Combination administration of bone marrow and Anti-ceramide antibodies

Abstract

The present disclosure provides (i) methods of improving hematopoietic stem cell transplantation using anti-ceramide antibodies or antigen-binding fragments thereof, for treating various non-malignant and malignant diseases; and (ii) compositions for such treatments.

Description

COMBINATION ADMINISTRATION OF BONE MARROW AND ANTI¬CERAMIDE ANTIBODIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 587,942, filed October 4, 2023, the content of which is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under AI106283, AI133598 and CA008748 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (CERA_023_01WO_SeqList_ST26.xml; Size: 80,001 bytes; and Date of Creation: October 3, 2024) are herein incorporated by reference in its entirety.BACKGROUND

[0004] Hematopoietic stem cell transplantation (HSCT), also known as bone marrow transplantation (BMT), has been widely used for treating various malignant diseases and non- malignant diseases (e.g., autoimmune diseases). However, traditional HSCT requires a large amount of hematopoietic stem cells (typically at least 2 * 106CD34+ hematopoietic stem cells per kilogram of the patient’s body weight) and carries a high risk of graft failure or poor graft function, leading to significant morbidity and mortality. Thus, there is an urgent need for strategies to improve the efficacy of HSCT and / or lower the amount of hematopoietic stem cells required for such procedures.SUMMARY

[0005] In one aspect, the disclosure provides methods of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population and a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof. In some embodiments, engraftment of the hematopoietic cell population is enhanced compared to a control engraftment not receiving the therapeutic agent.

[0006] In some embodiments, step c) is performed after step b). In some embodiments, the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours after step b). In some embodiments, the therapeutic agent is administered at least 1, 2, 3, 4, 5, 6, or 7 days after step b).

[0007] In some embodiments, the therapeutic agent is administered concurrently with the hematopoietic cell population in step c).

[0008] In some embodiments, the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours before the hematopoietic cell population in step c). In some embodiments, the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours after the hematopoietic cell population in step c).

[0009] In some embodiments, the hematopoietic cell population is an autologous hematopoietic cell population. In some embodiments, the hematopoietic cell population is an allogenic hematopoietic cell population.

[0010] In some embodiments, the conditioning in step b) comprises ablating the patient’s native bone marrow cell population. In some embodiments, the ablating of the patient’s native bone marrow cell population comprises marrow-ablative chemical treatment. In some embodiments, the ablating of the patient’s native bone marrow cell population comprises administration of a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of chlorambucil, cyclophosphamide, ifosfamide, melphalan, streptozocin, carmustine, lomustine, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, 5 -fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, daunorubicin, doxorubicin, epirubicin, idarubicin, SN-38, camptothecin, topotecan, 9- nitrocamptothecin, 9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX- 895 If, MAG-CPT, amsacrine, etoposide, etoposide phosphate, teniposide, paclitaxel, and docetaxel. In some embodiments, the marrow-ablative chemical treatment is administration of an alkylating agent to the native bone marrow. In some embodiments, the alkylating agent is melphalan.

[0011] In some embodiments, the ablating of the patient’s native bone marrow cell population comprises administration of ablative radiation. In some embodiments, the ablative radiation is not administered to the patient’s intestines. In some embodiments, the ablative radiation is not sufficient to trigger Gastrointestinal Acute Radiation Syndrome (GI-ARS). In someembodiments, the ablative radiation comprises a dose of less than 13 Gy. In some embodiments, the dose of marrow-ablative chemical treatment, the chemotherapeutic agent, or the ablative radiation is higher than that in a control engraftment not receiving the therapeutic agent comprising the anti-ceramide antibody or the antigen binding fragment thereof.

[0012] In some embodiments, the conditioning in step b) comprises reduced-intensity conditioning (RIC) or nonmyeloablative conditioning.

[0013] In some embodiments, the method reduces the recurrence of the hematologic malignancy in the patient.

[0014] In some embodiments, engraftment is measured by patient’s white blood cell count returning to a pre-transplant level within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s white blood cell count of at least 5 K / uL within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s neutrophil count returning to a pre-transplant level within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s neutrophil blood cell count of at least 1 K / uL within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s platelet count returning to a pre-transplant level within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s platelet count of at least 300 K / uL within 90 days posttransplant. In some embodiments, engraftment is measured by the patient surviving 30 days post-transplant.

[0015] In one aspect, the disclosure provides methods of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient: (i) a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; and (ii) a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof.

[0016] In some embodiments, the patient receiving the therapeutic agent and the reduced hematopoietic cell population exhibits a hematopoietic cell engraftment level that is higher than that of a control patient receiving the reduced hematopoietic cell population but not receiving the therapeutic agent.

[0017] In some embodiments, the reduced hematopoietic cell population comprises no more than 1.5 million, no more than 1.2 million, no more than 1.0 million, no more than 0.9 million,no more than 0.8 million, no more than 0.7 million, no more than 0.6 million, or no more than 0.5 million, CD34+ hematopoietic stem cells per kilogram of the patient’s body weight.

[0018] In some embodiments, engraftment is measured by patient’s white blood cell count returning to a pre-transplant level within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s white blood cell count of at least 5 K / uL within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s neutrophil count returning to a pre-transplant level within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s neutrophil blood cell count of at least 1 K / uL within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s platelet count returning to a pre-transplant level within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s platelet count of at least 300 K / uL within 90 days posttransplant. In some embodiments, engraftment is measured by the patient surviving 30 days post-transplant.

[0019] In one aspect, the disclosure provides methods of improving engraftment of hematopoietic cells in a patient in need thereof, the method comprising administering a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof in addition to the administration of the hematopoietic cells.

[0020] In some embodiments, the patience has a chronically injured marrow substratum or suffers from a hemoglobinopathy. In some embodiments, the patience has sickle cell disease.

[0021] In some embodiments, the hematopoietic cell population administered to the patient is an autologous hematopoietic cell population. In some embodiments, the hematopoietic cell population administered to the patient is an allogenic hematopoietic cell population.

[0022] In one aspect, the disclosure provides methods of hematopoietic stem cell engraftment, said method comprising: administering to the patient a hematopoietic cell population and a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof; wherein the therapeutic agent is administered concurrently with or following administration of the hematopoietic cell population.

[0023] In some embodiments, engraftment of the hematopoietic cell population is enhanced compared to a control engraftment not receiving the therapeutic agent.

[0024] In some embodiments, the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours after administration of the hematopoietic cell population. In someembodiments, the therapeutic agent is administered at least 1, 2, 3, 4, 5, 6, or 7 days after administration of the hematopoietic cell population. In some embodiments, the therapeutic agent is administered concurrently with the hematopoietic cell population.

[0025] In some embodiments, the hematopoietic cell population is an autologous hematopoietic cell population. In some embodiments, the hematopoietic cell population is an allogenic hematopoietic cell population.

[0026] In some embodiments, engraftment is measured by patient’s white blood cell count returning to a pre-transplant level within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s white blood cell count of at least 5 K / uL within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s neutrophil count returning to a pre-transplant level within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s neutrophil blood cell count of at least 1 K / uL within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s platelet count returning to a pre-transplant level within 90 days post-transplant. In some embodiments, engraftment is measured by patient’s platelet count of at least 300 K / uL within 90 days posttransplant. In some embodiments, engraftment is measured by the patient surviving 30 days post-transplant.

[0027] In some embodiments, the method reduces the likelihood of graft versus host disease compared to a control method without the administration of the therapeutic agent comprising the anti -ceramide antibody or the antigen binding fragment thereof.

[0028] In some embodiments, the anti-ceramide antibody or the antigen binding fragment thereof comprises an immunoglobulin heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), and an immunoglobulin light chain variable region (VL) comprising a light chain complementarity determining region (CDR) 1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3), wherein: (a) the HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 1 and 2; (b) the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, and 6; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the HCDR1 comprises SEQ ID NO: 1 and the HCDR2 comprises SEQID NO: 3. In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 18 and the VL comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 22.

[0029] In some embodiments, the therapeutic agent is an anti-ceramide single chain variable fragment (scFv). In some embodiments, the therapeutic agent is not for treating or preventing a Graft-versus-host disease (GVHD). In some embodiments, the anti-ceramide antibody or the antigen binding fragment thereof is a bispecific antibody or a bispecific T cell engager (BiTE).

[0030] In some embodiments, the hematopoietic cell population comprises genetically engineered hematopoietic cells.

[0031] In one aspect, the disclosure provides compositions comprising: a) a hematopoietic cell population graft; and b) an anti-ceramide antibody or the antigen binding fragment thereof. In some embodiments, the hematopoietic cell population is an autologous hematopoietic cell population. In some embodiments, the hematopoietic cell population is an allogenic hematopoietic cell population.

[0032] In some embodiments, the composition is for administration to a patient, and wherein the hematopoietic cell population comprises fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight. In some embodiments, the composition comprises no more than 1.5 million, no more than 1.2 million, no more than 1.0 million, no more than 0.9 million, no more than 0.8 million, no more than 0.7 million, no more than 0.6 million, or no more than 0.5 million, of CD34+ hematopoietic stem cells per kilogram of the patient’s body weight. In some embodiments, the antigen binding fragment of the anti- ceramide antibody is a single chain variable fragment (scFv).

[0033] In one aspect, the disclosure provides methods of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population; wherein the patient’s ceramide signaling pathway is blocked.

[0034] In one aspect, the disclosure provides methods of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cellpopulation; and c) administering to the patient a hematopoietic cell population; wherein endothelial cells in the patient are unable to form a ceramide-rich platform.

[0035] In one aspect, the disclosure provides methods of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population; wherein endothelial cells in the patient are temporarily blocked from forming a ceramide-rich platform.

[0036] In one aspect, the disclosure provides methods of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; wherein the patient’s ceramide signaling pathway is blocked.

[0037] In one aspect, the disclosure provides methods of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; wherein endothelial cells in the patient are unable to form a ceramide-rich platform.

[0038] In one aspect, the disclosure provides methods of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; wherein endothelial cells in the patient are temporarily blocked from forming a ceramide-rich platform.

[0039] In some embodiments, blocking the ceramide signaling pathway or the ceramide-rich platform formation comprises administering an anti-ceramide therapeutic agent. In some embodiments, the anti-ceramide therapeutic agent comprises an anti-ceramide antibody, imipramine, a statin (e.g., nystatin), an antisense nucleic acid, a small interfering RNA (siRNA), or a combination thereof. In some embodiments, the antisense nucleic acid or the siRNA reduces expression of ASMase, Bak, or Bax. In some embodiments, the blocking ofthe ceramide signaling pathway or the ceramide-rich platform (CRP) formation is evaluated by a 2-dimensional CRP incidence analysis or a 3-dimensional CRP volumetric analysis.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings illustrate one or more embodiments of the present disclosure and, together with the written description, serve to explain the principles of exemplary embodiments of the present disclosure.

[0041] FIG. 1 shows the extended survival of C57BL / 6J mice treated with the combination of BMT and anti-ceramide 6B5 scFv after 15 Gy, an LD9o dose for the gastrointestinal -acute radiation syndrome (GI-ARS). Only co-administration of syngeneic bone marrow cells (5*106cells / 25 gm mouse) intravenously at 16 h plus anti-ceramide 6B5 scFv (150 pg / 25 gm mouse) subcutaneously at 24 h post 15 Gy WBI prevents lethality from both the hematopoietic-acute radiation syndrome (H-ARS) and GI-ARS. 90-day Kaplan -Mei er survival data are presented. Upper panels represent three independent studies. Lower panel represents the collation of the 3 studies. Numbers in parentheses represent animals / group.

[0042] FIG. 2 shows the ability of anti-ceramide 6B5 scFv to prevent crypt-villus unit depletion after 15 Gy. Representative images of 5 pm H&E-stained sections of small intestinal circumferences (jejunum at 10 cm from the Ligament of Treitz) and bone marrow (tibia, femur) obtained at time of death from the cohorts of mice treated with 15 Gy with or without BMT or anti-ceramide 6B5 scFv. Scale bars: 40 pm.

[0043] FIG. 3A-3C show the ability of anti-ceramide 6B5 scFv+BMT to repopulate bone marrow and prevent crypt- villus unit depletion at 10 days post-irradiation. C57BL / 6J mice were treated at 8-10 weeks of age with 15 Gy followed at 24 hours with 150 ug 6B5 scFv / 25 g mouse s.c. with or without BMT. Mice were sacrificed at 10 days post radiation by hypercapnia asphyxiation and 5 pm small intestinal sections (from the jejunum at 10 cm from the Ligament of Treitz) were stained with H&E. Representative images of jejunal circumferences are shown in FIG. 3A and quantified in FIG. 3B as percent surviving crypts per circumference (n=20 / group). FIG. 3C shows representative images of bone marrow (tibia, femur) from cohort treated with 15 Gy+BMT+scFv. Scale bars: 40 pm. Data from the cohort of mice that died on day 7 from FIG. 2 are provided for comparison. ***p value<0.01 by paired t-test.

[0044] FIG. 4 shows charts demonstrating that the combination of BMT and anti-ceramide 6B5 scFv normalize complete blood counts in mice subjected to 15 Gy. CBC analysis ofirradiated C57BL / 6J mice was performed on Day 4, 8, 12, 28 and 90 post irradiation. Differential blood populations including WBCs, neutrophils, platelets and RBCs were measured in 15 Gy, 15 Gy+BMT, 15 Gy+1 scFv and 15 Gy+BMT+scFv cohorts. The dark background represents the normal range of blood components in each graph.

[0045] FIG. 5 is a chart showing that anti-ceramide scFv enhances BMT engraftment (survival rate) at 14 Gy, the upper limit dose for induction of the H-ARS in C57BL / 6J mice. Percent survival of mice treated with 14 Gy+BMT vs. 14 Gy+BMT+scFv was assessed at 90 days post-radiation. Bone marrow cells (0.5-5.0 x 106cells / 25 g mouse) were administered intravenously at 16-18 h post-radiation. scFv was delivered subcutaneously at 24 h postradiation. Numbers of mice per group are indicated in parentheses. Mean survival values are presented.

[0046] FIG. 6 is a chart showing serum biochemistries of C57BL / 6J mice treated with 15 Gy+BMT+anti -ceramide 6B5 scFv prior to sacrifice at 90 days. Serum biochemistry panel of the cohort of mice treated with 15 Gy+BMT+anti-ceramide scFv obtained at time of sacrifice on day 90. Blood was immediately obtained after euthanasia through heart puncture. Serum biochemical analysis was performed using a Beckman Coulter AU 680 clinical chemistry analyzer on 100 pl samples. Abbreviations: BUN-Blood Urea Nitrogen; CREA-Creatinine; ALP -Alkaline phosphatase; ALT-Alanine transaminase; AST-Aspartate transferase; GGT- Gamma-glutamyl transferase; TBIL-Total Bilirubin; DBIL-Direct bilirubin; IBIL-Indirect bilirubin; TP-Total protein; ALB-Albumin; GLOB-Globulin, P-Phosphate; Ca-Calcium; GLU-Glucose; CHOL-Cholesterol; TRIG-Triglycerides; CK-Creatine Kinase; TCO2-Total carbon dioxide; Na-Sodium; K-Potassium; CL-Chloride.

[0047] FIG. 7 shows representative H&E images of vital organs isolated from the cohort of mice treated with 15 Gy+BMT+anti-ceramide 6B5 scFv obtained at complete necropsy on day 90 post irradiation. At euthanasia organs were surgically removed and placed in formalin, stained, and evaluated. Representative images of 5-pm sections of identified tissues from a representative mouse are shown.

[0048] FIG. 8 shows representative H&E images of additional organs isolated from the cohort of mice treated with 15 Gy+BMT+anti-ceramide 6B5 scFv obtained at complete necropsy on day 90 post irradiation. At euthanasia, organs were surgically removed and placed in formalin. Representative images of 5-pm sections of identified tissues from a representative mouse are shown.

[0049] FIG. 9 shows representative H&E images of vital organs isolated from the cohort of 90-day age matched unirradiated control mice. At euthanasia, organs were surgically removed and placed in formalin. Representative images of 5-pm sections of identified tissues from a representative mouse are shown.

[0050] FIG. 10 shows representative H&E images of additional organs isolated from the cohort of 90-day age matched unirradiated control mice. At euthanasia organs were surgically removed and placed in formalin. Representative images of 5-pm sections of identified tissues from a representative mouse are shown.

[0051] FIG. 11 are charts showing complete blood counts and serum biochemistries of age- matched unirradiated control mice. Upper panels: CBC analysis of age-matched C57BL / 6J mice was performed on day 90. Differential blood populations were measured including red blood cells (RBCs), white blood cells (WBCs), neutrophils, and platelets. The gray region represents the normal range of blood components in each graph delimited by arrows. Bottom panel: serum biochemistry panel of a subset of the cohort of age-matched control mice on day 90.

[0052] FIG. 12 are images showing the lack of fibrosis in lungs, liver and kidneys at 90 days in the 15 Gy+BMT+anti -ceramide scFv cohort. The upper panel shows representative images of 5-pm tissue sections of lung, liver and kidney from the 15 Gy+BMT+scFv cohort stained with Masson’s Tri chrome at necropsy on day 90, captured at 20X magnification. Scale bars: 100 pm. The lower panel shows high-resolution representative images of 5-pm tissue kidney sections from 1 the 15 Gy+BMT+scFv cohort stained with Masson’s tri chrome at necropsy on day 90, captured at 60X magnification. These images each show a blood vessel with normal perivascular collagen (blue) and no evidence of fibrosis of glomeruli (Left: longitudinal; Right: transverse sections). Scale bars: 20 pm.

[0053] FIG. 13 are images showing Normal appearing small intestinal microvessels in the 15 Gy+BMT+anti-ceramide scFv cohort at day 90. Representative images of CD31 -stained capillaries (e.g., those marked by black arrow heads) in the lamina propria, submucosa, and mesentery of the small intestines of the 15 Gy+BMT+scFv cohort of mice at the time of sacrifice on day 90. Arrows indicate capillaries captured at 40X magnification.DETAILED DESCRIPTION

[0054] The details of the disclosure are set forth in the accompanying description below.Although methods and materials similar or equivalent to those described herein can be usedin the practice or testing of the present disclosure, illustrative methods, and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated herein by reference in their entireties for all purposes.Definitions

[0055] The term “antibody” refers to an immunoglobulin (Ig) molecule capable of binding to a specific target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one epitope recognition site located in the variable region of the Ig molecule. As used herein, the term encompasses intact polyclonal or monoclonal antibodies and antigen-binding fragments thereof. For example, a native immunoglobulin molecule is comprised of two heavy chain polypeptides and two light chain polypeptides. Each of the heavy chain polypeptides associate with a light chain polypeptide by virtue of interchain disulfide bonds between the heavy and light chain polypeptides to form two heterodimeric proteins or polypeptides (i.e., a protein comprised of two heterologous polypeptide chains). The two heterodimeric proteins then associate by virtue of additional interchain disulfide bonds between the heavy chain polypeptides to form an immunoglobulin protein or polypeptide.

[0056] The term “antigen-binding fragment” as used herein refers to a polypeptide fragment that contains at least one complementarity-determining region (CDR) of an immunoglobulin heavy and / or light chain that binds to at least one epitope of the antigen of interest. In this regard, an antigen-binding fragment of the herein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a variable heavy chain (VH) and variable light chain (VL) sequence from antibodies that specifically bind ceramide. Antigen-binding fragments include proteins that comprise a portion of a full length antibody, generally the antigen binding or variable region thereof, such as Fab, F(ab’)2, Fab’, Fv fragments, minibodies, diabodies, single domain antibody (dAb), single-chain variable fragments (scFv), multispecific antibodies formed from antibody fragments, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or fragment of the required specificity. In certain embodiments of the disclosure, an antigen-binding fragment, rather than an intact antibody, is used to increase tissue penetration or tumor penetration. In other embodiments, antigen-binding fragments are further modified to increase serum half-life.

