# Patent

## Title
Using phage epitopes to profile the immune response

## Abstract

The present disclosure provides compositions and methods for using one or more polypeptide probes to profile an immune response. The polypeptide probe can be used to detect one or more antibodies from a sample. Furthermore, the present disclosure provides methods and compositions for characterizing a cancer based on the detection of one or more antibodies, such as autoantibodies.

## Background
CROSS-REFERENCE
This application is a continuation of U.S. application Ser. No. 13/050,544, filed Mar. 17, 2011, which claims the benefit of U.S. provisional application Ser. No. 61/314,750, filed Mar. 17, 2010, which is incorporated herein by reference in its entirety.


BACKGROUND
It is desirable to improve cancer detection, prognostic prediction, monitoring, and therapeutic decisions. For example, when cancer is identified at the earliest stages, the probability of cure is very high and therefore diagnostic screening tests that can detect these early stages are crucial.
One example in which early detection can be beneficial is prostate cancer (PCA). PCA is a leading cause of male cancer-related death, second only to lung cancer (Abate-Shen and Shen, Genes Dev 14:2410 (2000); Ruijter et al., Endocr Rev, 20:22 (1999)). Prostate cancer is typically diagnosed with a digital rectal exam and/or prostate specific antigen (PSA) screening. An elevated serum PSA level can indicate the presence of PCA. PSA is used as a marker for prostate cancer because it is secreted only by prostate cells. A healthy prostate will produce a stable amount—typically below 4 nanograms per milliliter (ng/ml), or a PSA reading of “4” or less—whereas cancer cells produce escalating amounts that correspond with the severity of the cancer. A level between 4 and 10 ng/ml may raise a doctor's suspicion that a patient has prostate cancer, while amounts above 50 ng/ml may show that the tumor has spread elsewhere in the body.
The advent of prostate specific antigen (PSA) screening has led to earlier detection of PCA and significantly reduced PCA-associated fatalities. However, a major limitation of the serum PSA test is a lack of prostate cancer sensitivity and specificity, especially in the intermediate range of PSA detection (4-10 ng/ml). Elevated serum PSA levels are often detected in patients with non-malignant conditions such as benign prostatic hyperplasia (BPH) and prostatitis, and provide little information about the aggressiveness of the cancer detected. Coincident with increased serum PSA testing, there has been a dramatic increase in the number of prostate needle biopsies performed (Jacobsen et al., JAMA 274:1445 (1995)). This has resulted in a surge of equivocal prostate needle biopsies (Epstein and Potter J. Urol., 166:402 (2001)).
Thus, development of biomarkers to detect cancer, with improved sensitivity and specificity is advantageous.
SUMMARY
Provided herein are methods and compositions for screening for, or characterizing, a cancer in a subject. In one embodiment, an antibody profiling panel comprising: a plurality of polypeptide probes, wherein at least one of the polypeptide probes comprises a full-length or fragment of a protein encoded by a gene listed in Tables 1, 2, 3, or 4; and each of the probes in the plurality of polypeptide probes is capable of being specifically bound by an antibody, is disclosed herein. In another embodiment, an antibody profiling panel comprising: a plurality of polypeptide probes, wherein at least one of the polypeptide probes comprises a sequence listed in Tables 1, 2, 3, or 4 or a sequence encoded by a sequence listed in Tables 1, 2, 3, or 4; and each of the probes in the plurality of polypeptide probes is capable of being specifically bound by an antibody, is disclosed herein. In one embodiment the subject is a human. In one embodiment the antibody is an autoantibody. In another embodiment the antibody is a human autoantibody. In one embodiment the presence of a human autoantibody that binds to a polypeptide probe is indicative of cancer (e.g. an expression level for one or more autoantibodies is indicative of the presence, absence, or stage of the cancer). In another embodiment the quantity or level of a human autoantibody that binds to a polypeptide probe is indicative of cancer. In one embodiment the cancer is a prostate, lung, breast or colon cancer.
In one embodiment, the polypeptide probe comprises a polypeptide sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof. In another embodiment, the polypeptide probe comprises a polypeptide sequence encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof.
In yet another embodiment, the polypeptide probe comprises the full-length or a fragment of a protein that is encoded by DCHS1 (SEQ ID NO: 29), Centrosomal Protein (CEP 164) (SEQ ID NO: 30), KBTBD6 (SEQ ID NO: 31), RPS19 (SEQ ID NO: 32), RPL34 (SEQ ID NO: 33), Hemk1 (SEQ ID NO: 34), eIF4G1 (SEQ ID NO: 35), BMI1 (SEQ ID NO: 36), BRD2 (SEQ ID NO: 37), RP3-323M22 (Nucleolin) (SEQ ID NO: 38), SFRS14 (SEQ ID NO: 39), LOC388789 (SEQ ID NO: 40), RNA binding motif protein 6 (genomic DNA sequence) (SEQ ID NO: 41), BRMSL1 (SEQ ID NO: 42), NKX3-1 (SEQ ID NO: 43), RPSA (SEQ ID NO: 44), Cytochrome C Oxidase 5 subunit (SEQ ID NO: 45), FAM53B (SEQ ID NO: 46), a fragment of the UTR region of chromosome 11 (Homo sapiens genomic DNA, chromosome 11 clone: CTD-2579L12, NTs 149521-151500) (SEQ ID NO: 47), MAPKKK9 (SEQ ID NO: 48) cDNA clone XR_113641.1 (Homo sapiens hypothetical LOC643783, transcript variant 2 (LOC643783), partial miscRNA) (SEQ ID NO: 49), PSA (SEQ ID NO: 50), H2aa4 (SEQ ID NO: 51). UBE2I (SEQ ID NO: 52), TIMP2 (SEQ ID NO: 53), WDR77 (SEQ ID NO: 54), a fragment of Deaminase Domain Cont 1 (Human DNA sequence from clone RP1-20N2 on chromosome 6q24 Contains the gene for a novel protein similar to yeast and bacterial cytosine deaminase, NTs 48121-50100) (SEQ ID NO: 55), Lamin A/C (SEQ ID NO: 85), Lsm3 (SEQ ID NO: 86), a fragment of cDNA clone Chromosome 19, which encompasses the nucleic acid sequence for DAZ associated protein (Homo sapiens chromosome 19 clone CTB-25B13, NTs 20521-22500) (SEQ ID NO: 87), ADAM metallopetidase domain 9 (SEQ ID NO: 88), AZGP1 (SEQ ID NO: 89), Desmocolin 3 (SEQ ID NO: 90), PERP (SEQ ID NO: 91), Chromosome 3 UTR region ropporin/RhoEGF (Homo sapiens 3 BAC RP11-783D3 (Roswell Park Cancer Institute Human BAC Library) NTs 178621-180600) (SEQ ID NO: 92), Cox5a (SEQ ID NO: 93), a Mitochondrion sequence (Homo sapiens isolate PD047 mitochondrion, NTs 4801-6780) (SEQ ID NO: 94), MYH9 (SEQ ID NO: 95), ASND1 (SEQ ID NO: 96), Cathepsin F (SEQ ID NO: 97), Mastermind-like 2 (Homo sapiens genomic DNA, chromosome 11q clone:RP11-82212, NTs 157801-159780) (SEQ ID NO: 98), CSNK2A2 (SEQ ID NO: 99), AURKAIP1 (SEQ ID NO: 100), a fragment of Chromosome 4 (Homo sapiens BAC clone RP11-327O17 from 4, NTs 107401-109380) (SEQ ID NO: 101), ARF6 (SEQ ID NO: 102), JAG1 (Human DNA sequence from clone RP1-278O22 on chromosome 20 Contains two novel genes, NTs 26161-26140) (SEQ ID NO: 103), a Mitochondrion sequence (Homo sapiens isolate PD047 mitochondrion, NTs 2041-4020) (SEQ ID NO: 104), a fragment of Chromosome 20 (Human DNA sequence from clone RP1-278O22 on chromosome 20 Contains two novel genes, NTs 25321-27300) (SEQ ID NO:105), a fragment of Chromosome 6 UTR region (Human DNA sequence from clone RP3-523G1 on chromosome 6p22.3-24.1, NTs 34621-36600) (SEQ ID NO: 106), a fragment of MAPKKK5 (SEQ ID NO: 107), RASA1 (SEQ ID NO: 108), Hsp90b (SEQ ID NO: 109), ribosomal protein S6 (RPS6) (SEQ ID NO: 110), or a fragment of Homo sapiens chromosome 3 (Homo sapiens 3 BAC RP13-616I3 (Roswell Park Cancer Institute Human BAC Library) NTs 22921-24900) (SEQ ID NO: 111).
In one embodiment, the antibody profiling panel comprises a plurality of polypeptide probes, wherein at least one of the polypeptide probes comprises a full-length or fragment of a protein listed in Table 1, or a polypeptide sequence selected from SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, or 141, and each of said probes in said plurality of polypeptide probes is capable of being specifically bound by an antibody. In one embodiment, one or more of the polypeptide probes can comprise SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7. In another embodiment, one or more of the polypeptide probes can comprise a polypeptide encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, or 21. In one embodiment, the antibody profiling panel can further comprise a full-length or fragment of a protein listed in Tables 2, 3, or 4. In another embodiment, the antibody profiling panel, one of the polypeptide probes can comprise SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14. In another embodiment, one or more of the polypeptide probes can comprise a polypeptide encoded by SEQ ID NO: 22, 23, 24, 25, 26, 27, or 28. In one embodiment the antibody is an autoantibody. In another embodiment the antibody is a human autoantibody. In one embodiment the presence of a human autoantibody that binds to a polypeptide probe is indicative of cancer (e.g. an expression level for one or more autoantibodies is indicative of the presence, absence, or stage of the cancer). In another embodiment the quantity or level of a human autoantibody that binds to a polypeptide probe is indicative of cancer. In one embodiment the cancer is a prostate, lung, breast or colon cancer.
In one embodiment, the plurality of probes comprise a polypeptide probe comprising a full-length or fragment of a protein encoded by CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, Deaminase Domain, FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789. In one embodiment, the polypeptide probe comprises SEQ ID NO: 2, 5, 9, 11, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or a fragment thereof. In another embodiment, the polypeptide probe comprises a polypeptide sequence encoded by SEQ ID NO: 16, 19, 70, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, or a fragment thereof.
In one embodiment, the plurality of probes comprise a polypeptide probe comprising a full-length or fragment of a protein encoded by CEP164, RPL34, BRMSL1, NKX31, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, or Deaminase Domain. In one embodiment, the plurality of probes comprise a polypeptide probe comprising a polypeptide sequence selected from SEQ ID NOs. 2, 5, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 68, or 69. In one embodiment, the plurality of probes comprises a polypeptide probe comprising a polypeptide sequence encoded by SEQ ID NO: 16, 19, 70, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, or 84.
In one embodiment, the plurality of probes comprise a polypeptide probe comprising a full-length or fragment of a protein encoded by FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789. In one embodiment, the plurality of probes comprise a polypeptide probe comprising a polypeptide sequence selected from SEQ ID NO: 9, 11, 14, or 60. In one embodiment, the plurality of probes comprises a polypeptide probe comprising a polypeptide sequence encoded by SEQ ID NO: 23, 25, 28, 71, or 75.
In another embodiment, an antibody profiling panel comprising: a plurality of polypeptide probes, wherein at least one of the polypeptide probes comprises a full-length or fragment of a protein that is DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, or Hemk1; and each of the probes in the plurality of polypeptide probes is capable of being specifically bound by an antibody, is disclosed herein. In another embodiment, the plurality of probes further comprise a polypeptide probe comprising a full-length or fragment of a protein encoded by eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789. In one embodiment, the polypeptide probe comprises a sequence listed in Table 1 or 2, such as SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or a fragment thereof. In one embodiment the antibody is an autoantibody. In another embodiment the antibody is a human autoantibody. In one embodiment the presence of a human autoantibody that binds to a polypeptide probe is indicative of cancer (e.g. an expression level for one or more autoantibodies is indicative of the presence, absence, or stage of the cancer). In another embodiment the quantity or level of a human autoantibody that binds to a polypeptide probe is indicative of cancer. In one embodiment the cancer is a prostate, lung, breast or colon cancer.
In another embodiment, one or more of the probes is displayed by a phage. In one embodiment, the one or more probes is attached to a substrate, such as attached via a phage. In another embodiment, the substrate is an array. In yet another embodiment, the panel comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different probes. In one embodiment, the panel characterizes a cancer, such as prostate cancer, with at least 80% sensitivity and specificity. In another embodiment, the panel screens for a cancer, such as prostate cancer, with at least 80% sensitivity and specificity.
Also provided herein is a method of characterizing or screening a subject for a cancer, such as prostate cancer, lung cancer, breast cancer or colon cancer. In one embodiment, the method comprises detecting in a sample obtained from a subject a presence or level of one or more antibodies to one or more polypeptide probes comprising a full-length or a fragment of a protein encoded by DCHS1, CEP164, KBTBD6, RPS19, RPL34, SFRS14, RNA binding protein 6, or Hemk1; and characterizing or identifying, the prostate cancer based on a presence or level of the one or more antibodies. In one embodiment, the method further comprises detecting a presence, absence or level of one or more antibodies to one or more polypeptide probe comprising a full-length or a fragment of a protein encoded by eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789. In one embodiment the antibody is an autoantibody. In another embodiment the antibody is a human autoantibody.
In another embodiment, the method comprises detecting in a sample obtained from a subject a presence or level of one or more antibodies to one or more polypeptide probes comprising a full-length or a fragment of a protein encoded by CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, or Deaminase Domain; and characterizing the prostate cancer based on a presence or level of the one or more antibodies. In one embodiment, the method further comprises detecting a presence, absence or level of one or more antibodies to one or more polypeptide probe comprising a full-length or a fragment of a protein encoded by FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789. In one embodiment the subject is a human. In one embodiment the antibody is an autoantibody. In another embodiment the antibody is a human autoantibody. In one embodiment the presence of a human autoantibody that binds to a polypeptide probe is indicative of cancer (e.g. an expression level for one or more autoantibodies is indicative of the presence, absence, or stage of the cancer). In another embodiment the quantity or level of a human autoantibody that binds to a polypeptide probe is indicative of cancer. In one embodiment the cancer is a prostate, lung, breast or colon cancer.
Also provided herein is a method of obtaining a biopsy, wherein a determination of whether a biopsy should be obtained is based on detecting an expression level for an antibody. In one embodiment, a subject suspected of having cancer based on an expression level of an antibody is recommended to have a biopsy obtained. In another embodiment, a biological sample is obtained from a subject with a PSA level of greater than about 2.5 ng/ml, and the sample is contacted with one or more probes for an antibody, and based on the expression level of an antibody, a biopsy is obtained or recommended for the subject. In one embodiment, the subject has a PSA level between about 2.5 ng/mL and about 10 ng/mL. In one embodiment the subject is a human. In one embodiment the antibody is an autoantibody. In another embodiment the antibody is a human autoantibody.
In one embodiment, the method further comprises contacting a biological sample obtained from the subject with one or more probes for a second antibody when the biopsy provides a positive result for a cancer, such as prostate cancer, and based on the expression level of the second antibody, a prognosis or theranosis is provided. In one embodiment the subject is a human. In one embodiment the second antibody is an autoantibody. In another embodiment the second antibody is a human autoantibody.
Also provided herein is a method of characterizing, identifying, or screening for a cancer in a subject. In one embodiment, the method comprises detecting an expression level for one or more antibodies, wherein the expression level of the one or more antibodies is indicative of the presence, absence, or stage of the cancer. In another embodiment, the indication is whether the cancer is aggressive or indolent. In one embodiment, the method of identifying a cancer as aggressive or indolent comprises: obtaining a positive biopsy result for cancer from the subject; contacting a biological sample obtained from the subject with one or more probes for an antibody; detecting an expression level for the antibody; and characterizing or identifying the cancer as aggressive or indolent based on the expression level of the antibody. In one embodiment the subject is a human. In one embodiment the antibody is an autoantibody. In another embodiment the antibody is a human autoantibody. In one embodiment the presence of a human autoantibody that binds to a polypeptide probe is indicative of cancer (e.g. an expression level for one or more autoantibodies is indicative of the presence, absence, or stage of the cancer). In another embodiment the quantity or level of a human autoantibody that binds to a polypeptide probe is indicative of cancer. In one embodiment the cancer is a prostate, lung, breast or colon cancer.
INCORPORATION BY REFERENCE
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.



BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
FIG. 1 is a schematic depicting detecting in a sample from a subject with PSA levels greater than 2.5 ng/mL the expression of one or more autoantibodies (“Autoantibody Test I”). If the result of the Autoantibody Test I is negative, a biopsy is not recommended to be obtained from the subject for further analysis. If result of the Autoantibody Test II is positive, then a biopsy is obtained. If the biopsy is positive for prostate cancer, expression of one or more autoantibodies is detected from a sample from the subject to characterize the cancer as aggressive or indolent, and a prognosis or theranosis provided.
FIG. 2 lists the nucleic acid sequence for DCHS1 (SEQ ID NO: 29).
FIG. 3 lists the nucleic acid sequence for Centrosomal Protein (CEP 164) (SEQ ID NO: 30).
FIG. 4 lists the nucleic acid sequence for KBTBD6 (SEQ ID NO: 31).
FIG. 5 lists the nucleic acid sequence for RPS19 (SEQ ID NO: 32).
FIG. 6 lists the nucleic acid sequence for RPL34 (SEQ ID NO: 33).
FIG. 7 lists the nucleic acid sequence for Hemk1 (SEQ ID NO: 34).
FIG. 8 lists the nucleic acid sequence for eIF4G1 (SEQ ID NO: 35).
FIG. 9 lists the nucleic acid sequence for BMI1 (SEQ ID NO: 36).
FIG. 10 lists the nucleic acid sequence for BRD2 (SEQ ID NO: 37).
FIG. 11 lists the nucleic acid sequence for RP3-323M22 (Nucleolin) (SEQ ID NO: 38).
FIG. 12 lists the nucleic acid sequence for SFRS14 (SEQ ID NO: 39).
FIG. 13 lists the nucleic acid sequence for LOC388789 (SEQ ID NO: 40).
FIG. 14 lists the nucleic acid sequence for RNA binding motif protein 6 (genomic DNA sequence) (SEQ ID NO: 41).
FIG. 15 lists the nucleic acid sequence for BRMSL1 (SEQ ID NO: 42).
FIG. 16 lists the nucleic acid sequence for NKX3-1 (SEQ ID NO: 43).
FIG. 17 lists the nucleic acid sequence for RPSA (SEQ ID NO: 44).
FIG. 18 lists the nucleic acid sequence for Cytochrome C Oxidase 5 subunit (SEQ ID NO: 45).
FIG. 19 lists the nucleic acid sequence for FAM53B (SEQ ID NO: 46).
FIG. 20 lists the nucleic acid sequence for a fragment of the UTR region of chromosome 11 (Homo sapiens genomic DNA, chromosome 11 clone: CTD-2579L12, NTs 149521-151500) (SEQ ID NO: 47).
FIG. 21 lists the nucleic acid sequence for MAPKKK9 (SEQ ID NO: 48).
FIG. 22 lists the nucleic acid sequence for cDNA clone XR_113641.1 (Homo sapiens hypothetical LOC643783, transcript variant 2 (LOC643783), partial miscRNA) (SEQ ID NO: 49).
FIG. 23 lists the nucleic acid sequence for PSA (SEQ ID NO: 50).
FIG. 24 lists the nucleic acid sequence for H2aa4 (SEQ ID NO: 51).
FIG. 25 lists the nucleic acid sequence for UBE2I (SEQ ID NO: 52).
FIG. 26 lists the nucleic acid sequence for TIMP2 (SEQ ID NO: 53).
FIG. 27 lists the nucleic acid sequence for WDR77 (SEQ ID NO: 54).
FIG. 28 lists the nucleic acid sequence for a fragment of Deaminase Domain Cont 1 (Human DNA sequence from clone RP1-20N2 on chromosome 6q24 Contains the gene for a novel protein similar to yeast and bacterial cytosine deaminase, NTs 48121-50100) (SEQ ID NO: 55).
FIG. 29 lists the nucleic acid sequence for Lamin A/C (SEQ ID NO: 85).
FIG. 30 lists the nucleic acid sequence Lsm3 (SEQ ID NO: 86).
FIG. 31 lists the nucleic acid sequence for a fragment of cDNA clone Chromosome 19, which encompasses the nucleic acid sequence for DAZ associated protein (Homo sapiens chromosome 19 clone CTB-25B13, NTs 20521-22500) (SEQ ID NO: 87).
FIG. 32 lists the nucleic acid sequence for ADAM metallopetidase domain 9 (SEQ ID NO: 88).
FIG. 33 lists the nucleic acid sequence for AZGP1 (SEQ ID NO: 89).
FIG. 34 lists the nucleic acid sequence for Desmocolin 3 (SEQ ID NO: 90).
FIG. 35 lists the nucleic acid sequence for PERP (SEQ ID NO: 91).
FIG. 36 lists the nucleic acid sequence for Chromosome 3 UTR region ropporin/RhoEGF (Homo sapiens 3 BAC RP11-783D3 (Roswell Park Cancer Institute Human BAC Library) NTs 178621-180600) (SEQ ID NO: 92).
FIG. 37 lists the nucleic acid sequence for Cox5a (SEQ ID NO: 93).
FIG. 38 lists the nucleic acid sequence for a Mitochondrion sequence (Homo sapiens isolate PD047 mitochondrion, NTs 4801-6780) (SEQ ID NO: 94).
FIG. 39 lists the nucleic acid sequence for MYH9 (SEQ ID NO: 95).
FIG. 40 lists the nucleic acid sequence for ASND1 (SEQ ID NO: 96).
FIG. 41 lists the nucleic acid sequence for Cathepsin F (SEQ ID NO: 97).
FIG. 42 lists the nucleic acid sequence for Mastermind-like 2 (Homo sapiens genomic DNA, chromosome 11q clone:RP11-82212, NTs 157801-159780) (SEQ ID NO: 98).
FIG. 43 lists the nucleic acid sequence for CSNK2A2 (SEQ ID NO: 99).
FIG. 44 lists the nucleic acid sequence for AURKAIP1 (SEQ ID NO: 100).
FIG. 45 lists the nucleic acid sequence for a fragment of Chromosome 4 (Homo sapiens BAC clone RP11-327O17 from 4, NTs 107401-109380) (SEQ ID NO: 101).
FIG. 46 lists the nucleic acid sequence for ARF6 (SEQ ID NO: 102).
FIG. 47 lists the nucleic acid sequence for JAG1 (Human DNA sequence from clone RP1-278O22 on chromosome 20 Contains two novel genes, NTs 26161-26140) (SEQ ID NO: 103).
FIG. 48 lists the nucleic acid sequence for a Mitochondrion sequence (Homo sapiens isolate PD047 mitochondrion, NTs 2041-4020) (SEQ ID NO: 104).
FIG. 49 lists the nucleic acid sequence for a fragment of Chromosome 20 (Human DNA sequence from clone RP1-278O22 on chromosome 20 Contains two novel genes, NTs 25321-27300) (SEQ ID NO:105).
FIG. 50 lists the nucleic acid sequence for a fragment of Chromosome 6 UTR region (Human DNA sequence from clone RP3-523G1 on chromosome 6p22.3-24.1, NTs 34621-36600) (SEQ ID NO: 106).
FIG. 51 lists the nucleic acid sequence for a fragment of MAPKKK5 (SEQ ID NO: 107).
FIG. 52 lists the nucleic acid sequence for RASA1 (SEQ ID NO: 108).
FIG. 53 lists the nucleic acid sequence for Hsp90b (SEQ ID NO: 109).
FIG. 54 lists the nucleic acid sequence for ribosomal protein S6 (RPS6) (SEQ ID NO: 110).
FIG. 55 lists the nucleic acid sequence for a fragment of Homo sapiens chromosome 3 (Homo sapiens 3 BAC RP13-616I3 (Roswell Park Cancer Institute Human BAC Library) NTs 22921-24900) (SEQ ID NO: 111).



DETAILED DESCRIPTION
The compositions and methods of the present disclosure relate to compositions and methods for characterizing a cancer or screening for a cancer. Provided herein are tests which can be used to analyze a presence or absence of an antibody from a subject, such as a subject being tested or screened for a cancer. In one embodiment, an antibody is an autoantibody. In another embodiment, the test comprises a single antigen, thus detecting only an antibody that binds to that antigen. In another embodiment, a panel of antigens is constructed such that the panel tests for a presence of one or more antibodies which specifically bind to two or more antigens derived from proteins associated with a specific cancer, such as lung cancer, prostate cancer, or ovarian cancer. By detecting an antibody to a protein associated with a disease state, the compositions and methods provided herein allow for the characterization of a cancer.
A cancer is characterized for a subject using a composition or method disclosed herein. In one embodiment, a subject is an individual or patient. In one embodiment, a subject is a human. In another embodiment, a subject is a cancer patient. In one embodiment, a subject exhibits no symptom of cancer, such as no symptoms of prostate cancer. In another embodiment, a subject has no detectable symptom of cancer, such as no detectable symptoms for prostate cancer. In yet another embodiment, a subject exhibits a symptom of cancer, such as a symptom for prostate cancer. In one embodiment, a subject is a human. In another embodiment, a subject is an individual. In yet another embodiment, a subject is a patient, such as a cancer patient.
Characterizing a cancer, or screening for a cancer, can include detecting the cancer (including pre-symptomatic early stage detecting), determining the prognosis, diagnosis, or theranosis of the cancer, or determining the stage or progression of the cancer. In one embodiment, a prognosis is predicting or giving a likelihood of outcome of a disease or condition, such as an extent of malignancy of a cancer, a likelihood of survival, or expected life expectancy, such as in an individual with prostate cancer. In another embodiment, a prognosis is a prediction or likelihood analysis of cancer progression, cancer recurrence, or metastatic spread or relapse.
In one embodiment, the diagnosis is prediction or likelihood an individual or subject has a disease or condition, such as prostate cancer. In one embodiment, the individual is an asymptomatic individual. In another embodiment, the individual is a symptomatic individual.
In one embodiment, a theranosis is a therapy selected based on an outcome of determining a binding of one or more antibodies from a sample from a subject to an antigen or polypeptide probe as described herein. In one embodiment, a theranosis is identifying an appropriate treatment or treatment efficacy for a cancer. In one embodiment, a theranosis is modifying a treatment. In another embodiment, a theranosis is selecting a treatment regimen. In yet another embodiment, a theranosis is discontinuing or not selecting a particular treatment regimen. In one embodiment a treatment regimen or therapeutic agent is selected based on the presence or absence of an autoantibody that binds to polypeptide probes described herein. In one embodiment the autoantibody is a human aautoantibody. In one embodiment a treatment regimen or therapeutic agent is excluded based on the presence or absence of an autoantibody that binds to polypeptide probes described herein. In one embodiment the autoantibody is a human aautoantibody.
In yet another embodiment, characterizing or screening for a cancer is detecting the cancer, such as pre-symptomatic early stage detecting. In one embodiment, characterizing a cancer is determining the stage or progression of the cancer, such as early-stage, late-stage or advanced stage of cancer. Characterizing or screening for a cancer can also be determining the likelihood or possibility an individual has a cancer. Characterizing or screening for a cancer can also be identification of a cancer, such as determining whether expression of one or more antibodies is indicative of the cancer.
In one embodiment, an antigen panel is used to detect a presence of one or more antibodies to one or more proteins, antigens, mimotopes, or epitopes. In one embodiment, one or more polypeptide probes described herein is a protein or fragment thereof. In another embodiment, one or more polypeptide probes described herein comprises an antigen, mimotope, or epitope. A “mimotope” can mimic the epitope of a protein or peptide. In one embodiment, the mimotope is structurally similar to an antigen or epitope of an expressed protein, but is unrelated or weakly related at the protein sequence level.
In one embodiment, the antigen panel comprises one or more polypeptide probes comprising a polypeptide sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof. In another embodiment, the antigen panel comprises one or more polypeptide probes comprising a sequence encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof. In yet another embodiment, the polypeptide probe comprises the full-length or a fragment of a protein that is encoded by SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or a fragment thereof.
In one embodiment, the antigen panel comprises one or more polypeptide probes derived from one or more proteins encoded by one or more genes selected from: CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, Deaminase Domain, FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789. In one embodiment, detection of one or more antibodies is used to detect a presence of prostate cancer in a subject.
In one embodiment, the antigen panel comprises one or more polypeptide probes derived from one or more proteins encoded by one or more genes selected from: DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, Hemk1, eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, and LOC388789. In one embodiment, detection of one or more antibodies is used to detect a presence of prostate cancer in a subject.
A cancer can also be characterized by determining a presence or absence, or level, of one or more antibodies in a sample. In one embodiment, a sample is obtained from a subject. The subject can be a mammal, including, but not limited to, humans, non-human primates, rodents, and the like. In another embodiment, a sample is a biological fluid. The biological fluid can be, but not limited to, peripheral blood, sera, or plasma. The sample can be ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, or bronchopulmonary aspirates.
In one embodiment, the level, presence, or absence of an antibody can be determined by detecting the binding of one or more antibodies to a polypeptide probe. In one embodiment, an antibody is an autoantibody. An autoantibody refers to an antibody produced by a host (with or without immunization) and directed to a host antigen (such as a tumor antigen). Tumor-associated antigens recognized by humoral effectors of the immune system are an attractive target for diagnostic and therapeutic approaches to human cancer.
The binding of an antibody with a polypeptide probe can be specific, such that the interaction of the autoantibody with the polypeptide probe is dependent upon a presence of a particular structure (i.e., the antigenic determinant or epitope) of the polypeptide probe. Antigenic determinates or epitopes can comprise amino acids in linear or non-linear sequence in a polypeptide probe and can also comprise one or more amino acids which are in proximity to each other via protein folding (e.g., conformational epitopes). Thus, a single polypeptide or protein can potentially be bound by multiple antibodies which recognize different epitopes. In some instances, known epitopes of a particular polypeptide can be used as a probe to detect for a presence, absence or level of autoantibodies which bind a particular epitope
The polypeptide probe can be an antigen identified through serologic identification of antigens, for example by recombinant expression cloning (SEREX), such as described by Kim et al., Biotech. Lett. (2004); 26: 585-588. Generally, in this method, an antigen can be identified by screening expression cDNA libraries from human solid tumors with sera of autologous patients. This type of screening of a cDNA expression library by conventional methods typically requires the preparation of a large number of membrane filters blotted with bacteriophage plaques that are then searched with a specific probe. In the case of the SEREX experiments, the screening is performed using sera from cancer patients, which can be in very limited quantities.
A polypeptide probe for detecting an antibody can also be identified by phage-display technology, which can be based on the insertion of foreign nucleotide sequences into genes encoding for various capsid proteins of T7 phage, resulting in a heterogeneous mixture of phages, each displaying the different peptide sequence encoded by a corresponding insert. A physical link between a displayed fusion protein and DNA encoded for it make this phage target selectable. The phage target can express or display a polypeptide probe, which can be used to detect antibodies that are produced by a subject, or autoantibodies, which can then be used to detect or characterize a cancer. The polypeptide probe can be displayed by a phage and used to detect an antibody from a sample obtained from a subject. In one embodiment, an antibody is an autoantibody.
Polypeptide Probes
Provided herein is a composition and method for detecting one or more antibodies in a sample using one or more polypeptide probes. Polypeptide is used in its broadest sense and can include a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. The subunits can be linked by peptide bonds. The polypeptides can be naturally occurring, processed forms of naturally occurring polypeptides (such as by enzymatic digestion), chemically synthesized or recombinantly expressed. The polypeptides for use in the methods of the present invention can be chemically synthesized using standard techniques. The polypeptides can comprise D-amino acids (which are resistant to L-amino acid-specific proteases), a combination of D- and L-amino acids, β amino acids, or various other designer or non-naturally occurring amino acids (e.g., β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids, etc.) to convey special properties. Synthetic amino acids can include ornithine for lysine, and norleucine for leucine or isoleucine. In addition, the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare polypeptides with novel properties. For example, a polypeptide can be generated that incorporates a reduced peptide bond, i.e., R1—CH2—NH—R2, where R1 and R2 are amino acid residues or sequences. A reduced peptide bond can be introduced as a dipeptide subunit. Such a polypeptide can be resistant to protease activity, and can possess an extended half-life in vivo. A polypeptide can also include a peptoid (N-substituted glycines), in which the one or more side chains are appended to nitrogen atoms along the molecule's backbone, rather than to the α-carbons, as in amino acids. Polypeptide and peptide are intended to be used interchangeably throughout this application, i.e. where the term peptide is used, it can also include polypeptide and where the term polypeptides is used, it can also include peptide.
In one embodiment, a polypeptide probe can be a fragment or portion of a larger protein. A fragment can range in size from two amino acid residues to the entire amino acid sequence minus one amino acid. In one embodiment, a polypeptide probe is a fragment of an untranslated region (UTR) of a protein, such as a fragment that is encoded by a nucleic sequence that is a UTR region of a gene, such as the 5′ or 3′ UTR of a gene.
The fragment can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in size. In one embodiment, the fragment is less than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in size. A polypeptide probe useful in the compositions and methods herein, regardless of size, is capable of specific interaction with an antibody, such as an autoantibody.
In one embodiment, a polypeptide probe can be a fragment of a protein encoded by a gene, or a region upstream or downstream of a coding sequence, such as a UTR region, of a gene listed in Table 1, Table 2, Table 3 or Table 4. In one embodiment, the polypeptide probe comprises a polypeptide sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof. In another embodiment, the polypeptide probe comprises a polypeptide sequence encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof. In yet another embodiment, the polypeptide probe comprises the full-length or a fragment of a protein that is encoded by SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or a fragment thereof.
In one embodiment, a polypeptide probe is a fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene.
In one embodiment, the gene can be CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, or Deaminase Domain. In another embodiment, the gene is FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789.
In another embodiment, a polypeptide probe comprises SEQ ID NO: 2, 5, 9, 11, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or a fragment thereof. In another embodiment, a polypeptide probe comprises a polypeptide encoded by SEQ ID NO: 16, 19, 23, 25, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or a fragment thereof.
In one embodiment, the gene can be DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, or Hemk1. In another embodiment, the gene is eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789. A polypeptide probe can comprise a peptide sequence, or fragment thereof, such as those listed in Tables 1, 2, 3 or 4.
In one embodiment, a polypeptide probe comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or a fragment thereof. In another embodiment, a polypeptide probe comprises a polypeptide encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or a fragment thereof.












TABLE 1





Clone

NCBI
Gene
Peptide
Clone DNA Sequence


ID
Gene
Designation
Sequence
Sequence
(Encoding Peptide Sequence)







2E11
DCHS1
AB384634.1
FIG. 2
PQTTAPRRAR
AGCTTTCGCTAGAGACGCCTCCATA



(protoc-

(SEQ ID
PRRS (SEQ ID
AGTCACTTGCCCGTTGGCCCCCACG



adherin-

NO: 29)
NO: 1)
ATCGGGGTCGGTTGCTCGCAGGGC



16



TGAGCAGAGATGTGCCAGGAGGGT



pre-



TGTTCTCACGCAAGAGGACGCTGT



cursor)



ACTCCTGCTGCTGGAAAGTAGGCG







CCTCGTCGTTGACGTCAGCGACACT







GACGGTCAGGACCTGCGTGGCCGA







GCGCGGCGGGGAGCCGTGGTCTGA







GG (SEQ ID NO: 15)


 


1B4A
Centro-
NM_014956.4
FIG. 3
PVSSSGSYSTP
TGGAGGAGAGGCTGGGCTGCCCCA



somal

(SEQ ID
IRKSLRRAAPP
AGCCCCTGCTCAGGGCCTCAGAAG



Protein

NO: 30)
FRA (SEQ ID
CCATACACCTTCACTCTGATTGTGC



(CEP


NO: 2)
TCATCAAGGCCCAGCATGCAGGAG



164)



GCTCAAAGTAGCTTTTGGCTTGGGT



(Minus



GTTGACGAGAAGAGAGGTAACCTG



strand)



GGGTCATTCTTGACACGTTCCAGCC







ACCTCCGGTTGGCCTCAATTATGCC







CTGAAAGGTGGTGCTGCCCGCCTC







AGGGACTTGCGAATGGGAGTGCTG







TAGGAGCCGGAGCTGCTCACTGG







(SEQ ID NO: 16)


 


37A8
KBTBD6
NM_152903.4
FIG. 4
SSFSPLN (SEQ
GAATTCGTCATTCTCACCTTTGAAT





(SEQ ID
ID NO: 3)
TAAAGCTTAGACTAAATAGTAATA





NO: 31)

TATCGTGGGAAGGATTTTGGTTTTG







TGATATTTCTGTGAATTAAGGAATA







GATGTTAACCATTATTTTGTAGAAA







AGTGATTTGTATGTGGTTAATTATA







AATAAAACTGGTACCAGAA 







(SEQ ID NO: 17)


 


4H10
RPS19
NM_001022.3
FIG. 5
AARRPHDAW
TTTATTAACCCAGCATGGTTTGTTC





(SEQ ID
SYCKRREPAG
TAATGCTTCTTGTTGGCAGCTGCCA





NO: 32)
VXQSSGSLPQ
CCTGTCCGGCGATTCTGTCCAGATC






KVREAESPRM
TCTTTGTCCCTGAGGTGTCAGTTTG






GGYRQAGQA
CGGCCGCCATCTTGGTCCTTTTCCA






QRACSLR
CCATTTTCAGCCCCTCCAGGGCTTG






(SEQ ID NO: 4)
GAGGACCCGGCGGGCCACACTCTT







GGAGCCTCGGCTGAAGTGGCTGGG







CATGACGCCGTTTCTCTGACGTCCC







CCATAGATCTTGGTCATGGAGCCA







ACCCCAGCGCCACCCCGGAGGTAC







AGGTGCCGCGCTGTGNAAGCAGCT







CGCGTGTAGAACCAGTTCTCATCGT







AGGGAGCAAGCTCTTTGTGCTTGGC







CAGCTTGACGGTATCCACCCATTCG







GGGACTTTCAGCTTCCCGGACTTTT







TGAGGAAGGCTGCCAGAGCTCTGA







CNAACTCCTGCTGGTTCACGTCTTT







TACAGTAACTCCAGGCATCGTGCG







GCCTCCGCGCTGC 







(SEQ ID NO: 18)


 


3D10
RPL34
NM_033625.2
FIG. 6
QARLFIFITQK
TTCTCGAGTGCGGCCGCAGCTTGGG





(SEQ ID
SFIFLFSFLTLC
TATGGAGACATATCATATAAGTAA





NO: 33)
LCLQHFHNDF
TGCTAGGGTCNGTGGTAGGAAGTT






LLLDKESTLD
TTTTCATAGGAGGTGTATGAGTTGG






PVTNTFSTHG
TCGTAGCGGAATCGGGGGTATGCT






TKTLLLTSLFL
GTTCGAATTCATAAGAACAGGGAG






(SEQ ID NO: 5)
GTTAGAAGTAGGGTCTTGGTTCCAT







GTGTGCTAAATGTGTTCGTGACAGG







ATCAAGCGTGCTTTCCTTATCGAGG







AGCAGAAAATCGTTGTGAAAGTGT







TGAAGGCACAAGCACAGAGTCAGA







AAGCTAAATAAAAAAATGAAACTT







TTTTGAGTAATAAAAATGAAAAGA







CGCGCTTGA (SEQ ID NO: 19)


 


40A3
RNA
NT_022517.18
FIG. 14
LRGITKNDRN
CTCTGAGGGGCATCACCAAAAATG



binding

(SEQ ID
FNRKIHLNWISK
ACAGGAATTTCAACAGGAAGATAC



protein

NO: 41)
(SEQ ID NO: 6)
ATCTGAATTGGATCTCGAAATAAG



6



GAGTTTGTGTAAGAGAAAAGGAGG



(Minus



ACACAAGCAAGGAGACACAAAAG



strand)



ACAATTTGTCCAAGAGAGTAGTAG







TAGAAACTGACAAAGGTAAGGCTG







CTTGGTGGCCGGGTGCAGTGACTC







ACGCCTGTAATCCCAGCACTTTGGG







AGGCCAAGGCGGGTGGATCACCTG







AGGTCAGGAGTTCGAGACCACCCT







GACCAACAGGTGAAACCCCTCTCT







ACTAAAAATACAAACATTAGCCCA







TAGTCCCAGCTACTGGGGAGGCTG







AGGCAGGAGAATCGCTTGAACCTG







GGAGGCGGAGGTTGCAGTGAGCCA







AGATCGTGCCATTGCACTCCAGCCT







GGGCGACAGAATGAGACTGTCTCA







AAACAAAAGGAAAAAAAAAA 







(SEQ ID NO: 20)


 


25C4
Hemk1
NM_016173.3
FIG. 7
RGCCAGIRCT
CACTTCTTCAAGCTCCAACACAAAT



(minus

(SEQ ID
(SEQ ID NO: 7)
GCTGCCTCCTTTAGGATGCCTGCTC



strand)

NO: 34)

TGTGCTCTCCCTGCCTCCCCTAGCC







CATACCTCTGCTGGCACCTTCTGTA







CCATGCCTTCAGAAACCTTCTTATC







CCCCTCATCTCTGGGGCCCCCTGTG







GATCTGGCATACCCAAGTTCAGTA







AATGTCTATCAGTAAGCTGATGGTA







CATGCATTTTCTAGAATAGAGCTGG







GACTTCCCATGTGGCCCACATCTGA







CCTGGCAGCCCATGTATTCCGGTCA







TTAGGGATGGGAAGCCATGAGGAC







CTGGCCTTCTGCCCGACCCAGGCAG







CCATTCAAGTTGAGCAATGGCCACT







TCGAAGACTCAAGTGCACCTGATC







CCTGCGCAACAGCCAC 







(SEQ ID NO: 21)





















TABLE 2







NCBI
Gene
Peptide
Clone DNA Sequence


Clone
Gene
Designation
Sequence
Sequence
(Encoding Peptide Sequence)







24E1
eIF4G1
NM_182917.3
FIG. 8
IRDPNQGG
TTCTTCTACAGACATTTGTATAGT





(SEQ ID
KDITEEIMS
TGTCATAGTGTCCCCAGGAATAG





NO: 35)
GARTASTP
AGAGGACTGCGAGATTAGGCTCA






TPPQTGGG
GACCCCGGTTCCAAGACTGGGGA






LEPQANGE
TGGTGATGGGGTCGGAGAAGGCG






TPQVAVIV
ACGAAGGCTGGGATTCTGAAGGG






RPDDRSQG
CTATGCTCTGGGCCAGGCAGCCC






AIIADRPGL
TGGCCGGTCAGCAATGATTGCTC






PGPEHSPSE
CCTGTGACCGGTCATCTGGCCGG






SQPSSPSPT
ACAATGACAGCAACCTGGGGCGT






PSPSPVLEP
CTCCCCATTAGCTTGAGGCTCCAG






GSEPNLAV
ACCGCCTCCCGTCTGGGGAGGGG






LSIPGDTM
TGGGTGTGGAGGCAGTGCGGGCC






TTIQMSVEE
CCAGACATGATCTCCTCTGTGATA






(SEQ ID NO: 8)
TCCTTTCCTCCTTGGTTTGGATCT







CGAATTCGGATC 







(SEQ ID NO: 22)


 


3C4
5′-UTR
BC011652.2
FIG. 9
GGGRGAG
ATCACAAATAGGACAATACTTGC



BMI1

(SEQ ID
GGRGAGA
TGGTCTCCAGGTAACGAACAATA





NO: 36)
GGGRPEAA
CACGTTTTACAGAAGGAATGTAG






(SEQ ID NO: 9)
ACATTCTATTATGGTTGTGGCATC







AATGAAGTACCCTCCACAAAGCA







CACACATCAGGTGGGGATTTAGC







TCAGTGATCTTGATTCTCGTTGTT







CGATGCATTTCTGCTTGATAAAAA







ATCCCGGAAAGAGCAGCCGGCGC







GAGGCGATCGAAGCGGGCGGAA







AAGACAATGAAAGTTAAAAGTCG







TTCAGCAGAAAATGAATGCGAGC







CAAGCGGCCATCTTGAAGCGAGC







TGCAGACGCCGCTGTCAATGGGC







AACCAGCGCGGCCCCGAGCAGCC







GCGGCCGCCACGCTCGTCTCATG







CCGCCTCCGGCCGGCCTCCTCCTG







CTCCGGCGCCTCGGCCTCCTCCGG







CGCCTCGGCCTCCTCCTCCTCCGC







CTCCGCCTCGACCTCCAACGCCTC







CTCCTCCGGGGCCTCCTCCTCCTC







CTCCTCGGC (SEQ ID NO: 23)


 


8A6
BRD2
BX908719.9
FIG. 10,
ESRPMSYD
TGTAGGGCTTCCGGGGTTTCTTAC





(SEQ ID
EKRQLSLDI
GTAGGCAGGAAAGGACATAGCGC





NO: 37)
NKLPGEKL
TCAAGCTCTCTAAGTGTGGATGG






GRVVHIIQ
CTTGAGTGTTTCAAAATCAATCTC






AREPSLRD
AATCTCTTCTGGGTTTGAATCACG






SNPEEIEID
TAAAGAGGGCTCCCTGGCTTGGA






FETLKPSTL
TTATATGCACAACTCGGCCCAGCT






RELERYVL
TCTCCCCAGGTAATTTGTTGATGT






SCLRKKPR
CCAGGCTCAGCTGCCGCTTCTCAT






KPYSTYEM
CGTAACTCATGGGCCTGCTCTC






RFISWF
(SEQ ID NO: 24)






(SEQ ID NO: 10)



 


15F1
RP3-
NM_005381.2
FIG. 11
LVSILLTKTIY
TTACTGTTACCTGATCAATGACAG



323M22

(SEQ ID
(SEQ ID NO: 11)
AGCCTTCTGAGGACATTCCAAGA



(Nucleolin)

NO: 38)

CAGTATACAGTCCTGTGGTCTCCT







TGGAAATCCGTCTAGTTAACATTT







CAAGGGCAATACCGTGTTGGTTTT







GACTGGATATTCATATAAACTTTT







TAAAGAGTTGAGTGATAGAGCTA







ACCCTTATCTGTAAGTTTTGAATT







TATATTGTTTCATCCCATGTACAA







AACCATTTTTTCCTACAAATAGTT







TGGGTTTTGTTGTTGTTTCTTTTTT







TTGTTTTGTTTTTGTTTTTTTTTTTT







TTGCGTTCGTGGGGTTGTAAAAG







AAAAGAAAGCAGAATGTTTTATC







ATGGTTTTTGCTTCAGCGGCTTTA







GGACAAATTAAAAG 







(SEQ ID NO: 25)


 


6E2
SFRS14
NM_001017392.3
FIG. 12
KAECFKNL
AAGCAGAGTGCTTTAAAAATTTG





(SEQ ID
IVKKQKSL
ATAGTAAAAAAGCAAAAATCTCT





NO: 39)
CSGFKEHL
GTGCTCTGGTTTTAAGGAACATTT






NEASILAQ
GAATGAGGCAAGCATTTTAGCAC






VSVSSSKR
AGGTTTCTGTTTCAAGTTCAAAGA






VWKSWEN
GAGTCTGGAAAAGTTGGGAAAAT






LISSFMVW
TTAATATCATCTTTTATGGTGTGG






NPAHLIISIP
AATCCTGCCCATTTGATTATTTCT






NLEKTSDL
ATCCCAAATCTTGAAAAAACATC






SMMSKLA
AGACTTATCTATGATGTCAAAGCT






AALE 
(SEQ ID NO: 26)






(SEQ ID NO: 12)



 


12B2
5′-UTR
BC011652.2
FIG. 9
QRSGRDNG
AAGCTTATTATCTCATCATCAGTT



BMI1

(SEQ ID
DVGAGAPF
ATAATTCTCTTATCTTCATCTGCA





NO: 36)
RLSSTSQPR
ACCTCTCCTCTATCTTCATTAGAG






RIKPIAPPP
CCATTGGCAGCATCAGCAGAAGG






RAPSPEXG
ATGAGCTGCATAAAAATCCCTTCT






AGGGGGG
TCTCTTCATTTCATTTTTGAAAAG






RGGGGGGP
CCCTGGAACTAATTTGTATACAAT






GGGGVGG
ATCTTGGAGAGTTTTATCTGACCT






RGGGGGG
TATATTCAGTAGTGGTCTGGTCTT






GGRGAGG
GTGAACTTGGACATCACAAATAG






GRGAGAG
GACAATACTTGCTGGTCTCCAGGT






GGRPEAA
AACGAACAATACACGTTTTACAG






(SEQ ID NO: 13) 
AAGGAATGTAGACATTCTATTAT







GGTTGTGGCATCAATGAAGTACC







CTCCACAAAGCACACACATCAGG







NGGGGATTTAGCTCAGTGATCTT







GATTCTCGTTGTTCGATGCATTTC







TGCTTGATAAAAAATCCCGGAAA







GAGCAGCCGGCGCGAGGCGATCG







AAGCGGGCGGAAAAGACAATGA







AAGTTAAAAGTCGTTCAGCAGAA







AATGAATGCGAGCCAAGCGGCCA







TCTTGAAGCGAGCTGCAGACGCC







GCTGTCAATGGNCAACCAGCGCG







GCCCCGAGCAGCCGCGGCCGCCA







CGCTCGTCTCATGCCGCCTCCGGC







CGGCCTCCTCCTGCTCCGGCGCCT







CGGCCTCCTCCGGCGCCTCGGCCT







CCTCCTCCTCCGCCTCCGCCTCGA







CCTCCAACGCCTCCTCCTCCGCTT







GAATTCGGATCCCCGAGCATCAC







ACCTGACTGGAATACGAACAGCT







CCACATNCNGT 







(SEQ ID NO: 27)


 


21D10
Homo
BC150559.1
FIG. 13
PASASILAG
TTGGGCGTTCAGAGAGTTCACTG



sapiens

(SEQ ID
VPMYRNEF
GGTACTTCACTTGCTGAGCCATCC



hypo-

NO: 40)
TAWYRRM
TTTTGGTCTACTGACGACTTCGCC



thetical


SVVYGIGT
ATTGTCCGGCTATAGTAAAGCAG



LOC388789


WSVLGSLL
TGAGCCCAACACAGACCAGGTGC



(LOC388789)


YYSRTMA
CGATCCCGTAGACCACCGACATC






KSSVDQKD
CGCCGGTACCAGGCCGTGAACTC






GSASEVPS
ATTTCGATACATGGGTACGCCAG






ELSERPSLR
CGAG (SEQ ID NO: 28)






PHSSN







(SEQ ID NO: 14)





















TABLE 3







NCBI
Gene
Peptide
Clone DNA Sequence


Gene
Clone
Designation
Sequence
Sequence
(Encoding Peptide Seqence)







8E10
BRMSL1
NM_032352.3
FIG. 15
APRTRTLR
TCGTCGAGGCTCCTGCTCCTGTGA





(SEQ ID
ARRSPRME
CTCTCGAGCAGCCAGAGGCTCCT





NO: 42)
IAQKWMM
ACCTCTATCGAGTCTTTACCTACT






KTVKEEEW
ACTTCTGACACTTTCTTCTTCTTA






NVWMKCPI
CCTTACAAACCTACTTTACAGGTT






LKNSLPIS
AGAACTTTTTGTCAAATGGCTAG






KINFIKND
AGTTTCTAGTTGAAATATTTCTTG






(SEQ ID 
CTAATTCAGTCCACCTACGTTTTG






NO: 56)
ATGTTCTTCAGTATCGACCTTTTC







GTGGTCTTATGAACCTTGGCGACC







GTTGAAATGTCCTTTTATACGTTT







AAGCATGTTTCCATCGTCCTTAGA







TATCTCTCGAGACGAATCTTAGAC







ATTTCTTGTTTATACTTACACTTT







AAGTTCGAA (SEQ ID NO: 70)


 


1D10
5′-UTR-BMI1
NM_005180.5
FIG. 9
GGRGGGG
GGAGGTCGAGGCGGAGGCGGAG





(SEQ ID
GGGGRGA
GAGGAGGAGGCCGAGGCGCCGG





No: 36)
GGGRGAG
AGGAGGCCGAGGCGCCGGAGCA






AGGGRPEAA
GGAGGAGGCCGGCCGGAGGCGG






(SEQ ID
CATGAGACGAGCGTGGCGGCCGC






NO: 172)
GGCTGCTCGGGGCCGCGCTGGTT







GNCCATTGACAGCGGCGTCTGCA







GCTCGCTTCAAGATGGCCGCTTG







GCTCGCATTCATTTTCTGCTGAAC







GACTTTTAACTTTCATTGTCTTTTC







CGCCCGCTTCGATCGCCTCGCGCC







GGCTGCTCTTTCCGGGATTTTTTA







TCAAGCAGAAATGCATCGAACAA







CGAGAATCAAGATCACTGAGCTA







AATCCCCNCCTGATGTGTGTGCTT







TGTGGAGGGTACTTCATTGATGCC







ACAACCATAATAGAATGTCTACA







TTCCTTCTGTAAAACGTGTATTGT







TCGTTACCTGGAGACCAGCAAGT







ATTGTCCTATTTGTGATGTCCAAG







TTCACAAGACCAGACCACTACTG







AATATAAGGTCAGATAAAACTCT







CCAAGATATTGTATACAAATTAG







TTCCAGGGCTTTTCAAAAATGAA







ATGAAGAGAAGAAGGGATTTTTA







TGCAGCTCATCCTTCTGCTGATGC







TGCCAATGGCTCTAATGAAGATA







GAGGAGGACGGTTGCAGATGAAG







ATAAGAGAATTATAANCTGATGA







TGAGATAATAAGGCTTGCGGCCG







CACTCGAGAAACAGT 







(SEQ ID NO: 71)


 


1H2
NKX3-1
NM_0067167.3
FIG. 16
GTNQRREG
GGAGAGAGGGAAAATCAAGTGGT





(SEQ ID
KSSGIFQHFV
ATTTTCCAGCACTTTGTATGATTT





NO: 43
(SEQ ID
TGGATGAGTTGTACACCCAAGGA






NO: 57)
TTCTGTTCTGCAACTCCATCCTCC







TGTGTCACTGAATATCAACTCTGA







AAGAGCAA (SEQ ID NO: 72)


 


4H9
RPSA
NM_002295.4
FIG. 17
GKWCHAC
CGGGAAATGGTGCCACGCATGCG





(SEQ ID
AELPEPAST
CAGAACTTCCCGAGCCAGCATCC





NO: 44)
TSNPLSELP
ACCACATCAAACCCACTGAGTGA






CCCMGWQ
GCTCCCTTGTTGTTGCATGGGATG






CPHSAEEN
GCAATGTCCACATAGCGCAGAGG






LCYTAQW
AGAATCTGTGTTACACAGCGCAA






(SEQ ID 
TGGTAGGTAGGTTAACATAAGAT






NO: 58)
GCCTCCGTGAGAGGCTGGTGGTC







AGCCCTGGGGTCAGTAACCACAA







GAAGCCGTGGCTCCCGGAAGGCT







GCCTGGATCTGGTTAGTGAAGGT







TCCAGGAGTGAAGCGGCCAGCAA







TTGGAGTGGCTCCAGTGGCAGCA







GCAAACTTCAGCACAGCCCTCTG







GCCAGTATTCCTGGAGGATATAA







CACTGACATCAGCAGGGTTTTCA







ATGGCAACAATTGCACGAGCTGC







CAGCAGAAGCTT 







(SEQ ID NO: 73)


 


5B1
Cytochrome
NM_004255.3
FIG. 18
INTLVTYD
GATAAACACACTTGTTACCTATG



C Oxidase 5

(SEQ ID
MVPEPKIID
ATATGGTTCCAGAGCCCAAAATC



Subunit

NO: 45)
AALRACRR
ATTGATGCTGCTTTGCGGGCATGC






LNDFASTV
AGACGGTTAAATGATTTTGCTAGT






RILEVVKD
ACAGTTCGTATCCTAGAGGTTGTT






KAGPHKEI
AAGGACAAAGCAGGACCTCATAA






YPYVIQEL
GGAAATCTACCCCTATGTCATCCA






RPTLNELGI
GGAACTTAGACCAACTTTAAATG






STPEELGL
AACTGGGAATCTCCACTCCGGAG






DKV (SEQ ID
GAACTGGGCCTTGA






NO: 59)
CAAAGTGTAAACCGCATGGATGG







GCTTCCCCAAGGATTTATTGACAT







TGCTACTTGAGTGTGAACAGTTAC







CTGGAAATACTGATGATAACATA







TTACCTTATTTGAACAAGTTTTCC







TTTATTGAGTACCAAGCCATGTAA







TGGTAACTTGGACTTTAATAAAA







GGGAAATGAGTTTGAACTGAAA







(SEQ ID NO: 74)


 


17B8
FAM53B
NM_014661.3
FIG. 19
EVHIKKKT
GGGAAGTCCACATTAAAAAGAAA





(SEQ ID
KQTLTNFQ
ACAAAACAAACCCTAACTAACTT





NO: 46)
MGLLVRG
CCAAATGGGTCTCCTGGTGCGGG






REWPCPGC
GGCGTGAGTGGCCGTGCCCTGGG






AACLSKLP
TGTGCTGCCTGTCTGAGCAAGCTT






(SEQ ID 
CCCTAGCTGTGGAACCCCGGGCC






NO: 60)
CCCTGCTGCGGGCTCTGCCTTGGT







GTCATGCCTGCTGCACCCCCGTTT







CCACTGACGTGCCGTCTGTGGCTA







TGGGGGTGGTCACTGGAATGACG







GTCACTCCAGACGTCAGCCGGCA







GGGATGCAGCAGGCTGGCCGCGC







A (SEQ ID NO: 75)


 


3C11
UTR-
AP003173.4
FIG. 20
DHSMVEFP
ATTCTATGGTGGAATTTCCAAGA



Region

(SEQ ID
RIIVYPQFG
ATAATTGTTTATCCTCAGTTTGGA



Chromosome

NO: 47)
VGNEG
GTAGGAAATGAAGGATAATTTTT



11


(SEQ ID 
TCCATTTCACCTCTATTGCAAATT






NO: 61)
TATTTTTTCAAGCCACACAAAAA







ATTGTCTAAGATAAAATGAGAAT







TATTCAGATCAATTCTGCAATGAT







ACAGGGAAGATGTGAAAGGAGG







GCTCAATGCAGAGTTGTGAAGTT







GAAAACCACTATTTCTGTTCTAAA







GACACAGTAAGCAGAGATCCATC







TCTCTTCAGGCATCCTGCTTCTCT







GCAGGTTACTTCTGCTTTAAGGAA







AGTACATTTTTAGAACAAAGCTT







(SEQ ID NO: 76)


 


3F6
MAPKKK9
NM_033141.2
FIG. 21
SSGSGESRL
TCAAGCGGGAGTGGAGAGAGTCG





(SEQ ID
QHSPSQSY
CCTACAGCATTCACCCAGCCAGT





NO: 48)
LCIPFPRGE
CCTACCTCTGTATCCCAT






DGDGPSSD
TCCCTCGTGGAGAGGATGGCGAT






GIHEEPTPV
GGCCCCTCCAGTGATGGAATCCA






NSATSTPQ
TGAGGAGCCCACCCCAGTCAACT






LTPTNSLK
CGGCCACGAGT






RGGAHHR
ACCCCTCAGCTGACGCCAACCAA






RCEVALLG
CAGCCTCAAGCGGGGCGGTGCCC






CGAVLAAT
ACCACCGCCGCTGCGAGGTGGCT






GLGFDLLE
CTGCTCGGCTG






AGKCQLLP
TGGGGCTGTTCTGGCAGCCACAG






LEEPEPPAR
GCCTAGGGTTTGACTTGCTGGAA






EEKKRREG
GCTGGCAAGTGCCAGCTGCTTCC






LFQRSSRPR
CCTGGAGGAGC






RSTSPPSRK
CTGAGCCACCAGCCCGGGAGGAG






LFKKEEHQ
AAGAAAAGACGGGAGGGTCTTTT






ACGRTRVT
TCAGAGGTCCAGCCGTCCTCGTC






S (SEQ ID
GGAGCACCAGC






NO: 62)
CCCCCATCCCGAAAGCTTTTCAAG







AAGGAGGAGCACCAAGCTTGCGG







CCGCACTCGAGTAACTAGTTAAC







CCCTTGGGGC







CTCTAAACGGGTCTTGAGGGGGT







TANCTNGTTACTCGNGTGCGGCC







GCNNGCTTGGTGCTCNNCNTTN







(SEQ ID NO: 77)


 


21H4
cDNA
XR_113641.1
FIG. 22
QKLCQAKE
ATCCCAGCACGGAGGCCCAGAAA



clone

(SEQ ID
KGMCMKK
ACTTTAAGATTTGAGTATTAATGT





NO: 49)
LRMLWEC
CTCAAGGTCAGGAGCAACCTCAA






QKLYSLGF*
GGCTAAAACTCAGATCTCAGGAC






(SEQ ID
TCAATTTCACAGAAGTTCCACTAT






NO: 63)
AAAGGCAATAATCTAAAGCTTTA







AATGATATGAAAATTTTGTAATA







AGAGTTCAGTATTTCTGCCAACAT







TGGCGCATGGATTGCAAAGTTCA







CAGGATTGAAAACACCATCGACA







TAATGGAAATTGAACAGCATCTG







ATTACTGAGTGCTATATCAGCAA







GTTAAAAGGATCTTTTGCATACCT







TTTAATGGTATATATCCTAAAACT







GAAGTGTTCAATATAGACATCCA







GATTGAAA (SEQ ID NO: 78)


 


4C4
PSA
M27274.1
FIG. 23
S E G R T V
TGTGTGGGTATGAGGGTATGAGA





(SEQ ID
T N K V S R
GGGCCCCTCTCACTCCATTCCTTC





NO: 50)
K Y T G
TCCAGGACATCCCTCCACTCTTGG






(SEQ ID 
GAGACACAGAGAAGGGCTGGTTC






NO: 64)
CAGCTGGAGCTGGGAGGGGCAAT







TGAGGGAGGAGGAAGGAGAAGG







GGGAAGGAAAACAGGGTATGGG







GGAAAGGACCCTGGGGAGCGAA







GTGGAGGATACAACCTTGGGCCT







GCAGGCCAGGCTACCTACCCACT







TGGAAACCCACGCCAAAGCCGCA







TCTACAGCTGAGCCACTCTGAGG







CCTCCCCTCCCCGGCGGTCCCCAC







TCAGCTCCAAAGTCTCTCTCCCTT







TTCTCTCCCACACTCTATCATCCC







CCGGATTCCTCTCTACTTGGTTCT







CATTCTTCCTTTGACTTCCTGATC







CTGTGTATTTTCGGCTCACCTTGA







TTTGTCACTGTTCTCCCCTC 







(SEQ ID NO: 79)


 


5A1
H2aa4
NM_001040874.1
FIG. 24
QRGSGQQE
ACGCGGCTCGGGGACAACAAGAA





(SEQ ID
DAHHPSSP
GACGCGCATCATCCCTCGTCACCT





NO: 51)
PAGHPQRR
CCAGCTGGCCATCCGCAACGACG






GTEQAAGQ
AGGAACTGAACAAGCTGCTGGGC






SHHRPGRR
AAAGTCACCATCGCCCAGGGCGG






LA (SEQ ID
CGTCTTGCCTAACATCCAGGCCGT






NO: 65)
ACTGCTCCCTAAGAAGACGGAGA







GTCACCACAAGGCAAAGGGCAAG







TGAGGCTGACGTCCGGCCCAAGT







GGGCCCAGCCCGGCCCGCGTCTC







GAAG (SEQ ID NO: 80)


 


1B4
UBE2I
NM_194259.1
FIG. 25
ILYPETLLK
TGTGGCATCGTCAAAAGGAAGGG





(SEQ ID
LLISLRRFW
ATTGGTTTGGCAAGAACTTGTTTA





NO: 52)
AEMMEFSR
CAACATTTTTGCAAATCTAAAGTT






YTIMSSEN
GCTCCATACAATGACTAGTCACCT






RDNLTSSFP
GGGGGGGTTGGGCGGGCGCCATC






N* (SEQ ID
TTCCATTGCCGCCGCGGGTGTGCG






NO: 66)
GTCTCGATTCGCTGAATTGCCCGT







TTCCATACAGGGTCTCTTCCTTCG







GTCTTTTGTATTTTTGATTGTTATG







TAAAACTCGCTTTTATTTTAATAT







TGATGTCAGTATTTCAACTGCTGT







AAAATTATAAACTTTTATACTTGG







GTAAGTCCCCCAGGGGCGAGTTC







CTCGCTCTGGGATGCAGGCATGC







TTCTCACCGTGCAGAGCTGCACTT







GGCCTCAGCTGGCTGTATGGAAA







(SEQ ID NO: 81)


 


18D3
TIMP2
NM_003255.4
FIG. 26
CSKHSSLL
ATGTTCTAAGCACAGCTCTCTTCT





(SEQ ID
LFSSCKQL
CCTATTTTCATCCTGCAAGCAACT





NO: 53)
KIFKIKFTL
CAAAATATTTAAAATAAAGTTTA






(SEQ ID 
CATTGTAGTTATTTTCAAATCTTT






NO: 67)
GCTTGATAAGTATTAAGAAATAT







TGGACTTGCTGCCGTAATTTAAAG







CTCTGTTGATTTTGTTTCCGTTTG







GATTTTTGGGGGAGGGGAGCACT







GTGTTTATGCTGGAATATGAAGTC







TGAGACCTTCGGTGCTGGGAACA







CACAAGAGTTGTTGAAAGTTGAC







AAGCAGACTGCGCATGTCTCTGA







TGCTTTGTATCATTCTTGAGCAAT







CGCTCGGTCCGTGGACAATAAAC







AGTATTATCAAAGAGAAAAAAAA







(SEQ ID NO: 82)


 


2B10
WDR77
NM_024102.2
FIG. 27
NSLPLFPPQ
GCCACTTTTCCCACCCCAAAACA





(SEQ ID
NSMGPDIF
GCATGGGGCCTGACATCTTCTGCC





NO: 54)
CPGPLSL
CTGGTCCCCTTTCTCTTGATGTGG






DVESLNAV
AAAGTCTGAATGCAGTATTTATA






FIDF* (SEQ
GACTTCTAAGGTTTTAAAATCCAG






ID NO: 68)
TATCAAGAAGAAAATCAGAAATA







CTGGTTGGTGAAATAAAGAGTTT







AGGCATTGTTGGCCTGTCTTTTTT







GAAGCATGTGTGTTATGTGTAGTT







AGATATATTTCACTTATGTGAGTC







ATCATGGTGTTGGTCTTGTAGCCC







ATTATTTTTCCTGTGCTTCCCCAG







CTTCCCAAAGTAGCTAGTTAGAA







CTTAAGGTAAATATTTATTCTTGG







GTTGGTGGAGTGGATATTGCCAG







TTAGGAGTCATGGATCAATTACT







GATTATATTGAAAGTAAATATAA







TCAATTATGTACTTTTGAGCTTTG







CAGGTTCAATTTAGGTAAAAATC







ACATTATGAAACTGGGAAAGTCT







GAAGGAATATGGGCAAAATATTT







CTCAGTAAAGCTT 







(SEQ ID NO: 83)


 


5F4
Deaminase
AL031320.1
FIG. 28
VSGSQRVK
GAGATGTAAGCGGCTCACAAAGG



Domain

(SEQ ID
YLLVNPLQ
GTGAAATATTTACTAGTTAACCCC



Cont 1

NO: 55)
KKFINPCY
CTTGCAGAAAAAGTTATCAACCC






RGF (SEQ
TTGCTACAGAGGATTTTAAAAAA






ID NO: 69)
TAAAATACAGCTTGTTCTATCTTT







AGCATCTAACTGGGGAAAAGAAT







CATAACATGTGAAAGAATAAATA







AGAAATTGTGCTAACAGTAAGGA







GTGTTATATGAAATATTACCTGAA







GAACATGAAACTTGAACTTGCTA







GAGATAGAGAATATTTAAAGAGG







CTAAGCAGAGCATTTCAGGGAAA







GGGCAAGAAGAAGCCTGGGTTGT







GTGTGAGGAAATCAGCTGACAGA







GGAGGAGACTATTAAGGAAGCAT







AAGGAAAGAAAGACAAAAAATT







GGGGTAAAAATATGTACGGCTTT







GAAAGCTT (SEQ ID NO: 84)





















TABLE 4







NCBI
Gene
Peptide
Clone DNA Sequence


Clone
Gene
Designation
Sequence
Sequence
(Encoding Peptide Sequence)







1G7
Lamin
BC014507.1
FIG. 29
SCGPSMRTRWS
AAGCTTCGCCTCCTTGGCTGCCAG



A/C

(SEQ ID
SIRRSWRRLILPS
CTGCTTCTGGAGCTGGCTGAGCTG





NO: 85)
WTMPGSLLRGT
GGCAGAGAGGCTGTCGATGCGGA






ATWWGLPTRSC
TGCGCGACTGCTGCAGCTCCTCGT






SSRASASTASLP
GGGCAGCCCCCACCAGGTTGCTG






SSASSRSSWQPR
TTCCTCTCAGCAGACTGCCTGGCA






RRSLRPHSS
TTGTCCAGCTTGGCAGAATAAGTC






(SEQ ID 
TTCTCCAGCTCCTTCTTATACTGC






NO: 112)
TCCACCTGGTCCTCATGCTGGGCC







CGCAG (SEQ ID NO: 142)


 


1B10
Lsm3
AJ238095.1
FIG. 30
MRNDRAASRQIT
AATGAGAAATGACCGAGCAGCTT





(SEQ ID
(SEQ ID 
CGAGGCAGATTACATGACTTATG





NO: 86)
NO: 113)
ATCTACATTTAAATATGATCTTGG







GAGATGTGGAAGAAACTGTGACT







ACTATAGAAATTGATGAAGAAAC







ATATGAAGAGATATATAAATCAA







CGAAACGGAATATTCCAATGCTC







TTTGTCCGGGGAGATGGCGTTGTC







CTGGTTGCCCCTCCACTGAGAGTT







GGCTGAAACAAAGAATTTGTCCT







GTATGGAAAACGGGAGACTTTGT







ACAGTGGCCTCTCTAAAAGTACA







AAACATTCATAAGAGAAACCTGC







ATACATTTTGATATTAAGAAATAA







TTCCGGGGATTCTCCACTCCTGAA







ATGAGTTGATTTGCAGATAACTCT







ACAACTTCTTAAGCTAAATGGTAT







TTTCATTTTTCTCAAGCTCTCCAA







TAAATATGACCACCAA







(SEQ ID NO: 143)


 


2D7
cDNA
AC027307.5
FIG. 31
LAHRPPCAEPDP
GGAGTTTCACTTTTGTTGCCCAGG



clone

(SEQ ID
GQRMELPAPVP
ATTGAGTGCAGTGCCCCGATCTTG



Chromo 19

NO: 87)
RPRGASKPRDG
GCTCACTACAACCTCTGCCTCCTG






TSSHCDMPNCQ
GGTTCAAGCGACTCTCCTGCCTCA






HPQGPGPAGEIR
GTGTCCTGAGTAGCTGGGATTAC






SRCRSCWLRAV
AGGCGTCTGCCACCACGCCCGGC






RCNPWLGR
TAATTTTGTATTTTTAGTAGAGAA






(SEQ ID 
CAGGTTTCACTATGTTGGTCAGGC






NO: 114)
TGGTCTTGAACTCCTGACCTCAGC







GCATCCAGAATTTTAGACGGGGC







CCCCAGGGTGAGGTCTTGGCACC







CTCCAGTAGAGAAGAAGGGACAT







GGGCCATACGTGGGGTGTCCTTTC







TGGGAGCCTTGCGTCCCTTACCTG







CCTAGCCAGGGATTGCACCTCAC







AGCACGCAGCCAGCAGGAACGGC







ACCGTGATCTGATTTCACCTGCGG







GCCCTGGGCCCTGGGGGTGTTGA







CAATTGGGCATATCACAGTGTGA







GCTAGTCCCGTCTCGGGGTTTGGA







GGCTCCACGTGGCCGTGGTACAG







GAGCAGGCAGTTCCATCCTCTGG







CCTGGATCAGGCTCTGCACACGG







AGGCCTGTGGGCCAG







(SEQ ID NO: 144)


 


1H3
ADAM
NR_027878.1
FIG. 32
NSGASGSRNFSS
TCGGCATAAAGTACCTCCTGGAA



metallo-

(SEQ ID
CSAEDFEK
GGAACCGACAGTCTTTACAACAG



peptidase

NO: 88)
(SEQ ID 
TCACCATATGCACACTCAGCAAA



domain 9


NO: 115)
TGATTTAAGCTTACAGGTACTTCC







TTCGCAGCAAGGGTCCAATTCAC







ATTCCTTTGGAGTACCACAGTCAC







ACTCTTCCCCAGCGTCCACCAACT







TATTACCACAGGAGGGAGCACTA







TAGGCTTCATCAGGCTTTGGAATA







TTAAGAAGGCAGTTTCCTCCTTTA







TTTAAAGTTACTTCTCAAAGTCCT







CTGCACTGCAACTGCTAAAGTTTC







TGGAACCCGATGCTCCTGAATTC







(SEQ ID NO: 145)


 


3F5
alpha-2
NM_001185.3
FIG. 33
SSVPPQDTAPYS
TCAAGCGTGCCCCCGCAGGACAC



glyco-

(SEQ ID
CHVQHSSLAQPL
AGCCCCCTACTCCTGCCACGTGCA



protein1

NO: 89)
VVPWEAS
GCACAGCAGCCTGGC



(AZGP1)


(SEQ ID 
CCAGCCCCTCGTGGTGCCCTGGG






NO: 116)
AGGCCAGCTAGGAAGCAAGGGTT







GGAGGCAATGTGGGATCTCAGAC







CCAGTAGCTGCCCTTCCTGCCTGA







TGTGGGAGCTGAACCACAGAAAT







CACAGTCAATGGATCCACAAGGC







CTGAGGAGCAGTGTGGGGGGACA







GACAGGAGGTGGATTTGGAGACC







GAAGACTGGGATGCCTGTCTTGA







GTAGACTTGGACCCAAAAAATCA







TCTCACCTTGAGCCCACCCCCACC







CCATTGTCTAATCTGTAGAAGCCG







GAAGCTTGCGGCCGCACTCGAGT







AACTAGTTAACCCCTTGGGGCCTC







TAAACGGGTCTTGAGGGGTTANC







TNGTTNCTCGNGTGCGGCCGCNN







GCTTCCGGCTTCTNCNGNTTNGNC







NNTG N







(SEQ ID NO: 146)


 


5F3
Hemk1
NM_016173.3
FIG. 7
VAVAQGSGALE
GTGGCTGTTGCGCAGGGATCAGG



(minus

(SEQ ID
SSKWPLLNLNG
TGCACTTGAGTCTTCGAAGTGGCC



strand)

NO: 34)
CLGRAEGQVLM
ATTGCTCAACTTGAATGGCTGCCT






ASHP
GGGTCGGGCAGAAGGCCAGGTCC






(SEQ ID 
TCATGGCTTCCCATCCCTAATGAC






NO: 117)
CGGAATACATGGGCTGCCAGGTC







AGATGTGGGCCACATGGGAAGTC







CCAGCTCTATTCTAGAAAATGCAT







GTACCATCAGCTTACTGATAGAC







ATTTACTGAACTTGGGTATGCCAG







ATCCACAGGGGGCCCCAGAGATG







AGGGGGATAAGAAGGTTTCTGAA







GGCATGGTACAGAAGGTGCCAGC







AGAGGTATGGGCTAGGGGAGGCA







GGGAGAGCACAGAGCAGGCATCC







TAAAGGAGGCAGCATTTGTGTTG







GAGCTTGAAGAAGTG







(SEQ ID NO: 147)


 


5F8
Desmo-
NG_016782.1
FIG. 34
SAFRGYLANNK
TAAGCTTTCATCTTCCCCAACCCT



collin 3

(SEQ ID
(SEQ ID 
GATGTCTTCCTATTCTCACTGATC





NO: 90)
NO: 118)
CCCCTACTGACTCAGCTTCACGCT







TCTTGATTATACCTCTCTCCTGTA







GAAAAGCCTTGGCTGGCTCTCCTT







TAGGATGAGAATAAATCCGAAAT







CCTTAGTGTAGCATTTAGAAGTCC







TATCTCCCACTTGTTTCTTAATATT







CTCTTCTCTAACACCGAACTTGTT







TCAAGCCTCTTTTCCAACACATGA







TTTCTTCTATTCTAAATCAATTTAT







TTATTATTTGCTAAATAGCCCCTA







AAC







(SEQ ID NO: 148)


 


1G12
DAZ
AC027307.5
FIG. 31
SLAHRPPCAEPD
GGCTAATTTTGTATTTTTAGTAGA



Associated
(this is for
(SEQ ID
PGQRMELPAPV
GAACAGGTTTCACTATGTTGGTCA



protein
a chromosome
NO: 87)
PRPRGASKPPRRD
GGCTGGTCTTGAACTCCTGACCTC




19 clone, not

(SEQ ID 
AGCGCATCCAGAATTTTAGACGG




the specified

NO: 119)
GGCCCCCAGGGTGAGGTCTTGGC




gene)


ACCCTCCAGTAGAGAAGAAGGGA







CATGGGCCATACGTGGGGTGTCC







TTTCTGGGAGCCTTGCGTCCCTTA







CCTGCCTAGCCAGGGATTGCACCT







CACAGCACGCAGCCAGCAGGAAC







GGCACCGTGATCTGATTTCACCTG







CGGGCCCTGGGCCCTGGGGGTGT







TTGACAATTGGGGCATATCACAG







TGTGAGCTAGTCCCGTCTCGGGG







GTTTGGAGGCTCCACGTGGCCGT







GGTACAGGAGCAGGCAGTTCCAT







CCTCTGGCCTGGATCAGGCTCTGC







ACACGGAGGCCTGTGGGCCAG







(SEQ ID NO: 149)


 


1G5
RPL34
NM_033625.2
FIG. 6
LFIFITQKSFIFLF
GTCTTTTCATTTTTATTACTCAAA



(Minus

(SEQ ID
SFLTLCLCLQHF
AAAGTTTCATTTTTTTATTTAGCTT



strand)

NO: 33)
HNDFLLLDKEST
TCTGACTCTGTGCTTGTGCCTTCA






LDPVTNTFSTHGT
ACACTTTCACAACGATTTTCTGCT






(SEQ ID 
CCTCGATAAGGAAAGCACGCTTG






NO: 120)
ATCCTGTCACGAACACATTTAGCA







CACATGGAACCAA







(SEQ ID NO: 150)


 


3C9
PERP
NM_022121.4
FIG. 35
PYQIYQVMIN
CTTACCAGATCTATCAGGTCATGA



(Minus

(SEQ ID
(SEQ ID 
TAAATTAGACCCAGTCCATCTTTC



strand)

NO: 91)
NO: 121)
AATCCAGTCTACTCTGGTTCTGAA







CATATAAACACAAAACACTACAG







ATTTATTAATATAGCATTTTCCCA







CACCCTAACCCTATAAAGAACTTT







AAAAGAGAAAATTTCATCTAAAT







ATTTCACACTTAAAGGAAAGCCTT







ACCAACTATGGCAACAGGTTTGG







ACCATGAAATAGTACTTTCCTAGA







TGACATATCGAGTCAACATGAAG







CCTTAGCTGAAATGAATGATTCA







GGATATTAATGAGAAATTCTCAC







AAATGATATGCATTTAGGAAATG







ATTTTGCTTTCCTTAAATAGTTCG







AAGGCTTGAAAATAAACTTTTTTT







TTGCATTTCTTTTAAAAGTT







(SEQ ID NO: 151)


 


3D11
Chromo-
AC117381.5
FIG. 36
VSTFLSRVGRVS
GTTTCCACATTCTTGTCAAGGGTT



some 3
(Homo
(SEQ ID
LLNFLPF
GGTAGGGTCAGTCTTTTAAATTTC



UTR
sapiens 3
NO: 92)
(SEQ ID 
TTGCCATTTTAGTGACTGTGCATT



region
BAC RP11-

NO: 122)
GGTATTTCATTGTGGTTTATTTGC



ropporin/ 
783D3)


ATGATGACTAATGCTCAACACCA



RhoEGF



ACTAATCATGTTGAGTATTTTTAA







TGTGCTTATTTGCCACTCATATAT







CTTCTTTGATGAAGTGTCTCTTCA







AATATTTTGCCCATTTAAAAACTG







TATTGATTCTTATTATTGAATTGC







AATAATTCTTTCTATCCGGATATA







TATCCTTTGCCAGATATGTGTATT







ACAAATGTTTTCTCCTAGCCTTCC







ACCTCAGCCTCCCAAGTAGCTGG







GAATGCAGGTGTGCACCACCACT







CCAGGGTTTTTTGTTGTTGTTGTT







GTTGTTTTTCTGTAGAGACAGGGT







CTTGCCATGCTGCCGAGGCTGCTC







TCAAACTCCTGGGATCAAGAAAT







CCTCCTGCCTCGGCCTCCCAAAGT







GCTGACATTACAAGCATGAGCCA







CTGTGCCTGGCTAACTTTTCATCT







TTTAAAGTAGTGTCTTGCAAAGA







ACAACATTTTAATGAAGTCCATTT







ATCAACTTTTTGATTCATTGTCCA







TGCTTTTTGCATAATAAGAAATCT







TTGCCTGCCTCAAAATTGCAAAGC







TT







(SEQ ID NO: 152)


 


3E4
Cox5a
NM_004255.3
FIG. 37
NTLVTYDMVPE
AACACACTTGTTACCTATGATATG





(SEQ ID
PKIIDAALRACR
GTTCCAGAGCCCAAAATCATTGA





NO: 93)
RLNDFASTVRIL
TGCTGCTTTGCGGGCATGCAGAC






EVVKDKAGPHK
GGTTAAATGATTTTGCTAGTACAG






EIYPYVIQELRPT
TTCGTATCCTAGAGGTTGTTAAGG






LNELGISTPEELG
ACAAAGCAGGACCTCATAAGGAA






LDKV
ATCTACCCCTATGTCATCCAGGAA






(SEQ ID 
CTTAGACCAACTTTAAATGAACTG






NO: 123)
GGAATCTCCACTCCGGAGGAACT







GGGCCTTGACAAAGTGTAACCGC







ATAATAAAAGGGAAATGAGTTTG







AACTG







(SEQ ID NO: 153)


 


4B11
Mito-
HQ113226.2
FIG. 38
PPSHHIPNLSLTK
GCCCCCATCTCATCATATACCAAA



chondrion

(SEQ ID
RKPSPHSLNLIH
TCTCTCCCTCACTAAACGTAAGCC



sequence

NO: 94)
HSRQLRWIKPNP
TTCTCCTCACTCTCTCAATCTTATC






ATQNLSILLNYP
CATCATAGCAGGCAGTTGAGGTG






HRMNNSSSTVQP
GATTAAACCAAACCCAGCTACGC






(SEQ ID 
AAAATCTTAGCATACTCCTCAATT






NO: 124)
ACCCACATAGGATGAATAATAGC







AGTTCTACCGTACAACCCTAACAT







AACCATTCTTAATTTAACTATTTA







TATTATCCTAACTACTACCGCA







(SEQ ID NO: 154)


 


4B3
MYH9
NM_002473.4
FIG. 39
SAGSCSSA
GGGTTCGTGTTCCTCAGCGTAGCC



(Minus

(SEQ ID
(SEQ ID 
ATCAGGCTTGGCCAGCTGCTCCTT



strand)

NO: 95)
NO: 125)
GTAAAGCTGCCCCACAGTGCGGA







ACATGCCCTTCCGCGTCTTGAAGG







CCCCGGGCAGTGCGGTCTCCGAC







ATGCCGGCCACCTGGTCCAGGCC







GATGATGCGGTCCACATCCTTCCA







CAGCTCCGAGACAAACTTGTCAG







AGGACTGGTGGAGCAGTGTGGCG







ATGTTGTCATTCAGGGGATCCATG







TTCTTCATCAGCCACTCGTCAGCT







TTGTAATCCACCTTGCCGGCATAG







TGGATAATGCAGAAATCAGCTTT







GTCCTTCAGCTGCTTGGGCTTCTG







GA







(SEQ ID NO: 155)


 


4D10
ASND1
NM_019048.2
FIG. 40
KLLFALQLWNL
AAATTACTTTTCGCCTTGCAGCTG





(SEQ ID
VLQPLLFCPNGP
TGGAACTTGGTCTTACAGCCTCTG





NO: 96)
CSLDQELQKWK
CTCTTCTGCCCAAACGGGCCATGC






KLMKRHLINVD
AGTTTGGATCAAGAATTGCAAAA






GSKSCP
ATGGAAAAAATTAATGAAAAGGC






(SEQ ID 
ATCTGATAAATGTGGACGGCTCC






NO: 126)
AAATCATGTCCTTAGAAAATCTTT







CTATTGAAAAGGAGACTAAATTG







TAATGTGATTCACAATGTAACAAT







ATAAAAATAAGTTTTTATATAATT







ATATAAAAGTAAGATACTCTGCT







GCTTTACTATTGTATAATAT







(SEQ ID NO: 156)


 


4D9
Cathepsin F
NM_003793.3
FIG. 41
EDDYSYQGHMQ
CAGAGGATGACTACAGCTACCAG





(SEQ ID
SCNFSAEKAKV
GGTCACATGCAGTCCTGCAACTTC





NO: 97)
YINDSVELSQNE
TCAGCAGAGAAGGCCAAGGTCTA






QKLAAWLAKRG
CATCAATGACTCCGTGGAGCTGA






PISVAINAFGMQ
GCCAGAACGAGCAGAAGCTGGCA






FYRHGISRPLRP
GCCTGGCTGGCCAAGAGAGGCCC






LCSPWLIDHAVL
AATCTCCGTGGCCATCAATGCCTT






LVGYGNRSDVP
TGGCATGCAGTTTTACCGCCACGG






FWAIKNSWGTD
GATCTCCCGCCCTCTCCGGCCCCT






WGEKGYYYLHR
CTGCAGCCCTTGGCTCATTGACCA






GSGACGVNTMA
TGCGGTGTTGCTTGTGGGCTACGG






SSAVVD
CAACCGCTCTGACGTTCCCTTTTG






(SEQ ID 
GGCCATCAAGAACAGCTGGGGCA






NO: 127)
CTGACTGGGGTGAGAAGGGTTAC







TACTACTTGCATCGCGGGTCCGGG







GCCTGTGGCGTGAACACCATGGC







CAGCTCGGCGGTGGTGGACTGAA







GAGGGGCCCCCAGCTCGGGACCT







GGTGCTGATCAGAGTGGCTGCTG







CCCCAGCCTGACATGTGTCCAGG







CCCCTCCCCGGGAGGTACAGCTG







GCAGAGGGAAAGGCACTGGTACC







TCAGGGTGAGCAGAGGGCACTGG







GCTGGGGCACAGCCCCTGCTTCCC







TGCACCCCATTCCCACCCTGAAGT







TCTGCACCTGCACCTTTGTTGAAT







TGTGGTAGCTTAGGAGGATGTCA







GGGTGAAGGGTGGTATCTTGGCA







GTTGAAGCTGGGGCAAGAACTCT







GGGCTTGGGTAATGAGCAGGAAG







AAAATTTTCTGATCTTAAGCCCAG







CTGTGTTCTGCCCCCGCTTTCCTC







TGTTTGATACTATAAATTTTCTGG







TTCCCTTGGATTTAGGGATAGTGT







CCCCCTCCATGTCCAGGAAACTTG







TAACCACCCTTTTCTAACAGCAAT







AAAGAGGGTCCTTGTCCCGAAAA







AAAAAAAAA







(SEQ ID NO: 157)


 


4F1
Mastermind-
AP000779.4
FIG. 42
GTNQRQTMENH
GGCAGACAATGGAAAACCATTGA



like 2
(Homo
(SEQ ID
(SEQ ID 
AAAGGATTAAACTGGGAAGTGAT




sapiens
NO: 98)
NO: 128)
ATGTTCTCTTTTGCATTTAAAAAG




genomic


ATCACCAATGGGGATATGGAGAA




DNA,


TGGTCTGGATAGGTCTTAAGACTA




Chromosome


GAGCCAGGAAGACATGTTAGAAG




11q)


GCTATCAATTGACCCTAAAGACA







CTGCTTCAATCCCTTTGATGACAG







TGAGTTTGCTTTCCCCAGAGATAG







CTTATTGGACCTCAGGACTGCTGT







GAGAAACAGAAAATGCTCCTTTA







CGTGTTGCCTGAAGTTAGGCTCAC







CGATTTGGGGCATGTTCTAATTCT







ACCAGCTAGGAACACACAGAATC







GCTTGTCAAACATTCTGAGTCAGA







TATGTCCTCCCTATGTCTTTTCTG







AGAAAGGCATACAGAAATTCCCA







GCTAAACATCACCAGTTCCCTCAT







TTGTTCCTCAGATGATATGGTCCA







TTCAAGTTTTGTAATCATCATGGG







GGTAGATGGAGGGTCCCAGTCCT







CACAACCATTCTGGTAATTTACTC







TTGAATTTACTGGTTCACATGTAT







CTATTTTGTAGTGTGGCTCCAGAAA







(SEQ ID NO: 158)


 


5D11
CSNK2A2
NM_001896.2
FIG. 43
SSCSEYNVRVAS
TCATCCTGCTCGGAGTACAATGTT





(SEQ ID
RYFKGPELLVD
CGTGTAGCCTCAAGGTACTTCAA





NO: 99)
YQMYDYSLDM
GGGACCAGAGCTCCTCGTGGACT






WSLGCMLASMI
ATCAGATGTATGATTATAGCTTGG






FRREPFFHGQDN
ACATGTGGAGTTTGGGCTGTATGT






YDQLVRIAKVL
TAGCAAGCATGATCTTTCGAAGG






GTEELYGYLKK
GAACCATTCTTCCATGGACAGGA






YHIDLDPHFNDI
CAACTATGACCAGCTTGTTCGCAT






LGQHSRKRWEN
TGCCAAGGTTCTGGGTACAGAAG






LSIVRTDTLSAL
AACTGTATGGGTATCTGAAGAAG






RP
TATCACATAGACCTAGATCCACA






(SEQ ID 
CTTCAACGATATCCTGGGACAAC






NO: 129)
ATTCACGGAAACGCTGGGAAAAC







TTATCCATAGTGAGAACAGACAC







CTTGTCAGCCCTGAGGCCCTAGAT







CTTCTGGACAAACTTCTGCGATAC







GACCATCAACAGAGACTGACTGC







CAAAGAGGCCATGGAGCACCCAT







ACTTCTACCCTGTGGTGAAGGAG







CAGTCCCAGCCTTGTGCAGACAA







TGCTGTGCTTTCCAGTGGTCTCAC







GGCAGCACGATGAAGACTGGAAA







GCGACGGGT







(SEQ ID NO: 159)


 


7A9
AURKAIP1
NM_001127230.1;
FIG. 44
AARLGPSLECW
CGGCCGCCCGCCTTGGCCCGTCTC




NM_001127229.1;
(SEQ ID
AAGSAGPFTAH
TGGAGTGCTGGGCAGCCGGGTCT




NM_0017900.2
NO: 100)
RRPAQVGRPLSL
GCGGGCCCCTTTACAGCACATCG




(transcript

ARGPSWSWRRC
CCGGCCGGCCCAGGTAGGGCGGC




variants)

WSPGRCPSAPW
CTCTCTCCCTCGCAAGGGGGCCCA






RAGSRPAASCPD
GCTGGAGCTGGAGGAGATGCTGG






WIPGPQGLWLH
TCCCCAGGAAGATGTCCGTCAGC






RNPTSVRPAR
CCCCTGGAGAGCTGGCTCACGGC






(SEQ ID 
CCGCTGCTTCCTGCCCAGACTGGA






NO: 130)
TACCGGGACCGCAGGGACTGTGG







CTCCACCGCAATCCTACCAGTGTC







CGCCCAGCCAGATAGGGGAAGGG







GCCGAGCAGGGGGATGAAGGCGT







CGCGGATGCGCCTCAAATTCAGT







GCAAAAACGTGCTGAAGATCCGC







CGGCGGAAGATGAA







(SEQ ID NO: 160)


 


3C1
Chromo-
AC096741.3
FIG. 45
GKERENIRTNT
GGCAGGGAAGGGAGAACATTAGG



some 4
(Homo
(SEQ ID
(SEQ ID 
ACAAATACCTAATGCACGCCAGG




sapiens BAC
NO: 101)
NO: 131)
CCCTANTAATCGTAGATGATGGG




clone RP11-


TTGATGGGTGTAGCAAACCACCA




327017)


TGGCACATGTATATCTATGTAACA







AACCTGCACATTCTGTACATGTAT







CCCAGAACTTCAAGTAAAATTTTA







AAAAATTCAAAAAAAGTAATAGG







AAAAGGGGAAACATCCACGTGAG







CAGTCCAGTTTCCCAATCTGGAAC







TTGGAGCTGTTCACCTGGTGGGTG







TTTGTGACTATTCAGACACAGACA







ACAAAGGCTACTCCAGATTGAAG







TGCACTGCTTACTTTCAGTGACCT







CATAGAACTACTCAACATTGTTTT







TGGTGATTCCTGTGCTATGGTTTG







AATGGCTCCGCTCCAAAACTCAG







GTGTTGCCAATGNGATGGTATTA







AGAAGTAGGGCATTTAAAAAACA







ACAACAGGCCTGGCGCGGTGGCC







CACGCCTGTAATCCCAGCACTTTG







GGAGGCTAAGGCGGGCGGATCAC







CGGAGGTCAGGAATTCAAAACCA







GCCTGGCCAACATGGCGAAACCC







TGTCTCTACTAAAAATACAAAAA







TTAGCCAGGCATGGTTGCGGGCG







CCTGTAATCCCGGCTACTCGGGA







GGCTGAGGCAGGGGAATCCTTGA







ACCCGGGA







(SEQ ID NO: 161)


 


3C3
ARF6
NM_001663.3
FIG. 46
PKCRLQRQYTG
GAAATGTAGACTGCAAAGGCAGT





(SEQ ID
KGGVGFVYEGV
ATACAGGAAAAGGTGGAGTGGGT





NO: 102)
(SEQ ID 
TTTGTTTATGAGGGTGTCTGAAAA






NO: 132)
CTAAAATTGAGCGGGATATCATG







GTATAGTTGGACAGTATTGGTCCT







TCACACTTTGGCCATATTGTATAA







TGGAGCTTTTACCAAAGATGTATG







AGAAGTGTAAGACTATAAAAAAA







TGAACTATTCAAAGTAAAACTCTT







AACAAACATTTTACTTAAAGCAG







ATGCAAAAGGGTATTCTCATGTA







GGCTCCTGTTGGTGCAGAGGGAT







TTTTTTGATTTCAGGATACAACTA







AAGTACGAAGTTCTCAGTTTCACT







TTAGTAGAAAGAGCTCTAGAAAT







GAGGCTGATAAACACATCTAAGA







ACACTGGTTGCTTTCTAAAATTTC







CAAAGCTCCACCATAAATGTAAT







TTTTAGTGTTTCAAATGATTGCAT







TTTAAAGTATATAAATATGGGTTA







TCCAATATCAATGCTATAGTAACA







TCCTGAAACAAAACAAGCACAAA







GGTATAAATGCCTAAACTGGAGG







AAGCTTG







(SEQ ID NO: 162)


 


3D1
3′ UTR
AL135937.22
FIG. 47
QTQTHTSAPLKC
CTCAGACTCAAACACACACCTCC



region
(Human DNA
(SEQ ID
QPWSFVEARICH
GCTCCCTTGAAGTGCCAGCCCTGG



JAG1
sequence
NO: 103)
GSQLVRCPVQH
AGCTTTGTTGAGGCTCGCATCTGC




from clone

PSRIS
CACGGGAGTCAGCTAGTACGTTG




RP1-278022 on

(SEQ ID 
CCCAGTTCAACATCCATCCAGGAT




chromosome 20)

NO: 133)
TTCATAGGAACTTGAGAATCATTG







TTTTTGGCTTGAATCCTGGGTTTG







AGGTTTCTTCGTGTAGGAATCTGA







AAAAAGGATTTGGAAACGTTGTT







GTCTCTAATCCCAAAGTATGTATC







TGGGAGGCTGCCTTCGCCATCACC







CACCTAATAACTCAGG







(SEQ ID NO: 163)


 


5A5
Mito-
HQ113226.2
FIG. 48
PRLHQXKANYI
AGACTTCACCAGTCAAAGCGAAC



chondrion

(SEQ ID
YSIDPIT
TACATATACTCAATTGATCCAATA



sequence

NO: 104)
(SEQ ID 
ACTTGACCAACGGAACAAGTTAC






NO: 134)
CCTAGGGATAACAGCGCAATCCT







ATTCTAGAGTCCATATCAACAATA







GGGTTTACGACCTCGATGTTGGAT







CAGGACATCCCGATGGTGCAGCC







GCTATTAAAGGTTCGTTTGTTCAA







CGATTAAAGTCCTACGTGATCTGA







GTTCAGACCGGAGTAATCCAGGT







CGGTTTCTATCTACTTCAAATTCC







TCCCTGTACGAAAGGACAAGAGA







AATAAGGCCTACTTCACAAAGCG







CCTTCCCCCGTAAATGATATCATC







TCAAGCTT







(SEQ ID NO: 164)


 


3E1
Chromo-
AL135937.22
FIG. 49
P Q T T A P R
CTCGCTCAAACACACACCTCCGCT



some 20

(SEQ ID
R A R P R R S
CCCTTGAAGTGCCAGCCCTGGAG





NO: 105)
(SEQ ID
CTTTGTTGAGGCTCGCATCTGCCA






NO: 135)
CGGGAGTCAGCTAGTACGTTGCC







CAGTTCAACATCCATCCAGGATTT







CATAGGAACTTGAGAATCATTGTT







TTTGGCTTGAATCCTGGGTTTGAG







GTTTCTTCGTGTAGGAATCTGAAA







AAAGGATTTGGAAACGTTGTTGT







CTCTAATCCCAAAGTATGTATCTG







GGAGGCTGCCTTCGCCATCACCC







ACCTAATAACTCAGGC 







(SEQ ID NO: 165)


 


5A9
Chromo-
AL034375.23
FIG. 50
G T I S I V C
ATTGTTTGTTGTTGGGGGTGTCTT



some 6

(SEQ ID
C W G C L C Q
TGTCAGCATCTAGTACAGTGCCTG



clone

NO: 106)
H L V Q C L A
GCAGATGGATGCTCAATAAATAT



UTR


D G C S I N I
TGATTTAATGGGTTATGAGGGTGT



region


D L M G Y E G
TAATATAAAATTAGCATTTATTCA



(Minus


V N I K L A F
GCAACTACTATGAGTCAGCCACT



strand)


I Q Q L L
GGGCTAAGTGGCTTACATGTTAA






(SEQ ID
GAACCTCACAGAAGCCAGGTGTG






NO: 136)
GTGGCTCACGCCTGTAATCCCAGC







ACTTTGGGAGGCTGAAGCGGGCA







GATCACCTGAGGTCAGGAGTTTG







AGTCCAGGCTGGCCAACGTGGTG







AAACCCCATCTCTACTAAAAATA







CAAAAATTAGCCAGTTGTGGTGG







CAGGCGCCTGTAGTCCCAGCCAC







TCAGGAGGCTAAGGCAGGAGAAT







AGCTGGAACCCGGGAGGTGGAGA







TTGCAGTGAGCCAAGATTGCACC







ACTGCACTCCAGCCTGGGTGACA







GAGTGAGACTCTGTCTCCAAAAA







AAAAAGAAAAAGAAAAAGAACC







TCCAGCAACCTAGTAGGTGAGCC







CGGTTACTCTTGTTTTACAGGTGA







GAAAATTGAGCCCTAGAGAAATA







AAGTAACTTGCTTCAGGTCTCATG







GTTAAGGGGAACCTGGGCCCTAA







CAGTCCACTTCCTGTACCTTCAAC







CACGGTTCTACCGCCTCCGCTAGG







AAATGGCCCGAGGACATTCCTTA







GCTGGCTTCAGCTTGCTCTTTTTC







CCCTGCGGTCCACCCCTG 







(SEQ ID NO: 166)


 


5H2
MAPKKK5
NG_011965.1
FIG. 51
G M S H H A W
AGAGGGAGTATAGGGCTGTGCAC





(SEQ ID
P R P S F F N
AGAGACTATGATGGCCGTGCTAA





NO: 107)
T E Y F
GGTAAGAGTATTGATAATGTAAG






(SEQ ID NO:
CATACTTCCTCTATCAACAATAAT






137)
TGTTAACAGCTGCTTCAAGCACTT







GATATTACCACTAGTTGTTAACTG







AATCAAGCATGTGCTCCAAGTTC







ACATTAATGTGAATTGAACAGCA







TTGTGTACGTACGAGGAGCTTCAT







GCAAGTGTTATACACTGCACTCAC







AAGTATTATGATCTTACTAAGCAT







TAGAAATACTCTGTGTTAAAGAA







GCTTGGTCTAGGCCAAGCGTGGT







GGCTCATGCCT (SEQ ID NO: 167)


 


1H5
RAS p21
BC020761.1
FIG. 52
D R R P G S F
GATCGGAGGCCAGGGTCCTTTGT



Protein

(SEQ ID
V L S F L S Q 
ACTTTCATTTCTTAGCCAGATGAA



activator

NO: 108)
Met N V V T
TGTTGTCACCCATTTTAGGATTAT



(RASA1)


H F R I I A
TGCTATGTGTGGAGATTACTACAT






Met C G D Y
TGGTGGAAGACGTTTTTCTTCACT






Y I G G R R F
GTCAGACCTAATAGGTTATTACA






S S L S D L I
GTCATGTTTCTTGTTTGCTTAAAG






G Y Y S H V S
GAGAAAAATTACTTTACCCAGTT






C L L K G E K
GCACCACCAGAGCCAGTAGAAGA






L L Y P V A P
TAGAAGGCGTGTACGAGCTATTC






P E P V E D R
TACCTTACACAAAAGTACCAGAC






R R V R A I L
ACTGATGAAATAAGTTTCTTAAA






P Y T K V P D
AGGAGATATGTTCATTGTTCATAA






T D E I S F L
TGAATTAGAAGATGGATGGATGT






K G D Met F
GGGTTACAAATTTAAGAACAGAT






I V H N E L E
GAACAAGGCCTTATTGTTGAAGA






D G W Met W
CCTAGTAGAAGAGGTGGGCCGGG






V T N L R T D
AAGAAGATCCACATGAAGGAAAA






E Q G L I V E
ATATGGTTCCATGGGAAGATTTCC






D L V E E V G
AAACAGGAAGCTT






R E E D P H E
(SEQ ID NO: 168)






G K I W F H G







K I S K Q E A







(SEQ ID 







NO: 138)



 


18H9
Hsp90b
Ay359878.1
FIG. 53
YFAYLISEQNEE
TGAAGTGG CAGCAGAGGAACC CA





(SEQ ID
NKINHNTQHPIL
ATGCTGCAGTTCCTGATGAGATCC





NO: 109)
LSRVREGMGLD
CCCCTCTCGAGGGCGATGAGGAT






TLSLLPSTQGQE
GCGTCTCGCATGGAAGAAGTCGA






REKNTRHQQGE
TTAGGTTAGGAGTTCATAGTTGGA






PGGTGALEAAV
AAACTTGTGCCCTTGTATAGTGTC






GAHGDTIQGHK
CCCATGGGCTCCCACTGCAGCCTC






FSNYELLT (SEQ
GAGTGCCCCTGTCCCACCTGGCTC






ID NO: 139)
CCCCTGCTGGTGTCTAGTGTTTTT







TTCCCTCTCCTGTCCTTGTGTTGA







AGGCAGTAAACTAAGGGTGTCAA







GCCCCATTCCCTCTCTCACTCTTG







ACAGCAGGATTGGATGTTGTGTA







TTGTGGTTTATTTTATTTTCTTCAT







TTTGTTCTGAAATTAAGTATGCAA







AATAA (SEQ ID NO: 169)


 


4D7
ribosomal
NM_001010.2
FIG. 54
C I V D A N L
GTTGCATTGTGGATGCAAATCTGA



protein 

(SEQ ID
S V L N L V I
GCGTTCTCAACTTGGTTATTGTAA



S6

NO: 110)
V K K G E K D
AAAAAGGAGAGAAGGATATTCCT



(RPS6)


I P G L T D T
GGACTGACTGATACTACAGTGCC






T V P R R L G
TCGCCGCCTGGGCCCCAAAAGAG






P K R A S R I
CTAGCAGAATCCGCAAACTTTTCA






R K L F N L S
ATCTCTCTAAAGAAGATGATGTCC






K E D D V R Q
GCCAGTATGTTGTAAGAAAGCCC






Y V V R K P L
TTAAATAAAGAAGGTAAGAAACC






N K E G K K P
TAGGACCAAAGCACCCAAGATTC






R T K A P K I
AGCGTCTTGTTACTCCACGTGTCC






Q R L V T P R
TGCAGCACAAACGGCGGCGTATT






V L Q H K R R
GCTCTGAAGAAGCAGCGTACCAA






R I A L K K Q
GAAAAATAAAGAAGAGGCTGCAG






R T K K N K E
AATATGCTAAACTTTTGGCCAAG






E A A E Y A K 
AGAATGAAGGAGGCTAAGGAGA






L L A K R Met
AGCGCCAGGAACAAATTGCGAAG






K E A K E K R
AGACGCAGACTTTCCTCTCTGCGA






Q E Q I A K R
GCTTCTACTTCTAAGTCTGAATCC






R R L S S L R
AGTCAGAAATAAGATTTTTTGAGT






A S T S K S E 
AACAAATAAATAAGATCAGA






S S Q K
(SEQ ID NO: 170)






(SEQ ID 







NO: 140)



 


36C4
Homo
AC128709
FIG. 55
L I C I S L M
CCTGGGCAGTGATTAGGTCATAA



sapiens
(Homo
(SEQ ID
A N D V E H L
AGGTGGAGTCCTCATGGATGGGA



chromo-
sapiens 3
NO: 111)
F M F I C H L  
TTAGTGTCTTTATAAAAGAGACCT



some 3
BAC RP13-

S
TTGCCATGTGAGGTTACAGTGAG



genomic
616I3)

(SEQ ID
AAGACATCTGTCTATGAAGAAAG



contig


NO: 141)
TGGGCCCTCACCAAACACAGTCT







GCTGGCACTTTGCACTTCAACTCC







CCAGCTTCCAGAACTGTAAGGAA







TATAAGTCTGTTGTTGGTAAGCCA







CCCGGTCTATGATATTTTGTTATA







GCAGCCCAAACAGACTAAGACAG







GTGACAAATAAACATGAAAAGAT







GTTCAACATCATTAGCCATTAGGG







AAATGCAGATTAAAA 







(SEQ ID NO: 171)









