Anti-ceramide antibody

Abstract

The present disclosure provides compositions of anti-ceramide antibodies and antigen-binding fragments thereof. The present disclosure also provides a method for preventing or inhibiting cell death in a subject in need thereof, the method comprising administering an anti-ceramide antibody or antigen-binding fragment to the subject. The subject in need thereof may be suffering from an autoimmune disease, GI syndrome, or GvHD.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 991,232, filed March 18, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Statement The sequence listing associated with this application is presented in text format, rather than as a paper manuscript, and is incorporated herein by reference. A computer-readable copy of the sequence listing is available at: Filename: CERA-012_01WO_ST25.txt, Recorded on: March 17, 2021, File Size: 68.1 kilobytes.

[0003] Field The present disclosure generally relates to anti-ceramide antibodies and methods of use thereof. In particular, the present disclosure relates to humanized anti-ceramide antibodies that inhibit cell death. Such antibodies are useful for treating and preventing gastrointestinal syndrome, graft-versus-host disease, and autoimmune diseases. [Background technology]

[0004] Acute radiation syndrome (ARS), sometimes known as radiation toxicity or radiation sickness, is an acute illness caused by the exposure of large parts of the body to high doses of penetrating radiation (e.g., high-energy X-rays, gamma rays, and neutrons) over a very short period of time. The chances of survival with this syndrome are extremely low due to devastating and irreversible changes in the gastrointestinal tract and bone marrow caused by radiation-induced cell death. Radiation is one of the most effective tools for the treatment of cancer. Unfortunately, a harmful side effect of radiation therapy is the extreme susceptibility of healthy cells in the bone marrow, hair follicles, epidermis, and gastrointestinal tract to radiation-induced cell death. Other types of cancer strategies, such as bone marrow transplantation, elicit damaging immune responses in the host, resulting in rejection or graft-versus-host disease (GvHD). Therefore, alternative strategies to reduce the incidence of cell death associated with radiation-induced GI syndrome and GvHD are urgently needed. Summary of the Invention

[0005] The present disclosure provides compositions of anti-ceramide antibodies and antigen-binding fragments thereof. In some embodiments, the anti-ceramide antibodies and antigen-binding fragments thereof are humanized antibodies or antigen-binding fragments thereof. In other embodiments, the anti-ceramide antibodies and antigen-binding fragments thereof are scFv. In some embodiments, the present disclosure further provides a method for preventing or inhibiting cell death in a subject in need thereof, comprising administering an anti-ceramide antibody or antigen-binding fragment to the subject. In some embodiments, the subject suffers from an autoimmune disease, GI syndrome, or GvHD. In some embodiments, the present disclosure provides an anti-ceramide antibody or antigen-binding fragment thereof, comprising an immunoglobulin heavy chain variable region (VH) comprising heavy chain complementarity determining region (CDR) 1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and an immunoglobulin light chain variable region (VL) comprising light chain complementarity determining region (CDR) 1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), wherein (a) HCDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1 and 2; (b) HCDR2 comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, and 6; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0006] In some embodiments, HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 3. In some embodiments, HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 4. In some embodiments, HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 5. In some embodiments, HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 6. In some embodiments, HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO: 3. In some embodiments, HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO: 4. In some embodiments, HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO: 5. In some embodiments, HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO: 6. In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 17, and the amino acid sequence of the VL consists of SEQ ID NO: 22.

[0007] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 23. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 17, and the amino acid sequence of the VL consists of SEQ ID NO: 23. In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 24. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 17, and the amino acid sequence of the VL consists of SEQ ID NO: 24.

[0008] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 18, and the amino acid sequence of the VL consists of SEQ ID NO: 22.

[0009] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 23. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 18, and the amino acid sequence of the VL consists of SEQ ID NO: 23.

[0010] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 24. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 18, and the amino acid sequence of the VL consists of SEQ ID NO: 24. In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 19, and the amino acid sequence of the VL consists of SEQ ID NO: 22.

[0011] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 23. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 19, and the amino acid sequence of the VL consists of SEQ ID NO: 23. In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 24. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 19, and the amino acid sequence of the VL consists of SEQ ID NO: 24.

[0012] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 20, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 20, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 20, and the amino acid sequence of the VL consists of SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 21, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 21, and the VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 21, and the amino acid sequence of the VL consists of SEQ ID NO: 22.

[0013] In some embodiments, the present disclosure provides an anti-ceramide antibody or antigen-binding fragment thereof, comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to a sequence selected from SEQ ID NOs: 17, 18, 19, 20, and 21; and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to a sequence selected from SEQ ID NOs: 22, 23, and 24.

[0014] In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 17, and the amino acid sequence of the VL consists of SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 23. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 17, and the amino acid sequence of the VL consists of SEQ ID NO: 23.

[0015] In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 24. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 24. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 17, and the amino acid sequence of the VL consists of SEQ ID NO: 24. In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 18, and the amino acid sequence of the VL consists of SEQ ID NO: 22.

[0016] In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 23. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 18, and the amino acid sequence of the VL consists of SEQ ID NO: 23. In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 24. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 24. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 18, and the amino acid sequence of the VL consists of SEQ ID NO: 24.

[0017] In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 19, and the amino acid sequence of the VL consists of SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 23. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 19, and the amino acid sequence of the VL consists of SEQ ID NO: 23.

[0018] In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 24. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 24. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 19, and the amino acid sequence of the VL consists of SEQ ID NO: 24. In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 20, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 20, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 20, and the amino acid sequence of the VL consists of SEQ ID NO: 22.

[0019] In some embodiments, the VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 21, and the VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 22. In some embodiments, the VH comprises an amino acid sequence 100% identical to SEQ ID NO: 21, and the VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. In some embodiments, the amino acid sequence of the VH consists of SEQ ID NO: 21, and the amino acid sequence of the VL consists of SEQ ID NO: 22. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a fully human antibody or antigen-binding fragment thereof. In some embodiments, the anti-ceramide antibody comprises one or more point mutations in the Fc domain of the antibody.

[0020] In some embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv). In some embodiments, the scFv comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 48-61. In some embodiments, the scFv comprises an amino acid sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 48-61. In some embodiments, the scFv consists of a sequence selected from the group consisting of SEQ ID NOs: 48-61. In some embodiments, the light chain variable region of the scFv is carboxy-terminal to the heavy chain variable region of the scFv. In some embodiments, the light chain variable region of the scFv is amino-terminal to the heavy chain variable region of the scFv. In some embodiments, the scFv comprises a linker polypeptide. In some embodiments, the linker polypeptide is between the light chain variable region and the heavy chain variable region of the scFv. In some embodiments, the linker polypeptide comprises a Gly4Ser linker. In some embodiments, the linker polypeptide has the formula (Gly4Ser) n [wherein n=1 to 5].

[0021] In some embodiments, the present disclosure provides an anti-ceramide single-chain variable fragment (scFv) comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 48-61. In some embodiments, the scFv comprises an amino acid sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 48-61. In some embodiments, the scFv consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-61. In some embodiments, the present disclosure provides a polynucleotide encoding an anti-ceramide antibody or antigen-binding fragment thereof described herein. In some embodiments, the present disclosure provides an anti-ceramide single-chain variable fragment (scFv) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 48 or 51. In some embodiments, the scFv comprises an amino acid sequence that is 100% identical to SEQ ID NO: 48 or 51. In some embodiments, the scFv consists of the amino acid sequence of SEQ ID NO: 48 or 51.

[0022] In some embodiments, the present disclosure provides a polynucleotide encoding an anti-ceramide single chain variable fragment (scFv) described herein, hi some embodiments, the present disclosure provides an expression vector comprising the polynucleotide. In some embodiments, the disclosure provides a host cell comprising a polynucleotide or expression vector described herein.

[0023] In some embodiments, the present disclosure provides a method for producing an anti-ceramide antibody or antigen-binding fragment thereof described herein, or an anti-ceramide single-chain variable fragment (scFv) described herein, comprising introducing an expression vector of embodiment 98 into a host cell. In some embodiments, the present disclosure provides a method of inhibiting apoptosis 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 described herein, or an anti-ceramide single-chain variable fragment (scFv) described herein. In some embodiments, the apoptosis is associated with a disease selected from the group consisting of graft-versus-host disease, radiation sickness, GI syndrome, and autoimmune disease. In some embodiments, the disease is radiation sickness or GI syndrome, and the anti-ceramide antibody or antigen-binding fragment thereof is administered before the subject is exposed to radiation. In some embodiments, the disease is graft-versus-host disease, and the anti-ceramide antibody or antigen-binding fragment thereof is administered before the subject undergoes a transplant. In some embodiments, the transplant is a bone marrow transplant.

[0024] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered intravenously, intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intrasynovially, intrathecally, orally, topically, or via inhalation. In some embodiments, the present disclosure provides a method for alleviating apoptosis in a subject with GI syndrome, comprising administering to the subject a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof of any one of embodiments 1-89, or an anti-ceramide scFv of any one of embodiments 94-96, after the subject has been exposed to penetrating radiation. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered shortly after the subject has been exposed to penetrating radiation. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered within 24 hours after the subject has been exposed to penetrating radiation. In some embodiments, the disclosure provides a method for inhibiting apoptosis in a subject with GvHD, comprising administering to the subject a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof of any one of embodiments 1-89, or an anti-ceramide scFv of any one of embodiments 94-96, before the subject undergoes a transplant, or after the subject has undergone a transplant and before the onset of GvHD. In some embodiments, the transplant is a bone marrow transplant.

[0025] The accompanying drawings illustrate one or more embodiments of the present disclosure and, together with the description, serve to explain the principles of the exemplary embodiments of the present disclosure. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 shows a dose-response curve for CD28-induced ceramide-rich platform (CRP) formation in T cells with increasing concentrations of 6B5 scFv. [Figure 2A] FIG. 1 shows a dose-response curve for radiation-induced T cell apoptosis with increasing concentrations of humanized 6B5 scFv. [Figure 2B] FIG. 1 shows dose-response curves for radiation-induced T cell apoptosis at increasing concentrations of VH4-VL1, VH5-VL1, and the parent scFv, murine 6B5 scFv. [Figure 2C] FIG. 1 shows dose-response curves for radiation-induced T cell apoptosis at increasing concentrations of VH4-VL1, VH5-VL1, and the parent scFv, murine 6B5 scFv. [Figure 2D] FIG. 1 shows dose-response curves for radiation-induced T cell apoptosis with increasing concentrations of VH4-VL1 and VH5-VL1. [Figure 2E] FIG. 1 shows the percentage of radiation-induced apoptotic T cells given the concentrations of VH4-VL1 and VH5-VL1. [Figure 3A]Figure 1 shows the number of apoptotic endothelial cells per villi over time in mice exposed to an LD90 dose of 15 Gray units (Gy). Numbers adjacent to the symbols represent the number of animals measured per time point. Gy: Gray, absorbed radiation unit. [Figure 3B]

[0023] Figure 1 shows the number of apoptotic endothelial cells per villi over time in mice exposed to an LD90 dose of 15 Gray units (Gy) and left untreated or treated with VH2-VL1 (SEQ ID NO: 51) 24 hours after irradiation. Numbers adjacent to the symbols represent the number of animals measured per time point. Gy: Gray absorbed radiation units. [Figure 4A] Figure 1 shows an amino acid sequence alignment for the humanized VH region. CDRs are underlined. Amino acids in the CDRs that differ from the parental CDR mouse CDR sequences are shown in bold. Amino acids in the framework regions that differ from the human germline framework sequences are shown in bold. [Figure 4B] Figure 1 shows an amino acid sequence alignment for the humanized VL region, with CDRs underlined. DETAILED DESCRIPTION OF THE INVENTION

[0027] Details about the present disclosure are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, exemplary methods and materials are described herein. Other features, objects, and advantages of the present disclosure will be apparent from the description and claims. In this specification and the appended claims, the singular also includes the plural unless the context clearly dictates otherwise. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited herein are incorporated herein by reference in their entirety.

[0028] definition 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 within the variable region of the Ig molecule. As used herein, the term "antibody" 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 heavy chain polypeptide 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., proteins 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.

