WO2025006452A9 - 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

Info

Publication number
WO2025006452A9
WO2025006452A9 PCT/US2024/035392 US2024035392W WO2025006452A9 WO 2025006452 A9 WO2025006452 A9 WO 2025006452A9 US 2024035392 W US2024035392 W US 2024035392W WO 2025006452 A9 WO2025006452 A9 WO 2025006452A9
Authority
WO
WIPO (PCT)
Prior art keywords
neu5gc
lewis
cancer
cell
tissue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2024/035392
Other languages
English (en)
Other versions
WO2025006452A2 (fr
WO2025006452A3 (fr
Inventor
Indu Venugopal
Shelby KNOCHE
Michael J. Mcguire
Kathlynn Corinne BROWN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SRI International Inc
Original Assignee
SRI International Inc
Stanford Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SRI International Inc, Stanford Research Institute filed Critical SRI International Inc
Publication of WO2025006452A2 publication Critical patent/WO2025006452A2/fr
Publication of WO2025006452A9 publication Critical patent/WO2025006452A9/fr
Publication of WO2025006452A3 publication Critical patent/WO2025006452A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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

  • 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.
  • 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 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 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 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 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
  • 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 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 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 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 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
  • 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,
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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 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
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • 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 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.
  • 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 IO 8 cells, or below, e.g., less than 1 x IO 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.
  • 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 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 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 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.
  • 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-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
  • 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 other therapeutic agent is an anti-tumor antibiotic (e.g., actinomycin D, doxorubicin, daunorubicin, Epirubicin, mitomycin, pelomycin, pingyangmycin, pirarubicin;), antiviral, anti-inflammatory agent, a cytokine, a hematopoietic growth factor, anti-cancer agent (including a chemotherapeutic agent), an immunomodulatory agent, an immunosuppressive agent, a steroid (e.g., a corticosteroid) or a pharmacologically active derivative thereof, a vitamin, calcium or a calcium supplement, and the like.
  • an anti-tumor antibiotic e.g., actinomycin D, doxorubicin, daunorubicin, Epirubicin, mitomycin, pelomycin, pingyangmycin, pirarubicin
  • antiviral anti-inflammatory agent
  • a cytokine e.g.,
  • 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.
  • 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.
  • 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
  • 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.
  • preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent.
  • 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
  • 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 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.
  • 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, 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: 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, 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, 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, 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: 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.
  • 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).
  • 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.
  • 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 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 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.
  • 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.
  • 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.)

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363510326P 2023-06-26 2023-06-26
US63/510,326 2023-06-26

Publications (3)

Publication Number Publication Date
WO2025006452A2 WO2025006452A2 (fr) 2025-01-02
WO2025006452A9 true WO2025006452A9 (fr) 2025-04-17
WO2025006452A3 WO2025006452A3 (fr) 2025-05-15

Family

ID=93940139

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/035392 Ceased WO2025006452A2 (fr) 2023-06-26 2024-06-25 Molécules de ciblage de neu5gc-lewisa et leurs utilisations contre le cancer

Country Status (1)

Country Link
WO (1) WO2025006452A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2524233A4 (fr) * 2010-01-15 2013-07-31 Univ California Compositions et procédés pour la détection du cancer
FI3218005T3 (fi) * 2014-11-12 2023-03-31 Seagen Inc Glykaanin kanssa vuorovaikutteisia yhdisteitä ja käyttömenetelmiä
AU2020357455B2 (en) * 2019-10-01 2026-04-09 Sri International Molecular guide system peptides and uses thereof

Also Published As

Publication number Publication date
WO2025006452A2 (fr) 2025-01-02
WO2025006452A3 (fr) 2025-05-15

Similar Documents

Publication Publication Date Title
JP7121724B2 (ja) ヒト抗体、医薬組成物及び方法
TWI780045B (zh) 抗體、醫藥組合物及方法
TW202023629A (zh) 用於調節單核球及巨噬細胞發炎表型之組合物及方法以及其免疫療法用途
US20230279112A1 (en) Methods of treating cancer using antibodies and molecules that bind to btn1a1 or btn1a1-ligands
US20200181264A1 (en) Bispecific antigen-binding molecule
TW201627324A (zh) 治療及檢測癌症之組合物及方法
TW201702269A (zh) 治療及檢測癌症之組合物及方法
US20250129159A1 (en) Methods of treating cancer using antibodies and molecules that immunospecifically bind to btn1a1
AU2019276647A1 (en) Combination therapy by using anti-Globo H or anti-SSEA-4 antibody with anti-negative immune check points antibody
CN105873950A (zh) 用于治疗和诊断癌症的抗s100a7抗体
US20250145715A1 (en) B7-h3 targeting fusion proteins and methods of use thereof
JP2022078202A (ja) Ｐｒｌ３抗体
WO2025006452A9 (fr) Molécules de ciblage de neu5gc-lewisa et leurs utilisations contre le cancer
JP2022513082A (ja) 免疫応答を調節するためのＩＲＥ１α－ＸＢＰ１シグナル伝達経路バイオマーカーの使用
US20050095237A1 (en) Methods of therapy and diagnosis using targeting of cells that express P2Y10
WO2004047612A2 (fr) Procédés de thérapie et de diagnostic
EP1572235A2 (fr) Procedes de therapie et de diagnostic par ciblage de cellules exprimant des proteines lax
WO2004049917A2 (fr) Procedes de therapie et de diagnostic utilisant le ciblage de cellules d'expression de ly-9
JP2011045339A (ja) ＴＧＦ−βＩＩ型受容体に結合する核酸およびその使用

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE