Compositions and methods for the treatment of cancer

Abstract

Compositions for the prevention and treatment of cancer, such as colorectal cancer or diseases associated with abnormal levels of β-1,4-galactosyltransferase-V (β-1,4-GalT-V), include at least one inhibitor of glycosphingolipid synthesis.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 231,694, filed August 10, 2021, which is incorporated by reference herein in its entirety.

[0002] Embodiments relate to compositions that inhibit glycosphingolipid synthesis and their use in the treatment of cancer, such as colorectal cancer.

[0003] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with Government support under Grant No. HL107153 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0004] Colorectal cancer (CRC) affects more than 1.4 million people and causes more than 690,000 deaths worldwide (P.Favoriti,et al.,Worldwide burden of colorectal cancer: a review,Updates Surg.68(1)(2016)7-11.doi.org / 10.1007 / s13304-016-0359-yH Brenner,et al.,Colorectal cancer,Lancet.383(9927)(2014)1490-1502.doi.org / 10.1016 / S0140-6736(13)61649-9.Ferlay,I.et al.,Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012,Int.J.Cancer.136(5)(2015)E359-E386.doi.org / 10.1002 / ijc.29210.M.Arnold,et al.,Global patterns and trends in colorectal cancer incidence and mortality,Gut.66(4)(2017)683-691.doi.org / 10.1136 / gutjnl-2015-310912), and third in prevalence of all cancer types (H.Brenner,C.Stock,M.Hoffmeister,Colorectal cancer screening:the time to act is now,BMC Med.13(2015)262.doi.org / 10.1186 / s12916-015-0498-x).Current methods for early detection of CRC are limited in availability, poor patient compliance, and low test specificity (T. Tanaka, et al., Biomarkers for colorectal cancer, Int. J. Mol. Sci. 11(9)(2010)3209-3225. doi.org / 10.3390 / ijms11093209; S. Hundt, U. Haug, H. Brenner, Blood markers for early detection of colorectal cancer: a systematic review, Cancer Epidemiol. Biomarkers Prev. 16(10)(2007)1935-1953. doi.org / 10.1158 / 1055-9965. EPI-06-0994; K. Simon, V. Balchen, Colorectal cancer development and advances in screening. Clin. Interv. Aging. 11 (2016) 967-976. doi.org / 10.2147 / CIA.S109285. TF Imperiale, et al., Multitarget stool DNA testing for colorectal-cancer screening, N. Engl. J. Med. 370 (2014) 1287-1297. doi.org / 10.1056 / NEJMoa1311194). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] P. Favoriti, et al., Worldwide burden of colorectal cancer: a review, Updates Surg.68(1)(2016)7-11.doi.org / 10.1007 / s13304-016-0359-y. [Non-Patent Document 2] H.Brenner,et al.,Colorectal cancer,Lancet.383(9927)(2014)1490-1502.doi.org / 10.1016 / S0140-6736(13)61649-9. [Non-licensed document 3] Ferlay,I.et al.,Cancer incidence and mortality worldwide:sources,methods and major patterns in GLOBOCAN 2012,Int.J.Cancer.136(5)(2015)E359-E386.doi.org / 10.1002 / ijc.29210.

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[0006] Embodiments of the present invention relate to compositions comprising and methods of use of inhibitors of glycosphingolipid synthesis. [Means for solving the problem]

[0007] In a first embodiment, a humanized antibody capable of specifically binding to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope is provided.

[0008] The humanized antibodies of the present invention are particularly useful in the treatment of cancer, particularly cancers that overexpress GalT-V, such as colorectal cancer, renal cancer and neuroblastoma.

[0009] In a second aspect, a method of treating cancer comprises administering to a subject in need thereof a composition comprising a therapeutically effective amount of an antibody, wherein the antibody specifically binds to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope, the antibody being selected from the group consisting of: (i) EVQLEQSGAELARPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARW and / or (ii) a heavy chain variable region sequence having at least 80% amino acid sequence identity to SDYDYWGQGTTLTVSS (SEQ ID NO: 3), and / or (ii) a light chain variable sequence having at least 80% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4). Preferably, a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis is also administered to the subject.

[0010] In a third aspect, a method of treating cancer comprises administering to a subject in need thereof a composition comprising a therapeutically effective amount of an antibody, wherein the antibody specifically binds to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope, the antibody being selected from the group consisting of: (i) EVQLEQSGAELARPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGTTLTV SS (SEQ ID NO: 3), and / or (ii) a light chain variable sequence having at least 83, 84, 85, 86, or 87% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4). Preferably, a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis is also administered to the subject.

[0011] In a fourth aspect, the method of treating cancer comprises administering to a subject in need thereof a therapeutically effective amount of (a) an antibody that specifically binds to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope, the antibody comprising: (i) EVQLEQSGAELARPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGT and / or (ii) a heavy chain variable region sequence having at least 90% or 95% amino acid sequence identity to TLTVSS (SEQ ID NO: 3), and / or (iii) a light chain variable sequence having at least 90% or 95% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4). Preferably, a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis is also administered to the subject.

[0012] In certain embodiments, the antibody comprises a heavy chain variable region sequence having the amino acid sequence set forth in SEQ ID NO:3. In certain embodiments, the antibody comprises a light chain variable region sequence having the amino acid sequence set forth in SEQ ID NO:4. In certain embodiments, the pharmaceutical composition further comprises one or more secondary therapeutic agents. In certain embodiments, the one or more secondary therapeutic agents comprise a chemotherapeutic agent, an anti-inflammatory agent, a cholesterol-lowering agent, insulin, an antibody, a peptide, an enzyme, an adjuvant, or a combination thereof. In certain embodiments, the pharmaceutical composition further comprises conjugating the antibody to a detectable agent, a radiotherapeutic agent, a toxin, a radioactive agent, a dye, a peptide, a polynucleotide, or a nanoliposome. In certain embodiments, the nanoliposome comprises the therapeutic agent(s). In certain embodiments, the pharmaceutical composition further comprises a peptide having at least 90% sequence identity to IGAQVYEQVLRSAYAKRNSSVND (SEQ ID NO:5).

[0013] In a fifth aspect, the pharmaceutical composition comprises a therapeutically effective amount of: (i) an antibody comprising (a) a heavy chain variable region sequence nucleic acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:3, and (b) a light chain variable region sequence nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:2, and / or (ii) a synthetic peptide comprising an amino acid sequence having at least 80%, 85%, 90% or 95% amino acid sequence to SEQ ID NO:5. In certain embodiments, the pharmaceutical composition may also comprise one or more adjuvants and / or one or more pharma- ceutically acceptable carriers. In certain embodiments, the antibody comprises (a) a heavy chain variable region nucleic acid sequence comprising SEQ ID NO:3, and / or (b) a light chain variable region nucleic acid sequence comprising SEQ ID NO:2, and / or a synthetic peptide amino acid sequence comprising SEQ ID NO:5.

[0014] In a sixth aspect, the pharmaceutical composition comprises a therapeutically effective amount of (a) an antibody that specifically binds to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope, the antibody having at least one of the following structure: (i) EVQLEQSGAELARPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGTTLTVSS (SEQ ID NO: 3). and / or (ii) a heavy chain variable region sequence having at least 80%, 85%, 90% or 95% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4). In certain preferred embodiments, the pharmaceutical composition may also comprise a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis. In certain embodiments, the antibody comprises a heavy chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the antibody comprises a light chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the at least one inhibitor of glycosphingolipid synthesis comprises D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), (1R,2R)-nonanoic acid (2-(2',3-dihydro-benzo(1,4)dioxin-6'-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl)-amide-L-tartrate (Genz-123346), imido sugar, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DMP), 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), lipid, ceramide, or combinations thereof, either unencapsulated or encapsulated by a biodegradable polymer.In certain embodiments, the inhibitor of glycosphingolipid synthesis is D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), either unencapsulated or encapsulated in a biodegradable polymer (BPD). In certain embodiments, the biodegradable polymer is comprised of polyethylene glycol and sebacic acid. In certain embodiments, the poly(amidoamine) dendrimer-based nanoplatform coupled to the antibody, D-PDMP peptide disclosed herein is useful in cancer detection and targeted therapy.

[0015] In a seventh embodiment, the antibody that specifically binds to the β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope is humanized. In certain embodiments, the antibody comprises (i) a heavy chain variable region sequence having at least 80%, 85%, 90% or 95% amino acid sequence identity to EVQLEQSGAELARPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGTTLTVSS (SEQ ID NO: 3), and / or (ii) a light chain variable sequence having at least 80%, 85%, 90% or 95% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4).

[0016] In an eighth aspect, the pharmaceutical composition comprises a therapeutically effective amount of a synthetic peptide comprising an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO:5, preferably at least one adjuvant, or at least one other pharma- ceutically acceptable carrier. In a particular embodiment, the synthetic peptide comprises SEQ ID NO:5.

[0017] In a ninth aspect, the expression vector comprises a heavy chain variable region sequence nucleic acid sequence having at least 80%, 85%, 90% or 95% sequence identity to gaagttcagctggagcagtctggggctgaactggctagacctggggcttcagtgaagttgtcctgtaggacttctggctacacctttacaaactactggatgcagtggattaaacagaggcctggacagggtctggaatggattggggctatgcatcctggacgtgcgtatattaggtacaaccagaagttccagggcaaggccacattgactgcagataaatcctccagcacagcttacatgcaactcaacagcttggcatctgaggactctgcggtctattactgtgcaagatggagtgactacgactactggggtcaaggcaccactctcacagtctcctca (SEQ ID NO: 1). In a specific embodiment, the vector comprises the nucleic acid sequence shown in SEQ ID NO: 1.

[0018] In a tenth embodiment, the expression vector comprises a light chain variable region sequence nucleic acid sequence having at least 80%, 85%, 90% or 95% sequence identity to gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactgggctctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttcctcggacgttcggtggaggcaccaagctggaaatcaaacgg (SEQ ID NO: 2). In a specific embodiment, the vector comprises the nucleic acid sequence shown in SEQ ID NO:2.

[0019] In an eleventh aspect, the expression vector comprises (i) a heavy chain variable region sequence nucleic acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:3, and / or (ii) a light chain variable region sequence nucleic acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:2.

[0020] In a twelfth embodiment, the expression vector comprises (i) a heavy chain variable region sequence nucleic acid sequence comprising SEQ ID NO:3, and (ii) a light chain variable region sequence nucleic acid sequence comprising SEQ ID NO:2.

[0021] In a thirteenth aspect, the synthetic peptide comprises an amino acid sequence having at least 80%, 85%, 90% or 95% of the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the synthetic peptide comprises SEQ ID NO:5.

[0022] In a fourteenth aspect, a method for generating an immune response against β-1,4-galactosyltransferase-V (β-1,4-GalT-V) in a subject in need thereof comprises administering a therapeutically effective amount of a synthetic peptide comprising an amino acid sequence having at least 80%, 85%, 90% or 95% amino acid sequence of SEQ ID NO:5, preferably together with an adjuvant or a pharma- ceutically acceptable carrier.

[0023] In a fifteenth aspect, a method of treating colorectal cancer comprises administering to a subject a pharmaceutical composition comprising an antibody comprising (a) a heavy chain variable region sequence nucleic acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:3, and / or (b) a light chain variable region sequence nucleic acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:2. In certain embodiments, the antibody comprises (a) a heavy chain variable region nucleic acid sequence comprising SEQ ID NO:3, and (b) a light chain variable region nucleic acid sequence comprising SEQ ID NO:2. In certain embodiments, the method further comprises administering a therapeutically effective amount of a synthetic peptide comprising an amino acid sequence having at least 80%, 85%, 90% or 95% amino acid sequence identity to SEQ ID NO:5, preferably together with at least one adjuvant or pharma- ceutically acceptable carrier. In certain embodiments, the synthetic peptide comprises SEQ ID NO:5. In certain embodiments, the method further comprises administering an anti-cancer agent, such as a chemotherapeutic agent, radiation therapy, a toxin, or a combination thereof. In certain embodiments, the anti-cancer agent is a chemotherapeutic agent or growth inhibitory agent, a targeted therapeutic agent, a T cell expressing a chimeric antigen receptor, an antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, an anti-neoplastic agent, a cancer vaccine, an adjuvant, and combinations thereof. In certain embodiments, the anti-cancer agent is a chemotherapeutic agent or growth inhibitory agent. For example, the chemotherapeutic agent or growth inhibitory agent may include alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, Lyn kinase inhibitors, Src kinase inhibitors, VEGF-R1 R2 inhibitors, EGF-R inhibitors, GSK-alpha kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents or antitumor antibiotics, proteasome inhibitors, anti-microtubule agents, EGFR antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof.

[0024] In certain embodiments, the anti-cancer agent is an adjuvant. Any substance that enhances an anti-cancer immune response, such as against a cancer-associated antigen, or aids in the presentation of cancer antigens to components of the immune system, may be considered an anti-cancer adjuvant of the present disclosure. In certain embodiments, the method further comprises administering at least one inhibitor of glycosphingolipid synthesis comprising D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), (1R,2R)-nonanoic acid (2-(2',3-dihydro-benzo(1,4)dioxin-6'-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl)-amide-L-tartrate (Genz-123346), an imido sugar, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DMP), 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), a lipid, a ceramide, or a combination thereof, either unencapsulated or encapsulated by a biodegradable polymer. In certain embodiments, the inhibitor of glycosphingolipid synthesis is D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) (e.g., including D-PDMP that may be mixed with a biodegradable polymer, e.g., unencapsulated or encapsulated in a biodegradable polymer (BPD)). In certain embodiments, the biodegradable polymer is comprised of polyethylene glycol and sebacic acid.

[0025] In a sixteenth aspect, a method for treating diabetes, atherosclerosis, obesity, an autoimmune disease, or a disease associated with abnormal levels of β-1,4-galactosyltransferase-V (β-1,4-GalT-V), such as systemic lupus erythematosus (SLE), renal cancer, lung cancer, melanoma, neuroblastoma, glioblastoma, lung, cancer, liver cancer, comprises administering to a subject in need thereof a pharmaceutical composition embodied herein; an expression vector embodied herein; a synthetic peptide embodied herein; or a combination thereof.

[0026] In a seventeenth aspect, a method of diagnosing and treating colorectal cancer comprises measuring the level of β-1,4-galactosyltransferase-V (β-1,4-GalT-V) and / or glycosphingolipids in a biological sample of a subject, where increased levels of β-1,4-GalT-V and / or GSLs are elevated when compared to healthy subjects, and where increased levels of β-1,4-GalT-V and / or GSLs are diagnostic of colorectal cancer, and administering to said subject diagnosed with colorectal cancer a pharmaceutical composition embodied herein; an expression vector embodied herein; a synthetic peptide embodied herein; or a combination thereof. In certain embodiments, the method further comprises measuring the level of a colorectal cancer tumor marker in combination with the level of β-1,4-GalT-V and / or GSLs. In certain embodiments, colorectal cancer tumor markers include NMT-1, APC, p53, NOTCH-1, B-CATENIN, and combinations thereof.

[0027] In an eighteenth embodiment, a method of monitoring tumor progression (including rectal or colorectal tumors or cancers) using fluorescently tagged GalT-V antibodies or GalT-V antibodies tagged with radioisotopes (e.g., [I],

[89] Zr, and other gamma emitting isotopes), or CF-750 tagged GATT-V antibodies.

[0028] In treating colorectal cancer in a subject, the treatment may include administering to a subject diagnosed with colorectal cancer a pharmaceutical composition disclosed herein; an expression vector embodied herein; or a synthetic peptide embodied herein, and measuring the levels of β-1,4-galactosyltransferase-V (β-1,4-GalT-V) and / or glycosphingolipids in the subject's biological sample, where a decrease in β-1,4-GalT-V and / or GSL levels (e.g., using fluorescently tagged glycosphingolipid antibodies) compared to baseline indicates a reduction in colorectal cancer cells and treatment of colorectal cancer. In certain embodiments, the dose of the composition administered to the subject is adjusted based on the progression of colorectal cancer.

[0029] In certain embodiments, the cancer being treated or monitored is Dukes' B (stage II) or Dukes' C (stage III) colorectal cancer.

[0030] In yet a further aspect, a method is provided for treating a patient suffering from or susceptible to macular degeneration, comprising administering to the subject an effective amount of one or more pharmaceutical compositions, peptides and / or expression vectors (including combinations thereof) disclosed herein. In certain aspects, the subject can be identified as suffering from macular degeneration, and one or more pharmaceutical compositions, peptides and / or expression vectors disclosed herein are administered to the identified subject. In certain preferred embodiments, the subject is a human.

[0031] In an additional aspect, a method is provided for treating a patient suffering from or susceptible to Alzheimer's disease, comprising administering to the subject an effective amount of one or more pharmaceutical compositions, peptides and / or expression vectors disclosed herein (including combinations thereof). In certain aspects, the subject may be identified as suffering from Alzheimer's disease, and one or more pharmaceutical compositions, peptides or expression vectors disclosed herein are administered to the identified subject. In certain preferred embodiments, the subject is a human.

[0032] In a further aspect, there is provided a method for treating a patient suffering from or susceptible to migraine headache or migraine pain, comprising administering to the subject an effective amount of one or more pharmaceutical compositions, peptides and / or expression vectors disclosed herein (including combinations thereof). In certain aspects, the subject may be identified as suffering from migraine headache or migraine pain, and one or more pharmaceutical compositions, peptides and / or expression vectors disclosed herein are administered to the identified subject. In certain preferred embodiments, the subject is a human.

[0033] In yet a further aspect, there is provided a method for treating a patient suffering from or susceptible to metabolic syndrome, comprising administering to the subject an effective amount of one or more pharmaceutical compositions, peptides and / or expression vectors disclosed herein (including combinations thereof). In certain aspects, the subject may be identified as suffering from metabolic syndrome, and one or more pharmaceutical compositions, peptides and / or expression vectors disclosed herein are administered to the identified subject. In certain preferred embodiments, the subject is a human.

[0034] Other aspects are described below.

[0035] definition The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the present invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising."

[0036] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value or range. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold or even within 2-fold, of a value. When a particular value is described in the present application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0037] The term "adjuvant" has its usual meaning in the field of vaccine technology, i.e., a substance or composition that 1) is not capable of initiating a specific immune response to the immunogen of a vaccine by itself, but 2) is nevertheless capable of enhancing the immune response to the immunogen. Or, in other words, vaccination with an adjuvant alone does not result in an immune response to the immunogen, vaccination with an immunogen may or may not result in an immune response to the immunogen, but vaccination with a combination of an immunogen and an adjuvant induces a stronger immune response to the immunogen than that induced by the immunogen alone.