[0029] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing 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, the antigen-binding fragments of the antibodies described herein can contain one, two, three, four, five, or all six CDRs of the variable heavy (VH) and variable light (VL) chains derived from an antibody that specifically binds to ceramide. Antigen-binding fragments include portions of full-length antibodies, generally proteins containing the antigen-binding or variable regions thereof, such as Fab, F(ab'), Fab', Fv fragments, minibodies, diabodies, single-domain antibodies (dAbs), single-chain variable fragments (scFvs), multispecific antibodies formed from antibody fragments, and any other modified configuration of an immunoglobulin molecule, which contain an antigen-binding site or antigen-binding fragment with 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, hi other embodiments, the antigen-binding fragments are further modified to increase serum half-life.

[0030] The term "F(ab)" refers to the two protein fragments resulting from the proteolytic cleavage of an IgG molecule by the enzyme papain. Each F(ab) comprises a covalent heterodimer of a VH chain 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. The term "F(ab')2" refers to the protein fragment of IgG produced by proteolytic cleavage by the enzyme pepsin. Each F(ab')2 fragment contains two F(ab') fragments and is therefore a bivalent antigen-binding fragment. An "Fv fragment" refers to a noncovalent VH::VL heterodimer comprising an antigen-binding site that retains much of the antigen recognition and binding capabilities 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 generated by preferential proteolytic cleavage of IgM, or in rare cases, IgG or IgA immunoglobulin molecules. More commonly, however, Fv fragments are derived using recombinant methods known in the art.

[0031] Also included herein are minibodies comprising an scFv joined to a CH3 domain (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, for example, Ward, E.S. 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.

[0032] The term "diabody" refers to a bispecific antibody in which a VH domain and a VL domain are expressed as a single polypeptide chain using a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing the domains to pair with the complementary domains on 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)). 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 desired targets based on automated high-throughput selection of B cells (see, e.g., WO06 / 079372).

[0033] 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. In some embodiments, the term "chimeric antibody," as used herein, refers to monoclonal antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to 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.

[0034] The term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of immunoglobulin heavy (VH) and light (VL) chains connected by a short linker peptide of 10 to 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. Numerous methods have been described to identify chemical structures for converting naturally aggregated (but chemically separated) light and heavy chain polypeptides from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405 by Huston et al.; and U.S. Pat. No. 4,946,778 by Ladner et al.

[0035] The term "antigen" refers to a molecule or portion of a molecule that can be bound by an antibody or antigen-binding fragment thereof and can be used to generate antibodies in an animal that are capable of binding to an epitope of the antigen. An antigen can have one or more epitopes. Embodiments herein contemplate the use of ceramide, or ceramide conjugated to a hapten, as an antigen. The term "epitope" refers to the region of an antigen to which an antibody binds. Epitopic determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0036] As used herein, "specifically binds to" means that the antibody or antigen-binding fragment thereof does not bind significantly to other components or antigens present in a mixture, but binds to the target molecule with at least 10 5 M -1 Reference herein to an anti-ceramide antibody refers to an antibody or antigen-binding fragment thereof that specifically binds to ceramide. Binding affinity (Ka) is defined as 1 / M or M -1 "Affinity" refers to the equilibrium association of a specific binding interaction, expressed in units of K. Antibodies or antigen-binding fragments thereof can be classified as "high affinity" antibodies or antigen-binding fragments thereof and "low affinity" antibodies or antigen-binding fragments thereof. A "high affinity" antibody or antigen-binding fragment thereof has a Ka of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 or at least 10 13 M -1 An antibody or antigen-binding fragment thereof with a "low affinity" refers to an antibody or antigen-binding fragment thereof with a Ka of 10 7 M -1 Below, 10 6 M -1 Below, 10 5 M -1 Alternatively, affinity can be defined as the equilibrium dissociation constant (Kd) for a particular binding interaction, in units of M (e.g., 10 -5 M~10 -13 M, or about 500 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, or about 5 nM). The affinity of binding domain polypeptides and single chain polypeptides in accordance with the present disclosure can be readily determined using routine techniques (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0037] In the art, "conservative substitution" is recognized as the substitution of one amino acid with another amino acid with similar properties. Exemplary conservative substitutions are well known in the art (see, for example, PCT Application Publication No. 97 / 09433, published March 13, 1997, p. 10; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).

[0038] As used herein, the term "derivative" refers to the modification of one or more amino acid residues of a peptide by chemical or biological means, with or without the involvement of an enzyme, for example, by glycosylation, alkylation, acylation, ester formation, or amide formation. As used herein, a polypeptide or polynucleotide from which another polypeptide or polynucleotide is derived is referred to as a "parent" or "reference" polynucleotide or polypeptide. For example, a humanized antibody may be derived from a murine antibody that is the parent antibody.

[0039] As used herein, the term "variant" or "variants" refers to polynucleotides or polypeptides whose sequences differ from those of a reference polynucleotide or polypeptide but retain the essential properties of the parent polynucleotide or polypeptide. Generally, the polynucleotide or polypeptide sequence of a variant is similar overall, and in many regions, identical, to that of the parent polynucleotide or polypeptide. For example, a variant polynucleotide or polypeptide may exhibit at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity compared to the parent polynucleotide or polypeptide.

[0040] 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 as the corresponding position in the comparison sequence, the sequences are said to be identical at this position. The percentage of sequence identity is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs, resulting in the number of identical positions. The number of identical positions is then divided by the total number of positions in the comparison region and multiplied by 100 to obtain the percentage of sequence identity. The percentage of sequence identity is determined by comparing two optimally aligned sequences across the comparison region. Comparison windows 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. Comparison windows 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 amino acids in length. To optimally align sequences for comparison purposes, portions of the polynucleotide or polypeptide sequences within the comparison window may contain additions or deletions, called gaps, while holding the reference sequence constant. An optimal alignment is one that, despite gaps, results in the greatest number of "identical" positions possible between the reference and comparison sequences.The percentage "sequence identity" between two sequences can be determined using the version of the "BLAST 2 Sequences" program, available as of September 1, 2004, from the National Center for Biotechnology Information, which incorporates the BLASTN program (for comparison of nucleotide sequences) and the BLASTP program (for comparison of polypeptide sequences), which are programs based on the algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing "Sequences," the parameters that were the default parameters as of September 1, 2004 may be used for all other required parameters, including, but not limited to, word size (3), gap opening penalty (11), gap extension penalty (1), gap dropoff (50), expectation value (10), and matrix selection. Two nucleotide or amino acid sequences are considered to have "substantially similar sequence identity" or "substantial sequence identity" if they 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 relative to each other.

[0041] The term "substantially identical" refers to a polypeptide sequence that contains a sufficient number of identical amino acids as a second polypeptide sequence such that the two polypeptide sequences have similar activity. Substantially identical polypeptides are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical in amino acid sequence.

[0042] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from a portion of an antibody capable of binding to cellular Fc receptors and / or the C1q component of complement, thereby mediating the antibody's effector functions. Fc stands for "fragment crystalline," a fragment of an antibody that readily forms protein crystals. Originally, significantly different protein fragments, described by proteolytic digestion, may define the overall general structure of immunoglobulin proteins. As originally defined, the Fc region is a homodimeric protein comprising two polypeptides associated by disulfide bonds, each containing a hinge region, a CH2 domain, and a CH3 domain. However, more recently, the terms "Fc region" or "Fc domain" have been applied to a single-chain monomeric entity consisting of a CH3, a CH2, and at least a portion of the hinge sufficient to form a disulfide-linked dimer with a second such chain. As used herein, the term "Fc region" or "Fc domain" refers, as such and depending on the context, to a dimeric form or to individual monomers that associate to form a dimeric protein. For reviews 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 naturally occurring sequence variants.

[0043] 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 (e.g., CH1, CH2, CH3) of an immunoglobulin. 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 constant domains. 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 derived from the light and heavy chains of an antibody, respectively. The variable binding regions are composed of distinct, well-defined subregions known as "complementarity-determining regions" (CDRs) and "framework regions" (FRs).

[0044] The term "immunoglobulin light chain constant region" (also referred to as "light chain constant domain" or "CL") is the constant region derived from the light chain of an antibody. The term "immunoglobulin heavy chain constant region" (also referred to as "heavy chain constant domain" or "CH") refers to the constant region derived from the heavy chain of an antibody. Depending on the antibody isotype, the CH is further divided into CH1, CH2, and CH3 domains (IgA, IgD, IgG) or CH1, CH2, CH3, and CH4 domains (IgE, IgM). As used herein, the term "complementarity determining region" or "CDR" refers to an immunoglobulin (antibody) molecule. There are three CDRs per variable domain: 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.

[0045] In some embodiments, "hinge" or "hinge region" refers to a polypeptide derived from an immunoglobulin hinge region and disposed between an antigen-binding domain (e.g., a ceramide-binding domain) and an immunoglobulin constant region within a polypeptide described herein. A "wild-type immunoglobulin hinge region" refers to the naturally occurring upper and middle hinge amino acid sequence found in an antibody heavy chain and inserted between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or between and connecting the CH1 and CH3 domains (for IgE and IgM). In certain embodiments, the wild-type immunoglobulin hinge region sequence is a human sequence and may include a human IgG hinge region (e.g., in addition to an IgG1 hinge region, an IgG2 hinge region, an IgG3 hinge region, or an IgG4 hinge region).

[0046] "Modified immunoglobulin hinge region" or "variant immunoglobulin hinge region" refers to a polypeptide hinge region with one or more mutations, substitutions, insertions, or deletions compared to a corresponding parent, wild-type immunoglobulin hinge region. Typically, a modified immunoglobulin hinge region that is a fragment of a wild-type immunoglobulin hinge region comprises the core hinge region of an IgG (e.g., a polypeptide comprising the sequence CXXC, where X is any amino acid), as disclosed in U.S. Patent Application Publication Nos. 2013 / 0129723 and 2013 / 0095097.

[0047] 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, an antibody binding fragment (e.g., a light chain variable region and a heavy chain variable region, Fab, scFv) is humanized. Non-human antigen-binding fragments have been "reshaped" (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), "hyperchimerized" (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 148:1149-1154), and "veneered" (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), including techniques known as CDR grafting (Jones et al., Nature 321:522 (1986)) and variations thereof. If derived from non-human sources, other regions of the antibody, such as the hinge region and constant region domains, may also be humanized.

[0048] As used herein, the term "pharmaceutically acceptable" generally refers to molecular entities and compositions that do not produce allergic or other serious adverse reactions when administered using routes known in the art. Molecular entities and compositions that are approved by a U.S. federal or state regulatory agency or are listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for use in mammals, and more particularly for use in humans, are considered "pharmaceutically acceptable."

[0049] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides comprising a polynucleotide may be RNA, DNA, or modified forms of any type of nucleotide, such as modified messenger RNA. The modifications include base modifications such as bromolysine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, and phosphoramidate. The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

[0050] As used herein, "polypeptide" or "protein" refers to a single, linear, and contiguous arrangement of covalently linked amino acids. A polypeptide may form one or more interchain disulfide bonds. The terms polypeptide and protein also encompass embodiments in which two polypeptide chains are linked together in a non-linear manner, such as via an interchain disulfide bond. As used herein, a protein or polypeptide may be an antibody or an antigen-binding antibody fragment. As used herein, the terms "transformation," "transfection," and "transduction" refer to the introduction of a polynucleotide into a cell. As used herein, the term "genetic transformation" refers to the introduction and incorporation of DNA, especially recombinant DNA, into a cell. The nucleic acid to be introduced may be introduced into the cell via an expression vector.

[0051] As used herein, the terms "treatment," "treating," or "ameliorating" refer to therapeutic or prophylactic / preventative treatment. Treatment is therapeutic if at least one symptom of a disease in the individual receiving the treatment is alleviated, or if the treatment may slow the progression of progressive disease in the individual or prevent the onset of further related disease. As used herein, the term "LD90" refers to the "90% lethal dose" or "median lethal dose" and is the amount of a substance required to kill 90% of a test population. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. As used herein, unless otherwise indicated, the term "and / or" refers to "and" or "or." Throughout this specification, unless the context requires otherwise, the word "comprisie" or variations such as "comprises" or "comprising" should 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.

[0052] Anti-ceramide antibody The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to ceramide. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. In other embodiments, the anti-ceramide antibody is a humanized 6B5 scFv antibody.

