Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • 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
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
• • 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
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
• • 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
  • G01N33/57415—Specifically defined cancers of breast

Definitions

• Globo H containing a hexasaccharide epitope, is expressed in various cancers, as well as in normal epithelial cells and glandular tissues. See Huang et al., Proc. Natl. Acad. Sci. USA 103:15-20 (2006), Wang et al., Proc. Natl. Acad. Sci. USA 33:11661-11666 (2008), and Chang et al., Proc. Natl. Acad. Sci. USA 5 33:11667- 11672 (2008). It has been reported that sera from breast cancer patients contain high levels of anti-Globo H antibodies. However, the level of these antibodies alone is not a reliable indication of breast cancer.
• the present invention is based on an unexpected discovery that the ratio of the level of antibodies against Globo H, its fragment Bb2, Bb3, or Bb4, to the level of antibodies against Gb5, another fragment of Globo H, is significantly higher in breast cancer patients than in cancer-free humans.
• this invention features a cancer diagnostic method, including (i) 5 providing a sample (e.g., a serum sample) containing antibodies from a subject (e.g., a human) who is suspected of having a cancer (e.g., breast cancer, melanoma, neuroblastoma, skin cancer, liver cancer, prostate cancer, ovary cancer, colon cancer, stomach cancer, lung cancer, and pancreas cancer), (ii) incubating the sample with Gb5 and one or more of Globo H, Bb2, Bb3, and Bb4 to allow binding of these o molecules to antibodies in the sample, (iii) measuring both the amount of Gb5-bound antibodies and the amount of Globo H-bound, Bb2-bound, Bb3 -bound, or Bb4-bound antibodies, and (iv) determining whether the subject has the cancer based on the ratio of the amount of Globo H-bound, Bb2-bound, Bb3-bound, or Bb4-bound antibodies to the amount of Gb
• Globo H, Gb5, Bb2, Bb3, and Bb4 can be immobilized on a supporter device to form a glycan array.
• the array contains Gb5 and one of Globo H, Bb2, Bb3, and Bb4. In another example, it contains all of these molecules. 5 Also within the scope of this invention is use of Gb5 and one or more of
• Fig.l is a diagram depicting the structures of the hexasaccharide epitope in
• Panel A the structure of the hexasaccharide epitope.
• Panel B the structures of the hexasaccharide epitopes and its seven fragments.
• Fig. 2 is a diagram showing use of glycan array, which mimics cancer cell o surface, for detecting anti-glycan antibodies.
• a method of diagnosing cancer by detecting the amounts of antibodies against Gb5 and one or more of Globo H, Bb2, Bb3, Bb4 in a subject suspected of having cancer, e.g., a human who is genetically susceptible to o cancer, and determining whether the subject has cancer based on any of the level ratios mentioned above.
• the antibodies mentioned above can be IgG, IgM, IgE, IgA, or IgD, or a mixture thereof.
• Globo H is a glycan containing the hexasaccharide epitope shown in Fig. 1, Panel A, and, optionally, a non-sugar moiety. Its fragment (e.g., Gb5, Bb2, Bb3, and Bb4) is a glycan containing a fragment of the hexasaccharide epitope and if 5 applicable, the non-sugar moiety. Seven fragments of the hexasaccharide epitope are shown in Fig. 1, Panel B. These oligosaccharides can be prepared by routine methods. See, e.g., Huang et al, Proc. Natl. Acad. ScL USA 103:15-20 (2006).
• a non-sugar linker such as an alkylamine, e.g., (CH 2 )SNH 2 , or an alkylazide, e.g., (CH 2 )sN3, which can be covalently bonded to a0 supporting device (e.g., a polymer substrate) made of various materials, such as glass, plastic, nylon, metal, or silicon.
• a0 supporting device e.g., a polymer substrate
• a supporting device e.g., a polymer substrate
• Each of Globo H, Gb5, Bb2, Bb3, and Bb4 can be spotted at a defined address on the supporting device so as to form a glycan array.
