WO2023113596A1 - Diagnostic et traitement du cancer colorectal - Google Patents

Diagnostic et traitement du cancer colorectal Download PDF

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Publication number
WO2023113596A1
WO2023113596A1 PCT/NL2022/050715 NL2022050715W WO2023113596A1 WO 2023113596 A1 WO2023113596 A1 WO 2023113596A1 NL 2022050715 W NL2022050715 W NL 2022050715W WO 2023113596 A1 WO2023113596 A1 WO 2023113596A1
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Prior art keywords
glycan
crc
core
subject
marker
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PCT/NL2022/050715
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English (en)
Inventor
Manfred Wuhrer
Katarina MADUNIC
Gerritje S.M. LAGEVEEN-KAMMEIJER
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ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n. LUMC)
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Priority claimed from NL2030124A external-priority patent/NL2030124B1/en
Application filed by ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n. LUMC) filed Critical ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n. LUMC)
Publication of WO2023113596A1 publication Critical patent/WO2023113596A1/fr

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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/38—Post-translational modifications [PTMs] in chemical analysis of biological material addition of carbohydrates, e.g. glycosylation, glycation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • CRC colorectal cancer
  • CRC is one of the leading malignancies worldwide with over 900,000 deaths in 2020
  • Conventional therapeutic strategies include chemotherapy, radiation therapy, and surgery.
  • due to poor screening strategies and lack of symptoms in early stages most cases are detected at an advanced stage, leading to unsuccessful treatment.
  • glycosylation of proteins and lipids is a hallmark of cancer 4 - 5 originating from changes in the expression of genes that encode for glycosyltransferases, changes in their enzymatic activity, their mislocalisation in the endoplasmic reticulum or Golgi apparatus, availability of substrates or nucleotide sugar donors as well as competition between the enzymes 4 .
  • the characteristic glycan alterations in cancer include specific aberrant expression of incomplete carbohydrate structures or de novo expression of carbohydrate antigens also known as tumor-associated carbohydrate antigens Aberrant cancer glycosylation has already found its way into the clinics as a serological marker for several cancers, namely the presence of a sialyl Lewis A antigen (CA19-9) in the sera of cancer patients.
  • the present disclosure is based on the inventors’ finding of new structural glycan features and O-glycosylation markers associated with CRC. Specifically, as shown in the Examples section of the present disclosure, and in Figures 8 and 15, the inventors have identified that the presence and/or increased expression of core 2 glycans with a2-3 sialylation, core 2 glycans with Sialyl Lewis X (SLeX), and/or Sialyl Lewis A (SLeA) allows differentiation between cancer and normal colon mucosa rendering these structural features useful tools for CRC diagnosis.
  • SLeX Sialyl Lewis X
  • SeA Sialyl Lewis A
  • Fig 15f the inventors have found that the expression of core 2 glycans with a2-3 sialylation is on average about 6.9-fold greater in CRC samples as compared to normal colon mucosa. Additionally, as shown in Fig 15e, the inventors have found that in normal colon mucosa core 2 glycans with SLeX and/or SLeA are substantially absent but readily present in CRC samples.
  • the inventors have also identified seven new glycosylation markers specific for CRC. These markers have one or more of the structural glycan features mentioned above. These seven markers are H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • markers Six of these markers were found by the inventors to be solely associated with CRC samples. Additionally, the marker H2N2S1d was found to be predominantly associated with CRC samples. These seven markers were not only detected in primary CRC samples, but also samples from metastatic CRC sites (such as liver metastases), rendering these markers a useful diagnostic tool for both, primary and metastatic, CRC. Whilst all seven markers disclosed herein are useful in the context of CRC diagnoses, the inventors have identified that 94% of the tested CRC samples had at least one of the markers selected from group consisting of H2N2F1S1c and H2N2S1d. Accordingly, these two markers may be of particular relevance in CRC diagnostics.
  • the markers disclosed herein may be useful therapeutic targets. This is because alterations in glycosylation are a proven hallmark in many diseases (including cancers). In cancers aberrant protein glycosylation has been shown to actively drive tumor development and progression. Additionally, targeting glycans may have several benefits compared to proteins. Namely, the markers are expressed on cell surfaces directly accessible to therapeutics and can be carried by multiple proteins, reflecting the overall glycosylation phenotype of the cell, providing a broader tumor targeting strategy 46 . Accordingly, the present disclosure also provides therapeutics and compositions related to the CRC markers disclosed herein. Such therapeutics may include antibodies against said markers, adoptive cell therapies and antibody-drug conjugates.
  • the present invention provides a method of diagnosing CRC in a subject, or a method for determining if a subject is at risk of CRC, the method comprising the step of detecting the presence or absence of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and a core 2 glycan with SLeA in a sample from the subject, wherein the presence of at least one marker comprising the glycan structural feature is indicative of the subject having CRC, or being at risk of developing CRC, optionally wherein the marker is selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the present invention provides a method of diagnosing CRC in a subject, or a method for determining if a subject is at risk of CRC, the method comprising the step of detecting the presence or absence of at least one marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject, wherein the presence of at least one of the markers is indicative of the subject having CRC or being at risk of developing CRC.
  • the step of detecting the presence may comprise determining the levels of at least one of the markers, and optionally comparing the levels to a reference level, wherein an increase in the levels as compared to a reference level is indicative of the subject having CRC or being at risk of developing CRC.
  • the method of diagnosing CRC in a subject, or determining if a subject is at risk of developing CRC may further comprise providing the subject with an effective amount of a treatment for CRC if the subject is diagnosed with CRC or determined to be at risk of developing CRC.
  • the present invention provides a method for monitoring CRC progression or remission in a subject, the method comprising: a) Detecting the presence or absence of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SleX, and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after a time interval; c) comparing the results obtained in step b) with the results obtained in step a), wherein: i) the presence in step a)
  • the present invention provides a method for monitoring CRC progression or remission in a subject, the method comprising: a) determining the levels of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after a time interval; and c) comparing the levels determined in step b) with the levels determined in step a), wherein: i) an increase in the levels determined in step
  • the present invention provides a method for monitoring the therapeutic effect of a treatment regimen for CRC, the method comprising: a) detecting the presence or absence of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after treatment for a time interval; and c) comparing the results obtained in step b) with the results obtained in step a), wherein: i) the absence in step
  • the present invention provides a method for monitoring the therapeutic effect of a treatment regimen for CRC, the method comprising: a) determining the levels of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after treatment for a time interval; and c) comparing the levels determined in step b) with the levels determined in step a), wherein an increase or no change in the levels determined in
  • the present invention provides a method for monitoring a subject’s compliance or adherence with a prescribed treatment regimen for CRC, the method comprising: a) detecting the presence or absence of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after the prescribed start of the treatment regimen; and c) comparing the results obtained in step b) with the results obtained in step a), wherein
  • the present invention provides a method for monitoring a subject’s compliance or adherence with a prescribed treatment regimen for CRC, the method comprising: a) determining the levels of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after the prescribed start of the treatment regimen; and c) comparing the levels determined in step b) with the levels determined in step a), wherein an increase
  • the present invention provides a method for monitoring CRC progression or remission in a subject, the method comprising: a) detecting the presence or absence of at least one marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after a time interval; c) comparing the results obtained in step b) with the results obtained in step a), wherein: i) the presence in step a) and absence in step b) is indicative of CRC remission; or ii) the absence in step a) and presence in step b) is indicative of CRC progression.
  • the present invention provides a method for monitoring CRC progression or remission in a subject, the method comprising: a) determining the levels of at least one marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after a time interval; and c) comparing the levels determined in step b) with the levels determined in step a), wherein: i) an increase in the levels determined in step b) as compared to the levels determined in step a) is indicative of CRC progression; or ii) a decrease in the levels determined in step b) as compared to the levels determined in step a) is indicative of CRC remission.
  • the present invention provides a method for monitoring the therapeutic effect of a treatment regimen for CRC, the method comprising: a) detecting the presence or absence of at least one marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after treatment for a time interval; and c) comparing the results obtained in step b) with the results obtained in step a), wherein: i) the absence in step a) and presence in step b) is indicative of a lack of therapeutic effect; or ii) the presence in step a) and the presence in step b) is indicative of a lack of therapeutic effect.
  • the present invention provides a method for monitoring the therapeutic effect of a treatment regimen for CRC, the method comprising: a) determining the levels of at least one marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after treatment for a time interval; and c) comparing the levels determined in step b) with the levels determined in step a), wherein an increase or no change in the levels determined in step b) as compared to the levels determined step a) is indicative of a lack of therapeutic effect.
  • the present invention provides a method for monitoring a subject’s compliance or adherence with a prescribed treatment regimen for CRC, the method comprising: a) detecting the presence or absence of at least one marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after the prescribed start of the treatment regimen; and c) comparing the results obtained in step b) with the results obtained in step a), wherein: i) the presence in step a) and absence in step b) is indicative of the subject having complied or adhered with the prescribed treatment; or ii) the absence in step a) and absence in step b) is indicative of the subject having complied or adhered with the prescribed treatment;
  • the present invention provides a method for monitoring a subject’s compliance or adherence with a prescribed treatment regimen for CRC, the method comprising: a) determining the levels of at least one marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after the prescribed start of the treatment regimen; and c) comparing the levels determined in step b) with the levels determined in step a), wherein an increase in the levels determined in step b) as compared to the levels determined step a) is indicative of the subject not having complied or adhered with the prescribed treatment.
  • any of the methods described herein may comprise detecting the presence or absence, or determining the levels of at least two, at least three, at least four, at least five, at least six or all seven markers selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the at least two markers are H2N2F1S1c and H2N2S1d.
  • the at least three markers comprise H2N2F1S1c, H2N2S1d and at least one marker selected from the group consisting of H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the at least four markers comprise H2N2F1S1c, H2N2S1d and at least two markers selected from the group consisting of H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the at least five markers comprise H2N2F1S1c, H2N2S1d and at least three markers selected from the group consisting of H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the at least six markers comprise H2N2F1S1c, H2N2S1d and at least four markers selected from the group consisting of H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the sample may be a fluid sample or a solid sample.
  • the solid sample may be a tissue biopsy sample.
  • the tissue biopsy sample may be of a suspected primary CRC site, and/or a metastatic CRC site.
  • the metastatic CRC site may be the liver, lymph nodes, lungs, bones, brain and/or spinal cord.
  • the fluid sample may be selected from the group consisting of whole blood, plasma, serum, feces, urine, and saliva.
  • the CRC may be selected from the group consisting of adenocarcinoma, neuroendocrine carcinoma, mucinous adenocarcinoma.
  • the mucinous adenocarcinoma may be signet-ring cell adenocarcinoma.
  • the invention further provides use of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA as a biological marker for CRC, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the invention provides use of a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a as a biological marker for CRC.
  • two, three, four, five, six or all seven markers may be used.
  • the two markers are H2N2F1S1c and H2N2S1d.
  • the marker may be used in combination with a further marker for CRC.
  • the further marker may be selected from the group consisting of a KRAS mutation, BRAF mutation, NRAS mutation, P I K3CA mutation, LIGT1A1 mutation, PTEN mutation, HER2 mutation, carcinoembryonic antigen (CEA), NTRK fusion, TMB, and microsatellite instability.
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA for use as a medicament, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA for use as a medicament,
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a for use as a medicament.
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA for use in the treatment or prevention of CRC, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA for use in
  • the binding region may specifically bind to the glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA.
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a for use in the treatment or prevention of CRC.
  • a marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a for use in the treatment or prevention of CRC.
  • the binding agent may be a protein, a peptide, a nucleic acid, a dendrimer, a small molecule, an adoptive cell, and/or an antibody.
  • the binding agent may be conjugated to a further moiety, such as a therapeutic moiety and/or a diagnostic moiety.
  • the conjugated binding agent is an antibody (i.e. antibody conjugate).
  • the therapeutic moiety may be selected from the group consisting of a small molecule therapeutic agent, a peptide, a protein, a polymer, an siRNA, a microRNA, and a nanoparticle, or a combination thereof.
  • the diagnostic moiety may be selected from the group consisting of a radionuclide , a fluorescent protein, a photosensitizer, a dye, an enzyme, a magnetic bead, a metallic bead, a colloidal particle, an electron-dense reagent, a biotin, a digoxigenin, and a hapten, or a combination thereof.
  • the protein may be an antibody or a fragment thereof.
  • the antibody or fragment thereof may be monoclonal or polyclonal.
  • the antibody or fragment thereof may be human or humanised.
  • the nucleic acid may be DNA, RNA, DNA aptamer, RNA aptamer, peptide nucleic acids (PNAs), or locked nucleic acids (LNAs).
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • the binding agent may comprise two or more binding regions, wherein each of the binding regions specifically bind a different marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the present invention provides a kit for use in any one of the methods described herein, wherein the kit comprises a detectably labelled binding agent comprising a binding region, wherein the binding region specifically binds to a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA as a biological marker for CRC, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S
  • the present invention further provides a kit for use in any one of the methods described herein, wherein the kit comprises a detectably labelled binding agent comprising a binding region, wherein the binding region specifically binds to a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • a detectably labelled binding agent comprising a binding region, wherein the binding region specifically binds to a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the binding agent may bind to one or more of the markers selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • kits described herein may further comprise a detectably labelled binding agent and/or primers for detecting a further marker for CRC.
  • the further marker may be a KRAS mutation, BRAF mutation, NRAS mutation, PIK3CA mutation, LIGT1A1 mutation, PTEN mutation, HER2 mutation, carcinoembryonic antigen (CEA), NTRK fusion, TMB, microsatellite instability, and/or a glycosylation marker.
  • the further glycosylation marker may be selected from the group consisting of H1 N4, H3N3F2S1Su1c, H2N2S1Su1 b, H2N2S2Su1 b, H1 N3F1S1 b, H2N2F1S2Su1 b, H2N2F1Su1a, H3N3F2S2b, H3N3F2Su1b, H3N4F2S2a, and H3N4F2S2b.
  • the present invention provides a method of treating or preventing CRC in a subject, the method comprising providing the subject with a therapeutically effective amount of a binding agent as described herein.
  • the first binding region may specifically bind H2N2F1S1c
  • the second binding region may specifically bind H2N2S1d.
  • the binding agent that comprises two or more binding regions may be a multivalent antibody or fragment thereof.
  • a multivalent antibody may be a bivalent, trivalent or tetravalent antibody.
  • the present invention provides a screening method for identifying a binding agent that is suitable for use in the treatment or prevention of CRC, the method comprising the step of contacting a test agent with a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA, and determining whether the test agent specifically binds to the marker, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the present invention provides a screening method for identifying a binding agent that is suitable for use in the treatment or prevention of CRC, the method comprising the step of contacting a test agent with a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a, and determining whether the test agent specifically binds to the marker.
  • a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a binding agent as described herein and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
  • Figure 1 shows a schematic workflow for /V- (optional) and O-glycomics from microdissected FFPE tissues. Approximately 20 000 cells were extracted from tissues by laser capture microdissection and lysates were used for protein immobilization on PVDF membrane followed by an overnight /V-glycan release by PNGase F digestion. After removal of the released /V-glycans, O-glycans were released by reductive p-elimination, purified by cation exchange chromatography (CEX) and graphitized carbon solid phase extraction (SPE) packed in a 96-well filter plates and finally analyzed by PGC nano-LC-ESI-MS/MS. Created with BioRender.com
  • CEX cation exchange chromatography
  • SPE graphitized carbon solid phase extraction
  • FIG. 2 shows representative images of FFPE specimens undergoing laser capture microdissection (LCM).
  • Figure 3 shows technical variation of samples.
  • PC1 and PC2 score plot (a) together with PC2 and PC3 score plot (b) based on relative abundances of all glycans in all measured samples as well as technical (TECH1/2/3) replicates and QC pools QC 14/21/37/64/80) display close clustering of scores showing robustness of the method.
  • the top three principal components explain 39.7 % of the variation within the data.
  • Figure 4 shows glycan specificity among cancer, tumor microenvironment controls and healthy mucosa controls.
  • Bars are shade/pattern coded by terminal core structures (A) or terminal epitopes present on the glycans (B). Counts are displayed on top of each bar.
  • FIG. 5 shows that O-glycomic signatures separate cancer from healthy colon mucosa.
  • PCA model based on relative abundances (%) of individual glycans as well as calculated structural glycan features. Separation between cancer, microenvironment controls and healthy mucosa is illustrated in the A) PCA score plot of PC1 against PC2.
  • B) The PCA loading plot displays the variables that drive the separation in the PCA model.
  • the TACAs driving the separation of cancer and control samples are depicted in italic grey.
  • the top two principal components explain 40.5% of the variation within the data.
  • TM cancer
  • C normal mucosa
  • S stroma
  • Figure 6 shows region specific O-glycan structures. Venn diagram depicting glycans expressed in more than 15% of the cancer epithelia-TACAs (left section, cancer), 15% of the normal colon mucosa (right section, normal mucosa), and a selection of the structures expressed in both regions (intersection).
  • Figure 7 shows glycan specificity among different types of cancer and healthy mucosa controls.
  • Figure 8 shows expression of SLeX/A epitopes on different glycan cores.
  • SleX/A expression shows statistically significant upregulation in cancer carried by core 2 glycans A).
  • Normal colon mucosa shows expression of SLeX/A epitopes, but carried by core 3 glycans, and shows statistically significant downregulation in cancer B).
  • SLeX/A epitopes carried by core 4 are expressed on a limited subset of both cancer and normal colon mucosa samples, with no significant difference C).
  • Core 1 glycans show no expression of SLeX/A epitopes in both cancer and normal mucosa D). Differences between groups were tested using Wilcoxon-Mann-Whitney non-parametric statistical test. P-values were adjusted for multiple testing using Benjamini- Hochberg method. # no p-value is reported as there is no variance in the control group.
  • Figure 9 shows hierarchical clustering based on structural glycan features.
  • the graph illustrates the clustering of samples based on calculated structural glycan features shared between individual glycan structures.
  • Figure 10 shows the association of glycomics signatures with cancer characteristics.
  • PCA model based on relative abundances (%) of individual glycans as well as calculated structural glycan features and clustering based on A) Microsatellite instability B) Differentiation grade C) Dukes stage D) Invasion to lymph node or distant organ. No clear clustering based on traits was observed.
  • Figure 11 shows the association of glycomics signatures with cancer characteristics. PCA model based on relative abundances (%) of individual glycans as well as calculated structural glycan features and clustering based on A) Stroma tumor ratio B) Immune infiltration C) Cancer location D) Sex. No clear clustering based on traits was observed.
  • Figure 12 shows a comparison of mRNA expression of glycosyltransferases in colorectal carcinoma and normal mucosa.
  • the figure illustrates the difference in expression of glycosyltransferases involved in the biosynthesis of observed O-glycans which show statistically significant difference in expression in colorectal cancer compared to normal colon mucosa.
  • the panel labelled LCM illustrates data from microdissected colorectal cancer tissues (LCM), whereas the panel labelled TCGA contains data from TCGA dataset, without enrichment for cancer or normal epithelial cells. The datasets were obtained from previous studies.
  • Figure 13 shows an example of O-glycan chromatographic profile from adenocarcinoma A) and normal colon mucosa B) from the same patient (T8 vs C8).
  • A) The adenocarcinoma from patient 8 is characterized by specific expression of core 2 glycans, carrying terminal Lewis X and sialyl Lewis X/A antigens, or just terminal a2-3 sialylation of the galactose. TACAs are circled with shaded background.
  • B) Normal colon mucosa from the same patient shows expression of a diversity of core 3 structures, in most cases carrying a a2-6 linked sialic acid linked to the core GalNAc. Core 3 glycans are carrying terminal Sda antigens, as well as Lewis X/A and sulpho Lewis X/A epitopes. TACAs are not detected in the normal colon mucosa from the same patient.
  • Figure 14 shows tumor associated carbohydrate antigens identified in colorectal cancer.
  • the figure shows a subset of the glycans overexpressed in cancer, detected in over 33 % of the cancers and undetectable in normal colon mucosa samples (a, b, c, d, e, f) or detected in maximum 1 normal mucosa (g).
  • Either one of the two TACAs in h) is detected in 94% of the cancers.
  • Figure 15 shows structural glycan features that differentiate between cancer and normal colon mucosa.
  • Core 2 glycans, a2-3 sialylation, core 2 glycans with SLeX/A and terminal a2-3 sialylation of Gal, (A, B, E, F) together with 1 individual glycan trait (I) show statistically significant upregulation in cancer.
  • core 3 glycans, a2-6 sialylation of the core GalNAc, Sda antigen, core 3 glycans with terminal a2-3 sialylation of Gal, core 3 glycans with a2-6 sialylation of the core GalNAc and core 3 glycans with SLeX/A show statistically significant downregulation in cancer (C, D, G, H, J, K).
  • Lewis X/A and sulfo Lewis X/A antigen show statistically significant downregulation in cancer (L, M). Differences between groups were tested using Wilcoxon-Mann-Whitney non-parametric statistical test. P-values were adjusted for multiple testing using Benjamini-Hochberg method. # no p-value is reported as there is no variance in the control group.
  • Figure 16 shows proposed biosynthetic model explaining the differences in glycosylation of colorectal cancer and normal colon mucosa.
  • the most abundant structures in cancer (upper core 1 and core 2 panel) and normal colon mucosa (core 4, 3 and 5 panels) are depicted together with genes encoding for the GTs involved in their biosynthesis.
  • Pathways upregulated in cancer are depicted in upper panels (core 1 and core 2), whereas pathways downregulated in cancer are marked in lower panels (core 3 and core 5).
  • Core 4 panel show no statistically significant difference between normal mucosa and cancer.
  • Glycosyltransferase genes upregulated in cancer are labelled with light grey, downregulated in cancer are presented in dashed rectangles.
  • Figure 17 A) to G) shows negative ionization mode CID MS/MS spectra of the markers disclosed herein.
  • the experiment is performed on an Amazon speed ion-trap MS (Bruker Daltonik).