[0053] As used herein, the term "ceramide" refers to a lipid second messenger molecule composed of sphingosine and fatty acids. Ceramides regulate intracellular stress signaling through the reorganization of the cell membrane and the formation of lateral Lo (liquid-ordered) phase microdomains (a type of "raft" referred to as "ceramide-rich platforms"). These CRPs cluster activated receptor molecules (e.g., members of the TNF-receptor Hooper family, including Fas), thereby providing a feed-forward mechanism that ultimately results in signal amplification and transduction. The formation of CRPs is particularly important for Fas-mediated cell death, and ceramide-rich regions on the plasma membrane of target cells are crucial for susceptibility to cytotoxic T lymphocyte (CTL)-induced cell death. Exemplary ceramides are glycosylceramide, galactosylceramide, and gangliosides (oligosaccharide-linked ceramides).

[0054] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to ceramide, and comprises an immunoglobulin heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), and an immunoglobulin light chain variable region (VL) comprising a light chain complementarity determining region (CDR) 1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3), wherein HCDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1 and 2; HCDR2 comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, and 6; HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0055] The CDRs of exemplary anti-ceramide antibodies are shown in Table 1. [Table 1]

[0056] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is an scFv comprising an immunoglobulin heavy chain variable region (VH) comprising heavy chain complementarity determining region (CDR) 1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and an immunoglobulin light chain variable region (VL) comprising light chain complementarity determining region (CDR) 1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), wherein HCDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1 and 2; HCDR2 comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, and 6; HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 3; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0057] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 5; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0058] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 6; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0059] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 6; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0060] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 3; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0061] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 5; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 6; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0062] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 3; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0063] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 5; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain comprises or consists of an amino acid sequence that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-21.

[0064] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VL chain comprises or consists of an amino acid sequence that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-24.

[0065] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-21. and the VL chain comprises or consists of an amino acid sequence that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 24.

[0066] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-21. and the VL chain comprises or consists of an amino acid sequence that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 24.

[0067] [Table 2]

[0068] In some embodiments, the anti-ceramide antibody or antigenic fragment thereof comprises the VH amino acid sequence of SEQ ID NO: 17 and the VL amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or antigenic fragment thereof comprises the VH amino acid sequence of SEQ ID NO: 17 and the VL amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 17 and the amino acid sequence of a VL of SEQ ID NO: 24. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 18 and the amino acid sequence of a VL of SEQ ID NO: 22. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 18 and the amino acid sequence of a VL of SEQ ID NO: 23. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 18 and the amino acid sequence of a VL of SEQ ID NO: 24. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 19 and the amino acid sequence of a VL of SEQ ID NO: 22. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 19 and the amino acid sequence of a VL of SEQ ID NO: 23. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 19 and the amino acid sequence of a VL of SEQ ID NO: 24. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 20 and the amino acid sequence of a VL of SEQ ID NO: 22. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 20 and the amino acid sequence of a VL of SEQ ID NO: 23. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 20 and the amino acid sequence of a VL of SEQ ID NO: 24. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 21 and the amino acid sequence of a VL of SEQ ID NO: 22. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 21 and the amino acid sequence of a VL of SEQ ID NO: 23. In some embodiments, the antibody or antigenic fragment thereof comprises the amino acid sequence of a VH of SEQ ID NO: 21 and the amino acid sequence of a VL of SEQ ID NO: 24.

[0069] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 17, and an amino acid sequence of VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO:22. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO: 17 and the amino acid sequence of the VL consists of SEQ ID NO: 22.

[0070] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of a VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to SEQ ID NO: 17, and an amino acid sequence of a VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 23. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of a VH consisting of SEQ ID NO: 17, and an amino acid sequence of a VL consisting of SEQ ID NO: 23.

[0071] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 17, and an amino acid sequence of VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO:24. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO: 17 and the amino acid sequence of the VL consists of SEQ ID NO: 24.

[0072] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 18, and an amino acid sequence of VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 22. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO: 18 and the amino acid sequence of the VL consists of SEQ ID NO: 22.

[0073] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of a VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 18, and an amino acid sequence of a VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO:23. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO: 18 and the amino acid sequence of the VL consists of SEQ ID NO: 23.

[0074] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 18, and an amino acid sequence of VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO:24. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO: 18 and the amino acid sequence of the VL consists of SEQ ID NO: 24.

[0075] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 19, and an amino acid sequence of VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO:22. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO: 19 and the amino acid sequence of the VL consists of SEQ ID NO: 22.

[0076] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 19, and an amino acid sequence of VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO:23. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO: 19 and the amino acid sequence of the VL consists of SEQ ID NO: 23.

[0077] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of a VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 19, and an amino acid sequence of a VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO:24. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO: 19 and the amino acid sequence of the VL consists of SEQ ID NO: 24. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 20, and an amino acid sequence of VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 22. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO:20 and the amino acid sequence of the VL consists of SEQ ID NO:22.

[0078] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an amino acid sequence of VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO:21, and an amino acid sequence of VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO:22. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO:21 and the amino acid sequence of the VL consists of SEQ ID NO:22.

[0079] In some embodiments, the anti-ceramide antigen-binding fragment is an scFv comprising an amino acid sequence of a VH that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO: 18, and an amino acid sequence of a VL that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identical to SEQ ID NO:22. In some embodiments, the anti-ceramide scFv comprises a VH chain and a VL chain, wherein the amino acid sequence of the VH chain consists of SEQ ID NO:18 and the amino acid sequence of the VL consists of SEQ ID NO:22.

[0080] In some embodiments, the anti-ceramide antigen-binding fragment is an scFv. In some embodiments, the anti-ceramide scFv comprises an amino acid sequence that is at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-61. In some embodiments, the anti-ceramide scFv consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-61. In some embodiments, the anti-ceramide scFv comprises or consists of the amino acid sequence of SEQ ID NO: 48 or 51.

[0081] In some embodiments, the anti-ceramide scFv comprises the following structure: VH-linker-VL or VL-linker-VH. As used herein, the term "linker" generally refers to a short polypeptide sequence connecting two subdomains of a polypeptide. Non-limiting examples of linkers include flexible linkers containing glycine-serine repeats, and linkers derived from (a) the interdomain region of a transmembrane protein (e.g., a type I transmembrane protein); (b) the stalk region of a type II C-lectin; or (c) an immunoglobulin hinge. In some embodiments, the linker provides a spacer function compatible with the interaction of the two binding subdomains so that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. In certain embodiments, the linker is composed of 5 to about 35 amino acids, e.g., about 15 to about 25 amino acids. Exemplary linkers are shown in Table 3.

[0082] [Table 3]

[0083] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is produced with an N-terminal signal sequence that facilitates secretion of the scFv protein. In some embodiments, the signal sequence comprises SEQ ID NO: 75. Those skilled in the art will understand that these signal sequences are not part of the mature secreted protein. Thus, in some embodiments, the signal sequence of SEQ ID NO: 75 is cleaved from the mature protein of the anti-ceramide antibody or antigen-binding fragment.

[0084] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is produced with a C-terminal tag sequence that facilitates purification during production. In some embodiments, the C-terminal tag comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is produced without a C-terminal tag sequence. Thus, in some embodiments, the amino acid sequence of the anti-ceramide antibody or antigen-binding fragment thereof does not comprise the C-terminal tag of SEQ ID NO: 76.

[0085] Exemplary amino acid sequences of anti-ceramide scFvs are presented in Table 4 below. CDRs of the scFvs are indicated by underlined text. Linker sequences are indicated by italicized text. Signal sequences are indicated by bolded text. C-terminal tag sequences are indicated by bolded and italicized text.

[0086] [Table 4] TIFF0007809067000005.tif244170

[0087] In some embodiments, the anti-ceramide antibody is a full-length antibody comprising a full-length antibody light chain and a full-length antibody heavy chain. In some embodiments, the anti-ceramide antibody is an IgG isotype (e.g., IgG1, IgG2, IgG3, IgG4). In some embodiments, the Fc domain of the anti-ceramide antibody comprises a wild-type IgG amino acid sequence. Such sequences are known in the art (see, e.g., Shields et al., J Biol Chem, (2001) 276:9; 6591-6604). In some embodiments, the CH2 or CH3 domain of the Fc domain comprises one or more amino acid mutations that alter the function and / or stability of the antibody. For example, in some embodiments, the Fc domain of the anti-ceramide antibody described herein lacks or has minimal effector function, while retaining the ability to bind to some Fc receptors, such as the neonatal Fc receptor (FcRn), and retaining a relatively long half-life in vivo. In some embodiments, the described anti-ceramide antibodies do not result in or substantially reduce the induction of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement activation, and / or complement-dependent cytotoxicity (CDC). Such mutations are known in the art (see, for example, Shields et al., J Biol Chem, (2001) 276:9;6591-6604; Arduin et al., Mol Immunol (2015) 63:2;456-463; Vafa et al., Methods (2014) 65:1;114-126).

[0088] In some embodiments, the described anti-ceramide antibodies are IgG1 isotype antibodies, in which the IgG1 constant region has mutations at one or more of the following positions: 228 (S228), 234 (L234), 235 (L235), 237 (G237), 297 (N297), 318 (E318), 320 (K320), 322 (K322), or any combination thereof (numbering according to EU). In some embodiments, the IgG1 Fc domain has mutations L234A and L235A. In some embodiments, the IgG1 Fc domain has mutation S228P.

[0089] Unless otherwise specified, the positions of amino acid residues in immunoglobulin molecules used herein are numbered according to EU numbering (Ward et al., 1995 Therap. Immunol. 2:77-94). Other numbering systems for amino acid positions in antibodies are known in the art, such as the IMGT system (Brochet et al., Nucl. Acids Res. (2008) 36, W503-508) and the Kabat numbering system (Kabat, Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: Public Health Service, National Institutes of Health (1991)). Methods and information for converting between one numbering system and another are known in the art. See, for example, the IMGT Scientific Chart - Correspondence between C numberings, available at imgt.org. In certain embodiments, anti-ceramide antibodies and their antigen-binding fragments can be prepared using standard molecular biology methods to select antibodies with desired specificity. In some embodiments, anti-ceramide antibodies and their antigen-binding fragments are produced using recombinant DNA technology. Procedures for recombinant protein expression and purification are well established in the art.

[0090] Polynucleotide and Protein Expression Methods The present disclosure also includes polynucleotides (e.g., DNA or RNA) encoding the anti-ceramide antibodies and antigen-binding fragments thereof of the present disclosure. In some embodiments, the polynucleotides encode polypeptides substantially identical to those listed in Tables 1, 2, 3, and 4. In some embodiments, the polynucleotides encode polypeptides that are at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to those listed in Tables 1, 2, 3, and 4. The polynucleotides of the present disclosure also include complementary nucleic acids. In some cases, the sequences will be perfectly complementary (no mismatches) when aligned. In other cases, there may be up to about 20% mismatches in the sequences. The polynucleotide sequences presented herein may be developed using codon optimization, degenerate sequences, silent mutations, and other DNA methods to optimize expression in a particular host, and the present disclosure encompasses such sequence modifications.

[0091] In some embodiments, the disclosure provides an isolated polynucleotide encoding an anti-ceramide antibody or antigen-binding fragment thereof, the isolated polynucleotide being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 65-73. In some embodiments, the disclosure provides an isolated polynucleotide encoding an anti-ceramide antibody or antigen-binding fragment thereof, the isolated polynucleotide comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 65-73.

[0092] In some embodiments, the polynucleotide of the present disclosure is inserted into a nucleic acid vector. The nucleic acid vector may be a viral vector or a non-viral vector, such as a plasmid. Vectors include, but are not limited to, plasmids, phagemids, cosmids, transposons, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. In some embodiments, the vector is a plasmid selected from pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). In some embodiments, the vector is selected from the group consisting of vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984, and WO95 / 00655); adeno-associated virus (see, e.g., U.S. Pat. No. 7,078,387; Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al,, PNAS 94:6916 6921 , 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir.(1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and the like. Examples of vectors are the pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.