[0038] As used herein, the term "administering" refers to any manner of transferring, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such agent, including, but not limited to, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

[0039] As used herein, the term "agent" is meant to encompass any molecule, chemical entity, composition, drug, therapeutic, chemotherapeutic, or biological agent capable of modulating the expression or activity of β1,4-galactosyltransferase V (BGA). This term includes small molecule compounds, antisense oligonucleotides, siRNA reagents, antibodies, Fab, Fab', F(ab')2 fragments, Fv fragments, single chain antibodies, antibody mimetics (e.g., DARPins, affibody molecules, affilins, affitins, anticalins, avimers, finomers, Kunitz domain peptides, and monobodies), antibody fragments having epitope recognition sites such as peptoids, aptamers, enzymes, peptide organic or inorganic molecules, natural or synthetic compounds, and the like. Agents can be assayed according to the methods of the present invention during clinical trials, preclinical trials, or at any stage after FDA approval.

[0040] As used herein, the term "antibody" includes all species, including human and humanized antibodies, and the antigenic target may be from any species. Thus, for example, an antibody that binds to an antigen "X" may be mouse anti-human X, human anti-human X; humanized anti-human X, goat anti-human X; goat anti-mouse X; rat anti-human X; mouse anti-rat X, etc. The combination of antibodies generated against a particular species against an antigenic target from another species, such as "X", or even against the same species (e.g., in an autoimmune or inflammatory response), is not limited and all species are embodied in the present invention. The term antibody is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies (including human antibodies, humanized antibodies, or chimeric antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab', F'(ab)2, Fv, single chain antibodies, diabodies) capable of binding to an antigen, comprising the aforementioned complementarity determining regions (CDRs), so long as they exhibit the desired biological activity. Examples of bispecific antibodies include a combination of a GalT-V antibody with another antibody, such as lactosulceramide, Lyn kinase, Src kinase, VEGF-R1, R2, EGF-R GSK-alpha kinase.

[0041] "Antisense oligonucleotide" or "antisense compound" refers to an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide, it binds to another RNA target by RNA-RNA interaction and changes the activity of the target RNA. An antisense oligonucleotide can upregulate or downregulate the expression and / or function of a particular polynucleotide. The definition is meant to include any foreign RNA or DNA molecule that is useful from a therapeutic, diagnostic, or other standpoint. Such molecules include, for example, antisense RNA or antisense DNA molecules, interfering RNA (RNAi), microRNA, decoy RNA molecules, siRNA, enzymatic RNA, short hairpin RNA (shRNA), therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of a target nucleic acid. Thus, these compounds can be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

[0042] As used in this specification and the appended claims, the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise.

[0043] As used herein, the term "chemotherapeutic agent" consistent with its use in the art refers to one or more agents known to treat cancer or known to contribute to the treatment of cancer or having known characteristics. In particular, chemotherapeutic agents include proapoptotic agents, cytostatic agents and / or cytotoxic agents. In some embodiments, the chemotherapeutic agent may be or may include alkylating agents, anthracyclines, cytoskeletal disrupting agents (e.g., the following microtubule targeting moieties, e.g., taxanes, maytansine and analogs thereof), epothilones, histone deacetylase inhibitors (HDACs), topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), kinase inhibitors, nucleotide analogs or nucleotide precursor analogs, peptide antibiotics, platinum-based drugs, retinoids, vinca alkaloids, and / or analogs that share related antiproliferative activity. In some embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, aviristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hirubin, The chemotherapeutic agent may be or may include droxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. In some embodiments, the chemotherapeutic agent may be utilized in the context of an antibody-drug conjugate.In some embodiments, the chemotherapeutic agent is hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumumab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-SME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mafodotin, and lorvotuzumab mertansine.

[0044] As used herein, the term "combination therapy" refers to a situation in which two or more different pharmaceutical agents are administered in an overlapping regimen, such that the subject is exposed to both agents simultaneously. When used in combination therapy, two or more different agents can be administered simultaneously or separately. This administration in combination can include simultaneous administration of two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more agents can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more agents can be administered simultaneously, where the agents are in separate formulations. In another embodiment, one or more additional agents can be administered after administration of a first agent. In separate administration protocols, two or more agents can be administered minutes apart, or hours apart, or days apart.

[0045] As used herein, the terms "comprising," "comprise," or "comprised," and variations thereof, with respect to defined or described elements, such as an item, composition, apparatus, method, process, system, etc., are meant to be inclusive or open-ended, allowing for additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements, or equivalents thereof, as appropriate, and that other elements may be included and fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0046] As used herein, the term "diagnosis" refers to determining whether a subject has or will develop a disease, disorder, symptom, or condition, and / or a qualitative determination of a quantitative probability. For example, in diagnosing cancer, diagnosis can include a determination regarding the risk, type, stage, grade, or other classification of cancer. In some cases, for example, as described herein, diagnosis can be or include a determination regarding prognosis and / or likely response to one or more general or specific therapeutic agents or regimens.

[0047] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and the animal's health continues to deteriorate if the disease is not improved. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but the animal's health is less favorable than it would be without the disorder. Left untreated, the disorder does not necessarily cause further deterioration of the animal's health. A disease or disorder is "alleviated" when the severity of the symptoms of the disease or disorder, the frequency with which such symptoms are experienced by the patient, or both, are reduced.

[0048] A "dosing regimen" (or "therapeutic regimen"), as that term is used herein, is a set of unit doses (typically two or more) that are administered individually to a subject, typically separated by a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may include one or more doses. In some embodiments, the dosing regimen includes multiple doses, each separated from the other by the same length of time, and in some aspects, the dosing regimen includes multiple doses and at least two different periods that separate the individual doses. In some embodiments, the dosing regimen is or has correlated with a desired therapeutic outcome when administered across a population of patients. As used herein, a "controlled release dosage formulation" refers to a formulation of a drug that provides an extended release at a specific, controllable rate.

[0049] By "effective amount" is meant the amount required to improve the symptoms of a disease compared to an untreated patient. The effective amount of active compound(s) used to practice the present invention for the therapeutic treatment of a disease will vary depending on the mode of administration, the age, weight and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen. Such an amount is referred to as an "effective" amount. The determination of a therapeutically effective amount, as well as other factors related to the effective administration of the compounds of the present invention to a subject of the present invention, including dosage form, route of administration, and frequency of administration, may depend on the specifics of the condition encountered, including the subject and condition being treated or addressed, the severity of the condition in the particular subject, the particular compound being used, the particular route of administration being used, the frequency of administration, and the particular formulation being used. The determination of a therapeutically effective treatment regimen for a subject of the present invention is within the level of ordinary skill in the medical or veterinary field. In clinical use, the effective amount may be the amount recommended by the U.S. Food and Drug Administration or an equivalent foreign agency. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration.

[0050] "High affinity" for an antibody means a K D is 1×10 -7M or less, preferably 5×10 -8 M or less, more preferably 1×10 -8 M or less, and even more preferably 5×10 -9 M or less, more preferably 1×10 -9 "High affinity" binding refers to an antibody with a binding affinity of 10 M or less. However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is defined as an antibody with a binding affinity of 10 -6 M or less, 10 -7 M or less, or 10 -8 K below M D It refers to an antibody having the following structure:

[0051] The terms "enhancement," "enhance," "enhances," or "enhancing" refer to an increase in a particular parameter (e.g., an increase of at least about 1.1-fold, 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, or even 15-fold or more) and / or an increase in a particular activity of at least about 5%, 10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%.

[0052] As used herein, the term "in combination" in the context of administering a treatment to a subject refers to the use of two or more treatments for therapeutic benefit.The term "in combination" in the context of administering can also refer to the prophylactic use of a treatment to a subject when used with at least one additional treatment.The use of the term "in combination" does not limit the order in which treatments (e.g., a first and a second treatment) are administered to a subject. The treatment can be administered prior to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously, or subsequently (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the second treatment to a subject who has had, has, or is susceptible to cancer. The treatments are administered to the subject within a series of time intervals so that the therapies can act together. In certain embodiments, the treatments are administered to a subject within a series of time intervals that provide a greater benefit than if they were administered otherwise. Any additional treatments can be administered in any order with other further treatments.

[0053] As used herein, an "inhibitor" of glycosphingolipid synthesis or glucosylceramide synthesis inhibits the synthesis of these molecules, including those involved in the synthesis cycle. Inhibition of the synthesis of these molecules can be measured by any standard assay. See, for example, the methods in the Examples section below.

[0054] As used herein, "inhibition" or "reduction" of β1,4-galactosyltransferase V refers to reducing the amount of β1,4-galactosyltransferase V in a cell by more than about 20%, 40%, 60%, 80%, 85%, 90%, 95%, or 100%. The amount of β1,4-galactosyltransferase V can be determined by well-known methods, including, but not limited to, densitometer, fluorometer, radiography, luminometer, antibody-based methods, and activity measurements.

[0055] The terms "inhibit," "attenuate," "reduce," or "suppress" refer to a decrease in a particular parameter (e.g., an increase of at least about 1.1-fold, 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, or even 15-fold or more) and / or a decrease or reduction of a particular activity by at least about 5%, 10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%. These terms are intended to be relative to a reference or control.

[0056] As used herein, "K assoc " or "K a " is intended to refer to the association rate of a particular antibody-antigen interaction, whereas as used herein, "K dis " or "K d As used herein, the term "K" is intended to refer to the off-rate of a particular antibody-antigen interaction. D The term "K d Against K a (i.e., K d / K a ) and expressed as a molar concentration (M). D Values ​​can be determined using methods well established in the art. D A preferred method for determining is by using surface plasmon resonance, for example, by using a biosensor system such as a BIACORE™ system.

[0057] As used herein, "modulate", "modulates" or "modulation" refers to enhancing (e.g., increasing) or inhibiting (e.g., attenuating, decreasing or suppressing) a particular activity or level (e.g., amount of mRNA, amount of protein, expression of a marker, amount of GSL, etc.). The level determined compared to a control level can be an increased level. As used herein, the term "increased" with respect to a level (e.g., protein or mRNA level) refers to any % increase over a control level. In various embodiments, the increased level may be at least or about 5% increase, at least or about 10% increase, at least or about 15% increase, at least or about 20% increase, at least or about 25% increase, at least or about 30% increase, at least or about 35% increase, at least or about 40% increase, at least or about 45% increase, at least or about 50% increase, at least or about 55% increase, at least or about 60% increase, at least or about 65% increase, at least or about 70% increase, at least or about 75% increase, at least or about 80% increase, at least or about 85% increase, at least or about 90% increase, at least or about 95% increase, compared to the control level. The level determined may be a decreased level. As used herein, the term "decreased" with respect to a level (e.g., protein or mRNA level) refers to any % decrease below the control level. In various embodiments, a decreased level can be at least or about a 5% decrease, at least or about a 10% decrease, at least or about a 15% decrease, at least or about a 20% decrease, at least or about a 25% decrease, at least or about a 30% decrease, at least or about a 35% decrease, at least or about a 40% decrease, at least or about a 45% decrease, at least or about a 50% decrease, at least or about a 55% decrease, at least or about a 60% decrease, at least or about a 65% decrease, at least or about a 70% decrease, at least or about a 75% decrease, at least or about a 80% decrease, at least or about a 85% decrease, at least or about a 90% decrease, or at least or about a 95% decrease compared to a control level.

[0058] The terms "prevent" and "prevention" as used herein in relation to the occurrence of a disease, disorder, or condition refer to reducing the risk of developing a disease, disorder, or condition; delaying the onset of a disease, disorder, or condition; delaying the onset of one or more characteristics or symptoms of a disease, disorder, or condition; and / or reducing the frequency and / or severity of one or more characteristics or symptoms of a disease, disorder, or condition. Prevention can refer to prevention in a particular subject or a statistical effect on a population of subjects. Prevention can be considered complete if the onset of a disease, disorder, or condition has been delayed for a predetermined period of time.

[0059] As used herein, the term "prognosis" refers to a qualitative determination of the quantitative probability of at least one possible future outcome or event. As used herein, a prognosis can be a determination of the likely course of a disease, disorder, or condition, such as cancer, in a subject, a determination regarding a subject's life expectancy, or a determination regarding a treatment, such as a response to a particular treatment.

[0060] As used herein, the term "prognostic information" refers to information useful for providing a prognosis. Prognostic information may include, but is not limited to, biomarker status information.

[0061] As used herein, the term "sample" refers to a biological sample obtained for the purpose of in vitro evaluation. In embodiments, the sample may include a bodily fluid. In some embodiments, the bodily fluid includes, but is not limited to, whole blood, plasma, serum, lymph, breast milk, saliva, mucus, semen, cell extract, inflammatory fluid, cerebrospinal fluid, vitreous fluid, tears, vitreous humor, aqueous humor, or urine obtained from a subject. In some embodiments, the sample is a composite panel of two or more bodily fluids. In exemplary embodiments, the sample includes blood or a fraction thereof (e.g., plasma, serum, or a fraction obtained by leukapheresis).

[0062] As used herein, the terms "prevent," "preventing," and "prevention" in the context of administration of a therapy to a subject refer to the prevention or inhibition of the recurrence, onset, and / or development of a disease or disorder, or a symptom thereof, in a subject resulting from the administration of a therapy (e.g., a prophylactic agent) or combination of therapies (e.g., a combination of prophylactic agents).

[0063] By "reduce" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100% compared to a reference.

[0064] "Reference" means a standard or control condition.

[0065] As used herein, an antibody that "specifically binds" to a polypeptide or epitope has a binding capacity of 1×10 -7 M or less, or 5 x 10 -8 M or less, or 3 x 10 -8 M or less, preferably 1×10 -8 M or less, or 5 x 10 -9 K below M D The term "specific binding" or "specifically binds" when used in reference to the interaction of a protein with an antibody or surrogate protein scaffold or peptoid or aptamer means that the interaction is dependent on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the protein; in other words, the antibody recognizes and binds to a particular protein structure, rather than the protein in general. Thus, an antibody that "specifically binds" or is "specific for" a particular polypeptide or epitope on a particular polypeptide is an antibody that binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0066] As used herein, a "sustained release dosage formulation" is a formulation of a drug designed to release the drug at a predetermined rate to maintain a constant drug concentration for a specified period of time with minimal side effects. Optionally, the period is 30 minutes or more, e.g., 2-4 hours or more, e.g., 3-8 hours or more, e.g., 4-24 hours, or for example, 1-3 days or more, e.g., 2-7 days or more, e.g., 4-14 days or more, e.g., 7 days or more, e.g., 14 days to 1 month or more.

[0067] As used herein, "treating" or "treatment" of a condition, disease or disorder, or symptoms associated with a condition, disease or disorder, refers to an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or symptoms, whether partial or total, attenuation of the severity of a condition, disorder or disease, stabilization of the condition of a condition, disorder or disease, prevention of the onset of a condition, disorder or disease, prevention of the progression of a condition, disorder or disease, delay or slowing of the progression of a condition, disorder or disease, delay or slowing of the onset of a condition, disorder or disease, improvement or alleviation of the condition of a condition, disorder or disease, and remission. "Treating" may also mean inhibiting the progression of a condition, disorder or disease, slowing the progression of a condition, disorder or disease temporarily, but in some cases includes permanently halting the progression of a condition, disorder or disease.

[0068] Ranges provided herein are understood to be shorthand for all values ​​within that range, for example, a range of 1 to 50 is understood to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values ​​between the aforementioned integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9. With respect to subranges, "nested sub-ranges" extending from either end of the range are specifically contemplated. For example, nested sub-ranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0069] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0070] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0071] [Figure 1]Figure 1A-1I are a series of immunostains and graphs demonstrating that CRC tissues are strongly immunoreactive with anti-β-1,4-GalT-V antibodies and show increased LCS activity and B4GALT5 expression. (Figure 1A) Normal colon score 2 (20x magnification). (Figure 1B) Cytoplasmic staining of the endothelium (20x magnification). (Figure 1C) Colon cancer case score 1 (20x magnification). (Figure 1D) Colon cancer case score 2 (20x magnification). (Figure 1E) Colon cancer case score 3 (20x magnification). (Figure 1F) CRC tissues overexpress β-1,4-GalT-V. Visually normal CRC tissues (50 mg each) were homogenized in RIPA buffer and centrifuged at 1000 rpm. The supernatant was used to measure β-1,4-GalT-V mass by ELISA. The assay was then performed in triplicate with N=10 for both normal and tumor samples. Mean ± SEm values ​​are shown (*P=0.0340). Statistical significance was determined using an unpaired t-test. (Figure 1G) Increased LCS activity in colorectal tumors. Mean ± SEm, **P=0.0052. (Figure 1H) APC, NMT1 and TP53 genes showed increased expression in tumors compared to that of normal samples. (Figure 1I) B4GALT5 specifically showed increased expression, whereas B4GALT6 and UGCG were relatively unincreased. Mean ± SEM, N≧4 for both normal and tumor samples from patients. Ordinary one-way ANOVA was used for statistical analysis.

[0072] [Diagram 2]Figures 2A-G are a series of graphs demonstrating that LacCer mass is increased in CRC tissues. Visually normal CRC tissues (50 mg each) were homogenized in chloroform-methanol (2:1) in the presence of internal sphingolipid standards. Lipid extracts were then subjected to LC-MS to investigate changes in the levels of sphingolipid species in CRC or normal tissues using (Figure 2A) Cer, (Figure 2B) DHCer, (Figure 2C) GalCer and GlcCer, (Figure 2D) dihydroGalCer / dihydroGlcCer, (Figure 2E) LacCer, and (Figure 2F) DHLacCer. Among the GSLs investigated, only LacCer levels (Figure 2E) were statistically and significantly increased in colorectal tumors (*P=0.0112). For Cer and dihydroGalCer / dihydroGlcCer, N=10 (normal) and N=9 (tumor); for DHCer, N=9 (normal) and N=10 (tumor); and for GalCer and GlcCer, LacCer, and DHLacCer, N=10 (both normal and tumor). (Figure 2G) Normal and tumor tissues were assessed for sphingomyelin and DHSM levels by LC-MS. No statically significant differences were found in normal vs. tumor sphingomyelin values. In contrast, tumor samples showed significantly elevated DHSM values ​​compared to normal (**P=0.0059). For sphingomyelin, N=10 (normal) and N=6 (tumor), and for DHSM, N=10 (both normal and tumor). Statistical significance was determined using mean ± SEm values ​​and unpaired t-tests.

[0073] [Diagram 3]Figures 3A-H are a series of graphs and fluorescent stainings demonstrating that pharmacological inhibition of GSL synthesis dose-dependently reduces proliferation and decreases β-1,4-GalT-V protein expression in HCT-116. D-PDMP showed a dose- and time-dependent decrease in HCT-116 cell proliferation at (Figure 3A) 24 h and (Figure 3B) 96 h, with a maximally effective dose of 20 μM, compared to controls. *P≦0.05, **P≦0.01, ***P≦0.001. No differences were observed in UGCG immunofluorescence of D-PDMP-treated cells (Figure 3D) compared to UGCG immunofluorescence of controls (Figure 3C) at 24 h. However, D-PDMP treatment at (Figure 3F) 24 h and (Figure 3H) 96 h reduced GalT-V fluorescence (E and G, respectively) compared to that of untreated controls.

