gels may also contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays may also contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons such as butane and propane.

[0096] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof may be administered in combination with a pharma- ceutically acceptable carrier and one or more of the following second pharmacological agents: local anesthetics (e.g., lidocaine, prilocaine, etc.), topical anti-inflammatory agents (e.g., naproxen, pramoxicam, etc.), corticosteroids (e.g., cortisone, hydrocortisone, etc.), antipruritic agents (e.g., loperamide, difilerenoxalate, etc.), divalent and trivalent metal ions (e.g., manganese, calcium, strontium, nickel, lanthanum, etc.), or combinations thereof. The composition may be formulated with at least one of the following: an agent that interferes with activation of peripheral sensory neurons, including agents that interfere with activation of peripheral sensory neurons, including agents that interfere with activation of peripheral sensory neurons, including agents that interfere with activation of peripheral sensory neurons, including agents that interfere with activation of peripheral sensory neurons, including agents that interfere with activation of peripheral sensory neurons, including agents that interfere with activation of peripheral sensory neurons, including agents that interfere with activation of peripheral sensory neurons, including agents that interfere with activation of peripheral sensory neurons, including agents that interfere with activation of peripheral sensory neurons,

[0097] The anti-ceramide antibodies and antigen-binding fragments thereof described herein can be administered to a subject by one or more routes of administration. Possible routes of administration include, for example, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, parenteral, intranasal, intrapulmonary, transdermal, intrathecal, oral, topical, and intralesional routes. In some embodiments, the route of administration is selected from the group consisting of topical, intralesional, subcutaneous, transdermal, intramuscular, intravenous, and parenteral administration.

[0098] In some embodiments, administration of the anti-ceramide antibodies and antigen-binding fragments thereof, or pharmaceutical compositions thereof disclosed herein comprises or consists of topical administration. In some embodiments, administration of the anti-ceramide antibodies and antigen-binding fragments thereof, or pharmaceutical compositions thereof disclosed herein comprises or consists of intralesional administration. In some embodiments, administration of the anti-ceramide antibodies and antigen-binding fragments thereof, or pharmaceutical compositions thereof disclosed herein comprises or consists of subcutaneous administration. In some embodiments, administration of the anti-ceramide antibodies and antigen-binding fragments thereof, or pharmaceutical compositions thereof disclosed herein comprises or consists of transdermal administration. In some embodiments, administration of the anti-ceramide antibodies and antigen-binding fragments thereof, or pharmaceutical compositions thereof disclosed herein comprises or consists of intramuscular administration. In some embodiments, administration of the anti-ceramide antibodies and antigen-binding fragments thereof, or pharmaceutical compositions thereof disclosed herein comprises or consists of intravenous administration. In some embodiments, administration of the anti-ceramide antibodies and antigen-binding fragments thereof disclosed herein, or pharmaceutical compositions thereof, comprises or consists of parenteral administration.

[0099] For prophylactic and therapeutic purposes, anti-ceramide antibodies and antigen-binding fragments thereof can be administered to a subject in a single bolus delivery, via continuous delivery over an extended period of time (e.g., continuous transdermal delivery), or in a repeated dosing protocol (e.g., hourly, daily, weekly, monthly, or yearly).

[0100] In some embodiments, the methods provided herein include administering a therapeutically effective amount of an anti-ceramide antibody or an antigen-binding fragment thereof. The therapeutically effective dose, dosage, or amount defined above refers to the amount of an anti-ceramide antibody or an antigen-binding fragment thereof required to produce a particular physiological effect, such as prevention or alleviation of one or more symptoms of a wound, or enhanced wound healing. The determination of a therapeutically effective dosage in this context is typically based on animal model studies followed by human clinical trials, and is guided by determining an effective dosage and administration protocol that significantly reduces the occurrence or severity of wounds or enhances wound healing in model subjects. The effective dose of the composition of the present disclosure will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other medications administered, whether the treatment is preventive or therapeutic, and the specific activity of the composition itself and its ability to induce a desired response in an individual. Typically, the dosing regimen is adjusted to provide an optimal therapeutic response, i.e., to optimize safety and efficacy.

[0101] In some embodiments, the dose of the anti-ceramide antibody or antigen-binding fragment thereof is about 0.1 μg to 100 mg / kg, or 1 μg / kg to about 50 mg / kg, or 10 μg to 5 mg / kg. In some embodiments, an effective amount of the anti-ceramide antibody or antigen-binding fragment thereof is about 1 μg / kg to about 20 mg / kg, about 10 μg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 5 mg / kg. The anti-ceramide antibodies and antigen-binding fragments thereof described herein may also be administered at a dosage of about 0.001 to about 10 milligrams (mg) per kilogram of body weight (mpk), given as a single dose or two or more doses. For administration to human adult patients, a therapeutically effective amount may be administered in a dose range of 0.2 mg to 800 mg per dose, including, but not limited to, 0.2 mg per dose, 0.5 mg per dose, 1 mg per dose, 5 mg per dose, 10 mg per dose, 25 mg per dose, 100 mg per dose, 200 mg per dose, and 400 mg per dose, and one or more doses may be administered over a course of treatment. In some embodiments, the total daily dosage of the anti-ceramide antibodies and antigen-binding fragments thereof described herein may range from about 1 mg to about 2 g, about 100 mg to about 1.5 g, or about 200 mg to about 1200 mg.

