CN113933509A - Antibody assay - Google Patents

Antibody assay Download PDF

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Publication number
CN113933509A
CN113933509A CN202010677754.6A CN202010677754A CN113933509A CN 113933509 A CN113933509 A CN 113933509A CN 202010677754 A CN202010677754 A CN 202010677754A CN 113933509 A CN113933509 A CN 113933509A
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China
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tumor marker
ssx1
hud
sox2
mage
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Inventor
安德烈亚·默里
贾里德·艾伦
菲利普·冈宁
伊莎贝尔·麦克唐纳
塞利娜·帕尔西-科瓦尔斯卡
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Oncimmune Ltd
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Oncimmune Ltd
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Priority to CN202010677754.6A priority Critical patent/CN113933509A/en
Priority to CN202180062774.5A priority patent/CN116324412A/en
Priority to US18/005,568 priority patent/US20230266331A1/en
Priority to JP2023502699A priority patent/JP2023533815A/en
Priority to EP21743191.5A priority patent/EP4182693A2/en
Priority to PCT/EP2021/069668 priority patent/WO2022013321A2/en
Priority to KR1020237004771A priority patent/KR20230068378A/en
Publication of CN113933509A publication Critical patent/CN113933509A/en
Pending legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4748—Details p53

Abstract

And (4) measuring the antibody. The present invention relates generally to the field of antibody detection, and in particular to methods comprising detecting autoantibodies associated with lung cancer in a sample comprising a body fluid of a patient. In particular, the present invention relates to a method for detecting lung cancer in a mammalian subject by detecting three or more autoantibodies in a test sample, wherein three of said autoantibodies are immunologically specific for the tumor marker antigens p53, p62 and SSX 1. The invention also relates to in vitro methods of determining autoantibody profiles, methods of diagnosing and treating lung cancer, methods of predicting response to lung cancer treatment, use of a set of three or more tumour marker antigens for detecting lung cancer, and kits for detecting autoantibodies.

Description

Antibody assay
Technical Field
The present invention relates generally to the field of antibody detection, and in particular to methods comprising detecting autoantibodies associated with lung cancer in a sample comprising a body fluid of a patient.
Background
Many diagnostic, prognostic, and/or monitoring assays rely on the detection of biomarkers for a particular disease state or disease susceptibility. Such biomarkers are typically proteins or polypeptides that are characteristic of a particular disease or associated with disease susceptibility, and are often used to detect cancer, including lung cancer.
Lung cancer is the most common cancer worldwide and the most common cause of death from cancer, with 176 ten thousand deaths recorded globally in 2018 (WHO face sheet-https:// www.who.int/news-room/face-sheets/detail/cancer). Cancer is often diagnosed when symptoms become apparent, when the tumor is usually in an advanced stage (III or IV). Thus, more than 50% of all patients die within 12 months after diagnosis. Early diagnosis triples the 5-year survival rate to 56% if the tumor is found to be localized, but unfortunately only 16% of lung cancers are diagnosed at a localized stage.
Antibodies and in particular autoantibodies may be used as biomarkers of disease or disease susceptibility. Autoantibodies are naturally occurring antibodies directed against an antigen that is recognized as a foreign body by the immune system of an individual, even if the antigen is actually derived from the individual. It may be present in the circulation as circulating free autoantibodies or in the form of circulating immune complexes consisting of autoantibodies bound to their target proteins. In certain instances, the difference between the wild-type protein expressed by a "normal" cell and the altered protein form produced by a diseased cell or during disease can cause the altered protein to be recognized by the individual's immune system as "non-self" and thereby elicit an immune response in the individual. This may be a humoral (i.e., B cell-mediated) immune response, resulting in the production of autoantibodies immunologically specific for the altered protein.
WO99/58978 describes methods for detecting/diagnosing cancer based on assessing the immune response of an individual to two or more different tumour markers. These methods generally comprise contacting a sample of bodily fluid taken from an individual with a set of two or more different tumor marker antigens, each from a separate tumor marker protein, and detecting the formation of a complex of tumor marker antigens that bind to circulating autoantibodies immunologically specific for the tumor marker proteins. The presence of such circulating autoantibodies is taken as an indication of the presence of cancer.