[0057] The term “F(ab)” refers to two of the protein fragments resulting from proteolytic cleavage of IgG molecules by the enzyme papain. Each F(ab) comprises a covalent heterodimer of the VH chain and 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 and may contain a small portion of Fc. Each Fab’ fragment is a monovalent antigen-binding fragment.

[0058] The term “F(ab’)2” refers to a protein fragment of IgG generated by proteolytic cleavage by the enzyme pepsin. Each F(ab’)2 fragment comprises two F(ab’) fragments and is therefore a bivalent antigen-binding fragment.

[0059] An “Fv fragment” refers to a non-covalent VH::VL heterodimer which includes an antigen-binding site that retains much of the antigen recognition and binding capabilities of the native antibody molecule, but lacks the CHI and CL domains contained within a 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, the Fv fragment can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art.

[0060] Minibodies comprising a scFv joined to a CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See e.g., Ward, E. S. et al., Nature 341, 544- 546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879- 5883, 1988); PCT / US92 / 09965; WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0061] The term “diabody” refers to a bispecific antibody in which VH and VL domains are expressed in a single polypeptide chain using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and Poljak et al., Structure 2: 1121-23 (1994)).

[0062] The term “nanobody” or a “single domain antibody” refers to an antigen-binding fragment consisting of a single monomeric variable antibody domain. The Nanoclone methodis a method for generating Nanobodies against a desired target based on automated high- throughput selection of B-cells (See, WO 2006 / 079372).

[0063] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0064] In some embodiments, the term “chimeric antibody” as used herein refers to a monoclonal antibody in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0065] The term “single chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker (e.g., a short linker peptide of ten to about 25 amino acids). Huston etal. (1988) Proc. Nat. Acad. Sci. USA S5(16):5879-5883. In some embodiments, the linker can connect the N-terminus of the VH with the C-terminus of the VL, or the N-terminus of the VL with the C-terminus of the VH. A number of methods have been described to discern chemical structures for converting the naturally aggregated — but chemically separated — light and heavy polypeptide chains from an antibody V region into an scFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.,- and U.S. Pat. No. 4,946,778, to Ladner et al.

[0066] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by an antibody or an antigen-binding fragment thereof and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes. Embodiments herein contemplate the use of ceramide, or ceramide conjugated to a hapten, as an antigen.

[0067] The term “epitope” refers to a region of an antigen that is bound by an antibody. Epitope determinants can include chemically active surface groupings of molecules such asamino acids, sugar side chains, phosphoryl, or sulfonyl and may have specific three- dimensional structural characteristics, and / or specific charge characteristics.

[0068] Herein, the term “specifically binds” refers to the ability of an antibody or antigenbinding fragment thereof to bind a target antigen with a binding affinity (Ka) of at least 105M'1while not significantly binding other components or antigens present in a mixture. Reference to an anti-ceramide antibody herein refers to an antibody or antigen-binding fragment thereof that specifically binds to ceramide.

[0069] Binding affinity (Ka) refers to an equilibrium association of a particular interaction expressed in the units of 1 / M or M’1. Antibodies or antigen-binding fragments thereof can be classified as “high affinity” antibodies or antigen-binding fragments thereof and “low affinity” antibodies or antigen-binding fragments thereof. “High affinity” antibodies or antigen-binding fragments thereof refer to those antibodies or antigen-binding fragments thereof with a Ka of at least 107M’1, at least 108M’1, at least 109M’1, at least IO10M’1, at least 1011M’1, at least 1012M’1, or at least 1013M’1. “Low affinity” antibodies or antigenbinding fragments thereof refer to those antibodies or antigen-binding fragments thereof with a Ka of up to 107M’1, up to 106M’1, up to 105M’1. Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10'5M to 10'13, 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). Affinities of binding domain polypeptides and single chain polypeptides according to the present disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51 :660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[0070] A “conservative substitution” is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well-known in the art (see, e.g., PCT Application Publication No. WO 97 / 09433, page 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).

[0071] As used herein, the term “derivative” refers to a modification of one or more amino acid residues of a peptide by chemical or biological means, either with or without an enzyme, e.g., by glycosylation, alkylation, acylation, ester formation, or amide formation.

[0072] As used herein, a polypeptide or polynucleotide from which another polypeptide or polynucleotide is derived from is referred to as the “parental” or “reference” polynucleotide or polypeptide. For example, a humanized antibody can be derived from a parental murine antibody.

[0073] The term “variant” or “variants” as used herein refers to a polynucleotide or polypeptide with a sequence differing from that of a reference polynucleotide or polypeptide, but retaining essential properties of the parental polynucleotide or polypeptide. Generally, variant polynucleotide or polypeptide sequences are overall closely similar, and, in many regions, identical to the parental polynucleotide or polypeptide. For instance, a variant polynucleotide or polypeptide may exhibit at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% at least about 99%, or at least about 99.5% sequence identity compared to the parental polynucleotide or polypeptide.

[0074] As used herein, the term “sequence identity” refers to a relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparator sequence, the sequences are said to be “identical” at that position. The percentage sequence identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of identical positions. The number of identical positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of sequence identity. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The comparison window for polynucleotide sequences can be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleic acids in length. The comparison window for polypeptide sequences can be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids in length. In order to optimally align sequences for comparison, the portion of a polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant. An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of “identical” positions between thereference and comparator sequences. Percentage “sequence identity” between two sequences can be determined using the version of the program “BLAST 2 Sequences” which was available from the National Center for Biotechnology Information as of September 1, 2004, which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing “BLAST 2 Sequences,” parameters that were default parameters as of September 1, 2004, can be used for word size (3), open gap penalty (11), extension gap penalty (1), gap dropoff (50), expect value (10) and any other required parameter including but not limited to matrix option. Two nucleotide or amino acid sequences are considered to have “substantially similar sequence identity” or “substantial sequence identity” if the two sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to each other.

[0075] The term “substantially identical” refers to a polypeptide sequence that contains a sufficient number of identical amino acids to a second polypeptide sequence such that the first and second polypeptide sequence have similar activity. Polypeptides that are substantially identical are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical in amino acid sequence.

[0076] “Fc region” or “Fc domain” refers to a polypeptide sequence corresponding to or derived from the portion of an antibody that is capable of binding to Fc receptors on cells and / or the Clq component of complement, thereby mediating the effector function of an antibody. Fc stands for “fragment crystalline,” the fragment of an antibody that will readily form a protein crystal. Distinct protein fragments, which were originally described by proteolytic digestion, can define the overall general structure of an immunoglobulin protein. As originally defined in the literature, the Fc region is a homodimeric protein comprising two polypeptides that are associated by disulfide bonds, and each comprising a hinge region, a CH2 domain, and a CH3 domain. However, more recently the term has been applied to the single chain monomer component consisting of CH3, CH2, and at least a portion of the hinge sufficient to form a disulfide-linked dimer with a second such chain. As such, and depending on the context, use of the terms “Fc region” or “Fc domain” will refer herein to either the dimeric form or the individual monomers that associate to form the dimeric protein. For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V(Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31 :169-217, 1994. As used herein, the term Fc domain includes variants of naturally occurring sequences.

[0077] The term “immunoglobulin constant region” or “constant region” refers to a peptide or polypeptide sequence that corresponds to or is derived from part or all of one or more constant domains of an immunoglobulin (e.g., CHI, CH2, CH3). In certain embodiments, the constant region does not comprise a CHI domain. In certain embodiments, the constant domains making up the constant region are human

[0078] The terms “light chain variable region” (also referred to as “light chain variable domain” or “VL”) and “heavy chain variable region” (also referred to as “heavy chain variable domain” or “VH”) refer to the variable binding region from an antibody light and heavy chain, respectively. The variable binding regions are made up of discrete, well-defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs).

[0079] The term “immunoglobulin light chain constant region” (also referred to as “light chain constant region” or “CL”) is a constant region from an antibody light chain.

[0080] The term “immunoglobulin heavy chain constant region” (also referred to as “heavy chain constant region” or “CH”) refers to the constant region from the antibody heavy chain. The CH is further divisible, depending on the antibody isotype into CHI, CH2, and CH3 (IgA, IgD, IgG), or CHI, CH2, CH3, and CH4 domains (IgE, IgM).

[0081] As used herein, the term “complementarity determining region” or “CDR” refer to an immunoglobulin (antibody) molecule. There are three CDRs per variable domain: CDR1, CDR2 and CDR3 in the variable domain of the light chain and CDR1, CDR2 and CDR3 in the variable domain of the heavy chain.

[0082] In some embodiments, a “hinge” or a “hinge region” refers to a polypeptide derived from an immunoglobulin hinge region and located between an antigen-binding domain (e.g., a ceramide-binding domain) and an immunoglobulin constant region in a polypeptide described herein. A “wild-type immunoglobulin hinge region” refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CHI and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CHI and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody. In certain embodiments, a wild type immunoglobulin hinge region sequence is human, and can comprise a human IgG hinge region (e.g., and IgGl, IgG2, IgG3, or IgG4 hinge region).

[0083] An “altered immunoglobulin hinge region” or “variant immunoglobulin hinge region” refers to a hinge region polypeptide with one or more mutations, substitutions, insertions, or deletions compared to a corresponding parental wild-type immunoglobulin hinge region. Typically, an altered immunoglobulin hinge region that is a fragment of a wild type immunoglobulin hinge region comprises an IgG core hinge region (e.g., a polypeptide comprising the sequence C-X-X-C, wherein X is any amino acid) as disclosed in U.S. Patent Application Publication Nos. 2013 / 0129723 and 2013 / 0095097.

[0084] As used herein, the term “humanized” refers to an antibody or antigen-binding fragment thereof derived from a non-human species that retains the antigen-binding properties of the original non-human antibody. In some embodiments, the binding fragments of an antibody (e.g., light and heavy chain variable regions, Fab, scFv) are humanized. Non- human antigen-binding fragments can be humanized using techniques known as CDR grafting (Jones et al., Nature 321 :522 (1986)) and variants thereof, including “reshaping” (Verhoeyen, et al., 1988 Science 239: 1534-1536; Riechmann, et al., 1988 Nature 332:323- 337; Tempest, et al., Bio / Technol 1991 9:266-271), “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 -CD 18 antibodies.” In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312). If derived from a non-human source, other regions of the antibody, such as the hinge region and constant region domains, can also be humanized.

[0085] As used herein, the term “pharmaceutically acceptable” refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans are considered to be “pharmaceutically acceptable.”

[0086] The term “polynucleotide” as referred to herein means single-stranded or doublestranded nucleic acid polymers. In certain embodiments, the nucleotides comprising the polynucleotide can be RNA or DNA or a modified form of either type of nucleotide, such as a modified messenger RNA. Said modifications may include, but are not limited to, base modifications such as bromouridine, ribose modifications such as arabinoside and 2', 3'-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term “polynucleotide” specifically includes single and double stranded forms of DNA.

[0087] As used herein, a “polypeptide” or “protein” refers to a single, linear, and contiguous arrangement of covalently linked amino acids. Polypeptides can form one or more intrachain disulfide bonds. The terms polypeptide and protein also encompass embodiments where two polypeptide chains link together in a non-linear fashion, such as via an interchain disulfide bond. Herein, a protein or polypeptide may be an antibody or an antigen-binding fragment of an antibody.

[0088] As used herein, the term “transformation,” “transfection,” and “transduction” refer to the transfer of a polynucleotide into a cell. As used herein, the term “genetic transformation” refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell. The transferred nucleic acid can be introduced into a cell via an expression vector.

[0089] As used herein, the terms “treatment,” “treating,” or “ameliorating” refers to either a therapeutic treatment or prophylactic / preventative treatment. A treatment is therapeutic if at least one symptom of disease in an individual receiving treatment improves or a treatment can delay worsening of a progressive disease in an individual or prevent onset of additional associated diseases.

[0090] As used herein, the term “LD90” refers to “Lethal Dose, 90%” or “median lethal dose” and is the amount of a substance required to kill 90% of a test population.

[0091] As used in this specification, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.

[0092] As used in this specification, the term “and / or” is used in this disclosure to either “and” or “or” unless indicated otherwise.

[0093] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0094] As used in this specification, the term "about" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as10% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length, inclusive of the endpoints. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth, unless otherwise apparent from context that it is impossible to extend the boundary beyond certain points (e.g., below 0% or above 100%).

[0095] As used in this specification, the term “therapeutically effective amount” refers to an amount of a therapeutic agent (e.g., a hematopoietic stem cell population, an anti-ceramide antibody or the antigen binding fragment thereof) that achieves the desired biologic or therapeutic effect, namely an amount that prevents, reduces or ameliorates one or more symptoms of the enumerated diseases being treated or prevented.

[0096] The present disclosure provides the unexpected finding that (i) transplanted hematopoietic stem cells and (ii) an anti-ceramide antibody or the antigen binding fragment thereof synergistically restore the bone marrow in patients, drastically increasing the engraftment efficiency of the transplanted hematopoietic stem cells. Further, post-radiation administration of both (i) transplanted hematopoietic stem cells and (ii) an anti-ceramide antibody or the antigen binding fragment thereof synergistically limits radiation damages to the patients. Without being bound by any particular theory, it is hypothesized that the prevention or reversal of microvascular tissue injury by the anti-ceramide therapy helps improve the efficacy of hematopoietic stem cells engraftment. Such synergistic effects bring various benefits to hematopoietic stem cell transplantation procedures, such as lowering the required amount of hematopoietic stem cells and allowing more thorough pre-transplant conditioning of the patient to remove residual undesirable bone marrow cells.Hematopoietic stem cell transplantation (HSCT)

[0097] The terms “hematopoietic stem cell transplantation” (HSCT), “Hematopoietic cell transplantation” (HCT), and “bone marrow transplantation” (BMT) are used interchangeably throughout this specification and refer to the administration of a population of cells comprising hematopoietic stem cells to a patient in need thereof. Such stem cells may be collected peripherally (e.g., via apheresis) or via bone marrow harvests. In some embodiments, the cells are administered intravenously. Once administered, the hematopoietic stem cells can move through the bloodstream to reconstitute the patient's bone marrow, where the donor cells grow and produce new blood cells (e.g., red blood cells, white blood cells, and platelets), a process termed “engraftment.”

[0098] In some embodiments, the HSCT is an autologous HSCT, in which the stem cells are derived from the patient himself or herself. In some embodiments, autologous HSCT is used for treating an autoimmune disease. In some embodiments, autologous HSCT reset the patient’s immune system, particularly the T cells. In some embodiments, autologous HSCT is used for treating multiple myeloma (e.g., following induction therapy), non-Hodgkin lymphoma (NHL), or metastatic germ cell tumors. In some embodiments, the cancer is a relapsed cancer.

[0099] In some embodiments, the HSCT is an allogenic HSCT, in which the stem cells are derived from someone other than the patient. The terms “allogenic” and “allogeneic” are used interchangeably throughout the specification. In some embodiments, the allogenic HSCT is used for treating a cancer (e.g., through a graft-versus-tumor (GVT) effect). In some embodiments, the allogenic HSCT is for replacing dysfunctional bone marrow or blood cells of the patient in nonmalignant conditions. In some embodiments, the allogeneic stem cells are collected peripherally or from the bone marrow. In some embodiments, the pre-transplant conditioning in the allogenic HSCT is myeloablative. In some embodiments, the pretransplant conditioning in the allogenic HSCT is not myeloablative.

[0100] In some embodiments, the hematopoietic stem cells are derived from bone marrow.

[0101] In some embodiments, the hematopoietic stem cells are derived from peripheral blood. In some embodiments, obtaining peripheral blood stem cells comprises mobilizing the stem cells. In some embodiments, mobilizing the stem cells comprises pre-treatment with filgrastim or plerixafor followed by apheresis.

[0102] In some embodiments, the hematopoietic stem cells are derived from umbilical cord blood.

[0103] In general, traditional HSCT requires at least 2 * 106CD34+ hematopoietic stem cells / kg for successful engraftment, and the recommended dose by the American Society for Blood and Marrow Transplant (ASBMT) is 3-5 x 106CD34+ cells / kg (see Visram et al., Blood Cancer J. 2018 Jan; 8(1): 14).

[0104] In allogeneic HSCT, a major complication is Graft-versus-host disease (GVHD), an immunologic process wherein donor T cells attack host tissue that they recognize as foreign. Autologous HSCT, on the other hand, typically carries no risk of GVHD.

[0105] GVHD is typically divided into acute and chronic forms, each affecting specific organ systems and possessing distinctive clinical and histologic manifestations. Acute GVHD occurs most frequently in the first 100 days after HSCT, although particularly with nonmyeloablative conditioning, a syndrome of ‘late’ acute GVHD has been recognized. The major targets of alloreactivity in acute GVHD are the skin, the GI tract and (less frequently) the liver. Typically, patients undergoing T-cell-replete HCT receive immunosuppressive prophylaxis against GVHD; without such prophylaxis, the incidence and severity of GVHD would be prohibitive, even in the HLA-identical setting. The most widely used prophylactic regimens for acute GVHD combine a calcineurin inhibitor (CSP or tacrolimus) with several doses of post-transplant methotrexate. Tacrolimus may be more effective than CSP in preventing acute GVHD. Other approaches to the prevention of acute GVHD include the use of T-cell depletion.

[0106] In comparison with acute GVHD, chronic GVHD is more poorly characterized and understood. The course of chronic GVHD is highly variable; many patients have eventual resolution of chronic GVHD and are able to discontinue all immunosuppressive treatment as tolerance develops, while others have severe and treatment-refractory manifestations. The manifestations of chronic GVHD differ significantly from those of acute GVHD. Chronic GVHD can affect nearly any organ system and its manifestations typically resemble those of classical autoimmune diseases, such as lupus, Sjogren syndrome and systemic sclerosis. First- line treatment for chronic GVHD typically includes a corticosteroid. Chronic GVHD, similar to acute GVHD, is generally understood as a T-cell-mediated phenomenon. However, the description of autoantibodies in chronic GVHD, directed against targets, such as PDGF receptor and various minor histocompatibility antigens, suggests a possible complementary role of B cells in chronic GVHD.Method of treatment

[0107] The present disclosure provides methods of hematopoietic stem cell transplantation (HSCT) to a patient in need thereof, said methods comprising administering to the patient (i) a hematopoietic stem cell population and (ii) a therapeutic agent comprising an anti -ceramide antibody or the antigen binding fragment thereof. In some embodiments, the therapeutic agent comprising an anti -ceramide antibody or the antigen binding fragment thereof is administered concurrently with or following administration of the hematopoietic cell population.

[0108] The present disclosure provides methods of improving engraftment of hematopoietic stem cells in a patient in need thereof, the method comprising administering a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof in addition to the administration of the hematopoietic cells. In some embodiments, the engraftment improvement is measured by at least one of the successful engraftment criteria described infra.