[0102] In some embodiments, the dose of anti-ceramide antibody or antigen-binding fragment thereof is about 0.1 μg / cm2 of wound surface area. 2 ~100mg / cm 2 , or 1 μg / cm 2 ~about 50mg / cm 2 , or 10 μg / cm 2 ~5mg / cm 2 In some embodiments, an effective amount of an anti-ceramide antibody or antigen-binding fragment thereof is about 1 μg / cm 2 ~about 20mg / cm 2 , about 10μg / cm 2 ~ approx. 10mg / kgcm 2 , or about 0.1 mg / cm 2 ~about 5mg / cm 2The anti-ceramide antibodies and antigen-binding fragments thereof described herein also provide a therapeutically effective amount of ceramide per square centimeter (cm ) of wound, given as a single dose or as two or more doses. 2 For administration to human adult patients, a therapeutically effective amount may be administered at a dosage of about 0.001 to about 10 milligrams (mg) per dose, including, but not limited to, 0.2 mg per dose, 0.5 mg per dose, 1 mg per dose, 5 mg per dose, 10 mg per dose, 25 mg per dose, 100 mg per dose, 200 mg per dose, and 400 mg per dose, and one or more doses may be administered in a course of treatment. In some embodiments, the total daily dosage of the anti-ceramide antibodies and antigen-binding fragments thereof described herein may range from about 1 mg to about 2 g, about 100 mg to about 1.5 g, or about 200 mg to about 1200 mg.

[0103] In some embodiments, the anti-ceramide antibody, antigen-binding fragment thereof, or composition comprising same may be formulated at a concentration of about 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL. The concentration may be 0.1-1 mg / mL, 1-5 mg / mL, 5-10 mg / mL, or 10-50 mg / mL. In some embodiments, the disclosed antibody, fragment, or composition may be administered at a dose of about 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1 mg. The dose volume can be about 0.005 mL, 0.01 mL, 0.02 mL, 0.03 mL, 0.04 mL, 0.05 mL, 0.06 mL, 0.07 mL, 0.08 mL, 0.09 mL, or 0.1 mL.

[0104] The anti-ceramide antibodies and antigen-binding fragments thereof described herein can be administered at different times of the day. In one embodiment, a dose can be administered in the evening. In another embodiment, a dose can be administered in the morning. The dosage can be administered in single or multiple administrations, including, for example, multiple weekly, biweekly, monthly, or yearly administrations. In some embodiments, a single dose of anti-ceramide antibody or antibody fragment is administered to a subject in need thereof. In some embodiments, a patient may receive two or more doses of anti-ceramide antibody therapy. In some embodiments, a patient may receive two or more doses of anti-ceramide antibody therapy, where successive doses are separated by at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 1 week. In some embodiments, a patient may receive two or more doses of anti-ceramide antibody therapy, where successive doses are separated by a period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 1 month, at least 2 months, or at least 3 months. In some embodiments, two or more doses may be administered to a patient in need thereof, separated by periods of about 1 week to about 2 weeks, about 2 weeks to about 4 weeks, about 1 month to about 2 months, about 2 months to about 4 months, or about 1 month to about 6 months. In some embodiments, the duration of administration is at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. In some embodiments, administration may be irregular, as indicated by monitoring clinical symptoms of the disorder.

[0105] Dosage of pharmaceutical compositions comprising anti-ceramide antibodies and antigen-binding fragments thereof can be varied by the attending physician to maintain a desired concentration at the target site. Higher or lower concentrations can be selected based on the mode of delivery. Anti-ceramide antibodies or antigen-binding fragments thereof may be administered at any time during the subject's life. Administration may occur during the inflammatory, proliferative, or remodeling phase of wound healing, or a combination thereof. In some embodiments, administration occurs during the inflammatory phase of wound healing. In some embodiments, administration occurs during the proliferative phase of wound healing. In some embodiments, administration occurs during the remodeling phase of wound healing.

[0106] Treatment Methods and Uses In some embodiments, the disclosure provides a method of treating, preventing, or alleviating a wound in a subject, comprising administering to the subject a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the wound is a chronic wound. In some embodiments, the wound is a diabetic wound.

[0107] In some embodiments, the disclosure provides a method of enhancing wound healing in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the wound is a chronic wound. In some embodiments, the wound is a diabetic wound.