[0074] [Figure 4] Figures 4A-H are a series of graphs demonstrating that D-PDMP treatment reduces the levels of several sphingolipids in HCT-116. HCT-116 cells (105) were seeded on sterile (100 mm2) plastic Petri dishes in 10 mL of medium for 24 h. The medium was then replaced with 2% serum-containing medium with or without D-PDMP (10 μM). After 24 h, the medium was removed and total lipids were extracted with hexane-isopropanol (3:2 by volume) in the presence of sphingolipid internal standards and subjected to MS. D-PDMP treatment (indicated as D10 on the x-axis) reduced the levels of (Figure 4A) Cer, (Figure 4B) DHCer, (Figure 4C) monohexosylceramide, (Figure 4D) dihydroGlc / galceramide, (Figure 4E) dihexosylceramide, (Figure 4F) DHLacCer, but not (Figure 4G) sphingomyelin or (Figure 4H) DHSM at 10 μM compared to control values ​​(indicated as C on the x-axis). Data represent the mean ± SEm, N = 3 biological replicates for control and (Figure 4A-H) 10 μM D-PDMP, and unpaired t-tests were used for statistical analysis. *P ≤ 0.05, **P ≤ 0.01]\

[0075] [Diagram 5]5A-5D are a series of immunostains demonstrating that CRC tissues are strongly immunoreactive with anti-β-1,4-GalT-V antibodies.

[0076] [Figure 6] FIG. 6 shows immunostaining of colon cancer sections with GalT-V antibody.

[0077] [Figure 7] FIG. 7 is a schematic diagram showing the sphingolipid synthesis pathway.

[0078] [Figure 8] FIG. 8 is a graph demonstrating that treatment with GalT-V antibodies against GalT-V dose-dependently reduces proliferation ion HCT-116 cells. HCT-116 cells were seeded in 96-well trays (1×104 cells / well) and grown in medium supplemented with 10% fetal bovine serum. After 24 hours, fresh medium containing 3H-thymidine (5 μCi / ml) and various dilutions of GalT-V monoclonal antibodies was added. Treatment with D-PDMP (5 μM) served as a positive control. After 24 hours of incubation, incorporation of radioactivity into DNA was measured by scintillation spectroscopy. GalT-V antibodies dose-dependently reduced HCT-116 cell proliferation (P**≦0.01, P***≦0.001 and P****≦0.0001 (N=5)).

[0079] [Figure 9] Figure 9 is a graph demonstrating that treatment with GalT-V antibody against GalT-V reduces proliferation in mouse colorectal cancer cells in a dose-dependent manner. Mouse colorectal cancer cells were seeded in 96-well trays (1x104 cells / well), grown in minimal essential medium supplemented with 10% fetal bovine serum, and treated as described in Figure 8 above. Note that GalT-V antibody reduced MC-38 cell proliferation in a dose-dependent manner (N=5).

[0080] [Figure 10]Figures 10A-10I are a series of photographs and graphs demonstrating that VEGF-induced tube formation was attenuated by antibodies against β-GalT-V and lactosylceramide antibodies. Human umbilical vein endothelial cells were incubated with various dilutions of β-GalT-V or LacCer antibodies for 1 hour, followed by treatment with VEGF for 6 hours. Tube formation assays were then performed. The letters on the treatment axis of the graphs (Figure 10I) represent the treatments shown in Figures 10A-10H.

[0081] [Figure 11] 11A-11D are a series of photographs demonstrating that treatment with β-1,4 GalT-V antibody or biopolymer-encapsulated D-PDMP prevented tumor growth in normal female mice. 32-week-old normal female mice (C57BL6) were shaved. The dorsal area was cleaned with alcohol swabs and injected with 100 μL of a suspension of colorectal cancer cells HCT-116 (4×106). One week later, 100 μL of a monoclonal antibody against B-1,4 GalT-V (FIG. 11A, FIG. 11B) or a β-1,4 GalT-V inhibitor (5 mpk of biopolymer-encapsulated D-PDMP (FIG. 11C, FIG. 11D) was injected at the tumor cell injection site for 3 weeks. If hair had grown on the back, the dorsal area was shaved again with Nair and the mice were photographed. Note that no tumor growth was observed in the treated mice (FIG. 11A-11D).

[0082] [Figure 12] FIG. 12 is a schematic diagram outlining how β-galactosyltransferase (β-GalT-V) may contribute to colorectal cancer and the response to the novel approach herein to prevent it.

[0083] [Figure 13] FIG. 13 is a schematic diagram of the GalT-V antibody treatment model of Example 4.

[0084] [Figure 14] FIG. 14 shows results indicating that treatment with GalT-V antibody did not alter body weight in NOD-SCID mice.

[0085] [Figure 15] FIG. 15 (including FIG. 15A-B) shows that treatment with GalT-V antibody dose-dependently reduced tumor volume in NOD-SCID mice inoculated with HCT-116 cells.

[0086] [Figure 16] FIG. 16 (including FIGS. 16A-C) shows optical imaging of mice bearing HCT-116 rectal orthotopic tumors.

[0087] [Figure 17] FIG. 17 shows q-RT-PCR analysis of gene expression of B4GALT-V, CEA and NMT-1 in CRC mice.

[0088] [Figure 18] FIG. 18 (including FIG. 18A-FIG. 18B) shows an ELISA assay in FIG. 18A demonstrating that treatment with GalT-V-Ab reduced the mass of GalT-V in plasma, and HPTLC and densitometry analyses in FIG. 18B demonstrating that treatment with GalT-V-Ab reduced the mass of LacCer in tumor tissue compared to placebo.

[0089] [Figure 19] FIG. 19 shows the cell surface localization of GalT-V antibodies as determined by confocal microscopy (Example 5).

[0090] [Figure 20] FIG. 20 shows internalized GalT-V antibody (37° C.) as determined by confocal microscopy.

[0091] [Figure 21] FIG. 21 shows [89Zr]GalT-V antibody binding in human coronary artery endothelial cells (HCAEC) and human colorectal cancer cells.

[0092] [Figure 22] FIG. 22 shows [89Zr]GalT-V antibody binding in human coronary artery endothelial cells (HCAEC) and human colorectal cancer cells.

[0093] [Diagram 23] FIG. 23 shows that D-PDMP inhibits zirconium-tagged GalT-V antibody binding in human colorectal cancer cells.

[0094] [Figure 24] FIG. 24 shows the specificity of binding and internalization of [89Zr]GalT-V antibody in human colorectal cancer cells.

[0095] [Diagram 25] FIG. 25 shows the time-dependent binding and internalization of [89Zr]GalT-V antibody in human colorectal cancer cells.

[0096] [Figure 26-27] FIG. 26 and FIG. 27 show in vivo xenofluorescence images of human CRC tumor-bearing mice at specific time periods.

[0097] [Figure 28] FIG. 28 (including FIGS. 28A-C) shows the distribution of CF-750 GalT-V antibody fluorescence in individual tissues from subcutaneous / xenograft tumor-bearing mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] The present invention is based in part on the discovery that β-galactosyltransferase (β-GalT-V) plays a role in human CRC and its inhibition also reduces tumor cell proliferation. Samples from colorectal cancer subjects were found to be immunoreactive to β-1,4-GalT-V antibodies. In addition, β-1,4-GalT-V mass, mRNA expression, enzyme activity, and GSL end product levels were evaluated. The effect of GSL glycosyltransferase inhibitors in human CRC cell lines was examined. These results, which are described in detail in the Examples section, provide new insights into the pathogenesis of CRC and reveal promising detection / prognosis biomarkers for CRC. Applications include biomarkers useful for screening cancer, particularly colorectal cancer and precursors of colorectal cancer (e.g., advanced adenomas). Compositions for use in the treatment of cancer, such as colorectal cancer, are also described.

[0099] Colorectal cancer includes, but is not limited to, colon cancer, rectal cancer, and combinations thereof. Colorectal cancer includes metastatic colorectal cancer and non-metastatic colorectal cancer. Colorectal cancer includes cancer located in the proximal part of the colon and cancer located in the distal part of the colon. Colorectal cancer includes any of the various possible stages of colorectal cancer known in the art, including, for example, stage I, stage II, stage III, and stage IV colorectal cancer (e.g., stage 0, I, IIA, IIB, IIC, IIIA, IIIB, IIIC, IVA, IVB, and IVC). Colorectal cancer includes all stages of the tumor / node / metastasis (TNM) staging system. With respect to colorectal cancer, T can refer to whether the tumor has grown into the wall of the colon or rectum and if so, how many layers; N can refer to whether the tumor has spread to lymph nodes and, if so, how many lymph nodes and where they are located; and M can refer to whether the cancer has spread to other parts of the body and, if so, to what extent. Specific stages of T, N, and M are known in the art. T stages can include TX, T0, Tis, T1, T2, T3, T4a, and T4b; N stages can include NX, N0, N1a, N1b, N1c, N2a, and N2b; and M stages can include M0, M1a, and M1b. Additionally, grades of colorectal cancer can include GX, G1, G2, G3, and G4. Various means of staging cancer, particularly colorectal cancer, are well known in the art and are summarized, for example, at cancer.net / cancer-types / colorectal-cancer / stages.

[0100] In certain embodiments, the present disclosure includes screening for early stage colorectal cancer.Early stage colorectal cancer may include, for example, colorectal cancer that is localized within a subject, for example, in that they have not yet spread to lymph nodes of the subject, for example lymph nodes close to the cancer (stage NO), and have not spread to distant sites (stage M0).Early stage cancer includes, for example, colorectal cancer corresponding to stages 0-IIC.

[0101] Thus, colorectal cancer includes, inter alia, pre-malignant colorectal cancer (e.g., advanced adenoma) and malignant colorectal cancer. The methods and compositions of the present disclosure are useful for screening colorectal cancer in all its forms and stages, including but not limited to those named herein or otherwise known in the art, and all subsets thereof. Thus, those skilled in the art will understand that all references to colorectal cancer provided herein include, but are not limited to, colorectal cancer in all its forms and stages, including but not limited to those named herein or otherwise known in the art, and all subsets thereof.

[0102] Thus, in certain embodiments, pharmaceutical compositions for the prevention and treatment of cancer, such as colorectal cancer, comprise the administration of an inhibitor of glycosphingolipid synthesis to a subject in need thereof.

[0103] In certain embodiments, the method of treating cancer includes providing a therapeutically effective amount of (a) an antibody that specifically binds to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope, the antibody having an amino acid sequence that is at least 90% identical to: (i) EVQLEQSGAELARPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGTTLTVSS (SEQ ID NO: 3). The method includes administering to a subject in need thereof a composition comprising (i) an antibody comprising a heavy chain variable region sequence having at least 90% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4), and (b) a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis. In certain embodiments, the antibody comprises a heavy chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the antibody comprises a light chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 4.

[0104] In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of (i) an antibody comprising (a) a heavy chain variable region sequence nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:3, and (b) a light chain variable region sequence nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:2, (ii) a synthetic peptide comprising an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO:5, and (iii) an adjuvant. In certain embodiments, the antibody comprises (a) a heavy chain variable region nucleic acid sequence comprising SEQ ID NO:3, and (b) a light chain variable region nucleic acid sequence comprising SEQ ID NO:2, and the synthetic peptide amino acid sequence comprising SEQ ID NO:5.

[0105] In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of (a) an antibody that specifically binds to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope, the antibody having a sequence similar to at least (i) EVQLEQSGAELARPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGTTLTVSS (SEQ ID NO: 3). and (ii) an antibody comprising a heavy chain variable region sequence having at least 90% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4); and (b) a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis. In certain embodiments, the antibody comprises a heavy chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the antibody comprises a light chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 4.

[0106] In certain embodiments, the pharmaceutical composition further comprises at least one inhibitor of glycosphingolipid synthesis comprising D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), (1R,2R)-nonanoic acid (2-(2',3-dihydro-benzo(1,4)dioxin-6'-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl)-amide-L-tartrate (Genz-123346), an imido sugar, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DMP), 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), a lipid, a ceramide, or a combination thereof, unencapsulated or encapsulated by a biodegradable polymer. In certain embodiments, the inhibitor of glycosphingolipid synthesis is D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), either unencapsulated or encapsulated in a biodegradable polymer (BPD). In certain embodiments, the biodegradable polymer is comprised of polyethylene glycol and sebacic acid.

[0107] In certain embodiments, the pharmaceutical composition further comprises one or more secondary therapeutic agents. In certain embodiments, the one or more secondary therapeutic agents comprise a chemotherapeutic agent, an anti-inflammatory agent, a cholesterol-lowering agent, insulin, an antibody, a peptide, an enzyme, an adjuvant, or a combination thereof. In certain embodiments, the pharmaceutical composition further comprises conjugating the antibody to a detectable agent, a radiotherapeutic agent, a toxin, a radioactive agent, a dye, a peptide, a polynucleotide, or a nanoliposome. In certain embodiments, the nanoliposome comprises the therapeutic agent(s).

[0108] In certain embodiments, the pharmaceutical composition further comprises a peptide having at least 90% sequence identity to IGAQVYEQVLRSAYAKRNSSVND (SEQ ID NO:5).

[0109] In certain embodiments, the composition comprises a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis and / or a therapeutically effective amount of an antibody that specifically binds to β1,4-galactosyltransferase V (BGA), its isoforms or peptides.

[0110] In certain embodiments, the composition comprises a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis and / or a therapeutically effective amount of an agent that modulates the expression or activity of β1,4-galactosyltransferase V (BGA), its isoforms or peptides. In certain embodiments, the agent inhibits the expression or activity of β1,4-galactosyltransferase V (BGA), its isoforms or peptides.

[0111] Examples of lipids include, but are not limited to, fatty acids, free fatty acids, cholesterol, sterol esters, triglycerides, diglycerides, glycerides, wax esters, squalene, ceramides, lipids, phospholipids, glycolipids, linoleic acid, or combinations thereof.

[0112] In another embodiment, the method of treating cancer comprises administering to a subject in need thereof a therapeutically effective amount of an inhibitor of glycosphingolipid synthesis, a lipid, or a combination thereof. In certain embodiments, the inhibitor of glycosphingolipid synthesis is D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) that is unencapsulated, unbound, or encapsulated in a biodegradable polymer (BPD). In certain embodiments, the biodegradable polymer is comprised of polyethylene glycol and sebacic acid.

[0113] Combination therapy In certain embodiments, the pharmaceutical composition comprises an anti-cancer agent, such as a chemotherapeutic agent, radiation therapy, a toxin, or a combination thereof.

[0114] In certain embodiments, the anti-cancer agent is a chemotherapeutic agent or growth inhibitor, a targeted therapeutic agent, a T cell expressing a chimeric antigen receptor, an antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, an anti-neoplastic agent, a cancer vaccine, an adjuvant, and combinations thereof. In certain embodiments, the anti-cancer agent is a chemotherapeutic agent or growth inhibitor. For example, the chemotherapeutic agent or growth inhibitor may include an alkylating agent, an anthracycline, an anti-hormonal agent, an aromatase inhibitor, an anti-androgen, a protein kinase inhibitor, a lipid kinase inhibitor, an antisense oligonucleotide, a ribozyme, an antimetabolite, a topoisomerase inhibitor, a cytotoxic agent or an antitumor antibiotic, a proteasome inhibitor, an anti-microtubule agent, an EGFR antagonist, a retinoid, a tyrosine kinase inhibitor, a histone deacetylase inhibitor, and combinations thereof. In certain embodiments, the anti-cancer agent is an adjuvant. Any substance that enhances an anti-cancer immune response, such as against a cancer-associated antigen, or aids in the presentation of cancer antigens to components of the immune system may be considered an anti-cancer adjuvant of the present disclosure.

[0115] Chemotherapy: In general, cancer treatment also includes various combination therapies that involve both chemotherapy and radiation therapy-based treatment.Combination chemotherapy includes, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabine, navelbine, famcil-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, temazolomide (aqueous form of DTIC), or any analog or derivative variant of the above.The combination of chemotherapy and biological therapy is known as biochemotherapy. Chemotherapy can also be administered in successive low doses, a technique known as metronomic chemotherapy.

[0116] Still further combination chemotherapies include, for example, alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, metoledopa, uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs, KW-2 189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongiostatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trophosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gumol and calicheamicin omegaol); dynemicins (including dynemicin A); bisphosphonates such as clodronate; esperamicin;Also included are neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autarmicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, doxorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolidine-doxorubicin, and deoxydoxorubicin). including epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, pofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and doxorubicin; metabolic antagonists such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; fludarabine, 6-mercaptopropionate, and the like; Purine analogues such as thiampurine, thiamiprine, thioguanine, etc.; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenergics such as mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; enyluracil; amsacrine; bestra Bucil;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diazicon;Elformitin;Elliptinium acetate;Epothilone;Toglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidanmol;Nitrelin;Pentostatin;Fenamet;Pirarubicin;Rosoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK polysaccharide complex;Razoxane;Rhizoxin;Schizophyllan;Spirogermanium;Tenuazonic acid;Triazicon;2,2',2"-Trichlorotriethylamine;Tricothecenes (especially T-2 toxin, veracrine A, roridin A and anguidin);Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Mitolactol;Pipobroman;Gacytosine;Arabinoside ("Ara-C");Cyclophosphamide;Taxoids such as paclitaxel and docetaxel gemcitabine;6-Thioguanine;Mercaptopurine;Platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin;Vinblastine;Platinum;Etoposide (VP-16);Ifos These include: familide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000; difluoromethylchlornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In certain embodiments, one or more chemotherapeutic agents may be used in combination with the compositions provided herein.;

[0117] Radiation Therapy: Other agents that cause DNA damage and are widely used in cancer treatment include what are commonly known as gamma rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also known, such as microwave and UV radiation. All of these agents most likely cause widespread damage to DNA, the precursors of DNA, DNA replication and repair, and the building and maintenance of chromosomes. Dose ranges for X-rays range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells.

[0118] Immunotherapy: Immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors can be, for example, antibodies specific for some marker on the surface of tumor cells. Antibodies can function alone as therapeutic effectors or can recruit other cells to actually effect cell death. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic drugs, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply function as targeting agents. Alternatively, effectors can be lymphocytes carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells, as well as genetically engineered variants of these cell types that have been modified to express chimeric antigen receptors.

