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
  • 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
• • 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
  • G01N33/57449—Specifically defined cancers of ovaries
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

• This invention relates to biomarkers of cancer, e.g., pancreatic, lung, breast, colon, or ovarian cancer, based on relative immunoreactivity of different IgG subclasses of autoantibodies, autoantibodies to defined antigens, e.g., antigens with specific subcellular localization.
• CA125 is neither sensitive nor specific for de novo ovarian cancer detection, since it is elevated in less than 50% of women with stage I disease.
• CA125 has poor specificity, which is shown by its elevation in benign and malignant breast and colon disease, peritoneal irritants, and benign gynecologic diseases, among others (Bast RC, Badgwell D, Lu Z, Marquez R, Rosen D, Liu J, Baggerly KA, Atkinson EN, Skates S, Zhang Z, Lokshins A, Menon U, Jacobs I, Lu K., Int J Gynecol Cancer 2005, 15 (suppl 3): 274-281).
• CA125 Due to CA125's limited expression in early stage ovarian cancers and its association with nonmalignant pathologies, CA125, at best, exhibits a positive predictive value of 57% (Nossov V, Amneus M, Su F, Lang J, Janco JMT, Reddy ST, Farias-Eisner R., Am J Obstet Gynecol 2008, 199: 215-223).
• SELDI-TOF-MS profiling has been successfully used to differentiate ovarian, breast, prostate, and liver cancers from healthy controls (Zhang H, Kong B, Qu X, Jia L, Deng B, Yang Q., Gynecol Oncol, 102:61-66, 2006).
• SELDI-TOF-MS profiling of serum was significantly better than the current standard serum biomarker CA125 at distinguishing patients with ovarian cancer from those with benign ovarian disease and from healthy controls (Petricoin EF, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steinberg SM., Lancet, 359:572-577, 2002). While these initial studies on SELDI-TOF-MS profiling are promising, translating this approach into a routine diagnostic test remains difficult. A drawback of MS techniques is that some proteins of importance may be masked by more abundant proteins in the MS as well as in the analysis of the spectrometric output. The greatest challenge in current MS approaches is the dynamic range rather than sensitivity.
• the calculated minimum tumor size leading to a positive test result was 116.7mm3 using CA125 and ovarian cancer (Lutz AM, Willmann JK, Cochran FY, Ray P, Gambhir SS., PLoS Medicine 2008, 5: 1287-1297). Since this model assumed uniform antigen production by all tumor cells, that 10% of the secreted biomarker (based on in vitro studies) reached the circulation, and that CA125 was not cleared from the blood or degraded, this calculated minimum size may be significantly underestimated.
• circulating tumor- reactive IgG can be demonstrated soon after initial tumor development and well in advance of palpable tumor or circulating tumor antigens (Taylor DD, Gercel-Taylor C, Oncol Rep 1998 Nov-Dec, 5(6): 1519-24; Nesterova M, Johnson N, Cheadle C, Cho- Chung YS., Biochim Biophys Acta 2006, 1762: 398-403).
• patterns of reactivity for the four IgG subclasses differ in ovarian cancer. Further, the antigenic components from different cellular compartments (membrane, nuclear or cytosol) also differ. Several of the tumor-derived antigens exhibiting shared recognition or stage-associated recognition were identified by MS to define recognition patterns of early and late stage cancers, e.g., ovarian cancer.
• detecting or staging e.g., for aiding in detecting or staging
• cancer e.g., pancreatic, lung, breast, colon, or ovarian cancer
• the methods include obtaining a sample comprising antibodies, e.g., IgG-type antibodies, from the subject; contacting the sample with one or more ovarian tumor-associated antigens, under conditions sufficient for the formation of antibody-antigen complexes; and detecting the formation of the antibody-antigen complexes, wherein the presence of complexes indicates the presence of autoantibodies against the tumor-associated antigens, and the presence of autoantibodies indicates the presence or stage of cancer, e.g., pancreatic, lung, breast, colon, or ovarian cancer, in the subject.
• the cancer is ovarian cancer.
• each of the one or more tumor associated antigens is classified as either expressed in the nucleus or cytoplasm of tumor cells, e.g., pancreatic, lung, breast, colon, or ovarian tumor cells.