[0108] subject Subjects for treatment with the methods disclosed herein include subjects having or at risk of developing a wound (e.g., a diabetic wound). Subjects having a wound (e.g., a diabetic wound) can have an early or late stage disease.

[0109] In some embodiments, the subject has previously undergone one or more treatments for a wound (e.g., a diabetic wound) but has failed to respond to the previous treatment. In such embodiments, "failed to respond" refers to the failure of the previous treatment to alleviate and / or improve the wound (e.g., a diabetic wound). In some embodiments, the previous therapy may have shown some results but may not have achieved the desired performance or may have ceased to show effectiveness after some period of time.

[0110] Treatment Readouts Wound healing can be measured by a variety of means. In some embodiments, wound healing is measured by the change in total surface area of ​​the wound. In some embodiments, wound healing is measured by the change in the length (length and width) of the major axes of the wound. In some embodiments, wound healing is measured by the wound margin distance from the wound center. In some embodiments, wound healing is measured by the change in the wound perimeter. In some embodiments, wound healing is measured by the change in the surface area to perimeter (S / P) ratio. Assessment of these parameters can be done in a variety of ways, for example, by computer-assisted planimetry. In some embodiments, the extent of wound healing is represented by the ratio of the value over time of any one of these parameters compared to the initial value.

[0111] Methods of enhancing wound healing using anti-ceramide antibodies or antigen-binding fragments thereof are provided. The effectiveness of such treatments can be characterized, assessed, measured, and / or monitored based on several parameters.

[0112] In some embodiments, the improvement is measured by a decrease in the time to achieve a particular degree of wound healing. For example, if the wounds of the control group take 25 days to heal and the wounds of the treatment group take only 20 days to heal, wound healing is enhanced / accelerated by such a measurement by 20%. In some embodiments, the improvement is measured by an increase in the relative degree of wound healing at a pre-determined time point. For example, if, at 20 days after wounding, the treatment group achieves an average of 50% or so wound healing (e.g., as measured by total surface area) and the aforementioned group achieves an average of only 40% or so wound healing, wound healing is enhanced / accelerated by such a measurement by 25%. Those skilled in the art will readily recognize an appropriate control group (e.g., control wound). In some embodiments, the control wound receives the same treatment, except for the anti-ceramide antibody or antigen-binding fragment thereof (e.g., as recited in the claims). In some embodiments, the control wound receives a standard of care treatment. In all cases, reference to a control group is meant to indicate that the recited property (e.g., enhanced wound healing) is the result of the use of an anti-ceramide antibody or antigen-binding fragment thereof.

[0113] In some embodiments, the methods provided herein enhance wound healing in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% (including all ranges and subranges therebetween) compared to a control wound, as measured by a decrease in the time it takes for the wound to improve according to one of the wound healing parameters at a pre-determined degree. In some embodiments, the methods provided herein enhance wound healing by at least 10%. In some embodiments, the methods provided herein enhance wound healing by at least 20%. In some embodiments, the methods provided herein enhance wound healing by at least 30%. In some embodiments, the methods provided herein enhance wound healing by at least 50%. In some embodiments, the methods provided herein enhance wound healing by at least 70%. In some embodiments, the wound healing parameter is the total surface area of ​​the wound, the perimeter of the wound, the S / P ratio of the wound, or the margin distance from the center of the wound. In some embodiments, the wound healing parameter is the total surface area of ​​the wound. In some embodiments, the preset extent is about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction. In some embodiments, the preset extent is 50%. In some embodiments, the preset extent is 95%. In some embodiments, the enhanced wound healing is measured by the time taken to achieve a 50% reduction in the total surface area of ​​the wound vs. the mortality. In some embodiments, the enhanced wound healing is measured by the time taken to achieve a 95% reduction in the total surface area of ​​the wound vs. the mortality. In some embodiments, the enhanced wound healing is measured by the time taken to achieve complete closure of the wound vs. the mortality. In some embodiments, the wound is a chronic wound. In some embodiments, the wound is a diabetic wound.

[0114] In some embodiments, the methods provided herein enhance wound healing in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, or at least 10-fold (including all ranges and subranges therebetween) as compared to a control wound, as measured by an increase in the relative extent of wound healing at a pre-determined time point according to one of the wound healing parameters. In some embodiments, the methods provided herein enhance wound healing by at least 10%. In some embodiments, the methods provided herein enhance wound healing by at least 20%. In some embodiments, the methods provided herein enhance wound healing by at least 30%. In some embodiments, the methods provided herein enhance wound healing by at least 50%. In some embodiments, the methods provided herein enhance wound healing by at least 70%. In some embodiments, the wound healing parameter is the total surface area of ​​the wound, the perimeter of the wound, the S / P ratio of the wound, or the margin distance from the center of the wound. In some embodiments, the wound healing parameter is the total surface area of ​​the wound. In some embodiments, the preset time point is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after wounding. In some embodiments, the preset time point is at the end of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, or 20 weeks post-wounding. In some embodiments, the preset time point is 10 days post-wounding. In some embodiments, the preset time point is 20 days post-wounding. In some embodiments, the preset time point is 30 days post-wounding.In some embodiments, enhanced wound healing is measured by total wound surface area mortality at 10 days post-wounding. In some embodiments, enhanced wound healing is measured by total wound surface area mortality at 20 days post-wounding. In some embodiments, enhanced wound healing is measured by total wound surface area mortality at 30 days post-wounding. In some embodiments, the wound is a chronic wound. In some embodiments, the wound is a diabetic wound.