  • the present disclosure is based on the inventors’ surprising finding of new glycan structural features as well as new glycosylation markers that are specific for CRC which renders them useful diagnostic tools.
  • the markers disclosed herein i.e. the markers comprising the glycan structural features, which may be the glycosylation markers described herein) are also useful new therapeutic targets for CRC.
  • the new glycan structural features are core 2 glycans with a2-3 sialylation and core 2 glycans with SLeX/A.
  • core 2 glycans with SLeX/A refers to either one or both of the structural features core 2 glycans with SLeX or core 2 glycans with SLeA.
  • the inventors have identified that these structural features (core 2 glycans with a2-3 sialylation and core 2 glycans with SLeX/A) are either substantially found only on glycosylation markers in CRC samples (i.e. the inventors did not detect them in healthy colon samples) or are significantly upregulated in CRC samples as compared to healthy samples.
  • the new glycosylation markers identified by the inventors are specific O-glycan isoforms which the inventors also found to be predominantly present in CRC samples (primary and/or metastatic) but not healthy tissues.
  • the glycan isoforms are H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • These new markers have one or more of the glycan structural features mentioned above, that is they are core 2 glycans with a2-3 sialylation, and optionally are core 2 glycans with SLeX/A.
  • these seven glycosylation markers are examples of markers comprising the glycan structural features of core 2 glycans with a2-3 sialylation, and optionally core 2 glycans with SLeX/A.
  • the markers disclosed herein i.e. the markers of the glycan structural features, which may be the glycosylation markers
  • the markers disclosed herein may be used for diagnosing CRC or determining if a subject is at risk of CRC.
  • Two of these markers, H2N2F1S1c, H2N2S1d, may be particularly useful in this context. This is because the inventors have surprisingly shown, that despite large glycosylation marker variability in CRC, at least one of these two markers is present in 94% of the tested CRC samples (obtained from both primary and metastatic CRC sites).
  • markers can therefore be used to diagnose or determine the risk of developing CRC, as well as to monitor CRC progression or remission, monitor the therapeutic effect of a treatment regimen for CRC, and/or monitor a subject’s compliance or adherence with a prescribed treatment regimen for CRC.
  • One or more (e.g. two, three, four, five, six or seven) of these markers can advantageously be used in any of the methods described herein.
  • binding agents that specifically bind one or more of the markers disclosed herein.
  • the present invention provides a method of diagnosing CRC in a subject, or a method for determining if a subject is at risk of CRC, the method comprising the step of detecting the presence or absence of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA in a sample from the subject, wherein the presence of at least one marker comprising the glycan structural feature is indicative of the subject having CRC, or being at risk of developing CRC, optionally wherein the marker is selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • glycan structural feature refers to the monosaccharide composition and linkages configuration of a portion of a glycosylation marker.
  • the glycan structural feature may be a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and/or a core 2 glycan with SLeA.
  • glycosylation markers H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a have one or more of these glycan structural features.
  • H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a have a core 2 glycan with a2-3 sialylation.
  • the marker the marker of a core 2 glycan with a2-3 sialylation may be a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the glycosylation markers H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a have a core 2 glycan with SLeX/A.
  • the marker of a core 2 glycan with SLeX/A may be a marker selected from the group consisting of H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • a “core 2 glycan” is a glycan that has a p 1-6 linked /V-acetylglucosamine linked to the reducing end /V-acetylgalactosamine.
  • a “core 2 glycan with a2-3 sialylation” as used herein is a glycan structural feature that has a 1-6 linked /V-acetylglucosamine linked to the reducing end /V-acetylgalactosamine and a2-3 linked /V-acetylneuraminic acid to a terminal galactose of the glycan.
  • a “core 2 glycan with SLeX” as used herein is a glycan structural feature that has a p 1-6 linked N-acetylglucosamine linked to the reducing end N-acetylgalactosamine and a sialyl Lewis X.
  • Sialyl Lewis X is /V-acetylneuraminic acid a2-3 - galactose pi-4 -(Fucose a1-3) /V- acetylglucosamine.
  • a “core 2 glycan with SLeA” as used herein is a glycan structural feature that has a p 1-6 linked N-acetylglucosamine linked to the reducing end N-acetylgalactosamine and a sialyl Lewis A.
  • Sialyl Lewis A is /V-acetylneuraminic acid a2-3 - galactose pi -3 -(Fucose a1-4) /V-acetylglucosamine.
  • the present invention provides a method of diagnosing CRC in a subject, or a method for determining if a subject is at risk of CRC, the method comprising the step of detecting the presence or absence of at least one marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject, wherein the presence of at least one of the markers is indicative of the subject having CRC or being at risk of developing CRC.
  • subject (which may be referred to herein as “individual” or “patient” where context permits) defines any subject, particularly a mammalian subject, who may be believed to have CRC or believed to be at risk of CRC.
  • Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees.
  • the subject is a human.
  • the subject can be symptomatic (e.g., the subject presents symptoms associated with CRC).
  • Symptoms associated with CRC include, but are not limited to, changes in bowel habits, constipation, diarrhoea, alternating diarrhoea and constipation, rectal bleeding or blood in stool, abdominal bloating, abdominal cramps, abdominal discomfort, gas pains, feeling of incomplete bowel emptying, thinner than normal stools, unexplained weight loss, unexplained loss of appetite, nausea, vomiting, anaemia, jaundice, weakness, and fatigue or tiredness.
  • Colorectal cancer symptoms may also include pain, fracture, decreased alertness, shortness of breath, difficulty breathing, coughing, chest wall pain, extreme fatigue, increased abdominal girth, swelling of the feet and hands, yellowing or itch skin, bloating, swollen belly, pain, confusion, memory loss, headache, blurred or double vision, difficulty with speech, difficulty with movement, and/or seizures.
  • the subject has CRC (and the method diagnoses, identifies, (or detects) that the subject has CRC).
  • the terms “diagnose” “identify”, and “detect” can be used interchangeably.
  • the subject may have localised CRC. In other examples, the subject has CRC.
  • the subject can be asymptomatic (e.g., the subject does not present symptoms associated with CRC).
  • the asymptomatic subject may have a CRC risk factor.
  • the method may be considered as a method for determining the risk of developing CRC.
  • CRC risk factors include, but are not limited to the presence or growth of polyps within the colon and rectum, a high fat diet, family history of ulcerative colitis and/or CRC, inflammatory bowel disease, and/or Crohn's disease.
  • colonal cancer refers to or describe the physiological condition that is typically characterized by unregulated cell growth.
  • colonal cancer or “CRC” includes the well-accepted medical definition that defines colorectal cancer as a medical condition characterised by cancer of cells of the intestinal tract below the small intestine (i.e., any portion of the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, or rectum).
  • colon cancer also further includes medical conditions which are characterized by cancer of cells of the duodenum and small intestine (jejunum and ileum).
  • the term CRC as used herein includes a CRC selected from the group consisting of consisting of adenocarcinoma, neuroendocrine carcinoma, mucinous adenocarcinoma.
  • the mucinous adenocarcinoma may be signet-ring cell adenocarcinoma.
  • the markers described herein and more specifically at least one of the two markers selected from the group consisting H2N2F1S1c and H2N2S1d, were found to be present in 94% of the tested cancer samples. Only one tested CRC sample was found to not have either one of these markers. This was a sample of a poorly differentiated adenocarcinoma (specifically a classified as a solid type, without glandular formation adenocarcinoma).
  • CRC may exclude a poorly differentiated adenocarcinoma (such as a solid type, without glandular formation adenocarcinoma).
  • CRC is not adenocarcinoma.
  • a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialy lation , and/or a core 2 glycans with SLeX/A the CRC is not adenocarcinoma.
  • CRC may be primary or metastatic.
  • metal or “metastasised” refers to colorectal cancer cells localized in a part of the body other than the duodenum, small intestine (jejunum and ileum), large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon, and rectum.
  • Primary CRC refers to colorectal cancer cells that are localized in the duodenum, small intestine (jejunum and ileum), large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon, and/or rectum.
  • the CRC may be invasive CRC.
  • the invasive cancer may be Dukes stage C and D.
  • the inventors have found that some of the markers disclosed herein may differentiate between different types of CRC. Accordingly, merely by way of example the presence or increased levels of a marker selected from the group consisting of H2N2F1S1c, H2N2F1S1f, and H2N2S1d may be indicative of invasive, metastatic adenocarcinoma.
  • the presence or increased levels of a marker selected from the group consisting of H2N2F1S1c and H2N2F1S1Su1e may be indicative of invasive non-metastatic adenocarcinoma.
  • the presence or increased levels of the marker H2N2S1d may be indicative of non-invasive adenocarcinoma.
  • the presence or increased levels of a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a may be indicative of partly signet ring adenocarcinoma.
  • the marker indicative of partly signet ring adenocarcinoma may be H2N2F1S1f.
  • the presence or increased levels of a marker selected from the group consisting of H2N2F1S1d, H2N2F1S1f, H2N2S1d, H2N2F1S2b and H3N3F2S2 may be indicative of mucinous adenocarcinoma.
  • the marker indicative of mucinous adenocarcinoma may be H2N2F1S1f and/or H2N2S1d.
  • the presence or increased levels of a marker selected from the group consisting of H2N2F1S1c, H2N2F1S1f and H2N2F1S2b may be indicative of neuroendocrine carcinoma.
  • the marker indicative of neuroendocrine carcinoma may be H2N2F1S1c and/or H2N2F1S2b.
  • the methods described are in vitro methods that are performed using a sample that has already been obtained from the subject (i.e. the sample is provided for the method, and the steps taken to obtain the sample from the subject are not included as part of the method).
  • the methods may therefore include the step of providing a biological sample from a subject.
  • the sample may be a fluid or solid sample.
  • the solid sample may be a tissue biopsy sample.
  • the tissue biopsy may be from a site suspected of being affected by, or at risk of CRC (primary or metastatic).
  • the site may be for example a colorectal lesion (such a polyp).
  • the metastatic CRC site may be the liver, lymph nodes, lungs, bones, brain and/or spinal cord.
  • the liquid sample may be selected from the group consisting of whole blood, plasma, serum, feces, urine, and saliva.
  • the tissue biopsy may be of epithelia and/or stroma.
  • the may have been subjected to /V-glycan release and/or O-glycan release from sample proteins.
  • the sample may have undergone protein solubilization prior to the step of detecting the presence or absence, or determining the levels of the markers.
  • the methods described herein may include the step of solubilizing the proteins in the sample prior to the step of detecting the presence or absence, or determining the levels of the markers.
  • Methods for protein solubilization will be known to those skilled in the art. An exemplary method is provided in the Examples section of the present disclosure.
  • Some of the methods provided herein include the step of detecting the presence or absence of at least one marker, for example a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the step of “detecting the presence or absence” refers to determining whether the marker is or is not present in the sample. It will be appreciated that when the absence of the marker is detected, this may be as a result of very low levels of the marker in the sample, such that the method of detecting does not register the presence of the sample. Therefore the term “absence” as used herein may mean the marker is substantially absent. When measuring the presence or absence, no quantitative assessment regarding the levels of the marker is necessary. Exemplary methods for measuring the presence or absence of the markers disclosed herein are provided in the Examples section of the present disclosure.
