WO2025006452A2 - Molécules de ciblage de neu5gc-lewisa et leurs utilisations contre le cancer - Google Patents

Molécules de ciblage de neu5gc-lewisa et leurs utilisations contre le cancer Download PDF

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Publication number
WO2025006452A2
WO2025006452A2 PCT/US2024/035392 US2024035392W WO2025006452A2 WO 2025006452 A2 WO2025006452 A2 WO 2025006452A2 US 2024035392 W US2024035392 W US 2024035392W WO 2025006452 A2 WO2025006452 A2 WO 2025006452A2
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neu5gc
lewis
cancer
cell
tissue
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WO2025006452A3 (fr
WO2025006452A9 (fr
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Indu Venugopal
Shelby KNOCHE
Michael J. Mcguire
Kathlynn Corinne BROWN
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SRI International Inc
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SRI International Inc
Stanford Research Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Definitions

  • the disclosure relates to the discovery of Neu5Gc-Lewis a as a biomarker for cancer cells, exosomes and tissues and provides methods for diagnosis, treatment, and drug targeting based on the same, as well as molecules that specifically bind to Neu5Gc- Lewis a .
  • Biomarkers play a crucial role in cancer research, diagnosis, prognosis, and treatment. They may help identify the presence of cancer at an early stage, even before symptoms become apparent. Early detection allows for timely intervention and potentially better treatment outcomes. Biomarkers may also provide valuable information about the prognosis or likely outcome of a particular cancer. They may help determine the aggressiveness of the disease, the risk of recurrence, and the overall survival rate. Moreover, biomarkers allow for targeted drug delivery, which significantly improves efficacy of cancer therapy. Drug targeting allows for the delivery of therapeutic agents directly to cancer cells or tumor sites, minimizing damage to healthy tissues.
  • Targeted therapies may be combined with other targeted agents or conventional chemotherapy drugs to create synergistic effects.
  • combination therapies may enhance treatment efficacy and overcome limitations associated with single-agent therapies.
  • Current targeting agents are primarily either small-molecule drugs or monoclonal antibodies. Small molecule drugs may enter cancer cells easily, but they cannot target cancer cells specifically. On the other hand, some monoclonal antibodies have the ability to target specific proteins found on the surface of cancer cells, but they generally do not have the ability to internalize into them. There is a need for molecules that may serve as disease biomarkers and do both - target as well as internalize into cancer cells.
  • the disclosure provides that Neu5Gc-a-2,3-Gal-P-l,3-(Fuc- a-l,4)-GlcNAc-P, or Neu5Gc-Lewis a , is a biomarker for cancer cells and tissues. Accordingly, the disclosure provides that Neu5Gc-Lewis a may be used as a biomarker for early cancer detection and drug targeting. Furthermore, the disclosure also provides agents that may specifically target cancer cells by specifically binding Neu5Gc-Lewis a , such as peptides and antibodies.
  • these peptides comprise MGS5, which may be MGS5_V1 and MGS5_V2.
  • the disclosure provides that both the MGS5_V1 and MGS5_V2 peptides may be conjugated to a therapeutic agent (such as an anti-cancer drug), allowing it to specifically target the tumor cells in a patient. This has several advantages including improved therapeutic efficacy while avoiding systemic toxicity.
  • Embodiment 1 A method of detecting Neu5Gc-Lewis a on the surface and/or extracellular matrix of a cell or tissue, comprising (a) obtaining or having obtained a cell or tissue sample; (b) detecting whether the Neu 5 Gc-Lewi s a is present on the surface and/or extracellular matrix of the cell or tissue by contacting the cell or tissue sample with a molecule known to bind Neu5Gc-Lewis a and that does not bind to any other form ofNeu5Gc or any other Sialyl-Lewis a , including free Neu5Gc, also referred to henceforth as a Neu5Gc-Lewis a -binding molecule, and detecting binding between Neu5Gc-Lewis a and the Neu5Gc-Lewis a -binding molecule on the surface and/or extracellular matrix of the cell or tissue, optionally wherein the cell or tissue sample is obtained from a subject; and, optionally further comprising a molecule
  • Embodiment 2 The method of embodiment 1, wherein the Neu 5 Gc-Lewi s a - binding molecule comprises a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer.
  • the Neu 5 Gc-Lewi s a - binding molecule comprises a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer.
  • Embodiment 3 The method of embodiment 1 or 2, wherein the peptide has at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO:1 or SEQ ID NO:2.
  • Embodiment 4 The method of embodiment 1, 2, or 3, wherein the Neu5Gc- Lewis a -binding molecule does not bind any other form of Neu5Gc or any other Sialyl- Lewis 21 , including free Neu5Gc.
  • Embodiment 5 The method of any one of embodiments 1 to 4, wherein the binding takes place in vitro.
  • Embodiment 6 The method of any one of embodiments 1 to 5, wherein the cell is a cancer cell.
  • Embodiment 7 A method of identifying a cell, exosome or tissue as a cancer cell, exosome or tissue or diagnosing cancer in a subject, comprising detecting the presence of Neu5Gc-Lewis a in a cell, exosome or tissue sample from the subject, wherein the presence of Neu 5 Gc-Lewi s a in the cell, exosome or tissue indicates the presence of a cancer cell, exosome or tissue and diagnosis of cancer in the subject, optionally further comprising administering an anti-cancer therapy to the subject.
  • Embodiment 8 The method of embodiment 7, wherein the presence of Neu5Gc- Lewis a in the cell, exosome or tissue is detected with a Neu5Gc-Lewis a -binding molecule that does not bind to any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • Embodiment 9 The method of embodiment 8, wherein the Neu5Gc-Lewis a - binding molecule comprises a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer.
  • Embodiment 10 The method of embodiment 9, wherein the peptide has at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • Embodiment 11 The method of any one of embodiments 7 to 10, wherein the cancer is pancreatic cancer, breast cancer, colon cancer, brain cancer, endometrial cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, stomach cancer, or urinary bladder cancer.
  • Embodiment 12 The method of embodiment 11, wherein the presence of Neu5Gc-Lewis a discriminates between normal and cancerous tissue.
  • Embodiment 13 A method of targeted delivery of a cargo molecule to a cell and/or tissue, comprising contacting the cell or tissue with a Neu5Gc-Lewis a -binding molecule linked to a cargo, directly or through a linker, wherein the Neu5Gc-Lewis a - binding molecule (preferably, a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer) has been previously determined not to bind to any other form of Neu5Gc or any other Sialyl- Lewis 3 , including free Neu5Gc, other than Neu5Gc-Lewis a , preferably wherein the cell or tissue is a cancer cell or tissue and/or preferably wherein the cargo is an anti-cancer therapy/agent.
  • a Neu5Gc-Lewis a -binding molecule linked to a cargo, directly or through
  • Embodiment 14 The method of embodiment 13, wherein the Neu5Gc-Lewis a - binding molecule comprises a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2 to detect Neu5Gc-Lewis a on the cell or tissue.
  • Embodiment 15 The method of embodiment 13 or 14, wherein the cancer is pancreatic cancer, breast cancer, colon cancer, brain cancer, endometrial cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, stomach cancer, or urinary bladder cancer; preferably, wherein the targeted delivery of the cargo to said cancer is therapeutic against said cancer.
  • Embodiment 16 The method of any one of embodiments 13 to 15, wherein the cargo comprises a molecule selected from peptides, nucleic acids (e.g., siRNA, antisense oligonucleotides, microRNA, or shRNA), proteins (including, but not limited to, streptavidin, phycoerythrin, a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, a ribonuclease (RNAse), a deoxyribonuclease (DNAse)), a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, granulocytemacrophage colony-stimulating factor (GMCSF), a transcription factor, a transposon, a reverse transcriptase, a viral interferon antagonist), fluor
  • Embodiment 17 A method of detecting extracellular or cell-surface or tissuebound Neu5Gc-Lewis a in a subject, comprising (a) obtaining or having obtained a sample of solid tissue, blood, serum, plasma, lavage, urine, milk, CSF, etc.
  • Neu5Gc-Lewis a is present in the sample by contacting the sample with a Neu5Gc-Lewis a -binding molecule, preferably a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer, wherein the molecule does not bind any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc, and detecting binding between the Neu5Gc-Lewis a -binding molecule and the Neu5Gc-Lewis a in the sample; optionally, further comprising administering an anti-cancer therapy to the subject.
  • a Neu5Gc-Lewis a -binding molecule preferably a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g.,
  • Embodiment 18 The method of embodiment 17, wherein the presence of extracellular, cell-surface or tissue-bound Neu5Gc-Lewis a indicates the presence of cancer in the subject.
  • Embodiment 19 The method of embodiment 17 or 18, wherein the Neu5Gc- Lewis a -binding molecule comprises a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • Embodiment 20 The method of any one of embodiments 17 to 19, wherein detection of extracellular or cell-surface or tissue-bound Neu5Gc-Lewis a may discriminate cancer patients from cancer-free individuals and/or monitor cancer progression in the subject.
  • Embodiment 21 The method of any one of embodiments 17 to 20, wherein the cancer is pancreatic cancer, breast cancer, colon cancer, brain cancer, endometrial cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, stomach cancer, or urinary bladder cancer.
  • Embodiment 22 A method of detecting whether a disease is associated with the presence of Neu5Gc-Lewis a , comprising (a) obtaining or having obtained diseased tissue from a subject and (b) contacting the diseased tissue with a Neu5Gc-Lewis a -binding molecule and detecting the binding between the Neu5Gc-Lewis a -binding molecule, preferably a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer, and the Neu5Gc-Lewis a in the diseased tissue, wherein the molecule does not bind to any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • a Neu5Gc-Lewis a preferably a peptide, antibody, nucleic acid, a carbohydrate, a lipid
  • Embodiment 23 A method of detecting atherosclerosis in a tissue, comprising contacting the tissue with a Neu5Gc-Lewis a -binding molecule, preferably a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer, and detecting the binding between the Neu5Gc-Lewis a -binding molecule and the Neu5Gc-Lewis a in the vessel wall in the tissue, thereby detecting atherosclerosis in the vessel wall, wherein the molecule does not bind any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • a Neu5Gc-Lewis a -binding molecule preferably a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (
  • Embodiment 24 The method of embodiment 22 or 23, wherein the Neu5Gc- Lewis a -binding molecule comprises a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • Embodiment 25 A composition comprising a Neu5Gc-Lewis a -binding molecule, preferably a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer, and a carrier or diluent, wherein the Neu5Gc-Lewis a -binding molecule does not bind to any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • a Neu5Gc-Lewis a -binding molecule preferably a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer, and a carrier or diluent, wherein the Neu5Gc-Lewis a -binding molecule does
  • Embodiment 26 The composition of embodiment 25, wherein the Neu5Gc- Lewis a -binding molecule comprises a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • Embodiment 27 The composition of embodiment 25 or 26, wherein the Neu5Gc- Lewis a -binding molecule is chemically modified.
  • Embodiment 28 The composition of embodiment 27, wherein the chemical modification comprises pegylation, acetylation, a d-amino acid, acylation, ADP- ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and/or transfer-RNA mediated addition of amino acids to
  • Embodiment 29 The composition of any one of embodiments 25 to 28, wherein the Neu5Gc-Lewis a -binding molecule comprises a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • Embodiment 30 The composition of any one of embodiments 25 to 29, wherein the Neu5Gc-Lewis a -binding peptide comprises a Neu5Gc-Lewis a -binding fragment of MGS5.
  • Embodiment 31 The composition of any one of embodiments 25 to 30, wherein the Neu5Gc-Lewis a -binding peptide comprises multiple MGS5 peptide moieties, Neu5Gc-Lewis a -binding fragments ofMGS5 moieties, and/or Neu5Gc-Lewis a -binding peptides moieties comprising an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to MGS5.
  • Embodiment 32 The composition of embodiment 31, wherein the multiple peptide moieties are linked to each other through a linker.
  • Embodiment 33 The composition of embodiment 32, wherein the linker comprises PEG.
  • Embodiment 34 A chimeric antigen receptor (“CAR”) wherein the CAR targets Neu5Gc-Lewis a , preferably wherein the CAR comprises an anti-Neu5Gc-Lewis a antibody, or a Neu5Gc-Lewis a -binding fragment thereof, or an Neu5Gc-Lewis a -binding peptide in addition to or instead of the antigen receptor (“AR”), wherein the antibody and peptide have been previously determined not to bind any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • CAR chimeric antigen receptor
  • Embodiment 35 A cell comprising the CAR of embodiment 34, preferably wherein the cell is a T lymphocyte (T cell, e.g., CD4+ T cells or CD8+ T cell), cytotoxic lymphocytes (CTL), macrophages or natural killer (NK) cell.
  • T cell e.g., CD4+ T cells or CD8+ T cell
  • CTL cytotoxic lymphocytes
  • NK natural killer
  • Embodiment 36 The cell of embodiment 35, wherein the Neu5Gc-Lewis a - binding peptide comprises a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • Embodiment 37 A method of treating cancer in a subject in need thereof, comprising administering an anti-cancer therapy to the subject wherein the subject has been selected for treatment because the subject has been diagnosed as having cancer by a method comprising contacting a cell, exosome or tissue sample from the subject with a Neu5Gc-Lewis a -binding molecule and detecting the binding of the molecule to Neu5Gc- Lewis a in the cell, exosome or tissue sample, thereby diagnosing the subject has having cancer.
  • Embodiment 38 The method of embodiment 37, wherein the Neu5Gc-Lewis a - binding molecule comprises a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer, wherein the molecule does not bind any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • the Neu5Gc-Lewis a - binding molecule comprises a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer, wherein the molecule does not bind any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • Embodiment 39 The method of embodiment 37 or 38, wherein the Neu5Gc- Lewis a -binding molecule comprises a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • Embodiment 40 A method of removing Neu5Gc-Lewis a from milk comprising contacting the milk with a Neu5Gc-Lewis a -binding molecule, preferably comprising a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2 and then removing the Neu5Gc-Lewis a -binding molecule -bound Neu5Gc-Lewis a from the milk.