[0115] In some embodiments, the treatments of the present disclosure are effective in treating superficial wounds, such as those to the skin, hi some embodiments, the treatments of the present disclosure are effective in treating internal wounds, such as wounds that may result from trauma or surgery.

[0116] In some embodiments, the treatment is administered after an injury (e.g., wound) has occurred, hi some embodiments, the treatments of the present disclosure are administered prophylactically, such as before undertaking an activity that may cause a wound, or before a surgical procedure. EXAMPLES

[0117] Example 1: Anti-ceramide antibodies accelerate wound healing in a mouse model of diabetes. To study the effects of anti-ceramide antibodies on diabetic wound healing, a standardized punch biopsy-based murine superficial wound model was used at the MSU core facility.

[0118] In this study, mice were divided into four experimental groups: (a) 1) Diabetic mice (6 animals) treated with anti-ceramide antibody 2A2 (diabetic+2A2); (b) 2) diabetic mice (6 animals) without anti-ceramide antibody 2A2 (diabetic); (c) 3) control mice (3 animals) treated with anti-ceramide antibody 2A2 (control+2A2); (d) 4) Control mice (5 animals) without anti-ceramide antibody 2A2 (control).

[0119] Wounds were introduced with a 5 mm biopsy needle on the upper back of control (nondiabetic) and STZ-induced diabetic SKH1 hairless mice and either 1 mg of 2A2 anti-ceramide antibody or vehicle control (PBS) was injected by IV injection every 4 days. Representative photographs of wound sites at 0, 1, 4, 8, 10, 12, 13, 16, 20, and 25 days after wounding are shown in Figure 1A-B.

[0120] Figure 2 shows the average rate of wound healing in each experimental group, as represented by the size of the lesion over time. To normalize the size of the lesion, the original size of the lesion in each case was set to 1. In both diabetic and non-diabetic mice, application of 2A2 antibody accelerated the wound healing process, and this effect was more pronounced in the diabetic mice group.

[0121] Figure 3A shows the mean wound closure calculated as a percentage of the original wound size on day 10. In both control and diabetic groups, mice injected with 2A2 achieved a greater degree of wound closure after 10 days than mice injected with the PBS vehicle control.

[0122] Figure 3B shows the average number of days it took for mice in each experimental group to achieve 95% wound closure (compared to the original wound size). In the diabetic mouse group, application of 2A2 antibody significantly reduced the length of time it took to achieve 95% healing.

[0123] Overall, this study demonstrates that application of anti-ceramide antibodies significantly accelerates the healing of wounds, particularly diabetic wounds.

[0124] Research Protocol: The protocol for the animal studies carried out in Example 1 is given below.

[0125] STUDY OBJECTIVE:To investigate the effect of anti-ceramide antibody 2A2 on wound healing when administered intravenously (IV) once every 4 days to male SKH1 mice undergoing streptozotocin (STZ)-induced type I diabetes.

[0126] Male SKH1 mice weighing 20-25 g were allowed to acclimate for approximately 2 weeks. Animals were divided into control (C) and diabetes-induced (D) mice. Diabetes was produced by intraperitoneal injection of streptozotocin (65 mg / kg body weight) for 5 consecutive days. Two weeks after the last injection, blood glucose was measured from a drop of blood taken from the dorsalis pedis vein, and diabetes was confirmed by a blood glucose value of more than 300 mg per dl. Weight loss, polyuria, water and food intake were monitored daily, and NPH insulin injections (0-2 units / day) were provided based on the clinical status of the animals. Wounds were introduced as described previously after diabetes was confirmed.

[0127] Animals were randomized into two groups for IV injection treatment with vehicle or 2A2.

[0128] Treatment was initiated on the day of wound creation. IV dosing continued every 4 days until the wound was completely healed (approximately 30 days). Control (C) animals were wounded on the same day as the diabetic animals and randomized into two groups for IV injection treatment with vehicle or 2A2. IV treatment was initiated on the day of wound creation. Dosing continued every 4 days until the wound was completely healed (approximately 15 days). The IV dose of 2A2 antibody was 1 mg / 25 g mouse every 4 days.