• This array mimicking the surface of a cancer cell that expresses the oligosaccharide epitopes contained in Globo H, Gb5, Bb2, Bb3, and Bb4 (see Fig. 2), can be used to5 detect the levels of antibodies that bind to the oligosaccharide epitopes.
• the glycan array described herein is incubated with an antibody-containing sample from a subject suspected of having a cancer.
• the sample include, but are not limited to, sera, saliva and lymph node fluids.
• the array is washed to remove unbound antibodies and then o incubated with a labeled secondary antibody that specifically bind to the antibodies of interest, which can be human IgG, IgA, IgD, IgE, or IgM.
• the glycan array is washed again to remove unbound secondary antibody molecules and the intensities of the signal released from the bound secondary antibody molecules correspond to the levels of the target antibodies.
• the subject When a higher level ratio of anti-Globo H/anti-Gb5, 5 anti-Bb2/anti-Gb5, anti-Bb3/anti-Gb5, or anti-Bb4/anti-Gb5 is observed in a subject, the subject is diagnosed as having of the cancer or at risk for developing the cancer.
• Fig. 1 were prepared according to the one-pot programmable protocol described in Hung et al, Proc. Natl. Acad. ScL USA 103-15-20 (2006). These oligosaccharides were covalently attached onto a NHS-coated glass slide, which was purchased from
• VK-9 a mouse IgG anti-Globo H monoclonal antibody
• A488, anti-mouse/human Gb5 monoclonal antibody • VK-9, a mouse IgG anti-Globo H monoclonal antibody, and • A488, anti-mouse/human Gb5 monoclonal antibody.
• Each of the antibodies was incubated with the glass slide (to which the oligosaccharides attached) described above in 0.05% Tween 20/PBS buffer (pH 7.4) in a humidifying chamber with shaking for Ih.
• the slide was then washed, in turn, three times with 0.05% Tween 20/ PBS buffer (pH 7.4), three times with PBS buffer (pH 7.4), and three times with water.
• the slide was incubated with
• VK9 specifically bound to Globo H and Bb4
• Mbrl specifically bound to Globo H and BB4 and also bound to Bb3 at a lower affinity
• A488 specifically bound to Gb5.
• the glycan array is capable of capturing antibodies that bind to Globo H and/or its fragments.
• Example 2 Use of A Glycan Array for Detecting Antibodies against Globo H and Its Fragments in Breast Cancer Patients
• Plasma samples from breast cancer patients and healthy individuals were diluted 1 :20 with 0.05% Tween 20/3% BSA/PBS buffer (pH 7.4) and incubated with the glycan array slide described in Example 1 above in a humidifying chamber with shaking for Ih. After being washed with 0.05% Tween 20/ PBS buffer (pH 7.4), PBS buffer (pH 7.4), and water, each for three times, the slide was incubated with Cy3 -conjugated goat anti-human IgM or IgG antibody in the humidifying chamber with shaking for Ih. The slide was then washed three times with 0.05% Tween20/PBS buffer (pH 7.4), three times with PBS buffer (pH7.4), and three times with H 2 O. After being dried, the slide was scanned at 595nm (for Cy3 -conjugated secondary antibody) with a microarray fluorescence chip reader (ArrayWorx microarray reader).
• ArrayWorx microarray reader microarray
• the level ratios of Globo H-bound IgG/Gb5-bound IgG (GH/Gb5 IgG) and Globo H-bound IgM/Gb5-bound IgM (GH/Gb5 IgM) were much higher in the plasma samples of the breast cancer patients then in the plasma samples of the healthy individuals.
• the ratios of Bb2/Gb5 IgG, Bb4/Gb5 IgM, Bb3/Gb5 IgM, and Bb2/Gb5 IgM of the cancer patients were significantly higher than those of the healthy individuals.
• Example 3 Use of Glycan Array for Monitoring Immune Responses Induced by
• mice (6-week-old female BALB/c mice, BioLASCO, Taiwan) were immunized subcutaneously with the Globo H-KLH vaccine (Optimer Pharmaceuticals, Inc., San Diego, Ca.) once every week for three weeks. Control mice were injected with phosphate buffer saline (PBS). Serum samples were collected from the treated mice 10 days after the last immunization.
• PBS phosphate buffer saline

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