  • the methods described herein may involve determining the levels of at least one marker, for example a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the level of a marker is typically “determined” by measuring the level of the marker in the sample. The term “determining” can therefore be replaced with the term “measuring” or “determining by measuring” herein.
  • determining may include sending a clinical sample(s) to a commercial laboratory for measurement of the levels in the biological fluid sample, or the use of commercially available assay kits for measuring the levels in the biological sample. Exemplary kits and suppliers will be apparent to a person of skill in the art.
  • the level of marker present in the sample may be determined by e.g. assaying the amount of marker present in the sample. Assays for measuring the amount of a specified marker are well known in the art and include direct or indirect measures.
  • Assays for measuring the levels of markers disclosed herein may include PGC-LC-MS/MS, high pH anion exchange chromatography, fluorophore assisted carbohydrate electrophoresis (FACE), sequential exoglycosidase digestion, mass spectrometry, NMR, and/or gel electrophoresis. More suitably, the levels may be measured by PGC-LC-MS/MS, more suitably by Amazon speed ion-trap MS.
  • FACE fluorophore assisted carbohydrate electrophoresis
  • the methods described herein may comprise detecting the presence or absence of, or determining level of, at least one marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • any of the methods described herein may comprise detecting the presence or absence, or determining the levels of at least two, at least three, at least four, at least five, at least six or all seven markers selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the at least two markers are H2N2F1S1c and H2N2S1d.
  • the at least three markers comprise H2N2F1S1c, H2N2S1d and at least one marker selected from the group consisting of H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the at least four markers comprise H2N2F1S1c, H2N2S1d and at least two markers selected from the group consisting of H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the at least five markers comprise H2N2F1S1c, H2N2S1d and at least three markers selected from the group consisting of H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the at least six markers comprise H2N2F1S1c, H2N2S1d and at least four markers selected from the group consisting of H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • marker or “biomarker” are used herein interchangeably, and refer to markers of glycan structural features selected from the group consisting of core 2 glycans with a2-3 sialylation, core 2 glycans with SLeX, and core 2 glycans with SLeA. It will be appreciated that a marker comprising a glycan structural feature selected from the group consisting of core 2 glycans with a2-3 sialylation, core 2 glycans with SLeX, and core 2 glycans with SLeA may be the glycan structural feature itself (i.e.
  • the glycosylation marker having such a glycan structural feature may be selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and/or H3N3F2S2a.
  • the term “marker” refers to a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and a core 2 glycan with SLeA, a glycosylation marker having such a glycan structural feature, and/or a glycosylation marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and/or H3N3F2S2a.
  • glycosylation marker refers to a glycan molecule.
  • the glycosylation marker may also be referred to as “a glycan”.
  • the glycan molecule may be an O-glycan molecule.
  • the O-glycan molecule may be selected from the group consisting H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and/or H3N3F2S2a.
  • markers described herein are differentially present in a sample taken from a subject having CRC as compared to a subject not having the disease.
  • markers may also be referred to as “tumor-associated carbohydrate antigens” or “TACAs”.
  • TACAs tumor-associated carbohydrate antigens
  • a marker is differentially present if the mean or median level of the biomarker in the different groups is calculated to be statistically significant. Common tests for statistical significance include, among others, t-test (e.g., student t-test), ANOVA, Kruskal-Wallis, Wilcoxon, Mann- Whitney, Receiver Operating Characteristic (ROC curve), accuracy and odds ratio.
  • Biomarkers alone or in combination, provide measures of relative risk that a subject belongs to one phenotypic status or another. Therefore, they are useful as markers for disease (diagnostics) or risk of disease, monitoring disease progression or remission, therapeutic effectiveness of a drug, as well as monitoring subject’s adherence to a prescribed treatment, giving rise to further aspects of the invention that are described herein.
  • glycosylation markers used in the methods described herein are provided in Table 1.
  • fragmentation spectra also known as fragmentation patterns
  • the skilled person would be able to determine whether a detected glycan is one of the glycans disclosed herein by determining the glycan mass, and/or fragmentation spectra and comparing the spectra shown in Fig 17.
  • H2N2F1S1c, H2N2F1S1f and H2N2F1S1d which are different isomers of the same glycan
  • a sample may need to be analysed using high resolution mass spectrometry (for example by Amazon speed ion-trap MS; Bruker Daltonik) in order to obtain a fragmentation spectrum showing the peaks visible in Figure 17 A-G.
  • H is hexose
  • N is /V-acetylhexosamine
  • F is deoxyhexose
  • S is /V-acetylneuraminic acid
  • Su is sulphate modification
  • a/b/c/d/e/f are internal designations.
  • H2N2F1S1d may also be referred to as “H2N2F1S1” having the structure as shown in Table 1 and/or a fragmentation spectrum as shown in Figure 17C.
  • the fragmentation spectrum may be obtained by Amazon speed ion-trap MS.
  • the methods described herein including a method of diagnosing CRC in a subject, or a method for determining if a subject is at risk of CRC, may include the step of determining the levels of one or more of the markers, for example markers selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and/or H3N3F2S2a.
  • the method may further include comparing the levels to a reference value.
  • the reference value may be derived from a control sample.
  • a control sample may be from an individual that does not have and/or is not at risk of having CRC.
  • An increase in the levels as compared to a reference value may indicate that the subject has CRC or is at risk of having CRC.
  • the control sample may be assayed at the same time, before or after, separately or simultaneously with the test sample (i.e. the sample from the subject to be diagnosed, or determined if at risk of CRC).
  • the reference value that is used in the comparison with the sample may be a value that is calculated as an average or median of more than one (e.g. two or more, five or more, ten or more, a group etc.) of control samples.
  • the control sample may be a sample that originated from (i.e. is a mix of) more than one (e.g. two or more, five or more, ten or more, a group etc.) individual that is not suffering from CRC.
  • the reference value may be obtained from a reference database, which may be used to generate a predetermined cut off value, i.e. a score that is statistically predictive of CRC. References from databases are typically derived from a group of control samples.
  • the reference value may be obtained from a sample from the same individual as the test sample, but at an earlier time point. This is particularly relevant for the methods described herein that monitor CRC progression or remission in a subject, determine the therapeutic effect of a treatment regimen for CRC, and/or determine a subject’s compliance or adherence with a prescribed treatment regimen for CRC.
  • the reference level (which may be obtained from the measurement in step a) of said methods) is used to determine any changes in the presence or absence or level of the marker over a time interval for the same subject.
  • the methods described herein such as the method of diagnosing CRC in a subject, or a method for determining if a subject is at risk of CRC, may further comprise the step of treating a subject that has been diagnosed or determined to be at risk of CRC.
  • the step of treating comprises selecting, and optionally administering, a treatment regimen for the subject based on the diagnosis (i.e., detection of the presence of one or more markers disclosed herein, or based on the comparison of the levels of the one or more markers with the reference levels).
  • Treatment can include, for example, surgery radiofrequency ablation, cryosurgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, or a combination thereof.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom.
  • Treatment therefore encompasses a reduction, slowing or inhibition of the symptoms of CRC, for example of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to the symptoms before treatment.
  • appropriate treatment may include surgery and/or therapy.
  • Surgical procedures and therapies used to treat CRC will be well known to those skilled in the art, who will also appreciate that the type of treatment will vary depending on the particular form of CRC that the subject has, is suspected of having, is at risk of developing, or is suspected of being at risk of developing.
  • a therapeutic agent When a therapeutic agent is administered, it may be administered in an amount and/or for a duration that is effective to treat the CRC or to reduce the likelihood (or risk) of CRC developing in the future.
  • An effective amount is a dosage of the therapeutic agent sufficient to provide a medically desirable result.
  • the effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and the like factors within the knowledge and expertise of the health care practitioner. For example, an effective amount can depend upon the degree to which a subject has abnormal levels of certain analytes (e.g., markers as described herein) that are indicative of CRC.
  • therapeutic agents described herein are used to treat and/or prevent CRC.
  • they may be used prophylactically in subjects at risk of developing CRC (for example with CRC polyps).
  • an effective amount is that amount which can lower the risk of, slow or perhaps prevent altogether, the development of CRC.
  • the medications or treatments can be administered to the subject by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be by infusion or by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration.
  • the medications may also be given in e.g. tablet form or in solution. Several appropriate medications and means for administration of the same are well known for treatment of CRC.
  • the therapeutic agent provided to the subject may be selected from the group consisting of 5-Fluorouracil (5-FU), Capecitabine (Xeloda), Irinotecan (Camptosar), Oxaliplatin (Eloxatin), Trifluridine and tipiracil (Lonsurf), Oxaliplatin (Eloxatin), Raltitrexed (Tomudex), Bevacizumab (Avastin), Ramucirumab (Cyramza), Pembrolizumab (Keytruda), Nivolumab (Opdivio), Panitumumab (Vectibix), Pembrolizumab, Regorafenib (Stivarga), Capecitabine (Xeloda), and Ipilimumab (Yervoy).
  • 5-Fluorouracil 5-Fluorouracil
  • Capecitabine Xeloda
  • Irinotecan Camptosar
  • Oxaliplatin Trifluridine and tip
  • the methods described herein may further comprise detecting the presence or absence, or determining the levels of one or more further markers.
  • the further marker may be, for example a KRAS mutation, BRAF mutation, NRAS mutation, PIK3CA mutation, UGT 1A1 mutation, PTEN mutation, HER2 mutation, carcinoembryonic antigen (CEA), NTRK fusion, TMB, and/or microsatellite instability.
  • the further marker may be a glycosylation marker.
  • such a further glycosylation marker may be selected from the group consisting of H1 N4, H3N3F2S1Su1c, H2N2S1Su1 b, H2N2S2Su1 b, H1 N3F1S1 b, H2N2F1S2Su1 b, H2N2F1Su1a, H3N3F2S2b, H3N3F2Su1 b, H3N4F2S2a, and H3N4F2S2b.
  • the inventors have found that these markers were present in at least 15% of the tested CRC samples, but not present in normal colon controls. Structures of these glycosylation markers are provided in the Table 4 of the present specification.
  • the present invention provides a method for monitoring CRC progression or remission in a subject, the method comprising: a) detecting the presence or absence of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after a time interval; c) comparing the results obtained in step b) with the results obtained in step a), wherein: i) the presence in step a) and
  • the present invention provides a method for monitoring CRC progression or remission in a subject, the method comprising: a) determining the levels of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after a time interval; and c) comparing the levels determined in step b) with the levels determined in step a), wherein: i) an increase in the levels determined in step
  • the present invention provides a method for monitoring CRC progression or remission in a subject, the method comprising: a) detecting the presence or absence of at least one marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after a time interval; c) comparing the results obtained in step b) with the results obtained in step a), wherein: i) the presence in step a) and absence in step b) is indicative of CRC remission; or ii) the absence in step a) and presence in step b) is indicative of CRC progression.
