Anti-ceramide antibodies

Abstract

This disclosure presents compositions of anti-ceramide antibodies and their antigen-binding fragments. [Solution] This disclosure further presents a method for preventing or inhibiting cell death in a subject requiring such prevention, comprising the step of administering an anti-ceramide antibody or antigen-binding fragment to the subject. Subjects requiring such prevention may be suffering from autoimmune diseases, GI syndrome, or GvHD.

Description

[Technical Field]

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

[0002] Statements regarding sequence listings The sequence listing associated with this application is presented in text format, not in paper form, and is incorporated herein by reference. A computer-readable copy of the sequence listing is: filename: CERA-012_01WO_ST25.txt, date recorded: March 17, 2021, file size: 68.1 kilobytes.

[0003] field This disclosure relates in general to anti-ceramide antibodies and their uses. In particular, this disclosure relates to humanized anti-ceramide antibodies that inhibit cell death. Such antibodies are useful for treating and preventing gastrointestinal syndromes, graft-versus-host diseases, and autoimmune diseases. [Background technology]

[0004] Acute radiation syndrome (ARS), also known as radiotoxicity or radiation sickness, is an acute illness caused by exposure to a large portion 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. Due to destructive and irreversible changes in the gastrointestinal tract and bone marrow caused by radiation-induced cell death, the chances of survival with this syndrome are extremely low. Radiation is one of the most effective tools for treating cancer. Unfortunately, a major adverse side effect of radiotherapy is the high 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, result in rejection or graft-versus-host disease (GvHD) as a result of triggering a destructive immune response in the host. Therefore, alternative strategies are urgently needed to reduce the incidence of radiation-induced GI syndrome and GvHD-associated cell death. [Overview of the Initiative]

[0005] This disclosure presents compositions of anti-ceramide antibodies and their antigen-binding fragments. In some embodiments, the anti-ceramide antibody and its antigen-binding fragment are humanized antibodies or their antigen-binding fragments. In other embodiments, the anti-ceramide antibody and its antigen-binding fragment are scFv. In some embodiments, this disclosure further presents a method for preventing or inhibiting cell death in a subject requiring it, comprising the step of 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 disclosure presents an anti-ceramide antibody or an antigen-binding fragment thereof 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), 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 includes sequence number 1 and HCDR2 includes sequence number 3. In some embodiments, HCDR1 includes sequence number 1 and HCDR2 includes sequence number 4. In some embodiments, HCDR1 includes sequence number 1 and HCDR2 includes sequence number 5. In some embodiments, HCDR1 includes sequence number 1 and HCDR2 includes sequence number 6. In some embodiments, HCDR1 includes sequence number 2 and HCDR2 includes sequence number 3. In some embodiments, HCDR1 includes sequence number 2 and HCDR2 includes sequence number 4. In some embodiments, HCDR1 includes sequence number 2 and HCDR2 includes sequence number 5. In some embodiments, HCDR1 includes sequence number 2 and HCDR2 includes sequence number 6. In some embodiments, VH includes an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 17, and VL includes 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, VH includes an amino acid sequence that is 100% identical to SEQ ID NO: 17, and VL includes an amino acid sequence that is 100% identical to SEQ ID NO: 22. In some embodiments, 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.

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

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

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

[0010] In some embodiments, 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 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, VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. In some embodiments, 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. In some embodiments, 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 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, VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. In some embodiments, 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.

[0011] In some embodiments, 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 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, VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 23. In some embodiments, 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. In some embodiments, 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 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, VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. In some embodiments, 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.

[0012] In some embodiments, 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 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, VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 20, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. In some embodiments, 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. In some embodiments, 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 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, VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 21, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. In some embodiments, 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.

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

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

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

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

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

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

[0019] In some embodiments, VH includes an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 21, and VL includes an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 22. In some embodiments, VH includes an amino acid sequence that is 100% identical to SEQ ID NO: 21, and VL includes an amino acid sequence that is 100% identical to SEQ ID NO: 22. In some embodiments, 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. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a fully human antibody or its antigen-binding fragment. In some embodiments, the anti-ceramide antibody contains one or more point mutations within 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 includes 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 includes 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 located on the carboxy-terminal side relative to the heavy chain variable region of the scFv. In some embodiments, 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. In some embodiments, the scFv includes a linker polypeptide. In some embodiments, the linker polypeptide is located between the light chain variable region and the heavy chain variable region of the scFv. In some embodiments, the linker polypeptide includes a Gly4Ser linker. In some embodiments, the linker polypeptide is of the formula (Gly4Ser) n Includes [arrays where n=1 to 5].

[0021] In some embodiments, the disclosure presents anti-ceramide single-chain variable fragments (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. 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 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-61. In some embodiments, this disclosure presents polynucleotides encoding anti-ceramide antibodies or antigen-binding fragments thereof, as described herein. In some embodiments, the disclosure presents anti-ceramide single-chain variable fragments (scFv) comprising amino acid sequences that are 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, this disclosure presents polynucleotides encoding anti-ceramide single-chain variable fragments (scFv) as described herein. In some embodiments, this disclosure presents expression vectors comprising the said polynucleotides. In some embodiments, this disclosure presents host cells containing polynucleotides or expression vectors described herein.

[0023] In some embodiments, the disclosure presents a method for producing an anti-ceramide antibody or an antigen-binding fragment thereof, or an anti-ceramide single-chain variable fragment (scFv), as described herein, comprising the step of introducing the expression vector of Embodiment 98 into host cells. In some embodiments, the Disclosure presents a method for inhibiting apoptosis in a subject requiring such inhibition, comprising the step of administering to the subject a therapeutically effective dose of an anti-ceramide antibody or its antigen-binding fragment, or an anti-ceramide single-chain variable fragment (scFv), as described herein. In some embodiments, apoptosis is associated with a disease selected from the group consisting of graft-versus-host disease, radiation disease, GI syndrome, and autoimmune disease. In some embodiments, the disease is radiation disease or GI syndrome, and the anti-ceramide antibody or its antigen-binding fragment 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 its antigen-binding fragment 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 its antigen-binding fragment is administered intravenously, intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intrasynovally, intrathecally, orally, topically, or by inhalation. In some embodiments, the Disclosure presents a method for mitigating apoptosis in a subject with GI syndrome, comprising the step of administering to the subject, after the subject has been exposed to penetrating radiation, a therapeutically effective dose of an anti-ceramide antibody or its antigen-binding fragment as described in any one of Embodiments 1 to 89, or an anti-ceramide scFv as described in any one of Embodiments 94 to 96. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered immediately after the subject is exposed to penetrating radiation. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered within 24 hours after the subject is exposed to penetrating radiation. In some embodiments, the Disclosure presents a method for inhibiting apoptosis in a subject with GvHD, comprising the step of administering to the subject a therapeutically effective dose of an anti-ceramide antibody or its antigen-binding fragment as described in any one of Embodiments 1 to 89, or an anti-ceramide scFv as described in any one of Embodiments 94 to 96, before the subject undergoes transplantation or after the subject has undergone transplantation and before the onset of GvHD. In some embodiments, the transplantation is a bone marrow transplant.

[0025] The accompanying drawings illustrate one or more embodiments of the present disclosure and, together with the descriptive text, are used to illustrate the principles of the exemplary embodiments of the present disclosure. [Brief explanation of the drawing]

[0026] [Figure 1] This figure shows the dose-response curve for the formation of a CD28-inducible ceramide-rich platform (CRP) in T cells when the concentration of 6B5 scFv is increased. [Figure 2A] This figure shows the dose-response curve for radiation-induced T cell apoptosis when the concentration of humanized 6B5 scFv is increased. [Figure 2B] This figure shows dose-response curves for radiation-induced T cell apoptosis when the concentrations of VH4-VL1, VH5-VL1, and the parental scFv, mouse 6B5 scFv, were increased. [Figure 2C] This figure shows dose-response curves for radiation-induced T cell apoptosis when the concentrations of VH4-VL1, VH5-VL1, and the parental scFv, mouse 6B5 scFv, were increased. [Figure 2D] This figure shows the dose-response curves for radiation-induced T cell apoptosis when the concentrations of VH4-VL1 and VH5-VL1 are increased. [Figure 2E] This figure shows the percentage of radiation-induced apoptotic T cells given concentrations of VH4-VL1 and VH5-VL1. [Figure 3A]This figure shows the number of apoptotic endothelial cells per villi over time in mice exposed to an LD90 dose of 15 Gray units (Gy). The number adjacent to the symbol represents the number of animals measured per time point. Gy: Gray is an absorbed radiation unit. [Figure 3B] This figure shows the number of apoptotic endothelial cells per villi over time in mice exposed to an LD90 dose of 15 Gray units (Gy), 24 hours after irradiation, either left untreated or treated with VH2-VL1 (SEQ ID NO: 51). The number adjacent to the symbol represents the number of animals measured per time point. Gy: Gray is an absorbed radiation unit. [Figure 4A] This figure shows the amino acid sequence alignment of the humanized VH region. The CDR is underlined. Amino acids of the CDR that differ from the parent mouse CDR sequence are shown in bold. Amino acids of the framework region that differ from the human germline framework sequence are also shown in bold. [Figure 4B] This figure shows the amino acid sequence alignment for the humanized VL region. CDR is underlined. [Modes for carrying out the invention]

[0027] Further details regarding this disclosure are provided in the appendices below. In the practice or trial of the present invention, similar or equivalent methods and materials may be used, but only exemplary methods and materials are described herein. Other features, purposes, and advantages of this disclosure will be apparent from the description and claims. In this specification and the appendices, singular nouns also include plural nouns, unless the context explicitly indicates otherwise. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. All patents and publications referenced herein are incorporated herein by reference in their entirety.

[0028] definition The term "antibody" refers to an immunoglobulin (Ig) molecule capable of binding to specific targets, such as carbohydrates, polynucleotides, lipids, and polypeptides, via 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 intact monoclonal antibodies and their antigen-binding fragments. 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 via an interchain disulfide bond between the heavy-chain polypeptide and the light-chain polypeptide to form a two-heterodimeric protein or polypeptide (i.e., a protein composed of two heterogeneous polypeptide chains). The two-heterodimeric protein then associates via a further 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 the heavy and / or light chain of an immunoglobulin that binds to at least one epitope of the antigen of interest. In this regard, the antigen-binding fragments of antibodies described herein may include all one, two, three, four, five, or six CDRs of variable heavy chain (VH) and variable light chain (VL) sequences derived from an antibody that specifically binds to ceramide. Antigen-binding fragments include portions of full-length antibodies, generally proteins containing their antigen-binding or variable regions, such as Fab, F(ab')2, Fab', Fv fragments, minibodies, diabodies, single-domain antibodies (dAb), single-chain variable fragments (scFv), polyspecific antibodies formed from antibody fragments, and any other modified configurations of immunoglobulin molecules, which contain an antigen-binding site or antigen-binding fragment having the required specificity. In certain embodiments of this disclosure, antigen-binding fragments are used, rather than intact antibodies, to increase tissue permeability or tumor permeability. In other embodiments, the antigen-binding fragments are further modified to extend the serum half-life.