[0109] The present disclosure provides methods of reducing the dose of hematopoietic stem cells in a patient in need thereof (e.g., in a HSCT), by administering a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof. In some embodiments, the patient receiving the therapeutic agent and the reduced hematopoietic cell population exhibits enhanced hematopoietic cell engraftment (e.g., as measured by at least one of the successful engraftment criteria described infra) compared to a control patient receiving the reduced hematopoietic cell population but not receiving the anti-ceramide antibody or the antigen binding fragment thereof.

[0110] The present disclosure provides methods of hematopoietic stem cell engraftment, comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population. In some embodiments, the patient’s ceramide signaling pathway is blocked before, during, and / or after the administration of the hematopoietic cell population. In some embodiments, the endothelial cells in the patient are unable to form a ceramide-rich platform before, during, and / or after the administration of the hematopoietic cell population. In some embodiments, the endothelial cells in the patient are temporarily blocked from forming a ceramide-rich platform before, during, and / or after the administration of the hematopoietic cell population.[OHl] The present disclosure provides methods of reducing a dose size of hematopoietic cells, comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) administering to the patient: (i) a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight. In some embodiments, the patient’s ceramide signaling pathway is blocked before, during, and / or after the administration of the hematopoietic cell population. In some embodiments, the endothelial cells in the patient are unable to form a ceramide-rich platform before, during, and / or after the administration of the hematopoietic cell population.In some embodiments, the endothelial cells in the patient are temporarily blocked from forming a ceramide-rich platform before, during, and / or after the administration of the hematopoietic cell population.

[0112] In some embodiments, blocking the ceramide signaling pathway or the ceramide-rich platform formation comprises administering an anti-ceramide therapeutic agent.

[0113] In some embodiments, the anti-ceramide therapeutic agent comprises an an anti- ceramide antibody, imipramine, a statin (e.g., nystatin), an antisense nucleic acid, a small interfering RNA (siRNA), or a combination thereof. In some embodiments, the antisense nucleic acid or the siRNA reduces expression of ASMase, Bak, or Bax.

[0114] In some embodiments, the blocking of the ceramide signaling pathway or the ceramide-rich platform (CRP) formation is evaluated by a 2-dimensional CRP incidence analysis or a 3-dimensional CRP volumetric analysis.

[0115] In some embodiments, the reduced dose of hematopoietic stem cells comprises less than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight. In some embodiments, the reduced dose of hematopoietic stem cells comprises less than 1.9 million, less than 1.8 million, less than 1.7 million, less than 1.6 million, less than 1.5 million, less than 1.4 million, less than 1.3 million, less than 1.2 million, less than 1.1 million, less than 1.0 million, less than 0.9 million, less than 0.8 million, less than 0.7 million, less than 0.6 million, less than 0.5 million, less than 0.4 million, less than 0.3 million, less than 0.2 million, or less than 0.1 million, CD34+ hematopoietic stem cells per kilogram of the patient’ s body weight. In some embodiments million, the reduced dose of hematopoietic stem cells comprises 0.1-0.2 million, 0.2-0.3 million, 0.3-0.5 million, 0.5-0.7 million, 0.7-1 million, 1- 1.2 million, 1.2-1.5 million, 1.5-1.6 million, 1.6-1.7 million, 1.7-1.8 million, 1.8-1.9 million, 1.9-2 million, 0.1-0.3 million, 0.2-0.5 million, 0.3-0.7 million, 0.5-1 million, 0.7-1.2 million, 1-1.5 million, 1.2-1.6 million, 1.5-1.7 million, 1.6-1.8 million, 1.7-1.9 million, 1.8-2 million, 0.1-0.5 million, 0.2-0.7 million, 0.3-1 million, 0.5-1.2 million, 0.7-1.5 million, 1-1.6 million, 1.2-1.7 million, 1.5-1.8 million, 1.6-1.9 million, 1.7-2 million, 0.1-0.7 million, 0.2-1 million, 0.3-1.2 million, 0.5-1.5 million, 0.7-1.6 million, 1-1.7 million, 1.2-1.8 million, 1.5-1.9 million, 1.6-2 million, 0.1-1 million, 0.2-1.2 million, 0.3-1.5 million, 0.5-1.6 million, 0.7-1.7 million, 1-1.8 million, 1.2-1.9 million, 1.5-2 million, 0.1-1.2 million, 0.2-1.5 million, 0.3-1.6 million, 0.5-1.7 million, 0.7-1.8 million, 1-1.9 million, 1.2-2 million, 0.1-1.5 million, 0.2-1.6 million, 0.3-1.7 million, 0.5-1.8 million, 0.7-1.9 million, 1-2 million, 0.1-1.6 million, 0.2-1.7million, 0.3-1.8 million, 0.5-1.9 million, 0.7-2 million, 0.1-1.7 million, 0.2-1.8 million, 0.3- 1.9 million, 0.5-2 million, 0.1-1.8 million, 0.2-1.9 million, 0.3-2 million, 0.1-1.9 million, 0.2- 2 million, or 0.1-2 million, CD34+ hematopoietic stem cells per kilogram of the patient’s body weight.

[0116] In some embodiments, the reduced dose of hematopoietic stem cells comprises about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight, including all ranges and subranges therebetween.

[0117] The hematopoietic stem cell population may be administered to a subject in a therapeutically effective amount. The hematopoietic stem cell population may be administered to subject, for example, by intramuscular, subcutaneous, intravenous, intra- atrial, intra-articular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, or oral administration. Ins some embodiments, the hematopoietic stem cell population is administered intravenously.

[0118] In some embodiments, the therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof is administered concurrently with the hematopoietic stem cell population.

[0119] In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or the antigen binding fragment thereof is administered before the administration of the hematopoietic stem cell population. In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof is administered at least about 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more, hours before the administration of the hematopoietic stem cell population, including all ranges and subranges therebetween. In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof is administered about 0-1, 0-2, 0- 3, 0-4, 0-6, 0-12, 0-24, 1-2, 1-3, 1-4, 1-6, 1-12, 1-24, 2-3, 2-4, 2-6, 2-12, 2-24, 3-4, 3-6, 3-12, 3-24, 4-6, 4-12, 4-24, 6-12, 6-24, or 12-24, hours before the administration of the hematopoietic stem cell population. In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof is administered at least 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days before the administration of the hematopoietic stem cell population, including all ranges and subranges therebetween. In some embodiments, the therapeutic agent comprising the anti-ceramideantibody or antigen binding fragment thereof is administered about 0-1, 0-2, 0-3, 0-4, 0-5, 0- 7, 0-14, 0-21, 1-2, 1-3, 1-4, 1-5, 1-7, 1-14, 1-21, 2-3, 2-4, 2-5, 2-7, 2-14, 2-21, 3-4, 3-5, 3-7, 3-14, 3-21, 4-5, 4-7, 4-12, 4-21, 5-7, 5-14, 5-21, 7-14, 7-21, or 14-21, days before the administration of the hematopoietic stem cell population.

[0120] In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or the antigen binding fragment thereof is administered after the administration of the hematopoietic stem cell population. In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof is administered at least about 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more, hours after the administration of the hematopoietic stem cell population, including all ranges and subranges therebetween. In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof is administered about 0-1, 0-2, 0- 3, 0-4, 0-6, 0-12, 0-24, 1-2, 1-3, 1-4, 1-6, 1-12, 1-24, 2-3, 2-4, 2-6, 2-12, 2-24, 3-4, 3-6, 3-12, 3-24, 4-6, 4-12, 4-24, 6-12, 6-24, or 12-24, hours after the administration of the hematopoietic stem cell population. In some embodiments, the therapeutic agent comprising the anti- ceramide antibody or antigen binding fragment thereof is administered at least 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the administration of the hematopoietic stem cell population, including all ranges and subranges therebetween. In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof is administered about 0-1, 0-2, 0-3, 0-4, 0-5, 0-7, 0-14, 0-21, 1-2, 1-3, 1-4, 1-5, 1-7, 1-14, 1-21, 2-3, 2-4, 2-5, 2-7, 2-14, 2-21, 3-4, 3-5, 3-7, 3-14, 3-21, 4-5, 4-7, 4-12, 4- 21, 5-7, 5-14, 5-21, 7-14, 7-21, or 14-21, days after the administration of the hematopoietic stem cell population.

[0121] In some embodiments, the hematopoietic cell population is an autologous hematopoietic cell population.

[0122] In some embodiments, the hematopoietic cell population is an allogenic hematopoietic cell population.

[0123] In some embodiments, the hematopoietic stem cell population used in the method of the disclosure comprises genetically engineered hematopoietic stem cells. In some embodiments, the method of the disclosure comprises genetic engineering the hematopoietic stem cell population. Genetic engineering may be performed, for example, using genome editing endonuclease, such as zinc finger nucleases (ZFN), transcription activator-likeeffector-based nuclease (TALEN), and CRISPR / Cas systems. Additional descriptions of such genetic engineering can be found, for example, in US 2020 / 0030381 and US 2018 / 0155717, the content of each of which is incorporated by reference in its entirety for all purposes.

[0124] In some embodiments, the genetic engineering corrects one or more genetic defects in the parental cells that the genetically engineered hematopoietic stem cells are derived from. In some embodiments, the genetically engineered hematopoietic cell population is an autologous hematopoietic cell population.

[0125] In some embodiments, the genetic engineering makes the hematopoietic stem cells more suitable for transplantation. For example, the genetic engineering may reduce the risk of graft-versus-host disease (GVHD) after the transplantation of the hematopoietic stem cell population, which may be particularly useful for allogenic HSCT.

[0126] In some embodiments, the method of the disclosure comprises conditioning the patient for receiving the hematopoietic stem cells. Typically, the conditioning step is performed before the transplant.

[0127] In some embodiments, the conditioning is a myeloablative conditioning (MAC). In some embodiments, myeloablative conditioning fully ablates the marrow. In some embodiments, myeloablative conditioning employs chemotherapy and / or radiation. In some embodiments, the chemotherapy uses one or more alkylators. In some embodiments, the alkylator comprises busulfan, cyclophosphamide, and / or fludarabine. In some embodiments, the alkylator comprises thiotepa, cytarabine, etoposide, melphalan, or combinations thereof. In some embodiments, radiation comprises total body irradiation (TBI) or total lymphoid irradiation (TLI). In some embodiments, MAC regimen is employed for acute diseases such as acute lymphoblastic leukemia.

[0128] In some embodiments, the conditioning is a reduced-intensity conditioning (RIC) or nonmyeloablative conditioning. RIC is generally similar to MAC but with significant dose reductions. With HSCT for hematologic malignancies like acute leukemia, the goal with RIC is to kill a fair number of malignant cells (and make “immunological space” for the incoming stem cells) and rely on GVT effects for the rest. More descriptions of reduced-intensity conditioning (RIC) or nonmyeloablative conditioning can be found, for example, in Gyurkocza et al., Expert Rev Hematol. 2010 Jun; 3(3): 285-299.

[0129] In some embodiments, the conditioning comprises administering a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is selected from the groupconsisting of chlorambucil, cyclophosphamide, ifosfamide, melphalan, streptozocin, carmustine, lomustine, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, 5- fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, daunorubicin, doxorubicin, epirubicin, idarubicin, SN-38, camptothecin, topotecan, 9-nitrocamptothecin, 9- aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, amsacrine, etoposide, etoposide phosphate, teniposide, paclitaxel, docetaxel, and combinations thereof.

[0130] In some embodiments, the conditioning comprises administering an alkylating agent (i.e., alkylator). In some embodiments, the alkylator comprises busulfan, cyclophosphamide, fludarabine, thiotepa, cytarabine, etoposide, and / or melphalan. In some embodiments, the alkylator comprises melphalan.

[0131] In some embodiments, the conditioning comprises radiation. In some embodiments, the radiation is does not trigger Gastrointestinal Acute Radiation Syndrome (GI-ARS). In some embodiments, the dose of the radiation is less than 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 Gy, including all ranges and subranges therebetween. In some embodiments, the dose ofthe radiation is about 10-11, 10-12, 10-13, 10-14, 10-15, 11-12, 11-13, 11-14, I lls, 12-13, 12-14, 12-15, 13-14, 13-15, or 14-15 Gy. In some embodiments, the dose of the radiation is less than 15, 15.5, 16, 16.5, or 17 Gy, including all ranges and subranges therebetween. In some embodiments, the dose of the radiation is about 15-16, 15-17, or 16- 17 Gy. In some embodiments, the radiation is not administered to the patient’s intestines.

[0132] In some embodiments, the conditioning regimen required prior to transplantation, designed to ablate or suppress the patient's immune system, renders the patient susceptible to neoplastic relapse or infection. Recent use of unrelated and HLA non-identical donors has unfortunately increased the incidence of GvHD. While removal of T cells from the donor marrow graft ameliorates GvHD, this strategy increases graft failure rates and markedly diminishes the therapeutically-beneficial graft-versus-tumor effect. As such, overall survival does not improve. Further, despite strong pre-clinical data, attempts to improve GvHD outcomes by diminishing inflammatory cytokine action by adding TNF antagonists to corticosteroids, the standard of care for acute GvHD, has provided limited therapeutic benefit. Thus, allowing a harsher conditioning regimen may benefit the patients receiving HSCT by reducing the rate of relapse.

[0133] In some embodiments, the dose of the chemical treatment, the chemotherapeutic agent, or the radiation in the conditioning step of the method is higher than that in a control method not receiving the therapeutic agent comprising the anti-ceramide antibody or the antigen binding fragment thereof. In some embodiments, the dose is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7- fold, at least 10-fold, at least 15-fold, or at least 20-fold, higher than the dose in a control method not receiving the therapeutic agent comprising the anti-ceramide antibody or the antigen binding fragment thereof, including all ranges and subranges therebetween.

[0134] In some embodiments, the method reduces the recurrence / relapse of the malignancy in the patient. In some embodiments, the reduction in recurrence / relapse at least partially results from the higher dose of the chemical treatment, the chemotherapeutic agent, or the radiation used in the conditioning step of the method that in a control method.

[0135] In some embodiments, the method of the disclosure comprises administering the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof after the conditioning of the patient for receiving the hematopoietic stem cells. In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof is administered at least about 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more, hours after the conditioning of the patient is completed, including all ranges and subranges therebetween. In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof is administered about 0-1, 0-2, 0-3, 0-4, 0-6, 0-12, 0-24, 1-2, 1-3, 1-4, 1-6, 1-12, 1-24, 2-3, 2- 4, 2-6, 2-12, 2-24, 3-4, 3-6, 3-12, 3-24, 4-6, 4-12, 4-24, 6-12, 6-24, or 12-24, hours after the conditioning of the patient is completed. In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof is administered at least 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the conditioning of the patient is completed, including all ranges and subranges therebetween. In some embodiments, the therapeutic agent comprising the anti-ceramide antibody or antigen binding fragment thereof is administered about 0-1, 0-2, 0-3, 0-4, 0-5, 0-7, 0-14, 0-21, 1-2, 1- 3, 1-4, 1-5, 1-7, 1-14, 1-21, 2-3, 2-4, 2-5, 2-7, 2-14, 2-21, 3-4, 3-5, 3-7, 3-14, 3-21, 4-5, 4-7, 4-12, 4-21, 5-7, 5-14, 5-21, 7-14, 7-21, or 14-21, days after the conditioning of the patient is completed.

[0136] In some embodiments, the anti-ceramide antibody or the antigen binding fragment thereof is administered at a dose of about 1 ng / kg body weight / day to about 100 mg / kg body weight / day. In some embodiments, the range of the anti-ceramide antibody or the antigen binding fragment thereof administered is from about 1 ng / kg body weight / day to about 1 pg / kg body weight / day, 1 ng / kg body weight / day to about 100 ng / kg body weight / day, 1 ng / kg body weight / day to about 10 ng / kg body weight / day, 10 ng / kg body weight / day to about 1 pg / kg body weight / day, 10 ng / kg body weight / day to about 100 ng / kg body weight / day, 100 ng / kg body weight / day to about 1 pg / kg body weight / day, 100 ng / kg body weight / day to about 10 pg / kg body weight / day, 1 pg / kg body weight / day to about 10 pg / kg body weight / day, 1 pg / kg body weight / day to about 100 pg / kg body weight / day, 10 pg / kg body weight / day to about 100 pg / kg body weight / day, 10 pg / kg body weight / day to about 1 mg / kg body weight / day, 100 pg / kg body weight / day to about 10 mg / kg body weight / day, 1 mg / kg body weight / day to about 100 mg / kg body weight / day and 10 mg / kg body weight / day to about 100 mg / kg body weight / day, including all ranges and subranges therebetween. Dosages within this range can be achieved by single or multiple administrations, including, e.g., multiple administrations per day or daily, weekly, bi-weekly, or monthly administrations. The anti-ceramide antibody or antigen-binding fragment thereof may be administered, as appropriate or indicated, as a single dose by bolus or by continuous infusion, or as multiple doses by bolus or by continuous infusion. Multiple doses may be administered, for example, multiple times per day, once daily, every 2, 3, 4, 5, 6 or 7 days, weekly, every 2, 3, 4, 5 or 6 weeks or monthly, including all ranges and subranges therebetween. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.

[0137] For administration to a human patient (e.g., an adult patient), the therapeutically effective amount of the anti-ceramide antibody or the antigen binding fragment thereof may be administered in doses in the range of 0.0006 mg to 1000 mg per dose, including but not limited to 0.0006 mg per dose, 0.001 mg per dose, 0.003 mg per dose, 0.006 mg per dose, 0.01 mg per dose, 0.03 mg per dose, 0.06 mg per dose, 0.1 mg per dose, 0.3 mg per dose, 0.6 mg per dose, 1 mg per dose, 3 mg per dose, 6 mg per dose, 10 mg per dose, 30 mg per dose, 60 mg per dose, 100 mg per dose, 300 mg per dose, 600 mg per dose and 1000 mg per dose, and multiple, usually consecutive daily doses may be administered in a course of treatment.

[0138] Dosage of the the anti-ceramide antibody or antigen-binding fragment thereof can be varied by the attending clinician to maintain a desired concentration at a target site. Forexample, if an intravenous mode of delivery is selected, local concentration of the agent in the bloodstream at the target tissue can be between about 0.01-50 nM, sometimes between about 1.0 nM and 10, 15, or 25 nM depending on the subject's status and projected measured response. Higher or lower concentrations can be selected based on the mode of delivery, e.g., trans-epidermal delivery versus delivery to a mucosal surface. Dosage should also be adjusted based on the release rate of the administered formulation, e.g., nasal spray versus powder, sustained release oral or injected particles, transdermal formulations, etc. To achieve the same serum concentration level, for example, slow-release particles with a release rate of 5 nM (under standard conditions) would be administered at about twice the dosage of particles with a release rate of 10 nM.

[0139] The anti-ceramide antibody or antigen-binding fragment thereof may be administered to a subject in a therapeutically effective amount. The anti-ceramide antibody or antigenbinding fragment thereof may be administered to subject, for example, by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, or oral administration. It can be administered to a subject in a single bolus delivery, via continuous delivery over an extended time period, or in a repeated administration protocol.

[0140] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a scFv.

[0141] In some embodiments, the method comprises providing and / or preparing a patient in need of the hematopoietic stem cells before the conditioning and administration of HSC / anti- ceramide therapy.

[0142] In some embodiments, engraftment of the hematopoietic cell population is enhanced compared to a control engraftment not receiving the anti-ceramide antibody or the antigen binding fragment thereof.