  • the present invention provides a method for monitoring CRC progression or remission in a subject, the method comprising: a) determining the levels of at least one marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after a time interval; and c) comparing the levels determined in step b) with the levels determined in step a), wherein: i) an increase in the levels determined in step b) as compared to the levels determined in step a) is indicative of CRC progression; or ii) a decrease in the levels determined in step b) as compared to the levels determined in step a) is indicative of CRC remission.
  • the methods for monitoring CRC progression or remission in a subject are based the fact that observing whether at least one of the markers disclosed herein is present or absent, or indeed determining the levels of the one or more markers disclosed herein may provide useful information regarding CRC status in the subject and/or information regarding therapeutic effect of a treatment regimen provided to a subject.
  • the subject may be one that has been diagnosed with CRC and is receiving treatment for CRC.
  • the subject may be one that does not have CRC but is believed to be at risk of CRC.
  • Such a subject may be receiving preventive or prophylactic treatment for CRC.
  • the presence of the one or more markers in step a) but absence in step b) may be indicative of CRC remission.
  • the presence of one or more markers in step a) and in step b) may be indicative of a lack of remission.
  • the absence of one or more markers in step a) and presence in step b) may be indicative of CRC progression.
  • the absence of one or more marker in step a) and step b) may be indicative of a lack of progression.
  • progression may encompass a noncancerous state developing into CRC, or worsening of symptoms of cancer and/or development of a lower CRC stage into a higher CRC stage.
  • remission may encompass a reduction in or disappearance of signs and symptoms of cancer.
  • an increase in the levels determined in step b) as compared to the levels determined in step a) may be indicative of CRC progression; or a decrease in the levels determined in step b) as compared to the levels determined in step a) may be indicative of CRC remission.
  • “decrease”, “decreased” “reduced”, “reduction” or 'down- regulated”, “lower” are all used herein generally to mean a decrease by a statistically significant amount.
  • “reduced”, “reduction”, “decreased” or “decrease” means a decrease by at least 10% as compared to a reference level/control, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e.
  • any decrease between 10-100% as compared to a reference level/control or at least about a 0.5-fold, or at least about a 1 .0-fold, or at least about a 1.2-fold, or at least about a 1.5-fold, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10- fold decrease, or any decrease between 1.0-fold and 10-fold or greater as compared to a reference level.
  • the terms “increased”, “increase” or “up-regulated”, “higher” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased” or “increase” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 0.5-fold, or at least about a 1.0-fold, or at least about a 1 .2-fold, or at least about a 1 .5-fold, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 1.0-fold
  • Disease progression or remission may be indicated by an increase or decrease of the levels of one or more markers described herein over a time interval, respectively.
  • the subject may have CRC (and suitably may be treated for CRC), or be monitored for the development CRC.
  • a subject that is monitored for the development of CRC may be believed to be at risk of CRC, or may have been previously diagnosed and subsequently successfully treated for CRC (such a subject may be monitored for relapse of CRC).
  • a subject that is monitored for the development or relapse of CRC may receive preventive or prophylactic treatment.
  • Monitoring for the progression of CRC in a subject over time assists in the earliest possible identification of disease progression (e.g. a worsening in disease status or disease symptoms). Such monitoring naturally involves the taking of repeated samples over time.
  • the method may therefore be repeated at one or more time intervals for a particular subject and the results compared to monitor the development, progression or improvement in CRC of that subject over time, wherein a change in the amount of level (or appearance or disappearance) of the one or more marker tested for in sample is indicative of a change in the progression of the CRC in the subject.
  • the marker may comprise a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and core 2 glycan with SLeA .
  • the marker may be selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • disease progression or relapse may be indicated by an increase in the levels of two, three, four, five, six, or seven of the markers selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • disease progression or relapse may be indicated by an increase in the level of one or two markers selected from the group consisting of H2N2F1S1c and H2N2S1d.
  • disease progression or relapse may be indicated by the presence of one or more marker disclosed herein.
  • the marker may comprise a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and core 2 glycan with SLeA .
  • the marker may be selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a, wherein said marker was not detected to be present (i.e. was absent or substantially absent) at an earlier time point.
  • disease progression or relapse may be indicated by the presence of two, three, four, five, six, or seven of the markers selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2, wherein said markers were not detected to be present (i.e. were absent or substantially absent) at an earlier time point.
  • disease progression or relapse may be indicated by the presence of at least one or two markers selected from the group consisting of H2N2F1S1c and H2N2S1d, wherein at an earlier time point, said markers were not detected to be present (i.e. were absent or substantially absent).
  • disease remission may be indicated by a decrease in the level of one or more marker disclosed herein.
  • the marker may comprise a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and core 2 glycan with SLeA .
  • the marker may be selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • disease remission may be indicated by a decrease in the levels of two, three, four, five, six, or seven of the markers selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • disease remission may be indicated by a decrease in one or two markers selected from the group consisting of H2N2F1S1c and H2N2S1d.
  • disease remission may be indicated by the absence of one or more marker disclosed herein.
  • the marker may comprise a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and core 2 glycan with SLeA .
  • the marker may be selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a, wherein said marker was present at an earlier time point.
  • disease remission may be indicated by the absence of two, three, four, five, six, or seven of the markers selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2, wherein said markers were present at an earlier time point.
  • disease remission may be indicated by the absence of at least one or two markers selected from the group consisting of H2N2F1S1c and H2N2S1d, wherein at an earlier time point, said markers were present.
  • Suitable time intervals for monitoring disease progression can easily be identified by a person of skill in the art and will depend on the specific form and stage of CRC being monitored. As a nonlimiting example, the method may be repeated at least every six months, or at least every year, or whenever clinically needed, i.e. in case of a significant change in CRC symptoms.
  • the present invention provides a method for monitoring the therapeutic effect of a treatment regimen for CRC, the method comprising: a) detecting the presence or absence of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after treatment for a time interval; and c) comparing the results obtained in step b) with the results obtained in step a), wherein: i) the absence in step
  • the present invention provides a method for monitoring the therapeutic effect of a treatment regimen for CRC, the method comprising: a) detecting the presence or absence of at least one marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after treatment for a time interval; and c) comparing the results obtained in step b) with the results obtained in step a), wherein: i) the absence in step a) and presence in step b) is indicative of a lack of therapeutic effect; or ii) the presence in step a) and the presence in step b) is indicative of a lack of therapeutic effect.
  • step a) and presence in step b) may be indicative of a lack of therapeutic effect in a subject who was determined to be at risk of developing CRC or had very early stages of CRC, and the sample in step a) was obtained prior to CRC developing or at very early stages of CRC.
  • the presence in step a) and the presence in step b) may be indicative of a lack of therapeutic effect in a subject who was diagnosed to have CRC.
  • the present invention provides a method for monitoring the therapeutic effect of a treatment regimen for CRC, the method comprising: a) determining the levels of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after treatment for a time interval; and c) comparing the levels determined in step b) with the levels determined in step a), wherein an increase or no change in the levels determined in step
  • the present invention provides a method for monitoring the therapeutic effect of a treatment regimen for CRC, the method comprising: a) determining the levels of at least one marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after treatment for a time interval; and c) comparing the levels determined in step b) with the levels determined in step a), wherein an increase in the levels determined in step b) as compared to the levels determined step a) is indicative of a lack of therapeutic effect.
  • no change in the levels of the one or more markers may be indicative of a lack of therapeutic effect or indicative of a therapeutic effect depending on the subject’s CRC status.
  • a subject that does not have CRC but may be determined to be at risk, for example due to presence of colorectal polyps
  • no change in levels may be indicative of the prophylactic or preventive treatment having a therapeutic effect.
  • no change in the levels, or a lack of a decrease in the levels, of the one or more markers may be indicative of a lack of therapeutic effect.
  • step a) may first be performed using a sample that was obtained from the subject at a time point before the treatment regimen for CRC began.
  • step a) may first be performed using a biological fluid sample that was obtained from the subject at the same time as commencing the treatment regimen, or at a time point after the treatment regimen for CRC began.
  • the method can therefore be used to determine the therapeutic effect of a treatment regimen for CRC from the outset (i.e. from the start of the regimen) or from a time point after the treatment regimen has started (i.e. determining the therapeutic effect of a treatment regimen for CRC during the treatment regimen itself).
  • the method can also be useful as a screening tool for determining if specific regimens or treatment modalities have a therapeutic effect on CRC.
  • the tested regimens or treatment modalities may be new regimens or treatment modalities, modified regimens or treatment modalities, or known regimens or treatment modalities that need further testing.
  • a treatment modality is e.g. a drug or medicament that is useful or suspected to be useful in the treatment of CRC.
  • a treatment regimen may be identified as having a therapeutic effect if it results in a delay in disease progression or a delay in the development of symptoms (e.g. over a treatment period).
  • a treatment regimen may also be identified as having a therapeutic effect if it results in an improvement in disease status or symptoms (e.g. over a treatment period). Methods for determining if the treatment regimen has a therapeutic effect are well known in the art.
  • a treatment period refers to a time interval over which treatment occurs (e.g. 1 month, 3 months, 6 months, 1 year, 2 years, etc). Suitable time intervals for monitoring an improvement in disease status or symptoms (e.g. during treatment of the subject) can easily be identified by a person of skill in the art and will depend on the specific form of CRC being monitored. As a non-limiting example, the method may be repeated at least every six months, or at least every year, or at least every two years, or more frequently as required.
  • a treatment regimen may be said to have a therapeutic effect if the levels of the markers disclosed herein, for example one, two, three, four, five, six or seven of the markers selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a are decreased (if the subject is diagnosed with CRC) in step b) as compared to step a), or don’t change or don’t substantially change (if the subject is believed to be at risk of CRC) in step b) as compared to step a).
  • a treatment regimen may be said to have a therapeutic effect if the levels of one or two markers selected from the group consisting of H2N2F1S1c and H2N2S1d are decreased (if the subject is diagnosed with CRC) in step b) as compared to step a), or don’t change or don’t substantially change (if the subject is believe to be at risk of CRC) in step b) as compared to step a).
  • the present invention provides a method for monitoring a subject’s compliance or adherence with a prescribed treatment regimen for CRC, the method comprising: a) detecting the presence or absence of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after the prescribed start of the treatment regimen; and c) comparing the results obtained in step b) with the results obtained in step a), wherein:
  • the present invention provides a method for monitoring a subject’s compliance or adherence with a prescribed treatment regimen for CRC, the method comprising: a) determining the levels of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX and core 2 glycan with SLeA in a sample from a subject, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a; b) repeating step a) using a sample obtained from the subject after the prescribed start of the treatment regimen; and c) comparing the levels determined in step b) with the levels determined in step a), wherein an increase in
  • the present invention provides a method for monitoring a subject’s compliance or adherence with a prescribed treatment regimen for CRC, the method comprising: a) detecting the presence or absence of at least one marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after the prescribed start of the treatment regimen; and c) comparing the results obtained in step b) with the results obtained in step a), wherein: i) the presence in step a) and absence in step b) is indicative of the subject having complied or adhered with the prescribed treatment; or ii) the absence in step a) and absence in step b) is indicative of the subject having complied or adhered with the prescribed treatment;
  • the present invention provides a method for monitoring a subject’s compliance or adherence with a prescribed treatment regimen for CRC, the method comprising: a) determining the levels of at least one marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a in a sample from the subject; b) repeating step a) using a sample obtained from the subject after the prescribed start of the treatment regimen; and c) comparing the levels determined in step b) with the levels determined in step a), wherein an increase in the levels determined in step b) as compared to the levels determined step a) is indicative of the subject not having complied or adhered with the prescribed treatment.