[0030] The term "F(ab)" refers to two protein fragments obtained from the proteolytic cleavage of an IgG molecule by the enzyme papain. Each F(ab) contains a covalent heterodimer of the VH chain and the VL chain, and includes 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, which may contain a small portion of Fc. Each Fab' fragment is a monovalent antigen-binding fragment. The term "F(ab')2" refers to a protein fragment of IgG produced by proteolytic cleavage mediated by the enzyme pepsin. Each F(ab')2 fragment contains two F(ab') fragments and is therefore a bivalent antigen-binding fragment. "Fv fragments" refer to non-covalent VH::VL heterodimers that contain an antigen-binding site that retains most of the antigen recognition and antigen-binding ability of a native antibody molecule, but lack 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 can be prepared by preferential proteolytic cleavage of IgM, but in rare cases, they can also be prepared by preferential proteolytic cleavage of IgG or IgA immunoglobulin molecules. More generally, however, Fv fragments are derived using recombinant methods known in the art.

[0031] This specification also includes minibodies containing scFv ligated to the CH3 domain (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). For example, see Ward, ES et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0032] The term "diabody" refers to a bispecific antibody in which the VH and VL domains 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 pairing the domains with complementary domains on another chain and creating two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993); and Poljak et al., Structure 2:1121-23 (1994)). The terms "nanobody" or "single-domain antibody" refer to antigen-binding fragments consisting of a single monomeric variable antibody domain. The Nanoclone method is a method for generating nanobody against a desired target based on automated high-throughput selection of B cells (see, for example, WO06 / 079372).

[0033] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies; that is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may exist in small amounts. In some embodiments, the term “chimeric antibody” as used herein refers to a monoclonal antibody in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, insofar 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 the heavy (VH) and light (VL) chains of immunoglobulins, linked by a short linker peptide of 10–25 amino acids (Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879–5883). The linker may connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. Numerous methods have been described for identifying the chemical structure to convert naturally aggregated (but chemically separated) light and heavy chain polypeptides into scFv molecules that fold from the V region of an antibody into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patents 5,091,513 and 5,132,405 by Huston et al., and U.S. Patent 4,946,778 by Ladner et al.

[0035] The term "antigen" refers to a molecule or part of a molecule that can be bound by an antibody or its antigen-binding fragment, and that can also be used to produce an antibody capable of binding to the epitope of this antigen in an animal. An antigen may have one or more epitopes. Embodiments herein envision 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. Epitope determinants may include a group of chemically active surface molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and may possess specific three-dimensional structural and / or specific charge characteristics.

[0036] In this specification, "specifically binds to ~" means that the antibody or its antigen-binding fragment does not significantly bind to other components or antigens present in the mixture, but binds to the target molecule at least 10 times. 5 M -1 This refers to the ability to bind by binding affinity (Ka). In this specification, references to anti-ceramide antibodies refer to antibodies or their antigen-binding fragments that specifically bind to ceramide. Binding affinity (Ka) refers to the equilibrium association due to a specific binding interaction, expressed in units of 1 / M or M. An antibody or its antigen-binding fragment can be classified as an antibody or its antigen-binding fragment with "high affinity" and an antibody or its antigen-binding fragment with "low affinity". An antibody or its antigen-binding fragment with "high affinity" means that Ka is at least 10 -1 M 7 M -1 M 8 M -1 M 9 M -1 M 10 M -1 M 11 M -1 M 12 M -1 or at least 10 13 M -1 refers to an antibody or its antigen-binding fragment. An antibody or its antigen-binding fragment with "low affinity" means that Ka is 10 7 M -1 or less, 10 6 M -1 or less, 10 5 M -1 or less, refers to an antibody or its antigen-binding fragment. Alternatively, affinity can also be defined as the equilibrium dissociation constant (Kd) in units of M for a specific binding interaction (e.g., 10 -5 M to 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 the binding domain polypeptide and the single-chain polypeptide according to the present disclosure can be easily determined using conventional techniques (see, for example, Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51:660; and U.S. Patent Nos. 5,283,173, 5,468,614, or equivalents).

[0037] In the art, a “conservative substitution” is recognized as the substitution of one amino acid with another amino acid having similar properties. Exemplary conservative substitutions are well known in the art (see, for example, PCT application publication 97 / 09433, p. 10, published March 13, 1997; 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 enzymes, such as glycosylation, alkylation, acylation, esterification, or amide formation. Polypeptides or polynucleotides used herein that are derived from another polypeptide or polynucleotide are referred to as “parent” polynucleotides or “reference” polynucleotides, or as “parent” polypeptides or “reference” polypeptides. For example, a humanized antibody may be derived from a parent antibody, which is a mouse antibody.

[0039] As used herein, the term "mutant" refers to a polynucleotide or polypeptide whose sequence differs from that of a reference polynucleotide or reference polypeptide, but which retains the fundamental properties of the parent polynucleotide or parent polypeptide. Generally, the polynucleotide or polypeptide sequence of a mutant is, overall, similar to, and identical to, the parent polynucleotide or parent polypeptide in many regions. For example, a mutant polynucleotide or mutant 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 parent 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. Sequences are said to be identical at a position if that 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 sequence identity percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs, thereby obtaining 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 sequence identity percentage. The sequence identity percentage is determined by comparing two optimally aligned sequences across the comparison region. The comparison region for polynucleotide sequences may 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 nucleic acid lengths or greater. The comparison region for polypeptide sequences may 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 amino acid lengths or greater. For comparison purposes, to align sequences in the most optimal way, portions of polynucleotide or polypeptide sequences within the comparison region may contain additions or deletions, known as gaps, while maintaining a constant reference sequence. Optimal alignment is one that, despite the presence of gaps, results in the maximum possible number of "identical" positions between the reference sequence and the comparison sequence.The "sequence identity" percentage 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. This program incorporates the BLASTN program (for nucleotide sequence comparison) and the BLASTP program (for polypeptide sequence comparison), both based on the algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When using Sequences, the default parameters as of September 1, 2004, may be used for any other required parameters, including but not limited to word size (3), gap start penalty (11), gap extension penalty (1), gap drop-off (50), expected value (10), and matrix selection. Two nucleotide or amino acid sequences are considered to have "substantially similar sequence identity" or "substantially similar 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 with respect to each other.

[0041] The term "substantially identical" refers to polypeptide sequences that contain a sufficient number of the same amino acids as the second polypeptide sequence, such that the first polypeptide sequence and the second polypeptide sequence 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 their amino acid sequences.

[0042] The term "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to, or derived from, a portion of an antibody capable of binding to the Fc receptor and / or the C1q component of complement on a cell, thereby mediating the effector function of the antibody. Fc stands for "Fc (fragment crystalline)," which is an antibody fragment that readily forms protein crystals. Originally described by proteolytic digestion, distinctly different protein fragments can define the overall general structure of immunoglobulin proteins. As defined in the original literature, the Fc region is a homodimeric protein containing two polypeptides associated by disulfide bonds, each containing a hinge region, a CH2 domain, and a CH3 domain. However, more recently, the term "Fc region" or "Fc domain" has been applied to single-chain monomeric components consisting of CH3, CH2, a second such chain, and at least a portion of the hinge sufficient to form a disulfide-linked dimer. In this specification, the terms “Fc region” or “Fc domain” refer, as such, and depending on the context, to individual monomers in dimeric form or associating to form a dimeric protein. For a review of the structure and function of immunoglobulins, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, the term Fc domain includes variants of naturally occurring sequences.

[0043] The terms “immunoglobulin constant region” or “constant region” refer to a peptide or polypeptide sequence that corresponds to, or is derived from, some 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 the CH1 domain. In certain embodiments, the constant domains constituting 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 variable binding regions derived from the light and heavy chains of an antibody, respectively. The variable binding regions consist of separate, 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 a 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 the CH1, CH2, and CH3 domains (IgA, IgD, IgG) or the CH1, CH2, CH3, and CH4 domains (IgE, IgM). As used herein, the terms “complementarity-determining region” or “CDR” refer to an immunoglobulin (antibody) molecule. There are three CDRs per variable domain: CDR1, CDR2, and CDR3 within the variable domain of the light chain, and CDR1, CDR2, and CDR3 within the variable domain of the heavy chain.

[0045] In some embodiments, “hinge” or “hinge region” refers to a polypeptide derived from the hinge region of an immunoglobulin and located between the antigen-binding domain (e.g., a ceramide-binding domain) and the constant region of the immunoglobulin within the polypeptides described herein. “Wild-type immunoglobulin hinge region” refers to a naturally occurring upper and central hinge amino acid sequence found in the heavy chain of an antibody, inserted between the CH1 domain and the CH2 domain (for IgG, IgA, and IgD) to connect them, or inserted between the CH1 domain and the CH3 domain (for IgE and IgM) to connect them. 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 this, an IgG1 hinge region, an IgG2 hinge region, an IgG3 hinge region, or an IgG4 hinge region).

[0046] A “modified immunoglobulin hinge region” or “mutant immunoglobulin hinge region” refers to a polypeptide of a hinge region that has one or more mutations, substitutions, insertions, or deletions compared to its corresponding parent, wild-type immunoglobulin hinge region. Typically, a modified immunoglobulin hinge region is a fragment of a wild-type immunoglobulin hinge region, and the core hinge region of IgG includes the core hinge region of IgG (e.g., a polypeptide containing the sequence CXXC [wherein X is any amino acid]) as disclosed in U.S. Patent Application Publications 2013 / 0129723 and 2013 / 0095097.

[0047] As used herein, the term “humanized” refers to an antibody or its antigen-binding fragment derived from a non-human species that retains the antigen-binding properties of the original non-human antibody. In some embodiments, the antibody-binding fragment (e.g., the light chain variable region and the heavy chain variable region, Fab, scFv) is humanized. Non-human antigen-binding fragments undergo "resizing" (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), "hyperchimerization" (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154), and "veneering" (Mark, et al., “Derivation of therapeutically active humanized and veneered anti-CD18 antibodies.”). Cellular adhesion can be humanized using known techniques, including CDR grafting (Jones et al., Nature 321:522 (1986)) and its variations, as described in: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312). When derived from non-human sources, other regions of the antibody, such as the hinge region and constant region domains, can also be humanized.

[0048] As used herein, the term “pharmaceutically acceptable” generally refers to molecular entities and compositions that, when administered using routes known in the art, do not produce allergic reactions or other serious adverse reactions. For use in mammals, and more specifically for use in humans, molecular entities and compositions that are approved by a U.S. federal or state regulatory agency, or listed in the United States Pharmacopeia or any other generally recognized pharmacopoeia, are considered “pharmaceutically acceptable.”

[0049] As used herein, the term “polynucleotide” means 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 of these nucleotides, such as modified messenger RNA. Such modifications include base modifications such as bromulidine, ribose modifications such as arabinosides and 2',3'-dideoxyribose, and internucleotide ligation modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladetes, and phosphoramidates. Specifically, the term “polynucleotide” includes single-stranded and double-stranded forms of DNA.