Assays that measure the immune response of an individual to the presence of a tumor marker protein based on autoantibody production provide an alternative to direct measurement or detection of tumor marker protein in bodily fluids. Such an assay essentially constitutes an indirect detection of the presence of a tumor marker protein. The nature of the immune response means that it is likely that autoantibodies can be elicited by very small amounts of circulating tumour marker protein and thus indirect methods which rely on detecting an immune response to a tumour marker will be more sensitive than methods for directly measuring tumour markers in bodily fluids. Thus, an assay method based on detection of autoantibodies may be particularly valuable early in the disease process and may also involve screening asymptomatic patients, for example to identify individuals "at risk" of developing disease in a population of asymptomatic individuals in the screen. Furthermore, methods based on detection of autoantibodies can be particularly valuable early in the disease process and can also be used to identify individuals who have developed a disease among a population of symptomatic individuals.
Diagnostic tests for early detection of lung cancer have been developed and are commercially available in many areas. A test consisting of a set of 7 Tumor marker antigens (p53, SOX2, NY-ESO-1, GBU4-5, CAGE, MAGE-A4 and HuD) (EarlyCDT Lung; manufactured by oncommune Limited, Nembum, England) was validated (Chapman et al, 2012, Tumor Biol, 33: 1319-. The test underwent the largest randomized control trial for early detection of lung cancer using biomarkers. A successful National Health Service (NHS) ECLS trial on 12, 209 high-risk smokers in scotland demonstrated that EarlyCDT Lung reduced the incidence of advanced Lung cancer and unclassified cases at diagnosis compared to standard clinical practice.
Another diagnostic Test using a panel of Seven tumor marker antigens (p53, GAGE7, PGP95, CAGE, MAGE-A1, SOX2, GBU4-5) has been developed specifically for detecting lung Cancer in the human population of China (Ren et al, 2017, Oncoimmunology, 7(2)), and is marketed in China (Seven Kinds of Autoantibodies Test Kit (ELISA); manufactured by Handgzhou Cancer Probe Biotechnology Company ("Cancer Probe") in Hangzhou, China).
However, there is still a need for diagnostic tests with improved sensitivity and specificity in order to improve the early detection of lung cancer in different ethnic groups, and therefore research on new tumor marker antigen sets is being conducted.
Disclosure of Invention
The present application describes a novel set of tumor marker antigens that can be used to detect autoantibodies associated with lung cancer. It was surprisingly found that a core set of three tumor marker antigens contributes to most of the performance of tests based on these new antigen sets. The addition of multiple other tumor marker antigens enhances performance, especially when directed to a diverse ethnic group. By detecting autoantibodies against these novel tumor marker antigen sets, the inventors have designed an effective and non-invasive method of lung cancer screening and corresponding kits.
The inventors of the present application have screened a set of tumor marker antigens and developed an antigen marker set suitable for relatively accurately predicting lung cancer. The inventors have surprisingly found that a set of three or more tumor marker antigens comprising p53, p62 and SSX1 provides improved performance in the detection of lung cancer compared to existing diagnostic tests based on detecting autoantibodies in human samples.
According to a first aspect, the present invention provides a method of detecting lung cancer in a mammalian subject by detecting three or more autoantibodies in a test sample (test sample) comprising a bodily fluid from the mammalian subject, wherein three of the autoantibodies are immunologically specific for the tumor marker antigens p53, p62 and SSX1, and wherein the method comprises the steps of:
(a) contacting the test sample with a set of three or more tumor marker antigens, wherein three of the tumor marker antigens are p53, p62, and SSX 1; and
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample,
wherein the presence of a complex comprising at least p53, p62, and SSX1 is indicative of the presence of lung cancer.
In certain embodiments, the set of three or more tumor marker antigens comprises p53, p62, and SSX1, and one or more tumor marker antigens selected from the group consisting of: HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H and alpha-enolase-1.
In certain embodiments, four or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of four or more tumor marker antigens, wherein four of the tumor marker antigens are p53, p62, SSX1, and HuD, and wherein the presence of a complex comprising at least p53, p62, SSX1, and HuD indicates the presence of lung cancer.
In certain embodiments, five or more autoantibodies are detected, wherein the method comprises the steps of: (a) contacting the test sample with a set of five or more tumor marker antigens, wherein five of the tumor marker antigens are p53, p62, SSX1, HuD, and MAGE a4, and wherein the presence of a complex comprising at least p53, p62, SSX1, HuD, and MAGE a4 is indicative of the presence of lung cancer.
In certain embodiments, the set of five or more tumor marker antigens comprises p53, p62, SSX1, HuD, and MAGE a4, and one or more tumor marker antigens selected from the group consisting of: SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8 and KRAS-G13C/Q61H.