• the tumor-associated antigens expressed in the nucleus are selected from the group consisting of heterogeneous nuclear ribonucleoprotein (HNRNP A2/B 1), non-metastatic cells 1/non-metastatic cells 2 (NME1/NME2), zinc finger DHHC-type containing 7 isoform 2, survivin, p53, p73, nucleophosmin (B23), synovial sarcoma X common antigen or breakpoint proteins 2 and 4 (SSX2, SSX4), and homeobox A7 (HoxA7).
• HNRNP A2/B 1 heterogeneous nuclear ribonucleoprotein
• NME1/NME2 non-metastatic cells 1/non-metastatic cells 2
• B23 zinc finger DHHC-type
• the tumor- associated antigens expressed in the cytoplasm are selected from the group consisting of pyridoxal kinase, galectin-1, heat shock protein 90, peroxiredoxin, glucose regulated protein 78, and proCathepsin D.
• GRP78 glucose
• the presence of autoantibodies that bind specifically to PLAP indicates the presence of ovarian cancer in the subject. In some embodiments, the presence of autoantibodies that bind specifically to one or more of Mucl6, p53, PLAP and survivin indicates that the subject has stage III or IV ovarian cancer. In some embodiments, the presence of autoantibodies to survivin indicates the presence of lung or colon cancer in the subject.
• the presence of autoantibodies that bind specifically to one or more of heterogeneous nuclear ribonucleoprotein (HNRNP A2/B1) and non-metastatic cells 1/non-metastatic cells 2 (NME1/NME2) in the nucleus, and/or the presence of one or both of pyridoxal kinase, galectin-1 and heat shock protein 90 in the cytosol, indicates that the subject has stage I ovarian cancer.
• HNRNP A2/B1 heterogeneous nuclear ribonucleoprotein
• NME1/NME2 non-metastatic cells 1/non-metastatic cells 2
• the presence of autoantibodies that bind specifically to one or more of zinc finger DHHC-type containing 7 isoform 2, survivin, p53, or p73 in the nucleus, and/or the presence of peroxiredoxin in the cytosol indicates that the subject has stage III ovarian cancer.
• the presence of autoantibodies that bind specifically to one or more of nucleophosmin (B23), synovial sarcoma X breakpoint proteins (SSX2, SSX4), or HoxA7 in the nucleus, and/or the presence of glucose regulated protein 78 in the endoplasmic reticulum, and/or the presence of proCathepsin D in the lysosome indicates that the subject has cancer, e.g., ovarian cancer.
• the tumor-associated antigens are bound to a substrate, e.g., a solid surface or a bead.
• the tumor-associated antigens are isolated from cytoplasm of cells that are known to be cancer cells, e.g., ovarian cancer cells, or isolated from nuclei of cells that are known to be cancer cells.
• the methods further include communicating information regarding the presence of the autoantibodies to a health care provider or to the subject. In some embodiments, the methods further include administering a treatment (as is known in the art) for the cancer to the subject.
• the invention provides methods (e.g., in vitro methods) of staging (e.g., for aiding in staging) ovarian cancer in a subject.
• the methods include obtaining a sample comprising IgG-type antibodies from the patient; contacting the sample with ovarian tumor-derived antigens, under conditions sufficient for the formation of antibody-antigen complexes; determining the subclass of the IgG antibodies bound to the antigens; and determining the relative immunoreactivity of the subclasses, wherein the relative immunoreactivity of the subclasses indicates whether the subject has early stage, middle stage, or advanced ovarian cancer.
• the subclasses are IgGl, IgG2, IgG3 and IgG4.
• IgG2>IgG3>IgGl>IgG4 indicates a diagnosis of middle stage ovarian cancer
• the subject is a human, e.g., a human known to have or suspected of having ovarian cancer.
• the sample comprises serum from the subject.
• FIGs. lA-B are representative western immunoblots of patient immunoreactivity with proteins isolated from specific cellular compartments of ovarian tumor cells: cytosol (designated as C), membrane (designated as M) and nuclear (designated as N).