[0129] Photographic wound measurements were taken immediately after surgery and every other day thereafter to calculate the percentage healing time. At the end of the experiment, wounds were excised, fixed, and analyzed for dermal thickness, matrix deposition, revascularization, and immune cell infiltration.

[0130] Example 2: Further experimental validation. The experiment performed in Example 1 was repeated with additional mouse subjects. The results from this experiment are provided in Tables 3-6 and Figures 3 and 5A-D. [Table 3] [Table 4] [Table 5] [Table 6]

[0131] Figure 4 shows the average rate of wound healing in each experimental group over time. The Y-axis shows wound size, normalized to the initial size of each wound as 1. The X-axis shows the number of days.

[0132] 5A-D are bar graphs showing the average number of days it took for mice to achieve 25%, 50%, 75%, and 90% healing for each experimental group, respectively.

[0133] This experiment further validates the treatment of mice with anti-ceramide antibodies in a statistically significant acceleration of wound healing, particularly in diabetic wounds.

[0134] Example 3: Local antibody administration The antibody therapeutic acceleration of wound healing described in Examples 1-2 is believed to be due to localized ceramide signaling effects at the wound site. Thus, topical administration of the anti-ceramide antibodies and / or antigen-binding fragments thereof of the present disclosure is expected to show similar effects as described in Examples 1-2. The effects of these antibodies and antigen-binding fragments are tested in animal models such as mice. The experiments are performed similarly to those described in Examples 1-2, except that the antibodies are administered locally via lotion, save, or other topical formulations known to those skilled in the art. In another experiment performed similarly to those described in Examples 1-2, wounds are treated via subcutaneous injection of the antibodies or antigen-binding fragments thereof. Topical administration of other ceramide signaling inhibitors, such as imipramine, is tested on wound healing according to the methods of Examples 1 and 2. Wounds treated with the antibodies and / or antigen-binding fragments thereof (or imipramine) are expected to accelerate / improve wound healing in control and / or diabetic animals.

[0135] Example 4: Internal Injury Additional confirmatory experiments will be performed to demonstrate the ability of the presently disclosed treatment to enhance / accelerate internal injury. Experiments will be performed in animal models in which control and diabetic subjects are given internal injury and healing from the injury is tracked over time in subjects treated with / without vehicle or with the anti-ceramide antibodies or imipramine of the present disclosure. Healing will be tracked via any of the techniques known to those skilled in the art, including visual assessment by sonogram, MRI, CT, or surgical means.

[0136] Example 5: Further validation of the disclosed antibodies and antigen-binding fragments thereof The present disclosure illustrates the effect of anti-ceramide antibodies and / or antigen-binding fragments thereof through validation experiments of the 2A2 antibody. The experiments of Examples 1-2 are repeated using any one of the additional antibodies and antigen-binding fragments disclosed herein, or other antibodies / antigen-binding fragments that can bind to ceramide or inhibit the formation of ceramide-rich platforms. For example, the wound healing experiments described in Examples 1-2 are repeated using 6b5 mice (US2019-0389970, which is incorporated herein by reference), and 6b5 humanized (PCT / US2021 / 022914 and its corresponding US Provisional Application No. 62 / 991,232, both of which are incorporated herein by reference), antibodies, and fragments thereof.

[0137] Further Numbered Embodiments Further embodiments of the present invention are provided in the following numbered embodiments.

[0138] Embodiment 1. A method of treating or preventing a wound in a subject in need thereof, comprising administering to the subject an anti-ceramide antibody, or an antigen-binding fragment thereof.

[0139] Embodiment 2. A method of enhancing wound healing in a subject in need thereof, comprising administering to the subject an anti-ceramide antibody, or antigen-binding fragment thereof.

[0140] Embodiment 3. The method of embodiment 1 or 2, wherein the wound is a chronic wound or a diabetic wound.

[0141] Embodiment 4. The method of embodiment 1 or 2, wherein the wound is a chronic wound.

[0142] Embodiment 5. The method of embodiment 1 or 2, wherein the wound is a diabetic wound.

[0143] Embodiment 5.1 The method of embodiment 1 or 2, wherein the wound is an external wound.

[0144] Embodiment 5.2 The method of embodiment 1 or 2, wherein the wound is an internal wound.

[0145] Embodiment 5.3 The method of embodiment 1 or 2, wherein the wound is on endothelial tissue.

[0146] Embodiment 6. The method of any one of embodiments 1-5.3, wherein the route of administration is selected from the group consisting of topical administration, intralesional administration, subcutaneous administration, transdermal administration, intramuscular administration, intravenous administration, and parenteral administration.

[0147] Embodiment 7. The method of embodiment 6, wherein the route of administration is topical administration.

[0148] Embodiment 8. The method of embodiment 6, wherein the route of administration is intravenous administration.