In certain embodiments, the set of five or more tumor marker antigens comprises, or consists of, one of the tumor marker antigen sets selected from:
(i)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE；
(ii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CK20；
(iii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE；
(iv)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE、CK20；
(v)p53、p62、SSX1、HuD、MAGE A4、NY-ESO-1、CAGE、CK20；
(vi)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE、CK20；
(vii)p53、p62、SSX1、HuD、MAGE A4、SOX2、CK20、CK8、KRAS-G13C/Q61H；
(viii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20、CK8、p53-95、KRAS-G13C/Q61H；
(ix) p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, KRAS-G13C/Q61H; and
(x)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、GBU4-5、CK8、KRAS-G13C/Q61H。
in certain embodiments, seven or more autoantibodies are detected, the method comprising the steps of: (a) contacting a test sample with a set of seven or more tumor marker antigens, wherein seven of the tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE is indicative of the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting a test sample with a set of eight or more tumor marker antigens, wherein eight of the tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, and CK20 indicates the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting a test sample with a set of eight or more tumor marker antigens, wherein eight of the tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, and CAGE, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, and CAGE indicates the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting a test sample with a set of eight or more tumor marker antigens, wherein eight of the tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, CAGE, and CK20 is indicative of the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting a test sample with a set of eight or more tumor marker antigens, wherein eight of the tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, NY-ESO-1, CAGE, and CK20 is indicative of the presence of lung cancer.
In certain embodiments, nine or more autoantibodies are detected, the method comprising the steps of: (a) contacting a test sample with a set of nine or more tumor marker antigens, wherein nine of the tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, CAGE, and CK20 indicates the presence of lung cancer.
In certain embodiments, nine or more autoantibodies are detected, the method comprising the steps of: (a) contacting a test sample with a set of nine or more tumor marker antigens, wherein nine of the tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H is indicative of the presence of lung cancer.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting a test sample with a set of eleven or more tumor marker antigens, wherein eleven of the tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CK20, CK8, p53-95, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CK20, CK8, p53-95, and KRAS-G13C/Q61H is indicative of the presence of lung cancer.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting a test sample with a set of eleven or more tumor marker antigens, wherein eleven of the tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H is indicative of the presence of lung cancer.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting a test sample with a set of eleven or more tumor marker antigens, wherein eleven of the tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H is indicative of the presence of lung cancer.
In certain embodiments, the method further comprises the steps of:
(c) detecting the amount of specific binding between the tumor marker antigen and autoantibodies present in the test sample,
wherein the presence or absence of the autoantibody is based on a comparison between the amount of specific binding observed and a predetermined cut-off value (cut-off).
In certain embodiments, the tumor marker antigen is provided in a plurality of different amounts, and wherein the method comprises the steps of:
(a) contacting the test sample with a plurality of different amounts of the tumor marker antigen;
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample;
(c) detecting the amount of specific binding between said tumor marker antigen and said autoantibody;
(d) plotting or calculating a curve of the amount of specific binding versus the amount of tumor marker antigen for each amount of tumor marker antigen used in step (a); and
(e) determining the presence or absence of the autoantibody based on the amount of specific binding between the tumor marker antigen and the autoantibody at each different amount of tumor marker antigen used.
In certain embodiments, the method further comprises the steps of:
(d1) calculating a secondary curve parameter from the curve plotted or calculated in step (d); and
(e) determining the presence or absence of the autoantibody based on a combination of:
(i) the amount of specific binding between said autoantibody and said tumor marker antigen determined in step (b); and
(ii) the secondary curve parameter determined in step (d 1).
In a second aspect, the present invention provides an in vitro method of determining the autoantibody profile of an individual having lung cancer by detecting three or more autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, wherein the three autoantibodies are immunologically specific for the tumour marker antigens p53, p62 and SSX1, the method comprising the steps of:
a) contacting the test sample with a set of three or more tumor marker antigens, wherein the three tumor marker antigens are p53, p62, and SSX 1; and
b) determining the presence or absence of a complex of a tumour marker antigen which binds to autoantibodies present in the test sample, wherein the method is repeated to establish a profile of autoantibody production.
In a third aspect, the present invention provides a method of diagnosing and treating lung cancer in a mammalian subject by detecting three or more autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein the three autoantibodies are immunologically specific for the tumour marker antigens p53, p62 and SSX1, the method comprising the steps of:
(a) contacting the test sample with a set of three or more tumor marker antigens, wherein the three tumor marker antigens are p53, p62, and SSX 1;
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample;
(c) diagnosing the subject as having lung cancer when a complex comprising at least tumor marker antigens p53, p62, and SSX1 that bind to autoantibodies present in the test sample is detected; and
(d) administering a lung cancer treatment to the diagnosed subject.