[0093] In some embodiments, a polynucleotide of the disclosure is inserted into a nucleic acid vector and operably linked to one or more regulatory sequences that control transcription, such as a promoter, enhancer, terminator, inducer, or repressor.Non-limiting examples of suitable eukaryotic promoters (promoters functional in eukaryotic cells) include the cytomegalovirus (CMV) immediate-early promoter, herpes simplex virus (HSV) thymidine kinase promoter, simian virus 40 (SV40) promoters (e.g., the early SV40 promoter and the late SV40 promoter), spleen focus-forming virus (SFFV) promoter, and long terminal repeat (LTR) promoters derived from retroviruses. repeats) (e.g., Moloney murine leukemia virus (MoMLV) LTR promoter or Rous sarcoma virus (RSV) LTR), herpes simplex virus (HSV) (thymidine kinase) promoter, H5 promoter, P7.5 promoter, and P11 promoter from vaccinia virus, elongation factor 1 alpha (EF1α) promoter, early growth response 1 (EGR1) promoter, ferritin H (FerH) promoter, ferritin L (FerL) promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, heat shock 70 kDa protein 5 (HSPA5) promoter, heat shock protein 90 kDa beta, member 1 (HSP90B1) promoter, heat shock protein 70 kDa (HSP70) promoter, β-kinase (β-kin) promoter, and the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C (UBC) promoter, phosphoglycerate kinase 1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deleted dl587rev primer binding site substitution (MND) promoter, and eukaryotic promoter derived from mouse metallothionein-1 (mouse metallothionein-1).

[0094] In some embodiments, the vector is introduced into a host cell to express the anti-ceramide antibody or its antigen-binding fragment. Thus, proteins for use in the present disclosure can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are cell types that are transformed or transfected with exogenous DNA and grown in culture, including bacterial cells, fungal cells, and cultured higher eukaryotic cells (including cultured cells of multicellular organisms), particularly cultured mammalian cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into various host cells are disclosed by Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Ausubel et al., Short Protocols in Molecular Biology (4th ed., John Wiley & Sons, 1999).

[0095] Gene products encoded by the polynucleotides of the present disclosure are expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian, and mammalian systems. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster blue-green algae cells (CHO-K1; ATCC CCL61; CHODG44; CHO DXB11 (Hyclone, Logan, UT); see also, e.g., Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650), and mouse embryonic cells (NIH-3T3; ATCC CRL 1658). Additional suitable cell lines are known in the art and are available from public depositories such as the American Type Culture Collection, Manassas, Virginia. Introduction of the DNA construct may use any convenient method, including, for example, conjugation, bacterial transformation, calcium precipitation of DNA, electroporation, fusion, transfection, infection with viral vectors, biolistics, and the like.

[0096] For example, for recombinant expression of the anti-ceramide antibodies or antigen-binding fragments thereof described herein, an expression vector will generally contain a nucleic acid segment operably linked to a promoter, encoding one or more of the amino acid sequences presented in Tables 1, 2, 3, and 4. The expression vector is introduced into host cells by conventional techniques, and the host cells are then cultured by conventional techniques to produce the encoded polypeptide to make the corresponding anti-ceramide antibody or antigen-binding fragment thereof.

[0097] To direct a recombinant protein into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence) is provided in the expression vector. The secretory signal sequence may be the native secretory signal sequence for the recombinant protein, may be from another secreted protein, or may be synthesized de novo. The secretory signal sequence is operably linked to the DNA sequence encoding the polypeptide, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are generally positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere within the DNA sequence of interest (see, e.g., U.S. Pat. Nos. 5,037,743 and 5,143,830). In some embodiments, the secretory signal sequence is SEQ ID NO: 75.

[0098] Cultured mammalian cells are suitable hosts for producing the recombinant polypeptides and proteins (eg, anti-ceramide antibodies and antigen-binding fragments thereof) of the present disclosure. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran-mediated transfection (Ausubel et al., supra), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, in US Pat. Nos. 4,713,339; 4,784,950; 4,579,821; and 4,656,134.

[0099] Host cells transformed or transfected to produce the polypeptides and proteins (e.g., ceramide-binding polypeptides) of the present disclosure are cultured according to conventional procedures in a culture medium containing nutrients and other components required for growth of the selected host cells. A variety of suitable media, including chemically defined and complex media, are known in the art and generally include a carbon source, a nitrogen source, basic amino acids, vitamins, and minerals. The medium may also contain components such as growth factors or serum, as required. Growth media generally select for cells containing the exogenously added DNA, for example, by drug selection or by deficiency of essential nutrients complemented by a selectable marker carried on a vector or co-transfected into the host cells.

[0100] The anti-ceramide antibodies and antigen-binding fragments thereof of the present disclosure can be purified by conventional protein purification methods, typically via a combination of chromatographic methods. See generally Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988); Scopes, Protein Purification: Principles and Practice (Springer-Verlag, New York 1994). Proteins containing the Fc region of an immunoglobulin can be purified by affinity chromatography against immobilized Protein A or Protein G. Further purification steps, such as gel filtration, can be used to obtain the desired level of purity or to effect desalting, buffer exchange, etc.

[0101] Methods for producing humanized antibodies The present disclosure provides humanized anti-ceramide antibodies and their antigen-binding fragments. The humanized antibodies have the same or similar binding specificity and affinity as mouse antibodies or other non-human antibodies that serve as starting materials for constructing humanized antibodies. A preferred antibody for humanization is the mouse 6B5 antibody, described in U.S. Patent Application Publication No. 2017-03334133, which is incorporated herein by reference in its entirety. The VH and VL sequences of the mouse 6B5 antibody are listed in Table 5 below. CDRs are indicated by underlined text. Linker sequences are indicated by italicized text. Signal sequences are indicated by bolded text. C-terminal tag sequences are indicated by bolded and italicized text.

[0102] [Table 5]

[0103] Substitution of mouse CDRs into a human variable domain framework is most likely to result in the retention of their correct spatial orientation if the human variable domain framework adopts a conformation that is the same as or similar to the mouse variable framework from which the CDRs were derived. This is achieved by human variable domains derived from human antibodies whose framework sequences share a high degree of sequence identity with the mouse variable framework domain from which the CDRs were derived. The heavy and light chain variable framework regions may be derived from the same or different human antibody sequences. The human variable framework region sequence may be that of a naturally occurring human antibody or a consensus sequence of several human antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993); and Carter et al., WO92 / 22653.

[0104] Once the donor mouse immunoglobulin CDRs and suitable acceptor human framework regions have been identified, the next step is to determine which, if any, residues from these components should be modified to optimize the properties of the resulting humanized antibody. Generally, substitution of human amino acid residues with mouse residues should be minimized, as the introduction of mouse residues increases the risk that the antibody will induce an anti-mouse antibody (HAMA) response in humans. Art-recognized methods for determining immune response can be performed to monitor HAMA responses in specific patients or during clinical trials.

[0105] In some embodiments, the selection of amino acid residues for modification is determined in part by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are described herein. Generally, molecular models are created starting from solved structures of immunoglobulin chains or domains thereof. The chains to be modeled are compared with chains or domains whose three-dimensional structures have been solved for amino acid sequence similarity, and the chains or domains showing the greatest sequence similarity are selected as the starting point for constructing the molecular model. Chains or domains that share at least 50% sequence identity are selected for modeling, and preferably, chains or domains that share at least 60%, 70%, 80%, 90%, or more sequence identity are selected for modeling. The solved starting structure is modified to allow for differences between the actual amino acids within the chains or domains of the modeled immunoglobulin and the amino acids within the chains or domains in the starting structure. The modified structures are then assembled into composite immunoglobulins. Finally, the model is refined by energy minimization and verifying that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0106] The selection of amino acid residues for modification may also be determined in part by consideration of the characteristics of the amino acid at a particular position, and in part by empirical observations of the effects of substitution or mutagenesis of particular amino acids. Further exemplary humanization methods that can be used to humanize the immunoglobulins of the present disclosure are described, for example, in Presta et al., J. Immunol., 151: 2623-2632 (1993); Carter et al., Proc. Natl. Acad. Sci. USA., 89: 4285-4289 (1992); Couto et al., Cancer Res., 55: 5973s-77s (1995); O'Conner et al., Protein Eng., 11: 321-328 (1998); and Antibody Engineering—Methods and Protocols by Lo, Vol. 248 (2004). Typically, the CDR regions in a humanized antibody are identical to the corresponding CDR regions of the mouse donor antibody. For example, in some embodiments, the amino acid sequences of the CDRs of humanized antibodies and fragments thereof are identical to the amino acid sequences of the CDRs of the mouse 6B5 antibody (e.g., SEQ ID NOS: 11-16).

[0107] In some embodiments, it may be desirable to modify one or more CDR regions to modify the antibody antigen-binding specificity and / or reduce the immunogenicity of the antibody. In some embodiments, one or more residues in the CDRs are altered to modify binding, to achieve a more favorable binding on-rate, a more favorable binding off-rate, or both, so that an ideal binding constant is achieved. Using this strategy, for example, 10 10 M -1Antibodies with high binding affinities, equal to or exceeding this level, can be achieved. The affinity maturation methods described herein can be used to alter parent CDR regions followed by screening the resulting binding molecules for desired binding changes. The methods can also be used to alter parent CDRs to reduce immunogenicity, minimizing or avoiding potential human anti-mouse antibody (HAMA) reactions. Thus, changes in binding affinity and immunogenicity upon CDR alteration can be monitored and assessed to achieve an antibody optimized for the best combination of binding and low immunogenicity (see, e.g., U.S. Pat. No. 6,656,467 and U.S. Patent Application Publication No. 20020164326). Furthermore, in some cases, one or more conservative amino acid substitutions of CDR residues can be made without significantly affecting the binding affinity of the resulting humanized immunoglobulin. Conservative substitutions are contemplated combinations such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr.

[0108] In some embodiments, humanized anti-ceramide antibodies and antigen-binding fragments thereof comprise one or more mutations compared to the corresponding CDR sequences of the murine 6B5 antibody (e.g., compared to one or more of SEQ ID NOs: 11-16). Exemplary mutations to the CDR amino acid sequences of the murine 6B5 CDRs are shown in bold text in Figure 4A.

[0109] The framework regions of humanized immunoglobulins are usually substantially identical, and more usually identical, to the framework regions of the human antibodies from which they are derived. In some embodiments, the humanized antibodies of the present disclosure comprise variable region framework sequences selected from human antibody genes (e.g., germline antibody gene segments) that contain one or more canonical CDR structural types that are the same as or similar to the canonical CDR structural types of the corresponding non-human antibody (e.g., a murine antibody) being humanized. See U.S. Patent No. 6,881,557 and Tan et al., Journal of Immunol 169:1119-1125 (2002) (incorporated by reference in their entirety for all purposes). Of course, many of the amino acids in the framework regions make little or no direct contribution to the specificity or affinity of an antibody. Thus, many individual conservative substitutions for framework residues can be tolerated without significantly altering the specificity or affinity of the resulting humanized immunoglobulin. Framework residues can be analyzed to determine which, if any, residues should be substituted to optimize the properties of the resulting humanized antibody. For example, computer modeling can be used to identify residues that are likely to directly or indirectly affect binding to the antigen.

[0110] Thus, in one embodiment, the variable framework regions of a humanized anti-ceramide antibody or antigen-binding fragment thereof share at least 85% sequence identity to human variable framework region sequences, or a consensus of such sequences. In some embodiments, the variable framework regions of a humanized anti-ceramide antibody or antigen-binding fragment thereof share at least 90%, preferably 95%, and more preferably 96%, 97%, 98%, or 99% sequence identity to human variable framework region sequences, or a consensus of such sequences.

[0111] In some embodiments, the humanized anti-ceramide antibody or antigen-binding fragment thereof has at least 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , or 10 10 M -1 The humanized antibody exhibits a specific binding affinity of approximately 100%. Typically, the upper limit of binding affinity of a humanized antibody for an antigen is within 3-, 4-, or 5-fold of that of the parent immunoglobulin. The lower limit of binding affinity is also often within 3-, 4-, or 5-fold of that of the parent immunoglobulin. Alternatively, the binding affinity can be compared to that of a humanized antibody without substitutions (e.g., an antibody having donor CDRs and acceptor FRs but no substitutions). In such cases, the binding of the optimized antibody (antibody with substitutions) is preferably at least 2- to 3-fold, or 3- to 4-fold, that of the unsubstituted antibody. To make the comparison, the activity of various antibodies can be determined, for example, by BIACORE (i.e., surface plasmon resonance using unlabeled reagents) or competitive binding assays.