  • a Neu5Gc-Lewis a -binding molecule preferably comprising a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2 and then removing the Neu5Gc-Lewis a -binding molecule -bound Neu5Gc
  • Embodiment 41 A method of removing Neu5Gc-Lewis a from cell culture medium or cell culture medium components comprising contacting the medium or medium components with a Neu5Gc-Lewis a -binding molecule comprising a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2 and then removing the Neu5Gc-Lewis a -binding molecule-bound Neu5Gc-Lewis a from the medium or medium components.
  • Embodiment 42 A method of reducing stem cell differentiation in vitro comprising culturing/expanding the cells in culture medium previously contacted with a Neu5Gc-Lewis a -binding molecule comprising a peptide having at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2 and subsequently depleted of Neu5Gc- Lewis a -binding molecule-bound Neu5Gc-Lewis a .
  • FIG. 1 Neu5Gc-Lewis a (R represents additional linked glycosidic side chains that do not compose the core structure of Neu5Gc-Lewis a and/or proteins).
  • FIG. 2 Glycan array showing MGS5_V1 and MGS5_V2 binding Neu5Gc- Lewis a .
  • Array positive controls spotted biotin
  • MGS5_V1 and MGS5_V2 were incubated on the array for 3 hours and binding was visualized using Cy3-Streptavidin.
  • FIGs. 3A-3F IHC results showing selective binding of MGS5 V2 to cancer tissue and thus identification of Neu5Gc-Lewis a as a marker for cancer tissue.
  • FIG. 3 A normal/healthy tissue
  • FIG. 3B developmental stages of Pancreatic Ductal Adenocarcinoma (PDAC) and metastasized PDAC sites
  • FIG. 3C, 3D, 3F PDAC, different magnifications
  • FIG. 3E Malignant/cancer tissues.
  • FIG. 4 Pan02 cells treated with 125 nM of MGS5_V2 with 0.1 U/ml, 0.2 U/ml, and 0.4 U/ml of sialidase Vibro cholerae) resulted in reduced cellular uptake of MGS5 V2 compared to that of the cells just treated with MGS5_V2. Error bars represent standard error of the mean from three independent experiments. Statistical analysis was done using a one-way ANOVA with multiple comparisons. The asterisks indicate the following P values: * p ⁇ 0.05; ** p ⁇ 0.01; **** p ⁇ 0.0001.
  • FIG. 5 Structure of AcMGS5 dimer conjugated to Alexa Fluor 647 dye.
  • the copies of the AcMGS5 peptide are linked covalently to the branches (a-amino group and e-amino groups) of the lysine core through a PEG12 linker.
  • the conjugate to a cargo molecule is formed by reaction of the thiol group (highlighted by arrow) on the dimer core with the appropriate reactive chemical group on the cargo, in this example maleimide AF647.
  • FIG. 6 Schematic of the use of MGS5 for capture and quantitation of exosomes from serum of tumor bearing mouse. DETAILED DESCRIPTION
  • “no more than” includes each value less than the stated value.
  • “no more than 100 monomers” includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72,
  • the terms “plurality,” “at least two,” “two or more,” “at least second,” and the like, are understood to include but not limited to at least 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,
  • the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. In one embodiment, “about” or “approximately” may mean within one or more than one standard deviation per the practice in the art. “About” or “approximately” may mean a range of up to 10% (i.e., ⁇ 10%).
  • “about” may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value.
  • about 5 mg may include any amount between 4.5 mg and 5.5 mg.
  • the terms may mean up to an order of magnitude or up to 5-fold of a value.
  • any concentration range, percentage range, ratio range or integer range is to be understood to be inclusive of the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one- hundredth of an integer), unless otherwise indicated.
  • a “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a therapeutic agent” or “agent” described in the specification is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. Such terms may be used interchangeably.
  • the ability of a therapeutic agent to promote disease regression may be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
  • Therapeutically effective amounts and dosage regimens may be determined empirically by testing in known in vitro or in vivo (e.g., animal model) systems.
  • the methods and compounds as described herein are useful for both prophylactic and therapeutic treatment.
  • treating or treatment includes prevention; delay in onset; diminution, eradication, or delay in exacerbation of signs or symptoms after onset; and prevention of relapse.
  • a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer.
  • Prophylactic administration may occur for several days to years prior to the manifestation of symptoms of an infection.
  • Prophylactic administration may be used, for example, in the chemopreventative treatment of subjects presenting precancerous lesions, those diagnosed with early-stage malignancies, and for subgroups with susceptibilities (e.g., family, racial, and/or occupational) to particular cancers.
  • Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein after cancer is diagnosed.
  • the term "combination” refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present disclosure and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “agent”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic effect.
  • a combination partner e.g., another drug as explained below, also referred to as “therapeutic agent” or “agent”
  • the single components may be packaged in a kit or separately.
  • One or both of components e.g., powders or liquids
  • co-administration or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.
  • a “patient” or a “subject” as used herein includes any human who is afflicted with a cancer or any disorder, particularly one that increases the level of the biomarker Neu5Gc-Lewis a .
  • the terms “subject” and “patient” are used interchangeably herein.
  • epitope refers to an antigenic determinant that interacts with (is bound by) a specific antigen binding site in the variable region of an antibody molecule (the paratope).
  • a single antigen such as, but not limited to, a polypeptide
  • different antibodies may bind to different epitopes on an antigen and may have different biological effects depending on which epitope is bound.
  • epitope also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody.
  • Epitopes may be defined as a structural epitope (the portion of the antigenic determinant that is contacted by the CDR loops of an antibody) or a functional epitope (a subset of a structural epitope comprising those energetic residues centrally located in the structural epitope and directly contribute to the affinity of the antibody-epitope interaction). Epitopes may become immunologically available after fragmentation or denaturation of an antigen (a cryptotope). Epitopes may be linear or conformational (composed of nonlinear amino acids brought together in a folded three-dimensional structure).
  • Epitopes may include residues that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope typically includes at least 3 to 15 amino acids.
  • antibody is used herein in the broadest sense and encompasses various antibody structures and antibody fragments so long as they exhibit the desired antigenbinding activity and fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins may be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes, e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • the heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • antibody structures include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multi-specific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affibodies, Fab, Fab', F(ab')2, and Fv fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), minibodies, domain antibodies
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human frame regions (FRs).
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions (HVRs) (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • HVRs hypervariable regions
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non- human antibody refers to an antibody that has undergone humanization.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and HVRs.
  • FRs conserved framework regions
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds to the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624- 628 (1991).
  • antigen-binding fragment refers to a molecule other than an intact antibody, such molecule comprises a portion of the intact antibody and binds to the antigen to which the intact antibody binds.
  • antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ,diabodies, dAb, linear antibody, single-chain antibodies (e.g., scFv); single-domain antibodies; antigen-binding fragments of bivalent or bispecific antibodies; camelid antibodies; single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and other fragments with desired antigen (e.g., Neu5Gc-syalyl Lewis-a)-binding ability.
  • desired antigen e.g., Neu5Gc-syalyl Lewis-a
  • antigen-binding site refers to the part of an antibody molecule that comprises determinants that form an interface that binds to a polypeptide, or an epitope thereof.
  • the antigen-binding site typically includes one or more loops (e.g., of at least four amino acids or amino acid mimics) that form an interface that binds to a polypeptide.
  • the antigen-binding site of an antibody molecule includes at least one or two CDRs and/or hypervariable loops, or more typically at least three, four, five or six CDRs and/or hypervariable loops.
  • CDR refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions.
  • CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.
  • CDRs may be referred to as Kabat CDRs.
  • Sub-portions of CDRs may be designated as LI, L2 and L3 or Hl, H2 and H3 where the "L” and the "H” designate the light chain and the heavy chains regions, respectively.
  • regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs.
  • Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J.
  • CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.
  • the methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDR.
  • the term “specifically binds” refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context.
  • the term, “specifically binds”, refers to the ability of the antibody to bind to a specific antigen with a degree of affinity or avidity, compared with an appropriate reference antigen or antigens, that enables the antibody to be used to distinguish the specific antigen from others, e.g., to an extent that permits preferential targeting to certain cells, e.g., muscle cells, through binding to the antigen, as described herein.
  • an antibody specifically binds to a target if the antibody has a KD (affinity) for binding the target of at least about 10-4M, 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-1 IM, 10-12M, 10-13M, or less, preferably as measured by the method of the Examples (biolayer interferometry (BLI).
  • KD affinity
  • linker refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches one molecule to another (e.g., a peptide to another peptide).
  • linkers include a divalent radical such as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as: — (CR2) nO (CR2) n— , repeating units of alkyloxy (e.g., polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g., polyethyleneamino); and diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide.
  • linkers may comprise one or more amino acid residues, such as valine, phenylalanine, Cys, lysine, and homolysine.
  • synthetic is generally used herein to refer to compounds or molecules, e.g., compounds described herein, that are not naturally occurring.
  • polypeptide refers to polymers of amino acids.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • the polypeptide may be isolated from natural sources, may be produced by recombinant techniques from a eukaryotic or prokaryotic host, or may be a product of synthetic procedures. In some embodiments, the polypeptide is greater than 50 amino acids in length.
  • peptide is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • nucleic acid refers to a polynucleotide sequence, or fragment thereof.
  • a nucleic acid may comprise nucleotides.
  • a nucleic acid may be exogenous or endogenous to a cell.
  • a nucleic acid may exist in a cell-free environment.
  • a nucleic acid may be a gene or fragment thereof.
  • a nucleic acid may be DNA.
  • a nucleic acid may be RNA.
  • a nucleic acid may comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase).
  • analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholines, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7- deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.
  • fluorophores e.g., rhodamine or fluorescein linked to the sugar
  • thiol containing nucleotides biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-gu
  • Nucleic acid may be used interchangeably.
  • a nucleic acid may comprise one or more modifications (e.g., a base modification, a backbone modification), to provide the nucleic acid with a new or enhanced feature (e.g., improved stability).
  • a nucleic acid may comprise a nucleic acid affinity tag.
  • a nucleic acid may comprise a nucleic acid mimetic.
  • the term “mimetic” may be intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the intemucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring may also be referred as being a sugar surrogate.
  • the heterocyclic base moiety or a modified heterocyclic base moiety may be maintained for hybridization with an appropriate target nucleic acid.
  • One such nucleic acid may be a peptide nucleic acid (PNA).
  • the sugar-b ackbone of a polynucleotide may be replaced with an amide containing backbone, in particular with an aminoethylglycine backbone.
  • the nucleotides may be retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • the backbone in PNA compounds may comprise two or more linked aminoethylglycine units which gives PNA an amide containing backbone.
  • the heterocyclic base moieties may be bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • aptamer refers to biomolecules that may be designed or selected to bind tightly to other ligands, for example using a technique called systematic evolution of ligands by exponential enrichment (SELEX; Tuerk C, Gold L: “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.” Science 1990, 249:505-510).
  • Aptamers may be peptides. Aptamers may also be nucleic acids.
  • Nucleic acid aptamers may for example be selected from pools of randomsequence oligonucleotides, with high binding affinities and specificities for a wide range of biomedically relevant targets, suggesting a wide range of therapeutic utilities for aptamers (Keefe, Anthony D., Supriya Pai, and Andrew Ellington. “Aptamers as therapeutics.” Nature Reviews Drug Discovery 9.7 (2010): 537-550). These characteristics also suggest a wide range of uses for aptamers as drug delivery vehicles (Levy-Nissenbaum, Etgar, et al. “Nanotechnology and aptamers: applications in drug delivery.” Trends in biotechnology 26.8 (2008): 442-449; and Hicke B J, Stephens A W.
  • RNA aptamers may also be constructed that function as molecular switches, responding to a que by changing properties, such as RNA aptamers that bind fluorophores to mimic the activity of green fluorescent protein (Paige, Jeremy S., Karen Y. Wu, and Samie R. Jaffrey. “RNA mimics of green fluorescent protein.” Science 333.6042 (2011): 642-646). It has also been suggested that aptamers may be used as components of targeted siRNA therapeutic delivery systems, for example targeting cell surface proteins (Zhou, Jiehua, and John J. Rossi.
  • a nucleic acid aptamer refers to single-stranded or double-stranded oligo-DNA, oligo-RNA or oligo-DNA/RNA or any analogue thereof that specifically binds to a target molecule such as a peptide.
  • aptamers display fairly high specificity and affinity for their targets. Aptamer production is described inter alia in U.S. Pat. No.
  • photoaptamer refers to an aptamer that contains one or more photoreactive functional groups that may covalently bind to or crosslink with a target molecule.
  • aptamer refers to an aptamer which includes L-DNA, L-RNA, or other lefthanded nucleotide derivatives or nucleotide-like molecules.
  • peptidomimetic refers to a non-peptide agent that is a topological analogue of a corresponding peptide.
  • Methods of rationally designing peptidomimetics of peptides are known in the art. For example, the rational design of three peptidomimetics based on the sulphated 8-mer peptide CCK26-33, and of two peptidomimetics based on the 11-mer peptide Substance P, and related peptidomimetic design principles, are described in Horwell 1995 (Trends Biotechnol 13: 132-134).
  • affinity refers to the strength of the sum of all noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to the intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen).
  • KD dissociation constant
  • association may mean that Neu5Gc-Lewis a is identifiable as being localized to the cell, exosome or tissue at a point in time.
  • label refers to detectable moieties, such as those whose molecular size is sufficient to induce detectable modifications in their physical and/or chemical properties; such detection may be achieved by optical methods (such as fluorescence, diffraction, surface plasmon resonance, surface variation and contact variation angles) or Physical methods such as atomic force spectroscopy and tunneling effect, and electron- dense substances, such as radioactive molecules (e.g. 32 P, 35 S or 125 1).
  • “Labels” and “reporter molecules” include fluorescent agents, chemiluminescent agents, chromogenic agents, quenching agents, radionucleotides, enzymes, substrates, cofactors, inhibitors, radioactive isotopes, magnetic particles, and other moieties known in the art. “Labels” or “reporter molecules” may generate a measurable signal and may be covalently or noncovalently joined to a ligand.