[0149] Embodiment 9 The method of any one of embodiments 1-8, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose.

[0150] Embodiment 10. The method of any one of embodiments 1-8, wherein the anti-ceramide antibody, or antigen-binding fragment thereof, is administered in two or more doses.

[0151] Embodiment 11. The method of embodiment 10, wherein the administration of successive doses is separated by at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 1 week.

[0152] Embodiment 12. The method of embodiment 10 or 11, wherein the duration of administration is at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks.

[0153] Embodiment 13 The method of any one of embodiments 1-12, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the inflammatory stage of wound healing.

[0154] Embodiment 14 The method of any one of embodiments 1-13, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative phase of wound healing.

[0155] Embodiment 15 The method of any one of embodiments 1-14, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the remodeling stage of wound healing.

[0156] Embodiment 16 The method of any one of embodiments 1 to 15, wherein the anti-ceramide antibody or antigen-binding fragment thereof is an antibody.

[0157] Embodiment 17 The method of any one of embodiments 1-15, wherein the anti-ceramide antibody, or antigen-binding fragment thereof, is a single chain variable fragment (scFv).

[0158] Embodiment 18. The method of any one of embodiments 1-17, wherein the method enhances wound healing by at least 10%, at least 20%, at least 30%, at least 50%, or at least 70% compared to a control wound.

[0159] Embodiment 19. The method of embodiment 18, wherein the enhanced wound healing is measured by total surface area mortality of the wound at 10, 20, or 30 days post-wounding.

[0160] Embodiment 20 The method of embodiment 19, wherein the enhanced wound healing is measured 10 days after wounding.

[0161] Embodiment 21. The method of embodiment 18, wherein the enhanced wound healing is measured by mortality in the time it takes to achieve a 50%, 70%, 90%, 95%, or 100% reduction in the total surface area of ​​the wound.

[0162] Embodiment 22 The method of embodiment 21, wherein the enhanced wound healing is measured by a 95% reduction in the total surface area of ​​the wound.

[0163] Embodiment 23 The method of any one of embodiments 1-22, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered prior to the onset of one or more symptoms of the wound.

[0164] Embodiment 24 The method of any one of embodiments 1-22, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered after onset of one or more symptoms of the wound.

[0165] Embodiment 25. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), a) VH comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1), an HCDR2 comprising the amino acid sequence of YNYPRDGSTKYNEKFKG (SEQ ID NO: 2), and an HCDR3 comprising the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3); b) Any one of the methods of embodiments 1 to 24, wherein the VL comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 comprising the amino acid sequence of SGSTLQS (SEQ ID NO: 5), and an LCDR3 comprising the amino acid sequence of QQHNEYPWT (SEQ ID NO: 6).

[0166] Embodiment 26 The method of any one of embodiments 1 to 25, wherein the VH comprises the amino acid sequence of SEQ ID NO:7 and the VL comprises the amino acid sequence of SEQ ID NO:8.

[0167] Embodiment 27 The method of any one of embodiments 1 to 26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a 6B5 antibody.

[0168] Embodiment 28 The method of any one of embodiments 1 to 26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a 6B5scFv antibody.

[0169] Embodiment 29. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), a) VH comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of NYWMH (SEQ ID NO: 33), an HCDR2 comprising the amino acid sequence of AIYPGDSDTSYNQKFKG (SEQ ID NO: 34), and an HCDR3 comprising the amino acid sequence of LYYGYD (SEQ ID NO: 35); b) Any one of the methods of embodiments 1 to 24, wherein the VL comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of KSSQSLIDSDGKTFLN (SEQ ID NO: 36), an LCDR2 comprising the amino acid sequence of LVSKLDS (SEQ ID NO: 37), and an LCDR3 comprising the amino acid sequence of WQGTHFPYT (SEQ ID NO: 38).

[0170] Embodiment 30. The method of any one of embodiments 1 to 29, wherein the VH comprises the amino acid sequence of SEQ ID NO: 39 and the VL comprises the amino acid sequence of SEQ ID NO: 40.

[0171] Embodiment 31 The method of any one of embodiments 1-24 and 29-30, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a 2A2 antibody.

[0172] Embodiment 32 The method of any one of embodiments 1-24 and 29-30, wherein the anti-ceramide antibody or antigen-binding fragment thereof is 2A2scFv.

[0173] Embodiment 33. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), a) VH comprises a heavy chain complementarity determining region 1 (HCDR1) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 43, an HCDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 47, and an HCDR3 comprising or consisting of the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3); b) Any one of the methods of embodiments 1 to 24, wherein the VL comprises a light chain complementarity determining region 1 (LCDR1) comprising or consisting of the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 comprising or consisting of the amino acid sequence of SGSTLQS (SEQ ID NO: 5), and an LCDR3 comprising or consisting of the amino acid sequence of QQHNEYPWT (SEQ ID NO: 6).