  • an increase in the levels determined in step b) as compared to the levels determined in step a) may be indicative of the subject not having complied or adhered with the prescribed treatment.
  • the subject does not have CRC (but is at risk of developing CRC) no change in the levels determined in step b) as compared to the levels determined in step a) may be indicative of the subject having complied or adhered with the prescribed treatment.
  • a decrease in the levels of determined in step b) as compared to step a) may be indicative of the subject having complied or adhered with the prescribed treatment.
  • the invention further provides use of a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA as a biological marker for CRC, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA as a biological marker for CRC, optionally wherein the marker is selected from the group consisting of H
  • the invention further provides use of a marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a as a biological marker for CRC.
  • a marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a as a biological marker for CRC. It will be appreciated that the use may be in the context of the methods described herein (for example CRC diagnostic and/or risk determination methods).
  • two, three, four, five, six or all seven markers may be used.
  • the two markers are H2N2F1S1c and H2N2S1d.
  • the markers may be used in combination with a further marker for CRC.
  • markers for CRC are known in the art.
  • these may include a KRAS mutation, BRAF mutation, NRAS mutation, PIK3CA mutation, LIGT1A1 mutation, PTEN mutation, HER2 mutation, carcinoembryonic antigen (CEA), NTRK fusion, TMB, and/or microsatellite instability.
  • CEA carcinoembryonic antigen
  • the markers described herein provide promising therapeutic targets for CRC.
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA , optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a
  • the present invention therefore provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the markers described herein provide promising therapeutic targets for CRC. Accordingly, the binding agents described herein may be used a medicament, for example in treating CRC. This gives rise to additional aspects of the invention.
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA for use as a medicament, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA for use as a medicament,
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a for use as a medicament.
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA for use in the treatment or prevention of CRC, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA for use in
  • the present invention provides a binding agent comprising a binding region, wherein the binding region specifically binds to a marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a for use in the treatment or prevention of CRC.
  • a marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a for use in the treatment or prevention of CRC.
  • the binding region may specifically bind to the glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA
  • the present invention provides a method of treating or preventing CRC in a subject, the method comprising providing the subject with a therapeutically effective amount of a binding agent as described herein.
  • prevention means the avoidance of the occurrence or re-occurrence of CRC. It will be appreciated that avoidance of progression of a precancerous colorectal lesion to CRC is CRC prevention.
  • binding agent refers to an agent that has a binding region that selectively binds to one or more of the markers disclosed herein. Selective binding means that under certain conditions the binding agent will not bind in a significant amount to other molecules (for example other glycan isoforms, proteins or peptides). In certain embodiments, the binding agent can only bind the markers disclosed herein.
  • binding agents described herein include, but are not limited to binding proteins (e.g. antibodies, antibody mimetics, camelid antibodies, duobodies), peptides, aptamers (e.g. nucleic acid aptamers, peptide aptamers, aptabodies, affimers), and small molecules, adoptive cells, and/or antibody-drug conjugates.
  • binding proteins e.g. antibodies, antibody mimetics, camelid antibodies, duobodies
  • peptides e.g. nucleic acid aptamers, peptide aptamers, aptabodies, affimers
  • Other appropriate binding agents are also well known and readily identifiable to a person of skill in the art using routine experimental procedures.
  • the binding agent may be conjugated to a further moiety.
  • a binding agent may be referred to as a binding agent conjugate.
  • the further moiety may be suitably, a therapeutic moiety and/or a diagnostic moiety.
  • the conjugated binding agent is an antibody (i.e. antibody conjugate).
  • conjugated chemically linked.
  • the chemical link may be a covalent link.
  • the binding agent for example antibody
  • further moiety such as a therapeutic moiety and/or a diagnostic moiety
  • the covalent link may be direct or indirect.
  • direct it is meant that the binding agent and further moiety are linked without a linker in between.
  • indirect it is meant that the binding agent and further moiety are linked by a linker.
  • linker may be a permanent linker or a liable linker.
  • the therapeutic moiety may be selected from the group consisting of a small molecule therapeutic agent, a peptide, a protein, a polymer, an siRNA, a microRNA, and a nanoparticle, or a combination thereof.
  • the therapeutic moiety may treat and/or prevent cancer, for example CRC.
  • the therapeutic moiety may treat and/or prevent cancer when conjugated to the binding agent, such as an antibody.
  • the therapeutic moiety may treat and/or prevent cancer upon cleavage from the binding agent, such as an antibody (for example in the vicinity of the cancer cell).
  • the glycan molecules described herein are markers associated with CRC specifically.
  • binding agent conjugates such an antibody conjugates may enable targeted delivery of drugs useful in the treatment of CRC to the CRC cells and/or the vicinity of CRC cells. Accordingly, the present invention further provides use of a binding agent as described herein for targeted delivery of a therapeutic moiety to CRC cells.
  • the term “diagnostic moiety” refers to a substance used for the analysis or detection of a disease, such as CRC.
  • the diagnostic moiety may be selected from the group consisting of a radionuclide, a fluorescent protein, a photosensitizer, a dye, an enzyme, a magnetic bead, a metallic bead, a colloidal particle, an electron-dense reagent, a biotin, a digoxigenin, and a hapten, or a combination thereof.
  • the binding agent conjugate comprises a binding agent and a diagnostic moiety it can be said that it is detectably labelled binding agent.
  • aptamer refers to nucleic acid aptamers and/or peptide aptamers.
  • examples of aptamers include affimers (an evolution of peptide aptamers) and aptabodies (formed by hybridisation of two DNA aptamers), which are also well known and readily identifiable to a person of skill in the art using routine experimental procedures.
  • the binding agent is a binding protein.
  • appropriate binding proteins include antibodies and antibody mimetics.
  • appropriate antibody mimetics include affibody molecules (including affimabs) affilins, peptide aptamers (including affimers), affitins, alphabodies, anticalins, avimers, DARPins, Fynomers, Kunitz domain peptides, monobodies, nanoCLAMPs etc.
  • antibody refers to molecules or active fragments of molecules that bind to known antigens, particularly it refers to immunoglobulin molecules and to immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site that specifically binds an antigen.
  • the immunoglobulin described herein can be of any class (IgG, IgM, I g D, I g E, IgA and IgY) or subclass (e.g. I gG 1 , I gG2 , I gG3, I gG4, I gA 1 and lgA2) of immunoglobulin molecule and based on heavy chain sequences from any species.
  • the species may be, but not limited to dogs, cats, horses, cows, pigs, guinea pigs, mice, rats and the like.
  • the species may be a primate (e.g. a non-human primate).
  • the species is a human.
  • the antibody may be a monoclonal antibody.
  • the term “monoclonal antibody” refers to an antibody that can be mass produced in the laboratory from a single clone and that recognizes only one antigen.
  • Monoclonal antibodies may be generated by any appropriate technique known in the art (e.g. by production in HEK or insect cells, or by generation of B cell hybridomas).
  • the antibody may be polyclonal.
  • humanized antibody or “humanized version of an antibody” refers to antibodies in which the framework or “complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin.
  • CDR complementarity determining regions
  • the CDRs of the VH and VL are grafted into the framework region of human antibody to prepare the “humanized antibody.” See e.g. Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S., et al., Nature 314 (1985) 268-270.
  • the heavy and light chain variable framework regions can be derived from the same or different human antibody sequences.
  • the human antibody sequences can be the sequences of naturally occurring human antibodies.
  • Human heavy and light chain variable framework regions are listed e.g. in Lefranc, M.-P., Current Protocols in Immunology (2000) — Appendix 1 P A.1 P.1-A.1 P.37 and are accessible via IMGT, the international ImMunoGeneTics information System® (http://imgt.cines.fr) or via http://vbase.mrc-cpe.cam.ac.uk, for example.
  • the framework region can be modified by further mutations.
  • Exemplary CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies.
  • such humanized version is chimerized with a human constant region.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences.
  • Human antibodies are well-known in the state of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374).
  • Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production.
  • Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.
  • the antibody may be selected from a Fab, bi-specific Fab2, tri-specific Fabs, scFv, bi-specific di-scFv, bi-specific scFv-Fc, bi-specific diabody, a tri-specific triabody, a single domain antibody or a bi-specific minibody.
  • single domain antibodies such as those found in camelids including, but not limited to, llamas and alpacas; and cartilaginous fish including, but not limited to, sharks which are widely known in the art.
  • single chain antibody refers to single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883 or a bi-specific single chain Fv (WO 03/11161).
  • Typical scFv linkers are well known in the art, are generally 10 to 25 amino acids in length and include glycines and serines.
  • VHH or nanobodies Single domain antibody fragments e.g. the functional antibodies produced by camelids that are devoid of light chains and wherein a single N-terminal domain is fully capable of antigen binding are also an example of a binding agent described herein.
  • Such fragments can also form bivalent VHH, or pentabodies (i.e. with 5 VHH domains).
  • a “di-ScFv” refers to a dimerized scFV.
  • minibodies are minimized antibody-like proteins comprising a scFv joined to a CH3 domain. See Hu et al., 1996, Cancer Res. 56:3055-3061.
  • the scFv can be joined to the Fc region, and may include some or the entire hinge region.
  • Fab or "Fab region” as used herein is meant the polypeptides that comprise the VH, CH1 , VH, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody or antibody fragment or fab fusion protein.
  • Fab fragment
  • Fab fragment
  • Fab fragment
  • Fab may refer to this region in isolation, or this region in the context of an antibody molecule described herein, as well as a full length immunoglobulin or immunoglobulin fragment.
  • a Fab region contains an entire light chain of an antibody.
  • a Fab region can be taken to define “an arm” of an immunoglobulin molecule. It contains the epitope-binding portion of that Ig.
  • the Fab region of a naturally occurring immunoglobulin can be obtained as a proteolytic fragment by a papaindigestion.
  • a “F(ab')2 portion” is the proteolytic fragment of a pepsin-digested immunoglobulin.
  • a “Fab' portion” is the product resulting from reducing the disulfide bonds of an F(ab')2 portion.
  • the terms “Fab”, “Fab region”, “Fab portion” or “Fab fragment” may further include a hinge region that defines the C-terminal end of the antibody arm (cf. above). This hinge region corresponds to the hinge region found C-terminally of the CH1 domain within a full length immunoglobulin at which the arms of the antibody molecule can be taken to define a Y.
  • the term hinge region is used in the art because an immunoglobulin has some flexibility at this region.
  • Fc fusion as used herein is meant a protein wherein one or more polypeptides is operably linked to Fc.
  • Fc fusion is herein meant to be synonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes) as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200).
  • An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general may be any protein, polypeptide or small molecule.