An antibody, such as an autoantibody, to one or more of a protein, or a fragment of a protein, encoded by a gene such as listed in Tables 1, 2, 3 or 4, or a polypeptide encoded by a UTR sequence of a gene such as one listed in Tables 1, 2, 3 or 4, can be detected according to one or more methods described herein and used to characterize a cancer, such as prostate cancer. Many of the proteins may have a role in various cancers, including prostate cancer. For example, the human DCHS1 protein (protocadherin-16 precursor) is believed to be a calcium-dependent cell adhesion protein found in the cell membrane of fibroblast cells. Without being bound by theory, DCHS1 is a cadherin, a class of type-1 transmembrane proteins. Cadherins typically play important roles in cellular adhesion, for example, by binding cells expressing similar cadherins to each other. Structurally, DCHS1 is thought to contain 27 cadherin repeats (extracellular calcium ion-binding domains). DCHS1 expression has been associated with certain cancers, potentially playing a role in tumor adherence (see, e.g., Sjöblom, et. al. Science, (2006) 314:268-274).
Another of the proteins, CEP164 is believed to be a centrosomal protein which binds chromatin and plays a role in the DNA damage-activated signaling cascade. It is known to interact with ataxia telangiectasia mutated (ATM) and ATM/Rad3-related (ATR) kinases which phosphorylate CEP164 upon replication stress, ultraviolet radiation (UV), and ionizing radiation (IR). CEP164 also plays a role in cell cycle regulation, specifically at the G2/M checkpoint and in nuclear division (see, e.g., Sivasubramaniam et al., Genes & Dev. (2008); 22(5):687-600). As CEP 164 plays a role in genome stabilization, misregulation or mutation of this gene and/or protein can play a role in certain cancers.
In a further example, the human KBTBD6 (kelch repeat and BTB (POZ) domain containing 6) is a protein expressed in a wide variety of normal tissues. Its expression and/or misregulation has also been noted in multiple cancer types, including prostate, ovarian, kidney and lung tumors. The function of the protein is not currently known, however, the presence of the kelch repeat and BTB domain suggest that the protein is involved in protein-protein interactions and actin filament organization.
Certain ribosomal proteins, such as RPS19 and RPL34 have also been associated with certain cancers. RPS19 (ribosomal protein S19) encodes a ribosomal protein that is a component of the 40S subunit. Located in the cytoplasm as part of the ribosomal complex, mutations in this gene are associated with Diamond-Blackfan anemia, suggesting a non-ribosomal function for the protein in erythropoietic differentiation. RPS19 protein is also known to interact with fibroblast growth factor-2 (see, e.g., Soulet et al., Biochem. Biophys. Res. Commun. (2001); 289:591-596). Increased expression of RPS19 has been associated with some cancers, but the role of RPS19 in cancer development is unknown. RPL34 (60S Ribosomal protein L34) is a ribosomal protein that is a component of the 60S subunit and is located in the cytoplasm. Expression of the gene encoding the RPL34 protein is known to be regulated by c-MYC and has been shown to have increased expression in primary invasive and metastatic breast cancer cells and colorectal cancer cells (see, e.g., Zucchi et al., Proc. Nat'l Acad. Sci., (2004); 101:18147-18152; Sjöblom, et. al. Science, (2006) 314:268-274).
Certain nucleic acid-binding proteins, such as RMB6 and HEMK1 have also been associated with certain cancers when misregulated and/or mutated. RBM6 (RNA binding protein 6) is a cytosolic protein that binds to poly-G homopolymers in vitro, but its function in vivo is not currently known. The protein thought to be phosphorylated (potentially by ATM or ATR) in its active form. The gene encoding the protein, without being bound by theory, is located in a portion of the genome, modifications of which are associated with cancerous transformation, such as lung carcinomas. Additionally, translocations of the gene which result in aberrant fusion proteins have been reported to be associated with cancer cells (see, e.g., Gu et al., Blood, (2007); 110:323-333). The human HEMK1 (HEMK methyltransferase family protein 1) protein is an S-adenosylmethionine-dependent methyltransferase and is also thought to bind nucleic acids. HEMK1 is considered a tumor-suppressor, misregulation of which is associated with various cancers, including prostate cancer, pancreatic cancer and liver cancer (see, e.g., U.S. Pat. App. Pub. No. 2008/0213791).
Thus one or more polypeptide probes, such as a fragment of a protein encoded by a gene, or a polypeptide encoded by a sequence of a UTR region of a gene, such as a gene listed in Tables 1, 2, 3 or 4, can be used to detect one or more antibodies, such as autoantibodies, from a sample from a subject. In one embodiment, the polypeptide probe comprises a polypeptide sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof. In another embodiment, the polypeptide probe comprises a polypeptide sequence encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof. In yet another embodiment, the polypeptide probe comprises the full-length or a fragment of a protein that is encoded by SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or a fragment thereof.
In one embodiment, a polypeptide probe is a fragment of a protein encoded by CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, or Deaminase Domain, or may be a polypeptide encoded by a UTR sequence of the gene, such as the 5′ or 3′ UTR sequence of CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, or Deaminase Domain. In one embodiment, a polypeptide probe can be a fragment of a protein encoded by FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789. In one embodiment, a polypeptide probe comprises a peptide sequence, or fragment thereof, such as those listed in Tables 1, 2, 3, and 4. The polypeptide probe can comprise SEQ ID NO: 2, 5, 9, 11, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or a fragment thereof, or a fragment thereof. In another embodiment, the polypeptide probe comprises a polypeptide encoded by SEQ ID NO: 16, 19, 23, 25, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or a fragment thereof.
In another embodiment, a polypeptide probe is a fragment of a protein encoded by DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, or Hemk1, or may be a polypeptide encoded by a UTR sequence of the gene, such as the 5′ or 3′ UTR sequence of DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, or Hemk1. In one embodiment, a polypeptide probe can be a fragment of a protein encoded by eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789. In one embodiment, a polypeptide probe comprises a peptide sequence, or fragment thereof, such as those listed in Tables 1 and 2. The polypeptide probe can comprise SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or a fragment thereof. In another embodiment, the polypeptide probe comprises a polypeptide encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or a fragment thereof.
Antibody Profiling Panel
Also provided herein is an antibody profiling panel. A panel as provided herein can be used to analyze one or more antibodies to a plurality of polypeptide probes, such as one or more autoantibodies. A panel allows for the simultaneous analysis of multiple antibodies, such as autoantibodies, to a plurality of polypeptide probes correlating with carcinogenesis and/or metastasis. For example, a panel can include markers identified as correlating with cancerous tissue, metastatic cancer, localized cancer that is likely to metastasize, pre-cancerous tissue that is likely to become cancerous, and pre-cancerous tissue that is not likely to become cancerous. Depending on the subject, panels may be analyzed alone or in combination in order to provide the best possible diagnosis and/or prognosis.
In one embodiment, an antibody profiling panel can comprise a plurality of polypeptide probes, wherein one or more of the probes is capable of binding an antibody. In another embodiment an antibody profiling panel can comprise a plurality of probes, wherein one or more of the probes is capable of binding an antibody that targets a foreign antigen. In another embodiment an antibody profiling panel can comprise a plurality of probes, wherein each of the probes is capable of binding an autoantibody.
In one embodiment, an antibody profiling panel comprises 2-100 probes, 50-200 probes, 100-500 probes 200-750 probes, 200-1000 probes, 2-5,000 probes or 2-10,000 probes. In one embodiment, an antibody profiling panel comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 polypeptide probes. In another embodiment, an antibody profiling panel comprises at least about 50, 100, 150, 200, 250, 500, 750, 1000, 5000, 10,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 75,000, or 100,000 polypeptide probes. In one embodiment, the probes are polypeptide probes. In another embodiment, the probes are molecules that mimic an epitope bound by a particular antibody.
An antibody profiling panel can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 polypeptide probes, wherein the polypeptide probes are a fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, such as genes listed in Tables 1, 2, 3, or 4. In one embodiment, the polypeptide probe comprises a polypeptide sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof. In another embodiment, the polypeptide probe comprises a polypeptide sequence encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof. In yet another embodiment, the polypeptide probe comprises the full-length or a fragment of a protein that is encoded by SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or a fragment thereof.
In one embodiment, an antibody profiling panel comprises a plurality of polypeptide probes, wherein at least a subset of the polypeptide probes is a fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, or Deaminase Domain. In one embodiment, the polypeptide probe can comprise a fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789.
In another embodiment, an antibody profiling panel comprises a plurality of polypeptide probes, wherein at least a subset of the polypeptide probes is a fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, or Hemk1. In one embodiment, the polypeptide probe can comprise a fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789.
In one embodiment, an antibody profiling panel comprises a plurality of polypeptide probes, wherein at least a subset of the polypeptide probes is a peptide sequence, or fragment thereof, as listed in Tables 1, 2, 3, or 4. In one embodiment, an antibody profiling panel comprises a plurality of polypeptide probes, wherein at least a subset of the polypeptide probes comprises a polypeptide sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof. In another embodiment, an antibody profiling panel comprises a plurality of polypeptide probes, wherein at least a subset of the polypeptide probes comprises a polypeptide sequence encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof. In yet another embodiment, the polypeptide probe comprises the full-length or a fragment of a protein that is encoded by SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or a fragment thereof.
In another embodiment, an antibody profiling panel comprises a plurality of polypeptide probes, wherein at least a subset of the polypeptide probes comprises SEQ ID NO: 2, 5, 9, 11, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or a fragment thereof, or a fragment thereof. In another embodiment, an antibody profiling panel comprises a plurality of polypeptide probes, wherein at least a subset of the polypeptide probes is encoded by SEQ ID NO: 16, 19, 23, 25, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or a fragment thereof.
In one embodiment, an antibody profiling panel comprises a plurality of polypeptide probes, wherein at least a subset of the polypeptide probes comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or a fragment thereof. In another embodiment, an antibody profiling panel comprises a plurality of polypeptide probes, wherein at least a subset of the polypeptide probes is encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or a fragment thereof.
In one embodiment, an antibody profiling panel can also comprise one or more polypeptide probes of the protein PSA, or fragment of PSA, in combination with one or more of the polypeptide probes discussed herein.
In one embodiment, an antibody profiling panel can comprise polypeptide probes including a full-length protein or fragment of PSA and one or more polypeptide probes comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof. In another embodiment, an antibody profiling panel can comprise polypeptide probes including a full-length protein or fragment of PSA and one or more polypeptide probes comprising a polypeptide sequence encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof. In yet another embodiment, the polypeptide probe comprises the full-length or a fragment of a protein that is encoded by SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or a fragment thereof.
In another embodiment, an antibody profiling panel can comprise polypeptide probes including a full-length protein or fragment of PSA and a full-length protein encoded by a gene, fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, or Deaminase Domain. In yet another embodiment, an antibody profiling panel can comprise a plurality of polypeptide probes, wherein the probes include a full-length protein or fragment of PSA and a full-length protein encoded by a gene, fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789.
In another embodiment, an antibody profiling panel can comprise polypeptide probes including a full-length protein or fragment of PSA and a full-length protein encoded by a gene, fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, or Hemk1. In yet another embodiment, an antibody profiling panel can comprise a plurality of polypeptide probes, wherein the probes include a full-length protein or fragment of PSA and a full-length protein encoded by a gene, fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789.
In another embodiment, an autoantibody profiling panel can comprise a plurality of polypeptide probes, wherein the probes includes a full-length protein or fragment of PSA and one or probes comprising a peptide sequence, or fragment thereof, as listed in Tables 1, 2, 3 and 4. In one embodiment, an autoantibody profiling panel can comprise a plurality of polypeptide probes, wherein the probes includes a full-length protein or fragment of PSA and one or more probes comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof. In another embodiment, an autoantibody profiling panel can comprise a plurality of polypeptide probes, wherein the probes includes a full-length protein or fragment of PSA and one or more probes comprising a polypeptide sequence encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof. In yet another embodiment, an autoantibody profiling panel can comprise a plurality of polypeptide probes, wherein the probes includes a full-length protein or fragment of PSA and one or more probes comprising the full-length or a fragment of a protein that is encoded by SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or a fragment thereof.
In another embodiment, an autoantibody profiling panel can comprise a plurality of polypeptide probes, wherein the probes includes a full-length protein or fragment of PSA and one or more probes comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or a fragment thereof.
In another embodiment, an autoantibody profiling panel can comprise a plurality of polypeptide probes, wherein the probes includes a full-length protein or fragment of PSA and one or more probes comprising SEQ ID NO: 2, 5, 9, 11, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or a fragment thereof, or a fragment thereof; or a polypeptide sequence encoded by a sequence selected from SEQ ID NOs. 16, 19, 23, 25, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or a fragment thereof.
In one embodiment, a PSA polypeptide probe can be combined with any two or more of the polypeptide probes described herein, such as a polypeptide probe derived from a protein encoded by a gene, fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, Deaminase Domain, FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789.
In another embodiment, a PSA polypeptide probe can be combined with any two or more of the polypeptide probes described herein, such as a polypeptide probe derived from a protein encoded by a gene, fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, Hemk1, eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789.
In yet another embodiment, a PSA polypeptide probe can be combined with at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of polypeptide probes disclosed herein, such as listed in Tables 1, 2, 3, and 4. In one embodiment, a polypeptide probe comprises SEQ ID NO: 2, 5, 9, 11, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or a fragment thereof, or a fragment thereof. In one embodiment, a polypeptide probe comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or a fragment thereof. In another embodiment, a polypeptide probe comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof.
In another embodiment, a polypeptide probe comprises a polypeptide encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or a fragment thereof. In another embodiment, a polypeptide probe comprises a polypeptide encoded by SEQ ID NO: 16, 19, 23, 25, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or a fragment thereof. In yet another embodiment a polypeptide probe comprises a polypeptide encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof.
In one embodiment, a polypeptide probe disclosed herein is attached to a substrate (e.g., glass slide chip or nanowell chip). A polypeptide probe can be directly or indirectly attached to the substrate. In one embodiment, a polypeptide probe is attached to a substrate via a phage. The substrate can be any physically separable solid to which a polypeptide probe can be directly or indirectly attached including, but not limited to, surfaces provided by microarrays and wells, particles such as beads, columns, optical fibers, wipes, glass and modified or functionalized glass, quartz, mica, diazotized membranes (paper or nylon), polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductive materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylics, polystyrene, copolymers of styrene or other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TEFLONTM, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, ceramics, conducting polymers (including polymers such as polypyrole and polyindole); micro or nanostructured surfaces such as nucleic acid tiling arrays, nanotube, nanowire, or nanoparticulate decorated surfaces; or porous surfaces or gels such as methacrylates, acrylamides, sugar polymers, cellulose, silicates, or other fibrous or stranded polymers.
The polypeptide probe can bound to a planar surface or to a particle, such as a bead or microsphere. In one embodiment, the polypeptide probe is attached to a bead. The bead can be a polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene, polyisoprene, polyurethane, polyvinyl acetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polyglycidylmethacrylate, polymethylmethacrylate, or copolymers, blends, composites, or combination thereof. The bead can have a diameter of between about 1 nm-1000 μm, 1 nm-500 μm, 5 nm-500 μm, or 10 nm-100 μm. In one embodiment, the bead has a diameter of between about 10 nm and 100 μm. In yet another embodiment, the bead has a diameter of less than about 1000 μm, 500 μm, 400 μm, 300 μm, 200 μm, or 100 μm.
In one embodiment, the bead is labeled or stained with more than one dye, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different dyes. In one embodiment, the bead is labeled or stained with two dyes. In another embodiment, the two dyes are hydrophobic. In another embodiment, the two dyes are fluorescent dyes, such as squaric acid-based dyes. In yet another embodiment, the squaric acid-based dyes are selected from cyclobutenedione derivatives, symmetrical and unsymmetrical squaraines, substituted cephalosporin compounds, fluorinated squaraine compositions, alkylalkoxy squaraines, or squarylium compounds. In another embodiment, the squaric acid-based dyes are selected from a red fluorescent dye and an orange fluorescent dye, such as the red fluorescent dye comprising 1,3-bis(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)methyl]-2,4-dihydro xycyclobutenediylium, bis(inner salt) and the orange fluorescent dye comprising 2-(3,5-dimethylpyrrol-2-yl)-4-(3,5-dimethyl-2H-pyrrol-2-ylidene)-3-hydroxy-2-cyclobuten-1-one.
In one embodiment, the substrate is coated using passive or chemically-derivatized coatings with any number of materials, including polymers, such as dextrans, acrylamides, gelatins or agarose. Such coatings can facilitate the use of the array with a biological sample.
Cancer Screening
A presence of an immune response to a specific protein expressed in cancerous cells can be indicative of a presence of cancer. Accordingly, the present invention provides a method (e.g., diagnostic or screening method) for detecting a presence of an antibody, such as an autoantibody, to a tumor or tumor-associated antigen. In one embodiment, the presence of an antibody in cancerous but not cancerous cells is indicative of the presence of cancer. In one embodiment, the antibody is an antibody to a tumor antigen.
A method or composition disclosed herein can find utility in the diagnosis, screening, or characterization of a cancer. In one embodiment, a presence of an antibody, such as an autoantibody, to a specific protein can be indicative of a cancer. In another embodiment, detection of an antibody in a sample, such as an autoantibody, can be indicative of a specific stage or sub-type of the same cancer. The information obtained by detecting an antibody as described herein can be used to determine a prognosis or theranosis, wherein an appropriate course of treatment can be determined. In another embodiment, a subject with a specific antibody or stage of cancer can respond differently to a given treatment than individuals lacking the antibody. The information obtained from a method disclosed herein can thus provide for the personalization of diagnosis and treatment.
In one embodiment, a cancer is characterized by detecting the level or presence or absence of an antibody, such as an autoantibody, in a sample. The cancer can be, but is not limited to, breast cancer, ovarian cancer, lung cancer, colon cancer, hyperplastic polyp, adenoma, colorectal cancer, high grade dysplasia, low grade dysplasia, prostatic hyperplasia, prostate cancer, melanoma, pancreatic cancer, brain cancer (such as a glioblastoma), hematological malignancy, hepatocellular carcinoma, cervical cancer, endometrial cancer, head and neck cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal cell carcinoma (RCC) or gastric cancer. The colorectal cancer can be CRC Dukes B or Dukes C-D. The hematological malignancy can be B-Cell Chronic Lymphocytic Leukemia, B-Cell Lymphoma-DLBCL, B-Cell Lymphoma-DLBCL-germinal center-like, B-Cell Lymphoma-DLBCL-activated B-cell-like, and Burkitt's lymphoma. The cancer can also be a premalignant condition, such as Barrett's Esophagus.
In one embodiment, a method for screening or characterizing a prostate cancer is provided. In one embodiment, the method can comprise detecting in a sample obtained from a subject a presence and/or level of one or more autoantibodies to one or more polypeptide probes comprising a polypeptide probe is a fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, Deaminase Domain, FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789. A polypeptide probe can also comprise a polypeptide sequence, or a fragment thereof, selected from Table 1, 2, 3 and 4, such as a polypeptide probe comprising polypeptide probe comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or a fragment thereof, or a polypeptide probe comprising a polypeptide encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or a fragment thereof. A polypeptide probe can also comprise SEQ ID NO: 12, 5, 9, 11, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or a fragment thereof, or a fragment thereof, or a polypeptide encoded by SEQ ID NO: 16, 19, 23, 25, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or a fragment thereof.
In one embodiment, the method can comprise detecting in a sample obtained from a subject a presence and/or level of one or more autoantibodies to one or more polypeptide probes comprising a polypeptide probe is a fragment of a protein encoded by a gene, or a fragment encoded by a sequence of a UTR region of a gene, wherein the gene is DCHS1, CEP164, KBTBD6, RPS19, RPL34, SFRS14, RNA binding protein 6, Hemk1, eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789. A polypeptide probe can also comprise a polypeptide sequence, or a fragment thereof, selected from Table 1 or Table 2, such as a polypeptide probe comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or a fragment thereof, or a polypeptide probe encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or a fragment thereof.
In yet another embodiment, the method can comprise detecting in a sample obtained from a subject a presence and/or level of one or more autoantibodies to one or more polypeptide probes comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof, or a fragment thereof; or polypeptide probe encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof; or polypeptide probe comprising full-length or a fragment of a protein that is encoded by SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or a fragment thereof.
Depending on the results, a cancer (or absence of cancer) can be characterized. For example, in a sample from a subject a presence or level of DCHS1, CEP164 and/or RPS19 autoantibodies is detected, indicating a presence of prostate cancer in the subject. Alternately, a method further comprises detecting a presence or level of one or more autoantibodies to one or more polypeptide probe comprising a fragment of eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789. The fragment of a protein encoded by eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789 can comprise a polypeptide sequence selected from Table 2.
A method disclosed herein can comprise detecting a plurality of antibodies, such as through the detection of binding of one or more antibodies that bind to a plurality of polypeptide probes. In one embodiment, the antibodies are autoantibodies. In another embodiment, the antibodies are antibodies to foreign antigens. In one embodiment, the method comprises detecting in a sample one or more antibodies that binds to a panel of polypeptide probes, wherein the panel comprises 2-100 probes, 50-200 probes, 100-500 probes 200-750 probes, 200-1000 probes, 2-5,000 probes or 2-10,000 probes. In another embodiment, the panel of polypeptide probes comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 polypeptide probes. In another embodiment, the panel comprises at least about 50, 100, 150, 200, 250, 500, 750, 1000, 5000, 10,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 75,000, or 100,000 polypeptide probes. In one embodiment, the panels comprises a plurality of polypeptide probes, wherein a subset of the probes comprise fragments of the same full-length protein, such that autoantibodies to different epitopes bind to the different probes and indicate a presence of an immune response, or antibody, to the full-length protein.
A panel comprising multiple polypeptide probes allow for the simultaneous analysis of multiple markers correlating with carcinogenesis and/or metastasis. In one embodiment, a panel includes markers identified as correlating with cancerous tissue, metastatic cancer, localized cancer that is likely to metastasize, pre-cancerous tissue that is likely to become cancerous, pre-cancerous tissue that is not likely to become cancerous, or any combination thereof. Depending on the subject, a panel can be analyzed alone or in combination in order to provide a diagnosis, prognosis, or theranosis. One or more markers for inclusion on a panel can be selected by screening for their diagnostic, prognostic, or theranostic value.
Any of the proteins listed in Tables 1, 2, 3 or 4, or proteins encoded by the genes listed in Tables 1, 2, 3 or 4, in any combination, can be utilized to detect a presence of an antibody, such as an autoantibody, in a subject. In one embodiment, the protein is encoded SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or a fragment thereof.
In one embodiment, detection of an autoantibody to a protein encoded by a gene, a fragment encoded by a sequence of a UTR region of a gene, or fragment of a protein encoded by a gene, wherein the gene is CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, Deaminase Domain, FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789, or any combination thereof, is indicative of a presence of prostate cancer in a subject. In another embodiment, any combination of two or more proteins (e.g., cancer markers) or fragments thereof is used to detect one or more autoantibodies (e.g., a panel consisting of one or more full-length or fragments of the polypeptides listed in Tables 1, 2, 3, and/or 4).
In another embodiment, detection of an autoantibody to a protein encoded by a gene, a fragment encoded by a sequence of a UTR region of a gene, or fragment of a protein encoded by a gene, wherein the gene is CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, Hemk1, eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, LOC388789, or any combination thereof, is indicative of a presence of prostate cancer in a subject. In another embodiment, any combination of two or more proteins (e.g., cancer markers) or fragments thereof is used to detect one or more autoantibodies (e.g., a panel consisting of one or more full-length or fragments of the polypeptides listed in Tables 1 and 2).
In one embodiment, the method comprises detecting one or more antibodies that bind to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 polypeptide probes, wherein the polypeptide probes are full-length or fragments of proteins encoded by the genes listed in Tables 1, 2, 3, and/or 4, or polypeptides encoded by the UTR sequence of the gene. In one embodiment, the antibody profiling panel comprises a plurality of polypeptide probes, wherein one or more polypeptide probes is a protein or fragment of a protein encoded by CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, Deaminase Domain, FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789, or any combination thereof. In another embodiment, the antibody profiling panel comprises a plurality of polypeptide probes, wherein one or more polypeptide probes is a protein or fragment of a protein encoded by DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, Hemk1, eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, LOC388789, or any combination thereof.
The cancer can be characterized with increased accuracy, such as with increased specificity, sensitivity, or both. The sensitivity can be determined by: (number of true positives)/(number of true positives+number of false negatives), whereas the specificity can be determined by: (number of true negatives)/(number of true negatives+number of false positives).
In one embodiment, the cancer can be characterized (e.g., detected, prognosed, etc.) with at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sensitivity. In another embodiment, the cancer can be characterized (e.g., detected, prognosed, etc.) with at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% specificity.
Specificity or sensitivity of detection can be altered by altering the polypeptide probe make-up of a panel. In one embodiment, sensitivity of a diagnostic, prognostic, or theranosstic assay (e.g., an antibody detection assay, such as an autoantibody detection assay) can be increased by increasing the number of probes, increasing the diversity of probes (e.g, utilizing probes comprising distinct epitopes from the same and/or different markers), or tailoring the probes to a particular subject or cancer to be diagnosed/prognosed. Furthermore, the confidence level for determining the specificity, sensitivity, or both, may be with at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% confidence.
A method and system disclosed herein can also comprise detecting a plurality of antibodies, such as through the detection of antibodies binding to a plurality of polypeptide probes, and characterizing or screening for a cancer with increased or greater specificity as compared to a characterization based on detection of antibodies that bind to less than the plurality of polypeptide probes. In one embodiment, the antibodies are autoantibodies. In another embodiment, the antibodies are to foreign antigens.
Two or more polypeptide probes can be used to diagnose a particular cancer. For example, a cancer can be diagnosed by measuring the binding of autoantibodies to two polypeptide probe. The number of polypeptide useful for diagnosing a cancer includes, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 polypeptide probes. In another embodiment, prostate cancer is diagnosed with 5 or more polypeptide probes. In one embodiment, prostate cancer is diagnosed with 5 polypeptide probes, which provides a diagnosis that has a higher sensitivity as compared to using less than the 5 polypeptide probes. In another embodiment, prostate cancer is diagnosed with 10 or more polypeptide probes. In another embodiment, a prostate cancer is diagnosed with 10 polypeptide probes, which provides a diagnosis that has a higher specificity as compared to using less than the 10 polypeptide probes.
Antibody Detection
The level, presence or absence of an antibody can be determined by detecting the binding of one or more autoantibodies to a polypeptide probe. Detection of an antibody can be either quantitative or qualitative. For quantitative assays, the amount of antibody detected can be compared to a control or reference to determine whether an antibody is overexpressed or underexpressed in a sample. For example, the control or reference can be a normal sample or a sample from a known disease state, such as a cancer sample.
Antibody binding to a polypeptide probe can be detected by techniques known in the art, such as, but not limited to, radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays. Any of the assays used can be quantitative or qualitative, as desired.
Detection of an antibody bound to a polypeptide probe can be detected using labeling technology. For example, one or more antibodies in a sample collected from a subject to be tested can be directly labeled (e.g., with a fluorescent or radioactive label) and exposed to a polypeptide probe or probe panel. Detection of a signal from the interaction can be achieved using methodology appropriate to the type of label used (e.g., fluorescent microscopy can be used to detect binding of a fluorescently labeled autoantibody to a polypeptide probe). In one embodiment, an autoantibody is detected by detecting binding of a labeled secondary antibody or other antibody-binding reagent which specifically binds to the antibody bound to the polypeptide probe (e.g., a “sandwich immunoassay”). Many methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. In one embodiment, the immunoassay described in U.S. Pat. Nos. 5,599,677, 5,672,480, or both, each of which is herein incorporated by reference, is used.
In one embodiment, automation is utilized to detect binding of one or more autoantibodies to a polypeptide probe or probe panels. Methods for the automation of immunoassays include those described in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is herein incorporated by reference. Analysis and/or presentation of results can also be automated. In one embodiment, a computer with software that analyzes raw data and generates a prognosis, diagnosis, or theranosis based on the level, presence or absence of antibody binding to one or more polypeptide probes is used. A computer-based analysis program can be used to translate the raw data generated by the detection assay (e.g., a presence, absence, or amount of antibody binding to one or more polypeptide probes) into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. In one embodiment, the data is transmitted over a network. In another embodiment, the data is accessible by a clinician.
Any method capable of receiving, processing, and transmitting the information to and from a laboratory conducting the assay, medical personnel, and a subject can be used. In one embodiment, a sample (e.g., a biopsy or a serum or urine sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. In one embodiment, the sample comprises a tissue or other biological sample and the subject visits a medical center to have the sample obtained and sent to the profiling center. In another embodiment, a subject collects the sample themself (e.g., a buccal swab) and directly sends it to a profiling center. In another embodiment, the sample comprises previously determined biological information. The information can be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication system). Upon being received by the profiling service, a sample can be processed and a profile produced (i.e., antibody level, presence or absence of antibody). A profile generated can be specific for the diagnostic, prognostic, or theranostic information desired for a subject. In one embodiment, a sample from a subject is analyzed for a presence or expression level of one or more antibodies to one or more proteins encoded by a gene, fragment of one or more proteins encoded by a gene, or fragment encoded by a UTR region of a gene, wherein the gene is CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, Deaminase Domain, FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789. In one embodiment, the antibodies are autoantibodies. In another embodiment, a sample from a subject is analyzed for a presence or expression level of one or more antibodies to one or more proteins encoded by a gene, fragment of one or more proteins encoded by a gene, or fragment encoded by a UTR region of a gene, wherein the gene is DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, Hemk1, eIF4G1, BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789. In one embodiment, the antibodies are autoantibodies.
Profile data can be prepared in a format suitable for interpretation by a treating clinician. In one embodiment, rather than providing raw expression data, the prepared format represents a diagnosis, screening or risk assessment (e.g., likelihood of metastasis or PSA failure or the development of high prostate specific antigen levels in a patient following prostate cancer therapy (e.g., surgery)) for the subject, along with recommendations for particular treatment options. The data can be displayed to the clinician by any suitable method. In one embodiment, the profiling service generates a report that is printed for the clinician (e.g., at the point of care). In another embodiment, the report is displayed to the clinician on a computer monitor.
In one embodiment, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis. In one embodiment, further analysis comprises converting the raw data to information useful for a clinician or subject, such as a patient. The central processing facility can provide the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can also control the fate of the data following treatment of a subject. In one embodiment, using an electronic communication system, the central facility provides data to the clinician, the subject, researchers, or any other individual. In one embodiment, a subject is able to directly access the data using the electronic communication system. In another embodiment, a subject chooses further intervention or counseling based on the result. In one embodiment, the data is used for research use. The data can be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease.
Antibody Test
The detection of one or more antibodies from a sample, such as described herein, can be used in conjunction with one or more other tests used for detecting or screening for cancer. The antibody detection can be used prior to, concurrent with, or subsequent to one or more other tests. In one embodiment, a genetic test for a mutation or expression level of one or more genes can be used in conjunction with determining the antibody profile of a subject.
Antibody detection can provide a non-invasive, inexpensive means for detecting or screening for a cancer. Thus, in one embodiment, the detection of a level, presence or absence of one or more antibodies can be used to determine whether a second sample or additional analysis of a sample from a subject is to be performed. In one embodiment, after detecting an expression level of one or more antibodies of sample obtained from subject to one or more polypeptide probes comprising a fragment of a protein encoded by, or a polypeptide encoded by a UTR sequence of, CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, Deaminase Domain, FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789, a biopsy can be recommended for the subject. In another embodiment, after detecting an expression level of one or more antibodies of sample obtained from subject to one or more polypeptide probes comprising a fragment of a protein encoded by, or a polypeptide encoded by a UTR sequence of, DCHS1, CEP164, KBTBD6, RPS19, RPL34, SFRS14, RNA binding protein 6, Hemk1, eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789, a biopsy can be recommended for the subject.
In another embodiment, an expression level for one or more antibodies from a subject can be detected, and based on the expression level of the one or more antibodies, the subject can be identified as suspected of having cancer. In one embodiment, the subject is characterized as having a high probability or likelihood of having cancer. Based on the detection or expression level of the one or more antibodies, a recommendation that a biopsy be obtained can be made for the subject. In another embodiment, if there is a lack of detection or expression of the one or more antibodies, further analysis is not recommended and a biopsy not be obtained. (see for example, FIG. 1, “Autoantibody Test I”)
In another embodiment, prior to detecting one or more antibodies from a subject, the subject is suspected of having cancer. The subject can have had a genetic test for a mutation or gene expression analysis, image analysis (such as magnetic resonance imaging (MRI), positron emission tomography (PET) scan, computerized tomography (CT) scan, nuclear magnetic resonance (NMR)), or biopsy, and have inconclusive or uncertain results. Thus, prior to further analysis and treatment for a suspected cancer, the subject can seek further verification of their likelihood of having a cancer, or their diagnosis, prognosis, or theranosis of a cancer.
In one embodiment, an antibody profiling panel described herein can be used in conjunction with a separate test which determines a presence or level of PSA (e.g., a serum PSA test). In one embodiment, the panels is utilized to diagnose or prognose a presence of a cancer (e.g., prostate cancer) in a subject. In one embodiment, a subject is suspected of having prostate cancer based on their PSA level, age, or both. A subject can be male and over 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 years of age. In another embodiment, the subject is between 30-80, 40-75, 45-75, or 50-75 years of age. In another embodiment, the subject had a PSA blood test, digital rectal exam, or both. In yet another embodiment, the subject may have a PSA level of at least about 1.0, 1.5, 2.0, 2.5, or 4.0 ng/ml. The subject can have a PSA level of between about 1.0-15 ng/ml, 2.0-15 ng/ml, or 2.5-10 ng/ml.
In one embodiment, a biological sample from a subject, such as a subject with a PSA level greater than about 2.5 ng/ml, is contacted with one or more probes for an antibody, such as one or more probes for an autoantibody. Based on the expression level of the antibody, a biopsy for the subject can be recommended (see for example FIG. 1, “Autoantibody Test I”). The antibody test can comprise detecting one or more antibodies in a sample that bind to a polypeptide probe as described herein. In another embodiment, the antibody test is an autoantibody test.
In one embodiment, the antibody binds a polypeptide probe comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or a fragment thereof. In another embodiment, the antibody binds a polypeptide probe comprising a polypeptide sequence encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or a fragment thereof. In yet another embodiment, the antibody binds a polypeptide probe comprising full-length or a fragment of a protein that is encoded by SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or a fragment thereof.
In one embodiment, the antibody binds a polypeptide probe comprising a full-length or fragment of a protein encoded by, or a polypeptide encoded by a CEP164, RPL34, BRMSL1, NKX3-1, RPSA, Cytochrome C oxidase 5 Subunit, UTR-region of chromosome 11, MAPKKK9, cDNA clone XR_113641.1, PSA, H2aa4, UBE2I, TIMP2, WDR77, Deaminase Domain, FAM53B, 5′UTR BMI1, RP3-323M22, or LOC388789. In one embodiment, a polypeptide probe comprises SEQ ID NO: 2, 5, 9, 11, 14, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or a fragment thereof, or a fragment thereof. In another embodiment, a polypeptide probe comprises a polypeptide encoded by SEQ ID NO: 16, 19, 23, 25, 28, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or a fragment thereof.
In another embodiment, the antibody binds a polypeptide probe comprising a full-length or fragment of a protein encoded by, or a polypeptide encoded by a UTR of, DCHS1, CEP164, KBTBD6, RPS19, RPL34, RNA binding protein 6, Hemk1, eIF4G1, 5′UTR BMI1, BRD2, RP3-323M22, SFRS14, or LOC388789. In one embodiment, a polypeptide probe comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or a fragment thereof. In another embodiment, a polypeptide probe comprises a polypeptide encoded by SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or a fragment thereof.
If a biopsy is recommended and the biopsy is positive for a cancer such as prostate cancer, a biological sample obtained from the subject can be contacted with one or more probes for an antibody, which can be the same or different, as those used in deciding whether to obtain a biopsy. Based on the expression level of antibodies in the sample, a prognosis for the cancer can be provided. (see for example, FIG. 1, “Autoantibody Test II”)
Thus, in one embodiment, a method of characterizing or screening for a cancer from a subject with a positive biopsy result is provided. In another embodiment, the subject has not yet provided a sample for detecting one or more antibodies. In yet another embodiment, the subject has provided an initial sample for detecting one or more antibodies and detection of the one or more antibodies is used in deciding whether a biopsy is obtained. Furthermore, in one embodiment, detection of one or more antibodies is used for a diagnosis, prognosis or theranosis of a cancer, such as prostate cancer. In one embodiment, the method comprises detecting an expression level for one or more antibodies, wherein the expression level of the one or more antibodies is indicative of the presence, absence, or stage of the cancer. In another embodiment, the indication is whether the cancer is aggressive or indolent.
In one embodiment, a cancer is classified based on the detection of one or more antibodies to one or more polypeptide probes disclosed herein. In one embodiment, the cancer is classified as aggressive or malignant. In another embodiment, the cancer is classified as indolent or benign. Furthermore, after classification, detection of one or more antibodies from a sample from the subject can be used to select a treatment or therapeutic for the cancer.
The present disclosure is not limited to the embodiments described above, but is capable of modification within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the present disclosure described herein.
EXAMPLES
Example 1
Probe Selection
Construction of T7 Phage Display Prostate Cancer cDNA Library
mRNA was isolated from total RNA following Novogen's Straight A's mRNA isolation protocol. OrientExpression cDNA synthesis and cloning system were used for the construction of T7 phage prostate cancer cDNA libraries.
To eliminate the 3′ bias inherent in oilgo(dT)-primed libraries, two libraries were constructed using directional oligo(dT) primer and random primer in parallel. After amplification, these two libraries were combined in same amount of titer.
Enrichment of Cancer Specific T7 Phage Library.
Protein A/G agarose beads (Pierce Biotechnology, Rockford, Ill.) were used to purify IgGs from the serum of prostate cancer patients. To enhance the selection of epitopes binding to IgGs specifically associated with prostate cancer, a dual procedure was performed.
First, a pre-clearing step was used to remove nonspecific clones by pre-absorbing the phage epitope libraries onto purified IgGs from normal serum pool from 10 control men. Next, the pre-cleared phage libraries were selected onto the pool of IgGs purified from the serum of 6 localized prostate cancer patients. In essence protein-A/G agarose beads provide a purification of the serum of IgGs. Fifty μl protein-A/G agarose beads were placed into 1.5 ml eppendorf tube and washed two times with 1× PBS. Washed beads were blocked with 4% nonfat milk at 4° C. for 1 hr. The beads were then incubated at 4° C. with 15 μl of pooled control sera at 1:30 dilution with 4% nonfat milk. After at least 2 hrs of incubation, the beads were washed three times with 1× PBS and then incubated with phage library ( ̃1010 phage particles) at 4° C. for at least 2 hrs. The mixture was centrifuged at 3000 rpm for 2 min. The beads with unspecifically bounded phage particles were discarded and the supernatant was collected for further immunoscreening.
Fifty μl fresh protein-A/G agarose beads were washed and blocked as same as above. The beads were then incubated at 4° C. for 3 hrs with 500 ml of PBS containing 15 ml patient sera pool at a 1:30 dilution. This amount of serum provides a three-fold molar excess of IgG to calculated number of protein-A/G binding capacity. The beads were washed three times with 1× PBS and then incubated with phage library supernatant from above allowed to react with the antibodies on the beads at 4° C. overnight. The mixture was centrifuged at 3000 rpm for 2 min and supernatant was discarded. The beads were then washed three times with 1× PBS.
To elute the bound phage 100 ml 1% SDS was used to strongly break up the antibody-antigen reaction without disrupting the T7 phage particles. The mixture of phage and elution buffer was incubated at room temperature for 10 min. The bound phages were removed from the beads by centrifugation at 8000 rpm for 8 min. Eluted phages were transferred to 10 ml BLT 5403 bacterial cells with OD600=0.6 ̃0.8 for amplification. Four or five cycles of affinity selections and biopanning were carried out with amplification of phage particles after each biopanning.
High Throughput Epitope Detection Using Phage Microarrays.
Random phage colonies were picked up and amplified in 96-well plates. Fresh phage lysates were spotted onto on FASTTM nitrocellulose coated glass slides (Schleicher & Schuell, Keene, N.H.). Extra T7 empty phage spots were spotted in quadruplicate as negative reference for normalizing the signal value from different slides. The arrays were dried overnight at room temperature. Before processing with serum, the arrays were rinsed briefly in a 4% nonfat milk/PBS with 0.1% tween-20 to remove unbound phage, then transferred immediately to 4% nonfat milk/PBS as a blocking solution for 1 hr at room temperature. Without allowing the array to dry, 2 ml of PBS containing human serum and T7-tag antibody (Novagen) at a dilution of 1:500 and 1:5000 respectively was applied to the surface in a screw-top slide hybridization tube.
The arrays were incubated at room temperature for 1 hour, and then washed gently three times in PBS/0.1% Tween-20 solution 10 min each. All washes were performed at room temperature. After washing, the arrays were incubated with 2 ml of PBS containing Cy3-labeled goat anti-mouse antibody and Cy5-labeled goat anti-human antibody (Jackson ImmunoResearch) at a dilution of 1:5,000 for both for 1 hr in the dark. Three washes were performed using PBS/0.1% Tween-20 solution with 10 min each. The arrays were then dried using a stream of compressed air and scanned using 532 nm and 635 nm lasers (Axon Laboratories).
Building Predictor and Validation of Biomarker Profile.
The arrays were quantified using GenePix software (Axon Laboratories). Raw ratios of each array were subtracted by median of ratios of the negative control spots with the observation that the signal for negative T7 empty phage on each chip correlates very well with the signal intensity for whole array. Then Z-transformation was applied to clones so that the mean of each clone is zero across arrays and the standard deviation is 1. Due to the fact a presence of antibodies specific to cancer was tested, epitopes with high reactivity in controls and low reactivity in patients were not expected. A GA/KNN algorithm, a machine learning language, was employed to calibrate the system. Briefly, the data set was randomly separated into a training set and a test set. In the training set, genetic algorithm (GA) was used to select optimized solutions (a subset of clones here) which had good fitness. The fitness was assessed by its ability to classify the training samples using the k-nearest neighbor (KNN) analysis (k=3 here). The fitness score was defined as the number of correctly classified training samples divided by the total number of training samples. The fitness score was specified to be equal or greater than 0.95. After getting 4000 optimized solutions, clones were ranked by their frequency in the solutions and top genes were used to predict the test samples. This cycle of sample partition, solution searching, clone ranking and test sample prediction was repeated 10 times and high-ranked clones were selected as optimized classifier.