[0119] The immunotherapy may be a cancer vaccine comprising one or more cancer antigens, in particular proteins or immunogenic fragments thereof, DNA or RNA encoding said cancer antigens, in particular proteins or immunogenic fragments thereof, cancer cell lysates, and / or protein preparations from tumor cells. As used herein, a cancer antigen is an antigenic substance present in cancer cells. In principle, any protein produced in cancer cells with an abnormal structure resulting from a mutation may act as a cancer antigen. In principle, cancer antigens may be products of mutated oncogenes and tumor suppressor genes, products of other mutated genes, overexpressed or aberrantly expressed cellular proteins, cancer antigens produced by oncogenic viruses, carcinoembryonic antigens, altered cell surface glycolipids and glycoproteins, or cell type specific differentiation antigens. Examples of cancer antigens include abnormal products of the ras and p53 genes. Other examples include tissue differentiation antigens, mutated protein antigens, oncogenic viral antigens, cancer testis antigens, and vascular or stromal specific antigens. Tissue differentiation antigens are antigens that are specific to a particular type of tissue. Mutant protein antigens are much more likely to be specific to cancer cells, since normal cells should not contain these proteins. Normal cells present normal protein antigens on their MHC molecules, while cancer cells present the mutant versions. Some viral proteins are involved in the formation of cancer, and some viral antigens are also cancer antigens.

[0120] In certain embodiments, a method of treating cancer comprises administering a therapeutically effective amount of a synthetic peptide comprising an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO:5, and at least one adjuvant. In certain embodiments, the synthetic peptide comprises SEQ ID NO:5. Administration of a therapeutically effective amount of SEQ ID NO:5 generates an immune response against β-1,4-galactosyltransferase-V (β-1,4-GalT-V). In certain embodiments, an adjuvant is also administered to the subject.

[0121] In certain embodiments, the immunotherapy may be an antibody, such as part of a polyclonal antibody preparation, or may be a monoclonal antibody. The antibody may be a humanized antibody, a chimeric antibody, an antibody fragment, a bispecific antibody, or a single chain antibody. The antibodies disclosed herein include antibody fragments, such as, but not limited to, Fab, Fab', and F(ab')2, Fd, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdfv), and fragments containing either the VL domain or the VH domain.

[0122] In certain embodiments, the antibody comprises (i) a heavy chain variable region sequence having at least 90% amino acid sequence identity to EVQLEQSGAELARPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGTTLTVSS (SEQ ID NO: 3), and / or (ii) a light chain variable sequence having at least 90% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4).

[0123] In certain embodiments, one or more antibodies are administered to a subject in need thereof as a combination therapy. Examples of monoclonal antibodies that may be used in combination with the compositions provided herein include, but are not limited to, trastuzumab (anti-HER2 / neu antibody); pertuzumab (anti-HER2 mAb); cetuximab (chimeric monoclonal antibody against epidermal growth factor receptor EGFR); panitumumab (anti-EGFR antibody); nimotuzumab (anti-EGFR antibody); zalutumumab (anti-EGFR mAb); necitumumab (anti-EGFR mAb); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptor bispecific antibody); rituximab (chimeric mouse / human anti-CD20 mAb); obinutuzumab (anti-CD20 mAb); ofatumumab (anti-CD20 mAb); tositumumab-I131 (anti-CD20 mAb); ibritumomab tiuxetan (anti-CD20 mAb); bevacizumab (anti-VEGF mAb); ramucirumab (anti-VEGFR2 mAb); ranibizumab (anti-VEGF mAb); aflibercept (extracellular domains of VEGFR1 and VEGFR2 fused to IgG1 Fc); AMG386 (angiopoietin-1 and -2 binding peptide fused to IgG1 Fc); dalotuzumab (anti-IGF-1R mAb); gemtuzumab ozogamicin (anti-CD33 mAb); alemtuzumab (anti-Campath-1 / CD52 mAb); brentuximab vedotin (anti-CD30 mAb); catumaxomab (bispecific mAb targeting epithelial cell adhesion molecule and CD3); naptumomab (anti-5T4 mAb); direntuximab (anticarbonic anhydrase IX); or farletuzumab (antifolate receptor).Other examples include antibodies such as Panorex™ (17-1A) (mouse monoclonal antibody); Panorex (17-1A) (chimeric mouse monoclonal antibody); BEC2 (amidiotypic mAb mimicking the GD epitope) (BCG used); tumor (Lym-1 monoclonal antibody); SMART M195 Ab, humanized 13' 1 LYM-1 (Oncolym), Ovarex (B43.13, anti-idiotypic mouse mAb); 3622W94 mAb that binds to the EGP40 (17-1A) pan-cancer antigen on adenocarcinoma; Zenapax (SMART anti-Tac (IL-2 receptor); SMART M195 Ab, humanized Ab, humanized); NovoMAb-G2 (pan-cancer specific Ab); TNT (chimeric mAb against histone antigen); TNT (chimeric mAb against histone antigen); Gliomab-H (monoclonal antibody-humanized Ab); GNI-250 Mab; EMD-72000 (chimeric EGF antagonist); Lymphoside (humanized IL.L.2 antibody); and MDX-260 bispecific, targeting GD-2, ANA Ab, SMART IDIO Ab, SMART ABL 364 Ab or ImmuRAIT-CEA. Examples of antibodies include those disclosed in U.S. Patent No. 5,736,167, U.S. Patent No. 7,060,808 and U.S. Patent No. 5,821,337.

[0124] Passive immunotherapy: There are several different approaches for passive immunotherapy of cancer. They can be broadly categorized as follows: injection of antibodies alone; injection of antibodies conjugated to toxins or chemotherapeutic agents; infusion of antibodies conjugated to radioisotopes; injection of anti-idiotypic antibodies; and finally, purging of the bone marrow of tumor cells.

[0125] Thus, in certain embodiments, a method of treating cancer includes providing a therapeutically effective amount of (a) an antibody that specifically binds to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope, the antibody having at least one of the following structures: (i) EVQLEQSGAELARPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGTTLTVSS (SEQ ID NO: 3). and (ii) a light chain variable region sequence having at least 90% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4), and one or more secondary therapeutic agents. In certain embodiments, the one or more secondary therapeutic agents comprise a chemotherapeutic agent, an anti-inflammatory agent, a cholesterol-lowering agent, insulin, an antibody, a peptide, an enzyme, an adjuvant, or a combination thereof. In certain embodiments, the pharmaceutical composition further comprises conjugating the antibody to a detectable agent, a radiotherapeutic agent, a toxin, a radioactive agent, a dye, a peptide, a polynucleotide, or a nanoliposome. In certain embodiments, the nanoliposome comprises the therapeutic agent(s).

[0126] Other Agents: It is believed that other agents may be used in combination with the compositions provided herein to improve the therapeutic efficacy of the treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1, RANTES and other chemokines. It is further envisioned that upregulation of cell surface receptors or their ligands (e.g., Fas / Fas ligand, DR4 or DR5 / TRAIL, etc.) will enhance the apoptosis-inducing ability of the compositions provided herein by establishing an autocrine or paracrine effect on the hyperproliferative cells. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with the compositions provided herein to improve the antiproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with the compositions provided herein to improve the efficacy of the treatment.

[0127] In further embodiments, the other agent may be one or more oncolytic viruses, such as oncolytic viruses engineered to express genes other than p53 and / or IL24, such as cytokines. Examples of oncolytic viruses include adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpes viruses, pox viruses, vaccinia viruses, vesicular stomatitis viruses, polio viruses, Newcastle disease viruses, Epstein-Barr viruses, influenza viruses, and reoviruses.

[0128] In certain embodiments, hormone therapy may also be used in combination with this embodiment or in combination with any other cancer treatments described above.The use of hormones may be used to reduce the level or block the effect of certain hormones.This treatment is often used in combination with at least one other cancer treatment as a treatment option or to reduce the risk of metastasis.

[0129] In some embodiments, the additional anti-cancer agent is a protein kinase inhibitor or a monoclonal antibody that inhibits a protein kinase or a receptor involved in a growth factor signaling pathway, such as an EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor, or BRAF inhibitor. Non-limiting examples of protein kinase or growth factor signaling pathway inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, sorafenib, sunitinib, trastuzumab, vandetanib, AP 23451, vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, alvocidib, genistein, selumetinib, AZD-6244, vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016 or mixtures thereof.

[0130] Additional cancer therapies include, for example, epidermal growth factor receptor (EGFR, EGFR1, ErbB-1, HER1), ErbB-2 (HER2 / neu), ErbB-3 / HER3, ErbB-4 / HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF binding protein (IGFBP), IGFR ligand family (IGF-1R); platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family , Vascular endothelial growth factor receptor (VEGFR) family, VEGF family; HGF receptor family; TRK receptor family; Ephrin (EPH) receptor family; AXL receptor family; Leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family; Angiopoietin 1, 2; Receptor tyrosine kinase-like orphan receptor (ROR) receptor family; Discoidin domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family transforming growth factor alpha (TGF-α), TGF-α receptor; transforming growth factor beta (TGF-β), TGF-β receptor; interleukin-13 receptor alpha 2 chain (1L13Ralpha2), interleukin-6 (IL-6), 1L-6 receptor, interleukin-4, IL-4 receptor, cytokine receptors, class I (hematopoietin family) and class II (interferon / 1L-10 family) receptors, tumor necrosis factor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily Milly (TNTRSF), cell death receptor family, TRAIL receptor; cancer testis (CT) antigen, lineage specific antigen, differentiation antigen, alpha-actinin-4, ARTC1, breakpoint cluster region-Abelson (Bcr-abl) fusion product, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), beta-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein, EFTUD-2, elongation factor 2 (ELF2),Ets mutant gene 6 / acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, low density lipid receptor / GDP-L fucose:beta-galactose 2-alpha-L fucosyltransferase; (LDLR / FUT) fusion protein, HLA-A2, arginine to isoleucine exchange at residue 170 of the alpha helix of the alpha 2 domain of the HLA-A2 gene (HLA-A*201-R170I), MLA-A11, heat shock protein 70-2 mutation (HSP70-2M), KI AA0205, MART2, ubiquitous melanoma mutation 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, myosin class 1, NFYC, OGT, OS-9, pml-RAR alpha fusion protein, PRDXS, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, triosephosphate isomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7 , 8, GnT-V (abnormal N-acetylglucosaminyltransferase V, MGATS), HERV-K-MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognized antigen on melanoma (CAMEL), MAGE-A1 (MAGE-1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, MAGE-C6, MAGE-C7, MAGE-C8, MAGE-C9, MAGE-C10, MAGE-C11, MAGE-C12, MAGE-C13, MAGE-C14, MAGE-C15, MAGE-C16, MAGE-C17, MAGE-C18, MAGE-C19, MAGE-C20, MAGE-C21, MAGE-C22, MAGE-C23, MAGE-C24, MAGE-C25, MAGE-C26, MAGE-C27, MAGE-C28, MAGE-C29, MAGE-C30, MAGE-C31, MAGE-C32, MAGE-C33, MAGE-C34, MAGE-C35, MAGE-C36, MAGE-C37, MAGE-C38, MAGE-C39, MAGE-C40, MAGE-C41, MAGE-C42, MAGE-C43, MAGE-C44, MAGE-C45, MAGE-C46, MAGE-C47, MAGE-C48, MAGE-C49 E-C2, mucin 1 (MUC1), MART-1 / Melan-A (MLANA), gp100, gp100 / Pme117 (S1LV), tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1 / LAGE-2, SAGE, Sp17, SSX-1, 2, 3, 4, TRP2-1NT2, carcinoembryonic antigen (CEA), kallikfein4, mammaglobm-A, OA1, prostate-specific antigen (PSA), prostate-specific membrane antigen, TRP-1 / gp75, TRP-2, adipophilin,Interferon-inducible protein absent in Niella norna 2 (AIM-2), BING-4, CPSF, cyclin D1, epithelial cell adhesion molecule (Ep-CAM), EpbA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250), intestinal carboxylesterase (iCE), alpha-fetoprotein (AFP), M-CSF, mdm-2, MUCI, p53 (TP53), PBF, FRAME, PSMA, RAGE-1, RNF43, RU2AS, SOX10, STEAP11, survivin (BIRCS), human telomerase reverse transcriptase (hTERT), telomerase, Wilms tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX15, FATE, TPTE, immunoglobulin idiotype, Bence Jones protein, estrogen receptor (ER), androgen receptor (AR), CD40, CD30, CD20, CD19, CD33, CD4, CD25, CD3, cancer antigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), β-human chorionic gonadotropin, 1-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enoJase, heat shock protein gp96, GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4), carcinogenic antigen peptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2), cyclophilin B (Cyp-B), human cine ring tumor-2 (HST-2), human papillomavirus (HPV) proteins (HPV-E6, HPV-E7, major capsid antigen or minor capsid antigen, others), Epstein-Barr-Bim (EBV) proteins (EBV latent membrane protein-LMP1, LMP2; others), hepatitis B or C virus proteins, and antibodies and peptides targeting HIV proteins,These may include polypeptides, small molecule inhibitors, siRNA, miRNA, or gene therapy.

[0131] Glycosphingolipid synthesis inhibitors: In certain embodiments, the method further comprises administering at least one inhibitor of glycosphingolipid synthesis comprising D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), (1R,2R)-nonanoic acid (2-(2',3-dihydro-benzo(1,4)dioxin-6'-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl)-amide-L-tartrate (Genz-123346), imido sugar, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DMP), 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), lipid, ceramide, or combinations thereof, either unencapsulated or encapsulated by a biodegradable polymer. In certain embodiments, the inhibitor of glycosphingolipid synthesis is D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) (e.g., including D-PDMP that may be mixed with a biodegradable polymer, e.g., unencapsulated or encapsulated in a biodegradable polymer (BPD)). In certain embodiments, the biodegradable polymer is comprised of polyethylene glycol and sebacic acid.

[0132] In certain embodiments, the composition comprises an inhibitor of glycosphingolipid synthesis, an inhibitor of glucosylceramide synthase, or a combination thereof. In certain embodiments, the compound that inhibits glucosylceramide synthesis is an iminosugar. In another embodiment, the imidosugar is N-butyldeoxynojirimycin, N-butyldeoxygalactonojirimycin (NB-DGJ), or N-nonyldeoxynojirimycin. In another embodiment, the inhibitor of glucosylceramide synthesis is 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DMP), D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, and structurally related analogs thereof. In another embodiment, the inhibitor of glucosylceramide synthesis is 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), and structurally related analogs thereof. In certain embodiments, the composition comprises D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), (1R,2R)-nonanoic acid (2-(2',3-dihydro-benzo(1,4)dioxin-6'-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl)-amide-L-tartrate (Genz-123346), imido sugar, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DMP), 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), lipid, ceramide, or combinations thereof, encapsulated by a biodegradable polymer.

[0133] Pharmaceutical preparations In certain embodiments, the pharmaceutical compositions embodied herein are formulated for systemic administration, e.g., orally, iv, im, etc., and comprise a therapeutically effective amount of an inhibitor of glycosphingolipid synthesis, e.g., a therapeutically effective amount of (a) an antibody that specifically binds to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope, the antibody comprising: (i) a heavy chain variable region sequence having at least 90% amino acid sequence identity to EVQLEQSGAELARPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGTTLTVSS (SEQ ID NO: 3); and (ii) an antibody comprising a light chain variable sequence having at least 90% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4), and / or a peptide comprising SEQ ID NO: 5, and / or D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), which may be mixed with a biodegradable polymer, e.g., D-PDMP unencapsulated or encapsulated in a biodegradable polymer (BPD), or a combination thereof.

[0134] The pharmaceutical composition may include a pharma- ceutically acceptable carrier. The term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, olive oil, gels (e.g., hydrogels), and the like. Saline is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0135] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin, the contents of which are incorporated herein by reference in their entirety. Such compositions will generally contain a therapeutically effective amount of the pharmaceutical agent and / or therapeutic compound (e.g., biopolymer encapsulated D-PDMP) in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0136] In embodiments, the pharmaceutical and / or therapeutic compounds are administered topically as immediate release or controlled release compositions, for example, by controlled dissolution and / or diffusion of the active substance. Dissolution or diffusion controlled release can be achieved by incorporating the active substance into a suitable matrix. The controlled release matrix can include one or more of biopolymers, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethyl cellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxymethacrylate, methacrylate hydrogel, 1,3 butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol and / or sebacic acid. In controlled release matrix formulations, the matrix material may also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, Carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbons. In certain embodiments, the controlled release composition is achieved via a transdermal patch.

[0137] The controlled release matrix may also be a hydrogel: a three-dimensional, hydrophilic or amphiphilic polymer network capable of incorporating large amounts of water. The network may be composed of homopolymers or copolymers that are insoluble due to the presence of covalent chemical or physical (e.g., ionic, hydrophobic interactions, entanglements) crosslinks. The crosslinks provide the network structure and physical integrity. Hydrogels exhibit a thermodynamic compatibility with water that allows them to swell in aqueous media. The chains of the network are connected in such a way that pores are present and a significant portion of these pores are between 1 nm and 1000 nm in size.

[0138] Hydrogels can be prepared by crosslinking hydrophilic biopolymers or synthetic polymers. Examples of hydrogels formed from physical or chemical crosslinking of hydrophilic biopolymers include, but are not limited to, hyaluronan, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin, agarose, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, poly(ethylene glycol) (PEO), poly(propylene glycol) (PPO), PEO-PPO-PEO copolymer (Pluronics), poly(phosphazene), poly(methacrylate), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymer, poly(ethyleneimine), and the like. See Hennink and van Nostrum, Adv. Drug Del. Rev. 54:13-36 (2002); Hoffman, Adv. Drug Del. Rev. 43:3-12 (2002); Cadee et al., J Control. Release 78:1-13 (2002); Surini et al., J. Control. Release 90:291-301 (2003); and U.S. Patent No. 7,968,085, each of which is incorporated by reference in its entirety. These materials consist of high molecular weight backbone chains of linear or branched polysaccharides or polypeptides.

[0139] The amount of the pharmaceutical composition of the present invention that is effective in treating or preventing atherosclerotic heart disease can be determined by standard clinical techniques.In addition, in vitro assays can be optionally used to help identify optimal dosage ranges.The exact dose to be used in the formulation can also depend on the route of administration and the severity of the disease, and should be determined according to the judgment of the practitioner and each patient's circumstances.Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems described herein or known to those skilled in the art.

[0140] Administration and Dosing Regimen Pharmaceutical agents and / or therapeutic compounds or compositions containing these agents / compounds may be administered in a manner compatible with the dosage formulation, and in such amounts as will be therapeutically effective, protective and immunogenic.

[0141] The agents and / or compositions may be administered via different routes, including but not limited to oral, oral gavage, parenteral, buccal and sublingual, rectal, aerosol, nasal, intramuscular, subcutaneous, intradermal, intraosseous, dermal, and topical. As used herein, the term parenteral includes, for example, intraocular, subcutaneous, intraperitoneal, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or other infusion techniques.