  • the term “ligand” refers to the molecule to which the label binds to and which binds or complexes with directly or indirectly.
  • the ligand is Neu5Gc-Lewis a , a molecular form or fragment thereof.
  • the ligand may be a peptide or an antibody.
  • the ligand is MGS5.
  • biomarker as used herein is meant a single molecule (e.g., Neu5Gc-Lewis a ), the levels or relative levels or ratios of which in a cell, exosome or tissue in a subject significantly change (either in an increased or decreased manner) from the level or relative levels present in a subject having one physical condition or disease or disease stage representative of another physical condition or disease stage, including “normal” or non-diseased stage.
  • Reference standard refers to the source of the reference biomarker levels. The “reference standard” is preferably provided by using the same assay technique as is used for measurement of the subject's biomarker levels in the reference subject or population, to avoid any error in standardization.
  • the reference standard is, alternatively, a numerical value, a predetermined cut point, a mean, an average, a numerical mean or range of numerical means, a numerical pattern, a ratio, a graphical pattern or a marker abundance profile or marker level profile derived from the same biomarker or biomarkers in a reference subject or reference population.
  • “Reference subject” or “Reference Population” defines the source of the reference standard.
  • the reference is a human subject or a population of subjects having no cancer or other disease, i.e., healthy controls or negative controls.
  • the reference is a human subject or population of subjects with one or more clinical indicators of cancer or other disease, but who did not develop the cancer or other disease.
  • the reference is obtained from the same test subject who provided a temporally earlier biological sample. That sample may be pre- or post-therapy or pre- or post-surgery.
  • sample or “biological sample” as used herein means any biological fluid or tissue that may or may not comprise Neu5Gc-Lewis a .
  • the most suitable samples for use in the methods and with the compositions are samples which require minimal invasion for testing, e.g., blood samples, including cerebrospinal fluid, serum, plasma, whole blood, and circulating tumor cells. It is also anticipated that other biological fluids, such as saliva or urine, vaginal or cervical secretions, and ascites fluids or peritoneal fluid may be similarly evaluated by the methods described herein. Also, circulating tumor cells or fluids containing them are also suitable samples for evaluation in certain embodiments of this disclosure.
  • the samples may include biopsy tissue, tumor tissue, surgical tissue, circulating tumor cells, or other tissue.
  • samples may further be diluted with saline, buffer or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means.
  • the samples may include biopsy tissue, surgical tissue, circulating tumor cells, or other tissue.
  • the sample is a tumor secretome, i.e., any fluid or medium containing the proteins secreted from the tumor. These shed proteins may be unassociated, associated with other biological molecules, or enclosed in a lipid membrane such as an exosome.
  • the sample is plasma.
  • the sample is a tissue sample, for example, tumor tissue, and may be fresh, frozen, or archival paraffin embedded tissue.
  • a biological sample is typically obtained from a eukaryotic organism, most preferably a mammal, or a bird, reptile, or fish.
  • a “subject” which may be subjected to the methods described herein may be any mammalian animals including human, dog, cat, cattle, goat, pig, swine, sheep and monkey; or a bird; reptile; or fish.
  • a subject is a human being; a human subject may be known as a “patient”
  • obtaining a biological sample it is herein meant to obtain a biological sample for use in methods described in this disclosure.
  • the degree of change (or differential expression) in Neu5Gc-Lewis a level may vary with each individual and is subject to variation with each population. For example, in one embodiment, a large change, e.g., 2-3 fold increase or decrease in Neu5Gc-Lewis a levels, is statistically significant. In another embodiment, a larger relative change of 10- fold increase or more is statistically significant.
  • the degree of change in Neu5Gc-Lewis a levels varies with the condition, such as type of cancer and with the size or spread of the cancer or solid tumor. The degree of change also varies with the immune response of the individual and is subject to variation with each individual.
  • a change at or greater than a 1 .2 fold increase or decrease in Neu5Gc-Lewis a levels is statistically significant.
  • a larger change e.g., at or greater than a 1.5 fold, greater than 1.7 fold or greater than 2.0 fold increase or a decrease in the level of Neu5Gc-Lewis a is statistically significant.
  • Neu5Gc-Lewis a level is significantly increased in biological samples which normally do not contain measurable levels of Neu5Gc-Lewis a , such increase in Neu5Gc-Lewis a level may alone be statistically significant.
  • the chance or different is significant when the fold-change in Neu5Gc-Lewis a levels compared to control level is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-fold different in the sample versus the control or in one sample versus another.
  • the differential Neu5Gc-Lewis a levels takes into account both increase and decreased levels.
  • Neu5Gc-Lewis a may be considered up or down-regulated if the differential expression meets a statistical threshold, a fold- change threshold, or both.
  • the criteria for identifying differential expression may comprise both a p-value of 0.001 and fold change of at least 1.2-fold (up or down).
  • Neu5Gc- Lewis a level is normally decreased or not significantly measurable in certain biological samples which normally do contain measurable Neu5Gc-Lewis a levels, such decrease in Neu5Gc-Lewis a may alone be statistically significant.
  • Neu5Gc-Lewis a levels are being evaluated or measured by immunohistochemistry, the presence versus absence of a signal indicating the presence vs absence of Neu5Gc-Lewis a is considered a significant change and a diagnostic of cancer (when present).
  • One of ordinary skill in the art is familiar with methods of relative quantification of immunohistochemistry signals for the change to be considered significant.
  • determining the level of Neu5Gc-Lewis a in a sample, control or reference, as described herein, shall refer to the quantification of the presence of Neu5Gc-Lewis a in the tested sample.
  • the level may be quantitative or qualitative.
  • the concentration of Neu5Gc-Lewis a in said samples may be directly quantified via measuring the amount of Neu5Gc-Lewis a as present in the tested sample.
  • it is also possible to quantify the amount of Neu5Gc-Lewis a for example by mass spectrometry. How to determine the level of Neu5Gc-Lewis a is well known to the skilled artisan.
  • the present disclosure shall not be restricted to any particular method for determining the level of Neu5Gc-Lewis a but shall encompass all means that allow for a quantification, or estimation, of the level of Neu5Gc-Lewis a , either directly or indirectly.
  • a quantitative “Level” in the context of the present disclosure is therefore a parameter describing the absolute amount of Neu5Gc-Lewis a , for example as absolute weight, volume, or molar amounts; or alternatively “level” pertains to the relative amounts, and preferably to the concentration of Neu5Gc-Lewis a in the tested sample, for example in mol/1, g/1, g/mol.
  • a qualitative “level” may be evaluated by immunohistochemistry, fluorescence activated cell sorting, and the like.
  • a “diagnosis” or the term “diagnostic” in the context of the present disclosure means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of “true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.”
  • the “specificity” of a diagnostic assay is 1 minus the false positive rate, where the “false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • the term “monitoring of a disease, condition or disorder” shall mean for the purpose of the present disclosure to observe disease progression in a patient who receives a therapy.
  • the patient during the therapy is regularly monitored for the effect of the applied therapy, which allows the medical practitioner to estimate at an early stage during the therapy whether the prescribed treatment is effective or not, and, therefore, to adjust the treatment regime accordingly.
  • prognosis refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a negative or poor prognosis is defined by a lower post -treatment survival term or survival rate. Conversely, a positive or good prognosis is defined by an elevated post-treatment survival term or survival rate. Usually, prognosis is provided as the time of progression free survival (PFS) or overall survival (OS). I Neu5Gc-sialyl Lewis a or Neu5Gc-Lewis a as a BIOMARKER
  • N-glycolyl neuraminic acid is among the most common sialic acid forms in nature.
  • Lewis a (Le a ) antigen is a blood group antigen and it is a carbohydrate found in glycolipids and glycoproteins on the surface of various cells.
  • N-glycolylneuraminic acid (Neu5Gc)-Lewis a is known as a dietary-derived glycan.
  • Neu5Gc itself is metabolically incorporated into human tissues from dietary sources (particularly red meat) and detected at even higher levels in some human cancers. But it was not known that the Neu5Gc-Lewis a is a more specific marker for cancer cells and tissues.
  • the disclosure provides evidence that Neu5Gc-Lewis a is present on the cell surface and extracellular matrix of cancer tissues and not in nondiseased tissues or healthy tissues such as brain, liver, lymph node, and pancreas. In other words, the disclosure provides that Neu5Gc-Lewis a is a biomarker for cancer.
  • Neu5Gc- Lewis a is superior to other forms of Neu5Gc as a cancer biomarker and cancer-target, at least because those other forms of Neu5Gc have been found to be present in some healthy tissues. Accordingly, in one embodiment, the disclosure provides methods of diagnosing cancer and targeting cancer tissues with a variety of molecules that bind Neu5Gc-Lewis a .
  • the presence of Neu5Gc-Lewis a in a cell, exosome or tissue may be used in the diagnosis or prognosis of cancer.
  • the disclosure provides a method of detecting Neu5Gc-Lewis a on the surface of a cell or on a tissue, comprising (a) obtaining or having obtained a cell or tissue; (b) detecting whether the Neu5Gc-Lewis a is present on the surface of the cell by contacting the cell with a molecule previously known to bind Neu5Gc-Lewis a , also referred to henceforth as 1 a Neu5Gc-Lewis a -binding molecule, and detecting binding between Neu5Gc-Lewis a and the Neu5Gc-Lewis a -binding molecule on the cell surface.
  • the disclosure provides a method of detecting Neu5Gc-Lewis a in the extracellular matrix (ECM) of a cell or tissue comprising (a) obtaining or having obtained a cell or tissue; (b) detecting whether the Neu5Gc-Lewis a is present on the extracellular matrix of the cell or tissue by contacting the cell or tissue with a Neu5Gc-Lewis a -binding molecule, and detecting binding between Neu5Gc-Lewis a and the Neu5Gc-Lewis a -binding molecule on the extracellular matrix of the cell or tissue.
  • the cell or tissue is from a subject having or at risk of cancer.
  • the method further comprises diagnosing the subject with cancer upon detection of Neu5Gc-Lewis a on the extracellular matrix of the cell or tissue.
  • the method further comprises administering a cancer treatment to the subject diagnosed as having cancer.
  • the disclosure provides a method of detecting Neu5Gc- Lewis a in an exosome released from a cell or tissue comprising (a) obtaining or having obtained a sample comprising an exosome released from a cell or tissue; (b) detecting whether the Neu5Gc-Lewis a is present on the exosome by contacting the exosome with a Neu5Gc-Lewis a -binding molecule, and detecting binding between Neu5Gc-Lewis a and the Neu5Gc-Lewis a -binding molecule on the exosome.
  • the sample is from a subject having or at risk of cancer.
  • the method further comprises diagnosing the subject with cancer upon detection of Neu5Gc-Lewis a on an exosome in the sample from the subject.
  • the method further comprises administering a cancer treatment to the subject diagnosed as having cancer.
  • the cell is a cancer cell.
  • the tissue is normal tissue.
  • the tissue is diseased tissue.
  • the tissue is suspected of comprising cancer tissue or is known to comprise cancer tissue.
  • the tissue comprises blood vessels.
  • the blood vessels are arteries.
  • the presence of Neu5Gc-Lewis a in either the cell surface, exosome and/or ECM of the cell or tissue identifies or diagnoses the cell or tissue as cancer tissue.
  • a significant increase in the level of Neu5Gc-Lewis a in the cell, exosome or tissue, relative to a healthy cell, exosome from a healthy cell or healthy tissue counterpart identifies the diagnoses the cell or tissue as cancer tissue.
  • the presence of Neu5Gc-Lewis a in the cell surface, exosome and/or ECM of the tissue comprising blood vessels identifies or diagnoses the Neu5Gc-Lewis a - positive area as atherosclerotic tissue.
  • the disclosure provides a method of detecting extracellular or cell-tissue-bound Neu5Gc-Lewis a in a subject, comprising (a) obtaining or having obtained a sample of solid tissue, blood, serum, plasma, lavage, urine, milk, CSF, or other tissue or fluid from the subject; and (b) detecting whether Neu5Gc-Lewis a is present in the sample by contacting the sample with a Neu5Gc-Lewis a binding molecule, preferably an antibody, peptide, or aptamer, wherein the molecule does not bind any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc, and detecting binding between the Neu5Gc-Lewis a -binding molecule and the Neu5Gc-Lewis a in the sample.
  • a Neu5Gc-Lewis a binding molecule preferably an antibody, peptide, or aptamer
  • the disclosure provides a method of detecting whether a disease is associated with the presence of Neu5Gc-Lewis a , comprising (a) obtaining or having obtained diseased tissue from a subject and (b) contacting the diseased tissue with a Neu5Gc-Lewis a -binding molecule and detecting the binding between the Neu5Gc- Lewis a -binding molecule, preferably an antibody, peptide, or aptamer, and the Neu5Gc- Lewis a in the diseased tissue, wherein the molecule does not bind to any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • the disclosure provides a method of detecting atherosclerosis in a tissue, comprising contacting the tissue with a Neu5Gc-Lewis a binding molecule, preferably an antibody, peptide, or aptamer, and detecting the binding between the Neu5Gc-Lewis a binding molecule and the Neu5Gc-Lewis a in the vessel wall in the tissue, thereby detecting atherosclerosis in the vessel wall, wherein the molecule does not bind any other form of Neu5Gc or any other Si alyl -Lewis 3 , including free Neu5Gc.
  • a Neu5Gc-Lewis a binding molecule preferably an antibody, peptide, or aptamer
  • the cancer may be any cancer.
  • the cancer comprises pancreatic cancer (including ductal adenocarcinoma); breast cancer (including infiltrating duct carcinoma, infiltrating lobular carcinoma, medullary carcinoma), colon cancer (including adenocarcinoma), endometrial cancer (including adenocarcinoma), liver cancer (including hepatocellular carcinoma and cholangiocarcinoma); lung cancer (including squamous cell carcinoma), ovarian cancer (including serous surface papillary carcinoma, poorly differentiated; serous adenocarcinoma, moderately differentiated, serous adenocarcinoma); prostate cancer (including adenocarcinoma), stomach cancer (including adenocarcinoma signet ring cell type); and/or urinary bladder cancer (transitional cell carcinoma).
  • pancreatic cancer including ductal adenocarcinoma
  • breast cancer including infiltrating duct carcinoma, infiltrating lobular carcinoma, medullary
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromo
  • the cancer comprises at least one tumor type selected from the group consisting of pancreatic cancer, breast cancer, biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, gliomas, astrocytomas, breast carcinoma, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal carcinoma, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinomas, gastrointestinal stromal tumors (GISTs), endometrial carcinoma, endometrial stromal sarcomas, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder carcinomas, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma
  • pancreatic cancer shall encompass benign or malignant forms of pancreatic cancer, as well as any particular type of cancer arising from cells of the pancreas (e.g., duct cell carcinoma, acinar cell carcinoma, papillary carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, mucinous carcinoma, giant cell carcinoma, mixed type pancreatic cancer, small cell carcinoma, cystadenocarcinoma, unclassified pancreatic cancers, pancreatoblastoma, and papillary- cystic neoplasm, and the like.)
  • cells of the pancreas e.g., duct cell carcinoma, acinar cell carcinoma, papillary carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, mucinous carcinoma, giant cell carcinoma, mixed type pancreatic cancer, small cell carcinoma, cystadenocarcinoma, unclassified pancreatic cancers, pancreatoblastoma, and papillary- cystic neoplasm, and the like.
  • the subject has not yet been determined to have a cancer
  • the subject has not yet been determined to harbor a cancer cell, or and/or (iii) the subject does not exhibit, or has not exhibited, a symptom associated with a cancer.
  • the presence of cancer is detected at a time period when the subject has not been diagnosed with a stage II cancer, has not been diagnosed with a stage I cancer, has not had a biopsy to confirm abnormal cellular growth, has not had a biopsy to confirm the presence of a tumor, has not undergone a diagnostic scan to detect a cancer, or any combination thereof.
  • said subject is a member of a population with a low risk, a medium risk, or a high risk of having the cancer based on one or more of the following factors: environmental factors, age, sex, medical history, medications, genetic factors, biochemical factors, biophysical factors, physiological factors, and/or occupational factors.
  • the disclosure provides methods of treatment according to the discovery that Neu5Gc-Lewis a is a biomarker for cancer. Accordingly, in one embodiment, the disclosure provides a method of cancer treatment in a subject in need thereof, whereby the subject is identified as having cancer and/or selected for treatment using the methods of the disclosure (i.e., detection of the presence of Neu5Gc-Lewis a in a subject’s tissue or body fluid and subsequently administered a cancer treatment).
  • the subject has previously received another treatment.
  • the levels of Neu5Gc-Lewis a in a subject’s tissue or body fluid are measured before, during, and after receiving treatment and are used to monitor treatment response.
  • the subject is said to have responded to the treatment when the circulating or tissue-bound levels of Neu5Gc-Lewis a in a subject’s tissue or body fluid decrease after treatment. In some embodiments, the decrease is statistically significant.
  • the disclosure provides a method for the prognosis, diagnosis, monitoring of a disease, and/or stratification of a condition or disorder in a subject, comprising the steps of (a) Providing a biological sample of said subject, (b) Determining the level of Neu5Gc-Lewis a in a cell and/or extracellular matrix or tissue in said biological sample, (c) Determining the level of Neu5Gc-Lewis a in second sample of the same kind obtained from the same subject, and (d) Comparing the determined level, in steps (b) and (c) with a reference sample or a reference value, and/or to each other, wherein a differential level of Neu5Gc-Lewis a is indicative of disease presence and/or disease progression or regression in said subject, when samples of steps b) and c) are biological samples from normal vs disease tissue, or different stages of treatment or before treatment, respectively.
  • the disease is cancer. In some embodiments, the disease therapy is adjusted based on the level of Neu5Gc-Lewis a in the samples. In one embodiment, the therapy is anti -cancer therapy. In one embodiment, the therapy is radiation. In one embodiment, the therapy is a therapeutic agent. In one embodiment, the therapy is combinations of the same. Non-limiting examples of therapeutic agents are described elsewhere in the application.
  • detection of extracellular or cell-tissue-bound Neu5Gc- Lewis a may discriminate cancer patients from cancer-free individuals and/or monitor cancer progression in the subject.
  • the efficacy of a treatment may be monitored by measuring the area of the diseased tissue in the subject that binds to the Neu5Gc-Lewis a binding molecule and/or the level of Neu5Gc-Lewis a in the tissue or body fluid, before and after exposure of the subject to the treatment.
  • the disclosure provides a method for monitoring the effectiveness of an anti-cancer therapy comprising determining the level of Neu5Gc- Lewis a in a sample from a subject treated with an anti-cancer therapy in accordance with the embodiments.
  • the levels of the Neu5Gc-Lewis a are significantly elevated as compared to a reference level, the subject is in need of additional anti-cancer therapy.
  • the level of Neu5Gc-Lewis a is not elevated as compared to a reference level, the subject is not in need of additional anti-cancer therapy.
  • a method for monitoring the effectiveness of an anti -cancer therapy comprising (a) determining the levels of a Neu5Gc-Lewis a in a sample from a subject before and after treatment with an anti-cancer therapy; and (b) identifying the subject as responsive to the therapy or not responsive to the therapy based on the change in levels of Neu5Gc-Lewis a .
  • the level of a Neu5Gc-Lewis a in a responsive subject should be reduced after therapy.
  • a subject identified as not responsive to the first anti cancer therapy may be administered a second anti-cancer therapy.
  • a threshold value may be obtained by performing the assay method on samples obtained from a population of patients having, for example, a certain type of cancer, and from a second population of subjects that do not have cancer.
  • a threshold value may be obtained by performing the assay method on samples obtained from a population of patients having, for example, an aggressive type of cancer associated with a reduced time of progression free survival (PFS) and/ or overall survival (OS), and from a second population of subjects that do have a rather mild form of cancer associated with an increased time of progression free survival (PFS) and/or overall survival (OS).
  • PFS time of progression free survival
  • OS overall survival
  • an increase or a decrease of a differential level, of the biomarker Neu5Gc-Lewis a may be indicative of a reduced time of progression free survival (PFS) and/or overall survival (OS). If decreased levels of the Neu5Gc-Lewis a are associated with poor prognosis, then also a decrease of Neu5Gc- Lewis a in said sample may be indicative of a reduced time of progression free survival (PFS) and/or overall survival (OS).
  • a population of patients may be followed for the time period of interest (e.g., six months following diagnosis or treatment, respectively), and then dividing the population into multiple groups, for example two groups: a first group of subjects that progresses to an endpoint (e.g., recurrence of disease, and/or death); and a second group of subjects that did not progress to the end point (e.g., no recurrence of disease, and/or no death).
  • endpoints e.g., recurrence of disease, and/or death
  • no recurrence of disease e.g., no recurrence of disease, and/or no death
  • Other suitable endpoints include, but are not limited to, 5-year mortality rates or progression to metastatic disease.
  • one or more thresholds may be selected that provide an acceptable ability to predict prognostic risk, diagnosis, treatment success, etc.
  • Receiver Operating Characteristic curves or “ROC” curves, are typically calculated by plotting the value of a variable versus its relative frequency in two populations (called arbitrarily “disease” and “normal” or “low risk” and “high risk”, for example). For any particular marker, a distribution of marker level for subjects with and without a disease may overlap.
  • a threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be “positive” and below which the test is considered to be “negative.”
  • the area under the ROC curve (AUC) is a measure of the probability that the perceived measurement may allow correct identification of a condition.
  • thresholds may be established by obtaining an earlier marker result from the same patient, to which later results may be compared.
  • the individuals act as their own “control group.”
  • markers that increase with disease severity or prognostic risk an increase over time in the same patient may indicate a worsening of disease or a failure of a treatment regimen, while a decrease over time may indicate remission of disease or success of a treatment regimen.
  • thresholds or reference values may be determined. This may be the case in so-called “tertile,” “quartile,” or “quintile” analyses.
  • the “disease” and “normal” groups (or “low risk” and “high risk”) groups may be considered together as a single population, and are divided into 3, 4, or 5 (or more) “bins” having equal numbers of individuals. The boundary between two of these “bins” may be considered “thresholds.”
  • a risk (of a particular prognosis or diagnosis for example) may be assigned based on which “bin” a test subject falls into.
  • stratification for the purposes of this disclosure shall refer to the advantage that the methods according to the disclosure render decisions for the treatment and therapy of the patient possible, whether it is the hospitalization of the patient, the use, effect and/or dosage of one or more drugs, a therapeutic measure or the monitoring of a course of the disease and the course of therapy or etiology or classification of a disease, e.g., into a new or existing subtype or the differentiation of diseases and the patients thereof.
  • stratification means in this context a classification of a pancreatic or breast cancer disease of an individual patient with regard of the metastatic status, or the presence or absence of circulating tumor cells.
  • the term “stratification” covers, in particular, the risk stratification with the prognosis of an outcome of a negative health event.
  • the presence of Neu5Gc-Lewis a in a cell, tissue, or fluid may be indicative of the presence of diseases other than cancer.
  • the disease is selected from infectious diseases, neurological disease, neurodegenerative disease, kidney disease, liver disease, heart disease, lung disease, gastric disease, ocular disease, bone disease, obesity, hematologic malignancies, autoimmune disorders, hypercholesterolemia, asthma, osteoporosis, inflammatory bowel disease, allograft rejection, drug reversal.
  • Neu5Gc-Lewis a in the diseased tissue or body fluid but not in the normal tissue or body fluid counterpart is an indication that Neu5Gc-Lewis a may be used as a biomarker in the detection of the disease.
  • a subject is identified as having one or more of those diseases using the methods of the disclosure and subsequently administered a therapy for the disease.
  • the disclosure provides a method of removing Neu5Gc- Lewis a from milk comprising contacting the milk with Neu5Gc-Lewis a -binding molecule and then removing the Neu5Gc-Lewis a -binding molecule -bound Neu5Gc-Lewis a from the milk.
  • the Neu5Gc-Lewis a -binding molecule comprises MGS5.
  • the disclosure provides a method of removing Neu5Gc- Lewis a from cell culture medium or cell culture medium components comprising contacting the medium of medium components with Neu5Gc-Lewis a -binding molecule and then removing the Neu5Gc-Lewis a -binding molecule -bound Neu5Gc-Lewis a from the medium.
  • the Neu5Gc-Lewis a -binding molecule comprises MGS5.
  • this method may be used in improving the quality and production of recombinantly produced proteins, including antibodies, where the presence of Neu5Gc is undesirable.
  • a method of reducing stem cell differentiation in vitro comprising culturing/expanding the cells in culture medium previously contacted with Neu5Gc- Lewis a -binding molecule and subsequently depleted of Neu5Gc-Lewis a -binding molecule -bound Neu5Gc-Lewis a .
  • the Neu5Gc-Lewis a -binding molecule comprises MGS5.
  • a Neu5Gc-Lewis a -binding molecule of the disclosure may be any molecule capable of binding Neu5Gc-Lewis a .
  • the Neu5GC-Lewis a -binding molecule is specific for Neu5Gc-Lewis a because it does not bind any other form of Neu5Gc or any other Si alyl -Lewis 3 , including free Neu5Gc.
  • the Neu5Gc-Lewis a -binding molecule comprises a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer.
  • the aptamer may be a nucleic acid-based aptamer or a peptide-based aptamer.
  • the antibody is selected from monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affibodies, Fab, Fab', F(ab')2, and Fv fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e g., anti-anti-Id antibodies), minibodies, domain antibodies
  • the peptide comprises a peptide that has at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • SEQ ID NO:1 is: LQWRRNFGVWARYRL
  • SEQ ID NO:2 is: LQWRRDDNVHNFGVWARYRL
  • the peptide of SEQ ID NO: 1 is also referred to as MGS5 V2 and the peptide of SEQ ID NO:2 is also referred to as MGS5_V1.
  • MGS5 relates to MGS5-V1 and MGS5-V2, indiscriminately.
  • the methods of the disclosure may be practiced with a peptide that has at least 75%, 80%, 85%, 90%, 95%, or 100% homology or identity to a peptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO:2 and still binds Neu5Gc-Lewis a specifically because it does not bind any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • the methods of the disclosure may be practiced with a peptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity to a peptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO:2, that specifically binds Neu5Gc-Lewis a specifically because it does not bind any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • the peptide has at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% homology or identity to a peptide comprising the sequence of SEQ ID NO:1 or SEQ ID NO:2, that specifically binds Neu5Gc-Lewis a specifically because it does not bind any other form of Neu5Gc or any other Sialyl-Lewis a , including free Neu5Gc.
  • the peptide is truncated and lacks up to 5, 4, 3, 2 or 1 contiguous or non-contiguous amino acids relative to a peptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO:2, that specifically binds Neu5Gc-Lewisa specifically because it does not bind any other form of Neu5Gc or any other Sialyl- Lewis a , including free Neu5Gc.
  • the methods are practiced with any one of the versions of MGS5.
  • MGS5 peptides can be used as multimers, such as dimers, trimers and tetramers.
  • the MGS peptides disclosed herein can be chemically conjugated to another MGS peptide, a cargo and/or another linker.
  • the one or more MGS peptides can be chemically conjugated to an agent (e.g., a cargo molecule).
  • the one or more MGS peptides can be conjugated to polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the number of PEG units can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more.
  • the number of PEG units can be of sufficient length to separate the one or more MGS peptides from one or more conjugated agent (e.g., cargo molecule) to prevent any steric interference between the one or more MGS peptides and the conjugated agent.
  • conjugated agent e.g., cargo molecule
  • MGS5 peptides can be modified.
  • modifying an MGS5 peptide comprises optimizing the peptide or stabilizing the peptide.
  • Optimized peptides can be obtained by applying modifications to the individual parental peptide sequences. Modification at the amino-terminus by acetylation (CH3CO-) and/or d-amino acids, such as d(Leu) can protect against degradation by peptidases.
  • the MGS5 peptide can have an N-terminal protection group.
  • the N-terminal protection group can be anything that prevents proteases from cleaving the amino acids from the N-terminus.