[0143] In some embodiments, the success of the engraftment is measured by patient’s white blood cell count returning to a pre-transplant level within about 30, 60, 90, 120, 180, or 365, days post-transplant, including any ranges or subranges therebetween. In some embodiments, the success of the engraftment is measured by patient’s white blood cell count returning to a pre-transplant level within about 90 days post-transplant. In some embodiments, the success of the engraftment is measured by patient’s white blood cell count of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 K / uL, including all ranges and subranges therebetween. In someembodiments, the success of the engraftment is measured by patient’s white blood cell count of at least about 5 K / uL within 90 days post-transplant.

[0144] In some embodiments, the success of the engraftment is measured by patient’s neutrophil count returning to a pre-transplant level within about 30, 60, 90, 120, 180, or 365, days post-transplant, including all ranges and subranges therebetween. In some embodiments, the success of the engraftment is measured by patient’s neutrophil count returning to a pretransplant level within about 90 days post-transplant. In some embodiments, the success of the engraftment is measured by patient’s neutrophil blood cell count of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 K / uL, including all ranges and subranges therebetween. In some embodiments, the success of the engraftment is measured by patient’s neutrophil blood cell count of at least about 1 K / uL within 90 days posttransplant.

[0145] In some embodiments, the success of the engraftment is measured by patient’s platelet count returning to a pre-transplant level within about 30, 60, 90, 120, 180, or 365, days posttransplant, including all ranges and subranges therebetween. In some embodiments, the success of the engraftment is measured by patient’s platelet count returning to a pre-transplant level within about 90 days post-transplant. In some embodiments, the success of the engraftment is measured by patient’s platelet count of at least about 1, 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 K / uL, including all ranges and subranges therebetween. In some embodiments, the success of the engraftment is measured by patient’s platelet count of at least about 300 K / uL within 90 days post-transplant.

[0146] In some embodiments, the success of the engraftment is measured by patient’s survival rate about 30, 60, 90, 120, 180, or 365, days post-transplant, including all ranges and subranges therebetween. In some embodiments, the success of the engraftment is measured by patient’s survival rate about 1, 2, 3, 4, or 5, years post-transplant, including all ranges and subranges therebetween.

[0147] In some embodiments, the success rate of the engraftment of the hematopoietic cell population using the method of the disclosure is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to a control engraftment not receiving the anti-ceramide antibody or the antigen binding fragment thereof, including all ranges and subranges therebetween.

[0148] In some embodiments, the success rate of the engraftment of the hematopoietic cell population using the method of the disclosure is increased by at least 10% compared to a control engraftment not receiving the anti -ceramide antibody or the antigen binding fragment thereof. In some embodiments, the success rate of the engraftment of the hematopoietic cell population using the method of the disclosure is increased by at least 20% compared to a control engraftment not receiving the anti -ceramide antibody or the antigen binding fragment thereof. In some embodiments, the success rate of the engraftment of the hematopoietic cell population using the method of the disclosure is increased by at least 30% compared to a control engraftment not receiving the anti -ceramide antibody or the antigen binding fragment thereof. In some embodiments, the success rate of the engraftment of the hematopoietic cell population using the method of the disclosure is increased by at least 40% compared to a control engraftment not receiving the anti -ceramide antibody or the antigen binding fragment thereof. In some embodiments, the success rate of the engraftment of the hematopoietic cell population using the method of the disclosure is increased by at least 50% compared to a control engraftment not receiving the anti -ceramide antibody or the antigen binding fragment thereof.

[0149] In some embodiments, the method reduces the likelihood of graft versus host disease (GVHD) compared to a control method without the administration of the therapeutic agent comprising the anti-ceramide antibody or the antigen binding fragment thereof. In some embodiments, the method reduces the rate of acute GVHD by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, in a patient population, as compared to the control method, including all ranges and subranges therebetween.

[0150] In some embodiments, the method reduces the rate of chronic GVHD by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, in a patient population, as compared to the control method, including all ranges and subranges therebetween.

[0151] In some embodiments, the method is for treating a non-malignant disease (e.g., an autoimmune disease). In some embodiments, the non-malignant disease is aplastic anemia (AA), pure red cell aplasia, Fanconi’s anemia, dyskeratosis congenita, Blackfan-Diamond syndrome, sickle cell disease, thalassemia, congenital amegakaryocytic thrombocytopenia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic syndromes, severe congenital neutropenia, chronic granulomatous disease, IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, common variable immunodeficiency, mucopolysaccharoidose (e.g., MPS-I or MPS-VI), osteopetrosis, or globoid cell leukodystrophie (e.g., Krabbe or Cerebral X-linked Adrenoleukodystrophy). In some embodiments, the method is for treating a chronically injured marrow substratum. In some embodiments, the method is for treating a hemoglobinopathy. In some embodiments, the method is for treating sickle cell disease.

[0152] In some embodiments, the method is for treating a cancer in the patient. In some embodiments, the treatment utilizes the graft-versus-tumor (GVT) effect.

[0153] In some embodiments, the cancer is leukemia. In some embodiments, the cancer is acute myelogenous leukemia (AML), acute promyelocytic leukemia (APL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS), a therapy-related AML / MDS, or myeloproliferative neoplasms (MPN).

[0154] In some embodiments, the cancer is a mature lymphoid neoplasm. In some embodiments, the cancer is multiple myeloma (MM), plasma cell leukemia, a lymphoblastic B cell NHL, diffuse large B cell lymphoma (DLBCL), a high-grade B cell lymphoma, follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt’s lymphoma, lymphoplasmacytic lymphoma (LPL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), T cell lymphomas, Hodgkin’s lymphoma (HL), or anaplastic large cell lymphoma.

[0155] Non-limiting examples of malignant and non-malignant diseases that can be treated by the HSCT based methods include acute and chronic leukemias, myelomas, solid tumors (R. J. Jones, Curr Opin Oncol 3 (2), 234 (1991); G. L. Phillips, Prog Clin Biol Res 354B, 171 (1990)), aplastic anemias and severe immunodeficiency's (R. P. Gale, R. E. Champlin, S. A. Feig et al., Ann Intern Med 95 (4), 477 (1981); G. M. Silber, J. A. Winkelstein, R. C. Moen et al., Clin Immunol Immunopathol 44 (3), 317 (1987)).Composition

[0156] The present disclosure provides compositions (e.g., pharmaceutical compositions) comprising (a) a hematopoietic stem cell population, and (b) an anti -ceramide antibody or the antigen binding fragment thereof.

[0157] In some embodiments, the hematopoietic cell population is an autologous hematopoietic cell population. In some embodiments, the hematopoietic cell population is an allogenic hematopoietic cell population.

[0158] In some embodiments, the hematopoietic stem cell population comprises genetically engineered hematopoietic stem cells. In some embodiments, the genetic engineering corrects one or more genetic defects in the parental cells that the genetically engineered hematopoietic stem cells are derived from. Such hematopoietic cell population can then be used for autologous transplantation. In some embodiments, the genetic engineering makes the hematopoietic stem cells more suitable for transplantation. For example, the genetic engineering may reduce the risk of graft-versus-host disease (GVHD) after the transplantation of the hematopoietic stem cell population.

[0159] In some embodiments, the hematopoietic stem cell population and the anti-ceramide antibody or the antigen binding fragment thereof are in a mixture. In some embodiments, the hematopoietic stem cell population, and the anti-ceramide antibody or the antigen binding fragment thereof, are kept separately in the pharmaceutical composition / kit.

[0160] In some embodiments, the pharmaceutical composition of the disclosure comprises a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier, diluent, or excipient. A carrier is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers, diluents, or excipients are well-known to those in the art. (See, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995).) Formulations can further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.

[0161] The composition (e.g., pharmaceutical composition) of the disclosure may be formulated in a dosage form selected from the group consisting of: an oral unit dosage form,an intravenous unit dosage form, an intranasal unit dosage form, a suppository unit dosage form, an intradermal unit dosage form, an intramuscular unit dosage form, an intraperitoneal unit dosage form, a subcutaneous unit dosage form, an epidural unit dosage form, a sublingual unit dosage form, and an intracerebral unit dosage form. The oral unit dosage form may be selected from the group consisting of: tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained-release formulations, aerosols, and sprays.

[0162] The pharmaceutical composition of the disclosure may be administered to a subject in a therapeutically effective amount. According to the methods of the present disclosure, the pharmaceutical composition may be administered to subjects by a variety of administration modes, including, for example, by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, and oral routes of administration. It can be administered to a subject in a single bolus delivery, via continuous delivery over an extended time period, or in a repeated administration protocol (e.g., on an hourly, daily, weekly, or monthly basis).

[0163] Effective doses of the compositions of the present disclosure vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, as well as the specific activity of the composition itself and its ability to elicit the desired response in the individual. Usually, the patient is a human, but in some diseases, the patient can be a nonhuman mammal. Typically, dosage regimens are adjusted to provide an optimum therapeutic response, i.e., to optimize safety and efficacy.

[0164] Determination of effective dosages is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of the subject disorder in model subjects.

[0165] In some embodiments, the composition is for a patient in need thereof. In some embodiments, the hematopoietic stem cell population of the composition comprises less than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight. In some embodiments, the hematopoietic stem cell population of the composition comprises less than 1.9 million, less than 1.8 million, less than 1.7 million, less than 1.6 million, less than 1.5 million, less than 1.4 million, less than 1.3 million, less than 1.2 million, less than 1.1 million,less than 1.0 million, less than 0.9 million, less than 0.8 million, less than 0.7 million, less than 0.6 million, less than 0.5 million, less than 0.4 million, less than 0.3 million, less than 0.2 million, or less than 0.1 million, CD34+ hematopoietic stem cells per kilogram of the patient’s body weight, including all ranges and subranges therebetween. In some embodiments million, the hematopoietic stem cell population of the composition comprises 0.1-0.2 million, 0.2-0.3 million, 0.3-0.5 million, 0.5-0.7 million, 0.7-1 million, 1-1.2 million, 1.2-1.5 million, 1.5-1.6 million, 1.6-1.7 million, 1.7-1.8 million, 1.8-1.9 million, 1.9-2 million, 0.1-0.3 million, 0.2-0.5 million, 0.3-0.7 million, 0.5-1 million, 0.7-1.2 million, 1-1.5 million, 1.2-1.6 million, 1.5-1.7 million, 1.6-1.8 million, 1.7-1.9 million, 1.8-2 million, 0.1- 0.5 million, 0.2-0.7 million, 0.3-1 million, 0.5-1.2 million, 0.7-1.5 million, 1-1.6 million, 1.2- 1.7 million, 1.5-1.8 million, 1.6-1.9 million, 1.7-2 million, 0.1-0.7 million, 0.2-1 million, 0.3- 1.2 million, 0.5-1.5 million, 0.7-1.6 million, 1-1.7 million, 1.2-1.8 million, 1.5-1.9 million, 1.6-2 million, 0.1-1 million, 0.2-1.2 million, 0.3-1.5 million, 0.5-1.6 million, 0.7-1.7 million, 1-1.8 million, 1.2-1.9 million, 1.5-2 million, 0.1-1.2 million, 0.2-1.5 million, 0.3-1.6 million, 0.5-1.7 million, 0.7-1.8 million, 1-1.9 million, 1.2-2 million, 0.1-1.5 million, 0.2-1.6 million, 0.3-1.7 million, 0.5-1.8 million, 0.7-1.9 million, 1-2 million, 0.1-1.6 million, 0.2-1.7 million, 0.3-1.8 million, 0.5-1.9 million, 0.7-2 million, 0.1-1.7 million, 0.2-1.8 million, 0.3-1.9 million, 0.5-2 million, 0.1-1.8 million, 0.2-1.9 million, 0.3-2 million, 0.1-1.9 million, 0.2-2 million, or 0.1-2 million, CD34+ hematopoietic stem cells per kilogram of the patient’s body weight.

[0166] In some embodiments, the anti-ceramide antibody or the antigen binding fragment thereof of the composition is about 1 ng / kg body weight to about 100 mg / kg body weight. In some embodiments, the range of the anti-ceramide antibody or the antigen binding fragment thereof administered is from about 1 ng / kg body weight to about 1 pg / kg body weight, 1 ng / kg body weight to about 100 ng / kg body weight, 1 ng / kg body weight to about 10 ng / kg body weight, 10 ng / kg body weight to about 1 pg / kg body weight, 10 ng / kg body weight to about 100 ng / kg body weight, 100 ng / kg body weight to about 1 pg / kg body weight, 100 ng / kg body weight to about 10 pg / kg body weight, 1 pg / kg body weight to about 10 pg / kg body weight, 1 pg / kg body weight to about 100 pg / kg body weight, 10 pg / kg body weight to about 100 pg / kg body weight, 10 pg / kg body weight to about 1 mg / kg body weight, 100 pg / kg body weight to about 10 mg / kg body weight, 1 mg / kg body weight to about 100 mg / kg body weight and 10 mg / kg body weight to about 100 mg / kg body weight.

[0167] In some embodiments, the anti-ceramide antibody or the antigen binding fragment thereof of the composition is about 1 ng / kg body weight, 10 ng / kg body weight, 20 ng / kg body weight, 30 ng / kg body weight, 40 ng / kg body weight, 50 ng / kg body weight, 60 ng / kg body weight, 70 ng / kg body weight, 80 ng / kg body weight, 90 ng / kg body weight, 100 ng / kg body weight, 150 ng / kg body weight, 200 ng / kg body weight, 250 ng / kg body weight, 300 ng / kg body weight, 350 ng / kg body weight, 400 ng / kg body weight, 450 ng / kg body weight, 500 ng / kg body weight, 550 ng / kg body weight, 600 ng / kg body weight, 650 ng / kg body weight, 700 ng / kg body weight, 750 ng / kg body weight, 800 ng / kg body weight, 850 ng / kg body weight, 900 ng / kg body weight, 950, 1 pg / kg body weight, 2 pg / kg body weight, 3 pg / kg body weight, 4 pg / kg body weight, 5 pg / kg body weight, 6 pg / kg body weight, 7 pg / kg body weight, 8 pg / kg body weight, 9 pg / kg body weight, 10 pg / kg body weight, 15 pg / kg body weight, 20 pg / kg body weight, 25 pg / kg body weight, 30 pg / kg body weight, 35 pg / kg body weight, 40 pg / kg body weight, 45 pg / kg body weight, 50 pg / kg body weight, 55 pg / kg body weight, 60 pg / kg body weight, 65 pg / kg body weight, 70 pg / kg body weight, 75 pg / kg body weight, 80 pg / kg body weight, 85 pg / kg body weight, 90 pg / kg body weight, 95 pg / kg body weight, 100 pg / kg body weight, 0.1 mg / kg body weight, 0.2 mg / kg body weight, 0.3 mg / kg body weight, 0.4 mg / kg body weight, 0.5 mg / kg body weight, 0.6 mg / kg body weight, 0.7 mg / kg body weight, 0.8 mg / kg body weight, 0.9 mg / kg body weight, 1 mg / kg body weight,1.5 mg / kg body weight, 2 mg / kg body weight, 2.5 mg / kg body weight, 3 mg / kg body weight,3.5 mg / kg body weight, 4 mg / kg body weight, 4.5 mg / kg body weight, 5 mg / kg body weight,5.5 mg / kg body weight, 6 mg / kg body weight, 6.5 mg / kg body weight, 7 mg / kg body weight,7.5 mg / kg body weight, 8 mg / kg body weight, 8.5 mg / kg body weight, 9 mg / kg body weight,9.5 mg / kg body weight, 10 mg / kg body weight, 10.5 mg / kg body weight, 11 mg / kg body weight, 11.5 mg / kg body weight, 12 mg / kg body weight, 12.5 mg / kg body weight, 13 mg / kg body weight, 13.5 mg / kg body weight, 14 mg / kg body weight, 14.5 mg / kg body weight, 15 mg / kg body weight, 15.5 mg / kg body weight, 16 mg / kg body weight, 16.5 mg / kg body weight, 17 mg / kg body weight, 17.5 mg / kg body weight, 18 mg / kg body weight, 18.5 mg / kg body weight, 19 mg / kg body weight, 19.5 mg / kg body weight, 20 mg / kg body weight, 20.5 mg / kg body weight, 21 mg / kg body weight, 21.5 mg / kg body weight, 22 mg / kg body weight,22.5 mg / kg body weight, 23 mg / kg body weight, 23.5 mg / kg body weight, 24 mg / kg body weight, 24.5 mg / kg body weight, 25 mg / kg body weight, 25.5 mg / kg body weight, 26 mg / kg body weight, 26.5 mg / kg body weight, 27 mg / kg body weight, 27.5 mg / kg body weight, 28 mg / kg body weight, 28.5 mg / kg body weight, 29 mg / kg body weight, 29.5 mg / kg bodyweight, 30 mg / kg body weight, 30.5 mg / kg body weight, 31 mg / kg body weight, 31.5 mg / kg body weight, 32 mg / kg body weight, 32.5 mg / kg body weight, 33 mg / kg body weight, 33.5 mg / kg body weight, 34 mg / kg body weight, 34.5 mg / kg body weight, 35 mg / kg body weight,35.5 mg / kg body weight, 36 mg / kg body weight, 36.5 mg / kg body weight, 37 mg / kg body weight, 37.5 mg / kg body weight, 38 mg / kg body weight, 38.5 mg / kg body weight, 39 mg / kg body weight, 39.5 mg / kg body weight, 40 mg / kg body weight, 40.5 mg / kg body weight, 41 mg / kg body weight, 41.5 mg / kg body weight, 42 mg / kg body weight, 42.5 mg / kg body weight, 43 mg / kg body weight, 43.5 mg / kg body weight, 44 mg / kg body weight, 44.5 mg / kg body weight, 45 mg / kg body weight, 45.5 mg / kg body weight, 46 mg / kg body weight, 46.5 mg / kg body weight, 47 mg / kg body weight, 47.5 mg / kg body weight, 48 mg / kg body weight,48.5 mg / kg body weight, 49 mg / kg body weight, 49.5 mg / kg body weight, or 50 mg / kg body weight, including all ranges and subranges therebetween.

[0168] Compositions (e.g., pharmaceutical compositions) of the disclosure can be supplied as a kit comprising a container that comprises the composition as described herein. A pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a pharmaceutical composition. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.Anti-ceramide antibodies and antigen-binding fragments thereof

[0169] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to ceramide. In some embodiments, the anti-ceramide antibody or antigen binding fragments thereof is humanized. In some embodiments, the anti-ceramide antibody is a humanized 6B5 scFv antibody.

[0170] The term “ceramide” as used herein refers to a lipid second messenger molecule comprised of sphingosine and a fatty acid. Ceramide regulates stress signaling within the cell through reorganization of the plasma membrane and formation of lateral liquid-ordered microdomains (a type of ‘raft’ termed ‘ceramide-rich platforms’). These CRPs serve as signaling platforms that cluster activated receptor molecules (e.g., members of the TNF- receptor superfamily including Fas), thereby providing feed forward mechanisms that ultimately result in signaling amplification and signal transduction. CRP formation isparticularly important for Fas-mediated cell death and ceramide-rich regions on the plasma membrane of target cells are critical for sensitivity to cytotoxic T lymphocyte (CTL)-induced cell death. Exemplary ceramides are glucosylceramides, galactosylceramides and gangliosides (oligosaccharide-linked ceramides).

[0171] Ceramide mediated raft clustering is involved in radiation-induced apoptosis and clonogenic cell death. It has long been accepted that the clonogenic compartment of the gastrointestinal (GI) mucosa is the specific and direct target for radiation in inducing GI damage.