[0112] Treatment The present application discloses a method of inhibiting cell death 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 of the present disclosure. In some embodiments, the antibody or antigen-binding fragment thereof is a humanized scFv that specifically binds to ceramide. In some embodiments, the cell death is associated with a disease selected from the group consisting of graft-versus-host disease, radiation sickness, GI syndrome, and autoimmune disease. In some further embodiments, the disease is radiation sickness or GI syndrome, and the anti-ceramide antibody or antigen-binding fragment thereof is administered before the subject is exposed to radiation.

[0113] Radiation targets both the gastrointestinal microvasculature and intestinal stem cell compartments. Microvascular endothelial dysfunction, detected as apoptosis 4 hours after radiation exposure, represents the primary lesion leading to GI syndrome. Endothelial dysfunction converts lesions into sublethal to lethal cycling crypt base columnar (CBC) cells, resulting in loss of regenerative crypts and enhanced GI toxicity. Immunohistochemistry and [H]TdR and BrdUrd labeling studies have revealed that crypt stem cell death does not occur acutely after radiation exposure. Rather, the earliest detectable response is a transient, dose-dependent delay in progression through the late S-phase checkpoint and mitotic arrest, apparently heralded by radiation-induced DNA double-strand breaks (DSBs). Rapid apoptotic death occurs in growth-arrested cells during the first 24 hours after irradiation at 12 Gy, accounting for 33% of total deaths. In mammalian cells, DNA DSBs activate DNA damage recognition / repair pathways and regulate cell cycle checkpoint activity. Arrest of intestinal stem cell mitosis is thought to represent a regulatory event in this pathway. Mitotic forms of death occur during the second 24 hours, representing 66% of total deaths. At this stage, no significant changes in crypt number throughout the intestinal periphery are evident; however, crypt size continues to decrease due to continued normal migration of transient / differentiated crypt cells from the crypts to the epithelial lining of the villi and loss of the villus apical end. Restoration of mitotic activity after 12–18 hours is associated with rapid depletion of crypt stem cell clonogens and an increase in crypt number throughout the intestinal periphery. Loss of stem cell clonogens leads to global collapse of the crypt-villus system, mucosal desquamation, and death due to GI syndrome.

[0114] Ceramide-mediated raft clustering is involved in radiation-induced apoptosis and clonogenic cell death. For many years, the clonogenic compartment of the gastrointestinal (GI) mucosa has been recognized as a specific and direct radiation target for the induction of GI injury.

[0115] The present disclosure is directed to methods for alleviating cell death in GI syndrome in a subject in need thereof. The method comprises administering a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the method comprises administering the anti-ceramide antibody or antigen-binding fragment thereof to the subject immediately after exposing the subject to penetrating radiation. In other embodiments, the method comprises administering the anti-ceramide antibody or antigen-binding fragment thereof to the subject within 1 hour after exposing the subject to penetrating radiation. In some embodiments, the method comprises administering the anti-ceramide antibody or antigen-binding fragment thereof to the subject within 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 18 hours after exposing the subject to penetrating radiation. In some embodiments, the method comprises administering the anti-ceramide antibody or antigen-binding fragment thereof to the subject within 24 hours after exposing the subject to penetrating radiation. In other embodiments, the method comprises administering the anti-ceramide antibody or antigen-binding fragment thereof to the subject within 30, 36, 42, 48, 54, 60, 66, or 72 hours after exposing the subject to penetrating radiation, hi other embodiments, the method comprises administering the anti-ceramide antibody or antigen-binding fragment thereof to the subject within 48, 36, 24, 18, 12, 10, 8, 6, 4, 2, or 1 hour, or within 45, 30, or 15 minutes before exposing the subject to penetrating radiation.

[0116] In some embodiments, the disease is graft-versus-host disease and the anti-ceramide antibody or antigen-binding fragment thereof is administered before the subject receives a transplant. In some embodiments, the transplant is a bone marrow transplant. In other embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered after the subject receives a transplant but before the onset of graft-versus-host disease. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered to a subject in need thereof after the onset of graft-versus-host disease in an amount effective to alleviate apoptosis in graft-versus-host disease.

[0117] The present disclosure also encompasses anti-ceramide antibodies and antigen-binding fragments thereof for the manufacture of a medicament for the treatment of a disorder characterized by the expression of ceramide (e.g., GI syndrome or GvHD). In some embodiments, the protein or polypeptide comprises an anti-ceramide binding domain and inhibits the formation of a ceramide-rich platform. In some embodiments, the present disclosure relates to a method for treating a disorder characterized by the expression of a ceramide-rich platform in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein or polypeptide (e.g., a ceramide-binding polypeptide) of the present disclosure that comprises a ceramide-binding domain that specifically binds to an epitope of human ceramide.

[0118] In some embodiments, the present disclosure provides a method of treating a patient with GI syndrome or GvHD, the method comprising administering to the patient an anti-ceramide antibody, or antigen-binding fragment thereof, comprising an amino acid sequence set forth herein (e.g., an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-61). In some embodiments, the present disclosure provides a method of treating a patient with GI syndrome or GvHD, the method comprising administering to the patient an anti-ceramide antibody, or antigen-binding fragment thereof, of SEQ ID NO: 48 or 51. In some embodiments, the anti-ceramide antibodies and antigen-binding fragments thereof prevent radiation-induced cell death or cell death associated with GvHD or autoimmune disease. Cell death can be measured by various means known in the art, including flow cytometry, immunofluorescence, and immunohistochemistry, which assess changes in marker expression in dying cells, cell counting, which assesses viability, and qPCR, which assesses changes in gene expression. In some embodiments, anti-ceramide antibodies and antigen-binding fragments thereof prevent cell death by inhibiting the formation of ceramide-rich platforms. For example, in some embodiments, anti-ceramide scFvs inhibit the formation of ceramide-rich platforms in irradiated Jurkat T cells (see Example 3 and Figure 1). In some embodiments, anti-ceramide antibodies or antigen-binding fragments thereof reduce the formation of ceramide-rich platforms by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0119] In some embodiments, anti-ceramide antibodies and antigen-binding fragments thereof inhibit cell death. For example, in some embodiments, anti-ceramide scFv inhibits cell death in irradiated Jurkat T cells (see Example 4 and Figures 2A-2D). In some embodiments, anti-ceramide antibodies or antigen-binding fragments thereof reduce cell death or apoptosis by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, for the treatment methods and uses described herein, the anti-ceramide antibody or antigen-binding fragment thereof is delivered in a manner consistent with conventional methods associated with the disease or disorder for which treatment is sought. In accordance with the disclosure herein, a therapeutically effective amount of a protein or polypeptide is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.

[0120] In preventive applications, pharmaceutical compositions or medicaments containing anti-ceramide antibodies or antigen-binding fragments thereof are administered to patients susceptible to or at risk of a particular disorder in an amount sufficient to eliminate the disorder, reduce the risk of developing the disorder, or delay the onset of the disorder. In therapeutic applications, compositions or medicaments containing proteins of the present disclosure are administered to patients suspected of or already suffering from such a disorder in an amount sufficient to cure the disorder or at least partially halt the symptoms and complications of the disorder. An amount sufficient to achieve this is referred to as a therapeutically effective dose or therapeutically effective amount. In both preventive and therapeutic regimens, the agent is typically administered in several doses until a sufficient response (e.g., inhibition of inappropriate angiogenic activity) is achieved. Typically, the response is monitored, and repeated administrations are administered when the desired response begins to wane.

[0121] For administration, anti-ceramide antibodies or antigen-binding fragments thereof can be formulated as pharmaceutical compositions. Pharmaceutical compositions can include (i) a ceramide-binding polypeptide; and (ii) a pharmaceutically acceptable carrier, diluent, or excipient. Pharmaceutical compositions containing ceramide-binding polypeptides can be formulated according to known methods for preparing pharmaceutically useful compositions in which a therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier, diluent, or excipient. A carrier is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other 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 comprise one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, and the like.

[0122] Pharmaceutical compositions comprising the polypeptides or proteins described herein may be formulated into a dosage form selected from the group consisting of oral unit dosage forms, intravenous unit dosage forms, intranasal unit dosage forms, suppository unit dosage forms, intradermal unit dosage forms, intramuscular unit dosage forms, intraperitoneal unit dosage forms, subcutaneous unit dosage forms, epidural unit dosage forms, sublingual unit dosage forms, and intracerebral unit dosage forms. The oral unit dosage form may be selected from the group consisting of tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained-release formulations, aerosols, and sprays. A pharmaceutical composition comprising an anti-ceramide antibody or its antigen-binding fragment can be administered to a subject in a therapeutically effective amount. According to the methods of the present disclosure, the anti-ceramide antibody or its antigen-binding fragment can be administered to a subject by various administration routes, including, for example, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, and oral routes. For prevention and treatment purposes, an agonist (e.g., a ceramide-binding polypeptide) can be administered to a subject by a single bolus delivery, continuous delivery over an extended period of time (e.g., continuous transdermal delivery), or by a repeated administration protocol (e.g., hourly, daily, weekly, or monthly).

[0123] The effective dose of the composition of the present disclosure varies 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 drugs administered, whether the treatment is a preventive or therapeutic treatment, and the specific activity of the composition itself and its ability to induce the desired response in an individual. Typically, the patient is a human, but for some diseases, the patient may be a non-human mammal. Typically, the administration regimen is adjusted to produce the optimal therapeutic response, i.e., to optimize stability and efficacy.

[0124] In this context, the determination of an effective dosage is typically guided by determining an effective dosage and administration protocol that significantly reduces the occurrence or severity of the target disorder in a model subject based on animal model studies and subsequent human clinical trials. Thus, as used herein, the term "therapeutically effective amount" refers to the amount of compound that achieves the desired biological or therapeutic effect, i.e., prevents, alleviates, or improves one or more symptoms of the listed disease to be treated or prevented. For example, the therapeutically effective amount of an antibody or antigen-binding fragment thereof will depend on the condition being treated, the severity and course of the condition, whether the antibody is administered for prevention or treatment, previous treatments, the patient's medical history and response to the antibody, the type of antibody or antigen-binding fragment used, and the discretion of the attending physician. An anti-ceramide antibody or antigen-binding fragment thereof may be administered to a patient at one time or over a series of treatments, and may be administered to a patient at any time after diagnosis. The anti-ceramide antibody or antigen-binding fragment thereof may be administered as the sole treatment or may be administered in conjunction with other drugs or therapies useful in treating the condition in question.

[0125] In some embodiments, a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof is between about 1 ng / kg body weight per day and about 100 mg / kg body weight per day. In some embodiments, the range of antibody administered is between about 1 ng / kg body weight per day and about 1 μg / kg body weight per day, 1 ng / kg body weight per day and about 100 ng / kg body weight per day, 1 ng / kg body weight per day and about 10 ng / kg body weight per day, 10 ng / kg body weight per day and about 1 μg / kg body weight per day, 10 ng / kg body weight per day and about 100 ng / kg body weight per day, 100 ng / kg body weight per day and about 1 μg / kg body weight per day, 100 ng / kg body weight per day and about 1 μg / kg body weight per day. and 10 mg / kg body weight per day to about 100 mg / kg body weight per day. Dosages within this range can be achieved by single or multiple administrations, including, for example, multiple daily administrations, or daily, weekly, biweekly, or monthly administrations. Anti-ceramide antibody or its antigen-binding fragment may be administered as a single dose by bolus injection or continuous infusion, or as multiple doses by bolus injection or continuous infusion, as appropriate or as indicated.Multiple doses may be administered, for example, multiple times daily, once daily, once every 2, 3, 4, 5, 6, or 7 days, once weekly, once every 2, 3, 4, 5, or 6 weeks, or once monthly.However, other dosing regimens may also be useful.The progress of this treatment can be easily monitored by conventional techniques.

[0126] For administration to adult human patients, a therapeutically effective amount may be administered at a dose ranging from 0.0006 mg to 1000 mg per dose, including, but not limited to, 0.0006 mg per dose, 0.001 mg per dose, 0.003 mg per dose, 0.006 mg per dose, 0.01 mg per dose, 0.03 mg per dose, 0.06 mg per dose, 0.1 mg per dose, 0.3 mg per dose, 0.6 mg per dose, 1 mg per dose, 3 mg per dose, 6 mg per dose, 10 mg per dose, 30 mg per dose, 60 mg per dose, 100 mg per dose, 300 mg per dose, 600 mg per dose, and 1000 mg per dose, and a course of treatment may involve multiple, typically daily, consecutive administrations. The anti-ceramide antibody or antigen-binding fragment thereof may be administered at different times of day. In one embodiment, the optimal therapeutic dose may be administered at night. In another embodiment, the optimal therapeutic dose may be administered in the morning. As expected, the dosage will depend on the size, age, and condition of the patient.