  • the role of the non-Fc part of an Fc fusion i.e., the fusion partner, is to mediate target binding, and thus it is functionally analogous to the variable regions of an antibody.
  • Virtually any protein or small molecule may be linked to Fc to generate an Fc fusion.
  • Protein fusion partners may include, but are not limited to, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
  • Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target.
  • Such targets may be any molecule, e.g., an extracellular receptor that is implicated in disease.
  • antibody fragments refers to a portion of a full length antibody, for example possibly a variable domain thereof, or at least an antigen binding site thereof.
  • antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • scFv antibodies are, e.g., described in Huston, J. S., Methods in Enzymol. 203 (1991) 46-88.
  • Antibody fragments can be derived from an antibody described herein by a number of art-known techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration.
  • binding proteins provided herein may be encoded by a nucleic acid molecule that has been codon optimized to increase protein expression in the appropriate host (e.g. HEK cells or insect cells). Codon optimization and protein production techniques are well known in the art and can readily be adapted for the production of binding proteins described herein.
  • an “adoptive cell” refers to a cell that may be genetically modified for use in a cell therapy treatment.
  • the adoptive cell may be an adoptive T-cell.
  • the term "adoptive T-cell” refers to an effector cell that is derived from a naive T-cell or activated T-cell capable of effector functions.
  • T-cell effector functions may involve the interaction of the adoptive T-cell with a target cell displaying specific antigen (such as the markers described herein). Upon interaction, effector proteins are released by T-cells by mechanisms that are activated by recognition of antigen on the target cell.
  • antibody conjugate refers to a conjugate of an antibody with a further moiety chemically bound to said antibody.
  • the first binding region may specifically bind H2N2F1S1c
  • the second binding region may specifically bind H2N2S1d.
  • the binding agent that comprises two or more binding regions may be a multivalent antibody or fragment thereof.
  • a multivalent antibody may be a bivalent, trivalent or tetravalent antibody.
  • a bivalent antibody may bind for example H2N2F1S1c and H2N2S1d.
  • the present invention provides a cell comprising a binding agent as described herein, optionally wherein the binding agent is a protein or nucleic acid as described herein.
  • the present invention provides a kit for use in any one of the methods described herein.
  • the kit may comprise a detectably labelled binding agent comprising a binding region, wherein the binding region specifically binds to a marker selected from the group consisting of H2N2F1S1C, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • suitable binding agents are provided hereinabove.
  • the binding agent may bind to one or more of the markers selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the binding agent may bind to two of the markers, for example H2N2F1S1c and H2N2S1d.
  • the kit may further comprise a detectably labelled binding agent and/or primers for detecting a further marker, for example a KRAS mutation, BRAF mutation, NRAS mutation, PIK3CA mutation, UGT1A1 mutation, PTEN mutation, HER2 mutation, carcinoembryonic antigen (CEA), NTRK fusion, TMB, and/or microsatellite instability.
  • a detectably labelled binding agent and/or primers for detecting a further marker for example a KRAS mutation, BRAF mutation, NRAS mutation, PIK3CA mutation, UGT1A1 mutation, PTEN mutation, HER2 mutation, carcinoembryonic antigen (CEA), NTRK fusion, TMB, and/or microsatellite instability.
  • the present invention provides a screening method for identifying a binding agent that is suitable for use in the treatment or prevention of CRC, the method comprising the step of contacting a test agent with a marker comprising a glycan structural feature selected from the group consisting of a core 2 glycan with a2-3 sialylation, a core 2 glycan with SLeX, and core 2 glycan with SLeA , and determining whether the test agent specifically binds to the marker, optionally wherein the marker is selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a.
  • the present invention provides a screening method for identifying a binding agent that is suitable for use in the treatment or prevention of CRC, the method comprising the step of contacting a test agent with a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a, and determining whether the test agent specifically binds to the marker.
  • a marker selected from the group consisting of H2N2F1S1c, H2N2S1d, H2N2F1S1f, H2N2F1S1d, H2N2F1S2b, H2N2F1S1Su1e and H3N3F2S2a
  • test agent is any compound the effect of which in the context of treatment or prevention of CRC is to be determined. It will be appreciated that any test agent that is determined to bind one of the markers described herein may be suitable for treating or preventing CRC.
  • the test agent may be an aptamer (e.g. nucleic acid aptamer, peptide aptamer, aptabody, affimer), binding protein (e.g. antibody, antibody mimetic, camelid antibody, duobody etc) or a small molecule.
  • aptamer e.g. nucleic acid aptamer, peptide aptamer, aptabody, affimer
  • binding protein e.g. antibody, antibody mimetic, camelid antibody, duobody etc
  • Other appropriate test agents will be well known and readily identifiable to a person of skill in the art.
  • the screening method may be a high throughput screening assay and/or involve the use of a screening library.
  • contacting means bringing the test agent into physical proximity with the one or more markers disclosed herein so that a physical and/or chemical interaction is possible, such that the test agent can bind the marker(s).
  • Appropriate conditions that allow specific interactions are well known to those skilled in the art. It will be appreciated that the conditions will depend on the test agent and/or markers used in the method, however the skilled person will routinely be able to adapt these conditions. Furthermore, sufficient time to allow interaction can be readily determined by the skilled person. It should be understood that a washing step may be performed so as to remove any test agents that have not bound the marker(s) to enable detection of those agents that can indeed bind the peptide. The skilled person will also routinely by able to determine if a tested agent has bound the markers(s).
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a binding agent as described herein and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
  • the pharmaceutical composition may contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds that are routinely found within pharmaceutical compositions.
  • pharmaceutically acceptable refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected nucleic acid sequence, vector, cell, binding agent or isolated peptide without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. a nucleic acid sequence, vector, cell, binding agent or isolated peptide as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.
  • Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation.
  • Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.
  • Diluents are diluting agents.
  • Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.
  • Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.
  • N- and O-glycans were consecutively released from the immobilized proteins, purified and analysed by PGC-LC- MS/MS.
  • the amount of detergent used for the lysis was decreased to 1 %, compared to the 4% used in the original FFPE tissue glycomics approach 17 .
  • the tedious protein precipitation step, using chloroform and methanol extraction could be avoided as this gave unreproducible protein yields from small sample amounts.
  • O- glycan detection the inventors used isopropanol enriched nitrogen gas to increase sensitivity and the quality of the MS/MS spectra, as described previously 20 . As depicted in Figure 3a and b the method showed very good precision.
  • adenocarcinoma AC
  • NEC neuroendocrine carcinoma
  • MUC mucinous adenocarcinoma
  • T2 mucinous adenocarcinoma
  • LCM epithelial tumor regions and mucosa regions from normal colon were collected and it was ensured that they were well separated from the surrounding submucosal and muscle layers of the colon and tumor stroma.
  • Stroma regions for a subset of stroma high cancers were also microdissected. O-glycosylation profiles were obtained for those regions separately.
  • TACAs a total of 140 O-glycans were detected in the cancer tissues. From those, 100 were solely observed in the cancers (TACAs), whereas 20 were shared between cancer and normal colon mucosa (Figure 4). The majority of the TACAs have a core 2 structure carrying sLeX/A antigens or a2-3 sialylation of galactose (Gal) ( Figure 4, 5b, and 6).
  • FIG. 14 a Six core 2 O-glycans that were not detected in normal mucosa, but exclusively found in more than 6 cancers (relative frequency > 33%) are depicted in Figure 14 a, b, c, d, e and f.
  • Sialylated core 2 O-glycan with terminal Lex antigen depicted in Figure 14 a was absent from normal mucosa and found in 72% of the cancers.
  • One core 2 glycan with terminal a2-3 sialylation of Gal on the 6 arm is found in one normal mucosa sample, and in 72% of cancers (Figure 14g).
  • at least one of the two TACAs depicted in Figure 14h were detected in 94% of the cancers in our study.
  • TACA expression is illustrated in Figure 13a which shows expression of four specific TACAs (circled) in the adenocarcinoma tissue from patient 8, and those glycans were not detected in the normal colon mucosa (Figure 13b).
  • Figure 15 shows expression of four specific TACAs (circled) in the adenocarcinoma tissue from patient 8, and those glycans were not detected in the normal colon mucosa ( Figure 13b).
  • This high specificity of TACA expression in cancer but not in presumably unaffected epithelium of the same patient was consistently observed across the entire dataset.
  • These data provide strong evidence that specific O-glycans as well as O-glycomic traits are significantly changed between CRC and normal colon mucosa tissue ( Figure 15). While core 2 glycans are overexpressed, core 3 O-glycans are downregulated in cancer ( Figure 15a and c).
  • Lewis X/A and sulfo Lewis X/A antigen show statistically significant downregulation in cancer (L, M) (Figure 15c, d, g, h, k, I, m).
  • a total of 12 glycans were shared between cancer, mucosa and microenvironment controls, mainly sialylated core 1 O-glycans, such as ubiquitous sialyl T, and disialyl T ( Figure 6 and 9) and core 2 types with terminal gal-a2-3 sialylation ( Figure 15i, Figure 4 and 6).
  • sialylTn antigen ST6GALNAC1 The main enzyme responsible for the biosynthesis of sialylTn antigen ST6GALNAC1 is downregulated in cancer, however, a higher expression of sialyIT n antigens can be due to a mutation in Cosme gene encoding for a chaperone required for the activity of core 1 synthase which could lead to a blockage of the alternative pathway 31 .
  • This competition between core 1 and core 3 synthase as well as core a2-6 sialylation has been described previously in colon cancer cells 32 .
  • B3GNT6 core 3 synthase
  • core 1 truncated glycans such as sialyl3-T by action of ST3GAL1, or branched glycans by the action of core 2 synthases GCNT1 and GCNT4.
  • Addition of a (31-4 galactose and a2-3 sialic acid on the 6 arm of the glycans forms a specific pathway in cancer, starting from a sialylated precursor ( Figure 14g) leading to biosynthesis of fucosylated TACAs carrying terminal sLeX antigen by the addition of a a1-3 linked fucose by FUT4 overexpressed in cancer.
  • Mucinous adenocarcinomas (T2 and T3) expressed 43 specific glycans (Figure 7), mostly carrying sialyl-LeX/A antigen including sialyl-dimeric LeX/A, on core 2 but also core 4 glycans ( Figure 5b and Figure 9). Both tumors were microsatellite instable (MSI) which are known to behave as low grade cancers 43 .
  • MSI microsatellite instable
  • Core 4 glycan expression can be explained by a strong downregulation of ST6GALNAC1 and 3 in cancer, leading to the branching of the core 3 precursors in cancers with expression of core 4 synthase GCNT3 ⁇ .
  • Neuroendocrine carcinoma showed specifically high expression of Sda antigen, compared to other types with low or no expression ( Figure 5b, 7 and 9).
  • TACAs on core 2 glycans were found in more cancers with lymph node invasion (LN I) or invasion to distant organs, including Dukes stage C and D, compared to cancers without invasion (Dukes stage B) ( Figure 14). Due to their absence or limited expression in the normal colon mucosa, and high specificity for invasive cancer, those TACAs can serve as promising targets for treatment of invasive CRC ( Figure 14).