[0142] In an embodiment, the pharmaceutical agent and / or therapeutic compound formulated according to the present invention is formulated and delivered to induce a systemic response. Thus, in an embodiment, the formulation is prepared by uniformly and intimately associating the active ingredient with a liquid carrier. Formulations suitable for administration include aqueous and non-aqueous sterile solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. The formulations may be provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a lyophilized (freeze-dried) condition requiring only the addition of a sterile liquid carrier, such as water, immediately prior to use. Extemporaneous solutions and suspensions may be prepared from sterile powders, granules, and tablets commonly used by those skilled in the art.

[0143] The agents and / or compositions may be administered in different forms, including, but not limited to, solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, liposomes, and the like.

[0144] Pharmaceutical and / or therapeutic compounds may be administered in a manner compatible with the dosage formulation, and in such amounts as may be therapeutically effective, immunogenic and protective. The amount administered will depend on the subject being treated, including, for example, the stage of the disease. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by those skilled in the art and may be on the order of one microgram to one milligram of active ingredient(s) per dose. Dosage may also depend on the route of administration and may vary with the size of the host.

[0145] The pharmaceutical agent and / or therapeutic compound should be administered to the subject in an amount effective to ameliorate, treat and / or prevent the disease. The specific dosage and treatment regimen for any particular subject may depend on a variety of factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, severity and course of the disease (including tumor size), symptoms or signs, the nature of the subject to the disease, symptoms or signs, method of administration, and the judgment of the treating physician. The actual dosage can be easily determined by those skilled in the art.

[0146] Exemplary unit dosage formulations are those containing a dose or unit of the administered ingredient, or an appropriate fraction thereof. It will be understood that, in addition to the ingredients mentioned herein, the formulations of the present invention may include other agents commonly used by those of skill in the art.

[0147] In certain embodiments, an antibody, isoform or peptide thereof that specifically binds to β1,4-galactosyltransferase V (BGA) is administered systemically, administered via endoscopy, or administered intraanally.

[0148] In certain embodiments, a composition comprising a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis and / or a therapeutically effective amount of an agent that modulates the expression or activity of β1,4-galactosyltransferase V (BGA), its isoforms or peptides is administered systemically or locally.

[0149] In certain embodiments, compositions comprising a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis and / or a therapeutically effective amount of an agent that modulates the expression or activity of β1,4-galactosyltransferase V (BGA), its isoforms or peptides are co-administered to a subject. The term "co-administration" refers to the simultaneous presence of two active agents in the blood of an individual. Co-administered active agents can be delivered simultaneously or sequentially.

[0150] Typically, in conventional systemic administration therapy, a therapeutically effective dose should result in a serum concentration of the compound of about 0.1 ng / ml to about 50-100 μg / ml. Pharmaceutical compositions typically provide a dosage of the compound of about 0.001 mg to about 2000 mg per kilogram of body weight per day. For example, dosages for systemic administration to a human patient may be 1-10 μg / kg, 20-80 μg / kg, 5-50 μg / kg, 75-150 μg / kg, 100-500 μg / kg, 250-750 μg / kg, 500-1000 μg / kg, 1-10 mg / kg, 5-50 mg / kg, 25-75 mg / kg, 50-100 mg / kg, 100-250 mg / kg, The dosage may be in the range of 50-100mg / kg, 250-500mg / kg, 500-750mg / kg, 750-1000mg / kg, 1000-1500mg / kg, 1500-2000mg / kg, 5mg / kg, 20mg / kg, 50mg / kg, 100mg / kg, 500mg / kg, 1000mg / kg, 1500mg / kg or 2000mg / kg. In an exemplary embodiment, the oral dosage for a human weighing 200kg is about 200mg / day. The pharmaceutical dosage unit form is prepared to provide about 1mg to about 5000mg, for example about 100 to about 2500mg of the compound or combination of essential ingredients per dosage unit form.

[0151] In general, a therapeutically effective amount of the compound in a dosage form will usually range from slightly less than about 0.025 mg / kg / day to about 2.5 g / kg / day of a patient, preferably from about 0.1 mg / kg / day to about 100 mg / kg / day or significantly more, depending on the compound used, the condition or infection being treated, and the route of administration, although exceptions to this dosage range may be contemplated by the present invention. It is to be understood that the present invention has both human and veterinary applications.

[0152] The agents and / or compositions are administered in one or more doses as needed to achieve the desired effect. Thus, the agents and / or compositions may be administered in one, two, three, four, five, or more doses. Furthermore, the doses may be separated by any period of time, for example, hours, days, weeks, months, and years.

[0153] The agents and / or compositions may be formulated as a liquid or a dry powder, or in the form of microspheres.

[0154] The agent and / or composition may be stored at a temperature of about -100°C to about 25°C depending on the storage period. The agent and / or composition may also be stored in a lyophilized state at different temperatures, including room temperature. The agent and / or composition may be sterilized by conventional means known to those skilled in the art. Such means include, but are not limited to, filtration. The composition may also be combined with other anti-atherosclerotic therapeutic agents.

[0155] The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form may vary depending upon the host treated and the particular mode of administration, in embodiments, the preparation may contain from about 0.1% to about 95% active compound (w / w), from about 20% to about 80% active compound, or any percentage therebetween.

[0156] In embodiments, the pH of the formulation may be adjusted with pharma- ceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.

[0157] In embodiments, the pharmaceutical carrier may be in the form of a sterile liquid preparation, for example, as a sterile aqueous or oleaginous suspension. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.

[0158] In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful for the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersants, which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and / or suspensions.

[0159] Other commonly used surfactants, such as TWEEN™ or SPAN™, and / or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma- ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0160] In embodiments, the agent and / or composition can be delivered in an exosome delivery system. Exosomes are small membrane vesicles that are released into the extracellular environment during fusion of multivesicular bodies with the plasma membrane. Exosomes are secreted by a variety of cell types, including hematopoietic cells, normal epithelial cells, and even some tumor cells.

[0161] In a particular embodiment, the biopolymer encapsulating the D-PDMP comprises polyethylene glycol (PEG) and sebacic acid (SA). Both PEG and SA are FDA approved. Polyethylene glycol-sebacic acid (PEG-SA) copolymer can be prepared as previously described (Fu J, et al. Biomaterials. 2002;23:4425-4433), and microparticles of D-PDMP encapsulated by PEG-SA copolymer are prepared by modifying the single emulsion solvent evaporation method. For scintigraphic tracking of the biopolymer, the PEG polymer is added at 45 mCi (810 kBq) of ( 125 I) radioiodinated with NaI. Radiolabeled PEG was then incorporated into PEG-SA biopolymers. PEG-SA copolymers can be prepared according to the literature procedure published by Fu and coworkers (Id.). Briefly, sebacic acid prepolymers are made by refluxing sebacic acid (SA) in acetic anhydride, followed by drying under high vacuum (evaporation), crystallization from dry toluene, washing with 1:1 anhydrous ethyl ether-petroleum ether, and finally air drying. PEG prepolymers are made by refluxing polyoxyethylene dicarboxylic acid in acetic anhydride, and the volatile solvents are removed under vacuum. The solid mass is extracted with anhydrous ether and air dried. Poly(PEG-SA) coblock polymers are then synthesized by melt polycondensation and characterized by proton NMR. It is noted that this copolymer has been extensively characterized for composition and structural identity (Aich U, et al. Glycoconjugate journal. 2010;27:445-459).

[0162] Encapsulation of D-PDMP in poly(PEG-SA) (to prepare the polymer-encapsulated drug, later called BPD) followed by melt polycondensation as described above for SA and PEG prepolymers, but with D-PDMP at a starting ratio of poly(PEG-SA) to D-PDMP of 70:30. Microparticles are then prepared using a single emulsion solvent evaporation method. Briefly, D-PDMP and PEG-SA are dissolved in chloroform (50 mg / mL) and emulsified in a 1.0% w / w aqueous poly(vinyl alcohol) solution under sonication conditions that keep the temperature below 25 °C. The particles are hardened by evaporating the chloroform at room temperature with stirring for 12 h. The particles are collected, washed three times with double distilled water via centrifugation at 2,600 × g (30 min), and lyophilized for 48 h until ready for use.

[0163] In certain embodiments, the D-PDMP is encapsulated in a multilamellar lipid vesicle comprising covalent crosslinks between the lipid bilayers, and at least two lipid bilayers in the multilamellar lipid vesicle are covalently crosslinked to each other by a thiolated biopolymer. In certain embodiments, the lipid bilayers are crosslinked via a functionalized lipid. In certain embodiments, one or more lipids comprise DOTAP, DOPE, DOBAQ, DOPC, or combinations thereof. In certain embodiments, the lipid is maleimide-functionalized or modified with dibenzocyclooctyne (DBCO). In certain embodiments, the thiolated biopolymer is selected from the group consisting of chitosan, polyglutamic acid, polyphosphazene, polyethyleneimine, polyalkylacrylic acid (e.g., polymethylmethacrylate, poly(ethylacrylic acid), poly(propylacrylic acid), or poly(butylacrylic acid), HA, pegylated azide-modified polyethyleneimine, branched polyethyleneimine, and diazide. In certain embodiments, the thiolated biopolymer comprises multiple sulfhydryl moieties.

[0164] Delivery of pharmaceuticals and / or therapeutic compounds using nanoparticles is also contemplated by the present invention. For example, the pharmaceuticals and / or compositions provided herein can contain nanoparticles having at least one or more drugs attached thereto, e.g., attached to the surface of the nanoparticle. The composition typically includes many nanoparticles, each nanoparticle having at least one or more drugs attached thereto. The nanoparticles can be colloidal metals. Colloidal metals include any water-insoluble metal particles or metal compounds dispersed in liquid water. Typically, colloidal metals are suspensions of metal particles in an aqueous solution. Any metal that can be made in colloidal form can be used, including gold, silver, copper, nickel, aluminum, zinc, calcium, platinum, palladium, and iron. In some cases, gold nanoparticles are used, prepared, for example, from HAuCl4. The nanoparticles can be of any shape and can range in size from about 1 nm to about 10 nm, e.g., from about 2 nm to about 8 nm, about 4 to about 6 nm, or about 5 nm. Methods for making colloidal metal nanoparticles, including gold colloidal nanoparticles from HAuCl4, are known to those skilled in the art. For example, the methods described herein as well as methods described elsewhere (e.g., U.S. Patent Application Publication Nos. 2001 / 005581; 2003 / 0118657; and 2003 / 0053983, which are incorporated herein by reference) are useful guides for making nanoparticles.

[0165] In certain cases, a nanoparticle can have two, three, four, five, six or more active agents attached to its surface. Typically, many molecules of active agent are attached to the surface of the nanoparticle at many positions. Thus, when a nanoparticle is described as having, for example, two active agents attached to it, the nanoparticle has two active agents attached to its surface, each with its own molecular structure. In some cases, one molecule of active agent can be attached to the nanoparticle via a single binding site or via multiple binding sites.

[0166] The active agent can be linked directly or indirectly to the surface of the nanoparticle. For example, the active agent can be linked directly to the surface of the nanoparticle or indirectly through an intervening linker.

[0167] Any type of molecule can be used as a linker. For example, the linker can be an aliphatic chain containing at least two carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more carbon atoms) and can be functionalized with one or more functional groups including ketones, ethers, esters, amides, alcohols, amines, ureas, thioureas, sulfoxides, sulfones, sulfonamides, and disulfides. When the nanoparticles include gold, the linker can be any thiol-containing molecule. The reaction of a thiol group with gold results in a covalent sulfide (-S-) bond. The design and synthesis of linkers are well known in the art.

[0168] In embodiments, the nanoparticles are linked to a targeting agent / moiety. The targeting function allows the nanoparticles to accumulate in the target at a higher concentration than in other tissues. In general, the targeting molecule can be one member of a binding pair that exhibits affinity and specificity for the second member of the binding pair. For example, an antibody or antibody fragment therapeutic agent can target the nanoparticle to a specific region or molecule of the body (e.g., a region or molecule for which the antibody is specific) while also performing a therapeutic function. In some cases, a receptor or receptor fragment can target the nanoparticle to a specific region of the body, such as the location of its binding pair member. Other therapeutic agents, such as small molecules, can similarly target the nanoparticle to receptors, proteins, or other binding sites that have affinity for the therapeutic agent.

[0169] When the compositions of the invention include one or more additional therapeutic or prophylactic agents, the therapeutic and additional agents should be present at dosage levels that are about 0.1-100%, or about 5-95%, of the dosage normally administered in a monotherapy regimen. The additional agents may be administered separately from the agents of the invention, as part of a multiple dose regimen. Alternatively, these additional agents may be part of a single dosage form, mixed together with the agents of the invention in a single composition.

[0170] Administration of the pharmaceutical agent and / or therapeutic compound of the present invention induces, for example, an anti-cancer response. Typically, the dosage can be adjusted within this range based on, for example, the subject's age, the subject's health and physical condition, the ability of the subject's immune system to produce an immune response, the subject's weight, the subject's sex, diet, time of administration, the degree of protection desired, and other clinical factors. Those skilled in the art can also easily address parameters such as biological half-life, bioavailability, route of administration and toxicity when formulating the agent and / or composition of the present invention. EXAMPLES

[0171] Example 1: Lactosylceramide synthase β-1,4-GalT-V: a novel target for the diagnosis and therapy of human colorectal cancer It was hypothesized that β-1,4-galactosyltransferase-V (β-1,4-GalT-V) may well play a role in human CRC and that its inhibition may also attenuate tumor cell proliferation. To test this hypothesis, we obtained colorectal tissue samples from unidentified cancer patients and assessed immunoreactivity to β-1,4-GalT-V antibodies. Furthermore, β-1,4-GalT-V mass, mRNA expression, enzyme activity, and GSL end product levels were evaluated. Finally, the effect of GSL glycosyltransferase inhibitors in human CRC cell lines was also examined. These results provide new insights into the pathogenesis of CRC and reveal promising detection / prognostic biomarkers for CRC. Furthermore, these findings demonstrate a viable target for future CRC treatment.

[0172] Materials and Methods Immunohistochemical (IHC) localization of β-1,4-GalT-V in human CRC tissues. IHC was performed on archived tissue from the Johns Hopkins Pathology Department after approval from the Institutional Review Board for research on human subjects. Retrieved tissue was sectioned from formalin-fixed paraffin-embedded blocks of colon cancer cases selected from 2-3 years of material. Four micron thick sections were cut and stained for IHC analysis. β-1,4-GalT-V staining was performed on an automated instrument using standard IHC methods. Briefly, sections were deparaffinized, hydrated, and prepared for staining.

[0173] Sections were incubated for 30 minutes with β-1,4-GalT-V mouse monoclonal antibody raised against GalT-V synthetic peptide, IGAQVYEQVLRSAYAKRNSSVND, SEQ ID NO:5 (1:600 ​​dilution). Secondary antibody anti-rabbit HRP was applied and a brown signal was developed using DAB chromogen detection (Cat. No. DS 9800, Leica Biosystems). Slides were then counterstained with hematoxylin, washed, dehydrated and coverslipped. Evaluation of IHC staining was performed by a blinded pathologist. Staining was scored based on the intensity of staining and the area of ​​stained tumor.

[0174] H-score determination of human CRC tissues. Tumor tissues were examined independently by two pathologists (RM and MA) of the team. Twenty-one cases of colon adenocarcinoma were selected from archived data at Johns Hopkins Hospital. Only primary colon cancer cases were included in this study. For case selection, archived cases were reviewed and selected based on reports from anatomic pathologists. Unstained sections were obtained from paraffin blocks, and IHC with GalT-V antibody was performed as described in the methods section. Results were evaluated after creating a scoring system of 0 to 3 (0 = no staining, 1 = very weak staining, 2 = moderate staining, and 3 = strong staining). The positive staining area on the tumor was also evaluated, which was used to calculate the H-score.

[0175] Gene expression analysis. Patient normal and CRC tissues were provided in collaboration with Dr. Bert Vogelstein (JHU). Total RNA was isolated from tissue samples using the RNAqueous-4PCR kit (Life Technologies) according to the manufacturer's instructions. TaqMan gene expression assays (Applied Biosystems) were used to determine the expression levels of adenomatous polyposis coli (APC, Hs01568269), N-myristoyltransferase-1 (NMT1, Hs00221506), tumor protein p53 (TP53, Hs01034249), UDP-Gal:βGlcNAc β-1,4-galactosyltransferase, polypeptide 5 (β-1,4-GalT-V, Hs00191142), UDP-Gal:βGlcNAc, β-1,4-galactosyltransferase, polypeptide 6 (β-1,4GalT-VI, Hs00191135), and UDP-glucose ceramide glucosyltransferase (UGCG, Hs00234293). cDNA was synthesized from the isolated RNA using the High Capacity cDNA Reverse Transcription Kit (Life Technologies 4374966) according to the manufacturer's protocol. TaqMan gene expression assays were performed by 7900HT Fast Real-Time PCR at The Genetic Resources Core Facility (Johns Hopkins Medical Institutions).

[0176] Measurement of lactosylceramide synthase (LCS) activity. LCS activity in visible normal and cancer tissues was measured according to our previously published methods. (20, 21) All assays were performed in triplicate for both 10 normal and 10 tumor samples, and mean values ​​± standard error of the measurement (SEm) were presented, with unpaired t-tests performed to determine statistical significance.

[0177] Measurement of β-1,4-GalT-V mass in CRC tissues. Approximately 10 mg of tissue was homogenized in radioimmunoprecipitation assay (RIPA) buffer and centrifuged at 10,000 rpm, and GalT-V mass in the supernatant was measured using ELISA as previously published ( 22 ).

[0178] Liquid chromatography-mass spectrometry (LC-MS). Sphingolipid levels in human CRC tissues and human cultured CRC cells (HCT-116) were measured by LC-MS as previously described (23). Visually normal CRC tissues (50 mg) were homogenized in chloroform-methanol (2:1) in the presence of sphingolipid internal standards. 5 100mm of HCT-116 cells 2 The cells were grown on sterile plastic Petri dishes at 100° C. Lipid extracts were subjected to LC-MS as described (24, 25).

[0179] Determining the effect of D-PDMP on IHC localization of glycosyltransferases in human CRC cells. HCT-116 cells were seeded (10 4 ), which were then placed into 6-well sterile plastic trays and grown for 24 hours in complete medium. The medium was then replaced with 2 mL of 2% serum-containing medium + 10 μM D-PDMP. After 24 and 96 hours of incubation, the medium was removed and the cells were fixed with ethanol, washed, incubated with antibodies against β-1,4-GalT-V or UGCG, and photographed.