[0174] Embodiment 34 The method of embodiment 33, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYNEKFQG (SEQ ID NO: 44).

[0175] Embodiment 35 The method of embodiment 33, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPREGSTKYNEKFQG (SEQ ID NO: 45).

[0176] Embodiment 36 The method of embodiment 33, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDVSTKYNEKFQG (SEQ ID NO: 46).

[0177] Embodiment 37 The method of embodiment 33, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYAEKFQG (SEQ ID NO: 47).

[0178] Embodiment 38 The method of embodiment 33, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYNEKFQG (SEQ ID NO: 44).

[0179] Embodiment 39 The method of embodiment 33, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPREGSTKYNEKFQG (SEQ ID NO: 45).

[0180] Embodiment 40 The method of embodiment 33, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDVSTKYNEKFQG (SEQ ID NO: 46).

[0181] Embodiment 41 The method of embodiment 33, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYAEKFQG (SEQ ID NO: 47).

[0182] Embodiment 42. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0183] Embodiment 43. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54.

[0184] Embodiment 44. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0185] Embodiment 45. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0186] Embodiment 46. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54.

[0187] Embodiment 47. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0188] Embodiment 48. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:53.

[0189] Embodiment 49. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:54.

[0190] Embodiment 50. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:55.

[0191] Embodiment 51. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0192] Embodiment 52. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54.

[0193] Embodiment 53. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0194] Embodiment 54. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0195] Embodiment 55. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54.

[0196] Embodiment 56. The method of embodiment 33, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0197] Embodiment 57 The method of any one of embodiments 33-56, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a humanized 6B5 (h6B5) antibody.

[0198] Embodiment 58 The method of any one of embodiments 33 to 56, wherein the anti-ceramide antibody or antigen-binding fragment thereof is an h6B5scFv antibody.

[0199] Embodiment 59. The method of any one of embodiments 1 to 24, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 48 and the VL comprises the amino acid sequence of SEQ ID NO: 53.

[0200] Embodiment 60. The method of any one of embodiments 1 to 24, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 48 and the VL comprises the amino acid sequence of SEQ ID NO: 55.

[0201] Embodiment 61. The method of any one of embodiments 1 to 24, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 49 and the VL comprises the amino acid sequence of SEQ ID NO: 53.

[0202] Embodiment 62. The method of any one of embodiments 1 to 24, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 49 and the VL comprises the amino acid sequence of SEQ ID NO: 54.

[0203] Embodiment 63. The method of any one of embodiments 1 to 24, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 50 and the VL comprises the amino acid sequence of SEQ ID NO: 53.

[0204] Embodiment 64. The method of any one of embodiments 1 to 24, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 50 and the VL comprises the amino acid sequence of SEQ ID NO: 54.

[0205] Embodiment 65. The method of any one of embodiments 1 to 24, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 51 and the VL comprises the amino acid sequence of SEQ ID NO: 53.

[0206] Embodiment 66. The method of any one of embodiments 1 to 24, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 52 and the VL comprises the amino acid sequence of SEQ ID NO: 53.

[0207] Embodiment 67 The method of any one of embodiments 59-66, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a humanized antibody.

[0208] Embodiment 68 The method of any one of embodiments 59-66, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a humanized scFv.

[0209] Embodiment 69. A method of treating or preventing a wound in a subject in need thereof, comprising administering imipramine or a salt thereof to the subject.

[0210] Embodiment 70. A method of enhancing wound healing in a subject in need thereof, comprising administering imipramine or a salt thereof to the subject.

[0211] Embodiment 71 The method of embodiment 70 or 71, wherein the wound is a chronic wound or a diabetic wound.

[0212] Embodiment 72 The method of embodiment 70 or 71, wherein the wound is a chronic wound.

[0213] Embodiment 73 The method of embodiment 70 or 712, wherein the wound is a diabetic wound.

[0214] Embodiment 74. The method of any one of embodiments 70-73, wherein the route of administration is selected from the group consisting of topical administration, intralesional administration, subcutaneous administration, transdermal administration, intramuscular administration, intravenous administration, and parenteral administration.

[0215] Embodiment 75. The method of embodiment 74, wherein the route of administration is topical administration.

[0216] Embodiment 76 The method of embodiment 74, wherein the route of administration is intravenous administration.

[0217] Embodiment 77 The method of any one of embodiments 70-76, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose.

[0218] Embodiment 78 The method of any one of embodiments 1-8, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses.

[0219] Embodiment 79. The method of embodiment 78, wherein the administration of consecutive doses is separated by at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 1 week.

[0220] Embodiment 80. The method of embodiment 78 or 79, wherein the duration of administration is at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks.

[0221] Embodiment 81 The method of any one of embodiments 70-80, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the inflammatory phase of wound healing.