[0172] The disclosure also encompasses anti-ceramide antibodies and antigen-binding fragments thereof for the manufacture of a medicament for treatment of a disorder characterized by expression of ceramide. In some embodiments, the protein or polypeptide comprises an anti-ceramide binding domain and inhibits formation of ceramide rich platforms.

[0173] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to ceramide and comprises an immunoglobulin heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), and an immunoglobulin light chain variable region (VL) comprising a light chain complementarity determining region (CDR) 1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3). In some embodiments, the HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 1 and 2; the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, and 6; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0174] The CDRs of exemplary anti-ceramide antibodies or antigen-binding fragments are shown in Table 1.Table 1: Exemplary anti-ceramide antibody sequences

[0175] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is an scFv 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 the HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 1 and 2; the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, and 6; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0176] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 3; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0177] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a)the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0178] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 5; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0179] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 6; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0180] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 6; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0181] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 3; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ IDNO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0182] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0183] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 5; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0184] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 6; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0185] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 3; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0186] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a)the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0187] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 5; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and (f) the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10.

[0188] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH and a VL chain, wherein the VH chain comprises or consists of an amino acid sequence that is at least about 85%%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-21.

[0189] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH and a VL chain, wherein the VL chain comprises or consists of an amino acid sequence that is at least about 85%%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-24.

[0190] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH and a VL chain, wherein the VH chain comprises or consists of an amino acid sequence that is at least about 85%%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:17-21, and the VL chain comprises or consists of an amino acid sequence that is at least about 85%%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-24.

[0191] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is an scFv comprising a VH and a VL chain, wherein the VH chain comprises or consists of an amino acid sequence that is at least about 85%%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-21, and the VL chain comprises or consists of an amino acid sequence that is at least about 85%%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-24.Table 2. Exemplary VH and VL Amino Acid Sequences

[0192] In some embodiments, the anti-ceramide antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 17 and a VL amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 17 and a VL amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 17 and a VL amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 18 and a VL amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 18 and a VL amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 18 and a VL amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or antigenfragment thereof comprises a VH amino acid sequence of SEQ ID NO: 19 and a VL amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 19 and a VL amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 19 and a VL amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 20 and a VL amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 20 and a VL amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 20 and a VL amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 21 and a VL amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or antigen-fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 21 and a VL amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody or antigenfragment thereof comprises a VH amino acid sequence of SEQ ID NO: 21 and a VL amino acid sequence of SEQ ID NO: 24.

[0193] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 17 and a VL 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 SEQ ID NO: 22. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 17 and the VL amino acid sequence consists of SEQ ID NO: 22.

[0194] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 17 and a VL 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 SEQ ID NO: 23. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH amino acid sequences consisting of 17 and a VL amino acid sequence consisting of 23.

[0195] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 17 and a VL 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 SEQ ID NO: 24. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 17 and the VL amino acid sequence consists of SEQ ID NO: 24.

[0196] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 18 and a VL 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 SEQ ID NO: 22. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 18 and the VL amino acid sequence consists of SEQ ID NO: 22.

[0197] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 18 and a VL 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 SEQ ID NO: 23. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 18 and the VL amino acid sequence consists of SEQ ID NO: 23.

[0198] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 18 and a VL 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 SEQ ID NO: 24. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 18 and the VL amino acid sequence consists of SEQ ID NO: 24.

[0199] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 19 and a VL 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 SEQ ID NO: 22. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 19 and the VL amino acid sequence consists of SEQ ID NO: 22.

[0200] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 19 and a VL 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 SEQ ID NO: 23. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 19 and the VL amino acid sequence consists of SEQ ID NO: 23.

[0201] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 19 and a VL 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 SEQ ID NO: 24. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 19 and the VL amino acid sequence consists of SEQ ID NO: 24.

[0202] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 20 and a VL 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 SEQ ID NO: 22. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 20 and the VL amino acid sequence consists of SEQ ID NO: 22.

[0203] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH 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 SEQ ID NO: 21 and a VL 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 SEQ ID NO: 22. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 21 and the VL amino acid sequence consists of SEQ ID NO: 22.

[0204] In some embodiments, the anti-ceramide antigen-binding fragment is an scFv comprising a VH 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 SEQ ID NO: 18 and a VL 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 SEQ ID NO: 22. In some embodiments, the anti-ceramide scFv comprises a VH chain and a VL chain, wherein the VH chain amino acid sequence consisting of SEQ ID NO: 18 and the VL amino acid sequence consists of SEQ ID NO: 22.

[0205] In some embodiments, the anti-ceramide antigen-binding fragment thereof is an scFv. In some embodiments, the anti-ceramide scFv comprises an amino acid sequence that is at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-61. In some embodiments, the anti-ceramide scFv consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-61 In some embodiments, the anti- ceramide scFv comprises or consists of the amino acid sequence of SEQ ID NOs: 48 or 51.

[0206] In some embodiments, the anti-ceramide scFv comprises the following structure: VH- linker-VL or VL-linker-VH. As used herein, the term “linker” generally refers to a short polypeptide sequence connecting two sub-domains of a polypeptide. Non-limiting examples of linkers include flexible linkers comprising glycine-serine repeats, and linkers derived from(a) an interdomain region of a transmembrane protein (e.g., a type I transmembrane protein);(b) a stalk region of a type II C-lectin; or (c) an immunoglobulin hinge. In some embodiments, a linker provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions. In certain embodiments, a linker is comprised of five to about 35 amino acids, for instance, about 15 to about 25 amino acids. Exemplary linkers are shown in Table 3.Table 3. Exemplary linkers

[0207] In some embodiments, the anti-ceramide antibody or antigen-binding fragments thereof is produced with an N-terminal signal sequence to promote secretion of the scFv protein. In some embodiments, the signal sequence comprises SEQ ID NO: 75. One of skill in the art will understand that these signal sequences are not a part of the mature, secreted protein. Therefore, in some embodiments, the signal sequence of SEQ ID NO: 75 is cleaved from the mature anti-ceramide antibody or antigen-binding fragment protein.

[0208] In some embodiments, the anti-ceramide antibody or antigen-binding fragments thereof are produced with a C-terminal tag sequence to facilitate purification during manufacturing. In some embodiments, the C-terminal tag comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-ceramide antibody or antigen-binding fragments thereof are produced without a C-terminal tag sequence. Therefore, in some embodiments, the amino sequences of the anti-ceramide antibody or antigen-binding fragments thereof do not comprise the C-terminal tag of SEQ ID NO: 76.

[0209] Exemplary amino acid sequences for the anti-ceramide scFvs are provided in Table 4 below. The CDRs of the scFvs are indicated by underlined text. Linker sequences are indicated by italicized text. Signal sequences are indicated by bold text. C-terminal tag sequences are indicated by bold and italicized text.Table 4. Amino Acid Sequences of Exemplary anti-ceramide scFvs

[0210] In some embodiments, the anti-ceramide antibody is a full-length antibody comprising a full length antibody light chain and a full length antibody heavy chain. In some embodiments, the anti-ceramide antibody is an IgG isotype (e.g., IgGl, IgG2, IgG3, IgG4). In some embodiments, the Fc domain of the anti-ceramide antibody comprises a wild-type IgG amino acid sequence. Such sequences are known in the art, see e.g., Shields et al., J Biol Chem, (2001) 276:9;6591-6604. In some embodiments, the CH2 or CH3 domain of the Fc domain comprises one or more amino acid mutations that alter the function and / or stability of the antibody. For example, in some embodiments, the Fc domain of an anti-ceramide antibody described herein lacks or has minimal effector functions while retaining the ability to bind some Fc receptors such as the neonatal Fc receptor (FcRn) and retaining a relatively long half-life in vivo. In some embodiments, anti-ceramide antibodies described do not resultin, or substantially reduce the induction of, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement activation, and / or complement-dependent cytotoxicity (CDC). Such mutations are well 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 a / ., Methods (2014) 65 : 1 ; 114-126.

[0211] In some embodiments, the anti-ceramide antibodies described are IgGl isotype antibodies, wherein the IgGl constant region has a mutation at one or more of the following positions: 228 (S228), 234 (L234), 235 (L235), 237 (G237), 297 (N297), 318 (E318), 320 (K320), 322 (K322), or any combination thereof (numbering according to EU). In some embodiments, the IgGl Fc domain has an L234A and L235A mutation. In some embodiments, the IgGl Fc domain has an S228P mutation. In some embodiments, the anti- ceramide antibodies described are IgG4 isotype antibodies, wherein the IgG4 antibodies comprise a human IgG4 constant region comprising a mutation of S228P according to the EU index (EU numbering scheme as in Kabat). In some embodiments, the IgG4 antibodies comprise any combination of VH and VL of the present disclosure (e.g., a VH and a VL selected from Table 2, such as VH1 / VL1, VH2 / VL1, VH3 / VL1, VH4 / VL1, VH5 / VL1, VH1 / VL2, VH2 / VL2, VH3 / VL2, VH4 / VL2, VH5 / VL2, VH1 / VL3, VH2 / VL3, VH3 / VL3, VH4 / VL3, or VH5 / VL3).

[0212] As used herein, unless otherwise provided, a position of an amino acid residue in an immunoglobulin molecule is numbered according to EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94). Other numbering systems for amino acid positions within antibodies are known in the art. For example, 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, 5thedition, Bethesda, MD: Public Health Service, National Institutes of Health (1991)). Methods and information to convert between one numbering system and the other are known in the art. See, for example, the IMGT Scientific Chart - Correspondence between C numberings, available at imgt.org.

[0213] In certain embodiments, the anti-ceramide antibodies and antigen-binding fragments thereof may be prepared using standard molecular biology techniques with regard to selecting antibodies that have a desired specificity. In some embodiments, the anti-ceramide antibodies and antigen-binding fragments thereof are produced using recombinant DNA technologies.Procedures for the expression and purification of recombinant proteins are well established in the art.

[0214] In some embodiments, the anti-ceramide antibodies and antigen-binding fragments thereof prevent cell death by inhibiting ceramide-rich platform formation. For example, in some embodiments, the anti-ceramide scFvs inhibit ceramide-rich platform formation in cells. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof reduces ceramide-rich platform formation 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%.

[0215] In some embodiments, the anti-ceramide antibodies and antigen-binding fragments thereof inhibit cell death. In some embodiments, the anti-ceramide antibody or antigenbinding fragment thereof reduces 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%.

[0216] In some embodiments, the anti-ceramide antibody or the antigen-binding fragment thereof is bispecific. As used herein, the term “bispecific” refers to an antibody construct that is at least bispecific — i.e., it comprises at least a first binding domain and a second binding domain, wherein the first binding domain binds to one antigen or target (e.g., a ceramide), and the second binding domain binds to another antigen or target (e.g., a non-ceramide antigen or target). Accordingly, in some embodiments, antibodies or antigen-binding fragments of the disclosure comprise specificities for at least two different antigens or targets. In some embodiments, such bispecific antibodies or antigen-binding fragments comprise specificities for two different antigens or targets. In some embodiments, such bispecific antibodies or antigen-binding fragments comprise specificities for more than two different antigens or targets, such as trispecific antibodies or antigen-binding fragments. In some embodiments, the anti-ceramide bispecific antibody or antigen binding fragment is a bispecific T-cell engager (“BiTE”). A BiTE typically comprises two independently different antibody regions (e.g., two single-chain variable fragments (scFv)), each of which binds a different antigen. One antibody region may engage effector cells (e.g., T cells) by binding an effector cell-specific antigen (e.g., CD3 molecule) and the second antibody region may bind a target cell (e.g., cancer cell or autoimmune-reactive cell) through a cell surface antigen (e.g., ceramide) expressed by said target cell. Binding of the BiTE to the two antigens will link theeffector cell (e.g., T cell) to the target cell and activate the effector cell (e.g., T cell) via effector cell-specific antigen signaling (e.g., CD3 signaling). The activated effector cell (e.g., T cell) will then exert cytotoxic activity against the target cell (e.g., tumor cells). In some embodiments, at least one of the binding domains of the anti-ceramide bispecific antibody or the antigen-binding fragment thereof comprises the VH and / or VL CDRs of the disclosure.

[0217] Further descriptions of anti-ceramide antibodies and antigen-binding fragments thereof can be found, for example, in U.S. Pat. No. 9,592,238, U.S. Pat. No. 10,450,385, US 2022 / 0118087, and WO 2021 / 188770, the content of each of which is incorporated by reference in its entirety for all purposes.Polynucleotides and Methods of Protein Expression

[0218] The disclosure also includes polynucleotides (e.g., DNA or RNA) encoding the anticeramide antibodies and antigen-binding fragments thereof of the present disclosure. In some embodiments, the polynucleotides encode a polypeptide that is substantially identical to a polypeptide listed in Tables 1, 2, 3 and 4. In some embodiments, the polynucleotides encode a polypeptide that is 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 about least 99%, or at about least 100% identical to a polypeptide listed in Tables 1, 2, 3 and 4. Polynucleotides of the disclosure also include complementary nucleic acids. In some instances, the sequences will be fully complementary (no mismatches) when aligned. In other instances, there can be up to about a 20% mismatch in the sequences. The polynucleotide sequences provided herein can be exploited using codon optimization, degenerate sequence, silent mutations, and other DNA techniques to optimize expression in a particular host, and the present disclosure encompasses such sequence modifications.

[0219] In some embodiments, the disclosure provides an isolated polynucleotide encoding an anti-ceramide antibody or antigen-binding fragment thereof, wherein said polynucleotide is 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 is 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 65-73. In some embodiments, the disclosure provides an isolated polynucleotide encoding an anti-ceramide antibody or antigen-binding fragment thereof, wherein said polynucleotide comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 65-73.

[0220] In some embodiments, the polynucleotides of the present disclosure are inserted into a nucleic acid vector. The nucleic acid vector may be a viral vector or a non-viral vector, e.g. a plasmid. Vectors include, without limitation, plasmids, phagemids, cosmids, transposons, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. In some embodiments, the vector is a plasmid selected from pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). In some embodiments, the vector is a viral vector selected from viral vectors based on 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; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191 ; WO 94 / 28938; WO 95 / 11984 and WO 95 / 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. (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); a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. Examples of vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5- DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus- mediated gene transfer and expression in mammalian cells.

[0221] In some embodiments, the polynucleotide is inserted into a nucleic acid vector and is operably linked to one or more regulatory sequences that control transcription, such as promoters, enhancers, terminators, inducers, or repressors. Exemplary promoters include Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, a viral simian virus 40 (SV40) (e.g., early and late SV40), a spleen focus forming virus (SFFV) promoter, long terminal repeats (LTRs) from retrovirus (e.g., aMoloney murine leukemia virus (MoMLV) LTR promoter or a a Rous sarcoma virus (RSV) LTR), a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl l promoters from vaccinia virus, an elongation factor 1 -alpha (EFla) promoter, early growth response 1 (EGR1) promoter, a ferritin H (FerH) promoter, a ferritin L (FerL) promoter, a Glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) promoter, a eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, a heat shock 70kDa protein 5 (HSPA5) promoter, a heat shock protein 90kDa beta, member 1 (HSP90B1) promoter, a heat shock protein 70kDa (HSP70) promoter, a P-kinesin (P-KIN) promoter, the human ROSA 26 locus (Irions et aL, Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C (UBC) promoter, a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer / chicken P-actin (CAG) promoter, a P-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter, and mouse metallothionein-1.

[0222] In some embodiments, the vector is introduced into a host cell for expression of the anti-ceramide antibody or antigen-binding fragment thereof. Accordingly, proteins for use within the present disclosure can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, 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 a variety of host cells are disclosed by Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001), and Ausubel et al.. Short Protocols in Molecular Biology (4th ed., John Wiley & Sons, 1999).

[0223] The gene product encoded by a polynucleotide of the disclosure is expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian, and mammalian systems. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44; CHO DXB11 (Hyclone, Logan, UT); see also, e.g., Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548)SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658). Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, Virginia. Introduction of the DNA construct can use any convenient method, including, e.g. conjugation, bacterial transformation, calcium-precipitated DNA, electroporation, fusion, transfection, infection with viral vectors, biolistics, and the like.

[0224] For example, for recombinant expression of an anti-ceramide antibody or antigenbinding fragment thereof as described herein, an expression vector will generally include a nucleic acid segment encoding one or more of the amino acid sequences provided in Tables 1, 2, 3 and 4, operably linked to a promoter. The expression vector is introduced to a host cell by conventional techniques, and the host cells are then cultured by conventional techniques to produce the encoded polypeptide(s) to produce the corresponding anti-ceramide antibodies or antigen-binding fragments thereof.

[0225] To direct a recombinant protein into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence) is provided in the expression vector. The secretory signal sequence can be that of the native form of the recombinant protein or can be derived from another secreted protein or synthesized de novo. The secretory signal sequence is operably linked to the polypeptide-encoding DNA sequence, z.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5’ to the DNA sequence encoding the polypeptide of interest, although certain signal sequences can be positioned elsewhere in the DNA sequence of interest (see, e.g., U.S. Patent Nos. 5,037,743 and 5,143,830). In some embodiments, the secretory signal sequence is SEQ ID NO: 75.

[0226] Cultured mammalian cells are suitable hosts for production of recombinant polypeptides and proteins of the present disclosure (e.g., anti-ceramide antibodies and antigen-binding fragments thereof). Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981 : Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1 :841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., supra), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993).The production of recombinant polypeptides in cultured mammalian cells is disclosed by, for example, U.S. Patent Nos. 4,713,339; 4,784,950; 4,579,821; and 4,656,134.

[0227] Transformed or transfected host cells to produce the polypeptides and proteins of the present disclosure (e.g., ceramide-binding polypeptides) are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, and minerals. Media can also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.

[0228] The anti-ceramide antibodies and antigen-binding fragments thereof of the present disclosure may be purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See generally Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988); Scopes, Protein Purification: Principles and Practice (Springer-Verlag, New York 1994). Proteins comprising an immunoglobulin Fc region can be purified by affinity chromatography on immobilized protein A or protein G. Additional purification steps, such as gel filtration, can be used to obtain the desired level of purity or to provide for desalting, buffer exchange, and the like.Methods of Humanized Antibody Generation

[0229] The present disclosure discloses humanized anti-ceramide antibodies and antigenbinding fragments thereof. Humanized antibodies have the same or similar binding specificity and affinity as a mouse or other nonhuman antibody that provides the starting material for construction of a humanized antibody. A preferred antibody for humanization is the murine 6B5 antibody described in US 2017-03334133, incorporated herein by reference in its entirety. The VH and VL sequences of the murine 6B5 antibody are provided below in Table 5. The CDRs are indicated by underlined text. Linker sequences are indicated by italicized text. Signal sequences are indicated by bold text. C-terminal tag sequences are indicated by bold and italicized text.Table 5. Murine 6B5 VH and VL sequences from anti-ceramide antibodies

[0230] The substitution of mouse CDRs into a human variable domain framework is most likely to result in retention of their correct spatial orientation if the human variable domain framework adopts the same or similar conformation to the murine variable framework from which the CDRs originated. This is achieved by obtaining the human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable framework domains from which the CDRs were derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human variable framework region sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences 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., WO 92 / 22653.

[0231] Having identified the CDRs of the murine donor immunoglobulin and appropriate human acceptor framework region, the next step is to determine which, if any, residues from these components should be modified to optimize the properties of the resulting humanized antibody. In general, substitution of human amino acid residues with murine residues should be minimized, because introduction of murine residues increases the risk of the antibody eliciting a human-anti-mouse-antibody (HAMA) response in humans. Art-recognized methods of determining immune response can be performed to monitor a HAMA response in a particular patient or during clinical trials.