• FIGs. 2A-C show representative western immunoblots of patient
• Representative sera were obtained from (2A) a patient with Stage I ovarian cancer, (2B) with Stage II ovarian cancer and (2C) with Stage III ovarian cancer.
• the representative immunoblots representing IgGl, IgG2, IgG3, and IgG4 for each stage utilized the same patient's serum.
• FIGs. 4A-C show 2-DIGE results of proteins recognized by Stage I and Stage III ovarian cancer patients.
• Cellular proteins immunopurified using affinity columns with IgG from patients with Stage I cancer were labeled with Cy2 (4A) and immunopurified using affinity columns with IgG from patients with Stage III cancer were labeled with Cy3 (4B).
• the gel was scanned using a Typhoon image scanner and each scan revealed one of the CyDye signals.
• ImageQuant software was used to generate an image presentation data including the single and overlay images (4C).
• examples of antigens linked with stage I disease are circled in medium grey (green in original)
• examples of components associated with Stage III were circled in dark grey (red in original)
• examples of shared components were circled in lightest grey (yellow in original).
• FIG. 5 is an exemplary clinical decision tree of ovarian cancer, indicating key sites for utility of tumor reactive IgG as biomarkers.
• FIG. 6 is an image of 2-DIGE image of tumor-derived antigens recognized by patients with ovarian cancer.
• Spots 1, 17-21, 28-32, and A-C are antigenic proteins recognized by IgG from ovarian cancer patients with Stage I disease.
• Spots 2-16 and 22-27 (red in original) are antigenic proteins recognized by IgG from ovarian cancer patients with Stage III disease.
• the brightest spots at top of image, just left of center; yellow in original) are antigens recognized by both.
• FIG. 7 shows a representative comparison of immunoreactive spots using sera from ovarian cancer patients with Stage I disease or Stage III disease. Upper panels present the 2-D gels results with the specific spots indicated. The lower panel presents the quantifications of those spots.
• Th2 cells scan host cells for mutations, which when detected, the mutated cells are eradicated. While significant literature alludes to cancer being associated with an immunosuppressed state, it may be more accurate that there is a Thl to Th2 shift associated with the development of cancer. Activation of Th2 cells is an antigen-specific mechanism of immunodepression, since Th2 cells have extreme sensitivity to the antigen by means of the T-cell receptor. In general, Th2-driven antibody responses recognizing tumor components are non-protective and may contribute to tumor progression by inhibiting the Thl cell-mediated immune response (Clerici M, Clerici E, Shearer GM, J Natl Cancer Inst 1996, 88(7):461-2).
• spontaneous tumors In contrast to the "strong" Thl immune response generated by transplantable tumors, spontaneous tumors elicit a quantitatively mild Th2 immune response. Thus, spontaneous tumors may not stimulate an appropriate immune response but rather elicit non-protective humoral immune responses that are not adequate for tumor eradication. Specifically, a "weak" immune response is a state in which immunological recognition of the tumor occurs but eradication is not achieved. While the induction of the non-protective humoral response in ovarian cancer fails to eradicate the tumor, the appearance of these tumor-reactive immunoglobulins, linked with this Th2 shift, appears to be characteristic of cancer and thus, may have utility as biomarkers of events associated with ovarian cancer development.
• IgG is the major effector molecule of the humoral immune response, accounting for approximately 75% of the total immunoglobulins in the circulation, expressing their activity during a secondary antibody response.
• the human IgG compartment consists of four distinct subclasses, designated IgGl, IgG2, IgG3 and IgG4 and their mean serum concentrations are 6.98mg/ml for IgGl, 3.80mg/ml for IgG2, 0.51mg/ml for IgG3, and 0.56mg/ml for IgG4.
• the principal biological activities of IgGs are related to their effector functions, including activation of complement and binding Fc receptors to mediate antibody- dependent cellular cytotoxicity. Although their heavy chains exhibit >95% sequence homology, IgG subclasses express unique profiles of effector activities (Ravetch TV, Bolland S. IgG Fc receptors. Annual Rev Immunol 2001; 19:275-290).
• IgG4 subclass appears to be characteristic of chronic antigen stimulation, such as observed in autoimmune disease; IgG4 exhibits restricted Fc receptor activation and does not activate Clq.