[0050] As used herein, “polypeptide” or “protein” refers to a single, linear, and continuous arrangement of covalently linked amino acids. Polypeptides 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 interchain disulfide bonds. In this specification, a protein or polypeptide may be a fragment of an antibody or an antigen-binding antibody. As used herein, the terms “transformation,” “transfection,” and “transduction” refer to the introduction of polynucleotides into cells. As used herein, the term “genetic transformation” refers to the introduction and integration of DNA, in particular recombinant DNA, into cells. The nucleic acids to be introduced may be introduced into cells via expression vectors.

[0051] As used herein, the terms “treatment,” “treating,” or “improving” refer to therapeutic or preventive / preventive treatments. A treatment is therapeutic if it alleviates at least one symptom of a disease in the individual being treated, or if it can delay the exacerbation of a progressive disease in the individual or prevent the onset of further related diseases. As used herein, the term "LD90" refers to the "90% lethal dose" or "median lethal dose," which is the amount of a substance required to kill 90% of the test population. Unless the content explicitly indicates otherwise, the singular forms “a,” “an,” and “that” as used in this specification and the accompanying claims also include plural referents. Unless otherwise indicated, the terms "and / or" as used herein mean "and" or "or". Throughout this specification, unless otherwise required by the context, the word “comprisie,” or variations such as “comprises” or “comprising,” should be understood to imply the inclusion of the 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 antibodies This disclosure presents an antibody or its antigen-binding fragment that specifically binds to ceramide. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment. In other embodiments, the anti-ceramide antibody is a humanized 6B5 scFv antibody.

[0053] As used herein, the term “ceramide” refers to a second messenger molecule in lipids, composed of sphingosine and fatty acids. Ceramides regulate intracellular stress signaling through the reorganization of cell membranes and the formation of lateral Lo (liquid-ordered) phase microdomains (a type of “raft” referred to as a “ceramide-rich platform”). These CRPs are used as signaling platforms, clustering activating receptor molecules (e.g., members of the TNF-receptor hooper family, including Fas), thereby resulting in a feedforward mechanism and ultimately leading to signal amplification and signaling. CRP formation is particularly important for Fas-mediated cell death, and ceramide-rich regions on the cell membrane of target cells are critical to susceptibility to cytotoxic T lymphocyte (CTL)-induced cell death. Exemplary ceramides include glycosylceramide, galactosylceramide, and gangliosides (oligosaccharide-bound ceramides).

[0054] In some embodiments, the disclosure presents an antibody or antigen-binding fragment comprising an immunoglobulin heavy chain variable region (VH) that specifically binds to ceramide and includes 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) that includes 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] Exemplary CDRs of anti-ceramide antibodies are shown in Table 1. [Table 1]

[0056] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is an scFv comprising an immunoglobulin heavy chain variable region (VH) containing 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) containing 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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which case (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which case (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which case (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which case (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which case (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which case (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which case (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which case (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 its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, in which case (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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the VH chain is identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 17-21 by 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%. The VL chain contains or consists of amino acid sequences that are 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.

[0066] In some embodiments, an anti-ceramide antibody or its antigen-binding fragment comprises a VH chain and a VL chain, wherein the VH chain is identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 17-21 by 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%. scFv containing or consisting of acid sequences, wherein the VL chain contains 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.

[0067] [Table 2]

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

[0069] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is from SEQ ID NO: 17 and the amino acid sequence of the VL chain is from SEQ ID NO: 22.

[0070] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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.

[0071] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is sequence number 17 and the amino acid sequence of the VL chain is sequence number 24.

[0072] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is from SEQ ID NO: 18 and the amino acid sequence of the VL is from SEQ ID NO: 22.

[0073] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is from SEQ ID NO: 18 and the amino acid sequence of the VL chain is from SEQ ID NO: 23.

[0074] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is sequence number 18 and the amino acid sequence of the VL chain is sequence number 24.

[0075] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is from SEQ ID NO: 19 and the amino acid sequence of the VL chain is from SEQ ID NO: 22.

[0076] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is from SEQ ID NO: 19 and the amino acid sequence of the VL chain is from SEQ ID NO: 23.

[0077] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is from SEQ ID NO: 19 and the amino acid sequence of the VL chain is from SEQ ID NO: 24. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is from SEQ ID NO: 20 and the amino acid sequence of the VL is from SEQ ID NO: 22.

[0078] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment comprises the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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 its antigen-binding fragment comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is from SEQ ID NO: 21 and the amino acid sequence of the VL is from SEQ ID NO: 22.

[0079] In some embodiments, the anti-ceramide antigen-binding fragment is an scFv comprising the amino acid sequence of VH, which 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 the amino acid sequence of VL, which 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, anti-ceramide scFv comprises a VH chain and a VL chain, in which case the amino acid sequence of the VH chain is sequence number 18 and the amino acid sequence of the VL chain is sequence number 22.

[0080] In some embodiments, the anti-ceramide antigen-binding fragment is an scFv. In some embodiments, the anti-ceramide scFv includes 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.

[0081] In some embodiments, anti-ceramide scFv comprises the following structures: VH-linker-VL or VL-linker-VH. As used herein, the term “linker” generally refers to a short polypeptide sequence that connects two subdomains of a polypeptide. Non-limiting examples of linkers include flexible linkers containing glycine-serine repeats, and linkers derived from (a) interdomain regions of transmembrane proteins (e.g., type I transmembrane proteins); (b) stalk regions of type II C-lectins; or (c) hinges of immunoglobulins. In some embodiments, the linker provides a spacer function for the interaction and compatibility of two binding subdomains so that the resulting polypeptide maintains specific binding affinity to antibodies containing the same light-chain and heavy-chain variable regions and to the same target molecule. In certain embodiments, the linker consists 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, an anti-ceramide antibody or its antigen-binding fragment is constructed with an N-terminal signal sequence that promotes the secretion of the scFv protein. In some embodiments, the signal sequence includes SEQ ID NO: 75. Those skilled in the art will understand that these signal sequences are not part of the mature secretory protein. Therefore, 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 its antigen-binding fragment is prepared with a C-terminal tag sequence to facilitate purification during manufacturing. In some embodiments, the C-terminal tag includes the amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is prepared without a C-terminal tag sequence. Therefore, in some embodiments, the amino acid sequence of the anti-ceramide antibody or its antigen-binding fragment does not include the C-terminal tag of SEQ ID NO: 76.

[0085] An exemplary amino acid sequence of anti-ceramide scFv is shown in Table 4 below. The CDR of scFv is indicated by the underlined text. The linker sequence is indicated by the italicized text. The signal sequence is indicated by the bold text. The C-terminal tag sequence is indicated by the bold and italicized text.

[0086] [Table 4] JPEG2026104852000006.jpg244170

[0087] In some embodiments, the anti-ceramide antibody is a full-length antibody comprising a full-length light chain and a full-length 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 contains a wild-type IgG amino acid sequence. Such sequences are known in the art (see, for example, Shields et al., J Biol Chem, (2001) 276:9;6591-6604). In some embodiments, the CH2 or CH3 domain of the Fc domain contains 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 certain Fc receptors, such as the neonatal Fc receptor (FcRn), and has a relatively long half-life in vivo. In some embodiments, the described anti-ceramide antibodies either fail to result in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement activation, and / or complement-dependent cytotoxicity (CDC), or substantially reduce their induction. 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 antibody is an IgG1 isotype antibody, in which case 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 follows 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 indicated, the positions of amino acid residues within immunoglobulin molecules used herein are numbered according to the EU numbering system (Ward et al., 1995 Therap. Immunol. 2:77-94). Other numbering systems for amino acid positions within antibodies are also known in the art, e.g., 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 may be prepared using standard molecular biology methods, with regard to the selection of antibodies having the desired specificity. In some embodiments, anti-ceramide antibodies and their antigen-binding fragments are produced using recombinant DNA technology. Procedures for the expression and purification of recombinant proteins are well established in the art.

[0090] Polymigration and protein expression methods This disclosure also includes polynucleotides (e.g., DNA or RNA) encoding the anti-ceramide antibodies and their antigen-binding fragments. In some embodiments, the polynucleotides encode polypeptides that are substantially identical to the polypeptides 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 the polypeptides listed in Tables 1, 2, 3, and 4. The polynucleotides of this disclosure also include complementary nucleic acids. In some cases, the sequences will be perfectly complementary (without 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 this disclosure encompasses such modifications of sequences.

[0091] In some embodiments, the disclosure presents isolated polynucleotides encoding an anti-ceramide antibody or its antigen-binding fragment, which are 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. In some embodiments, the disclosure presents isolated polynucleotides encoding an anti-ceramide antibody or its antigen-binding fragment, which include or consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs.

[0092] In some embodiments, the polynucleotides of this disclosure are inserted into nucleic acid vectors. Nucleic acid vectors may be viral vectors or non-viral vectors, such as plasmids. 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 phages or M13 phages, 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 may be 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. Patent 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.See (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 (see, e.g., mouse leukemia virus, splenic necrosis virus, and retroviral vectors such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus). Examples of vectors include the pClneo vector (Promega) for expression within mammalian cells; and pLenti4 / V5-DEST®, pLenti6 / V5-DEST®, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene introduction and expression within mammalian cells.

[0093] In some embodiments, the polynucleotides of the Disclosure are inserted into a nucleic acid vector and operably ligated to one or more regulatory sequences that control transcription, such as promoters, enhancers, terminators, inducers, or repressors.Appropriate eukaryotic promoters (promoters that function within eukaryotic cells) are not limited to the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase promoter, the monkey virus 40 (SV40) promoter (e.g., the early SV40 promoter and the late SV40 promoter), the spleen focus-forming virus (SFFV) promoter, and retrovirus-derived LTRs (long terminal promoters). (repeats) (e.g., Moloney mouse leukemia virus (MoMLV) LTR promoter, or Rous sarcoma virus (RSV) LTR), herpes simplex virus (HSV) (thymidine kinase) promoter, vaccinia virus-derived H5 promoter, P7.5 promoter, and P11 promoter, 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 70kDa protein 5 (HSPA5) promoter, heat shock protein 90kDa beta, member 1 (HSP90B1) promoter, heat shock protein 70kDa (HSP70) promoter, β-kin promoter, human ROSA 26 locus (Irions et al., Nature Biotechnology) This includes 25, 1477-1482 (2007), the ubiquitin C (UBC) promoter, the phosphoglycerate kinase 1 (PGK) promoter, the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, the β-actin promoter and the myeloproliferative sarcoma virus enhancer, the negative regulatory region deletion dl587rev primer-binding site substitution (MND) promoter, and the eukaryotic promoter derived from mouse metallothionein-1.