[0180] Determination of the effect of D-PDMP on cell proliferation. HCT-116 cells were seeded (10 4 100 μL / well) and grown in complete medium containing 10% fetal bovine serum for 24 h. The cultures were then changed to 2% serum-containing medium (100 μL) + 3 H-thymidine (5 μCi / mL) was used for replacement. After a further 24-h incubation, 3H-thymidine incorporation into DNA was measured by scintillation spectroscopy.

[0181] result Human CRC tissues show strong positive immunoreactivity to the β-1,4-GalT-V antibody. First, we investigated β-1,4-GalT-V immunoreactivity in 24 human CRC specimens using monoclonal antibodies. Normal colon tissues showed cytoplasmic localization of β-1,4-GalT-V with strongly positive immunostained endothelial cells in large and small vessels (Figure 1B). In colon adenocarcinomas (Figure 1C), approximately 50% of the diseased cells were found to express mild cytoplasmic β-1,4-GalT-V immunoreactivity. H-score analysis resulted in a total score of 100 in some cases, while in others it reached a total score of 200 (Figure 1D) and in others a total score of 300 (Figure 1E) (Table 1). Additional studies showed strong immunoreactivity in the perinuclear region in association with antigen localization to the Golgi apparatus, the cytoplasm, and, to a lesser extent, the inner surface of the cell surface.

[0182] Table 1: β-1,4-GalT-V immunohistochemical staining of tumor tissues. [Table 1]

[0183] Table 1 shows the evaluation of β-1,4-GalT-V immunohistochemical staining of normal and CRC tissue sections. Examination of immunostaining for GalT-V revealed various degrees of positivity in tumor cells: 1+ (15%; 3 / 20), 2+ (65%; 13 / 20), and 3+ (20%; 4 / 20). Staining was mostly observed in the tumor cytoplasm and only occasionally in the nuclei of tumor cells. When available, weak (1+) to moderate (2+) cytoplasmic staining was also observed in adjacent normal colonic mucosa in 79% of cases (15 / 19). In most cases, normal colonic mucosa was also stained at a mild to moderate level. H-score was determined as tumor intensity score% × total tumor area.

[0184] CRC tissues have increased protein mass, activity (lactosylceramide synthesis), and gene expression of β-1,4-GalT-V. ELISA revealed a significant increase (approximately 6.5-fold) of β-1,4-GalT-V in CRC tissues compared to visually normal areas (i.e., "adjacent normal") from the same tissue specimens (Figure 1F). A 2.25-fold increase in lactosylceramide synthase activity was observed in CRC samples compared to that of normal colonic epithelium (**P=0.0052, Figure 1G). Quantitative RT-PCR further revealed elevated expression of several genes previously associated with CRC, such as adenomatous polyposis coli (APC) (26), N-myristoyltransferase 1 (NMT1), and tumor protein p53 (TP53), compared to normal samples (Figure 1H). Furthermore, β-1,4-GalT-V (B4GALT5), but not β-1,4-galactosyltransferase, polypeptide 6 (B4GALT6) and UDP-glucose-ceramide β-1,4-glucosyltransferase (UGCG) showed specifically increased expression (Figure 1I). LC-MS revealed slight but moderately elevated levels of ceramide (Cer) (Figure 2A), dihydroceramide (DHCer) (Figure 2B), monoglycosylceramide (i.e., galactosylceramide (GalCer)) and glucosylceramide (GlcCer), Figure 2C), dihydroGalCer / dihydroGlcCer (Figure 2D), and dihydrolactosylceramide (DHLacCer) (Figure 2F) in tumors compared to normal colon specimens. However, among the GSLs investigated, only lactosylceramide (LacCer) levels were statistically and significantly increased in CRC tissues (Figure 2E, *P=0.0112).CRC samples also showed higher dihydrosphingomyelin (DHSM) (Figure 2G, **P=0.0059) and similar levels of sphingomyelin (Figure 2G) compared to normal tissues.

[0185] Inhibition of GSL synthesis dose-dependently reduces the proliferation of human CRC cells. HCT-116 cells were treated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP, Figure 3A), a potent inhibitor of UDP-glucose-cer:glucosyltransferase and LCS / 3-1,4-galactosyltransferase (GalT-V) activity (27, 28, 29). In CRC cells, D-PDMP showed a dose- and time-dependent (Figure 3A,B) decrease in proliferation compared to control cells, with the maximal effective dose found to be 20 μM.

[0186] D-PDMP treatment reduces β-1,4-GalT-V protein expression and activity (ie, sphingolipid synthesis) in human CRC cells. No difference was observed in UGCG immunofluorescence of D-PDMP-treated cells (Fig. 3D) compared to the control (Fig. 3C) at 24 h. However, D-PDMP treatment for 24 h (Fig. 3F) and 96 h (Fig. 3H) reduced GalT-V fluorescence compared to the control (Fig. 3E and Fig. 3G). LC-MS analysis of GSLs derived from HCT-116 cells treated with or without D-PDMP (10 μM) revealed reduced levels of Cer (Fig. 4A), DHCer (Fig. 4B), monohexosylceramide (Fig. 4C), dihydroGalCer / dihydroGlcCer (Fig. 4D), dihexosylceramide (Fig. 4E), DHLacCer (Fig. 4F), but not sphingomyelin (Fig. 4G) and DHSM (Fig. 4H), compared to control values.

[0187] Consideration Several key findings emerge from this study. First, β-1,4-GalT-V expression, protein abundance, and IHC staining were all observed to be significantly increased in human CRC tissues compared to visually normal tissues. Second, the activity of β-1,4-GalT-V (i.e., synthesis of lactosylceramide) in human CRC tumors was statistically significantly higher compared to control tissues. Third, inhibition of glycosphingolipid synthesis reduced immunostaining of β-1,4-GalT-V and, consequently, reduced cell proliferation in cultured human CRC (HCT-116) cells. Fourth, enhanced dihydrosphingolipid metabolism (Figure 2G) was noted in tumor tissues, with significantly increased levels of dihydrosphingomyelin compared to visually normal tissues.

[0188] Immunostaining with β-1,4-GalT-V antibody also enabled the pathologist to clearly distinguish between normal epithelial cells (Figure 1A) and cancerous epithelial cells (Figure 1C-E). After staining, tissues were evaluated for tumor adequacy, and 20 cases in which adequate tumor tissue was available for evaluation were selected. Examination of immunostaining for GalT-V revealed various degrees of positivity in tumor cells (Table 1): 1+ (15%; 3 / 20), 2+ (65%; 13 / 20), and 3+ (20%; 4 / 20). Staining was mostly observed in the tumor cytoplasm and only rarely in the nuclei of tumor cells. When available, weak (1+) to moderate (2+) cytoplasmic staining was also observed in adjacent normal colonic mucosa in 79% of cases (15 / 19). Furthermore, computerized (Asperion program) analysis of 20 such tissues provided a quantitative estimate of the immunostaining index, resulting in a substantial H-score of 100–300 (on a scale of 0–300) (21). These findings provide evidence that β-1,4-GalT-V immunostaining may indeed serve as a novel biomarker for CRC progression. Strong β-1,4-GalT-V immunostaining of capillary endothelial cells was also observed, consistent with our previous studies (30, 31). Within squamous epithelial cells, strong immunostaining was observed in the perinuclear region, suggesting that the antigen is concentrated within the Golgi apparatus (data not shown).

[0189] Cytoplasmic β-1,4-GalT-V immunostaining in both control and CRC tumor tissues provides evidence that β-1,4-GalT-V must exist in membrane-bound as well as soluble forms. Solubility allows the measurement of this antigen in various body fluids via non- or minimally invasive procedures. Brush border membranes of colonic epithelial cells also reacted positively to the antibody, providing evidence that in colorectal tissues GalT-V can be released in exosomes.

[0190] Previous studies have shown that β-1,4-galactosyltransferases share a common stem region. Because the stem region is short in β-1,4-GalT-1 (lactosamine synthase), most of the enzyme is localized in the cytoplasm of the Golgi and relatively little at the plasma membrane (32). In addition, it has been proposed that the number of hydroxylated amino acids that make up the stem region determines the localization of β-1,4-galactosyltransferase, although other factors may determine the localization of this protein. Further studies are needed to examine whether changes in the stem region allow for plasma membrane localization of β-1,4-GalT-V.

[0191] Moreover, the enrichment of β-1,4-GalT-V observed by IHC was further substantiated by quantitative measurements of β-1,4-GalT-V protein mass. There was a statistically significant increase in β-1,4-GalT-V mass in CRC tissues compared to control tissues (Figure 1F). The activity of lactosylceramide synthase (and thus lactosylceramide mass) was also significantly higher in CRC tissues compared to controls (Figure 1G). In contrast, the differences in the levels of other sphingolipids in tumor versus control tissues were not statistically significant (Figure 2A-G).

[0192] Another interesting finding of this study was that the dihydrosphingolipid pathway (Figure 7) was significantly active in tumor tissues (Figure 2G). For example, we observed that the masses of dihydroceramide, dihydroGlcCer / dihydroGalCer, and dihydroLacCer were all slightly elevated (but not significantly) in tumor tissues. However, the most significant difference between tumor tissues and visible normal tissues was that the level of dihydrosphingomyelin was markedly increased in tumor tissues (Figure 2G). Recent studies have suggested that dihydrosphingolipids, such as dihydroceramide, play an important role in autophagy (33, 34). However, the role of dihydrosphingomyelin in human CRC needs to be investigated.

[0193] The quantitative RT-PCR studies herein revealed increased B4GALT5 gene expression in tumor tissues, but comparatively not that of its isoform B4GALT6 or homolog UGCG (Figure 1I). These observations were confirmed using serial analysis of gene expression techniques to confirm the specific increase of β-1,4-GalT-V, but not β-1,4-GalT-VI, in CRC tumors (30). Thus, the increase in β-1,4-GalT-V gene and protein expression is specific to CRC tissues and may well serve as a biomarker for diagnosing this disease and measuring drug response. Previously, several other genes have been suggested to serve as CRC biomarkers, including NMT1, APC, and TP53 (35, 36, 37). Therefore, the expression of these genes was analyzed and upregulation of all three was observed in CRC tumors versus normal tissues (Figure 1H). Thus, upregulation of β-1,4-GalT-V could be feasibly added to the three-biomarker panel and thus enhance the predictive value for CRC diagnosis.

[0194] In HCT-116 cells, β-1,4-GalT-V immunostaining was also observed within the plasma membrane and the inner layer of the cytosol. Furthermore, immunoreactivity increased as cells grew from 24 to 96 h (Figure 3A-H), correlating with increased expression of β-1,4-GalT-V. In contrast, inhibition of glycosphingolipid synthesis with D-PDMP significantly attenuated GalT-V immunoreactivity in HCT-116 cells (Figure 3A-H), thus providing evidence that the treatment can reduce β-1,4-GalT-V protein mass and dihydroglycosylceramide / LacCer levels in these cells, and consequently, cell proliferation (Figure 3A-H).

[0195] Immunostaining of UGCG in HCT-116 cells was performed. Similarly, this antigen / enzyme was found to be localized in the perinuclear region and cytoplasm. However, treatment with D-PDMP did not reduce the immunoreactivity to anti-UGCG antibodies. Thus, the treatment may reduce glucosylceramide levels in HCT-116 cells by inhibiting enzyme activity.

[0196] In addition to human CRC tumors, HCT-116 cells were also found to have an active dihydrosphingolipid pathway. Treatment with D-PDMP reduced the levels of all dihydrosphingolipid species in our study, except for the level of dihydrosphingomyelin. Further mechanistic studies are required to further address this observation. The net result of D-PDMP treatment was also found to be a dose-dependent reduction in HCT-116 cell proliferation.

[0197] In summary, this study showed a specific increase in gene expression, protein levels, and enzyme activity of β-1,4-GalT-V in human CRC, concomitant with an increase in lactosylceramide content. These molecular and biochemical data were further substantiated by IHC and pathology studies. These findings demonstrate that the levels of β-1,4-GalT-V and lactosylceramide in human liquid biopsy specimens may complement other currently used biomarkers (e.g., NMT1, APC, and TP53), thus increasing the positive predictive value of CRC. Finally, inhibition of glycosphingolipid synthesis may be a novel approach to treat human colorectal cancer, and possibly other types of cancer.

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Zheng, J. Kollmeyer, H. Symolon, et al., Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy. Biochim. Biophys. Acta. 1758(12) (2006) 1864-84. https: / / doi.org / 10.1016 / j.bbamem.2006.08.009. (34) M.M. Siddique, Y. Li, B. Chaurasia, et al., Dihydroceramides: from bit players to lead actors, J. Biol. Chem. 290(25) (2015) 15371-9. https: / / doi.org / 10.1074 / jbc.R115.653204. (35) C.E. Ducker, J.J. Upson, K.J. French, et al., Two N-myristoyltransferase isozymes play unique roles in protein myristylation, proliferation, and apoptosis, Mol. Cancer Res. 3(8) (2005) 463-476. https: / / doi.org / 10.1158 / 1541-7786.MCR-05-0037. (36) T. Armaghany, J.D. Wilson, Q. Chu, et al., Genetic alterations in colorectal cancer, Gastrointest. Cancer Res. 5(1) (2012) 19-27. (37) XL Li, J. Zhou, ZR Chen, et al., p53 mutations in colorectal cancer- molecular pathogenesis and pharmacological reactivation, World J. Gastroenterol. 21(1) (2015) 84-93. https: / / doi.org / 10.3748 / wjg.v21.i1.84. (38) NS Radin, JA Shayman, J. Inokuchi, Metabolic effects of inhibiting glucosylceramide synthesis with PDMP and other substances, Adv. Lipid. Res. 26 (1993) 183-213. (39) J. Inokuchi, I. Mason, NS Radin, Antitumor activity via inhibition of glycosphingolipid biosynthesis. Cancer Lett. 38(1-2) (1987) 23-30. https: / / doi.org / 10.1016 / 0304-3835(87)90196-0. (40) J. Inokuchi, M. Jimbo, K. Momosaki, et al., Inhibition of experimental metastasis of murine Lewis lung carcinoma by an inhibitor of glucosylceramide synthase and its possible mechanism of action, Cancer Res. 50(20) (1990) 67316737.

[0199] Example 2: Immunotherapy using lactosylceramide synthase (β-1,4 GalT-V) antibodies to prevent colorectal cancer (CRC) in vivo. It is hypothesized that β-1,4 GalT-V plays an important role in human CRC, and manipulating this enzyme may be sufficient to attenuate tumor cell proliferation and metastasis by inhibiting angiogenesis. To test this hypothesis, we prepared a mouse monoclonal antibody against β-1,4 GalT-V and determined its effect on proliferation and angiogenesis in human and mouse CRC cells, human umbilical vein endothelial cells, and a mouse xenograft model of human CRC.

[0200] Materials and Methods A monoclonal antibody against a β-1,4 galactosyltransferase (GalT-V) peptide with the amino acid sequence (IGAQVYEQVLRSAYAKRNSSVND, SEQ ID NO:5) was prepared and characterized for its potency and used in ELISA, Western immunoblotting assays, and immunocapture of mouse and human tissues. The human colorectal cancer cell line (HCT-116) was a late gift from this institution from Dr. David Huso (Department of Comparative Medicine). The mouse colorectal cancer line MC-38 and thin tissue sections from mouse colorectal cancer were gifts from Dr. Cindy Sears in the Oncology Department of the institution. Human umbilical vein endothelial cells were purchased from Clonetics and cultured in the appropriate growth medium. Human microvascular endothelial cells were a gift from Stephanie Brindal in the same department. Vascular endothelial growth factor was purchased from R and D Inc. Matrigel, and all other reagents were purchased from Sigma-Aldrich. Biopolymer-encapsulated-PDMPs were prepared as described (6).

[0201] Cell proliferation assay 1×10 4 HCT-116 and MC-38 cells were seeded in 96-well sterile plastic trays and grown in 100 μL of Dulbecco's minimum essential medium containing 10% fetal bovine serum at 37 °C in a 5% CO2 air humidified incubator for 24 h. The medium was supplemented with 2% serum and 5 μCi / mL of DMSO. 3H) The medium was replaced with fresh medium supplemented with thymidine. Increasing dilutions of β-1,4 GalT-V antibody were added to the wells. In these experiments, human IgG or mouse IgG were used as negative controls, and D-PDMP (10 μM) or 1 uM BPD were used as positive controls. After 24 hours of incubation, the experiment was terminated and ( 3 H) Thymidine incorporation into DNA was measured by scintillation spectroscopy.

[0202] Angiogenesis assay Angiogenesis assays were performed using a commercially available kit from Chemicon Inc. (7).

[0203] Measurement of β-1,4 GalT-V activity The activity of β-1,4 GalT-V was measured in cells treated with and without β-1,4 GalT-V antibody as previously described (14).

[0204] Measurement of glycosphingolipids The masses of GSLs were determined by quantitative HPTLC as described ( 9 ).

[0205] Inhibition of tumor cell growth Normal male and female mice (C57 BL-6) were purchased from The Jackson Laboratory and fed with normal mouse chow. Semi-confluent cultures of HCT-116 cells were harvested and cell pellets were resuspended in medium supplemented with Matrigel at a ratio of 70:30 (by volume). The back hair of the mice was removed using a hair remover, Nair (Church and Dwight Co.), and the hairless skin area was cleaned with an alcohol swab. Then, 4 × 10 6 HCT cell suspension was injected subcutaneously. One week later, 100 μl of GalT-V antibody or 100 μL of BPD (1 mg / kg body weight) was injected subcutaneously at the tumor cell injection site daily for 3 weeks. As hair had grown in the shaved area, a Nair was used to remove the hair to expose the skin area, and the mice were photographed and documented.

[0206] immunohistochemistry Thin tissue sections were cut from mouse colorectal cancer tumor tissues and subjected to immunohistochemical staining with β-1,4 GalT-V antibody as previously described (8). Briefly, sections were deparaffinized, hydrated, and incubated with β-1,4 GalT-V antibody (1:600 ​​dilution) for 30 min. A second antibody, anti-rabbit HRP, was applied and a brown signal was developed using DAB chromogen detection (Leica Biosystems). Slides were then counterstained with hematoxylin, washed, dehydrated, coverslipped, and photographed (8).

[0207] result GalT-V antibodies are internalized into endothelial cells and HCT-116 cells in a time-dependent manner. In human microvascular endothelial cells, fluorescently tagged GalT-V was taken up at 4°C. Upon temperature shift to 37°C, a time-dependent increase in GalT-V antibody uptake into the cytoplasm and in the perinuclear region representing the Golgi apparatus was observed. Preincubation of cells with excess GalT-V peptide. Similarly, HCT-116 cells also took up fluorescently tagged β-1,4 GalT-V antibody in a similar temperature- and time-dependent manner. These studies demonstrated that β-1,4 GalT-V antibody is taken up and internalized by human endothelial cells and HCT-116 cells in a time- and temperature-dependent manner.