• the IgG2 subclass often predominates in responses to carbohydrate antigens and also exhibits restricted Fc receptor and Clq activation (Ravetch JV, Bolland S. IgG Fc receptors. Annual Rev Immunol 2001; 19:275-290).
• biomarkers have generally focused on patient extremes; advanced stage cancer versus completely normal volunteers. Based on these extremes, many biomarkers exhibit the specificity and sensitivity necessary for utility in screening and diagnosis. However, when benign disease, pre-malignant disease, early stage cancer and inflammatory pathologies are included, most biomarkers fail to reach adequate sensitivity and specificity for clinical utility. Jacobs and Menon calculated that to be an effective screening test, an assay needs to achieve a minimum of 99.6% specificity (Jacobs IJ, Menon U., Mol Cell Proteomics, 3 :355-366, 2004).
• Chatterjee et al (Chatterjee M, Mohapatra S, Ionan A, Bawa G, Ali-Fehmi R, Wang X, Nowak J, Ye B, Nahhas FA, Lu K, Witkin SS, Fishman D, Munkarah A, Morris R, Levin NK, Shirley NN, Tromp G, Abrams J, Draghici S, Tainsky MA., Cancer Res 2006, 66: 1181-1190) identified 65 different antigens and demonstrated reactivity in sera from 32 ovarian cancer patients and no reactivity in sera from healthy female controls and 14 patients with either benign disease or other malignant gynecologic diseases.
• Chatterjee et al (Chatterjee M, Mohapatra S, Ionan A, Bawa G, Ali-Fehmi R, Wang X, Nowak J, Ye B, Nahhas FA, Lu K, Witkin SS, Fishman D, Munkarah A, Morris R, Levin NK, Shirley , Tromp G, Abrams J, Draghici S, Tainsky MA., Cancer Res 2006, 66: 1181-1190) found only a sensitivity and specificity of 55% and 98%, respectively.
• stage III patients Despite advances in surgery and chemotherapy, the majority of patients with advanced ovarian cancer will recur within a median of 12-18 months after completing first-line therapy. The risk of recurrence varies based on several factors, including the stage at diagnosis, with approximately 90-95% of stage IV patients recurring. Further, 80-85% of stage III patients who are suboptimally debulked will recur, as will 70-80% of stage III patients optimally debulked and 30% of stage II patients. In contrast, less than 10% of stage I patients will recur.
• Recurrent ovarian cancer is invariably fatal and treatment of recurrent disease is palliative and is generally initiated with the goals of controlling disease-related symptoms, limiting treatment-related toxicity, maintaining quality of life, and prolonging survival (Herzog TJ., Clin Cancer Res 2004, 10:7439-7449).
• a biomarker is capable of identifying early stage disease with specificity greater than 99.6%.
• the immunoreactivity of IgG2 and IgG3 with nuclear and membrane antigens with all stages of ovarian cancer were greater than that observed for controls or patients with benign disease ( Figures 2 and 3).
• IgG subclasses recognizing specific tumor antigens provides superior biomarkers for identification of early cancers and allows for differentiation of benign versus malignant ovarian masses.
• a protein can be evaluated using methods known in the art, e.g., using quantitative immunoassay methods.
• high throughput methods e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al, Eds. Modern genetic Analysis, 1999; W. H. Freeman and Company, Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000,
• biomarkers such as tumor-reactive antibodies
• potential biomarkers can be used for screening by applying them to the detection of cancer in asymptomatic individuals in high risk populations or in the general population.
• potential biomarkers can be used for definitive diagnosis of individuals with suspicious or palpable masses, ultrasound-identified masses or symptoms of pelvic or abdominal pain.
• potential biomarkers can be used for disease monitoring or follow-up in individuals treated for ovarian cancer (by surgery and first-line
• biomarkers described herein can be use at one or more, or all three, of the above, or at other time points, e.g., as determined by a health care provider or insurance provider.
• ovarian cancer is used as an example herein, the methods described herein can also be used for other cancers, e.g., other cancers of epithelial origin, e.g., carcinomas, e.g., pancreatic, lung, breast, or colon cancer.