[0094] In some embodiments, the vector is introduced into host cells to express an anti-ceramide antibody or its antigen-binding fragment. Therefore, proteins for use within this disclosure can be produced in genetically modified host cells according to standard techniques. Suitable host cells are cell types transformed with or transfected with exogenous DNA, grown in culture, and include 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 in 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] The gene products encoded by the polynucleotides of this disclosure can be 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 fetal 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 cyanobacteria cells (CHO-K1; ATCC CCL61; CHODG44; CHO DXB11 (Hyclone, Logan, UT); see also, for example, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986), rat pituitary cells (GH1; ATCC CCL82), and HeLa S3 cells (ATCC This includes CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650), and mouse fetal cells (NIH-3T3; ATCC CRL 1658). Further suitable cell lines are known in the art and are available from publicly contracted institutions such as the American Type Culture Collection, Manassas, Virginia. The introduction of DNA constructs can be carried out using any convenient method, including, for example, conjugation, bacterial transformation, calcium precipitation of DNA, electroporation, fusion, transfection, viral vector infection, and biorhythmics.

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

[0097] A secretory signal sequence (also known as a reader sequence) is added to the expression vector to direct recombinant proteins to the host cell's secretory pathway. The secretory signal sequence may be a native secretory signal sequence for the recombinant protein, derived from another secretory protein, or synthesized de novo. The secretory signal sequence is operably ligated to the polypeptide-encoding DNA sequence; that is, the two sequences are linked within a proper reading frame, positioned to direct the newly synthesized polypeptide to the host cell's secretory pathway. The secretory signal sequence is generally positioned at the 5' end of the DNA sequence encoding the target polypeptide, although certain signal sequences may be positioned at other locations within the target DNA sequence (see, for example, U.S. Patents 5,037,743 and 5,143,830). In some embodiments, the secretory signal sequence is Sequence ID No. 75.

[0098] Cultured mammalian cells are suitable hosts for the production of recombinant polypeptides and recombinant proteins (e.g., anti-ceramide antibodies and their antigen-binding fragments) according to this 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 preparation of recombinant polypeptides in cultured mammalian cells is disclosed, for example, in U.S. Patents No. 4,713,339; No. 4,784,950; No. 4,579,821; and No. 4,656,134.

[0099] Host cells transformed or transfected to produce polypeptides and proteins of this disclosure (e.g., ceramide-binding polypeptides) are cultured in a culture medium containing nutrients and other components required for the growth of selected host cells, following standard procedures. In the art, a variety of suitable media are known, including known-composition media and complex media, and generally contain a carbon source, a nitrogen source, basic amino acids, vitamins, and minerals. The media may also contain components such as growth factors or serum, as required. Growth media are generally selected for cells containing exogenously added DNA, for example, by drug selection or by deficiencies in basic nutrients, supplemented by selection markers that are delivered on a vector or co-transfected into host cells.

[0100] The anti-ceramide antibodies and their antigen-binding fragments described herein can typically be purified by conventional protein purification methods, usually involving a combination of chromatographic techniques. See, for general information, 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 immunoglobulins can be purified by affinity chromatography against immobilized protein A or protein G. Further purification steps, such as gel filtration, may be used to obtain the desired level of purity or to result in desalting, buffer exchange, etc.

[0101] Method for producing humanized antibodies This disclosure discloses 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 provide 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 shown in Table 5 below. The CDR is indicated by underlined text. The linker sequence is indicated by italicized text. The signal sequence is indicated by bold text. The C-terminal tag sequence is indicated by bold and italicized text.

[0102] [Table 5]

[0103] The substitution of mouse CDRs with human variable domain frameworks is most likely to result in the preservation of their proper spatial orientation if the human variable domain framework adopts the same or similar conformation as the mouse variable framework from which the CDRs are derived. This is achieved by the framework sequence being derived from the mouse variable framework domain from which the CDRs are derived and from human variable domains derived from human antibodies exhibiting a high degree of sequence identity. The heavy-chain and light-chain variable framework regions may be derived from the same human antibody sequence or from different human antibody sequences. The human variable framework region sequence may be from a naturally occurring human antibody sequence or from 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 CDR of the donor mouse immunoglobulin and the appropriate acceptor human framework region have been identified, the next step is to determine which residues derived from these components should be modified to optimize the properties of the resulting humanized antibody, if any exist. In general, 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 human-to-human. Methods for determining immune responses that are recognized in the art may 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 partially determined by computer modeling. This specification describes computer hardware and software for creating three-dimensional images of immunoglobulin molecules. Generally, molecular models are created starting from elucidated structures of immunoglobulin chains or domains. The chain to be modeled is compared to a chain or domain whose three-dimensional structure has been elucidated for amino acid sequence similarity, and the chain or domain showing the greatest sequence similarity is selected as the starting point for constructing the molecular model. A chain or domain sharing at least 50% sequence identity is selected for modeling, preferably a chain or domain sharing at least 60%, 70%, 80%, 90%, or more sequence identity is selected for modeling. The elucidated starting structure is modified to allow for differences between the actual amino acids in the chain or domain of the immunoglobulin to be modeled and the amino acids in the chain or domain in the starting structure. The modified structure is then assembled into a complex immunoglobulin. Finally, the model is refined by verifying that the energy is minimized, all atoms are within a suitable distance from each other, and bond lengths and bond angles are within chemically acceptable limits.

[0106] The selection of amino acid residues for modification may also be partially determined by examining the characteristics of amino acids at specific positions, and may also be partially determined by empirical observations of the effects of substitution or mutagenesis of specific amino acids. Further exemplary humanization methods that may be used to humanize the immunoglobulins of this 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 region in a humanized antibody is identical to the corresponding CDR region in a mouse donor antibody. For example, in some embodiments, the amino acid sequence of the CDR in a humanized antibody and its fragments is identical to the amino acid sequence of the CDR in a 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 antibody-antigen binding specificity and / or reduce the immunogenicity of the antibody. In some embodiments, one or more residues of the CDR are modified to modify binding in order 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 even higher binding affinity can be achieved. The affinity maturation methods described herein can be used to modify the parent CDR region and then screen the resulting binding molecules for desired binding changes. The methods can also be used to reduce immunogenicity by modifying the parent CDR so that potential human anti-mouse antibody (HAMA) reactions are minimized or avoided. Thus, changes in binding affinity and immunogenicity in response to CDR modifications can be monitored and evaluated so that antibodies optimized for the best combination of binding and low immunogenicity are achieved (see, for example, U.S. Patent No. 6,656,467 and U.S. Patent Application Publication No. 20020164326). Furthermore, it is possible, in some cases, to perform one or more conserved amino acid substitutions of CDR residues without significantly affecting the binding affinity of the resulting humanized immunoglobulin. Conservative substitutions are intended combinations such as gly, ala;val, ile, leu;asp, glu;asn, gin;ser, thr;lys, arg; and phe, tyr.

[0108] In some embodiments, the humanized anti-ceramide antibody and its antigen-binding fragment contain one or more mutations compared to the corresponding CDR sequence of the mouse 6B5 antibody (e.g., compared to one or more of sequence numbers 11-16). Exemplary mutations in the CDR amino acid sequence of the mouse 6B5 CDR are shown in bold in Figure 4A.

[0109] The framework regions of humanized immunoglobulins are typically substantially identical, and more typically identical, to, the framework regions of the human antibodies from which they are derived. In some embodiments, the humanized antibodies of this disclosure include a variable region framework sequence selected from a human antibody gene (e.g., a germline antibody gene segment) that includes one or more canonical CDR structural types that are identical or similar to the CDR structural type that is canonical for the corresponding non-human antibody being humanized (e.g., a mouse antibody). See U.S. Patent No. 6,881,557 and Tan et al., Journal of Immunol 169:1119-1125 (2002) (incorporated in their entirety by reference for all purposes). Naturally, many amino acids within the framework region have little to no direct contribution to the specificity or affinity of the antibody. Therefore, many individual conservative substitutions of framework residues may be acceptable without significantly altering the specificity or affinity of the resulting humanized immunoglobulin. Framework residues, if present, can be analyzed to determine which residues should be substituted to optimize the properties of the resulting humanized antibody. For example, computer modeling can be used to identify residues that have a high probability of directly or indirectly affecting antigen binding.

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

[0111] In some embodiments, the humanized anti-ceramide antibody or its antigen-binding fragment is at least 10 against the antigen. 7 M -1 , 10 8 M -1 , 10 9 M -1 , or 10 10 M -1 It exhibits specific binding affinity. Typically, the upper limit of the binding affinity of a humanized antibody to an antigen is within 3, 4, or 5 times the binding affinity of the progenitor immunoglobulin. The lower limit of binding affinity is also often within 3, 4, or 1 / 5 of the binding affinity of the progenitor immunoglobulin. Alternatively, binding affinity can be compared to the binding affinity of a non-substituted humanized antibody (e.g., an antibody having a donor CDR and an acceptor FR, but without substitution). In such cases, the binding of the optimized antibody (substituted antibody) is preferably at least 2-3 times, or 3-4 times, the binding affinity of the non-substituted antibody. To make comparisons, the activity of various antibodies can be determined, for example, by BIACORE (i.e., surface plasmon resonance using unlabeled or reagent-based) or competitive binding assays.

[0112] Treatment method This application discloses a method for inhibiting cell death in a subject requiring such inhibition, comprising the step of administering to the subject a therapeutically effective dose of the anti-ceramide antibody or an antigen-binding fragment thereof of the present disclosure. In some embodiments, the antibody or the antigen-binding fragment thereof is a humanized scFv that specifically binds to ceramide. In some embodiments, 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 an anti-ceramide antibody or its antigen-binding fragment is administered to the subject before exposure to radiation.

[0113] Radiation targets both the gastrointestinal microvascular compartment and the intestinal stem cell compartment. Microvascular endothelial dysfunction, detected as apoptosis four hours after irradiation, represents the major lesion leading to GI syndrome. Endothelial dysfunction results in the loss of regenerative crypts and increased GI toxicity, as the lesions are converted into sublethal to lethal CBC (cycling crypt base columnar) cells. Immunohistochemical studies, as well as labeling studies with [3H]TdR and BrdUrd, 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 a late S phase checkpoint and mitotic arrest, seemingly preceded by radiation-induced DNA double-strand breaks (DSBs). Rapid apoptotic death occurs in cells that arrest proliferation within the first 24 hours after irradiation at 12 Gy, accounting for 33% of the total number of deaths. In mammalian cells, DNA dsb activates DNA damage recognition / repair pathways and regulates cell cycle checkpoint activity. Arrest of intestinal stem cell mitosis is thought to represent a regulatory event in this pathway. The mitotic morphology of death occurs during this second 24-hour period and accounts for 66% of the total number of dead cells. At this stage, although a significant change in crypt number across the perigutin is not apparent, a decrease in crypt size progresses due to the continued normal migration of transient / differentiated crypt cells from the crypts to the inner epithelial layer of the villous tissue, and the loss of villous apical stents. Recovery of mitotic activity after 12–18 hours is associated with rapid depletion of crypt stem cell chronogen and crypt number across the perigutin. The loss of stem cell chronogen leads to the overall collapse of the crypt-villous system, mucosal detachment, and death due to GI syndrome.