[0208] GalT-V antibodies inhibit proliferation in human and mouse colorectal cancer cells. 3 H-thymidine incorporation studies revealed that in human colorectal cancer cells, GalT-V antibodies dose-dependently reduced cell proliferation, and this inhibition was within the range of inhibition observed using D-PDMP, a pharmacological inhibitor of β-1,4 GalT-V.

[0209] Similarly, the β-1,4 GalT-V antibody also dose-dependently inhibited proliferation of mouse colorectal cells (Figure 8);MC-38 (Figure 9). No inhibition of cell proliferation was observed when cells were incubated with mouse IgG.

[0210] GalT-V antibody inhibits VEGF-induced angiogenesis in human umbilical vein endothelial cells. Figures 10A-10H show photographs of tube formation / angiogenesis in HUVECs. Figure 10I shows the corresponding quantification of angiogenesis. Incubation of HUVECs with VEGF / FGF significantly increased tube formation / angiogenesis (Figure 10B) compared to the control (Figure 10A). Treatment with β-1,4 GalT-V antibody showed a dose-dependent decrease in VEGF-induced angiogenesis in HUVECs (Figures 10C-10F), but not rabbit IgG (Figure 10G).

[0211] GalT-V antibodies prevent tumor growth in mice. Seven days after the mice were injected with HCT-16 cells, the mice were treated with and without β-1,4 GalT-V antibody daily for 3 weeks. In parallel, another group of mice was treated with BPD (5 mpk) daily for 3 weeks at the tumor cell injection site. It was observed that treatment with β-1,4 GalT-V antibody (FIG. 11A, FIG. 11B) or BPD (FIG. 11C, FIG. 11D) completely prevented tumor growth and progression.

[0212] Consideration This study leads to the following conclusions: (i) treatment with β-1,4 GalT-V antibody dose-dependently reduced proliferation in cultured human and mouse colorectal cancer cells, (ii) treatment of human umbilical vein endothelial cells with β-1,4 GalT-V antibody dose-dependently reduced VEGF-induced angiogenesis, and (iii) treatment with β-1,4 GalT-V antibody and BPD prevented tumor growth in a mouse xenograft model of colorectal cancer.

[0213] β-1,4 GalT-V is a member of a large family of galactosyltransferases whose function is to transfer galactose from UDP-galactose to glucosylceramide to form lactosylceramide (1). It also transfers galactose to the GlcNAc β-1,6 mannose group of highly branched N-glycans characteristic of tumor cells (2, 3). Of these products, LC has been shown to serve as an independent mitogenic and angiogenic agent, as well as being involved in cell migration, apoptosis, and cell adhesion (4). Most importantly, LC acts as a surrogate to mediate the action of growth factors such as VEGF, FGF, PDGF, EGF, and the proinflammatory cytokine TNF, resulting in the above phenotypes depending on the cell type (4). Importantly, these growth factor and TNFα-induced phenotypes can be alleviated by the use of the pharmacological agents BPD and D-PDMP (6, 9) and GalT-V genetic manipulation in vitro (7) and in vivo (15) (Figure 12). This is the first report showing that β-1,4 GalT-V immunotherapy is effective in reducing colorectal cancer growth and proliferation. The study herein shows that cellular β-1,4 GalT-V mRNA levels are specifically increased in human colorectal cancer tissue endothelium, and as a result, the mass of β-1,4 GalT-V protein also increases in a cancer stage-dependent manner. In normal colonocytes, β-1,4 GalT-V is found mostly in the cytoplasm / Golgi, but in colorectal cancer, it is present to some extent in the plasma membrane of human colorectal cancer cells in addition to the brush border membrane of epithelial cells (8). This allows β-1,4 GalT-V antibodies to bind to GalT-V protein. At 37°C, fluorescently tagged β-1,4 GalT-V antibodies were observed to bind to antigens in the majority of the cytoplasm of normal human endothelial cells and HCT-116 cells (8). Large amounts of unlabeled antibodies competitively inhibited uptake of the tagged antibodies, suggesting that binding and uptake of β-1,4 GalT-V antibodies is specific in cultured colorectal cells (data not shown).

[0214] Our previous study showed that LCs are generated due to growth factor-induced activation of β-1,4 GalT-V, an activated NAD(P)H oxidase, generating reactive oxygen species that act as signaling intermediates in the mitogen-activated protein kinase / c-fos pathway leading to cell proliferation. (4) In this study, we observed that treatment with β-1,4 GalT-V antibody reduced β-1,4 GalT-V enzyme activity and LC mass, thus attenuating cell proliferation in HCT-116 cells.

[0215] Monoclonal antibodies are a specific type of antibody / protein that has been created for therapeutic use. Such antibodies can be used in targeted therapy to block abnormal proteins in cancer cells. Some monoclonal antibodies bind specifically to cancer cells that express that protein, so monoclonal antibodies can be used in immunotherapy. Thus, by identifying the cancer cells, it allows the immune system to attack and destroy them. Another type of antibody can affect cancer growth by releasing the brakes on the immune system, destroying them. Research has shown that the programmed cell death (PD1) / programmed cell death ligand (PDL-1), CTLA-4 pathway is important for the immune system's ability to control cancer growth. Such pathways are called "immune checkpoints." Some types of cancers make fair use of these pathways to escape the immune system. In contrast, immune checkpoint inhibitors, such as pembrolizumab (Keytruda), are useful for identifying blockade by the PD-L1 protein, which acts like a protective shield in cancer cells. Recently, pembrolizumab was approved by the FDA to treat tumor-metastatic cancers that cannot be treated by chemotherapy as well as Merkel skin cancer due to Merkel polyomavirus infection. Therefore, this checkpoint inhibitor can target any tumor in the body and is therefore called a tumor-independent treatment. Nivolumab is a drug approved to treat CRC with M91-H or dMMR in patients after chemotherapy has failed. Interferons and interleukins are also used to fight cancer and develop the immune system to generate cancer-destroying cells. The 2-year success rate of immunotherapy was highest in stage IV lymphoma (82%) and only 38% in patients with stage IV CRC. β-1,4 GalT-V monoclonal antibody is of IgG type and may be well-served in targeted therapy to block excess amounts of β-1,4 GalT-V protein found in human CRC tissues and decorate the inner surface of the cell membrane of endothelial cells and cytoplasm of cultured human CRC cells (8).The use of humanized β-1,4 GalT-V monoclonal antibodies alone or in combination with BPD and / or other CRC drugs, such as nivolumab, may be a future direction of research to accelerate our therapeutic efforts to alleviate CRC. Because β-1,4 GalT-V protein is also overexpressed in renal cancer (9), multiple applications of this immunotherapy approach may be found.

[0216] References 1. Kolmakova A, Chatterjee S. Platelet derived growth factor recruits lactosylceramide to induce cell proliferation in UDPGal:GlcCer:β1 4Galactosyltransferase (GalT-V) mutant Chinese hamster ovary cells. Glycoconj J 2005; 22: 401-7. 2. J. Arango, M. Pierce, Comparison of N-acetylglucosaminyltransferase activities in Rous sarcoma-transformed baby hamster kidney (RS-BHK) and BHK cells, J. Cell. Biochem., 37 (2) (1988), pp. 225-231. 3. K. Shirane, T. Sato, K. Segawa, et a!. Involvement of beta-1,4-galactoslytransferase V in malignant transformation-associated changes in glycosylation, Biochem. Biophys. Res. Commun., 265 (2) (1999), pp. 434-438. 4. Chatterjee, S. and Pandey, A. The Yin and Yang of lactosylceramide metabolism: implications in cell function. Biochem.Biophys Acta. 1780:370-382, 2007. 5. Chatterjee, S. Kolmakova, A and Mohanraj,R. Regulation of lactosylceramide synthase; implications as a drug target. Curr. Drug Targets 9: 272-281, 2008. 6. D. Bedja, W. Yan, V. Lad, et al. Inhibition of glycosphingolipid synthesis reverses skin inflammation and hair loss in ApoE - / - mice fed western diet, Sci. Rep., 8 (2018), p. 11463. 7. M. Rajesh, A. Kolmakova, S. Chatterjee. Novel role of lactosylceramide in vascular endothelial growth factor-mediated angiogenesis in human endothelial cells, Circ. Res., 97 (8) (2005), pp. 796804. 8. S. Chatterjee, et al. Lactosylceramide synthase B-1,4GalT-V : A novel target for the diagnosis and therapy of human colorectal cancer. Bio Chem. Biophys. Res. Comm. 2019; 508, 380-400. 9. S. Chatterjee, N. Alsaeedi, J. Hou, et al., Use of a glycolipid inhibitor to ameliorate renal cancer in a mouse model, PloS One, 8 (5) (2013), Article e63726. 10. P. Favoriti, G. Carbone, M. Grego, et al. Worldwide burden of colorectal cancer: a review, Updates Surg, 68 (1) (2016), pp. 7-11. 11. S.I. Hakomori, W.T. Murakami, Glycolipids of hamster fibroblasts and derived malignant-transformed cell lines, Proc. Natl. Acad. Sci. U.S.A., 59 (1) (1968), pp. 254-261. 12. J.W. Dennis, S. Laferte, Oncodevelopmental expression of -GlcNAc beta 1-6Man alpha 1-6Man beta 1-branched asparagine-linked oligosaccharides in murine tissues and human breast carcinomas, Cancer Res., 49 (4) (1989), pp. 945-950. 13. S. Hakomori, Tumor malignancy defined by aberrant glycosylation and sphingo(glycol)lipid metabolism, Cancer Res., 56 (23) (1996), pp. 5309-5318. 14. YY Liu, RA Hill, YT Li, Ceramide glycosylation catalyzed by glucosylceramide synthase and cancer drug resistance, Adv. Cancer Res., 117 (2013), pp. 59-89. 15. Y. Wei, F. Zhou, Y. Ge, et al., β1,4-Galactosyltransferase V regulates self-renewal of glioma-initiating cell, Biochem. Biophys. Res. Commun., 396 (3) (2010), pp. 602-607.

[0217] Example 3: β-1,4 GalT-V monoclonal antibody to reduce atherosclerosis and weight loss in type II diabetic mice (db / db). Male type II diabetic mice (db / db) aged 11 weeks were purchased from Jackson Laboratory. We fed the mice with normal mouse chow and water. At 30 weeks of age, the mice were divided into two groups. The first group of mice (placebo) received saline (100 μL) by intraperitoneal injection daily for 6 weeks. The second group of mice was treated with GalT-V antibody (1 mg / kg body weight) for the same period. At the end of 36 weeks of age, the mice were weighed and various tissues were collected and frozen until further analysis. Next, to extract total lipids, about 10 mg of liver tissue was excised (internal standards of C12 ceramide and C12 sphingomyelin were added to confirm recovery), homogenized in acetonitrile, and centrifuged at 1000 rpm for several minutes. The clear supernatant was saved and the pellet was extracted repeatedly. The pooled supernatant was dried in N2 and reconstituted in chloroform-methanol (2:1 v / v). Appropriate aliquots of lipid extracts were loaded onto high performance thin layer chromatography plates. Standard neutral lipids consisting of cholesterol esters, triglycerides and cholesterol were also loaded to calibrate the plates. Plates were developed using heptane:ethyl ether and acetic acid (65:16:1 v / v) as solvent. Lipids were identified by exposing the plates to iodine vapor and photographed. Quantification of lipid mass was performed by densitometric analysis using individual lipid standard curves and two-tailed parametric t-tests.

[0218] The results demonstrated that diabetic mice treated with GalT-V monoclonal AB had significantly reduced cholesterol levels compared to placebo mice. The treatment also significantly reduced triglyceride levels compared to placebo mice. In addition, the treatment reduced the mice's body weight by approximately 20%.

[0219] Example 4: We showed that treatment with GalT-V AB in cultured human CRC cells (HCT-116) dose-dependently reduced cell proliferation and angiogenesis. [1] Furthermore, we demonstrated enrichment of CF-750-tagged GalT-V-Ab in xenograft tumors in a NOD-SCID mouse model of CRC.

[0220] We further wish to determine whether treatment with GalT-V-Ab affects tumor growth and metastasis in mouse orthotopic tumors in the mouse rectum.

[0221] method: Rectal cancer was treated with live human colorectal cancer cells (HCT-116) (1 × 10 in 50 uL in McCoy's medium) 6 The IL-16 expression was induced in NOD-SCID / immunocompromised mice by injecting 100 μL of PBS (placebo), 20 μg / kg GalT-V-Ab, and 200 μg / kg GalT-V-Ab by IV injection into the tail vein.

[0222] Figure 13 summarizes the scheme followed to study the effect of treatment with GalT-V antibodies in a mouse model of rectal cancer. Briefly, HCT-116 cells were grown in tissue culture to approximately 75% confluence. Cells were harvested using trypsin, centrifuged, and cell numbers were counted. 1 x 10 cells suspended in serum-free McCoy's medium were 6The cells were injected into the rectum of male NOD SCID mice (approximately 10 weeks old). Two weeks later, when rectal tumors were visible and quantified, treatment was initiated. Mice were divided into three groups: A. Placebo, B. 20ug GalT-V-Ab / kg body weight, and C. 200ug GalT-V-Ab / kg body weight. Treatments were administered IV into the tail vein every other day, and body weight and tumor size were recorded. After 4 weeks of treatment, mice were divided into two groups: 1. Some mice were used for tumor imaging, and 2. The remaining mice were euthanized and blood was collected to obtain plasma and various tissues. Half of the tumor tissue was preserved in formalin solution and used for lipid analysis as well as immunohistochemistry studies. The other half of the tumor tissue was snap frozen and used for molecular studies.

[0223] result: Treatment with GalT-AB antibody dose-dependently reduces rectal tumor volume in an orthotopic model of CRC in NOD-SCID mice.

[0224] The body weight of the mice did not change regardless of the treatment over the 4 weeks (Figure 14). Treatment with GalT-V-Ab had a dose- and time-dependent reduction in tumor volume. For example, after 2 weeks of treatment, we noted that tumor volume was reduced by approximately 38-41% in mice receiving 20ug / kg and 200ug / kg of antibody (Figure 15A). After 4 weeks of treatment, tumor volumes in mice treated with 20 and 200ug / kg of antibody were 32% and 41% lower, respectively, compared to placebo mice bearing rectal tumors (Figure 15B).

[0225] Molecular imaging of tumor volume reduction: After 4 weeks of treatment, mice were injected with 50 uL of CF-750 tagged carcinoembryonic antigen (CEA), an established tumor biomarker.Two hours later, whole mice were imaged, as shown in FIG.

[0226] In FIG. 16, an optical image of a mouse bearing an HCT-116 rectal orthotopic tumor is shown, showing in clockwise order: liver (1), colon (2), blood (3), brain (4), tumor indicated by black arrow (5), small intestine (6), spleen (7), heart (8), lungs (9), cecum (10), stomach (11), and kidneys (12).

[0227] HCT-116 human CRC tumor cells (1 x 10 6 ) were implanted into the rectum of NOD-SCID male mice (10 weeks old). Two weeks later, treatment was started by IV injection every other day for 4 weeks. Two hours after delivery of CF-750 antibody against carcinoembryonic antigen (CEA), mice were imaged using a Forager imaging machine (Figure 16A): control, mice without CEA-Ab, M1, mice treated with 200ug GalT-V Ab / Kg body weight, M2, mice given placebo, and M3, mice treated with 20ug GalT-V Ab / Kg body weight. After 24 hours, mouse tissues were harvested, placed in petri dishes, photographed (Figure 16B), and finally imaged (Figure 16C).

[0228] The imaging study involved preparing a CF-750 tagged antibody against human CEA and delivering it by IV injection into the tail vein. As shown in Figure 16A, whole body imaging of mice treated with placebo (M2 in Figure 16A) revealed extensive localization / concentration of tag in the rectum (green), liver (blue) and head (blue). Mice treated with 20ug GalT-V Ab had relatively little tag associated with these tissues (M3 in Figure 16A). However, upon treatment with 200ug GalT-V-Ab, most of the tag was concentrated in the rectal tumor (blue) (M1 in Figure 16A).

[0229] Figure 16B shows photographs of individual mouse tissues before and after imaging 24 hours after injection of CF-750-CEA-Ab (Figure 16C). The placebo tumor (Figure 16C, M2) accounted for the highest level of tags (red). The staining intensity was highest in the placebo liver (M2 in Figure 16C) compared to the GalT-V-Ab treatment group. Some tags were seen in the lungs, kidneys and blood (red). We also note that the intensity of staining was dose-dependently decreased in the tumor tissue (red) upon treatment with 20 μg (M3 in Figure 4C) and 200 μg GalT-V-Ab / kg (M1 in Figure 16C). Since we used intact CEA-Ab (IgG1), it entered the liver via the portal circulation and the kidneys and lungs. This observation is consistent with previous reports. In the future, we plan to use only the CF-750-Fab fragment of CEA-Ab, since it is taken up in tumor tissue vs liver at a ratio of >10:1.

[0230] Treatment with GalT-V antibody and expression of tumor marker genes: Quantitative analysis of gene expression by reverse transcription polymerase chain reaction (RT-PCR) shows that CEA and NMT-1 mRNA levels were similar in the placebo and treatment groups (ns). However, expression of B4 GALT-V was decreased in the 20U / kg group compared to controls and was not significant in the 200U / kg group. See Figure 17.

[0231] Treatment with GalT-V antibody reduced serum levels of GalT-V and tumor levels of lactosylceramide. Our previous study in human CRC tissues revealed that both the mass of GalT-V and the level of LacCer were increased compared to visible normal tissues from the same CRC patients. We also showed that treatment with D-PDMP, an inhibitor of GalT-V, reduced the level of GalT-V and the mass of LacCer in HCT-116 cells and reduced cell proliferation. Also, in human endothelial cells, treatment with D-PDMP and GalT-V-Ab reduced angiogenesis. Finally, in a mouse model of renal cancer treatment with D-PDMP, tumor volume and GalT-V mass were significantly reduced [2]. Therefore, we measured the mass of GalT-V in plasma and the level of LacCer in tumor tissue. As shown in Figure 18A, treatment with GalT-V-Ab significantly reduced the level of GalT-V in plasma. Furthermore, the LacCer level in the tumor tissue tended to decrease in the GalT-V-treated mice (FIG. 18B).