[0232] In some embodiments, the selection of amino acid residues for modification is determined, in part, by computer modeling. Computer hardware and software are described herein for producing three-dimensional images of immunoglobulin molecules. In general, molecular models are produced starting from solved structures for immunoglobulin chains ordomains thereof. The chains to be modeled are compared for amino acid sequence similarity with chains or domains of solved three-dimensional structures, and the chains or domains showing the greatest sequence similarity is / are selected as starting points for construction of the molecular model. Chains or domains sharing at least 50% sequence identity are selected for modeling, and preferably those sharing at least 60%, 70%, 80%, 90%, or more sequence identity are selected for modeling. The solved starting structures are modified to allow for differences between the actual amino acids in the immunoglobulin chains or domains being modeled, and those in the starting structure. The modified structures are then assembled into a composite immunoglobulin. Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from one another and that bond lengths and angles are within chemically acceptable limits.

[0233] The selection of amino acid residues for modification can also be determined, in part, by examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.

[0234] Additional exemplary humanization techniques that can be used for humanizing the immunoglobulins of the disclosure are described in, for example, 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).

[0235] Usually the CDR regions in humanized antibodies are identical to the corresponding CDR regions of the murine donor antibody. For example, in some embodiments, the CDR amino acid sequences of the humanized antibodies and fragments thereof are identical to the CDR amino acid sequences of the murine 6B5 antibody (e.g., SEQ ID NOs: 11-16).

[0236] In some embodiments, it may be desirable to modify one or more CDR regions to modify the antigen binding specificity of the antibody and / or reduce the immunogenicity of the antibody. In some embodiments, one or more residues of a CDR are altered to modify binding to achieve a more favored on-rate of binding, a more favored off-rate of binding, or both, such that an idealized binding constant is achieved. Using this strategy, an antibody having high binding affinity of, for example, I O'° M1or more, can be achieved. Affinity maturation techniques, as described herein, can be used to alter the parent CDR region(s) followed by screening of the resultant binding molecules for the desired change in binding. The method may also be used to alter the parent CDR to be less immunogenic such that apotential human anti-mouse antibody (HAMA) response is minimized or avoided. Accordingly, as CDR(s) are altered, changes in binding affinity as well as immunogenicity can be monitored and scored such that an antibody optimized for the best combined binding and low immunogenicity are achieved (see, e.g., U.S. Pat. No. 6,656,467 and U.S. Pat. Pub. US20020164326A1). Moreover, it is sometimes possible to make one or more conservative amino acid substitutions of CDR residues without appreciably 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.

[0237] In some embodiments, the humanized anti-ceramide antibodies and antigen-binding fragments thereof comprise one or more mutations relative to the corresponding CDR sequence of the murine 6B5 antibody e.g., relative to one or more of SEQ ID NOs: 11-16).

[0238] The framework regions of humanized immunoglobulins are usually substantially identical, and more usually, identical to the framework regions of the human antibodies from which they were derived. In some embodiments, humanized antibodies of the disclosure include a variable region framework sequence selected from human antibody genes (e.g., germline antibody gene segments) which include one or more canonical CDR structure types that are identical or similar to the canonical CDR structure types for the corresponding nonhuman antibody (e.g., murine) which is humanized. See, U.S. Pat. No. 6,881,557 and Tan et al., Journal of Immunol 169: 1119-1125 (2002) (incorporated by reference in their entirety for all purposes).

[0239] Of course, many of the amino acids in the framework region make little or no direct contribution to the specificity or affinity of an antibody. Thus, many individual conservative substitutions of framework residues can be tolerated without appreciable change of the specificity or affinity of the resulting humanized immunoglobulin. Framework residues can be analyzed to determine which, if any, residues should be substituted to optimize the properties of the resulting humanized antibody. For example, computer modeling can be used to identify residues which have a good probability of directly or indirectly influencing antigen binding.

[0240] Thus, in one embodiment the variable framework region of the humanized anti- ceramide antibody or antigen-binding fragment thereof shares at least 85% sequence identity to a human variable framework region sequence or consensus of such sequences. In some embodiments, the variable framework region of the humanized anti-ceramide antibody orantigen-binding fragment thereof shares at least 90%, preferably 95%, more preferably 96%, 97%, 98%, or 99% sequence identity to a human variable framework region sequence or consensus of such sequences.

[0241] In some embodiments, the humanized anti-ceramide antibody or antigen-binding fragment thereof exhibits a specific binding affinity for antigen of at least I O7M I O8M I O9M ', or I OI()M ' . Usually the upper limit of binding affinity of the humanized antibodies for antigen is within a factor of three, four, or five of that of the parental immunoglobulin. Often the lower limit of binding affinity is also within a factor of three, four, or five of that of the parent immunoglobulin. Alternatively, the binding affinity can be compared to that of a humanized antibody having no substitutions (e.g., an antibody having donor CDRs and acceptor FRs, but no FR substitutions). In such instances, the binding of the optimized antibody (with substitutions) is preferably at least two- to three-fold greater, or three- to four-fold greater, than that of the unsubstituted antibody. For making comparisons, activity of the various antibodies can be determined, for example, by BIACORE (i.e., surface plasmon resonance using unlabelled reagents) or competitive binding assays.EXAMPLES

[0242] The present disclosure is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and Tables, are incorporated herein by reference.Example 1: Anti-ceramide scFv prevents GI-ARS lethality and promotes extended mouse survival

[0243] The goal of this study was to investigate the radio-protective effects of anti-ceramide 6B5 scFv administration in animals. Radiation exposure in mammals can cause hematopoietic-acute radiation syndrome (H-ARS) and gastrointestinal-acute radiation syndrome (GI-ARS). Both H-ARS and GI-ARS can be independently lethal within weeks. The GI-ARS occurs subsequent to destruction of crypt / villus units that results in mucosal denudation, loss of nutrient adsorption and susceptibility to infection by resident bacterial flora. Clinically, the GI-ARS presents with anorexia, vomiting, diarrhea, dehydration, systemic infection, and in extreme cases, septic shock and death within days of exposure (3- 5).

[0244] Anti-ceramide 6B5 scFv was shown to mitigate ongoing endothelial cell apoptosis, preventing Lgr5+stem cell loss and lethality from the gastrointestinal-acute radiation syndrome (GI-ARS) with >80% survival at 30 days after an LD90 dose of 15 Gy in C57BL / 6J mice (11). In this initial study, animals were divided into the following cohorts: 15 Gy radiation alone; 15 Gy+BMT (5 xlO6cells / 25 g mouse); 15 Gy+6B5 scFv (150 pg / 25 g mouse); and 15 Gy+BMT+scFv.

[0245] Mice in all cohorts were provided a soft food diet in the form of liquid gels and water ad libitum and were monitored twice daily during the first week post radiation. Data are shown from 3 independent studies (FIG. 1, top panels) and as collated data (FIG. 1, bottom panel). Mice in the 15 Gy, 15 Gy+BMT, and 15 Gy+scFv groups exhibited clinical symptoms of acute radiation exposure including markedly reduced foraging and food consumption accompanied by bloody diarrhea, and group huddling. Mice in these groups died between 8- 10 days post irradiation.

[0246] In the post 15 Gy+BMT+scFv cohort, however, mice continued to forage actively, maintained a near normal eating and grooming pattern, and displayed frequent urination while developing rough fur for a period of one week, which resolved thereafter. After day 12, mice in the 15 Gy+BMT+scFv group displayed gradual improvement and at day 20 were returned to a hard kibble diet. All mice in the 15 Gy+BMT+scFv group that survived the acute radiation syndromes were alive at day 90 (except for 1 mouse in the 3rd study that died late from unknown causes) with behavioral patterns indistinguishable from unirradiated mice. Further, stratified (by study) log-rank tests show that the difference is statistically significant (p<0.001) when comparing either all four groups simultaneously, or 15Gy vs. 15Gy+BMT+scFv. These studies show that surviving the H-ARS and GI-ARS renders prolonged mouse survival.Example 2: Acute autopsy findings confirm the potential of anti-ceramide scFv to mitigate the GI-ARS

[0247] GI tracts, femurs and tibias from mice in Example 1 that succumbed to 15 Gy within the first 8-14 days were fixed in formalin at time of sacrifice for evaluation (FIG. 2). In the small intestine (jejunum at 10 cm from the Ligament of Treitz) of mice receiving 15 Gy or 15 Gy+BMT, villi were highly disorganized with poorly preserved crypts. In contrast, mice receiving 6B5 scFv at 24h post irradiation displayed a more normal architecture with better preserved crypts. Quantitative analysis of the extent of normal-appearing crypt-villus units inthe 15 Gy+scFv cohort revealed that 48 ± 21% (Mean ± 1 SD) of the small intestinal surface of mice was covered with crypt-villus units on Day 7 at the time of death. Whereas anticeramide 6B5 scFv appeared to improve GI tract recovery, all 6 images of bone marrow had complete absence of hematopoietic cells irrespective of BMT. Only endothelial cells, adipocytes and hemosiderin-laden macrophages were detected. Additionally, femurs from scFv-treated animals displayed intravascular colonies of bacteria, suggesting sepsis which is commonly observed as the ultimate cause of death in mice that have been irradiated and did not get their bone marrow reconstituted (28). Hence, these studies indicate that despite provision of 5 x 106marrow cells to mice at 16-hour after irradiation with 15 Gy there was no evidence of marrow reconstitution. A separate cohort of mice receiving 15 Gy+BMT±scFv, that otherwise would have survived, sacrificed on day 10 for comparison with the above cohorts, displayed 83 ± 7% relatively normal mucosal architecture (FIG. 3A and FIG. 3B) However, a complementary group of mice receiving only 15 Gy+scFv that survived for 10 days, displayed, at sacrifice, only 5 ± 3% normal-appearing structures indicating deterioration of crypt-villus architecture occurred after day 7. It is hypothesized based on these data that in the absence of hematopoietic elements nascent regenerative mucosa cannot be maintained, likely due to a combination of lack of reparative microvascular angiogenic cells and infection by resident bacteria, yielding crypt-villus unit deterioration between day 7-10 post irradiation.Example 3: Anti-ceramide scFv and BMT restore circulating hematopoietic elements post irradiation

[0248] Complete blood counts were performed on these cohorts of mice in the previous Examples up until the point of death. In the 15 Gy, 15 Gy+BMT and 15 Gy+scFv groups total white blood cell (WBC) counts, neutrophils and platelets were depleted by day 4 post irradiation and were undetectable 1 at day 8 (FIG. 4). A similar pattern was observed in mice receiving 15 Gy+BMT±scFv, however, in this cohort all hematopoietic elements returned to either normal or near normal levels by day 12 and remained at normal levels until these animals were sacrificed on day 90. Consistent with this latter finding, the 15 Gy+BMT±scFv cohort of mice sacrificed on day 10 displayed largely reconstituted marrow in the tibia and femur (FIG. 3C). Red blood cells (RBCs) appeared to be less affected by ionizing radiation as the small dip in RBCs in the irradiated and irradiated+BMT groups normalized by day 8. Altogether the autopsy findings suggest that anti-ceramide scFv might improve marrow reconstitution. To address this latter concept directly the radiation dose was lowered to 14 GyWBI, the highest dose, absent scFv provision, that BMT alone can prevent H-ARS lethality in C57BL / 6J mice, and dose-dependence of marrow reconstitution with and without scFv was examined. This strategy permits examination of the capability of anti-ceramide scFv to improve marrow reconstitution absent the potentially confounding possibility that prevention of GI lethality might improve marrow take. FIG. 5 shows that anti-ceramide scFv markedly enhances potency of bone marrow cells to confer survival from the H-ARS at 90 days post irradiation with a 3 -fold reduction of the number of marrow cells required for 50% survival, indicating the ability of anti-ceramide scFv to concomitantly mitigate GI-ARS and H-ARS lethality.Example 4: Anti-ceramide scFv+BMT treatment renders normal serum metabolic profile and tissue structure at day 90

[0249] The impact of scFv+BMT treatment on serum chemistries in mice surviving 90 days after 15 Gy was further investigated. No significant abnormalities in serum electrolytes were detected (FIG. 6). Furthermore, a comprehensive metabolic panel (CMP) indicated liver function was normal as liver enzymes [alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT)], bilirubin and serum albumin were within normal limits, and kidney function was maintained as BUN and creatinine levels were unaffected. Similarly, the serum lipid profile was within normal limits (triglycerides and cholesterol) and glucose levels were normal. Effectively these studies indicate that C57BL / 6J mice treated with 15 Gy, an LD90 dose for this strain for GLARS lethality, and anti-ceramide 6B5 scFv+BMT post irradiation display evidence of normally functioning major organs.

[0250] Mice in the 15 Gy+BMT+anti -ceramide 6B5 scFv group were sacrificed by CO2 asphyxiation at day 90 and subjected to full autopsies. Data from major organs and known radiosensitive tissues are shown in FIG. 7. Data from minor organs are shown in FIG. 8. Of note, specimens were analyzed from 42 separate sites and are shown for a single mouse in these two figures. Complete autopsy data (42 tissues, CBCs, and serum chemistries) from a cohort of 9 unirradiated age matched control mice sacrificed on day 90 are presented for comparison (FIG. 9-11). Only two minor abnormalities were observed in the 15 Gy+ BMT+anti -ceramide 6B5 scFv group at day 90: testicular atrophy and mild crypt dilation in the small intestines. The testicular lesion likely represents a permanent (non-reversible) radiation-induced injury of germ cells resulting in decreased sperm production consistent with the known sensitivity of germ cells to ionizing radiation (29). The mild small intestinal lesionsmay represent a remnant of radiation injury on intestinal crypts, but overall, the small intestinal mucosa was almost normal, suggesting that if radiation injury occurred in the intestine it was followed by almost complete recovery of this tissue. Furthermore, no differences between the 15 Gy+ BMT+anti -ceramide 6B5 scFv and age-matched unirradiated control groups were observed in vessel density, diameter, tortuosity, or distribution (FIG. 12). Vessels in both groups were morphologically normal, and no telangiectasia or other vascular lesions were observed. The only other abnormality detected was a grey discoloration of the hair coat, consistent with radiation injury of follicular melanocytes. Of note, there was no loss of acini in the salivary gland, which displayed normal architecture (FIG. 7, bottom row, third column), consistent with the ability to return these mice to a hard kibble diet. Further, there was no evidence of fibrosis in the lung, liver, or kidney as assessed by Masson’s Tri chrome staining (FIG. 13), nor was there evidence of cataracts (FIG. 8). No lesions could be attributed to administration of antibody. Overall, mitigation of an LD90 dose of 15 Gy for the GI-ARS with anti-ceramide 6B5 scFv and BMT results in a near normal mouse by behavior, appearance, serum chemistries, blood counts and metabolic profiling, and pathologically at autopsy.

[0251] All the above studies suggest that anti-ceramide therapy can achieve long-term multiorgan protection — e.g., avoiding systemic radiation injury, preventing the loss of salivary gland function after radiation, and mitigating liver injury. In particular, mice treated with 15Gy WBI and administered BMT+ anti-ceramide 6B5 scFv had prolonged survival of 90 days with no abnormal pathology in any major organ (small intestine, salivary glands liver, lungs, kidneys and heart) or alteration in organ function. Previous studies have shown that marrow stromal damage induced by WBI in the range of the GI-ARS impairs engraftment of hematopoietic stem cells. Data in the current Examples show that anti-ceramide antibody therapy can help mitigate this stromal incapacity. Notably, the use of anti-ceramide scFv markedly enhances the potency of bone marrow cells, allowing a 3-fold reduction of the number of marrow cells required to confer the same survival benefit as the control treatment of bone marrow transfer cells without the anti-ceramide therapy. Overall, the data supports the notion that anti-ceramide therapy promotes re-population of transplanted marrow — a previously unrecognized application of anti-ceramide therapy.

[0252] In the same way, anti-ceramide therapy may enhance engraftment of hematopoietic stem cells in the clinical setting of hematopoietic stem cell transplantation (HCT). Primary graft failure is defined as failure to achieve hematologic recovery with donor cell engraftmentwithin 28 days of transplant. It is a rare complication of HCT (1% of all grafts) but is almost universally fatal unless patients undergo a second HCT, which many times is not feasible (57). On the other hand, poor graft function is much more common and is associated with significant increases in morbidity and mortality (57,58). It is hypothesized that both of these adverse outcomes of HCT are amenable to anti-ceramide therapy. Furthermore, the current data suggest that anti-ceramide therapy enhances hematopoietic and potentially immune recovery in a variety of settings after HCT (e.g., allogeneic or autologous), such as enhancing recovery after cord blood transplantation (e.g., where stem cell dose is considered the main determinant of recovery) (59), improving engraftment of genetically-modified stem cells into a chronically injured marrow substratum (e.g., to facilitate cure of patients with sickle cell disease and other hemoglobinopathies) (60), or allowing delivery of higher doses of radiation and / or chemotherapy to moderate risk of relapse in older patients with high risk hematologic malignancies (e.g., those in which reduced intensity conditioning is associated with significant risk of recurrence) (61).

[0253] The current Examples further provide unexpected findings to support the notion that radiation-induced multi-organ damage may reflect a generic outcome of concomitant microvascular tissue injury, which may be reversed or prevented by anti-ceramide therapy. Previously, it was not possible to investigate impact of 15 Gy on multiple organs simultaneously because mice succumb to the GI-ARS so quickly, between 7-10 days, precluding assessment of organs that express damage at later times. Current data show that microvascular injury may play a determinant role in multi-organ radiation tissue injury in mammals, and anti-ceramide therapy may be effective in mitigating multiple adverse outcomes of radiation exposure by generic protection of the microvasculature.Example 5: Materials and Methods

[0254] Radiation, survival and complete necropsy studies: These studies used 8-12-week- old male C57BL / 6J mice 1 (The Jackson Laboratory, CT, USA). Mouse irradiation and treatment including BMT and scFv administration were performed as described previously (11). Briefly, C57BL / 6J mice were subjected to the LD90 dose of 15 Gy WBI using a Shepherd Mark-I unit (model 68, SN643, J. L. Shepherd & Associates) operating 137Cs sources at 1.5 Gy / minute. BM cells (5xl06 / 25 gm mouse) harvested from the femur and tibia of C57BL / 6J littermates, were washed, resuspended in culture medium, and delivered to irradiated mice by tail-vein injection. Anti-ceramide 6B5 scFv (150 pg / 25 gm mouse) wasadministered subcutaneously at 24 h post irradiation. Mice were followed for 90 days, and Kaplan Meier analysis used to evaluate survival. Agonal mice were sacrificed by CO2 asphyxiation. A 3-cm full circumference of jejunum was cut beginning at 10 cm from the Ligament of Treitz. Both femurs and tibial bones were collected from each mouse. Mice surviving until day 90 were subjected to complete necropsy and histopathological evaluation of all organs. Sections from all organs were stained with hematoxylin and eosin (H&E) and select organs (lung, liver, kidney) were also stained with Masson trichrome for collagen.

[0255] Complete Blood Count (CBC) and serological studies: CBC and serum chemistry profiles were evaluated for all mice on day 4 and 8 post irradiation. For mice surviving beyond 8 days, blood collection for CBC was performed on days 12, 28 and 90. An additional serum chemistry profile was performed on day 90. All CBC and serum chemistry testing was performed using ProCyte Dx Hematology Analyzer (IDEXX Laboratories, Westbrook, ME) & AU680 Clinical Chemistry Analyzer (Beckman Coulter Inc., Brea, CA).