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

[0115] This disclosure relates to a method for mitigating cell death in GI syndrome in subjects requiring such mitigation. The method comprises administering a therapeutically effective dose of an anti-ceramide antibody or an antigen-binding fragment thereof. In some embodiments, the method comprises administering the anti-ceramide antibody or an antigen-binding fragment to the subject immediately after the subject's exposure to penetrating radiation. In other embodiments, the method comprises administering the anti-ceramide antibody or an antigen-binding fragment to the subject within one hour after the subject's exposure to penetrating radiation. In some embodiments, the method comprises administering the anti-ceramide antibody or an antigen-binding fragment to the subject within two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or eighteen hours after the subject's exposure to penetrating radiation. In some embodiments, the method comprises administering the anti-ceramide antibody or an antigen-binding fragment to the subject within 24 hours after the subject's exposure to penetrating radiation. In other embodiments, the method includes administering the anti-ceramide antibody or its antigen-binding fragment to the subject within 30, 36, 42, 48, 54, 60, 66, or 72 hours after the subject's exposure to penetrating radiation. In other embodiments, the method includes administering the anti-ceramide antibody or its antigen-binding fragment to the subject within 48, 36, 24, 18, 12, 10, 8, 6, 4, 2, or 1 hour before the subject's exposure to penetrating radiation, or within 45, 30, or 15 minutes.

[0116] In some embodiments, the disease is graft-versus-host disease, and the anti-ceramide antibody or its antigen-binding fragment 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 its antigen-binding fragment 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 its antigen-binding fragment is administered to a subject in need after the onset of graft-versus-host disease, in a dose effective in mitigating apoptosis in graft-versus-host disease.

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

[0118] In some embodiments, the Disclosure presents a method for treating a patient with GI syndrome or GvHD, comprising the step of administering to the patient an anti-ceramide antibody or an antigen-binding fragment thereof, comprising an amino acid sequence expressed herein (for example, an amino acid sequence selected from the group consisting of SEQ ID NOs. 48 to 61). In some embodiments, the Disclosure presents a method for treating a patient with GI syndrome or GvHD, comprising the step of administering an anti-ceramide antibody or an antigen-binding fragment thereof, of SEQ ID NOs. 48 or 51. In some embodiments, anti-ceramide antibodies and their antigen-binding fragments prevent radiation-induced cell death or cell death associated with GvHD or autoimmune diseases. Cell death can be measured by various means known in the art, including flow cytometry, immunofluorescence, and immunohistochemistry to assess changes in marker expression in dead cells, cell counting to assess viability, and qPCR to assess changes in gene expression. In some embodiments, anti-ceramide antibodies and their antigen-binding fragments prevent cell death by inhibiting the formation of ceramide-rich platforms. For example, in some embodiments, anti-ceramide scFv inhibits the formation of ceramide-rich platforms in irradiated Jurkat T cells (see Example 3 and Figure 1). In some embodiments, anti-ceramide antibodies or their antigen-binding fragments 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 their antigen-binding fragments 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 their antigen-binding fragments 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 treatments and uses described herein, the anti-ceramide antibody or its antigen-binding fragment is delivered in a manner consistent with conventional methods associated with the disease or disorder for which treatment is required. In accordance with the disclosures herein, a therapeutically effective amount of protein or polypeptide is administered to a subject requiring such treatment for a sufficient duration and under conditions sufficient to prevent or treat the disease or disorder.

[0120] In prophylactic applications, a pharmaceutical composition or agent containing an anti-ceramide antibody or its antigen-binding fragment is administered to patients who are susceptible to a particular disorder or, conversely, at risk of developing it, in an amount sufficient to eliminate the disorder, reduce the risk of developing the disorder, or delay its development. In therapeutic applications, a composition or agent containing the protein of this disclosure is administered to patients suspected of having such a disorder or who already have it, in an amount sufficient to cure the disorder or at least partially cessate the symptoms of the disorder and its complications. The amount sufficient to achieve this is referred to as the therapeutically effective dose or therapeutic dose. In both prophylactic and therapeutic regimens, the agent is typically administered in several doses until a satisfactory response (e.g., inhibition of inadequate angiogenesis) is achieved. Typically, the response is monitored, and repeated doses are administered when the desired response begins to weaken.

[0121] For administration, anti-ceramide antibodies or their antigen-binding fragments may be formulated as pharmaceutical compositions. A pharmaceutical composition may comprise (i) a ceramide-binding polypeptide and (ii) a pharmaceutically acceptable carrier, diluent, or excipient. Pharmaceutical compositions comprising a ceramide-binding polypeptide may be formulated according to known methods for preparing pharmaceutically useful compositions in which the therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier, diluent, or excipient. A carrier is considered a "pharmaceutically acceptable carrier" if its administration is acceptable to the 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, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further contain one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc.

[0122] Pharmaceutical compositions comprising polypeptides or proteins described herein may be formulated into dosage forms selected from the group consisting of oral, intravenous, intranasal, suppository, intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual, and intracerebral dosage forms. Oral dosage forms 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 may be administered to a subject in a therapeutically effective dose. According to the methods of this disclosure, the anti-ceramide antibody or its antigen-binding fragment may be administered to a subject by various administration methods, including, for example, intramuscular, subcutaneous, intravenous, intraatrial, intra-articular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, and oral administration routes. For prevention and treatment purposes, an agonist (e.g., a ceramide-binding polypeptide) may be administered to a subject by single bolus delivery, via long-term continuous delivery (e.g., continuous transdermal delivery), or by a repeated-dose protocol (e.g., an hourly, daily, weekly, or monthly-based protocol).

[0123] The effective dose of the compositions disclosed herein varies depending on many different factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, whether other pharmaceuticals or treatments administered are prophylactic or therapeutic, and the specific activity of the composition itself and its ability to induce a desired response in an individual. Typically, the patient is human, but in some diseases, the patient may be a non-human mammal. Typically, the administration regimen is adjusted to produce the best possible therapeutic response, i.e., to optimize stability and efficacy.

[0124] In this context, the determination of an effective dose is typically guided by determining an effective dose and administration protocol that significantly reduces the incidence or severity of the target disorder in the model subjects, based on animal model studies and subsequent human clinical trials. Therefore, as used herein, “therapeutic effective dose” refers to the amount of compound that achieves the desired biological or therapeutic effect, i.e., the amount that prevents, reduces, or improves one or more symptoms of the listed disease being treated or prevented. For example, the therapeutic effective dose of an antibody or its antigen-binding fragment 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. Anti-ceramide antibodies or their antigen-binding fragments may be administered to the patient in a single dose or over a series of treatments, and may be administered to the patient at any point in time after diagnosis. Anti-ceramide antibodies or their antigen-binding fragments may be administered as a monotherapy or in combination with other drugs or treatments that are useful in treating the condition in question.

[0125] In some embodiments, the therapeutically effective dose of anti-ceramide antibody or its antigen-binding fragment is between approximately 1 ng and approximately 100 mg per kg of body weight per day. In some embodiments, the range of antibodies administered is approximately 1 ng to approximately 1 μg per kg of body weight per day, 1 ng to approximately 100 ng per kg of body weight per day, 1 ng to approximately 10 ng per kg of body weight per day, 10 ng to approximately 1 μg per kg of body weight per day, 10 ng to approximately 100 ng per kg of body weight per day, 100 ng to approximately 1 μg per kg of body weight per day, and 100 ng to approximately 1 μg per kg of body weight per day. The dosage ranges are approximately 10 pg, 1 μg to approximately 10 pg per kg of body weight per day, 1 μg to approximately 100 pg per kg of body weight per day, 10 pg to approximately 100 pg per kg of body weight per day, 10 pg to approximately 1 mg per kg of body weight per day, 100 μg to approximately 10 mg per kg of body weight per day, 1 mg to approximately 100 mg per kg of body weight per day, and 10 mg to approximately 100 mg per kg of body weight per day. Doses within this range can be achieved, for example, by single or multiple doses, including daily, weekly, bi-weekly, or monthly administrations. Anti-ceramide antibodies or their antigen-binding fragments may be administered as a single dose by bolus or serial infusion, or as multiple doses by bolus or serial 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 administration regimens may also be useful. The progress of this treatment can be easily monitored using standard techniques.

[0126] For administration to adult human patients, effective therapeutic doses may be administered in doses ranging from 0.0006 mg to 1000 mg per dose, including but not limited to 0.0006 mg, 0.001 mg, 0.003 mg, 0.006 mg, 0.01 mg, 0.03 mg, 0.06 mg, 0.1 mg, 0.3 mg, 0.6 mg, 1 mg, 3 mg, 6 mg, 10 mg, 30 mg, 60 mg, 100 mg, 300 mg, 600 mg, and 1000 mg per dose. Treatment courses may involve multiple, typically daily, consecutive doses. Anti-ceramide antibodies or their antigen-binding fragments may be administered at different times of the 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 patient's physique, age, and condition.

[0127] The dosage of a pharmaceutical composition containing an anti-ceramide antibody or its antigen-binding fragment may be varied by the attending physician to maintain the desired concentration at the target site. For example, when intravenous delivery is selected, the local concentration of the drug in the bloodstream at the target tissue may be between approximately 0.01 and 50 nM, and possibly between approximately 1.0 nM and 10, 15, or 25 nM, based on the estimated value of the subject's condition and response. Higher or lower concentrations may be selected based on the delivery method, e.g., transepidermal delivery as opposed to delivery to the mucosal surface. The dosage will also be adjusted based on the release rate of the administered formulation, e.g., powder, sustained-release oral particles or sustained-release injectable particles, transdermal formulations, etc., compared to a nasal spray. 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 its antigen-binding fragment may be supplied as a kit comprising a container containing the pharmaceutical composition described herein. The pharmaceutical composition may also be supplied, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder to be restored before injection. Alternatively, such a kit may include a dry powder dispenser, a liquid aerosol generator, or a sprayer for administering the pharmaceutical composition. Such a kit may further include written information regarding the indications and uses of the pharmaceutical composition. [Examples]

[0129] This disclosure should not be considered limiting, as further examples illustrate the following embodiments. All references, patents, and patent application publications, as well as figures and tables, cited throughout this application are incorporated herein by reference.

[0130] (Example 1) Humanization and production of 6B5 antibodies The goal of this study was to maximize the amount of human sequences in the humanized ceramide antibody while maintaining the original specificity of the mouse 6B5 antibody. Antibody Humanization Design: Humanization design of the mouse 6B5 parental antibody was performed using computer analysis. Briefly, homology modeling was used to generate the 3D structure of the parental 6B5 antibody, and a profile of the parental 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, interface position matching, similarly classified canonical CDR positions, and the presence of N-glycosylation sites. Humanized antibodies were then designed by creating multiple hybrid sequences by fusing selected portions of the parental antibody sequence with human framework sequences. Using the 3D structure of the parental 6B5 antibody, these humanized sequences were histologically analyzed visually and by computer modeling to isolate the sequences most likely to retain binding to the antigen. 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 humanized VL1 utilize their respective first frameworks, containing the maximum possible human sequence along with the minimum amount of parental antibody framework sequence. VH2 and VL2 use the same framework as VH1 and VL1, but contain additional parental sequences. VH3 and VL3 utilize their respective second frameworks, similar to humanized VH2 and humanized VL2, but also contain additional parental sequences fused with the human framework. Fully humanized antibodies were created by combining the humanized heavy chains and humanized light chains described above. The combinations of humanized heavy chains and humanized light chains were investigated in terms of their expression levels and antigen-binding affinity to identify antibodies that act similarly to the mouse 6B5 parental antibody. Calculation of Humanity Score for Manipulated Antibodies: We developed a tool for calculating the humanity score of manipulated 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 mouse 6B5 and humanized 6B5 are shown in Tables 7 and 8 below.