  • FFPE tissue sectioning is part of standard care in Pathology and many tissues are widely available and well preserved on room temperature for a long period of time. Tissue heterogeneity, however, often poses a problem for glycomics and transcriptomics analysis, due to presence of different cell populations, hampering the identification of cell specific signatures. Dissecting specific regions of the tissue by LCM largely overcomes this issue and enables analysis of glycosylation signatures from specific cells of interest 45 . However, it is essential that further improvements are made in regard to sensitivity of our current mass spectrometric methods, making single cell glycomics analysis possible to avoid any contamination from different cell populations.
  • TACAs are expressed on cell surface directly accessible to therapeutics and can be carried by multiple proteins, reflecting the overall glycosylation phenotype of the cell, providing a broader tumor targeting strategy 46 .
  • TACAs vascular endothelial growth factor-1
  • PSGL-1 P-selectin glycoprotein ligand-1
  • Cancer cells carrying the same ligands may employ this physiological mechanism for successful metastasis to distant organs which is why it is important to target these antigens, particularly in invasive carcinoma 22 .
  • TACAs are protein specific, and if the differences in their expression is related to mucin expression.
  • proteins carriers either adjuvants or pathological carriers such as mucins.
  • Aberrant expression of Tn antigen was found on MLIC1 membrane glycoprotein, and anti-Tn and T antigen carrying MLIC1 expressed on showed efficiency in phase I clinical trial on ovarian, breast and cervical cancer 49 but no effect on patient outcome in phase II clinical trial 50 .
  • Another monoclonal antibody targeting Tn-antigen on MLIC1 showed promising preclinical results for targeting breast cancer, however, the results need to be further validated by clinical trials 51 .
  • genetically modified T-cells expressing chimeric antigen receptors (CAR-s) targeting Tn glycoforms of MLIC1 showed promising cytotoxicity in xenograft models of T cell leukemia and pancreatic cancer 52 .
  • CAR-s chimeric antigen receptors
  • Hematoxylin cat. nr. GHS232
  • sodium dodecyl sulfate solution 20% cat. nr. 05030
  • trifluoroacetic acid TAA; cat. nr. 1.38178.0050
  • tris(hydroxymethyl)amino-methane cat. nr. 252859; lot#BCBM2559V
  • sodium borohydride NaBH4; cat. nr. 452882; lot nr. STBD8912V
  • hydrochloric acid HI; cat. nr. 258148; lot nr. SZBD3100V
  • DTT cat. nr. D0632; lot nr.
  • MultiScreen® HTS 96-multiwell plates (pore size 0.45m) with high protein-binding membrane (hydrophobic Immobilon-P PVDF membrane) and 96-well PP Microplate (cat. nr. 651201 ; lot nr. E1708385) were purchased from Millipore (Amsterdam, The Netherlands), 96-well PP filter plate (cat. nr. GF1100) was obtained from Orochem technologies (Naperville, II).
  • MembraneSlide 1.0 PEN (cat.nr. 415190-9041-000) and Adhesive Cap 500 pL tubes (cat.nr.
  • Human CRC paraffin tissue blocks were from the Department of Pathology at Leiden University Medical Center (LUMC, Leiden, The Netherlands). Paired primary tumor (T1-T12) and adjacent normal colon mucosa (C1-C12) from 12 patients were selected for the cohort. Additionally, 6 metastatic cancers obtained from liver metastases of different patients were included (M15- M21). The tissues were cut into 5 pm thick sections from paraffin blocks using a microtome, and mounted on glass slides for hematoxylin and eosin (HE) staining, or 10 pm thick sections on membrane PEN slides for laser capture microdissections (LCM). All slides were dried overnight at 37 °C and stored at 4 °C.
  • HE hematoxylin and eosin
  • Slides used for LCM were deparaffinized using xylene (three times for 5 min) and washed with absolute ethanol (two times for 2 min). The slides were then rehydrated in a series of ethanol by submerging in 85% ethanol, followed by 70% ethanol and distilled water. Haematoxylin was applied for 20 s. The slides were then washed with demineralized water for 2 minutes, and dehydrated with increasing ethanol series by submerging in 70% ethanol, followed by 85% ethanol and finally absolute ethanol. The slides were briefly air dried and stored at 4 °C. The slides used for pathologist annotation were stained with HE following the routine protocols.
  • Tumor and healthy epithelial tissue regions from HE slides were annotated by a pathologist by careful inspection under the microscope. Differentiation grade was determined by assessing glandular formation in the HE histological slides.
  • Tissue sections mounted on PEN membrane slides for LCM were carefully inspected under the microscope. Target areas marked on HE slides by the pathologist were located, and the laser was positioned in this area (Supplementary Figure 1). To ensure that comparable amounts of tissue were used for each sample, cells were counted in three different regions of the tissue in an area of approx. 2500 pm2. An average of the count was used to extrapolate the area needed to be cut in order to obtain samples containing approximately 20,000 cells. The large difference in cell size between hepatic and colon cells was taken into account by performing the LCM on 10 pm thick tissue sections, enabling the extraction of sufficient material for the analysis. The area cut for each tissue is listed in Supplementary Table S1.
  • LCM was performed using PALM RoboSoftware and collected in adhesive cap 500 pL tubes. Considering the healthy colon mucosa is surrounded by infiltrated lymphocytes that could not be separated from the epithelial cells successfully, the inventors also extracted lymphocyte follicles from different normal colon tissues and pooled them together to obtain a potentially confounding immune cell glycan profile (IM). Moreover, the inventors extracted the stroma controls for the tumors with high stroma infiltration that might have contamination coming from cancer associated fibroblasts and immune cells (ST4, ST6, ST7, ST11 , ST12, SM19, and SM21).
  • Glycan release and purification 100 pL of lysis buffer containing 100 mM Tris HCI, 0.1 M DTT, 100 mM NaCI, and 1% SDS was added to microdissected tissues collected in adhesive caps. The pieces were carefully collected in lysis buffer from the lid of the adhesive caps and transferred to a 1.5 mL Eppendorf tube. Prior to sonication using a Branson sonicator rod (three times for 15 s, with output power 2/10) the tubes were placed on ice. During sonication, the samples were kept on ice and left 20 seconds to cool down in between each cycle. Furthermore, the samples were incubated at 99 °C for 60 min with mild agitation (400 rpm).
  • PVDF membrane plate was preconditioned using 100 pL of 70% ethanol, until the membrane turned opaque, followed by a wash using 100 pL of MQ. Upon rewetting the membrane with 5 pL of 70% ethanol, 100 pL of carefully mixed sample lysates were loaded onto the membrane wells. Additionally, a tissue lysate (containing approximately 80,000 cells) was split into three separate samples (TECH1 , TECH2, and TECH3) and processed independently (different wells of the PVDF membrane plate) to check for technical variability. The plate was incubated at RT on a shaker for 20 min to ensure binding.
  • the unbound material was removed by centrifugation at 500 x g, and washed with 100 pL of MQ.
  • An additional, 40 pL of 0.5% PVP-40 in MQ was added to the PVDF membrane wells to block the membrane and incubated for 5 minutes on a shaker.
  • the membrane wells were extensively washed, two times with 100 pL of PBS, two times with 100 pL of 10 mM ammonium bicarbonate followed by two times 100 pL of MQ. Each time the washing agent was removed by centrifugation at 500 x g for 1 minute.
  • the samples Prior to analysis, the samples were resuspended in 15 pL of MQ and 1 pL of each sample was pooled together in a separate vial for quality check and method optimization (QC). The QC pool and a bovine submaxillary mucin standard were used to check instrument performance each day of measurement. A total of 6 pL of each tissue sample was injected for analysis (40%). A custom- made trap column (size 30 x 0.32 mm) packed with 5 pm particle size PGC stationary phase from Hypercarb PGC analytical column (size 100 x 4.6 mm, 5 pm particle size, Thermo Fisher Scientific, Waltham, MA) was used to load the samples using 100% buffer A (10 mM ammonium bicarbonate) at a loading flow of 6 pL/min.
  • buffer A 10 mM ammonium bicarbonate
  • the glycans were separated on a custom-made PGC column (100 mm x 75 pm, 3 pm particle size obtained from Thermo Fisher Scientific) at a 0.6 pL/min flow rate by applying a linear gradient from 1% to 50% of buffer B (acetonitrile, 10 mM ammonium bicarbonate) over 73 min. A constant column temperature of 45 °C was maintained.
  • the LC system was coupled to an amaZon ETD speed ESI ion trap MS using the CaptiveSprayTM source (Bruker Daltonics) with an applied capillary voltage of 1000 V in negative-ionization mode.
  • the drying gas (N2) temperature was set at 280°C and the flow to 3 L/min.
  • MS spectra were acquired in enhanced mode within a mass to charge ratio (m z) range of 380-1850, target mass of smart parameter setting was set to m/z 900, ion charge control (ICC) to 40,000 and maximum acquisition time to 200 ms.
  • MS/MS spectra were generated by collision-induced dissociation on the three most abundant precursors, applying an isolation width of 3 Th.
  • the fragmentation cut-off was set to 27% with 100% fragmentation amplitude using the Enhanced SmartFrag option (30-120% in 32 ms) and ICC was set to 150,000.
  • Extracted ion chromatograms including all observed charge states (1-and 2-) of the first three isotopes were used to integrate the area under the curve (AUC) for each individual glycan isomer using Compass DataAnalysis software (v.5.0). Peaks were manually picked and integrated. Relative quantitation was performed using the total area of all glycans within one sample as reference (100%). Glycan structures were identified by manual inspection of MS/MS spectra following known O-glycan biosynthetic pathways and available literature 41 ,54-57.
  • TCGA expression data was downloaded via the firebrowse.org website.
  • Table 3 Relative abundances of the TACAs with highest specificity in cancer. Each glycan isomer is labelled by its monosaccharide composition, with a letter indicating different isomers a-f. H- hexose; N- N-acetylhexosamine; F- deoxyhexose; S- N-acetylneuraminic acid;
  • Trinchera M. et al. Selectin Ligands Sialyl-Lewis a and Sialyl-Lewis x in Gastrointestinal Cancers. Biology 6, 16 (2017).
  • Trinchera M. et al.
  • the biosynthesis of the selectin-ligand sialyl Lewis x in colorectal cancer tissues is regulated by fucosyltransferase VI and can be inhibited by an RNA interference-based approach. Int. J. Biochem. Cell Biol. 43, 130-139 (2011).

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Abstract

L'invention concerne des méthodes de diagnostic du cancer colorectal (CRC), de détermination si un sujet présente un risque de CRC, des méthodes de suivi de la progression ou de la rémission du CRC chez un sujet, des méthodes de suivi de l'effet thérapeutique d'un régime thérapeutique du CRC, et des méthodes de suivi de l'observance ou de l'adhésion d'un sujet à un régime de traitement prescrit du CRC. La présente invention concerne également des agents de liaison destinés à être utilisés dans le traitement de celui-ci. L'invention concerne des glycanes de noyau 2 (avec sialylation alpha 2-3, sialyl Lewis X ou sialyl Lewis A) en tant que biomarqueurs pour des problèmes médicaux liés au cancer colorectal.
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