• other cancers of epithelial origin e.g., carcinomas, e.g., pancreatic, lung, breast, or colon cancer.
• Tumor derived cellular proteins were prepared for western blot analysis as follows. Total cellular proteins, including those from cellular compartments, were isolated from human ovarian tumor cell lines established in our laboratory from women with Stage IIIc cyst adenocarcinoma of the ovary (designated UL-B and UL-O). UL-0 cells were derived from a 48-year old Caucasian woman with a family history of breast/ovarian cancers (medical records indicated that the patient was BRCA1+), while UL-B was derived from 72 year old Caucasian woman, with no family history of cancer (Taylor DD, Gercel- Taylor C, Gynecol Oncol 2008, 1 10: 13-21).
• ovarian tumor cells are grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, ImM sodium pyruvate, 200mM L-glutamine, lOOmg/ml streptomycin and lOOIU/ml penicillin in a humidified 5% C02 atmosphere. Cell viability was evaluated by trypan blue exclusion and all cultures utilized for this study were >95% viable.
• Subcellular fractionations of proteins were performed according to the manufacturer's instructions (BioVision, Mountain View, CA). Tumor cell monolayers were extensive washed with 20mM sodium phosphate buffered saline (PBS), then removed from the monolayer by scraping and separated into fractions derived from the cytosol, membrane, and nucleus. The protein concentrations of each cellular fraction were determined using the Bradford microassay (Bio-Rad Laboratories, Hercules, CA). To assess appropriate fractionation, proteins markers of the membrane (placental type alkaline phosphatase and EpCAM), nuclear (histone H3) and cytoplasmic (GAPDH) fractions are evaluated by western immunoblotting.
• PBS sodium phosphate buffered saline
• Antibodies were obtained from Santa Cruz Biotechnology and were anti-PLAP (sc-47691), anti-EpCAM (sc-73491), anti- histone H3 (sc- 10809) and anti-GAPDH (sc-47724). Each subcellular fraction exhibited detectable bands on immunoblots only for their specific marker.
• Immunoreactivities for antigens, either greater or less than 40kD, from each cellular compartment were standardized using the pixel values of a control standard (HRP-anti-mouse Ig) included on each gel.
• the standardized pixel values were divided by the negative control lane on each gel, such that a lane with no immunoreactivity exhibits a value of 1.
• Duplicate gels were run for each patient and the resulting ratios from these gels were averaged. The mean values and standard deviations were calculated from the averages of all patients within each stage.
• 2D DIGE 2-dimensional difference in gel electrophoresis protein expression profiling was performed as follows.
• the immunoaffinity-isolated cellular proteins (300 ⁇ g) eluted from Stage I patients are labeled with Cy2, while proteins (300 ⁇ g) eluted from columns with Stage III patient-derived IgG were labeled with Cy3.
• the two samples were simultaneously separated on a single 2D gel, using isoelectric focusing (IEF) in the first dimension and SDS polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension.
• IEF isoelectric focusing
• SDS-PAGE SDS polyacrylamide gel electrophoresis
• the gel was scanned using a Typhoon image scanner. Each scan revealed one of the CyDye signals (Cy2 and Cy3).
• ImageQuant software was used to generate an image presentation data including the single and overlay images.
• the images were then subjected to DeCyder software analysis, which automatically located and analyzed multiplexed samples.
• the 2D gel spots were removed, washed to remove staining dye and inhibitory chemicals and dried to absorb maximum volume of digestion buffer.
• the dried 2D gel spots were rehydrated in digestion buffer containing sequencing grade modified trypsin (1 :30 by mass) and proteins were digested in-gel at 37°C.
• Digested peptides were extracted from gel with trifluoroacetic acid extraction buffer and the digested tryptic peptides were desalted using C-18 Zip-tips (Millipore). The desalted peptides were mixed with CHCA matrix (a-cyano-4-hydroxycinnamic acid) and spotted into wells of a MALDI plate.
• Mass spectra (MS) of the peptides in each sample were obtained using an Applied Biosystems 4700 Proteomics Analyzer. A minimum of 10 of the most abundant peptides for each sample were further subjected to fragmentation and tandem mass spectrometry (MS/MS) analysis. Protein identification was based on peptide fingerprint mass mapping and peptide fragmentation mapping (using MS/MS spectra). Combined MS and MS/MS spectra were submitted for database search using GPS Explorer software equipped with the MASCOT search engine to identify proteins from primary sequence databases.
• Figs. 1A-1B The results are shown in Figs. 1A-1B.
• the control samples failed to exhibit any reactivity with antigens derived from the cytosol, regardless of IgG subclass.
• the controls recognized a single band at 50kD in antigens derived from the membrane compartment in all IgG subclasses. These controls also recognized the 50kD antigen in the nuclear fraction, as well as two bands at 52 and 60kD in all IgG subclasses.
• the IgG3 subclasses also exhibit additional weak reactive bands.
• the 50kD band is recognized in the membrane and nuclear fractions with all IgG subclasses.
• An additional 34kD band is recognized in all fractions with all IgG subclasses.
• a group of bands between 15-30kD was recognized in the membrane fraction by IgGl, IgG3 and IgG4, with an additional group between 55-lOOkD being recognized in the nuclear fraction by all IgG subclasses.
• IgG2>IgG3>IgGl IgG4 ( Figure 3).
• IgG2 and IgG3 reactivity was greater for nuclear antigens, followed by membrane antigens and minor recognition of cytosol antigens.
• membrane antigens exhibited the greatest reactivity followed by cytosol antigens, with the least reactivity with nuclear antigens. The primary immunoreactivity was observed in antigens exhibiting molecular weights greater than 40kD.
• immunoreactivity was IgG2>IgG3>IgGl>IgG4 ( Figure 3).
• IgG subclasses For all IgG subclasses, reactivity was greater for nuclear antigens, followed by membrane antigens and minor recognition of cytosol antigens.
• IgGl and IgG4 no significant reactivity was observed by antigens derived from the cytosol, while for IgG2 and IgG3, lower reactivity (2.68 ⁇ 1.25 and 1.85 ⁇ 0.81, respectively) was observed primarily in the less than 40kD antigens.
• IgG2 ⁇ IgG3 IgG4>IgGl ( Figure 3). Except for intensity, the recognition patterns appeared to be identical for all IgG subclasses. For IgG2 and IgG3, reactivity was slightly greater for nuclear antigens, followed by membrane antigens and less recognition of cytosol antigens. For nuclear antigens, the level of reactivity was similar for the ⁇ 40kD and >40kD antigens for IgG2 (4.68 ⁇ 0.63 versus 4.71 ⁇ 0.72, respectively) and IgG3 (3.42 ⁇ 0.58 versus 4.08 ⁇ 0.67, respectively).
• the primary reactivity was observed with antigens less than 40kD: IgGl exhibited a 2.08-fold greater reactivity with ⁇ 40kD antigens (versus >40kD antigen), IgG2 exhibited a 2.27-fold greater reactivity, IgG3 exhibited a 1.64-fold greater reactivity, and IgG4 exhibited a 1.51 -fold greater reactivity. Antigens derived from the cytosol compartment exhibited a similar greater reactivity with the ⁇ 40kD antigens.
• the column was equilibrated with 0.2M triethanolamine, pH8.0 and 1ml cross-linking buffer containing 25mM DMP (dimethyl pimelidate dihydrochloride) was added and incubated at room temperature for 45 minutes. Then, 1ml blocking buffer (0.1M ethanolamine, pH 8.2) was added to the column and incubated for 1 hour at room temperature. The immunoaffinity column was washed twice with binding buffer, followed by addition of 1ml 0.1M glycine-HCl (pH 2.5) to elute antibody not cross-linked with DMP.
• DMP dimethyl pimelidate dihydrochloride
• clarified cell lysates were use to identify specific immunoreactivity.
• solubilized cellular proteins were applied to immunoaffinity columns constructed of pooled patient-derived IgG. Initially, the cellular proteins were pre-absorbed using an affinity column constructed on IgG from patients with benign disease. The non-binding proteins were either applied to affinity columns constructed of IgG from Stage I patients or from Stage III patients. These proteins were recirculated on the columns for 1 hour at room temperature and then washed with 10ml of binding buffer (20mM sodium phosphate, pH7.0) or until no material, absorbing at 280nm, appeared in the effluent.