[0256] Crypt rescue and bone marrow aplasia studies: Proximal jejunum and bone marrow specimens were collected, processed and stained as published (7). Tibial and femur samples were subjected to decalcification overnight. All samples were subjected to dehydration by exposure to ethanol and xylene mixture (1 : 1, v / v) for 30 min followed by embedding in paraffin. 5 -pm sections were stained with hematoxylin and eosin (H&E) and slides were cover slipped using mounting medium, allowed to dry overnight, scanned and images captured using CaseViewer software (3DHISTECH Ltd., Budapest, Hungary). Further, representative images were evaluated to determine extent of tissue damage and preservation.

[0257] Statistics: Stratified log-rank tests for statistical comparison of Kaplan Meier curves was performed using R software. P-values < 0.05 were considered significant.References

[0258] References listed infra correspond to reference numbering in text supra. All cited references, including those listed below are incorporated by reference in their entireties for all purposes.1. DiCarlo AL. Scientific research and product development in the united states to address injuries from a radiation public health emergency. J Radiat Res 2021;62:752-763.2. DiCarlo AL, Homer MJ, Coleman CN. United states medical preparedness for nuclear and radiological emergencies. J Radiol Prot 2021;41.3. Hendry JH, Potten CS, Roberts NP. The gastrointestinal syndrome and mucosal clonogenic cells: Relationships between target cell sensitivities, ld50 and cell survival, and their modification by antibiotics. RadiatRes 1983;96: 100-12.4. Hendry JH, Roberts SA, Potten CS. The clonogen content of murine intestinal crypts: Dependence on radiation dose used in its determination. RadiatRes 1992; 132: 115-9.5. Potten CS. A comprehensive study of the radiobiological response of the murine (bdfl) small intestine. Int J Radiat Biol 1990;58:925-73.6. Hua G, Wang C, Pan Y, et al. Distinct levels of radioresistance in lgr5(+) colonic epithelial stem cells versus lgr5(+) small intestinal stem cells. Cancer Res 2017;77:2124-2133.7. Martin ML, Adileh M, Hsu KS, et al. Organoids reveal that inherent radiosensitivity of small and large intestinal stem cells determines organ sensitivity. Cancer Res 2020;80: 1219- 1227.8. Paris F, Fuks Z, Kang A, et al. Endothelial apoptosis as 1 the primary lesion initiating intestinal radiation damage in mice. Science 2001;293:293-7.9. Rotolo J, Stancevic B, Zhang J, et al. Anti-ceramide antibody prevents the radiation gastrointestinal syndrome in mice. The Journal of clinical investigation 2012; 122: 1786-90.10. Rotolo JA, Kolesnick R, Fuks Z. Timing of lethality from gastrointestinal syndrome in mice revisited. Int J Radiat Oncol Biol Phys 2009;73:6-8.11. Rotolo JA, Fong CS, Bodo S, et al. Anti-ceramide single-chain variable fragment mitigates radiation gi syndrome mortality independent of DNA repair. JCI Insight 2021;6.12. Cassatt DR, Gorovets A, Karimi-Shah B, et al. A trans-agency workshop on the pathophysiology of radiation-induced lung injury. RadiatRes 2021.13. Wakabayashi T, Naito H, Suehiro JI, et al. Cdl57 marks tissue-resident endothelial stem cells with homeostatic and regenerative properties. Cell stem cell 2018;22:384-397. e6.14. Raveh-Amit H, Berzsenyi S, Vas V, et al. Tissue resident stem cells: Till death do us part. Biogerontology 2013;14:573-90.15. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249- 57.16. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996;86:353-64.17. Risau W. Mechanisms of angiogenesis. Nature 1997;386:671-4.18. Venkateswaran K, Shrivastava A, Agrawala PK, et al. Mitigation of radiation-induced gastro intestinal injury by the polyphenolic acetate 7, 8-diacetoxy-4-methylthiocoumarin in mice. Scientific Reports 2019;9: 14134.19. Romesser PB, Kim AS, Jeong J, et al. Preclinical murine platform to evaluate therapeutic countermeasures against radiation-induced gastrointestinal syndrome. Proceedings of the National Academy of Sciences 2019; 116:20672-20678.20. Booth C, Tudor G, Tudor J, et al. Acute gastrointestinal syndrome in high-dose irradiated mice. Health Phys 2012;103:383-99.21. Saha S, Bhanja P, Liu L, et al. Tlr9 agonist protects mice from radiation-induced gastrointestinal syndrome. PLOS ONE 2012;7:e29357.22. Saha S, Bhanja P, Kabarriti R, et al. Bone marrow stromal cell transplantation mitigates radiation-induced gastrointestinal syndrome in mice. PLOS ONE 201 l;6:e24072.23. Dong Y, Cheng Y, Hou Q, et al. The 1 protective effect of new compound xh-103 on radiation induced gi syndrome. OxidMed Cell Longev 2018;2018:3920147.24. Shao L, Yang W, Xu R, et al. Inhibition of mtorcl signaling protects kidney from irradiation4 induced toxicity via accelerating recovery of renal stem-like cells. Stem Cell Research & Therapy 2018,9:219.25. Sharma NK, Holmes-Hampton GP, Kumar VP, et al. Delayed effects of acute whole body lethal radiation exposure in mice pre-treated with bbt-059. Scientific Reports 2020; 10:6825.26. Marmary Y, Adar R, Gaska S, et al. Radiation-induced loss of salivary gland function is driven by cellular senescence and prevented by il6 modulation. Cancer Res 2016;76: 1170- 80.27. Kim J, Wang S, Hyun J, et al. Hedgehog signaling is associated with liver response to fractionated irradiation in mice. Cell Physiol Biochem 2016;40:263-276.28. Gugel M LK, White J, Monette S, Lipman N. Acute morbidity and mortality in highly immunocompromised mice following irradiation and hematopoietic cell line injection. . J Am Assoc Lab Anim Sci 2017;56:574-694.29. Kesari KK, Agarwal A, Henkel R. Radiations and male fertility. Reprod Biol Endocrinol 2018;16:l 18.30. Wang J, Albertson CM, Zheng H, et al. Short-term inhibition of adp-induced platelet aggregation by clopidogrel ameliorates radiation-induced toxicity in rat small intestine. Thromb Haemost 2002; 87: 122-8.31. Kim KT, Chae HS, Kim JS, et al. Thalidomide effect in endothelial cell of acute radiation proctitis. World J Gastroenterol 2008;14:4779-83.32. Kwak SY, Shim S, Park S, et al. Ghrelin reverts intestinal stem cell loss associated with radiation-induced enteropathy by activating notch signaling. Phytomedicine 2021;81 : 153424.33. Espinal A, Epperly MW, Mukherjee A, et al. Intestinal radiation protection and mitigation by second-generation probiotic lactobacillus-reuteri engineered to deliver interleukin-22. Int JMolSci 2022;23.34. Torres S, Thim L, Milliat F, et al. Glucagon-like peptide-2 improves both acute and late experimental radiation enteritis in the rat. IntJRadiat Oncol Biol Phys 2007;69: 1563-71.35. Burdelya LG, Krivokrysenko VI, Tailant TC, et al. An agonist of toll-like receptor has radioprotective activity in mouse and primate models. Science 2008;320:226-30.36. Keskek M, Gocmen E, Kilic M, et al. Increased expression of 1 cyclooxygenase-2 (cox- 2) in radiation-induced small bowel injury in rats. J Surg Res 2006;135:76-84.37. Ersin S, Tuncyurek P, Esassolak M, et al. The prophylactic and therapeutic effects of glutamine- and arginine-enriched diets on radiation-induced enteritis in rats. J Surg Res 2000;89: 121-5.38. Empey LR, Papp JD, Jewell LD, et al. Mucosal protective effects of vitamin e and misoprostol during acute radiation-induced enteritis in rats. Dig Dis Sci 1992;37:205-14.39. Wang J, Boerma M, Fu Q, et al. Simvastatin ameliorates radiation enteropathy development after localized, fractionated irradiation by a protein c-independent mechanism. Int J Radiat Oncol Biol Phys 2007;68: 1483-90.40. Deng W, Kimura Y, Gududuru V, et al. Mitigation of the hematopoietic and gastrointestinal acute radiation syndrome by octadecenyl thiophosphate, a small molecule mimic of lysophosphatidic acid. Radiat Res 2015;183:465-75.41. Zhang L, Sun W, Wang J, et al. Mitigation effect of an fgf-2 peptide on acute gastrointestinal syndrome after high-dose ionizing radiation. Int J Radiat Oncol Biol Phys 2010;77:261-8.42. Stenson WF. Prostaglandins and epithelial response to injury. Curr Opin Gastroenterol 2007;23: 107-10.43. Yoon SC, Park JM, JangHS, et al. Radioprotective effect of captopril on the mouse jejunal mucosa. Int J Radiat Oncol Biol Phys 1994;30:873-8.44. Ciorba MA, Stenson WF. Probiotic therapy in radiation-induced intestinal injury and repair. Ann N Y Acad Sci 2009; 1165:190-4.45. Patterson AM, Plett PA, Chua HL, et al. Development of a model of the acute and delayed effects of high dose radiation exposure in jackson diversity outbred mice; comparison to inbred c57bl / 6 mice. Health Phys 2020;119:633-646.46. Patterson AM, Vemula S, Plett PA, et al. Age and sex divergence in hematopoietic radiosensitivity in aged mouse models of the hematopoietic acute radiation syndrome. Radiat Res 2022;198:221-242.47. Patterson AM, Sellamuthu R, Plett PA, et al. Establishing pediatric mouse models of the hematopoietic acute radiation syndrome and the delayed effects of acute radiation exposure. Radiat Res 2021 ; 195 : 307-323.48. Chibly AM, Wong WY, Pier M, et al. Apkc^-dependent repression of yap is necessary for functional restoration of irradiated salivary glands with igf-1. Scientific Reports 33 2018;8:6347.49. Peng X, Wu Y, Brouwer U, et al. Cellular 1 senescence contributes to radiation-induced hyposalivation by affecting the stem / progenitor cell niche. Cell Death & Disease 2020;l l :854.50. Mizrachi A, Cotrim AP, Katabi N, et al. Radiation-induced microvascular injury as a mechanism of salivary gland hypofunction and potential target for radioprotectors. Radiat Res 2016;186: 189-95.51. Mezzaroma E, Di X, Graves P, et al. A mouse model of radiation-induced cardiomyopathy. Int J Cardiol 2012;156:231-3.52. Seemann I, Gabriels K, Visser NL, et al. Irradiation induced modest changes in murine cardiac function despite progressive structural damage to the myocardium and microvasculature. Radiother Oncol 2012;103: 143-50.53. Jackson IL, Vujaskovic Z, Down JD. Revisiting strain-related differences in radiation sensitivity of the mouse lung: Recognizing and avoiding the confounding effects of pleural effusions. Radiat Res 2010;173: 10-20.54. Ao X, Zhao L, Davis MA, et al. Radiation produces differential changes in cytokine profiles in radiation lung fibrosis sensitive and resistant mice. Journal of Hematology & Oncology 2009;2:6.55. Jackson IL, Xu P, Hadley C, et al. A preclinical rodent model of radiation-induced lung injury for medical countermeasure screening in accordance with the fda animal rule. Health Phys 2012;103:463-73.56. Hankey KG, Farese AM, Blaauw EC, et al. Pegfilgrastim improves survival of lethally irradiated nonhuman primates. Radiat Res 2015;183:643-55.57. Rondon G, Saliba RM, Khouri I, et al. Long-term follow-up of patients who experienced graft failure postallogeneic progenitor cell transplantation. Results of a single institution analysis. Biol Blood Marrow Transplant 2008;14:859-66.58. Wolff SN. Second hematopoietic stem cell transplantation for the treatment of graft failure, graft rejection or relapse after allogeneic transplantation. Bone Marrow Transplant 2002;29:545-52.59. Kong Y, Chang YJ, Wang YZ, et al. Association of an impaired bone marrow microenvironment with secondary poor graft function after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2013;19: 1465-73.60. Goyal S, Tisdale J, Schmidt M, et al. Acute myeloid leukemia case after gene therapy for sickle cell disease. N Engl J Med 2022;386: 138-147.61. Hourigan CS, Dillon LW, Gui G, et al. Impact of conditioning 1 intensity of allogeneic transplantation for acute myeloid leukemia with genomic evidence of residual disease. J Clin Oncol 2020;38: 1273-1283.62. Rotolo JA, Maj JG, Feldman R, et al. Bax and bak do not exhibit functional redundancy in mediating radiation-induced endothelial apoptosis in the intestinal mucosa. Int J Radiat Oncol Biol Phys 2008;70:804-15.FURTHER NUMBERED EMBODIMENTS

[0259] Further embodiments of the instant disclosure are provided in the numbered embodiments below:

[0260] Embodiment 1. A method of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population and a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof.

[0261] Embodiment 2. The method of Embodiment 1, wherein engraftment of the hematopoietic cell population is enhanced compared to a control engraftment not receiving the therapeutic agent.

[0262] Embodiment 3. The method of Embodiment 1 or 2, wherein step c) is performed after step b).

[0263] Embodiment 4. The method of Embodiment 3, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours after step b).

[0264] Embodiment 5. The method of Embodiment 3, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 5, 6, or 7 days after step b).

[0265] Embodiment 6. The method of any one of Embodiments 1-5, wherein the therapeutic agent is administered concurrently with the hematopoietic cell population in step c).

[0266] Embodiment 7. The method of any one of Embodiments 1-5, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours before the hematopoietic cell population in step c).

[0267] Embodiment 8. The method of any one of Embodiments 1-5, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours after the hematopoietic cell population in step c).

[0268] Embodiment 9. The method of any one of Embodiments 1-8, wherein the hematopoietic cell population is an autologous hematopoietic cell population.

[0269] Embodiment 10. The method of any one of Embodiments 1-9, wherein the hematopoietic cell population is an allogenic hematopoietic cell population.

[0270] Embodiment 11. The method of any one of Embodiments 1-10, wherein the conditioning in step b) comprises ablating the patient’s native bone marrow cell population.

[0271] Embodiment 12. The method of Embodiment 11, wherein the ablating of the patient’s native bone marrow cell population comprises marrow-ablative chemical treatment.

[0272] Embodiment 13. The method of Embodiment 11, wherein the ablating of the patient’s native bone marrow cell population comprises administration of a chemotherapeutic agent.

[0273] Embodiment 14. The method of Embodiment 13, wherein the chemotherapeutic agent is selected from the group consisting of chlorambucil, cyclophosphamide, ifosfamide, melphalan, streptozocin, carmustine, lomustine, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, 5 -fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, daunorubicin, doxorubicin, epirubicin, idarubicin, SN-38, camptothecin, topotecan, 9- nitrocamptothecin, 9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX- 895 If, MAG-CPT, amsacrine, etoposide, etoposide phosphate, teniposide, paclitaxel, and docetaxel.

[0274] Embodiment 15. The method of Embodiment 12, wherein the marrow-ablative chemical treatment is administration of an alkylating agent to the native bone marrow.

[0275] Embodiment 16. The method of Embodiment 15, wherein the alkylating agent is melphalan.

[0276] Embodiment 17. The method of any one of Embodiments 11-16, wherein the ablating of the patient’s native bone marrow cell population comprises administration of ablative radiation.

[0277] Embodiment 18. The method of Embodiment 17, wherein the ablative radiation is not administered to the patient’s intestines.

[0278] Embodiment 19. The method of Embodiment 17 or 18, wherein the ablative radiation is not sufficient to trigger Gastrointestinal Acute Radiation Syndrome (GI-ARS).

[0279] Embodiment 20. The method of Embodiment 17, wherein the ablative radiation comprises a dose of less than 13 Gy.

[0280] Embodiment 21. The method of any one of Embodiments 12-20, wherein the dose of marrow-ablative chemical treatment, the chemotherapeutic agent, or the ablative radiation is higher than that in a control engraftment not receiving the therapeutic agent comprising the anti-ceramide antibody or the antigen binding fragment thereof.

[0281] Embodiment 22. The method of any one of Embodiments 1-10, wherein the conditioning in step b) comprises reduced-intensity conditioning (RIC) or nonmyeloablative conditioning.

[0282] Embodiment 23. The method of Embodiment 21 or 22, wherein the method reduces the recurrence of the hematologic malignancy in the patient.

[0283] Embodiment 24. The method of any one of Embodiments 1-23, wherein engraftment is measured by patient’s white blood cell count returning to a pre-transplant level within 90 days post-transplant.

[0284] Embodiment 25. The method of any one of Embodiments 1-23, wherein engraftment is measured by patient’s white blood cell count of at least 5 K / uL within 90 days posttransplant.

[0285] Embodiment 26. The method of any one of Embodiments 1-23, wherein engraftment is measured by patient’s neutrophil count returning to a pre-transplant level within 90 days post-transplant.

[0286] Embodiment 27. The method of any one of Embodiments 1-23, wherein engraftment is measured by patient’s neutrophil blood cell count of at least 1 K / uL within 90 days posttransplant.

[0287] Embodiment 28. The method of any one of Embodiments 1-23, wherein engraftment is measured by patient’s platelet count returning to a pre-transplant level within 90 days posttransplant.

[0288] Embodiment 29. The method of any one of Embodiments 1-23, wherein engraftment is measured by patient’s platelet count of at least 300 K / uL within 90 days post-transplant.

[0289] Embodiment 30. The method of any one of Embodiments 1-23, wherein engraftment is measured by the patient surviving 30 days post-transplant.

[0290] Embodiment 31. A method of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient:(i) a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; and(ii) a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof.

[0291] Embodiment 32. The method of Embodiment 31, wherein the patient receiving the therapeutic agent and the reduced hematopoietic cell population exhibits a hematopoietic cell engraftment level that is higher than that of a control patient receiving the reduced hematopoietic cell population but not receiving the therapeutic agent.

[0292] Embodiment 33. The method of Embodiment 31 or 32, wherein the reduced hematopoietic cell population comprises no more than 1.5 million, no more than 1.2 million, no more than 1.0 million, no more than 0.9 million, no more than 0.8 million, no more than 0.7 million, no more than 0.6 million, or no more than 0.5 million, CD34+ hematopoietic stem cells per kilogram of the patient’s body weight.

[0293] Embodiment 34. The method of any one of Embodiments 32-33, wherein engraftment is measured by patient’s white blood cell count returning to a pre-transplant level within 90 days post-transplant.

[0294] Embodiment 35. The method of any one of Embodiments 32-33, wherein engraftment is measured by patient’s white blood cell count of at least 5 K / uL within 90 days posttransplant.

[0295] Embodiment 36. The method of any one of Embodiments 32-33, wherein engraftment is measured by patient’s neutrophil count returning to a pre-transplant level within 90 days post-transplant.

[0296] Embodiment 37. The method of any one of Embodiments 32-33, wherein engraftment is measured by patient’s neutrophil blood cell count of at least 1 K / uL within 90 days posttransplant.

[0297] Embodiment 38. The method of any one of Embodiments 32-33, wherein engraftment is measured by patient’s platelet count returning to a pre-transplant level within 90 days posttransplant.

[0298] Embodiment 39. The method of any one of Embodiments 32-33, wherein engraftment is measured by patient’s platelet count of at least 300 K / uL within 90 days post-transplant.

[0299] Embodiment 40. The method of any one of Embodiments 32-33, wherein engraftment is measured by the patient surviving 30 days post-transplant.