[0127] The dosage of a pharmaceutical composition containing an anti-ceramide antibody or its antigen-binding fragment can be varied by the attending physician to maintain a desired concentration at the target site. For example, if an intravenous delivery method is selected, the local bloodstream concentration of the drug at the target tissue can be between about 0.01 and 50 nM, and in some cases between about 1.0 nM and 10, 15, or 25 nM, as an estimated measure of the subject's condition and response. Higher or lower concentrations can be selected depending on the delivery method, e.g., transepidermal delivery versus delivery to a mucosal surface. Dosages should also be adjusted based on the release rate of the administered formulation, e.g., a nasal spray versus a powder, sustained-release oral or injectable particles, transdermal formulation, etc. To achieve the same serum concentration level, for example, sustained-release particles with a release rate of 5 nM (under standard conditions) would be administered at approximately twice the dose of particles with a release rate of 10 nM.

[0128] A pharmaceutical composition comprising an anti-ceramide antibody or an antigen-binding fragment thereof can be provided as a kit containing a container containing the pharmaceutical composition described herein. The pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder to be reconstituted before injection. Alternatively, such a kit can include a dry powder dispenser, a liquid aerosol generator, or a nebulizer for administering the pharmaceutical composition. Such a kit can further include written information about the indications and usage of the pharmaceutical composition. [Example]

[0129] The present disclosure is further illustrated by the following examples, which should not be construed as limiting. All references, patents, and patent application publications, as well as figures, cited throughout this application are hereby incorporated by reference.

[0130] Example 1 Humanization and production of 6B5 antibody The goal of this study was to maximize the amount of human sequence in the humanized ceramide antibody while retaining the original specificity of the murine 6B5 antibody. Antibody humanization design: Humanization design of the murine 6B5 parent antibody was performed using computational analysis. Briefly, homology modeling was used to generate a 3D structure of the parent 6B5 antibody, and a profile of the parent antibody was created based on the 3D model. Two human heavy chain (HC) acceptor frameworks and two human light chain (LC) acceptor frameworks were selected based on overall sequence identity across the frameworks, matching interface positions, similarly classified canonical CDR positions, and the presence of N-glycosylation sites. Humanized antibodies were then designed by creating multiple hybrid sequences in which selected portions of the parent antibody sequence were fused with the human framework sequences. Using the 3D structure of the parent 6B5 antibody, these humanized sequences were systematically analyzed by visual inspection and computational modeling to isolate sequences most likely to retain antigen binding. Three humanized HCs were designed based on two different human HC acceptor frameworks, and three humanized LCs were designed based on two different human LC acceptor frameworks. The framework combinations are shown in Table 6 below.

[0131] [Table 6]

[0132] Humanized VH1 and VL1 utilize the respective first frameworks and contain maximum human sequence with minimal parental antibody framework sequence. VH2 and VL2 use the same frameworks as VH1 and VL1 but contain additional parental sequence. VH3 and VL3 utilize the respective second frameworks and are similar to humanized VH2 and VL2 but also contain additional parental sequence fused to the human framework. The humanized heavy and light chains described above were combined to create a fully humanized antibody. The combinations of humanized heavy and light chains were examined for their expression levels and antigen-binding affinities to identify antibodies that behave similarly to the murine 6B5 parent antibody. Calculation of humanity scores for engineered antibodies: A tool was developed to calculate the humanity score of engineered humanized antibodies (Gao et al., BMC Biotechnology 2013, 13:55). The T20 score represents the degree of humanity of an antibody by analyzing the primary sequence of the variable region. The T20 scores for the heavy and light chains of murine 6B5 and humanized 6B5 are shown in Tables 7 and 8 below.

[0133] [Table 7]

[0134] [Table 8]

[0135] For the full-length HC, a score of 79 or greater indicates a human-like heavy chain, and for the full-length kappa LC, a score of 86 or greater indicates a human-like light chain. As shown in Tables 7 and 8, the humanity scores of full-length VH1, VL1, VL2, and VL3 indicated that these chains were human-like chains. The T20 score for a full-length antibody is generally significantly affected by the low degree of humanness of the CDR regions derived from the mouse 6B5 antibody, which are kept intact during humanization. Therefore, for humanized antibodies, the T20 scores for framework-only regions, i.e., regions without mouse CDR sequences, were also calculated. For the HC framework, a score of 84 or higher indicates a human-like heavy chain, and for the kappa LC framework, a score of 90 or higher indicates a human-like light chain. As shown in Tables 7 and 8, the humanity scores for the 6B5 heavy chain and 6B5 light chain (framework only) indicated that the VH and VL regions were human-like regions. Construction and small-scale production of humanized scFvs: The humanized VH and VL chains described above were reformatted into scFvs with an 8xHis tag at the C-terminus (SEQ ID NO: 76). For comparison, the mouse parental 6B5 scFv was expressed in parallel. To produce antibodies, E. coli BL21 (DE3) strain was transformed with the plasmids for the indicated scFvs, and the scFvs were then purified from lysed cells using a His-tag affinity purification column.

[0136] Example 2 Generation and analysis of humanized anti-ceramide scFv Expression of antibody constructs: The nucleotide sequences of anti-ceramide VH2 and anti-ceramide VL1 cloned into a high-expression mammalian vector (Lake Pharma) and the nucleic acid sequences of the cloned constructs were verified. Each construct was then scaled up to an appropriate amount for transfection, and the quality of the plasmid DNA was assessed by gel electrophoresis. CHO cells were seeded in shake flasks and expanded using serum-free, chemically defined medium. On the day of transfection, the expanded cells were seeded into fresh flasks with fresh medium, and the CHO cells were transiently transfected with each antibody expression construct using standard procedures known in the art. Cells were maintained as fed-batch cultures (C4611 and C4612, Medina) until the end of production. Conditioned medium from transient transfections was harvested and clarified by centrifugation and filtration. The antibodies were then purified by Protein L or IMAC purification, as described below.

[0137] Protein L affinity purification: The supernatant was loaded onto a Protein L column pre-equilibrated with binding buffer. Washing buffer was passed through the column until the OD280 value (NanoDrop, ThermoScientific) was zero. The target protein was eluted with a low pH buffer, fractions were collected, and the OD280 value of each fraction was recorded. Fractions containing the target protein were pooled and filtered through a 0.2 μm membrane. The protein concentration was calculated from the OD280 value and the calculated decay constant.

[0138] Purification of His-tagged proteins by IMAC: The supernatant was loaded onto an immobilized metal (nickel) affinity chromatography (IMAC) column pre-equilibrated with binding buffer. A wash buffer containing 40 mM imidazole was passed through the column until the OD280 value (NanoDrop, ThermoScientific) approached zero. The target protein was eluted with a linear gradient of increasing imidazole concentration up to 0.5 M. The eluate was collected in fractions, and the OD280 value of each fraction was recorded. Each fraction was analyzed by CE-SDS (SDS gel capillary electrophoresis) (LabChip GXII, Perkin Elmer). Fractions containing the target protein were pooled and dialyzed into a buffer specified by the supplier. The protein was filtered through a 0.2 μm membrane, and the protein concentration was calculated from the OD280 value (NanoDrop, ThermoScientific) and the calculated decay constant.

[0139] Antibody analysis: Size exclusion chromatography (SEC) was performed using a HiLoad 26 / 600 Superdex 200 column (GE Healthcare Life Sciences). Collected fractions were analyzed by CE-SDS (LabChip GXII, Perkin Elmer). Fractions containing the target protein were pooled and analyzed by SE-UPLC. A summary of the analysis reports for antibody construct 2766 using each purification method is shown in Table 9.

[0140] [Table 9]

[0141] Example 3 Humanized 6B5 scFv inhibits the formation of ceramide-rich platforms in vitro The humanized anti-ceramide scFvs generated in Examples 1 and 2 were then examined for their ability to inhibit the formation of ceramide-rich platforms. For cell culture and stimulation, Jurkat T lymphocytes (clone E6-1; TIB-152; ATCC) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS in a humidified incubator with 5% CO at 37°C. Cells were washed and resuspended in RPMI 1640 medium supplemented with 1% FBS or delipidated FBS. Briefly, cells were counted on a hemocytometer and maintained at 1 x 10 cells per 0.5 ml. 6 Cells were seeded into 24-well plates at 37°C. After 2 hours, cells were stimulated with 0.6 μg / ml of human anti-CD28 antibody (AF-342-PB; R&D systems). In addition, Jurkat T lymphocytes were pretreated with 50 ng / mL to 800 ng / mL of humanized 6B5 scFv for 1 hour before stimulation with anti-CD28 antibody (0.6 μg / mL; product number: AF-342-PB; R&D systems) for 45 seconds. Cells were then immediately fixed with 4% paraformaldehyde for 10 minutes at 4°C.

[0142] Ceramide-rich platform (CRP) was detected as previously described ( J.A. Rotolo et al., 2005 ; Stancevic et al., 2013 ). Nonspecific sites were blocked by incubating cells with 2% normal donkey serum (D9663; Sigma) on ice for 2 hours. After washing with PBS, cells were stained for surface ceramide overnight at 4°C using mouse anti-ceramide 1° Ab MID 15B4 IgM (1:50, Alexis Biochemicals). Irrelevant mouse IgM was used as an isotype control. Cells were then washed three times with PBST and stained with Cy3-conjugated anti-mouse IgM 2° Ab (1:400, Jackson ImmunoResearch) for 1 hour at 4°C. Cells were washed three times with PBST and mounted on glass slides using Vectashield fluorescent mounting media containing DAPI (Vector Laboratories). Platforms were imaged at room temperature using a Leica SP5 Inverted microscope with a HyD hybrid detector photon counter and an HCX PL APO CS 63.0x 1.40 oil objective lens with digital zoom at 7 for volumetric analysis and an HCX PL APO CS 40.0x 1.25 oil objective lens with digital zoom at 1 for incidence analysis. Z-stacks were collected using Leica LAS AF software.

[0143] Three-dimensional CRP volume analysis was performed using Imaris software (Bitplane AG, Zurich, Switzerland). Briefly, 10–12 individual Z-stacks for each sample were collected under optimized conditions, and the data were imported into Imaris software. Intensity normalization was performed for control and treated samples. Briefly, the mean intensity threshold for the z-column was obtained from the untreated control sample. Based on the mean intensity threshold, surfaces were created for untreated control, CD28-treated, and scFv-treated cells using the "Surfaces" function in Imaris. The mean intensity threshold ensured that only a certain range of intensity values ​​could be considered positive for surface volume quantification, while any values ​​outside this range were considered negative. The volume for each surface was generated using the surface volume function in Imaris. For final analysis, the individual volumes for each sample set were summarized and the mean value was calculated. To calculate IC50 values, the inhibitory effect of each antibody was normalized to a 0-100% CRP inhibition scale, where 0% represents the untreated control (CD28 antibody alone) and 100% represents the antibody treatment that achieved the best inhibitory effect. Data were plotted using Graph Pad Prism 8, and curves were fitted using nonlinear regression analysis. As shown in Figure 1, preincubation with different concentrations of 6B5 scFv inhibited the formation of CRP in CD28-activated T cells. VH2-VL1 was the most effective in preventing the formation of CRP, with IC 50 The IC value was 6.1 ng / mL. 50 The values ​​are shown in Table 10 below.

[0144] [Table 10]

[0145] Example 4 Humanized 6B5 scFv inhibits radiation-induced apoptosis in vitro The humanized anti-ceramide scFvs generated in Examples 1 and 2 were then examined for their ability to inhibit radiation-induced cell death.