[0222] Embodiment 82 The method of any one of embodiments 70-81, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative phase of wound healing.

[0223] Embodiment 83 The method of any one of embodiments 70-82, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the remodeling stage of wound healing.

[0224] Incorporation by Reference All references, documents, publications, patents, patent publications, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. However, the mention of any references, documents, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the general knowledge anywhere in the world.

Claims

1. A pharmaceutical composition for enhancing wound healing, comprising an anti-ceramide antibody or an antigen-binding fragment thereof.

2. A pharmaceutical composition for treating wounds, comprising an anti-ceramide antibody or an antigen-binding fragment thereof.

3. A pharmaceutical composition described in claim 1 or 2, wherein the wound is a diabetic wound.

4. A pharmaceutical composition described in any one of claims 1 to 3, which is administered locally.

5. A pharmaceutical composition described in any one of claims 1 to 3, which is administered intravenously.

6. A pharmaceutical composition described in any one of claims 1 to 5, administered as a single dose.

7. A pharmaceutical composition described in any one of claims 1 to 6, wherein the anti-ceramide antibody or its antigen-binding fragment is an antibody.

8. A pharmaceutical composition described in any one of claims 1 to 6, wherein the anti-ceramide antibody or its antigen-binding fragment is a single-chain variable fragment (scFv).

9. A pharmaceutical composition described in any one of claims 1 to 8, wherein the pharmaceutical composition enhances wound healing by at least 10%, at least 20%, at least 30%, at least 50%, or at least 70% compared to a control wound.

10. The enhancement of wound healing is a reduction in the total surface area of ​​the wound at 10, 20, or 30 days after wounding; or a reduction in the time it takes to achieve a 50%, 70%, 90%, 95%, or 100% reduction in the total surface area of ​​the wound; The pharmaceutical composition of claim 9, wherein the IL-10 is measured by

11. A pharmaceutical composition described in any one of claims 1 to 3 and 6 to 10, wherein the route of administration is selected from the group consisting of topical administration, intralesional administration, subcutaneous administration, transdermal administration, intramuscular administration, intravenous administration, and parenteral administration.

12. A pharmaceutical composition described in any one of claims 1 to 11, administered before the onset of one or more symptoms of the wound.

13. A pharmaceutical composition described in any one of claims 1 to 11, administered after the onset of one or more symptoms of the wound.

14. The anti-ceramide antibody or antigen-binding fragment thereof is a 2A2 antibody or antigen-binding fragment thereof comprising a variable heavy chain (V H ) and a variable light chain (V L ); a) the VH comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of NYWMH (SEQ ID NO: 33), a HCDR2 comprising the amino acid sequence of AIYPGDSDTSYNQKFKG (SEQ ID NO: 34), and a HCDR3 comprising the amino acid sequence of LYYGYD (SEQ ID NO: 35); b) The pharmaceutical composition according to any one of claims 1 to 13, wherein the V L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of KSSQSLIDSDGKTFLN (SEQ ID NO: 36), an LCDR2 comprising the amino acid sequence of LVSKLDS (SEQ ID NO: 37), and an LCDR3 comprising the amino acid sequence of WQGTHFPYT (SEQ ID NO: 38).

15. The pharmaceutical composition of claim 14, wherein the V H comprises the amino acid sequence of SEQ ID NO:39, and the V L comprises the amino acid sequence of SEQ ID NO:

40.

16. The anti-ceramide antibody or antigen-binding fragment thereof is a 6B5 antibody or antigen-binding fragment thereof comprising a variable heavy chain (V H ) and a variable light chain (V L ); a) said V H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 and 43, an HCDR2 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 44 to 47, and an HCDR3 comprising or consisting of the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3); b) The pharmaceutical composition according to any one of claims 1 to 13, wherein the VL comprises a light chain complementarity determining region 1 (LCDR1) comprising or consisting of the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 comprising or consisting of the amino acid sequence of SGSTLQS (SEQ ID NO: 5), and an LCDR3 comprising or consisting of the amino acid sequence of QQHNEYPWT (SEQ ID NO: 6).

17. The pharmaceutical composition of claim 16, wherein the HCDR1 comprises or consists of the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1), and the HCDR2 comprises or consists of the amino acid sequence YNYPREGSTKYNEKFQG (SEQ ID NO: 45).

18. The pharmaceutical composition of claim 17, wherein the V H comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:51, and the V L comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:

53.

19. The pharmaceutical composition of any one of claims 1 to 13, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and a variable light chain (V L ), wherein the V H comprises the amino acid sequence of SEQ ID NO:51, and the V L comprises the amino acid sequence of SEQ ID NO:

53.

20. A pharmaceutical composition described in any one of claims 1 to 19, wherein the anti-ceramide antibody or its antigen-binding fragment is a humanized antibody.

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