In a fourth aspect, the present invention provides a method of predicting response to a lung cancer treatment, the method comprising detecting three or more autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, wherein the three autoantibodies are immunologically specific for the tumour marker antigens p53, p62 and SSX1, the method comprising the steps of:
(a) contacting the test sample with a set of three or more tumor marker antigens, wherein the three tumor marker antigens are p53, p62, and SSX 1;
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample;
(c) detecting the amount of specific binding between the tumor marker antigen and autoantibodies present in the test sample; and
(d) comparing the amount of specific binding between the tumour marker antigen and the autoantibody with a previously established relationship between the amount of binding and the likely outcome of the treatment,
wherein a change in the amount of specific binding when compared to a control is predictive that the patient will or will not respond to lung cancer treatment.
In certain embodiments, the lung cancer treatment is selected from the group consisting of surgery, teleassisted thoracoscopic surgery, radiation therapy, chemotherapy, immunotherapy, radiofrequency ablation, biological therapy, cryotherapy, and photodynamic therapy.
In a fifth aspect, the present invention provides the use of a set of three or more tumour marker antigens for the detection of lung cancer in a mammalian subject by detecting autoantibodies immunologically specific for p53, p62 and SSX1 in a test sample comprising a bodily fluid from the mammalian subject.
In a sixth aspect, the present invention provides a kit for detecting autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:
(a) a set of three or more tumor marker antigens, wherein the three tumor marker antigens are p53, p62, and SSX 1;
(b) a reagent capable of detecting a complex of a tumor marker antigen bound to an autoantibody present in a test sample.
In certain embodiments, the kit further comprises:
(c) means (means) for contacting the tumour marker antigen with a test sample comprising a bodily fluid from a mammalian subject.
In certain embodiments, the means for contacting a tumor marker antigen with a test sample comprising a bodily fluid from a mammalian subject comprises the tumor marker antigen immobilized on a chip, slide, plate, well of a microtiter plate, bead, membrane or nanoparticle.
In certain embodiments, the kit is for detecting lung cancer.
In all aspects of the invention, the tumor marker antigen may be a naturally occurring protein or polypeptide, a recombinant protein or polypeptide, a synthetic peptide, a peptidomimetic, a polysaccharide or a nucleic acid.
In all aspects of the invention, the bodily fluid may be selected from the group consisting of plasma, serum, whole blood, urine, sweat, lymph, stool, cerebrospinal fluid, ascites, pleural effusion, semen, sputum, nipple aspirate, post-operative seroma, saliva, amniotic fluid, tears, and wound drainage fluid.
In all aspects of the invention, the method is preferably performed in vitro on a test sample comprising a bodily fluid obtained or prepared from a mammalian subject.
In all aspects of the invention, the mammalian subject is preferably a human.
In another aspect of the present invention, there is provided a method of detecting lung cancer in a mammalian subject by detecting autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein the autoantibodies are immunologically specific for a tumor marker antigen selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95 and CK8, and wherein the method comprises the steps of:
(a) contacting a test sample with a tumor marker antigen selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK 8; and
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample,
wherein the presence of the complex is indicative of the presence of lung cancer.
Drawings
FIG. 1A shows an exemplary plate package layout. If the antigen is coated at two concentrations (50 and 160nM), columns 1, 3, 5, 7 and 9 are 50nM, and columns 2, 4, 6, 8 and 10 are 160 nM.
FIG. 1B shows an exemplary board distribution layout. Each plate can run 5 to 10 specimens (specimen).
Figure 2 shows ROC curves for a panel of all 14 markers for cohort 2(98 lung cancer cases and 55 benign lung disease controls).
FIG. 3 shows ROC curves for a panel of 9 autoantibody markers for p53, p62, SSX1, HuD, MAGE-A4, SOX2, CK20, NY-ESO-1 and CAGE for cohort 2(98 lung cancer cases and 55 benign lung disease controls).
Figure 4 shows ROC curves for a panel of five autoantibody markers for p53, p62, SSX-1, HuD and MAGE a4 for cohort 2(98 lung cancer cases and 55 benign lung disease controls).
Figure 5 shows ROC curves for group 2(98 lung cancer cases and 55 benign lung disease controls) for a set of three autoantibody markers selected from p53, p62, SSX1 and HuD.