## Claims

1. A method for screening for prostate cancer, the method comprising:
(a) contacting a biological fluid sample obtained from the subject with an antibody profiling panel comprising a plurality of polypeptide probes comprising a polypeptide or epitope fragment of:
(i) amino acid sequence SEQ ID NO: 13 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 27,
(ii) amino acid sequence SEQ ID NO: 132 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 162,
(iii) amino acid sequence SEQ ID NO: 2 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 16,
(iv) amino acid sequence SEQ ID NO: 141 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 171,
(v) amino acid sequence SEQ ID NO: 129 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 159,
(vi) amino acid sequence SEQ ID NO: 118 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 148,
(vii) amino acid sequence SEQ ID NO: 122 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 152, and
(viii) amino acid sequence SEQ ID NO: 130 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 160,


wherein each of the probes in the plurality of polypeptide probes is attached to a substrate and is capable of being specifically bound by an autoantibody from a sample obtained from a subject; and
(b) detecting a level of a bound antibody, wherein the bound antibody is bound to one of the probes in the plurality of polypeptide probes in the panel, and wherein the level is indicative of the presence or absence of prostate cancer.

2. A method of screening for prostate cancer in a subject, wherein the subject exhibits a symptom of prostate cancer, the method comprising:
(a) obtaining a first biological fluid sample from the subject;
(b) contacting the first biological fluid sample obtained from the subject with a first panel comprising at least two polypeptide probes comprising a polypeptide or epitope fragment of:
(i) amino acid sequence SEQ ID NO: 132 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 162, and
(ii) amino acid sequence SEQ ID NO: 130 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 160; and

(c) detecting a level of autoantibodies bound to the polypeptide probes, wherein the level of the autoantibodies bound to the polypeptide probes are analyzed in combination to detect the presence of prostate cancer in the subject with a sensitivity of at least 80%.

3. The method of claim 2, further comprising confirming the detection of the presence of prostate cancer by obtaining a prostate tissue biopsy and analyzing the prostate tissue biopsy for the presence of prostate cancer.

4. The method of claim 2, further comprising selecting a treatment for prostate cancer based on the detection of the level of the autoantibodies.

5. The method of claim 2, wherein the symptom of prostate cancer is a PSA level that is from about 2.5 ng/mL to about 10 ng/mL.

6. The method of claim 3, further comprising:
(d) contacting a second biological fluid sample obtained from the subject with a second panel comprising one or more polypeptide probes for an autoantibody when the prostate tissue biopsy provides a positive result for cancer;
(e) detecting a level for the second autoantibody; and
(f) providing a prognosis or theranosis based on the level of the autoantibody.

7. The method of claim 2, wherein the first panel further comprises a polypeptide probe comprising a polypeptide or epitope fragment of:
(i) amino acid sequence SEQ ID NO: 13 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 27,
(ii) amino acid sequence SEQ ID NO: 2 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 16,
(iii) amino acid sequence SEQ ID NO: 141 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 171,
(iv) amino acid sequence SEQ ID NO: 129 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 159,
(v) amino acid sequence SEQ ID NO: 118 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 148, or
(vi) amino acid sequence SEQ ID NO: 122 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 152;

wherein each of the one or more polypeptide probes is attached to a substrate.

8. The method of claim 2, wherein the detecting the level is by an immunoassay.

9. The method of claim 7, wherein the first panel further comprises one or more polypeptide probes comprising a polypeptide or epitope fragment of amino acid sequence SEQ ID NO: 5, 11, 12, 14, 56, 58, 60, 62, 64, 67, 69, 115, 116, 123 or 140.

10. The method of claim 7, wherein the first panel further comprises one or more polypeptide probes comprising a polypeptide or epitope fragment of an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 19, 25, 26, 28, 70, 73, 75, 77, 79, 82, 84, 145, 146, 153, or 170.

11. The method of claim 2, wherein the first panel comprises at least 3, 4, 5, 6, 7 or 8 polypeptide probes.

12. The method of claim 6, wherein the first or second biological fluid sample is serum, urine, or a previous tissue biopsy.

13. The method of claim 6, wherein the second panel is the same as the first panel.

14. The method of claim 2, wherein the symptom of prostate cancer is a PSA level greater than about 2.5 ng/mL.

15. The method of claim 2, wherein the detection of the binding of the autoantibodies to the polypeptide probes detects the presence of prostate cancer in the subject with a specificity of at least 30%.

16. The method of claim 2, wherein the detection of the binding of the autoantibodies to the polypeptide probes detects the presence of prostate cancer in the subject with a specificity of at least 45%.

17. The method of claim 7, wherein the first panel comprises the polypeptide probe comprising the polypeptide or epitope fragment of amino acid sequence SEQ ID NO: 2 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 16.

18. The method of claim 7, wherein the first panel comprises the polypeptide probe comprising the polypeptide or epitope fragment of amino acid sequence SEQ ID NO: 141 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 171.

19. The method of claim 7, wherein the first panel comprises the polypeptide probe comprising the polypeptide or epitope fragment of amino acid sequence SEQ ID NO: 129 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 159.

20. The method of claim 7, wherein the first panel comprises the polypeptide probe comprising the polypeptide or epitope fragment of amino acid sequence SEQ ID NO: 118 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 148.

21. The method of claim 7, wherein the first panel comprises the polypeptide probe comprising the polypeptide or epitope fragment of amino acid sequence SEQ ID NO: 122 or an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 152.

22. The method of claim 7, wherein the substrate is an array.

23. The method of claim 1, wherein the substrate is an array.

24. The method of claim 1, wherein the panel further comprises one or more polypeptide probes comprising a polypeptide or epitope fragment of amino acid sequence SEQ ID NO: 5, 11, 12, 14, 56, 58, 60, 62, 64, 67, 69, 115, 116, 123 or 140.

25. The method of claim 1, wherein the panel further comprises one or more polypeptide probes comprising a polypeptide or epitope fragment of an amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 19, 25, 26, 28, 70, 73, 75, 77, 79, 82, 84, 145, 146, 153, or 170.

26. The method of claim 1, wherein the biological fluid sample comprises serum, urine, or a previous tissue biopsy.

27. The method of claim 1, wherein the biological fluid sample comprises serum.

28. The method of claim 2, wherein the first panel further comprises a polypeptide probe comprising a polypeptide or epitope fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 2, 141, 129, 118, and 122.

29. The method of claim 2, wherein the first panel further comprises a polypeptide probe comprising a polypeptide or epitope fragment of an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 27, 16, 171, 159, 148, and 152.