• binding buffer (20mM sodium phosphate, pH7.0
• Table 1 Mass spectrometric identification of spots from the 2-DIGE of tumor- derived antigens recognized by Stage I patients, Stage III patients or both.
• heterogeneous nuclear ribonucleoprotein HNRNP A2/B1
• non-metastatic cells 1/non-metastatic cells 2 NME1/NME2
• pyridoxal kinase, galectin-1 and heat shock protein 90 are generally expressed in the cytosol.
• zinc finger DHHC-type containing 7 isoform 2, survivin, p53 and p73 are localized to the nucleus and
• peroxiredoxin is present in the cytosol.
• nucleophosmin B23
• synovial sarcoma X breakpoint proteins SSX2, SSX4
• HoxA7 represent proteins generally localized to the nucleus
• glucose regulated protein 78 is localized to the endoplasmic reticulum
• proCathepsin D is present in the lysosome.
• solubilized cellular antigens from UL-6 were isolated based on their specific recognition by IgG from patients with early or late stage ovarian cancer. IgG from these patients was isolated on a protein G-Sepharose column and crosslinked using DMP. In a similar fashion, IgG from women with benign ovarian disease was also isolated and coupled to Protein G. Initially, the solubilized proteins were applied to the immunoaffinity column derived from benign disease. This removed those proteins reacting with IgG from these patients. The non- binding proteins were then applied to either the immunoaffinity columns prepared with early or late stage derived IgG. The columns were extensively washed and then eluted.
• the eluted proteins were compared by 2-DIGE.
• the reactive spots isolated from early stage patients was compared with late stage patients by overlaying the digitized images.
• the reactive spots were then quantified and spots expressing a 4-fold increase/decrease were defined by MS sequencing.
• Reactive spots include nucleophosmin, nucleoside diphosphate kinase, NME1-NME2, nuclear riboprotein A2/B 1, Zn-DHHC-containing 7, and aldose reductase isoforms.
• Proteins from the ovarian tumor cell lines were solubilized in 50mM Tris-HCl (pH7.5), containing 0.3% SDS, 2mM sodium orthovanadate, 200mM DTT, ImM sodium fluoride, lmM sodium pyrophosphate, ⁇ g/mL leupeptin, ⁇ g/mL aprotinin, ⁇ g/mL pepstatin, and ImM PMSF on ice.
• the solubilized proteins were applied to the immunosorbent column and incubated overnight at 4°C.
• An array assay for tumor antigen-reactive immunoglobulins was performed as follows. Purified exosomal proteins (250 ⁇ containing 20ng/mL protein) were applied to nitrocellulose membranes using a bio-dot microfiltration apparatus (Bio-Rad Laboratories, Hercules, CA). As controls, serially diluted human IgG was spotted onto each membrane as an internal positive control for standardizing blots, diluted mouse and rabbit Ig as a negative control, and peroxidase-conjugated Ig samples as a reagent control and for orientation. Membranes were blocked with 5% BSA and then washed 3 times with TBS plus 0.1% Tween-20 and twice with TBS.
• Sera (diluted 1 : 100) from known cancer patients and non-cancer bearing controls were incubated with the membranes overnight at 4°C, incubated with peroxidase-conjugated anti-human IgG, and visualized by ECL. The resulting film was imaged and analyzed using Kodak analysis software. Pixel values for all spots were obtained and comparisons between membranes were performed after standardization to the internal positive control. The cutoff for positive values was set as the maximum standardized pixel value of initial 20 control samples.

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• 2010
  • 2010-11-19 CA CA2781408A patent/CA2781408A1/en not_active Abandoned
  • 2010-11-19 WO PCT/US2010/057427 patent/WO2011063232A1/en not_active Ceased
  • 2010-11-19 JP JP2012540089A patent/JP2013511728A/ja active Pending
  • 2010-11-19 AU AU2010321787A patent/AU2010321787B2/en not_active Ceased
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• 2014
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