[0146] Apoptosis assays were performed as described (Haimovitz-Friedman et al., 1994; J.A. Rotolo et al., 2005). Jurkat T lymphocytes were resuspended in RPMI 1640 supplemented with 1% FBS and pretreated with 100 ng / mL, 200 ng / mL, 400 ng / mL, 600 ng / mL, or 800 ng / mL of humanized 6B5 scFv for 1 hour at 37°C before exposure to 10 Gy (approximately 1.5 Gy / min) of radiation. Irradiated Jurkat T cells were incubated at 37°C for 16 hours and then fixed in 4% paraformaldehyde. Cells were then washed with PBS, stained with 24 μg / ml of the fluorophore bisbenzimide trihydrochloride (Hoechst 33258; Sigma-Aldrich), and placed on glass slides for counting on an Olympus IMT-2 microscope at room temperature using a 40× DPlanApo40UV 0.850160 / 0.11-0.23 objective. Apoptosis was quantified using a fluorescence microscope based on morphological features of apoptotic nuclei, including chromatin condensation, segmentation, and the appearance of condensation and apoptotic bodies with a perinuclear rim. A minimum of 100 cells were counted per sample. Percent inhibition of apoptosis was determined by normalizing values ​​to the radiation-only control (Figure 2A). IC 50 Values ​​were calculated using nonlinear regression on GraphPad Prism v7 software.

[0147] As shown in Figure 2A, preincubation with different concentrations of 6B5 scFv inhibited apoptosis in irradiated Jurkat T cells. VH2-VL1 was the most effective in inhibiting radiation-induced apoptosis, with IC 50 The IC value for the various humanized 6B5 scFvs tested was 130.3 ng / mL. 50The values ​​are shown in Table 11 below.

[0148] [Table 11]

[0149] Using a similar experimental setup, two additional humanized 6B5 scFvs, VH4-VL1 (SEQ ID NO: 52) and VH5-VL1 (SEQ ID NO: 53), were examined for their ability to inhibit radiation-induced T cell apoptosis (Figures 2B-2E). In Figure 2B, VH4-VL1, VH5-VL1, and the parental murine 6B5 scFv at 25, 50, 100, 200, 400, 600, or 800 ng / mL were incubated with Jurkat T cells for 1 hour, and the ability of each scFv to inhibit radiation-induced apoptosis was normalized to the protective effect of the parental murine 6B5 scFv at 800 ng / mL. In Figure 2C, VH4-VL1, VH5-VL1, and the parent scFv, murine 6B5 scFv, at 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, or 800 ng / mL, were incubated with Jurkat T cells for 1 hour, and the ability of each scFv to inhibit radiation-induced apoptosis was normalized to the protective effect of VH5-VL1 at 800 ng / mL. In Figure 2D, VH4-VL1 and VH5-VL1 at 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 600 ng / mL, or 800 ng / mL were incubated with Jurkat T cells for 1 hour, and the ability of each construct to inhibit radiation-induced apoptosis was normalized to the protective effect of VH4-VL1 at 800 ng / mL. IC values ​​based on these sets of experiments were calculated. 50 The values ​​are presented in Table 12 below.

[0150] [Table 12] Figure 2E shows the protective effects of VH4-VL1 and VH5-VL1 at given concentrations (400 ng / mL and 800 ng / mL) under these conditions, as assessed by the percentage of radiation-induced apoptotic T cells. Overall, these results confirm that the humanized 6B5 scFv can effectively protect T cells against radiation-induced apoptosis.

[0151] Example 5 Treatment with humanized 6B5 scFv to prevent radiation-induced cell death in vivo Anti-ceramide VH2-VL1 (SEQ ID NO: 51) was investigated for in vivo treatment of mice exposed to lethal doses of radiation. A cesium irradiator was used to irradiate mice with an LD of 15 Gray units (Gy). 90 The dose was delivered to C57BL / 6 mice, and 24 hours after irradiation, the mice were either left untreated or treated intravenously with 6B5 VH2-VL1 (150 μg / 25 g). Mice were euthanized at different time points after irradiation, and continuous segments of jejunum proximal to the ligament of Treitz were isolated, fixed in 4% paraformaldehyde, and embedded in paraffin. Tissues were sectioned at 5 μm and apoptotic endothelial cells were identified using a TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) assay with MECA-32 antibody (a pan-endothelial cell marker). Dual-stained apoptotic endothelial cells were quantified.

[0152] 15 Gy LD 90Mice irradiated with this dose resulted in small intestinal endothelial cell death, which peaked 24 hours after irradiation (Figure 3A). Treatment with 6B5 VH2-VL1 24 hours after irradiation significantly reduced endothelial cell apoptosis compared to untreated mice (Figure 3B). Overall, these data indicate that inhibition of ceramide prevents endothelial cell death in the gastrointestinal tract in a mouse model of GI syndrome. Humanized anti-ceramide scFv may be beneficial for the treatment of diseases associated with increased cell death, including GI syndrome, GvHD, and autoimmunity.

[0153] Further Numbered Embodiments Further embodiments of the present disclosure are presented in the following numbered embodiments: Embodiment 1. An immunoglobulin comprising an immunoglobulin heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), and an immunoglobulin light chain variable region (VL) comprising a light chain complementarity determining region (CDR) 1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3); (a) HCDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1 and 2; (b) HCDR2 comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, and 6; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 10; An anti-ceramide antibody or an antigen-binding fragment thereof. Embodiment 2. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 3. Embodiment 3. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 4. Embodiment 4. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 5. Embodiment 5. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 6. Embodiment 6. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 1, wherein HCDR1 comprises SEQ ID NO:2 and HCDR2 comprises SEQ ID NO:3. Embodiment 7. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 1, wherein HCDR1 comprises SEQ ID NO:2 and HCDR2 comprises SEQ ID NO:4. Embodiment 8. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 1, wherein HCDR1 comprises SEQ ID NO:2 and HCDR2 comprises SEQ ID NO:5. Embodiment 9. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 1, wherein HCDR1 comprises SEQ ID NO:2 and HCDR2 comprises SEQ ID NO:6. Embodiment 10. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 22. Embodiment 11. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 10, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 17 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. Embodiment 12. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 10, wherein the amino acid sequence of VH consists of SEQ ID NO: 17 and the amino acid sequence of VL consists of SEQ ID NO: 22. Embodiment 13. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 23. Embodiment 14. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 13, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 17 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 23. Embodiment 15. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 13, wherein the amino acid sequence of VH consists of SEQ ID NO: 17 and the amino acid sequence of VL consists of SEQ ID NO: 23. Embodiment 16. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 17, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 24. Embodiment 17. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 16, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 17 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 24. Embodiment 18. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 16, wherein the amino acid sequence of VH consists of SEQ ID NO: 17 and the amino acid sequence of VL consists of SEQ ID NO: 24. Embodiment 19. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 22. Embodiment 20. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 19, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 18 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. Embodiment 21. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 19, wherein the amino acid sequence of VH consists of SEQ ID NO: 18 and the amino acid sequence of VL consists of SEQ ID NO: 22. Embodiment 22. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 23. Embodiment 23. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 22, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 18 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 23. Embodiment 24. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 22, wherein the amino acid sequence of VH consists of SEQ ID NO: 18 and the amino acid sequence of VL consists of SEQ ID NO: 23. Embodiment 25. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 18, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 24. Embodiment 26. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 25, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 18 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 24. Embodiment 27. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 25, wherein the amino acid sequence of VH consists of SEQ ID NO: 18 and the amino acid sequence of VL consists of SEQ ID NO: 24. Embodiment 28. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 22. Embodiment 29. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 28, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 19 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. Embodiment 30. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 28, wherein the amino acid sequence of VH consists of SEQ ID NO: 19 and the amino acid sequence of VL consists of SEQ ID NO: 22. Embodiment 31. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 23. Embodiment 32. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 31, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 19 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 23. Embodiment 33. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 31, wherein the amino acid sequence of VH consists of SEQ ID NO: 19 and the amino acid sequence of VL consists of SEQ ID NO: 23. Embodiment 34. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 19, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 24. Embodiment 35. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 34, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 19 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 24. Embodiment 36. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 34, wherein the amino acid sequence of VH consists of SEQ ID NO: 19 and the amino acid sequence of VL consists of SEQ ID NO: 24. Embodiment 37. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 20, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 22. Embodiment 38. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 37, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 20 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. Embodiment 39. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 37, wherein the amino acid sequence of VH consists of SEQ ID NO: 20 and the amino acid sequence of VL consists of SEQ ID NO: 22. Embodiment 40. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 1, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 21, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 22. Embodiment 41. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 40, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 21 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. Embodiment 42. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 40, wherein the amino acid sequence of VH consists of SEQ ID NO: 21 and the amino acid sequence of VL consists of SEQ ID NO: 22. Embodiment 43. An immunoglobulin comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to a sequence selected from SEQ ID NOs: 17, 18, 19, 20, and 21; an amino acid sequence in which the VL is at least 90%, 95%, or 97% identical to a sequence selected from SEQ ID NOs: 22, 23, and 24; An anti-ceramide antibody or an antigen-binding fragment thereof, comprising: Embodiment 44. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 22. Embodiment 45. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 44, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 17 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. Embodiment 46: An anti-ceramide antibody or antigen-binding fragment thereof according to any one of embodiments 43 to 45, wherein the amino acid sequence of VH consists of SEQ ID NO: 17 and the amino acid sequence of VL consists of SEQ ID NO: 22. Embodiment 47. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 23. Embodiment 48. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 47, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 17 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 23. Embodiment 49. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 43, 47, or 48, wherein the amino acid sequence of VH consists of SEQ ID NO: 17 and the amino acid sequence of VL consists of SEQ ID NO: 23. Embodiment 50. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 24. Embodiment 51. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 50, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 17 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 24. Embodiment 52. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 43, 50, or 51, wherein the amino acid sequence of VH consists of SEQ ID NO: 17 and the amino acid sequence of VL consists of SEQ ID NO: 24. Embodiment 53. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 22. Embodiment 54. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 53, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 18 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. Embodiment 55. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 43, 53, or 54, wherein the amino acid sequence of VH consists of SEQ ID NO: 18 and the amino acid sequence of VL consists of SEQ ID NO: 22. Embodiment 56. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 23. Embodiment 57. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 56, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 18 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 23. Embodiment 58. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 43, 56, or 57, wherein the amino acid sequence of VH consists of SEQ ID NO: 18 and the amino acid sequence of VL consists of SEQ ID NO: 23. Embodiment 59. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 24. Embodiment 60. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 59, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 18 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 24. Embodiment 61. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 43, 59, or 60, wherein the amino acid sequence of VH consists of SEQ ID NO: 18 and the amino acid sequence of VL consists of SEQ ID NO: 24. Embodiment 62. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 22. Embodiment 63. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 62, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 19 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. Embodiment 64. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 43, 62, or 63, wherein the amino acid sequence of VH consists of SEQ ID NO: 19 and the amino acid sequence of VL consists of SEQ ID NO: 22. Embodiment 65. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 23. Embodiment 66. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 65, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 19 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 23. Embodiment 67. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 43, 65, or 66, wherein the amino acid sequence of VH consists of SEQ ID NO: 19 and the amino acid sequence of VL consists of SEQ ID NO: 23. Embodiment 68. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 24. Embodiment 69. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 68, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 19 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 24. Embodiment 70. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 43, 68, or 69, wherein the amino acid sequence of VH consists of SEQ ID NO: 19 and the amino acid sequence of VL consists of SEQ ID NO: 24. Embodiment 71. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 20, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 22. Embodiment 72. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 71, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 20 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. Embodiment 73. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 43, 71, or 72, wherein the amino acid sequence of VH consists of SEQ ID NO: 20 and the amino acid sequence of VL consists of SEQ ID NO: 22. Embodiment 74. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43, wherein VH comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 21, and VL comprises an amino acid sequence at least 90%, 95%, or 97% identical to SEQ ID NO: 22. Embodiment 75. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 43 or 74, wherein VH comprises an amino acid sequence 100% identical to SEQ ID NO: 21 and VL comprises an amino acid sequence 100% identical to SEQ ID NO: 22. Embodiment 76. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 43, 74, or 75, wherein the amino acid sequence of VH consists of SEQ ID NO: 21 and the amino acid sequence of VL consists of SEQ ID NO: 22. Embodiment 77. The anti-ceramide antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76, which is a humanized antibody or antigen-binding fragment thereof. Embodiment 78. The anti-ceramide antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76, which is a fully human antibody or antigen-binding fragment thereof. Embodiment 79. An anti-ceramide antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 78, wherein the anti-ceramide antibody comprises one or more point mutations within the Fc domain of the antibody. Embodiment 80. The anti-ceramide antibody or antigen-binding fragment thereof of any one of embodiments 1 to 79, wherein the antigen-binding fragment is a single-chain variable fragment (scFv). Embodiment 81. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 80, wherein the scFv comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to an sequence selected from the group consisting of SEQ ID NOs: 48 to 61. Embodiment 82. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 80, wherein the scFv comprises an amino acid sequence that is 100% identical to an sequence selected from the group consisting of SEQ ID NOs: 48 to 61. Embodiment 83. An anti-ceramide antibody or antigen-binding fragment thereof described in embodiment 80, wherein the scFv consists of an array selected from the group consisting of SEQ ID NOs: 48 to 61. Embodiment 84. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 80, wherein the light chain variable region of the scFv is carboxy-terminal to the heavy chain variable region of the scFv. Embodiment 85. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 80, wherein the light chain variable region of the scFv is amino-terminal to the heavy chain variable region of the scFv. Embodiment 86. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 80 to 85, wherein the scFv comprises a linker polypeptide. Embodiment 87. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 86, wherein a linker polypeptide is located between the light chain variable region and the heavy chain variable region of the scFv. Embodiment 88. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 86 or 87, wherein the linker polypeptide comprises a Gly4Ser linker. Embodiment 89. The linker polypeptide has the formula (Gly4Ser) n The anti-ceramide antibody or antigen-binding fragment thereof according to any one of embodiments 86 to 88, comprising [in the sequence, n=1 to 5]. Embodiment 90. An anti-ceramide single chain variable fragment (scFv) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 48-61. Embodiment 91. The anti-ceramide scFv of embodiment 90, comprising an amino acid sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 48 to 61. Embodiment 92. The anti-ceramide scFv of embodiment 90, consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 61. Embodiment 93. A polynucleotide encoding the anti-ceramide antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 92. Embodiment 94. An anti-ceramide single chain variable fragment (scFv) comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 48 or 51. Embodiment 95. The anti-ceramide scFv of embodiment 94, comprising an amino acid sequence that is 100% identical to SEQ ID NO: 48 or 51. Embodiment 96. An anti-ceramide scFv according to embodiment 94, consisting of the amino acid sequence of SEQ ID NO: 48 or 51. Embodiment 97. A polynucleotide encoding the anti-ceramide single chain variable fragment (scFv) of any one of embodiments 94 to 96. Embodiment 98. An expression vector comprising the polynucleotide of embodiment 93 or 97. Embodiment 99. A host cell comprising a polynucleotide according to embodiment 93 or 97, or an expression vector according to embodiment 97. Embodiment 100. A method for producing an anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 89, or an anti-ceramide single-chain variable fragment (scFv) described in any one of embodiments 94 to 96, comprising the step of introducing the expression vector described in embodiment 98 into a host cell. Embodiment 101. A method for inhibiting apoptosis 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 described in any one of embodiments 1 to 88, or an anti-ceramide scFv described in any one of embodiments 94 to 96. Embodiment 102. The method of embodiment 101, wherein the apoptosis is associated with a disease selected from the group consisting of graft-versus-host disease, radiation sickness, GI syndrome, and autoimmune disease. Embodiment 103. The method of embodiment 102, wherein the disease is radiation sickness or GI syndrome, and the anti-ceramide antibody or antigen-binding fragment thereof is administered before the subject is exposed to radiation. Embodiment 104. The method of embodiment 102, wherein the disease is graft-versus-host disease and the anti-ceramide antibody or antigen-binding fragment thereof is administered before the subject receives a transplant. Embodiment 105. The method of embodiment 104, wherein the transplant is a bone marrow transplant. Embodiment 106. The method of embodiment 101, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered intravenously, intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intrasynovially, intrathecally, orally, topically, or via inhalation. Embodiment 107. A method for alleviating apoptosis in a subject with GI syndrome, comprising administering to the subject a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 89, or an anti-ceramide scFv described in any one of embodiments 94 to 96, after the subject has been exposed to penetrating radiation. Embodiment 108 The method of embodiment 107, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered immediately after the subject has been exposed to penetrating radiation. Embodiment 109. The method of embodiment 107, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered within 24 hours after the subject is exposed to penetrating radiation. Embodiment 110. A method for inhibiting apoptosis in a subject with GvHD, comprising administering to the subject a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 89, or an anti-ceramide scFv described in any one of embodiments 94 to 96, before the subject undergoes a transplant, or after the subject has undergone a transplant and before the onset of GvHD. Embodiment 111. The method of embodiment 110, wherein the transplant is a bone marrow transplant.