[0133] [Table 7]

[0134] [Table 8]

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

[0136] (Example 2) Preparation and analysis of humanized anti-ceramide scFv Antibody construct expression: 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 a transfection-appropriate amount, and the plasmid DNA quality was evaluated by gel electrophoresis. CHO cells were seeded in shake flasks and expanded using serum-free medium of known composition. On the day of transfection, the expanded cells were seeded into unused flasks with unused medium, and each antibody expression construct was transiently transfected into the CHO cells using standard procedures known in the art. The cells were maintained as fed-batch cultures (C4611 and C4612, Medna) until the end of the preparation. The conditioned medium from the transient transfection was collected 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. Wash buffer was passed through the column until the OD280 value (NanoDrop, ThermoScientific) was zero. The target protein was eluted with a low pH buffer, the fraction was collected, and the OD280 value of each fraction was recorded. The fractions containing the target protein were pooled and filtered through a 0.2 μm membrane. 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-equilibriumized with binding buffer. A washing buffer containing 40 mM imidazole was passed through the column until the OD280 value (NanoDrop, ThermoScientific) approached zero. The target protein was eluted by a linear gradient increasing the imidazole concentration to 0.5 M. The eluates were collected in fractions, and the OD280 value of each fraction was recorded. CE-SDS (SDS gel capillary electrophoresis) (LabChip GXII, Perkin Elmer) was performed on each fraction and analyzed. The fractions containing the target protein were pooled and dialyzed to the 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, Thermo Scientific) 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). The recovered fractions were analyzed by CE-SDS (LabChip GXII, Perkin Elmer). Fractions containing the target protein were pooled and analyzed by SE-UPLC. Table 9 shows a summary of the analysis reports for 2766 antibody constructs using each purification method.

[0140] [Table 9]

[0141] (Example 3) Humanized 6B5 scFv inhibits the formation of ceramide-rich platforms in vitro. Next, the humanized anti-ceramide scFv prepared in Examples 1 and 2 were investigated 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% CO2 at 37°C. The cells were washed and resuspended in RPMI 1640 medium supplemented with 1% FBS or defatted FBS. In summary, cells were counted using a hematologist, with 1 × 10⁶ cells per 0.5 ml. 6The cells were seeded in a 24-well plate at 37°C. After 2 hours, the cells were stimulated with 0.6 μg / ml human anti-CD28 antibody (AF-342-PB; R&D systems). In addition, Jurkat T lymphocytes were pre-treated with anti-CD28 antibody (0.6 μg / mL; model number: AF-342-PB; R&D systems) at a concentration of 50 ng / mL to 800 ng / mL humanized 6B5 scFv for 1 hour prior to the 45-second stimulation. The cells were then rapidly fixed at 4°C with 4% paraformaldehyde for 10 minutes.

[0142] Ceramide-rich platform (CRP) was detected as previously described (JA 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 using mouse anti-ceramide 1° Ab MID 15B4 IgM (1:50, Alexis Biochemicals) overnight at 4°C. Unrelated mouse IgM was used as an isotype control. Cells were then washed three times with PBST and stained with Cy3 conjugate 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). The platform was imaged at room temperature for volumetric analysis on a Leica SP5 inverted microscope with a HyD hybrid detector photon counter and on an HCX PL APO CS 63.0x 1.40 oil objective lens with digital zoom at 7, and for incidence analysis on an HCX PL APO CS 40.0×1.25 oil objective lens with digital zoom at 1. 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, for each sample, 10-12 individual Z-stacks were collected under optimized conditions, and the data was transferred to the Imaris software. Intensity normalization was performed for both control and treated samples. Briefly, the threshold for the mean intensity of the z-column was obtained from the untreated control sample. Based on the mean intensity threshold, surfaces were created for the untreated control CD28-treated cells and scFv-treated cells using the "Surfaces" function in Imaris. The mean intensity threshold ensures that only intensity values ​​within a specific range are considered positive for surface volume quantification, while all values ​​outside this range are considered negative. Volume for each surface is generated by the surface volume function in Imaris. For the final analysis, the individual volumes for each sample set are summarized, and the average value is calculated. To calculate the IC50 value, the inhibitory effect of each antibody was normalized against a CRP inhibition scale of 0-100% [in the scale, 0% represents the untreated control (CD28 antibody alone), and 100% represents the antibody treatment that achieved the best inhibitory effect]. The data was plotted using GraphPad Prism 8, and curves were fitted using nonlinear regression analysis. As shown in Figure 1, pre-incubation with different concentrations of 6B5 scFv inhibited CRP formation in CD28-activated T cells. VH2-VL1 was the most effective in preventing CRP formation. 50 The value was 6.1 ng / mL. IC for various humanized 6B5 scFv samples. 50 The values ​​are shown in Table 10 below.

[0144] [Table 10]

[0145] (Example 4) Humanized 6B5 scFv inhibits radiation-induced apoptosis in vitro. Next, we investigated the ability of the humanized anti-ceramide scFv prepared in Examples 1 and 2 to inhibit radiation-induced cell death.

[0146] Apoptosis assays were performed as described (Haimovitz-Friedman et al., 1994; JA Rotolo et al., 2005). Jurkat T lymphocytes were resuspended in RPMI 1640 supplemented with 1% FBS, pre-treated with 100 ng / mL, 200 ng / mL, 400 ng / mL, 600 ng / mL, or 800 ng / mL of humanized 6B5 scFv at 37°C for 1 hour, and then exposed 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. Next, the cells were washed with PBS, stained with bisbendimide trihydrochloride (Hoechst 33258; Sigma-Aldrich), a 24 μg / ml fluorophore, and placed on glass slides for counting on an Olympus IMT-2 microscope using a 40 × DPlanApo40UV 0.850160 / 0.11-0.23 objective lens at room temperature. Apoptosis was quantified using fluorescence microscopy based on the morphological features of apoptotic nuclei, including chromatin condensation, segmentation, and the appearance of condensation and apoptotic bodies with nuclear periphery. A minimum of 100 cells were counted per sample. The percentage of apoptosis inhibition was determined by normalizing the values ​​against a radiation-only control (Figure 2A). IC 50 The values ​​were calculated using nonlinear regression in GraphPad Prism v7 software.

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

[0148] [Table 11]

[0149] Using a similar experimental setup, two further humanized 6B5 scFvs, VH4-VL1 (SEQ ID NO: 52) and VH5-VL1 (SEQ ID NO: 53), were investigated for their ability to inhibit radiation-induced T cell apoptosis (Figures 2B-2E). In Figure 2B, VH4-VL1, VH5-VL1, and the parental scFv mouse 6B5 scFv were incubated with Jurkat T cells for 1 hour at 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 600 ng / mL, or 800 ng / mL. The ability of each scFv to inhibit radiation-induced apoptosis was normalized against the protective effect of the parental scFv mouse 6B5 scFv at 800 ng / mL. In Figure 2C, VH4-VL1, VH5-VL1, and the parental scFv, mouse 6B5 scFv, were incubated with Jurkat T cells for 1 hour at concentrations of 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, or 800 ng / mL. 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 were incubated with Jurkat T cells for 1 hour at concentrations of 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 600 ng / mL, or 800 ng / mL. The ability of each construct to inhibit radiation-induced apoptosis was normalized to the protective effect of VH4-VL1 at 800 ng / mL. ICs based on these experimental sets were obtained. 50 The values ​​are shown 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), assessed by the percentage of radiation-induced apoptotic T cells under these conditions. Overall, these results support the idea that humanized 6B5 scFv can effectively protect T cells from radiation-induced apoptosis.

[0151] (Example 5) Treatment to prevent radiation-induced cell death in vivo in patients with humanized 6B5 scFv. The treatment of anti-ceramide VH2-VL1 (SEQ ID NO: 51) in vivo with mice exposed to a lethal dose of radiation was investigated. Using a cesium irradiator, 15 Gray units (Gy) of LD50 were administered. 90 The dose was delivered to C57BL / 6 mice, and 24 hours after irradiation, the mice were either left untreated or intravenously treated with 6B5 VH2-VL1 (150 μg / 25 g). Mice were euthanized at different time points after irradiation, and continuous segments of the jejunum proximal to the ligament of Treitz were isolated, fixed in 4% paraformaldehyde, and embedded in paraffin. The tissue was sectioned to 5 μm using the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) assay with MECA-32 antibody (panendothelial cell marker) to identify apoptotic endothelial cells. Double-stained apoptotic endothelial cells were quantified.

[0152] 15Gy LD 90Mice irradiated with the specified dose resulted in small intestinal endothelial cell death, peaking 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 ceramide inhibition prevents endothelial cell death in the gastrointestinal tract in a GI syndrome mouse model. Humanized anti-ceramide scFv may be beneficial for treating diseases associated with increased cell death, including GI syndrome, GvHD, and autoimmune diseases.