  • the MGS5 peptides disclosed herein can be modified by acetylation on the N-terminus.
  • the N-terminal protection group can be, but is not limited to, PEG, Formyl, CH3-(CH)n-CO, Fluorophore, Fatty acid, alkyl amine, aryl groups, carbohydrates, sulfonamide, or carbamate.
  • hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (- 0.5); acidic amino acids: aspartate (+3.0 + 1), glutamate (+3.0 + 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (- 1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 + 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (- 3.4), phenylalanine (-2.5), and tyrosine (-2.3).
  • amino acid may be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein.
  • substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those that are within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.
  • Amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain, for example amino acids with basic side chains (e.g., lysine, arginine, histidine) ; acidic side chains (e.g., aspartic acid, glutamic acid) ; uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine) ; nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) ; beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).
  • basic side chains e.g., lysine, arg
  • Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, serine for threonine, and the like.
  • Proline which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., Leu, Vai, He, Gly, and Ala).
  • substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively.
  • GCG program package which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two peptides for which the percent sequence identity is to be determined.
  • sequences are aligned for optimal matching of their respective amino acid (the “matched span,” as determined by the algorithm.)
  • a standard comparison matrix see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 10915-10919 for the BLOSUM 62 comparison matrix
  • identity may be determined as percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.
  • the Neu5 Gc-Lewi sMhnding molecule is labeled.
  • the label facilitates the detection of the binding to Neu5Gc-Lewis a .
  • the label is a fluorescence label.
  • the label is biotinylated.
  • the label is a radiolabel.
  • the binding takes place in vivo.
  • the labeled Neu5Gc-Lewis a -binding molecule may be used to image a cancer or diseased tissue to which it binds. In some embodiments, the binding takes place in vitro.
  • the Neu5Gc-Lewis a -binding molecule is identified by screening a library of molecules for binding to Neu5Gc-Lewis a .
  • the molecules are a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer.
  • the libraries are combinatorial libraries.
  • the library is a chemical peptide library.
  • the library is a biological library.
  • the libraries are phage libraries. New molecule identification via library production and screening is a technique well described in the art.
  • the disclosure provides Neu5Gc-Lewis a -binding molecules that have been chemically or physically modified.
  • the chemical modification comprises pegylation (directly or through a linker), acetylation, a d-amino acid, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, proteolytic processing, phosphorylation, prenylation
  • Neu5Gc-Lewis a -binding molecules are incorporated into lipid vesicles or nanoparticles, covalently or non-covalently.
  • the nanoparticle comprises a porous inorganic nanoparticle, a metalorganic framework nanoparticle, or a porous organic nanoparticle.
  • the nanoparticle is a solid nanoparticle and the cargo may be disposed within (e.g., intermixed with) the material forming the nanoparticle or adsorbed to, or covalently or ionically bound to, the nanoparticle surface).
  • the nanocarrier comprises a lipid bilayer encasing (or surrounding or enveloping) the particle core.
  • the nanocarrier is a liposome and the cargo may be disposed within the liposome or nanoparticle.
  • the Neu5Gc- Lewis a -binding molecules are conjugated, directly or through a linker, to a cargo molecule.
  • the Neu5Gc-Lewis a -binding molecules is covalently linked to the cargo via a cleavable or non-cleavable linker (e.g., bifunctional, trifunctional linker, or multi-functional linker.
  • the Neu5Gc-Lewis a -binding molecule is linked to a cargo molecule or delivery vehicle.
  • the Neu5Gc-Lewis a -binding molecules binds to a cell surface molecule of a target cell of interest, thereby directing the cargo or delivery vehicle to the target cell.
  • the cargo or delivery vehicle is operably linked to a targeting molecule that comprises a targeting domain comprising at least one MGS5 peptide that promotes targeting of the cargo or delivery vehicle to a cancer cell.
  • the targeting domain comprises at least one MGS5 peptide selected from SEQ ID NO: 1 or SEQ ID NO:2, or a fragment or variant thereof.
  • a dimer of an MGS5 peptide may be conjugated to a PEG moiety on a lipid nanoparticle (LNP) to direct the LNP to a cancer cell.
  • LNP lipid nanoparticle
  • the LNP encapsulates a therapeutic agent for the treatment of the cancer.
  • linkers can conjugate or link two or more MGS peptides together, two or more linkers together, or a linker or peptide to a cargo.
  • a linker that conjugates two or more MGS peptides together can be referred to as a dimeric core (if linked to two peptides) or a tetrameric core (if linked to four peptides).
  • any length of PEG can be used.
  • any of PEG 1 - PEG30 can be used.
  • a PEG of length 1-5000 can be used.
  • any linker can be used in place of PEG 12 of the dimeric core or tetrameric core.
  • linkers comprising at least one reactive group capable of binding a C-terminus of a peptide; and at least one additional reactive group capable of chemically reacting with a moiety.
  • the linker has a length of up to PEG5000.
  • the length of the linker can be a single PEG all the way up to 5000 PEGs.
  • the linker between the peptide and the cargo can be longer than the linker between two MGS peptides.
  • the linker comprises two to four PEG linkers.
  • a linker with two PEG linkers can be referred to as a dimeric core.
  • a linker with four PEG linkers can be referred to as a tetrameric core.
  • a linker comprises at least two PEG linkers and a reactive group between at least two of the PEG linkers.
  • the reactive group connects at least two PEG linkers.
  • the linker comprises an amino acid, a peptide, an alkyl group, a maleimide, a thiol, hydrazone, or amide.
  • the amino acid can be a modified amino acid.
  • a modified amino acid can be a functionalized lysine, functionalized cysteine, functionalized glutamic acid, or functionalized aspartic acid.
  • the linker comprises biotin.
  • the reactive group can be, but is not limited to, carboxylic acid, acyl halides, sulfonyl halides, chloroformates, aldehydes, alkynes, alkynes (with No Acetylenic Hydrogen), amides and imides, amines, phosphines, and pyridines, anhydrides, azo, diazo, azido, hydrazine, and azide compounds, carbamates, epoxides, esters, sulfate esters, phosphate, esters, thiophosphate esters, and borate esters, halogenated organic compounds, isocvanates and isothiocyanates, ketones, oximes, sulfides (Organic).
  • a moiety can be a cargo.
  • a cargo can be, but is not limited to, a dye, an imaging agent, a therapeutic, a protein, a nucleic acid, an amino acid, a peptide, a lipid, an antibody, a radionuclide, carbohydrate or a nanoparticle.
  • the moiety can be a linker. Therefore, in some aspects, disclosed are linkers comprising linkers. For example, if the linker is a tetrameric core (i.e., first linker), the tetrameric core can comprise moiety that is a second linker.
  • a second linker can comprise the same elements or different elements from the tetrameric core.
  • a second linker conjugates a tetrameric core (i.e., third linker) to a dimeric core.
  • any of the first, second, or third linkers can comprise an amino acid, a peptide, an alkyl group, a maleimide, a thiol, a hydrazone, dibenzocyclooctyne, azide, or an amide.
  • the disclosure provides cells that have been modified to comprise a Neu5Gc-Lewis a binding molecule.
  • the molecule is on the cell surface.
  • the Neu5Gc-Lewis a binding molecule helps direct the cell to another cell or tissue (target cell or tissue) that comprises Neu5Gc-Lewis a on the cell surface and/or ECM.
  • the target cell is a cancer cell.
  • the cells are immune cells, which are then referred to as modified immune cells.
  • the cells are T cells or natural killer (NK) cells.
  • the Neu5Gc-Lewis a binding molecule, or a multimer (e.g., dimer) thereof is part of a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the cell is a CAR-T cell, a CAR-NK cell or a tumor infdtrating lymphocyte.
  • the modified immune cells expressing the CARs may be, e.g., T lymphocytes (T cells, e.g., CD4+ T cells or CD8+ T cells), cytotoxic lymphocytes (CTLs) or natural killer (NK) cells.
  • T lymphocytes used in the compositions and methods provided herein may be naive T lymphocytes or MHC-restricted T lymphocytes.
  • the T lymphocytes are tumor infiltrating lymphocytes (TILs).
  • T lymphocytes have been isolated from a tumor biopsy or have been expanded from T lymphocytes isolated from a tumor biopsy.
  • the T cells have been isolated from, or are expanded from T lymphocytes isolated from, peripheral blood, cord blood, or lymph.
  • Immune cells to be used to generate modified immune cells expressing a CAR may be isolated using art-accepted, routine methods, e.g., blood collection followed by apheresis and optionally antibody-mediated cell isolation or sorting.
  • the modified immune cells are preferably autologous to an individual to whom the modified immune cells are to be administered.
  • the modified immune cells are allogeneic to an individual to whom the modified immune cells are to be administered.
  • allogeneic T lymphocytes are used to prepare modified T lymphocytes
  • virus-specific T lymphocytes are selected for preparation of modified T lymphocytes; such lymphocytes will be expected to have a greatly reduced native capacity to bind to, and thus become activated by, any recipient antigens.
  • recipient-mediated rejection of allogeneic T lymphocytes may be reduced by co-admini strati on to the host of one or more immunosuppressive agents, e.g., cyclosporine, tacrolimus, sirolimus, cyclophosphamide, or the like.
  • compositions comprising these cells, which compositions may be as described above for the Neu5Gc-Lewis a binding molecules of the disclosure. These cells may be administered by any of the methods described elsewhere in the specification.
  • an effective amount or sufficient number of the isolated modified immune cells is present in the composition and introduced into the subject such that long term, specific, anti-tumor responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment.
  • the amount of T cells reintroduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the T cells are not present.
  • anti-tumor effective amount refers to an effective amount of CAR- expressing immune effector cells to reduce cancer cell or tumor growth in a subject.
  • each active agent included in the compositions described herein may vary in different applications.
  • the concentration of T cells desirably should be sufficient to provide in the subject being treated at least from about 1 x 10 6 to about 1 x 10 9 transduced T cells, even more desirably, from about 1 x 10 7 to about 5 x 10 8 transduced T cells, although any suitable amount may be utilized either above, e.g., greater than 5 x 10 8 cells, or below, e.g., less than 1 x 10 7 cells.
  • the dosing schedule may be based on well-established cell-based therapies (see, e.g., U.S. Pat. No. 4,690,915), or an alternate continuous infusion strategy may be employed.
  • the Neu5Gc-Lewis a -binding molecule binds to or targets a cell, provided that the cell comprises Neu5Gc-Lewis a . While healthy cells do not appear to comprise Neu5Gc-Lewis a , the disclosure provides that there may be embodiments where a cell does comprise Neu5Gc-Lewis a , in which case it may be targeted by the Neu5Gc-Lewis a -binding molecules of the disclosure.
  • exemplary cells may include polymorphonuclear cells (also known as PMN, PML, PMNL, or granulocytes), stem cells, embryonic stem cells, neural stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), human myogenic stem cells, muscle- derived stem cells (MuStem), embryonic stem cells (ES or ESCs), limbal epithelial stem cells, cardio- myogenic stem cells, cardiomyocytes, progenitor cells, immune effector cells, lymphocytes, macrophages, dendritic cells, natural killer cells, T cells, cytotoxic T lymphocytes, allogenic cells, resident cardiac cells, induced pluripotent stem cells (iPS), adipose-derived or phenotypic modified stem or progenitor cells, CD133+ cells, aldehyde dehydrogenase-positive cells (ALDH+), umbilical cord blood (UCB) cells, peripheral blood stem cells (PBSCs), neurons,
  • the target tissue may include liver, lungs, heart, spleen, pancreas, gastrointestinal tract, kidney, testes, ovaries, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye.
  • the target cell is a muscle cell (e.g., skeletal muscle cell), kidney cell, liver cell (e.g., hepatocyte), or a cardiac cell (e.g., cardiomyocyte).
  • the target cell is a cardiac cell, e.g., a cardiomyocyte (e.g., a quiescent cardiomyocyte), a hepatoblast (e.g., a bile duct hepatoblast), an epithelial cell, a T cell (e.g., a naive T cell), a macrophage (e.g., a tumor infiltrating macrophage), or a fibroblast (e.g., a cardiac fibroblast).
  • a cardiomyocyte e.g., a quiescent cardiomyocyte
  • a hepatoblast e.g., a bile duct hepatoblast
  • an epithelial cell e.g., a T cell (e.g., a naive T cell)
  • a macrophage e.g., a tumor infiltrating macrophage
  • a fibroblast e.g., a cardiac fibroblast
  • the target cell is a tumor-infiltrating lymphocyte, a T cell, a neoplastic or tumor cell, a virus-infected cell, a stem cell, a central nervous system (CNS) cell, a hematopoietic stem cell (HSC), a liver cell or a fully differentiated cell.
  • the target cell is a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a hepatocyte, a hematopoietic stem cell, a CD34+ hematopoietic stem cell, a CD 105+ hematopoietic stem cell, a CD117+ hematopoietic stem cell, a CD 105+ endothelial cell, a B cell, a CD20+ B cell, a CD 19+ B cell, a cancer cell, a CD 133+ cancer cell, an EpCAM+ cancer cell, a CD 19+ cancer cell, a Her2/Neu+ cancer cell, a GluA2+ neuron, a GluA4+ neuron, aNKG2D+ natural killer cell, a SLC1A3+ astrocyte, a SLC7A10+ adipocyte, or a CD30+ lung epithelial cell.
  • the target cell is an antigen presenting cell, an MHC class 11+ cell, a professional antigen presenting cell, an atypical antigen presenting cell, a macrophage, a dendritic cell, a myeloid dendritic cell, a plasmacyteoid dendritic cell, a CD1 lc+ cell, a CD1 lb+ cell, a splenocyte, a B cell, a hepatocyte, an endothelial cell, or a non-cancerous cell).
  • the disclosure also provides methods of targeting diseased cells and tissues and/or delivering a cargo such as, without limitation, therapeutic agents or diagnostic agents to the same.
  • the disclosure provides a method of targeted delivery of a cargo molecule to a cell known to, or suspected of, express Neu5Gc-Lewis a on its cell surface, comprising contacting the cell with a Neu5Gc-Lewis a binding molecule linked to a cargo, directly or through a linker (e.g., bifunctional, trifunctional linker, or multi-functional linker).
  • the linker may be a cleavable or non-cleavable linker.
  • the Neu5Gc-Lewis a binding molecule comprises a peptide, antibody, nucleic acid, a carbohydrate, a lipid, a small organic molecule, a lipid particle (e.g., nanoparticle), or an aptamer.
  • the Neu5Gc-Lewis a binding molecule binds specifically to Neu5Gc-Lewis a .
  • the Neu5Gc-Lewis a binding molecule has been previously determined not to bind to any other form of Neu5Gc or any other Si alyl -Lewi s a , other than Neu5Gc- Lewis a on the cell, including free Neu5Gc.
  • the cell is a cancer cell.
  • the tissue is cancer tissue.
  • the tissue is diseased tissue which has been identified as comprising areas comprising Neu5Gc-Lewis a on the cell surface and/or the ECM.
  • the tissue is atherosclerotic tissue.
  • the cargo is delivered to the cell surface or extracellular matrix. In some embodiments, the cargo is delivered intracellularly.
  • any cell, exosome or tissue may be targeted by a Neu5Gc-Lewis a -binding molecule, provided that it comprises Neu5Gc-Lewis a .
  • the Neu5Gc-Lewis a is on the cell surface.
  • the Neu5Gc-Lewis a is on an exosome.
  • the Neu5Gc-Lewis a is in the ECM.
  • the Neu5Gc-Lewis a is in a combination of any of the cell surface, an exosome or in the ECM.
  • the Neu5Gc-Lewis a is intracellular.
  • a cargo molecule is linked to a Neu5Gc-Lewis a binding molecule and is delivered to a cell, exosome or tissue comprising Neu5Gc-Lewis a .
  • the cargo molecule is delivered extracellularly and/or intracellularly.
  • the cargo comprises a labeling agent (e.g., a visualization agent, a diagnostic agent).
  • the cargo comprises a therapeutic agent.
  • the cargo comprises a molecule selected from peptides, nucleic acids (e.g., siRNA, antisense oligonucleotides, microRNA, shRNA, miRNA, shRNA, gRNA, mRNA, DNA, DNA plasmid, an oligonucleotide, and an analogue thereof), proteins (including, but not limited to, streptavidin, phycoerythrin, a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, a ribonuclease (RNAse), a deoxyribonuclease (DNAse), a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold,
  • nucleic acids e.g., siRNA
  • the linker comprises a polyglycine linker.
  • the linker is selected from the group consisting of beta alanine, cysteine, cysteamide bridge, poly glycine (such as G2 or G4), a PEG linker moiety, Aun (11- amino- undecanoic acid), Ava (5-amino pentanoic acid), and Ahx (aminocaproic acid).
  • the linker comprises a PEG linker moiety.
  • the PEG linker moiety consists of about one to ten (such as about 1-8, 2-7, 1-5, or 6-10) ethylene glycol units.
  • the molecular weight of the PEG linker moiety is about 0.05 kDa to about 0.5 kDa (such as about 0.05-0.1, 0.05-0.4, 0.1 -0.3. 0.05-0.25, 0.25-0.5 kDa).
  • the PEG linker moiety is a linear PEG.
  • the PEG linker moiety is a branched PEG.
  • the linker comprises a beta-alanine.
  • the linker comprises at least about two, three, or four glycines, optionally continuous glycines.
  • the linker further comprises a serine.
  • the linker comprises a GGGGS (SEQ ID NO:3) or SGGGG (SEQ ID NO:4) sequence. In some embodiments, the linker comprises a Glycine-b- Alanine motif. In some embodiments, the cargo may be attached to the Neu5Gc-Lewis a -binding molecule at an amino group, carboxylate group, or the side chain of any of the amino acids of the Neu5Gc-Lewis a -binding molecule (e.g., at the amino group, the carboxylate group, or the side chain.
  • a Neu5Gc-Lewis a -binding molecule e.g., a peptide
  • a bifunctional crosslinking reagent refers to a reagent that possesses two reactive groups one of which is capable of reacting with a Neu5Gc-Lewis a -binding molecule, while the other one is capable of reacting with the drug to link the Neu5Gc-Lewis a -binding molecule with the drug, thereby forming a conjugate.
  • any suitable bifunctional crosslinking reagent may be used in connection with the disclosure, so long as the linker reagent provides for retention of the drug, e.g., cytotoxicity, and targeting characteristics of the Neu5Gc-Lewis a -binding molecule.
  • the linker molecule joins the drug to the Neu5Gc-Lewis a -binding molecule through chemical bonds, such that the drug and the Neu5Gc-Lewis a -binding molecule are chemically coupled (e.g., covalently bonded) to each other.
  • the bifunctional crosslinking reagent comprises noncl eavable linkers.
  • a non-cleavable linker is any chemical moiety that is capable of linking a drug to aNeu5Gc-Lewis a -binding molecule in a stable, covalent manner.
  • a non-cleavable linker is not cleavable under physiological conditions, in particular inside the body and/or inside a cell.
  • non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase- induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the drug or the Neu5Gc-Lewis a -binding molecule remains active.
  • Suitable crosslinking reagents that form non-cleavable linkers between a drug and a Neu5Gc- Lewis a -binding molecule are well known in the art.
  • the drug is linked to the Neu5Gc-Lewis a -binding molecule through a thioether bond.
  • the linking reagent is a cleavable linker.
  • a cleavable linker is cleavable under physiological conditions, in particular inside the body and/or inside a cell.
  • suitable cleavable linkers include disulfide linkers, acid labile linkers, photolabile linkers, peptidase labile linkers, and esterase labile linkers.
  • Non-limiting examples of linkers include, but are not limited to, N-succinimidyl- 3-(2-pyridyldithio)butyrate (SPDB), N-succinimi dyl-3-(2 -pyridyl dithio)propionate (SPDP), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (Sulfo- SMCC), N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N- succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate) (LC- SMCC), 4-maleimidobutyric acid N-hydroxy succinimide ester (GMBS), 3- maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-
  • Disulfide containing linkers are linkers cleavable through disulfide exchange, which may occur under physiological conditions.
  • the linker is cleavable under reducing conditions (e.g., a disulfide linker).
  • disulfide linkers are known in the art, including, for example, those that may be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2- pyridyldithio)propi onate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)
  • SATA N-succinimidyl-5-acetylthioacetate
  • SPDP N-succinimidyl-3-(2- pyridyldithio)propi onate
  • SPDB N-succinimidyl-3-(2-pyridyldithio)butyrate
  • SMPT N-succinimidyl
  • Acid labile linkers are linkers cleavable at acidic pH.
  • certain intracellular compartments such as endosomes and lysosomes, have an acidic pH (pH 4- 5), and provide conditions suitable to cleave acid labile linkers.
  • Acid labile linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0.
  • a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like may be used.
  • Photolabile linkers are useful at the body surface and in many body cavities that are accessible to light.
  • Peptidase labile linkers may be used to cleave certain peptides inside or outside cells.
  • the cleavable linker is cleaved under mild conditions, i.e., conditions within a cell under which the activity of the cytotoxic agent is not affected.
  • the linker may be or may comprise, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • the peptidyl linker is at least two amino acids long or at least three amino acids long.
  • Cleaving agents may include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells.
  • a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue may be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly linker).
  • the peptidyl linker cleavable by an intracellular protease is a valinecitrulline (Val-Cit; vc) linker or a phenylalanine-lysine (Phe-Lys) linker.
  • One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.
  • the therapeutic agent comprises an agent that works as an anti-cancer agent.
  • the therapeutic agent comprises a cell of the disclosure, a therapeutic antibody or a chemotherapeutic agent.
  • the therapeutic agent is selected from the group consisting of an antibody or fragment thereof, a toxin, a radionuclide, an immunomodulator, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
  • the anti-cancer agent is a therapeutic antibody.
  • the antibody or functional fragment thereof is selected from the group consisting of anti-Her2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD33 antibody, anti-PD-Ll antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, anti-TNFa antibody, anti-CD28 antibody, anti-4- IBB antibody, anti-OX40 antibody, anti-GITR antibody, anti-CD27 antibody, antib-CD40 antibody, or anti-ICOS antibody, anti-CD25 antibody, anti-CD30 antibody, anti-CD3 antibody, anti-CD22 antibody, anti-CCR4 antibody, anti-CD38 antibody, anti-CD52 antibody, antiComplement C5 antibody, anti-F protein of RSV, anti-GD2 antibody, anti-GITR antibody, anti-Gly coprotein receptor lib/Illa antibody, anti-ICOS antibody, anti-IL2R antibody, anti-LAG3 antibody, anti-Integrin
  • the therapeutic agent is a “check point inhibitor.”
  • check-point inhibitor refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more checkpoint proteins.
  • checkpoint proteins regulate T cell activation or function. Numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 with its ligands PD-L1 and PD-L2. These proteins appear responsible for costimulatory or inhibitory interactions of T cell responses. Immune check-point proteins appear to regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses.
  • chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophy cin 8);
  • compositions/therapeutic formulations comprising Neu5Gc- Lewis a -binding molecule and another component, such as a carrier.
  • pharmaceutical compositions/therapeutic formulations comprising a Neu5Gc-Lewis a - binding molecule and an excipient and/or diluent.
  • the carrier is not a naturally existing compound.
  • the excipient is not a naturally existing compound.
  • the diluent is not a naturally existing compound.
  • the formulation comprising the Neu5Gc-Lewis a -binding molecule does not contain a naturally existing compound, except, optionally, water.
  • any references to “Neu5Gc-Lewis a -binding molecule” include the “naked” molecule as well as modifications to the “naked” molecule (i.e., modified Neu5Gc-Lewis a -binding molecule), including, for example, addition (direct or indirect linkage or conjugation) of a cargo molecule (e.g., a therapeutic agent, a label, etc.), incorporation into a lipid vesicle, chemical modifications, any other modifications to the “naked” molecule.
  • a cargo molecule e.g., a therapeutic agent, a label, etc.
  • the “naked” Neu5Gc-Lewis a -binding molecule is a peptide or an antibody and the peptide or antibody is chemically modified (e.g., acylation, pegylation, etc.) and conjugated, directly or through a linker, to a cargo molecule.
  • modifications and cargo molecules are described elsewhere in the application.
  • the disclosure provides pharmaceutical compositions of Neu5Gc-Lewis a -binding molecule to be used in accordance with the present disclosure.
  • the compositions are prepared for storage and/or administration by mixing a Neu5Gc-Lewis a -binding molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, diluents, excipients, or stabilizers (Remington's Pharmaceutical Sciences 23rd edition, Adejare, A., Ed. (2020)), in the form of lyophilized formulations or aqueous solutions.
  • acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • the formulation further comprises a surfactant.
  • the surfactant may, for example, be selected from a detergent, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (eg.
  • poloxamers such as Pluronic® F68, poloxamer 188 and 407, Triton X-100
  • polyoxyethylene sorbitan fatty acid esters polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives (tweens, e.g., Tween-20, Tween-40, Tween-80 and Brij-35), monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lectins and phospholipids (e.g., phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin), derivates of phospholipids (e.g., dipalmitoyl phosphatidic acid) and lysophospholipids (eg., dip
  • acylcamitines and derivatives N alpha. -acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, Nalpha.-acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, Nalpha-acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS (docusate sodium, CAS registry no [577-11-7]), docusate calcium, CAS registry no [128-49-4]), docusate potassium, CAS registry no [7491-09-0]), SDS (sodium dodecyl sulphate or sodium lauryl s
  • N-alkyl-N,N- dimethylammonio-1 -propanesulfonates 3-cholamido-l-propyldimethylammonio-l- propanesulfonate, cationic surfactants (quaternary ammonium bases) (e.g.
  • cetyltrimethylammonium bromide, cetylpyridinium chloride non-ionic surfactants (e.g., Dodecyl .beta.-D-glucopyranoside), poloxamines (e.g., Tetronic's), which are tetrafunctional block copolymers derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine, or the surfactant may be selected from the group of imidazoline derivatives, or mixtures thereof.
  • the surfactant is not a naturally existing compound.
  • Each one of these specific surfactants constitutes an alternative embodiment of the disclosure.
  • One embodiment provides for stable formulations of Neu5Gc-Lewis a -binding molecules, which comprise preferably a phosphate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative, as well as multiuse preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one the antibodies and/or target-binding fragments thereof in a pharmaceutically acceptable formulation.
  • a -binding molecules which comprise preferably a phosphate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative, as well as multiuse preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one the antibodies and/or target-binding fragments thereof in a pharmaceutically acceptable formulation.
  • Any suitable concentration or mixture may be used as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,
  • Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001- 0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2
  • the pharmaceutical composition comprises a Neu5Gc-Lewis a -binding molecule of the disclosure and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Additional examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the Neu5Gc-Lewis a -binding molecule.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the carrier include saline, Ringer's solution and dextrose solution.
  • the pH of the solution is from about 5 to about 8. In another embodiment, the pH is from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the Neu5Gc-Lewis a -binding molecule, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles.
  • a sustained release matrix is a matrix made of materials, usually polymers which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
  • the sustained release matrix desirably is chosen by biocompatible materials such as liposomes, polylactides (polylactide acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
  • compositions of this disclosure may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions e.g., tablets, pills, powders, liposomes and suppositories.
  • Dosage forms suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of Neu5Gc-Lewis a -binding molecule (the active ingredient) per unit or container.
  • the active ingredient will ordinarily be present in an amount of about 0.5-99.999% by weight based on the total weight of the composition.
  • compositions/formulations typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions may be prepared by incorporating the active compound (i.e., Neu5Gc-Lewis a -binding molecule) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the preferred dosage form depends on the intended mode of administration and therapeutic application.
  • Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with antibodies.
  • the most typical mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the antibody is administered by intravenous infusion or injection.
  • the antibody is administered by intramuscular or subcutaneous injection.
  • the Neu5Gc-Lewis a -binding molecules of the disclosure may be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • a Neu5Gc-Lewis a -binding molecule may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • routes of administration of any of the compositions disclosed herein include oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • a Neu5Gc-Lewis a -binding molecule may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the Neu5Gc-Lewis a -binding molecule may also be enclosed in a hard- or soft-shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
  • a Neu5Gc-Lewis a -binding molecule may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Neu5Gc- Lewis a -binding molecule To administer a Neu5Gc- Lewis a -binding molecule by other than parenteral administration, it may be necessary to coat the Neu5Gc-Lewis a -binding molecule with or co-administer the Neu5Gc-Lewis a - binding molecule with, a material to prevent its inactivation.
  • compositions are bioavailable and may be delivered orally.
  • Oral compositions may be tablets, troches, pills, capsules, and the like, and may also contain the following: binders such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of Wintergreen, or cherry flavoring may be added.
  • binders such as gum tragacanth, acacia, com starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • the unit dosage form When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar and the like.
  • a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and devices.
  • the compositions suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle may be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity may be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms may be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions may be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating a compound and/or agent disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • compounds and agents disclosed herein may be applied in as a liquid or solid. However, it will generally be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
  • a dermatologically acceptable carrier which may be a solid or a liquid.
  • Compounds and agents and compositions disclosed herein may be applied topically to a subject's skin to reduce the size (and may include complete removal) of malignant or benign growths, or to treat an infection site.
  • Compounds and agents disclosed herein may be applied directly to the growth or infection site.
  • the compounds and agents are applied to the growth or infection site in a formulation such as an ointment, cream, lotion, solution, tincture, or the like.
  • Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein may be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and may be determined by one of skill in the art. The dosage may be adjusted by the individual physician in the event of any counterindications.
  • Dosage may vary, and may be administered in one or more dose administrations daily, for one or several days.
  • doses of a peptide of from 1 ng to 1 mg, preferably from 10 ng to 100 pg, are formulated and administered.
  • doses of from 1 ng to 0.1 mg may be formulated and administered.
  • the therapeutically effective amount of Neu5Gc-Lewis a - binding molecule may be administered in an amount as a measure with regards to the weight of the patient in need thereof.
  • the Neu5Gc-Lewis a -binding molecule may be administered in an amount of about: 0.1 mg/kg to about 50 mg/kg, 0.1 mg/kg to about 40 mg/kg, 0.1 mg/kg to about 30 mg/kg, 0.1 mg/kg to about 25 mg/kg, 0.1 mg/kg to about 20 mg/kg, 0.1 mg/kg to about 15 mg/kg, 0.1 mg/kg to about 10 mg/kg, 0.1 mg/kg to about 7.5 mg/kg, 0.1 mg/kg to about 5 mg/kg, 0.1 mg/kg to about 2.5 mg/kg, or about 0.1 mg/kg to about 1 mg/kg.
  • the Neu5Gc-Lewis a -binding molecule may be administered in an amount of about: 0.5 mg/kg to about 50 mg/kg, 0.5 mg/kg to about 40 mg/kg, 0.5 mg/kg to about 30 mg/kg, 0.5 mg/kg to about 25 mg/kg, 0.5 mg/kg to about 20 mg/kg, 0.5 mg/kg to about 15 mg/kg, 0.5 mg/kg to about 10 mg/kg, 0.5 mg/kg to about 7.5 mg/kg, 0.5 mg/kg to about 5 mg/kg, 0.5 mg/kg to about 2.5 mg/kg, or about 0.5 mg/kg to about 1 mg/kg.
  • the Neu5Gc-Lewis a -binding molecule may be administered in an amount of about 0.5 mg/kg to about 5 mg/kg or about 0.1 mg/kg to about 10 mg/kg.
  • the Neu5Gc-Lewis a -binding molecule may be administered in an amount of about 0.1 mg/kg to about 20 mg/kg or about 0.1 mg/kg to about 30 mg/kg.
  • the Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 150 mg, or 200 mg.
  • the Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg.
  • the Neu5Gc-Lewis a - binding molecule may be administered at an amount of about 1000 mg to about 2000 mg.
  • the Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg to about 10 mg, 10 mg to about 20 mg, 25 mg to about 50 mg, 30 mg to about 60 mg, 40 mg to about 50 mg, 50 mg to about 100 mg, 75 mg to about 150 mg, 100 mg to about 200 mg, 200 mg to about 500 mg, 500 mg to about 1000 mg, 1000 mg to about 1200 mg, 1000 mg to about 1500 mg, 1200 mg to about 1500 mg, or 1500 to about 2000 mg.
  • the Neu5Gc-Lewis a -binding molecule may be administered in an amount of about 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL, 400 mg/mL, or 500 mg/mL.
  • the Neu5Gc-Lewis a -binding molecule is present in the combination in an amount of about: 1 mg/mL to about 10 mg/mL, 5 mg/mL to about 10 mg/mL, 5 mg/mL to about 15 mg/mL, 10 mg/mL to about 25 mg/mL; 20 mg/mL to about 30 mg/mL; 25 mg/mL to about 50 mg/mL, or 50 mg/mL to about 100 mg/mL.
  • the Neu5Gc-Lewis a -binding molecule may be administered, for example, once a day (QD), twice daily (BID), once a week (QW), twice weekly (BIW), three times a week (TIW), or monthly (QM) regularly on a continuous base or intermittent base such as BIW for 3 months then resume a month later.
  • the Neu5Gc-Lewis a -binding molecule may be administered BID.
  • the Neu5Gc-Lewis a -binding molecule may be administered TIW.
  • the Neu5Gc-Lewis a -binding molecule is administered 2 to 3 times a week.
  • the Neu5Gc-Lewis a -binding molecule is administered QD.
  • the Neu5Gc-Lewis a -binding molecule may be administered QD for about: 1 day to about 7 days, 1 day to about 14 days, 1 day to about 21 days, 1 day to about 28 days, or daily until disease progression or unacceptable toxicity.
  • the administration of Neu5Gc-Lewis a -binding molecule may, in part, depend upon the tolerance of the patient where greater tolerance may allow greater or more frequent administration. Alternatively, where a patient shows poor tolerance to Neu5Gc- Lewis a -binding molecule, a less amount of the compound or a less frequent dosing may be performed.
  • Neu5Gc-Lewis a -binding molecules may be administered in any regimen as described herein.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg, QD.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg, BIW.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg, TIW.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg, QW.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg, Q2W.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about 5 mg or about 10 mg, QD.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about 5 mg or about 10 mg, BIW.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about 5 mg or about 10 mg, TIW. In one embodiment, a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about 5 mg or about 10 mg, QW. In one embodiment, a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about 5 mg or about 10 mg, Q2W. Administration of a Neu5Gc-Lewis a -binding molecule may be continuous. Administration of a Neu5Gc-Lewis a -binding molecule may be intermittent.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg to about 10 mg, 1 mg to about 25 mg, 1 mg to about 50 mg, 5 mg to about 10 mg, 5 mg to about 25 mg, 5 mg to about 50 mg, 10 mg to about 25 mg, 10 mg to about 50 mg, 50 mg to about 100 mg, or 100 mg to about 200 mg, QD.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg to about 10 mg, 1 mg to about 25 mg, 1 mg to about 50 mg, 5 mg to about 10 mg, 5 mg to about 25 mg, 5 mg to about 50 mg, 10 mg to about 25 mg, 10 mg to about 50 mg, 50 mg to about 100 mg, or 100 mg to about 200 mg, BIW.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg to about 10 mg, 1 mg to about 25 mg, 1 mg to about 50 mg, 5 mg to about 10 mg, 5 mg to about 25 mg, 5 mg to about 50 mg, 10 mg to about 25 mg, 10 mg to about 50 mg, 50 mg to about 100 mg, or 100 mg to about 200 mg, TIW.
  • a Neu5Gc- Lewis a -binding molecule may be administered at an amount of about: 1 mg to about 10 mg, 1 mg to about 25 mg, 1 mg to about 50 mg, 5 mg to about 10 mg, 5 mg to about 25 mg, 5 mg to about 50 mg, 10 mg to about 25 mg, 10 mg to about 50 mg, 50 mg to about 100 mg, or 100 mg to about 200 mg, QW.
  • a Neu5Gc-Lewis a - binding molecule may be administered at an amount of about: 1 mg to about 10 mg, 1 mg to about 25 mg, 1 mg to about 50 mg, 5 mg to about 10 mg, 5 mg to about 25 mg, 5 mg to about 50 mg, 10 mg to about 25 mg, 10 mg to about 50 mg, 50 mg to about 100 mg, or 100 mg to about 200 mg, Q2W.
  • Administration of a Neu5Gc-Lewis a -binding molecule may be continuous.
  • Administration of a Neu5Gc-Lewis a -binding molecule may be intermittent.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 0.0001 mg/kg to about 200 mg/kg, 0.001 mg/kg to about 200 mg/kg, 0.01 mg/kg to about 200 mg/kg, 0.01 mg/kg to about 150 mg/kg, 0.01 mg/kg to about 100 mg/kg, 0.01 mg/kg to about 50 mg/kg, 0.01 mg/kg to about 25 mg/kg, 0.01 mg/kg to about 10 mg/kg, or 0.01 mg/kg to about 5 mg/kg, 0.05 mg/kg to about 200 mg/kg, 0.05 mg/kg to about 150 mg/kg, 0.05 mg/kg to about 100 mg/kg, 0.05 mg/kg to about 50 mg/kg, 0.05 mg/kg to about 25 mg/kg, 0.05 mg/kg to about 10 mg/kg, or 0.05 mg/kg to about 5 mg/kg, 0.5 mg/kg to about 200 mg/kg, 0.5 mg/kg to about 150 mg/kg,
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 0.0001 mg/kg to about 200 mg/kg, 0.001 mg/kg to about 200 mg/kg, 0.5 mg/kg to about 200 mg/kg, 0.5 mg/kg to about 150 mg/kg, 0.5 mg/kg to about 100 mg/kg, 0.5 mg/kg to about 50 mg/kg, 0.5 mg/kg to about 25 mg/kg, 0.5 mg/kg to about 10 mg/kg, or 0.5 mg/kg to about 5 mg/kg, BIW.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 0.0001 mg/kg to about 200 mg/kg, 0.001 mg/kg to about 200 mg/kg, 0.5 mg/kg to about 200 mg/kg, 0.5 mg/kg to about 150 mg/kg, 0.5 mg/kg to about 100 mg/kg, 0.5 mg/kg to about 50 mg/kg, 0.5 mg/kg to about 25 mg/kg, 0.5 mg/kg to about 10 mg/kg, or 0.5 mg/kg to about 5 mg/kg, TIW.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 0.0001 mg/kg to about 200 mg/kg, 0.001 mg/kg to about 200 mg/kg, 0.5 mg/kg to about 200 mg/kg, 0.5 mg/kg to about 150 mg/kg, 0.5 mg/kg to about 100 mg/kg, 0.5 mg/kg to about 50 mg/kg, 0.5 mg/kg to about 25 mg/kg, 0.5 mg/kg to about 10 mg/kg, or 0.5 mg/kg to about 5 mg/kg, QW.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 0.0001 mg/kg to about 200 mg/kg, 0.001 mg/kg to about 200 mg/kg, 0.5 mg/kg to about 200 mg/kg, 0.5 mg/kg to about 150 mg/kg, 0.5 mg/kg to about 100 mg/kg, 0.5 mg/kg to about 50 mg/kg, 0.5 mg/kg to about 25 mg/kg, 0.5 mg/kg to about 10 mg/kg, or 0.5 mg/kg to about 5 mg/kg, Q2W.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about 15 mg/kg to about 75 mg/kg, QD.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about 20 mg/kg to about 50 mg/kg. In still another example, a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about 0.001 mg/kg, 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, or 200 mg/kg.
  • a Neu5Gc-Lewis a -binding molecule may be continuous. Administration of a Neu5Gc-Lewis a -binding molecule may be intermittent. [0202] In one embodiment, a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg/kg to about 200 mg/kg, 1 mg/kg to about 150 mg/kg, 1 mg/kg to about 100 mg/kg, 1 mg/kg to about 50 mg/kg, 1 mg/kg to about 25 mg/kg, 1 mg/kg to about 10 mg/kg, or 1 mg/kg to about 5 mg/kg, QD.
  • a Neu5Gc- Lewis a -binding molecule may be administered at an amount of about: 1 mg/kg to about 200 mg/kg, 1 mg/kg to about 150 mg/kg, 1 mg/kg to about 100 mg/kg, 1 mg/kg to about 50 mg/kg, 1 mg/kg to about 25 mg/kg, 1 mg/kg to about 10 mg/kg, or 1 mg/kg to about 5 mg/kg, BIW.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg/kg to about 200 mg/kg, 1 mg/kg to about 150 mg/kg, 1 mg/kg to about 100 mg/kg, 1 mg/kg to about 50 mg/kg, 1 mg/kg to about 25 mg/kg, 1 mg/kg to about 10 mg/kg, or 1 mg/kg to about 5 mg/kg, TIW.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about: 1 mg/kg to about 200 mg/kg, 1 mg/kg to about 150 mg/kg, 1 mg/kg to about 100 mg/kg, 1 mg/kg to about 50 mg/kg, 1 mg/kg to about 25 mg/kg, 1 mg/kg to about 10 mg/kg, or 1 mg/kg to about 5 mg/kg, QW.
  • a Neu5Gc-Lewis a - binding molecule may be administered at an amount of about: 1 mg/kg to about 200 mg/kg, 1 mg/kg to about 150 mg/kg, 1 mg/kg to about 100 mg/kg, 1 mg/kg to about 50 mg/kg, 1 mg/kg to about 25 mg/kg, 1 mg/kg to about 10 mg/kg, or 1 mg/kg to about 5 mg/kg, Q2W.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about 15 mg/kg to about 75 mg/kg, QD.
  • a Neu5Gc- Lewis a -binding molecule may be administered at an amount of about 20 mg/kg to about 50 mg/kg.
  • a Neu5Gc-Lewis a -binding molecule may be administered at an amount of about 0.001 mg/kg, 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, or 200 mg/kg.