[0300] Embodiment 41. A method of improving engraftment of hematopoietic cells in a patient in need thereof, the method comprising administering a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof in addition to the administration of the hematopoietic cells.

[0301] Embodiment 42. The method of any one of Embodiments 1-41, wherein the patience has a chronically injured marrow substratum or suffers from a hemoglobinopathy.

[0302] Embodiment 43. The method of Embodiment 42, wherein the patience has sickle cell disease.

[0303] Embodiment 44. The method of any one of Embodiments 31-43, wherein the hematopoietic cell population administered to the patient is an autologous hematopoietic cell population.

[0304] Embodiment 45. The method of any one of Embodiments 31-43, wherein the hematopoietic cell population administered to the patient is an allogenic hematopoietic cell population.

[0305] Embodiment 46. A method of hematopoietic stem cell engraftment, said method comprising: administering to the patient a hematopoietic cell population and a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof; wherein the therapeutic agent is administered concurrently with or following administration of the hematopoietic cell population.

[0306] Embodiment 47. The method of Embodiment 46, wherein engraftment of the hematopoietic cell population is enhanced compared to a control engraftment not receiving the therapeutic agent.

[0307] Embodiment 48. The method of Embodiment 46 or 47, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours after administration of the hematopoietic cell population.

[0308] Embodiment 49. The method of Embodiment 46 or 47, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 5, 6, or 7 days after administration of the hematopoietic cell population.

[0309] Embodiment 50. The method of Embodiment 46 or 47, wherein the therapeutic agent is administered concurrently with the hematopoietic cell population.

[0310] Embodiment 51. The method of any one of Embodiments 46-50, wherein the hematopoietic cell population is an autologous hematopoietic cell population.

[0311] Embodiment 52. The method of any one of Embodiments 46-50, wherein the hematopoietic cell population is an allogenic hematopoietic cell population.

[0312] Embodiment 53. The method of any one of Embodiments 46-52, wherein engraftment is measured by patient’s white blood cell count returning to a pre-transplant level within 90 days post-transplant.

[0313] Embodiment 54. The method of any one of Embodiments 46-52, wherein engraftment is measured by patient’s white blood cell count of at least 5 K / uL within 90 days posttransplant.

[0314] Embodiment 55. The method of any one of Embodiments 46-52, wherein engraftment is measured by patient’s neutrophil count returning to a pre-transplant level within 90 days post-transplant.

[0315] Embodiment 56. The method of any one of Embodiments 46-52, wherein engraftment is measured by patient’s neutrophil blood cell count of at least 1 K / uL within 90 days posttransplant.

[0316] Embodiment 57. The method of any one of Embodiments 46-52, wherein engraftment is measured by patient’s platelet count returning to a pre-transplant level within 90 days posttransplant.

[0317] Embodiment 58. The method of any one of Embodiments 46-52, wherein engraftment is measured by patient’s platelet count of at least 300 K / uL within 90 days post-transplant.

[0318] Embodiment 59. The method of any one of Embodiments 46-52, wherein engraftment is measured by the patient surviving 30 days post-transplant.

[0319] Embodiment 60. The method of any one of Embodiments 1-59, wherein the method reduces the likelihood of graft versus host disease compared to a control method without the administration of the therapeutic agent comprising the anti-ceramide antibody or the antigen binding fragment thereof.

[0320] Embodiment 61. The method of any one of Embodiments 1-60, wherein the anti- ceramide antibody or the antigen binding fragment thereof comprises an immunoglobulin heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), and an immunoglobulin light chain variable region (VL) comprising a light chain complementarity determining region (CDR) 1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3), wherein:(a) the HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 1 and 2;(b) the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, and 6(c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7;(d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8;(e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and(f) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

[0321] Embodiment 62. The method of Embodiment 61, wherein the HCDR1 comprises SEQ ID NO: 1 and the HCDR2 comprises SEQ ID NO: 3.

[0322] Embodiment 63. The method of Embodiment 61 or 62, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 18 and the VL comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 22.

[0323] Embodiment 64. The method of any one of Embodiments 1-63, wherein the therapeutic agent is an anti-ceramide single chain variable fragment (scFv).

[0324] Embodiment 65. The method of any one of Embodiments 1-64, wherein the therapeutic agent is not for treating or preventing a Graft-versus-host disease (GVHD).

[0325] Embodiment 66. The method of any one of Embodiments 1-65, wherein the anticeramide antibody or the antigen binding fragment thereof is a bispecific antibody or a bispecific T cell engager (BiTE).

[0326] Embodiment 67. The method of any one of Embodiments 1-66, wherein the hematopoietic cell population comprises genetically engineered hematopoietic cells.

[0327] Embodiment 68. A composition comprising: a) a hematopoietic cell population graft; and b) an anti -ceramide antibody or the antigen binding fragment thereof.

[0328] Embodiment 69. The composition of Embodiment 68, wherein the hematopoietic cell population is an autologous hematopoietic cell population.

[0329] Embodiment 70. The composition of Embodiment 68, wherein the hematopoietic cell population is an allogenic hematopoietic cell population.

[0330] Embodiment 71. The composition of any one of Embodiments 68-70, wherein the composition is for administration to a patient, and wherein the hematopoietic cell population comprises fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight.

[0331] Embodiment 72. The composition of Embodiment 71, wherein the composition comprises no more than 1.5 million, no more than 1.2 million, no more than 1.0 million, no more than 0.9 million, no more than 0.8 million, no more than 0.7 million, no more than 0.6 million, or no more than 0.5 million, of CD34+ hematopoietic stem cells per kilogram of the patient’s body weight.

[0332] Embodiment 73. The composition of any one of Embodiments 68-72, wherein the antigen binding fragment of the anti-ceramide antibody is a single chain variable fragment (scFv).

[0333] Embodiment 74. A method of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; andc) administering to the patient a hematopoietic cell population; wherein the patient’s ceramide signaling pathway is blocked.

[0334] Embodiment 75. A method of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population; wherein endothelial cells in the patient are unable to form a ceramide-rich platform.

[0335] Embodiment 76. A method of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population; wherein endothelial cells in the patient are temporarily blocked from forming a ceramide-rich platform.

[0336] Embodiment 77. A method of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient: a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; wherein the patient’s ceramide signaling pathway is blocked.

[0337] Embodiment 78. A method of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; andb) administering to the patient: a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; wherein endothelial cells in the patient are unable to form a ceramide-rich platform.

[0338] Embodiment 79. A method of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient: a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; wherein endothelial cells in the patient are temporarily blocked from forming a ceramide-rich platform.

[0339] Embodiment 80. The method of any one of Embodiments 74-79, wherein blocking the ceramide signaling pathway or the ceramide-rich platform formation comprises administering an anti-ceramide therapeutic agent.

[0340] Embodiment 81. The method of Embodiment 80, wherein the anti-ceramide therapeutic agent comprises an anti-ceramide antibody, imipramine, a statin (e.g., nystatin), an antisense nucleic acid, a small interfering RNA (siRNA), or a combination thereof.

[0341] Embodiment 82. The method of Embodiment 81, wherein the antisense nucleic acid or the siRNA reduces expression of ASMase, Bak, or Bax.

[0342] Embodiment 83. The method of any one of Embodiments 74-82, wherein the blocking of the ceramide signaling pathway or the ceramide-rich platform (CRP) formation is evaluated by a 2-dimensional CRP incidence analysis or a 3-dimensional CRP volumetric analysis.

[0343] Sequence Listing of the Disclosure:

[0344] INCORPORATION BY REFERENCE

[0345] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

CLAIMS1. A method of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population and a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof.

2. The method of claim 1, wherein engraftment of the hematopoietic cell population is enhanced compared to a control engraftment not receiving the therapeutic agent.

3. The method of claim 1 or 2, wherein step c) is performed after step b).

4. The method of claim 3, wherein the therapeutic agent is administered at least 1, 2, 3,4. 6, 12, 18, or 24 hours after step b).

5. The method of claim 3, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 5, 6, or 7 days after step b).

6. The method of any one of claims 1-5, wherein the therapeutic agent is administered concurrently with the hematopoietic cell population in step c).

7. The method of any one of claims 1-5, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours before the hematopoietic cell population in step c).

8. The method of any one of claims 1-5, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours after the hematopoietic cell population in step c).

9. The method of any one of claims 1-8, wherein the hematopoietic cell population is an autologous hematopoietic cell population.

10. The method of any one of claims 1-9, wherein the hematopoietic cell population is an allogenic hematopoietic cell population.

11. The method of any one of claims 1-10, wherein the conditioning in step b) comprises ablating the patient’s native bone marrow cell population.

12. The method of claim 11, wherein the ablating of the patient’s native bone marrow cell population comprises marrow-ablative chemical treatment.

13. The method of claim 11, wherein the ablating of the patient’s native bone marrow cell population comprises administration of a chemotherapeutic agent.

14. The method of claim 13, wherein the chemotherapeutic agent is selected from the group consisting of chlorambucil, cyclophosphamide, ifosfamide, melphalan, streptozocin, carmustine, lomustine, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, 5- fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, daunorubicin, doxorubicin, epirubicin, idarubicin, SN-38, camptothecin, topotecan, 9-nitrocamptothecin, 9- aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, amsacrine, etoposide, etoposide phosphate, teniposide, paclitaxel, and docetaxel.

15. The method of claim 12, wherein the marrow-ablative chemical treatment is administration of an alkylating agent to the native bone marrow.

16. The method of claim 15, wherein the alkylating agent is melphalan.

17. The method of any one of claims 11-16, wherein the ablating of the patient’s native bone marrow cell population comprises administration of ablative radiation.

18. The method of claim 17, wherein the ablative radiation is not administered to the patient’s intestines.

19. The method of claim 17 or 18, wherein the ablative radiation is not sufficient to trigger Gastrointestinal Acute Radiation Syndrome (GI-ARS).

20. The method of claim 17, wherein the ablative radiation comprises a dose of less than 13 Gy.

21. The method of any one of claims 12-20, wherein the dose of marrow-ablative chemical treatment, the chemotherapeutic agent, or the ablative radiation is higher than that in a control engraftment not receiving the therapeutic agent comprising the anti-ceramide antibody or the antigen binding fragment thereof.

22. The method of any one of claims 1-10, wherein the conditioning in step b) comprises reduced-intensity conditioning (RIC) or nonmyeloablative conditioning.

23. The method of claim 21 or 22, wherein the method reduces the recurrence of the hematologic malignancy in the patient.

24. The method of any one of claims 1-23, wherein engraftment is measured by patient’s white blood cell count returning to a pre-transplant level within 90 days post-transplant.

25. The method of any one of claims 1-23, wherein engraftment is measured by patient’s white blood cell count of at least 5 K / uL within 90 days post-transplant.

26. The method of any one of claims 1-23, wherein engraftment is measured by patient’s neutrophil count returning to a pre-transplant level within 90 days post-transplant.

27. The method of any one of claims 1-23, wherein engraftment is measured by patient’s neutrophil blood cell count of at least 1 K / uL within 90 days post-transplant.

28. The method of any one of claims 1-23, wherein engraftment is measured by patient’s platelet count returning to a pre-transplant level within 90 days post-transplant.

29. The method of any one of claims 1-23, wherein engraftment is measured by patient’s platelet count of at least 300 K / uL within 90 days post-transplant.

30. The method of any one of claims 1-23, wherein engraftment is measured by the patient surviving 30 days post-transplant.

31. A method of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient:(i) a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; and(ii) a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof.

32. The method of claim 31, wherein the patient receiving the therapeutic agent and the reduced hematopoietic cell population exhibits a hematopoietic cell engraftment level that is higher than that of a control patient receiving the reduced hematopoietic cell population but not receiving the therapeutic agent.

33. The method of claim 31 or 32, wherein the reduced hematopoietic cell population comprises no more than 1.5 million, no more than 1.2 million, no more than 1.0 million, no more than 0.9 million, no more than 0.8 million, no more than 0.7 million, no more than 0.6 million, or no more than 0.5 million, CD34+ hematopoietic stem cells per kilogram of the patient’s body weight.

34. The method of any one of claims 32-33, wherein engraftment is measured by patient’ s white blood cell count returning to a pre-transplant level within 90 days post-transplant.

35. The method of any one of claims 32-33, wherein engraftment is measured by patient’ s white blood cell count of at least 5 K / uL within 90 days post-transplant.

36. The method of any one of claims 32-33, wherein engraftment is measured by patient’ s neutrophil count returning to a pre-transplant level within 90 days post-transplant.

37. The method of any one of claims 32-33, wherein engraftment is measured by patient’ s neutrophil blood cell count of at least 1 K / uL within 90 days post-transplant.

38. The method of any one of claims 32-33, wherein engraftment is measured by patient’ s platelet count returning to a pre-transplant level within 90 days post-transplant.

39. The method of any one of claims 32-33, wherein engraftment is measured by patient’ s platelet count of at least 300 K / uL within 90 days post-transplant.

40. The method of any one of claims 32-33, wherein engraftment is measured by the patient surviving 30 days post-transplant.

41. A method of improving engraftment of hematopoietic cells in a patient in need thereof, the method comprising administering a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof in addition to the administration of the hematopoietic cells.

42. The method of any one of claims 1-41, wherein the patience has a chronically injured marrow substratum or suffers from a hemoglobinopathy.

43. The method of claim 42, wherein the patience has sickle cell disease.

44. The method of any one of claims 31-43, wherein the hematopoietic cell population administered to the patient is an autologous hematopoietic cell population.

45. The method of any one of claims 31-43, wherein the hematopoietic cell population administered to the patient is an allogenic hematopoietic cell population.

46. A method of hematopoietic stem cell engraftment, said method comprising: administering to the patient a hematopoietic cell population and a therapeutic agent comprising an anti-ceramide antibody or the antigen binding fragment thereof; wherein the therapeutic agent is administered concurrently with or following administration of the hematopoietic cell population.

47. The method of claim 46, wherein engraftment of the hematopoietic cell population is enhanced compared to a control engraftment not receiving the therapeutic agent.

48. The method of claim 46 or 47, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 6, 12, 18, or 24 hours after administration of the hematopoietic cell population.

49. The method of claim 46 or 47, wherein the therapeutic agent is administered at least 1, 2, 3, 4, 5, 6, or 7 days after administration of the hematopoietic cell population.

50. The method of claim 46 or 47, wherein the therapeutic agent is administered concurrently with the hematopoietic cell population.

51. The method of any one of claims 46-50, wherein the hematopoietic cell population is an autologous hematopoietic cell population.

52. The method of any one of claims 46-50, wherein the hematopoietic cell population is an allogenic hematopoietic cell population.

53. The method of any one of claims 46-52, wherein engraftment is measured by patient’ s white blood cell count returning to a pre-transplant level within 90 days post-transplant.

54. The method of any one of claims 46-52, wherein engraftment is measured by patient’ s white blood cell count of at least 5 K / uL within 90 days post-transplant.

55. The method of any one of claims 46-52, wherein engraftment is measured by patient’ s neutrophil count returning to a pre-transplant level within 90 days post-transplant.

56. The method of any one of claims 46-52, wherein engraftment is measured by patient’ s neutrophil blood cell count of at least 1 K / uL within 90 days post-transplant.

57. The method of any one of claims 46-52, wherein engraftment is measured by patient’ s platelet count returning to a pre-transplant level within 90 days post-transplant.

58. The method of any one of claims 46-52, wherein engraftment is measured by patient’ s platelet count of at least 300 K / uL within 90 days post-transplant.

59. The method of any one of claims 46-52, wherein engraftment is measured by the patient surviving 30 days post-transplant.

60. The method of any one of claims 1-59, wherein the method reduces the likelihood of graft versus host disease compared to a control method without the administration of the therapeutic agent comprising the anti-ceramide antibody or the antigen binding fragment thereof.

61. The method of any one of claims 1-60, wherein the anti-ceramide antibody or the antigen binding fragment thereof comprises an immunoglobulin heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), and an immunoglobulin light chain variable region (VL) comprising a light chain complementarity determining region (CDR) 1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3), wherein:(a) the HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 1 and 2;(b) the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5, and 6;(c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 7;(d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 8;(e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 9; and(f) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

62. The method of claim 61, wherein the HCDR1 comprises SEQ ID NO: 1 and the HCDR2 comprises SEQ ID NO: 3.

63. The method of claim 61 or 62, wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 18 and theVL comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 22.

64. The method of any one of claims 1-63, wherein the therapeutic agent is an anticeramide single chain variable fragment (scFv).

65. The method of any one of claims 1 -64, wherein the therapeutic agent is not for treating or preventing a Graft-versus-host disease (GVHD).

66. The method of any one of claims 1-65, wherein the anti -ceramide antibody or the antigen binding fragment thereof is a bispecific antibody or a bispecific T cell engager (BiTE).

67. The method of any one of claims 1-66, wherein the hematopoietic cell population comprises genetically engineered hematopoietic cells.

68. A composition comprising: a) a hematopoietic cell population graft; and b) an anti -ceramide antibody or the antigen binding fragment thereof.

69. The composition of claim 68, wherein the hematopoietic cell population is an autologous hematopoietic cell population.

70. The composition of claim 68, wherein the hematopoietic cell population is an allogenic hematopoietic cell population.

71. The composition of any one of claims 68-70, wherein the composition is for administration to a patient, and wherein the hematopoietic cell population comprises fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight.

72. The composition of claim 71, wherein the composition comprises no more than 1.5 million, no more than 1.2 million, no more than 1.0 million, no more than 0.9 million, nomore than 0.8 million, no more than 0.7 million, no more than 0.6 million, or no more than 0.5 million, of CD34+ hematopoietic stem cells per kilogram of the patient’s body weight.

73. The composition of any one of claims 68-72, wherein the antigen binding fragment of the anti -ceramide antibody is a single chain variable fragment (scFv).

74. A method of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population; wherein the patient’s ceramide signaling pathway is blocked.

75. A method of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population; wherein endothelial cells in the patient are unable to form a ceramide-rich platform.

76. A method of hematopoietic stem cell engraftment, said method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; b) conditioning the patient’s native bone marrow cell population; and c) administering to the patient a hematopoietic cell population; wherein endothelial cells in the patient are temporarily blocked from forming a ceramide-rich platform.

77. A method of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; wherein the patient’s ceramide signaling pathway is blocked.

78. A method of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; wherein endothelial cells in the patient are unable to form a ceramide-rich platform.

79. A method of reducing a dose size of hematopoietic cells, the method comprising: a) providing and / or preparing a patient in need of a hematopoietic stem cell transplantation; and b) administering to the patient: a reduced hematopoietic cell population comprising fewer than 2 million CD34+ hematopoietic stem cells per kilogram of the patient’s body weight; wherein endothelial cells in the patient are temporarily blocked from forming a ceramide- rich platform.

80. The method of any one of claims 74-79, wherein blocking the ceramide signaling pathway or the ceramide-rich platform formation comprises administering an anti-ceramide therapeutic agent.

81. The method of claim 80, wherein the anti-ceramide therapeutic agent comprises an anti-ceramide antibody, imipramine, a statin (e.g., nystatin), an antisense nucleic acid, a small interfering RNA (siRNA), or a combination thereof.

82. The method of claim 81, wherein the antisense nucleic acid or the siRNA reduces expression of ASMase, Bak, or Bax.

83. The method of any one of claims 74-82, wherein the blocking of the ceramide signaling pathway or the ceramide-rich platform (CRP) formation is evaluated by a 2- dimensional CRP incidence analysis or a 3-dimensional CRP volumetric analysis.

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