[0153] Further numbered embodiments Further embodiments of this disclosure are presented in the numbered embodiments below: Embodiment 1. 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), (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 contains the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 contains the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 contains the amino acid sequence of SEQ ID NO: 9; (f)LCDR3 contains the amino acid sequence of SEQ ID NO: 10, Anti-ceramide antibody or its antigen-binding fragment. Embodiment 2. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 3. Embodiment 3. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 4. Embodiment 4. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 5. Embodiment 5. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO: 6. Embodiment 6. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO: 3. Embodiment 7. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO: 4. Embodiment 8. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO: 5. Embodiment 9. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO: 6. Embodiment 10. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein 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 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 its antigen-binding fragment according to Embodiment 10, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. Embodiment 12. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 10, wherein the amino acid sequence of VH is that of SEQ ID NO: 17, and the amino acid sequence of VL is that of SEQ ID NO: 22. Embodiment 13. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein 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 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. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 13, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 23. Embodiment 15. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 13, wherein the amino acid sequence of VH is that of SEQ ID NO: 17, and the amino acid sequence of VL is that of SEQ ID NO: 23. Embodiment 16. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein 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 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. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 16, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. Embodiment 18. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 16, wherein the amino acid sequence of VH is from SEQ ID NO: 17 and the amino acid sequence of VL is from SEQ ID NO: 24. Embodiment 19. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein 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 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 its antigen-binding fragment according to Embodiment 19, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. Embodiment 21. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 19, wherein the amino acid sequence of VH is that of SEQ ID NO: 18, and the amino acid sequence of VL is that of SEQ ID NO: 22. Embodiment 22. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein 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 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 its antigen-binding fragment according to Embodiment 22, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 23. Embodiment 24. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 22, wherein the amino acid sequence of VH is from SEQ ID NO: 18 and the amino acid sequence of VL is from SEQ ID NO: 23. Embodiment 25. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein 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 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. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 25, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. Embodiment 27. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 25, wherein the amino acid sequence of VH is from SEQ ID NO: 18 and the amino acid sequence of VL is from SEQ ID NO: 24. Embodiment 28. The anti-ceramide antibody or antigen-binding fragment thereof according to Embodiment 1, wherein 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 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. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 28, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. Embodiment 30. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 28, wherein the amino acid sequence of VH is from SEQ ID NO: 19 and the amino acid sequence of VL is from SEQ ID NO: 22. Embodiment 31. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein 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 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. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 31, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 23. Embodiment 33. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 31, wherein the amino acid sequence of VH is from SEQ ID NO: 19 and the amino acid sequence of VL is from SEQ ID NO: 23. Embodiment 34. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein 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 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. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 34, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. Embodiment 36. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 34, wherein the amino acid sequence of VH is from SEQ ID NO: 19 and the amino acid sequence of VL is from SEQ ID NO: 24. Embodiment 37. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein 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 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 its antigen-binding fragment according to Embodiment 37, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 20, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. Embodiment 39. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 37, wherein the amino acid sequence of VH is from SEQ ID NO: 20 and the amino acid sequence of VL is from SEQ ID NO: 22. Embodiment 40. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 1, wherein 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 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 its antigen-binding fragment according to Embodiment 40, wherein VH comprises an amino acid sequence identical to SEQ ID NO: 21, and VL comprises an amino acid sequence identical to SEQ ID NO: 22. Embodiment 42. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 40, wherein the amino acid sequence of VH is that of SEQ ID NO: 21, and the amino acid sequence of VL is that of SEQ ID NO: 22. Embodiment 43. comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to a sequence selected from sequence numbers 17, 18, 19, 20, and 21; The amino acid sequence in which VL is at least 90%, 95%, or 97% identical to the sequence selected from SEQ ID NOs. 22, 23, and 24. An anti-ceramide antibody or its antigen-binding fragment, including the above. Embodiment 44. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 22. Embodiment 45. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 44, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. Embodiment 46. An anti-ceramide antibody or its antigen-binding fragment according to any one of Embodiments 43 to 45, wherein the amino acid sequence of VH is from SEQ ID NO: 17 and the amino acid sequence of VL is from SEQ ID NO: 22. Embodiment 47. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 23. Embodiment 48. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 47, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 23. Embodiment 49. An anti-ceramide antibody or its antigen-binding fragment according to 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 its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 24. Embodiment 51. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 50, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. Embodiment 52. An anti-ceramide antibody or its antigen-binding fragment according to 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 its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 22. Embodiment 54. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 53, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. Embodiment 55. An anti-ceramide antibody or its antigen-binding fragment according to 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 its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 23. Embodiment 57. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 56, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 23. Embodiment 58. An anti-ceramide antibody or its antigen-binding fragment according to 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 its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 24. Embodiment 60. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 59, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. Embodiment 61. An anti-ceramide antibody or its antigen-binding fragment according to 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 its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 22. Embodiment 63. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 62, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. Embodiment 64. An anti-ceramide antibody or its antigen-binding fragment according to 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 its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 23. Embodiment 66. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 65, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 23. Embodiment 67. An anti-ceramide antibody or its antigen-binding fragment according to 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 its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 24. Embodiment 69. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 68, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24. Embodiment 70. An anti-ceramide antibody or its antigen-binding fragment according to 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 its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 20, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 22. Embodiment 72. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 71, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 20, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO: 22. Embodiment 73. An anti-ceramide antibody or its antigen-binding fragment according to 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 its antigen-binding fragment according to Embodiment 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 21, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 22. Embodiment 75. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 43 or 74, wherein VH comprises an amino acid sequence identical to SEQ ID NO: 21, and VL comprises an amino acid sequence identical to SEQ ID NO: 22. Embodiment 76. An anti-ceramide antibody or its antigen-binding fragment according to 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. An anti-ceramide antibody or antigen-binding fragment thereof, as described in any one of Embodiments 1 to 76, which is a humanized antibody or an antigen-binding fragment thereof. Embodiment 78. An anti-ceramide antibody or antigen-binding fragment thereof, as described in any one of Embodiments 1 to 76, which is a fully human antibody or an antigen-binding fragment thereof. Embodiment 79. An anti-ceramide antibody or its antigen-binding fragment according to any one of Embodiments 1 to 78, wherein the anti-ceramide antibody contains one or more point mutations within the Fc domain of the antibody. Embodiment 80. An anti-ceramide antibody or its antigen-binding fragment according to any one of Embodiments 1 to 79, wherein the antigen-binding fragment is a single-chain variable fragment (scFv). The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 80, wherein Embodiment 81.scFv includes 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 to 61. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 80, wherein Embodiment 82.scFv contains an amino acid sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 48 to 61. Embodiment 80, wherein Embodiment 83.scFv comprises a sequence selected from the group consisting of SEQ ID NOs. 48 to 61, wherein the anti-ceramide antibody or its antigen-binding fragment is described above. Embodiment 84. The anti-ceramide antibody or antigen-binding fragment thereof according to Embodiment 80, wherein the light chain variable region of the scFv is located on the carboxyl terminal side with respect to the heavy chain variable region of the scFv. Embodiment 85. The anti-ceramide antibody or antigen-binding fragment thereof according to Embodiment 80, wherein 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. Embodiment 86.scFv is an anti-ceramide antibody or antigen-binding fragment thereof according to any one of Embodiments 80 to 85, comprising a linker polypeptide. Embodiment 87. The anti-ceramide antibody or its antigen-binding fragment according to Embodiment 86, wherein the linker polypeptide is located between the light chain variable region and the heavy chain variable region of the scFv. Embodiment 88. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 86 or 87, wherein the linker polypeptide comprises a Gly4Ser linker. Embodiment 89. The linker polypeptide is of the formula (Gly4Ser) n An anti-ceramide antibody or its antigen-binding fragment according to any one of embodiments 86 to 88, comprising [n=1 to 5 in the sequence]. 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 to 61. Embodiment 91. The anti-ceramide scFv according to 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 according to Embodiment 90, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 61. Embodiment 93. A polynucleotide encoding an anti-ceramide antibody or its antigen-binding fragment 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 according to Embodiment 94, comprising an amino acid sequence that is 100% identical to SEQ ID NO: 48 or 51. Embodiment 96. The anti-ceramide scFv according to Embodiment 94, comprising the amino acid sequence of SEQ ID NO: 48 or 51. Embodiment 97. A polynucleotide encoding an anti-ceramide single-chain variable fragment (scFv) as described in any one of Embodiments 94 to 96. Embodiment 98. An expression vector comprising the polynucleotide described in Embodiment 93 or 97. Embodiment 99. A host cell containing the polynucleotide described in Embodiment 93 or 97, or the expression vector described in Embodiment 97. Embodiment 100. A method for producing an anti-ceramide antibody or its antigen-binding fragment according to any one of Embodiments 1 to 89, or an anti-ceramide single-chain variable fragment (scFv) according to any one of Embodiments 94 to 96, comprising the step of introducing the expression vector according to Embodiment 98 into a host cell. Embodiment 101. A method for inhibiting apoptosis in a subject requiring such inhibition, comprising the step of administering to the subject a therapeutically effective amount of an anti-ceramide antibody or its antigen-binding fragment according to any one of Embodiments 1 to 88, or an anti-ceramide scFv according to any one of Embodiments 94 to 96. Embodiment 102. The method according to Embodiment 101, wherein apoptosis is associated with a disease selected from the group consisting of graft-versus-host disease, radiation disease, GI syndrome, and autoimmune disease. Embodiment 103. The method according to Embodiment 102, wherein the disease is a radiation-induced illness or GI syndrome, and an anti-ceramide antibody or its antigen-binding fragment is administered before the subject is exposed to radiation. Embodiment 104. The method according to Embodiment 102, wherein the disease is graft-versus-host disease, and an anti-ceramide antibody or its antigen-binding fragment is administered to the subject before the transplant. Embodiment 105. The method according to Embodiment 104, wherein the transplant is a bone marrow transplant. Embodiment 106. The method according to Embodiment 101, wherein the anti-ceramide antibody or its antigen-binding fragment is administered intravenously, intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intrasynovally, intrathecally, or orally, topically, or by inhalation. Embodiment 107. A method for mitigating apoptosis in a subject with GI syndrome, comprising the step of administering to the subject, after the subject has been exposed to penetrating radiation, a therapeutically effective dose of an anti-ceramide antibody or its antigen-binding fragment according to any one of Embodiments 1 to 89, or an anti-ceramide scFv according to any one of Embodiments 94 to 96. Embodiment 108. The method according to Embodiment 107, wherein an anti-ceramide antibody or its antigen-binding fragment is administered immediately after the subject is exposed to penetrating radiation. Embodiment 109. The method according to Embodiment 107, wherein an anti-ceramide antibody or its antigen-binding fragment 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 the step of administering to the subject a therapeutically effective amount of an anti-ceramide antibody or its antigen-binding fragment according to any one of Embodiments 1 to 89, or an anti-ceramide scFv according to any one of Embodiments 94 to 96, before the subject undergoes transplantation or after the subject has undergone transplantation and before the onset of GvHD. Embodiment 111. The method according to 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 YNYPRDGSTKYNEKFQG (SEQ ID NO: 3), YN An anti-ceramide antibody or its antigen-binding fragment comprising an amino acid sequence selected from YPREGSTKYNEKFQG (SEQ ID NO: 4), YNYPRDVSTKYNEKFQG (SEQ ID NO: 5), and YNYPRDGSTKYAEKFQG (SEQ ID NO: 6); (c) HCDR3 comprising the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 7); (d) LCDR1 comprising the amino acid sequence of RASKSISKYLA (SEQ ID NO: 8); (e) LCDR2 comprising the amino acid sequence of SGSTLQS (SEQ ID NO: 9); and (f) LCDR3 comprising the amino acid sequence of QQHNEYPWT (SEQ ID NO: 10). Embodiment 113. The anti-ceramide antibody or its antigen-binding fragment according to 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 according to Embodiment 112, wherein HCDR1 comprises GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises YNYPREGSTKYNEKFQG (SEQ ID NO: 4). Embodiment 115. An anti-ceramide antibody or its antigen-binding fragment according to 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 its antigen-binding fragment according to 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 its antigen-binding fragment according to Embodiment 112, wherein HCDR1 comprises GYTFTDHTMH (SEQ ID NO: 2) and HCDR2 comprises YNYPRDGSTKYNEKFQG (SEQ ID NO: 3). Embodiment 118. The anti-ceramide antibody or its antigen-binding fragment 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 its antigen-binding fragment according to Embodiment 112, wherein HCDR1 comprises GYTFTDHTMH (SEQ ID NO: 2) and HCDR2 comprises YNYPRDVSTKYNEKFQG (SEQ ID NO: 5). Embodiment 120. An anti-ceramide antibody or its antigen-binding fragment according to Embodiment 112, wherein HCDR1 comprises GYTFTDHTMH (SEQ ID NO: 2) and HCDR2 comprises YNYPRDGSTKYAEKFQG (SEQ ID NO: 6).