  • Administration of a Neu5Gc- Lewis a -binding molecule may be continuous.
  • Administration of a Neu5Gc-Lewis a - binding molecule may be intermittent.
  • the Neu5Gc-Lewis a -binding molecule, or any one of the combinations described herein, may be administered in a regimen.
  • the regimen may be structured to provide therapeutically effective amounts of Neu5Gc-Lewis a -binding molecule, or any one of the combinations described herein, over a predetermined period of time (e.g., an administration time).
  • the regimen may be structured to limit or prevent side-effects or undesired complications of each of the components of Neu5Gc-Lewis a -binding molecule, or any one of the combinations described herein described herein.
  • the regimen may be structured in a manner that results in increased effect for both therapies of the combination (e.g., synergy).
  • Regimens useful for treating cancer may include any number of days of administration which may be repeated as necessary. Administration periods may be broken by a rest period that includes no administration of at least one therapy. For example, a regimen may include administration periods that include 2, 3, 5, 7, 10, 15, 21, 28, or more days. These periods may be repeated. For example, a regimen may include a set number of days as previously described where the regimen is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more times.
  • the regimens may include a rest period of at least 1, 2, 3, 5, 7, 10, or more days, where at least one therapy is no longer administered to a patient.
  • the rest period may be determined by, for example, monitoring the reaction of the patient to Neu5Gc-Lewis a -binding molecule, or any one of the combinations described herein or by measuring the efficacy of the treatment.
  • a rest period may be applicable to a single therapy, such that only one therapy of a combination described herein is discontinued in the rest period, but the other therapy(ies) are still administered. Rest periods may be applied to all the therapies administered to the subject such that the subject receives no therapy for a set period of time during the rest period.
  • the Neu5Gc-Lewis a -binding molecule may be formulated with or conjugated to another therapeutic agent.
  • This agent may be anything that may also be used in the indication for which the Neu5Gc-Lewis a -binding molecule is designed to work.
  • the Neu5Gc-Lewis a -binding molecule may be coadministered in combination with another therapeutic agent or treatment (e.g., radiation).
  • the therapeutic agent or treatment are administered before, concurrently, and/or after the administration of the Neu5Gc-Lewis a -binding molecule.
  • Co-administered means that two (or more) different treatments (e.g., a Neu5Gc-Lewis a -binding molecule and an antibiotic) are delivered to the subject during the course of the subject's affliction with the disease, e.g., the two or more treatments are delivered after the subject has been diagnosed with a disease and before the disease has been cured or eliminated or treatment has ceased for other reasons.
  • the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”.
  • the delivery of one treatment ends before the delivery of the other treatment begins.
  • the treatment is more effective because of combined administration.
  • the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment.
  • delivery is such that the reduction in a symptom (e.g., toxicity resulting from by administration of Neu5Gc-Lewis a -binding molecule), or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments may be partially additive, wholly additive, or greater than additive.
  • the delivery may be such that the effect of the first treatment delivered is still detectable when the second is delivered.
  • the molecules of the disclosure and the additional therapeutic agent may be administered simultaneously, in the same or in separate compositions, or sequentially.
  • the Neu5Gc-Lewis a -binding molecule or other molecules described herein may be administered first, and the therapeutic agent may be administered second, or the order of administration may be reversed.
  • the Neu5Gc-Lewis a -binding molecule or any other molecule described herein may be administered during periods of active disorder, or during a period of remission or less active disease.
  • the Neu5Gc-Lewis a -binding molecule or any other molecule described herein may be administered before another treatment, concurrently with the treatment, post-treatment, or during remission of the disorder (e.g., cancer).
  • the disclosure provides a kit for the diagnosis or prognosis of cancer and other diseases associated with the presence of Neu5Gc-Lewis a as a biomarker of the same.
  • the kit comprises a reference sample known to comprise Neu5Gc-Lewis a and means for detecting presence and level of Neu5Gc-Lewis a in a test sample.
  • the means comprises an anti-Neu5Gc-Lewis a antibody or peptide.
  • the means for detecting Neu5Gc-Lewis a comprises MGS5 or a peptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity to a peptide comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • a kit of the embodiments comprises a desialylating reagent such as a sialidase, a neuraminidase enzyme, or a mild acid, such as H2SO4.
  • kits of the embodiments include, without limitation, a microtiter plate, a detectable label, a dilution buffer (e.g., PBS or water), a solid phase carrier, a blocking solution, a chromogenic reagent, a calibrator, and a washing buffer.
  • the blocking solution may be one or more of BSA, bovine serum, skim milk, TBST and other components.
  • the chromogenic solution may be determined according to the labeled substance on the Neu5Gc-Lewis a -binding molecule (e.g., peptide, antibody, etc.), for example, when the labeled substance is horseradish peroxidase, the chromogenic reagent may be luminol.
  • a -binding molecule e.g., peptide, antibody, etc.
  • the presence of Neu5Gc-Lewis a in the sample is quantitated (for example, for monitoring disease progression or treatment effectiveness).
  • the quantitative assay is an ELISA assay, a radioimmunoassay (RIA), an immunoradiometric assay, a fluoroimmunoassay, a chemiluminescent immunoassay, a bioluminescent immunoassay, an enzyme multiplied immunoassay (EMIT), a cloned enzyme donor immunoassay (CEDIA), an immuno-PCR assay, a phosphor immunoassay, a quantum dot immunoassay, a solid phase light- scattering immunoassay, a surface effect immunoassay, or an immunoassay employing lateral flow test strips.
  • the specificity of the detection of the Neu5Gc-Lewis a biomarker may be confirmed by desialylating the sample with, for example, a sialidase.
  • the kit may further comprise a standard or control information so that the test sample may be compared with the control information standard to determine if the test amount of Neu5Gc-Lewis a detected in a sample is a diagnostic amount consistent with a diagnosis of a disease, condition or disorder, such as cancer, preferably pancreatic or breast cancer.
  • the immunodetection methods of the present disclosure have evident utility in the diagnosis and prognosis of conditions such as cancer wherein Neu5Gc-Lewis a is expressed, and wherein antibodies or other Neu5Gc-Lewis a -binding molecules exist that react (immunologically) with Neu5Gc-Lewis a .
  • a biological and/or clinical sample suspected of containing a specific disease associated with Neu5Gc-Lewis a is used.
  • these embodiments also have applications to non-clinical samples, such as in the titering of antigen or antibody samples, for example in the selection of hybridomas.
  • kits may be used to identify compounds that modulate expression of Neu5Gc-Lewis a in in vitro disease cell models and/or in in vivo animal models, such as in, e.g., pancreatic or breast cancer cells or in vivo animal models for, e.g., pancreatic or breast cancer.
  • the Glycan Array 300 was obtained from (RayBiotech Life, GA-Glycan-300). The glycan array glass slide assembly was allowed to equilibrate to room temperature for 30 minutes. The slide was then removed from the package along with the cover film and allowed to air dry at room temperature for another 2 hours. The array was blocked for 30 minutes using the sample diluent provided in the kit. MGS5_V2 was diluted using the sample diluent to a concentration of 1 pg/ml and added to the array. The array was incubated for 3 hours at room temperature with gentle rocking. Following the 3-hour incubation the array was washed a total of 7 times with the provided wash buffers.
  • the kit provided Cy3-Conjugated Streptavidin was added to the array for 1 hour at room temperature with gentle rocking. After the incubation the array was washed 5 times with the kit provided wash buffer. The array was disassembled and washed for 15 minutes with wash buffer, decanted, and then washed an additional five minutes. The array was washed with de-ionized water for 5 minutes. The array was then imaged using the Leica DMi8 microscope. Result: MGS5_V1 and MGS5_V2 are shown to strongly bind Neu5Gc-a-2,3-Gal-P-l,3-(Fuc-a-l,4)-GlcNAc (spotted in triplicate). FIG. 2.
  • Tissue Microarrays were obtained from Novus Biologies and the Cooperative Human Tissue Network (CHTN). Slides were dried for 1 hour at 60 °C. Tissues were deparaffmized by immersing in EZ-DeWax solution for 5 minutes, two times, with occasional agitation, and rinsed with water. Antigen retrieval was performed using citrate buffer pH 6.0 at 65°C for 10 minutes. Slides were washed three times for 5 minutes each with lx TBS.
  • CHTN Cooperative Human Tissue Network
  • BLOXALL® (Vector Laboratories, SP-6000-100) was applied to the tissues for 10 minutes to block any endogenous peroxidases, followed by a 5-minute wash using lx TBS. Slides were blocked for 30 minutes, followed by the addition of 25 nM MGS5-V2-HRP for 1 hour. Slides were washed 5 times for 5 minutes each using lx TBS + 0.1% Tween 20. To detect HRP signal, ImmPACT DAB solution (Vector Laboratories, SK-4105) was added to the tissues for 1 minute. Slides were rinsed in 0.05 M sodium bicarbonate, pH 9.6 for 10 minutes.
  • Tissues were incubated with DAB enhancing solution (Vector Laboratories, H-2200) for 15 seconds, followed by a 5- minute wash in water. Slides were dipped in Hematoxylin (Gill’s Formula) (Vector Laboratories, H-3401) for 3 minutes and rinsed in running tap water. Slides were differentiated by dipping 10 times in an acid rinse followed by 10 dips in tap water. Slides were incubated in a bluing solution for 1 minute and washed for 5 minutes in water. Rapid dehydration was performed using 2-propanol/ isopropyl alcohol, twice, for 1 minute each. Coverslips were mounted using VectaMount ® Express Mounting Medium (Vector Laboratories, H-5700-60) and allowed to dry. Slides were viewed and imaged using the Leica DMi8 microscope. FIG. 3.
  • Pan02 cells were plated at 75,000 cells/well in a 12-well plate and allowed to attach for 24 hours. Following attachment, cells were treated with various concentrations of sialidase Vibro cholerae) (Millipore Sigma, 11080725001) for 1 hour at 37°C and pH 6.0. Sialidase treatment was removed and replaced with 125 nM of MGS5_V2-647 and incubated for 1 hour at 37°C. Following the 1 hour incubation, cells were washed with lx PBS 3 times and prepared for flow cytometry. Result: Following sialidase treatment, MGS5 V2 showed up to a 50% reduction in cellular uptake by the murine pancreatic cancer cell line, Pan02. This result demonstrates MGS5_V2 dependence on Neu5Gc- Lewis a for cellular internalization. FIG. 4.
  • Retrogenix assay The following describes the detailed procedure for the Retrogenix assay, which was used to determine MGS5’s ability to bind to a variety of protein receptors.
  • test peptide or PBS only was added to slides of fixed untransfected HEK293 cells using either the sequential method (test peptide added to slides, washed and then addition of AF647 Streptavidin detection reagent) or preincubation method (test peptide pre-incubated with AF647 Streptavidin at a 4:1 molar ratio before addition to slides) of secondary addition. Binding to untransfected cells was assessed by fluorescence imaging.
  • Test peptide MGS5_V2 was added to each slide after cell fixation, using both the sequential and pre-incubation methods of secondary addition, giving a final concentration of 0.2 mg/mL. Detection of binding was performed by using AF647 Streptavidin. Fluorescent images were analyzed and quantitated (for transfection) using ImageQuant software (GE healthcare, Version 8.2). A protein ‘hit’ was defined as a duplicate spot showing a raised signal compared to background levels. This is achieved by visual inspection using the images gridded on the ImageQuant software. Hits were then classified as ‘strong, medium, weak or very weak’, depending on the intensity of the duplicate spots.
  • vectors encoding all hits identified in the Library screen were arrayed and expressed in HEK293 cells on new slides.
  • MGS5_V2 showed no specific interactions using either the sequential or pre-incubation methods of secondary addition on either fixed or live cell microarrays.
  • the disclosure provides methods for diagnosing and treating cancer and provides evidence that Neu5Gc-Lewis a is present in cancer cells and tissues but not in their healthy counterparts. However, the methods may be used to treat other diseases or conditions, provided that they are first identified as associated with the presence of Neu5Gc-Lewis a .
  • Neu5Gc-Lewis a presence be limited to cancer cells and tissues for purposes of drug targeting and diagnosis
  • the methods of the disclosure provide that the presence of Neu5Gc-Lewis a may be associated with, although it may be confirmed, a pathological cell in an organ or tissue selected from the group consisting of liver, lung, kidney, brain, intestine, spleen, heart, muscle, and lymph node.
  • the disease or condition is selected from the group consisting of diabetes, autoimmune diseases, hematological diseases, cardiac diseases, vascular diseases, inflammatory diseases, fibrotic diseases, viral infectious diseases, hereditary diseases, ocular diseases, liver diseases, lung diseases, muscle diseases, protein deficiency diseases, lysosomal storage diseases, neurological diseases, kidney diseases, aging and degenerative diseases, and diseases characterized by cholesterol level abnormality.
  • FIG. 5 shows an example of a conjugate formed by reaction of the thiol group (highlighted by arrow) on the dimer core with the cargo, in this example AF647.
  • FIG. 6 shows an example of an assay to detect Neu5Gc-Lewis a on exosomes.
  • An exemplary protocol may include the steps of: contacting a surface bound with an Neu5Gc-Lewis a binding molecule with a sample comprising an exosome (e.g., an ELISA plate coated with MGS5 peptide), to capture exosomes comprising Neu5Gc-Lewis a ; contacting the captured exosomes with anti-exosome antibody conjugated to a molecule for detection of the captured exosomes (e.g., an anti-CD63 antibody linked to a HRP enzyme); and detecting the binding of the secondary antibody to the captured exosomes (e.g., developed using HRP chromogenic substrate.)

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Abstract

La divulgation concerne la découverte de Neu5Gc-Lewisa en tant que biomarqueur pour des cellules et des tissus cancéreux et concerne des méthodes de diagnostic, de traitement et de ciblage pharmacologique basés sur celles-ci, ainsi que des molécules qui se lient spécifiquement à Neu5Gc-Lewisa.
PCT/US2024/035392 2023-06-26 2024-06-25 Molécules de ciblage de neu5gc-lewisa et leurs utilisations contre le cancer Ceased WO2025006452A2 (fr)

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