[0154] Embodiment 112. An immunoglobulin heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), and an immunoglobulin light chain variable region (VL) comprising a light chain complementarity determining region (CDR) 1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3), wherein (a) HCDR1 comprises an amino acid sequence selected from GYTFTDHTIH (SEQ ID NO: 1) and GYTFTDHTMH (SEQ ID NO: 2); (b) HCDR2 comprises an amino acid sequence selected from YNYPRDGSTKYNEKFQG (SEQ ID NO: 3), YN An anti-ceramide antibody or antigen-binding fragment thereof, comprising an amino acid sequence selected from YPREGSTKYNEKFQG (sequence number 4), YNYPRDVSTKYNEKFQG (sequence number 5), and YNYPRDGSTKYAEKFQG (sequence number 6); (c) HCDR3 comprises the amino acid sequence of GFITTVVPSAY (sequence number 7); (d) LCDR1 comprises the amino acid sequence of RASKSISKYLA (sequence number 8); (e) LCDR2 comprises the amino acid sequence of SGSTLQS (sequence number 9); and (f) LCDR3 comprises the amino acid sequence of QQHNEYPWT (sequence number 10). Embodiment 113. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 112, wherein HCDR1 comprises GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises YNYPRDGSTKYNEKFQG (SEQ ID NO: 3). Embodiment 114. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 112, wherein HCDR1 comprises GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises YNYPREGSTKYNEKFQG (SEQ ID NO: 4). Embodiment 115. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 112, wherein HCDR1 comprises GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises YNYPRDVSTKYNEKFQG (SEQ ID NO: 5). Embodiment 116. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 112, wherein HCDR1 comprises GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises YNYPRDGSTKYAEKFQG (SEQ ID NO: 6). Embodiment 117. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 112, wherein HCDR1 comprises GYTFTDHTMH (SEQ ID NO: 2) and HCDR2 comprises YNYPRDGSTKYNEKFQG (SEQ ID NO: 3). Embodiment 118. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 112, wherein HCDR1 comprises GYTFTDHTMH (SEQ ID NO: 2) and HCDR2 comprises YNYPREGSTKYNEKFQG (SEQ ID NO: 4). Embodiment 119. An anti-ceramide antibody or antigen-binding fragment thereof according to embodiment 112, wherein HCDR1 comprises GYTFTDHTMH (SEQ ID NO: 2) and HCDR2 comprises YNYPRDVSTKYNEKFQG (SEQ ID NO: 5). Embodiment 120. The anti-ceramide antibody or antigen-binding fragment thereof of embodiment 112, wherein HCDR1 comprises GYTFTDHTMH (SEQ ID NO: 2) and HCDR2 comprises YNYPRDGSTKYAEKFQG (SEQ ID NO: 6).

[0155] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein shall not be construed as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the general public knowledge in any country in the world.

[0156] [Table 13] TIFF0007809067000015.tif250165 TIFF0007809067000016.tif250164 TIFF0007809067000017.tif250165 TIFF0007809067000018.tif250165 TIFF0007809067000019.tif249165 TIFF0007809067000020.tif137170

Claims

1. an immunoglobulin heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3); and an immunoglobulin light chain variable region (VL) comprising a light chain complementarity determining region (CDR) 1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3); (a) HCDR1 comprises SEQ ID NO: 1; (b) HCDR2 comprises SEQ ID NO:4; (c) HCDR3 comprises SEQ ID NO:7; (d) LCDR1 comprises SEQ ID NO:8; (e) LCDR2 comprises SEQ ID NO: 9; (f) LCDR3 comprises SEQ ID NO: 10; An anti-ceramide antibody or an antigen-binding fragment thereof.

2. The anti-ceramide antibody or antigen-binding fragment thereof of claim 1, wherein VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 20 and VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:

22.

3. The anti-ceramide antibody or antigen-binding fragment thereof of claim 2, wherein VH comprises SEQ ID NO: 20 and VL comprises SEQ ID NO:

22.

4. The anti-ceramide antibody or antigen-binding fragment thereof according to claim 2, wherein VH consists of SEQ ID NO: 20 and VL consists of SEQ ID NO:

22.

5. The anti-ceramide antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a humanized antibody or antigen-binding fragment thereof.

6. The anti-ceramide antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the anti-ceramide antibody comprises one or more point mutations in the Fc domain of the antibody.

7. The anti-ceramide antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antigen-binding fragment is a single-chain variable fragment (scFv).

8. The anti-ceramide antibody or antigen-binding fragment thereof of claim 7, wherein the scFv (i) comprises an amino acid sequence at least 90% identical to SEQ ID NO: 49 or 52, or (ii) consists of SEQ ID NO: 49 or 52.

9. The anti-ceramide antibody or antigen-binding fragment thereof according to claim 7, wherein the light chain variable region of the scFv is located on the carboxy-terminal side relative to the heavy chain variable region of the scFv, or the light chain variable region of the scFv is located on the amino-terminal side relative to the heavy chain variable region of the scFv.

10. The anti-ceramide antibody or antigen-binding fragment thereof according to any one of claims 7 to 9, wherein the scFv comprises a linker polypeptide.

11. The anti-ceramide antibody or antigen-binding fragment thereof of claim 10, wherein a linker polypeptide is located between the light chain variable region and the heavy chain variable region of the scFv.

12. The linker polypeptide comprises: (i) Gly 4 Ser linker, or (ii) a linker of the formula (Gly 4 Ser) n The anti-ceramide antibody or antigen-binding fragment thereof according to claim 10 or 11, comprising: [in the sequence, n=1 to 5].

13. A polynucleotide encoding the anti-ceramide antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. An expression vector comprising the polynucleotide of claim 13.

15. A host cell comprising the polynucleotide of claim 13 or the expression vector of claim 14.

16. A method for producing the anti-ceramide antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, comprising the step of introducing into a host cell an expression vector comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof.

17. The anti-ceramide antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, for use in a method for inhibiting apoptosis in a subject in need thereof.

18. The anti-ceramide antibody or antigen-binding fragment thereof of claim 17, wherein apoptosis is associated with a disease selected from the group consisting of graft-versus-host disease, radiation sickness, GI syndrome, and autoimmune disease.

19. (i) the disease is radiation sickness or GI syndrome and the anti-ceramide antibody or antigen-binding fragment thereof is administered before the subject is exposed to radiation; or (ii) the disease is graft-versus-host disease, and the anti-ceramide antibody or antigen-binding fragment thereof is administered before the subject receives a transplant; The anti-ceramide antibody or antigen-binding fragment thereof according to claim 18.

20. The anti-ceramide antibody or antigen-binding fragment thereof of claim 19, wherein the transplant is a bone marrow transplant.

21. The anti-ceramide antibody or antigen-binding fragment thereof of claim 18, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered intravenously, intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intrasynovially, intrathecally, orally, or topically.

22. 13. The anti-ceramide antibody or antigen-binding fragment thereof of any one of claims 1 to 12, for use in a method for alleviating apoptosis in a subject with GI syndrome, the method comprising the step of administering a therapeutically effective amount of the anti-ceramide antibody or antigen-binding fragment thereof to a subject after the subject has been exposed to penetrating radiation.

23. The anti-ceramide antibody or antigen-binding fragment thereof of claim 22, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered (i) immediately after the subject is exposed to penetrating radiation or (ii) within 24 hours after the subject is exposed to penetrating radiation.

24. An anti-ceramide antibody or antigen-binding fragment thereof described in any one of claims 1 to 12 for use in a method for inhibiting apoptosis in a subject with GvHD, the method comprising the step of administering a therapeutically effective amount of the anti-ceramide antibody or antigen-binding fragment thereof to a subject before the subject undergoes a transplant or after the subject has undergone a transplant and before the onset of GvHD.

25. The anti-ceramide antibody or antigen-binding fragment thereof of claim 24, wherein the transplant is a bone marrow transplant.

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