[0155] Embedding by reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated in their entirety by reference for all purposes. However, no reference to any reference, article, publication, patent, patent publication, or patent application cited herein shall be understood as an endorsement, or any suggestion, of them constituting valid prior art or forming public knowledge in any country of the world.

[0156] [Table 13] JPEG2026104852000016.jpg250165 JPEG2026104852000017.jpg250164 JPEG2026104852000018.jpg250165 JPEG2026104852000019.jpg250165 JPEG2026104852000020.jpg249165 JPEG2026104852000021.jpg137170

Claims

1. It comprises an immunoglobulin heavy chain variable region (VH) containing 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) containing light chain complementarity-determining region (CDR) 1 (LCDR1), light chain CDR2 (LCDR2), and 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 contains the amino acid sequence of SEQ ID NO: 7; (d) LCDR1 contains the amino acid sequence of SEQ ID NO: 8; (e) LCDR2 contains the amino acid sequence of SEQ ID NO: 9; (f) LCDR3 contains the amino acid sequence of SEQ ID NO: 10, Anti-ceramide antibody or its antigen-binding fragment.

2. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO:

3.

3. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO:

4.

4. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO:

5.

5. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein HCDR1 comprises SEQ ID NO: 1 and HCDR2 comprises SEQ ID NO:

6.

6. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO:

3.

7. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO:

4.

8. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO:

5.

9. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein HCDR1 comprises SEQ ID NO: 2 and HCDR2 comprises SEQ ID NO:

6.

10. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

22.

11. The anti-ceramide antibody or its antigen-binding fragment according to claim 10, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 17, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

22.

12. The anti-ceramide antibody or its antigen-binding fragment according to claim 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.

13. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

23.

14. The anti-ceramide antibody or its antigen-binding fragment according to claim 13, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 17, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

23.

15. The anti-ceramide antibody or its antigen-binding fragment according to claim 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.

16. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

24.

17. The anti-ceramide antibody or its antigen-binding fragment according to claim 16, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 17, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

24.

18. The anti-ceramide antibody or its antigen-binding fragment according to claim 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.

19. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

22.

20. The anti-ceramide antibody or its antigen-binding fragment according to claim 19, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 18, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

22.

21. The anti-ceramide antibody or its antigen-binding fragment according to claim 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.

22. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

23.

23. The anti-ceramide antibody or its antigen-binding fragment according to claim 22, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 18, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

23.

24. The anti-ceramide antibody or its antigen-binding fragment according to claim 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.

25. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

24.

26. The anti-ceramide antibody or its antigen-binding fragment according to claim 25, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 18, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

24.

27. The anti-ceramide antibody or its antigen-binding fragment according to claim 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.

28. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

22.

29. The anti-ceramide antibody or its antigen-binding fragment according to claim 28, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 19, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

22.

30. The anti-ceramide antibody or its antigen-binding fragment according to claim 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.

31. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

23.

32. The anti-ceramide antibody or its antigen-binding fragment according to claim 31, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 19, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

23.

33. The anti-ceramide antibody or its antigen-binding fragment according to claim 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.

34. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

24.

35. The anti-ceramide antibody or its antigen-binding fragment according to claim 34, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 19, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

24.

36. The anti-ceramide antibody or its antigen-binding fragment according to claim 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.

37. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

22.

38. The anti-ceramide antibody or its antigen-binding fragment according to claim 37, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 20, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO:

22.

39. The anti-ceramide antibody or its antigen-binding fragment according to claim 37, wherein the amino acid sequence of VH is from SEQ ID NO: 20, and the amino acid sequence of VL is from SEQ ID NO:

22.

40. The anti-ceramide antibody or its antigen-binding fragment according to claim 1, wherein 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 VL comprises an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:

22.

41. The anti-ceramide antibody or its antigen-binding fragment according to claim 40, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 21, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

22.

42. The anti-ceramide antibody or its antigen-binding fragment according to claim 40, wherein the amino acid sequence of VH is the sequence of SEQ ID NO: 21, and the amino acid sequence of VL is the sequence of SEQ ID NO:

22.

43. It includes an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to a sequence selected from SEQ ID NOs: 17, 18, 19, 20, and 21; The amino acid sequence in which VL is at least 90%, 95%, or 97% identical to the sequence selected from SEQ ID NOs. 22, 23, and 24. An anti-ceramide antibody or its antigen-binding fragment, including the above.

44. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

22.

45. The anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 44, wherein VH comprises an amino acid sequence identical to SEQ ID NO: 17, and VL comprises an amino acid sequence identical to SEQ ID NO:

22.

46. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

47. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

23.

48. The anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 47, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 17, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

23.

49. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

50. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 17, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

24.

51. An anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 50, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 17, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

24.

52. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

53. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

22.

54. An anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 53, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 18, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

22.

55. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

56. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

23.

57. An anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 56, wherein VH comprises an amino acid sequence that is 100% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is 100% identical to SEQ ID NO:

23.

58. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

59. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 18, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

24.

60. An anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 59, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 18, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

24.

61. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

62. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

22.

63. An anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 62, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 19, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

22.

64. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

65. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

23.

66. An anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 65, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 19, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

23.

67. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

68. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 19, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

24.

69. An anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 68, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 19, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

24.

70. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

71. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 20, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

22.

72. An anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 71, wherein VH comprises an amino acid sequence identical to SEQ ID NO: 20, and VL comprises an amino acid sequence identical to SEQ ID NO:

22.

73. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

74. The anti-ceramide antibody or its antigen-binding fragment according to claim 43, wherein VH comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO: 21, and VL comprises an amino acid sequence that is at least 90%, 95%, or 97% identical to SEQ ID NO:

22.

75. An anti-ceramide antibody or its antigen-binding fragment according to claim 43 or 74, wherein VH comprises an amino acid sequence identical to that of SEQ ID NO: 21, and VL comprises an amino acid sequence identical to that of SEQ ID NO:

22.

76. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 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.

77. An anti-ceramide antibody or antigen-binding fragment according to any one of claims 1 to 76, which is a humanized antibody or an antigen-binding fragment thereof.

78. An anti-ceramide antibody or antigen-binding fragment according to any one of claims 1 to 76, wherein the antibody is a fully human antibody or an antigen-binding fragment thereof.

79. An anti-ceramide antibody or its antigen-binding fragment according to any one of claims 1 to 78, wherein the anti-ceramide antibody comprises one or more point mutations within the Fc domain of the antibody.

80. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 1 to 79, wherein the antigen-binding fragment is a single-chain variable fragment (scFv).

81. The anti-ceramide antibody or its antigen-binding fragment according to claim 80, wherein 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 to 61.

82. The anti-ceramide antibody or its antigen-binding fragment according to claim 80, wherein scFv comprises an amino acid sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 48 to 61.

83. The anti-ceramide antibody or its antigen-binding fragment according to claim 80, wherein scFv consists of a sequence selected from the group consisting of SEQ ID NOs. 48 to 61.

84. The anti-ceramide antibody or its antigen-binding fragment according to claim 80, wherein the light chain variable region of the scFv is located on the carboxyl terminal side with respect to the heavy chain variable region of the scFv.

85. The anti-ceramide antibody or its antigen-binding fragment according to claim 80, wherein the light chain variable region of the scFv is located on the amino-terminal side with respect to the heavy chain variable region of the scFv.

86. The anti-ceramide antibody or its antigen-binding fragment according to any one of claims 80 to 85, wherein the scFv comprises a linker polypeptide.

87. The anti-ceramide antibody or its antigen-binding fragment according to claim 86, wherein the linker polypeptide is located between the light chain variable region and the heavy chain variable region of the scFv.

88. Linker polypeptide, Gly 4 An anti-ceramide antibody or its antigen-binding fragment according to claim 86 or 87, comprising a Ser linker.

89. The linker polypeptide is of formula (Gly 4 Ser) n An anti-ceramide antibody or its antigen-binding fragment according to any one of claims 86 to 88, comprising [n = 1 to 5 in the sequence].

90. 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.

91. The anti-ceramide scFv according to claim 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.

92. The anti-ceramide scFv according to claim 90, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 61.

93. A polynucleotide encoding an anti-ceramide antibody or an antigen-binding fragment thereof, as described in any one of claims 1 to 92.

94. 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 sequence number 48 or 51.

95. The anti-ceramide scFv according to claim 94, comprising an amino acid sequence that is 100% identical to sequence number 48 or 51.

96. The anti-ceramide scFv according to claim 94, comprising the amino acid sequence of sequence number 48 or 51.

97. A polynucleotide encoding an anti-ceramide single-chain variable fragment (scFv) according to any one of claims 94 to 96.

98. An expression vector comprising a polynucleotide according to claim 93 or 97.

99. A host cell comprising a polynucleotide according to claim 93 or 97, or an expression vector according to claim 97.

100. A method for producing an anti-ceramide antibody or an antigen-binding fragment thereof according to any one of claims 1 to 89, or an anti-ceramide single-chain variable fragment (scFv) according to any one of claims 94 to 96, comprising the step of introducing the expression vector according to claim 98 into a host cell.

101. A method for inhibiting apoptosis in a subject requiring such inhibition, comprising the step of administering to the subject a therapeutically effective amount of an anti-ceramide antibody or its antigen-binding fragment according to any one of claims 1 to 88, or an anti-ceramide scFv according to any one of claims 94 to 96.

102. The method according to claim 101, wherein apoptosis is associated with a disease selected from the group consisting of graft-versus-host disease, radiation disease, GI syndrome, and autoimmune disease.

103. The method according to claim 102, wherein the disease is radiation sickness or GI syndrome, and the anti-ceramide antibody or its antigen-binding fragment is administered to the subject before exposure to radiation.

104. The method according to claim 102, wherein the disease is graft-versus-host disease, and an anti-ceramide antibody or its antigen-binding fragment is administered to the subject before the transplant.

105. The method according to claim 104, wherein the transplant is a bone marrow transplant.

106. The method according to claim 101, wherein the anti-ceramide antibody or its antigen-binding fragment is administered intravenously, intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intrasynovally, intrathecally, orally, topically, or by inhalation.

107. A method for mitigating apoptosis in a subject with GI syndrome, comprising the step of administering to the subject, after the subject has been exposed to penetrating radiation, a therapeutically effective amount of an anti-ceramide antibody or an antigen-binding fragment thereof according to any one of claims 1 to 89, or an anti-ceramide scFv according to any one of claims 94 to 96.

108. The method according to claim 107, wherein an anti-ceramide antibody or an antigen-binding fragment thereof is administered immediately after the subject is exposed to penetrating radiation.

109. The method according to claim 107, wherein an anti-ceramide antibody or an antigen-binding fragment thereof is administered within 24 hours after the subject is exposed to penetrating radiation.

110. A method for inhibiting apoptosis in a subject with GvHD, comprising the step of administering to the subject a therapeutically effective amount of an anti-ceramide antibody or its antigen-binding fragment according to any one of claims 1 to 89, or an anti-ceramide scFv according to any one of claims 94 to 96, before the subject undergoes transplantation or after the subject has undergone transplantation and before the onset of GvHD.

111. The method according to claim 110, wherein the transplant is a bone marrow transplant.

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