[0232] overview: 1. In an orthotopic mouse model of rectal cancer inoculated with live HCT-116 cells, there was a significant increase in tumor volume over a 6 week period. In mice given 2.20 ug / kg GalT-V Ab, the tumor volume was reduced by approximately 32% to 38% in a treatment- and time-dependent manner with GalT-V-Ab dose from 2 to 4 weeks. 3. Treatment with the higher dose of GalT-V-Ab reduced tumor volume by 41% compared to placebo tumor volume. 4. The biochemical and molecular basis of this observation was explained by our observation that the treatment targeted the antigen GalT-V by reducing its mass in the blood, and similarly targeted its product LacCer in tumor tissues by reducing GalT-V gene expression, at least when mice were treated with 20ug GalT-V Ab / kg. 5. Imaging studies reproduced the above observations. Moreover, imaging studies revealed that in placebo mice, 6 weeks after inoculation, tumor metastasis occurred in other tissues, such as the liver, kidneys and lungs, which are major tissues in the blood circulation. This was significantly reduced when mice were treated with low or high doses of GalT-V-Ab, and was markedly attenuated in mice treated with high doses of GalT-V-Ab.

[0233] Immunotherapy with GalT-V antibodies is an effective treatment for preventing or inhibiting the growth and metastasis of rectal tumors.

[0234] Example 5: The localization of GalT-V in human colorectal cancer cells and its co-localization with cell surface proteins and cellular organelle-specific biomarkers of the Golgi apparatus were determined.

[0235] In vitro confocal fluorescence images of HCT-116 cells were taken (see FIG. 19). Cell surface localization of labeled mouse monoclonal antibody (mAb) raised against human GalT-V (red, rhodamine, Sigma Aldrich) and monoclonal anti-caveolin-1 antibody raised in mouse (green, Alexa Fluor 488 NHS ester, ThermoFisher Scientific) was observed after 1 hour of incubation at 4° C. Colocalization of GalT-V and caveolin (yellow, note white arrow) when images were merged was also observed. No colocalization with monoclonal anti-Golgi 58k protein (green, Alexa Fluor 488 NHS ester, ThermoFisher Scientific) was observed at 4° C. when corresponding images were merged.

[0236] A mouse monoclonal antibody (mAb) against human GalT-V was tagged with rhodamine. Caveolin-1 Ab and Golgi Ab were also tagged with indocyanine green. These antibodies were then used to determine the localization of GalT-V using confocal microscopy (following the protocol from DOI: 10.1021 / acs.molpharmaceut.0c00457). Nuclei were stained with DAPI stain (ThermoFisher Scientific) which binds to DNA (blue). As shown in Figure 19 (top panel), GalT-V (red stain) and caveolin (green stain) antibodies at 4°C bound strongly to the cell surface of the human colorectal cancer cell line HCT-116. When these figures were merged (top panel on the far right), it was observed that the immunostaining of GalT-V and caveolin overlapped and changed color to yellow (indicated by white arrow). When cells were incubated with methyl-cyclodextrin, a blocker of internalization, the majority of GalT-V immunostaining was associated with the cell surface. In contrast, when cells were treated with an antibody against the Golgi apparatus (Figure 19, lower panel), no immunostaining was observed. Confocal microscopy images were taken on a Zeiss LSM 700 using 20x magnification and processed with Image-J.

[0237] Example 6: In vitro confocal fluorescence images of HCT-116 cells with fluorescently tagged antibodies against GalT-V (red), caveolin (green) and Golgi (green). See Figure 20. Intracellular internalization of GalT-V (red) and caveolin (green) antibodies was observed after 2 hours of incubation at 37°C. When the images were merged, limited colocalization of GalT-V and caveolin antibodies was observed (yellow, see white arrow).

[0238] The cells were then warmed to 37°C for 2 hours and then subjected to confocal microscopy. As shown in the top panel of Figure 20, GalT-V Ab (red staining) was internalized in the cells and associated with the cytoplasm. Since caveolin contained within coated pits is known to be internalized back to the cell surface, we observed cytoplasmic and cell surface immunostaining (green staining). Faint immunostaining with Golgi antibody (green staining) was observed in the bottom panel.

[0239] Example 7: Human coronary artery endothelial cells (HAEC) and human colorectal cancer cells (HCT-116) 89 Binding and internalization of Zr]GalT-V antibody Cell cultures were incubated with increasing concentrations of 89 The cells were incubated with [Zr]GalT-V antibody, washed, and radioactivity was measured in an automated gamma counter (1282 Compu-gamma CS, Pharmacia / LKB Nuclear, Inc.). Note that HCT-116 cells bound significantly more GalT-V antibody compared to HCAEC cells (n=3, p****(5ug / mL)=0.000053, p***(10ug / mL)=0.000805).

[0240] FIG. 21 shows the [ 89 Zr]GalT-V antibody binding.

[0241] Example 8: In human coronary artery endothelial cells (HCAEC) and human colorectal cancer cells (HCT-116) 89 Zr]GalT-V antibody binding Cell cultures were incubated with increasing concentrations of 89The cells were incubated with [Zr]GalT-V antibody, washed, and radioactivity was measured in a gamma counter. Note that HCT-116 cells bound and internalized significantly more amount of GalT-V antibody compared to HCAEC cells (n=3, p***(10ug / mL)=0.000124). The results are shown in Figure 22, which shows the [Zr]GalT-V antibody in human coronary artery endothelial cells (HCAEC) and human colorectal cancer cells (HCT-116) after 2 hours of incubation at 37°C. 89 Zr]GalT-V antibody binding.

[0242] Example 9: Cells were preincubated with or without the inhibitor GalT-V, D-PDMP (20 uM), and then [ 89 The binding and internalization of the [Zr]GalT-V antibody was measured. Since D-PDMP reduces the mass of GalT-V, 89 Zr]GalT-V antibody binding and internalization was also reduced (n=3, p**=0.0079). The results are shown in Figure 23, which shows that D-PDMP inhibits zirconium-tagged GalT-V antibody binding in human colorectal cancer cells (HCT-116).

[0243] Example 10: Cells were incubated with excess unlabeled (cold, 50ug / mL) GalT-V antibody and [ 89 The presence of unlabeled antibody was associated with increased expression of [Zr]GalT-V Ab in treated HCT-116 cells. 89 Note that Zn-Zn significantly attenuated the binding and internalization of Zn2+ (n=3, p****≦0.0001) in human colorectal cancer cells (HCT-116). 89 The specificity of binding and internalization of the [Zr]GalT-V antibody is shown in FIG.

[0244] Example 11: GalT-V antibodies are potent gamma emitters [ 89 The antibody was radiolabeled with [Zr] and is useful for in vitro studies as well as in vivo studies in mice. As shown in Figure 21, the [ 89[Zr]GalT-V Ab binding was concentration dependent. Furthermore, GalT-V Ab binding to HCT-116 cells was statistically significantly higher than that to HCAEC cells at both 4°C (Figure 21) and 37°C (Figure 22). D-PDMP, known to inhibit GalT-V activity and mass in HCT-116 cells, also bound less GalT-V Ab compared to untreated cells (Figure 23). 89 The specificity of [Zr]GalT-V Ab binding was assessed by incubating HCT cells with excess GalT-V antibody that could compete with the radiolabeled Ab. As shown in Figure 24, HCT-116 cells incubated with 50ug / mL cold unlabeled GalT-V showed significantly reduced binding (blue bars) compared to cells incubated with heat-radioactive GalT-V Ab (red bars). [Zr]GalT-V Ab binding to HCT-116 cells (Figure 25) 89 A time course study of [Zr]GalT-V Ab binding showed approximately linear binding from 30 min to 2 h in HCT-116 cells.

[0245] Example 12: In vivo xenofluorescence imaging of human CRC tumor-bearing mice In vivo xenofluorescence images of human CRC tumor-bearing mice were taken 5 hours after injection of CF-750-GalT-V antibody, showing the subcutaneous / xenotropic CRC tumors indicated by the black arrows shown in FIG.

[0246] 48 hours after injection of fluorescently tagged GalT-V antibody, a xenotropic CRC tumor is shown, indicated by a black arrow in Figure 27. Note that the intensity of CF-750-GalT-V antibody significantly increased in mice bearing xenotropic CRC tumors from 5 hours post-injection to 48 hours post-injection (blue spots).

[0247] As shown in FIG. 26, we were able to image xenotropic CRC tumors in mice within 5 hours after injection of CF-750-GalT-V antibody (indicated by black arrow). The bar on the right indicates the intensity of fluorescence. Deep blue and red represent very high and very low GalT-V antibody fluorescence intensity, respectively. Because the hair of these mice was autofluorescent, we were unable to image other internal organs, such as liver, spleen, etc. non-invasively after anesthesia in intact animals. We observed a time-dependent enrichment of fluorescence in the xenotropic tumors, as indicated by a color change from green to blue (compare FIG. 26 5 hours and FIG. 27 48 hours after injection with fluorescently tagged GalT-V antibody).

[0248] Example 13: Tissue distribution of CF-750 fluorescent GalT-V antibody in xenotropic tumor-bearing mice Figures 28A, B and C show the distribution of CF-750 GalT-V antibody fluorescence in individual tissues from subcutaneous / xenograft tumor-bearing mice: liver (1), colon (2), brain (3), tumor (4), sigmoid colon (5), large intestine (6), spleen (7), heart (8), lung (9), small intestine (10), cecum (11), stomach (12), kidney (13), muscle (14), and blood (15).

[0249] Seventy-two hours after injection of CF-750-GalT-V antibody, three xenotropic tumor-bearing mice (M1, M2, M3) were euthanized and individual organs were excised and photographed (Fig. 28A, B, C). Each organ was photographed first (left panel Fig. 28A, B, C), followed by xenotropic graft images. Note that the liver, spleen, and kidneys absorbed significant fluorescence. However, the tumor tissue (indicated by white arrows) accumulated the greatest amount of fluorescence.

[0250] In summary, our in vivo studies reveal that fluorescent tagging of GalT-V antibodies is a valuable reagent for non-invasive imaging of CRC tumors in mice. Over time, CF-750-GalT-V antibodies are primarily concentrated in tumor tissues. Significant fluorescence was also observed in the liver, kidneys and lungs, as these tissues are involved in the metabolism and subsequent excretion of antibodies. This additional information may be useful to determine the therapeutic efficacy of GalT-V antibodies in mitigating kidney and liver cancers, in addition to CRC. Also, CF-750-GalT-V antibodies can be used as a diagnostic tool in human CRC and other cancer tissues where GalT-V enrichment may occur.

[0251] Statistical analysis: Statistical analysis of data was performed using single / multiple unpaired t-test using GraphPad Prism 9 software.

[0252] Conclusion: 1. Immunohistochemical studies show that GalT-V colocalizes with caveolin on the cell surface in human HCT-116 cells. 2. Upon shifting the incubation temperature from 4°C to 37°C, GalT-V bound to the cell surface was internalized and present in the cytoplasm. Weak immunostaining was associated with the Golgi apparatus due to its low abundance. 3. [ 89 Binding and internalization studies using the [Zr]GalT-V antibody demonstrated concentration- and time-dependent binding to HCT-116 and HCAEC cells. 4. The binding was specific to the GalT-V antigen. 5. Binding was dependent on the cellular levels of GalT-V as follows: a. HCT-116 cells bound significantly more GalT-V antibody compared to HCAEC. b. D-PDMP-treated HCT-116 cells bound less than untreated cells. Our previous studies have shown that D-PDMP reduces GalT-V mass.

[0253] In human colorectal cells, GalT-V is localized to the cell surface and the corresponding 89Zr]GalT-V antibody binding and subsequent internalization.

[0254] Other embodiments From the above description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various applications and conditions, and such embodiments are also within the scope of the following claims.

[0255] All citations to sequences, patents, and publications in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A pharmaceutical composition comprising a therapeutically effective amount of an antibody, said antibody specifically binding to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope, said antibody comprising: a heavy chain variable region sequence having at least 80% amino acid sequence identity to EVQLEQSGAELARRPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGTTLTVSS (SEQ ID NO: 3); and / or A pharmaceutical composition comprising a light chain variable sequence having at least 80% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4).

2. The pharmaceutical composition of claim 1 , wherein the antibody comprises a heavy chain variable region sequence having the amino acid sequence set forth in SEQ ID NO:

3.

3. The pharmaceutical composition of claim 1 or 2, wherein the antibody comprises a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:

4.

4. The pharmaceutical composition of any one of claims 1 to 3, further comprising one or more secondary therapeutic agents.

5. The pharmaceutical composition of any one of claims 1 to 4, further comprising a peptide having at least 90% sequence identity with IGAQVYEQVLRSAYAKRNSSVND (SEQ ID NO: 5).

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the antibody is humanized.

7. The pharmaceutical composition of claim 6, wherein the antibody is a humanized antibody capable of specifically binding to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope.

8. A pharmaceutical composition comprising the humanized antibody of claim 7.

9. an expression vector comprising: gaagttcagctggagcagtctggggctgaactggctagacctggggcttcagtgaagttgtcctgtaggacttctggctacacctttacaa; actactggatgcagtggattaaacagaggcctggacaggtctggaatggattggggctatgcatcctggacgtgcgtatattaggtacaaccagaagttcc an expression vector comprising a heavy chain variable region sequence nucleic acid sequence having at least 80% sequence identity to agggcaaggccacattgactgcagataaatcctccagcacagcttacatgcaactcaacagcttggcatctgaggactctgcggtctattactgtgcaagatgggagtgactacgactactggggtcaaggcaccactctcacagtctcctca (SEQ ID NO: 1); or an expression vector, the expression vector comprising: agatagtgatggaaagacatatttgaattggttgttacagaggccagggccagtctccaaagcgcctaatctatctggtgtctaaactgggctctggagtcc an expression vector comprising a light chain variable region sequence nucleic acid sequence having at least 80% sequence identity to ctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagttttaattattgctggcaaggtacacatttttcctcggacgttcggtggaggcaccaagctggaaatcaaacgg (SEQ ID NO: 2); or An expression vector comprising: (i) a heavy chain variable region sequence nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:3; and (ii) a light chain variable region sequence nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

2.

10. A pharmaceutical composition comprising a therapeutically effective amount of a synthetic peptide comprising an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO:5 and at least one adjuvant; or A pharmaceutical composition comprising a therapeutically effective amount of (i) an antibody comprising (a) a heavy chain variable region sequence nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:3, and / or (b) a light chain variable region sequence nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:2; (ii) a synthetic peptide comprising an amino acid sequence having at least 80% amino acid sequence identical to SEQ ID NO:5; (iii) an adjuvant or a pharmaceutically acceptable carrier; or a pharmaceutical composition comprising A pharmaceutical composition comprising a therapeutically effective amount of (a) an antibody, the antibody specifically binding to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope, the antibody comprising: (i) a heavy chain variable region sequence having at least 80% amino acid sequence identity to EVQLEQSGAELARRPGASVKLSCRTSGYTFTNYWMQWIKQRPGQGLEWIGAMHPGRAYIRYNQKFQGKATLTADKSSSTAYMQLNSLASEDSAVYYCARWSDYDYWGQGTTLTVSS (SEQ ID NO: 3); and / or (ii) a light chain variable sequence having at least 80% amino acid sequence identity to DVVMTQTPPTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLGSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKR (SEQ ID NO: 4); and (b) a therapeutically effective amount of at least one inhibitor of glycosphingolipid synthesis; A pharmaceutical composition comprising:

11. A composition for use in a method for generating an immune response against β-1,4-galactosyltransferase-V (β-1,4-GalT-V) in a subject in need thereof, comprising a therapeutically effective amount of an amino acid sequence having at least 80% amino acid sequence identical to SEQ ID NO:5, and an adjuvant.

12. 1. A composition for use in a method of treating colorectal cancer in a subject suffering from or susceptible to colorectal cancer, the composition comprising an antibody that specifically binds to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope.

13. 1. A composition for use in a method of treating colorectal cancer in a subject suffering from or susceptible to colorectal cancer, the composition comprising a humanized antibody that specifically binds to a β-1,4-galactosyltransferase-V (β-1,4-GalT-V) epitope.

14. 11. A composition for use in a method for treating colorectal cancer in a subject suffering from or susceptible to colorectal cancer, the composition comprising a pharmaceutical composition, antibody, expression vector or peptide according to any one of claims 1 to 10.

15. A pharmaceutical composition for use in a method for treating colorectal cancer, comprising an antibody comprising (a) a heavy chain variable region sequence nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:3, and / or (b) a light chain variable region sequence nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

2.

16. 11. A composition for use in a method for treating diabetes, atherosclerosis, obesity, an autoimmune disease, or a disease associated with abnormal levels of β-1,4-galactosyltransferase-V (β-1,4-GalT-V), comprising the pharmaceutical composition, antibody, expression vector, or peptide of any one of claims 1 to 10, or a combination thereof.

17. 1. A composition for use in a method of diagnosing and treating colorectal cancer, comprising: measuring the level of β-1,4-galactosyltransferase-V (β-1,4-GalT-V) and / or glycosphingolipids in a biological sample from a subject, wherein increased levels of β-1,4-GalT-V and / or GSLs are elevated when compared to healthy subjects, and wherein elevated levels of β-1,4-GalT-V and / or GSLs are diagnostic of colorectal cancer; Administering to said subject diagnosed with colorectal cancer the pharmaceutical composition, antibody, expression vector or peptide of any one of claims 1 to 10, or a combination thereof, thereby treating colorectal cancer; 1. A composition for use in a method for diagnosing and treating colorectal cancer, comprising:

18. 1. A composition for use in a method for monitoring the progression and treatment of colorectal cancer in a subject, comprising: Administering to said subject diagnosed with colorectal cancer a pharmaceutical composition, antibody, expression vector or peptide according to any one of claims 1 to 10, or a combination thereof; measuring the levels of β-1,4-galactosyltransferase-V (β-1,4-GalT-V) and / or glycosphingolipids in a biological sample from the subject, wherein a decrease in β-1,4-GalT-V and / or GSL levels compared to baseline indicates a reduction in colorectal cancer cells and treatment of colorectal cancer; thereby monitoring the progression and treatment of colorectal cancer; 1. A composition for use in a method for monitoring the progression and treatment of colorectal cancer in a subject, comprising:

19. 11. A composition for use in a method of treating a subject suffering from or susceptible to suffering from macular degeneration, the composition comprising a pharmaceutical composition, antibody, expression vector or peptide, or a combination thereof, according to any one of claims 1 to 10.

20. 11. A composition for use in a method of treating a subject suffering from or susceptible to Alzheimer's disease, the composition comprising the pharmaceutical composition, antibody, expression vector or peptide of any one of claims 1 to 10, or a combination thereof.

21. 11. A composition for use in a method of treating a subject suffering from or susceptible to suffering from migraine or migraine pain, the composition comprising the pharmaceutical composition, antibody, expression vector or peptide of any one of claims 1 to 10, or a combination thereof.

22. 11. A composition for use in a method of treating a subject suffering from or susceptible to suffering from metabolic syndrome, the composition comprising the pharmaceutical composition, antibody, expression vector or peptide of any one of claims 1 to 10, or a combination thereof.

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