Figure 6 shows a ROC scattergram summary of the multiple cutoff solutions obtained using a simulated annealing based algorithm for a group of seven markers for cohort 3(148 lung cancer cases and 145 healthy controls).
Detailed Description
A.Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Without limiting any terminology, further description of some terminology used herein is provided below.
The term autoantibody as used herein refers to a naturally occurring antibody directed against an antigen that is recognized as foreign by the immune system of an individual even if the antigen actually originates from the individual. Generally, autoantibodies include antibodies directed against a variant form of a naturally occurring protein produced by diseased cells or during disease progression. Variant forms of a protein are derived from an individual, but may be considered "non-self" by the individual's immune system and therefore elicit an immune response in the individual in the form of autoantibodies that are immunologically specific for the variant protein. Such protein variants may include, for example, mutants having an altered amino acid sequence, optionally accompanied by alterations in secondary, tertiary or quaternary structure, truncated forms, splice variants, altered glycoforms, and the like. In other embodiments, the autoantibodies may be directed against proteins that are overexpressed in disease states or as a result of gene amplification or aberrant transcriptional regulation. Overexpression of proteins that are not normally encountered by cells of the immune system in significant amounts can trigger an immune response, resulting in the production of autoantibodies. In other embodiments, the autoantibodies may be directed against a fetal form of a protein that becomes expressed under a disease state. If fetal proteins, which are normally expressed at an early stage of development just before the immune system is functional, become expressed under a disease state, the fetal form expressed under the disease state in a fully developed person can be recognized by the immune system as "foreign", triggering an immune response, leading to the production of autoantibodies. In other embodiments, the autoantibodies may be directed to proteins expressed at different locations in a disease state. For example, a protein may be expressed at an internal location in a healthy individual, but at a surface-exposed location in a disease state, such that it is exposed to the circulation and thus the immune system in the disease state rather than in a healthy individual. Herein, the protein to which the autoantibody is directed is referred to as "tumor marker protein".
The term antigen as used herein refers to an immunospecific agent that complexes with autoantibodies present in a test sample. An antigen is a substance comprising at least one antigenic determinant or epitope capable of specific interaction with a target autoantibody desired to be detected, or any capture reagent that specifically interacts with a variable region or complementarity determining region of said autoantibody. The antigen is typically a naturally occurring or synthetic biological macromolecule such as a protein or peptide, polysaccharide or nucleic acid, and may include an antibody or fragment thereof, such as an anti-idiotypic antibody. A "tumor marker antigen" is an antigen that is elevated in a subject suffering from cancer (particularly lung cancer in this context). The terms "tumor marker antigen", "tumor antigen" and "antigen" will be used interchangeably herein.
The term different antigens as used herein encompasses antigens derived from different proteins or polypeptides (e.g., antigens derived from unrelated proteins encoded by different genes).
The term antigenic variant as used herein refers to a single antigen, for example an allele or other variant of a single protein antigen as defined above. Antigen variants are typically derived from a single gene, and different antigen variants may be expressed in different members of a population or under different disease states. Antigen variants may differ by amino acid sequence or by post-translational modifications (e.g., glycosylation, phosphorylation, or acetylation). In addition, the term "antigenic variant" encompasses antigenic mutations, such as amino acid substitutions, additions or deletions. Generally, an antigenic variant will comprise fewer than five (e.g., fewer than four, fewer than three, fewer than two, or one) mutations relative to the wild-type antigen.
When referring to a substance for testing for the presence of autoantibodies using the method of the invention, the term bodily fluid as used herein includes inter alia: plasma, serum, whole blood, urine, sweat, lymph, feces, cerebrospinal fluid, ascites, pleural effusion, semen, sputum, nipple aspirates, post-operative seroma, saliva, amniotic fluid, tears, or wound drainage fluid. As described above, the method of the present invention is preferably performed in vitro on a test sample comprising a body fluid taken from a test subject. The type of body fluid used may vary depending on the nature of the autoantibodies to be tested and the clinical situation in which the assay is used. In general, it is preferred to perform the assay on a serum or plasma sample. The test sample may also contain other components in addition to the body fluid, such as diluents, preservatives, stabilizers, buffers, and the like. Since the assay is performed on a body fluid sample, it is substantially non-invasive. This means that the assay can be repeated as many times as necessary, for example, to establish a profile of the patient's immune response throughout the course of the disease.
The terms mammalian subject and subject as used herein will be used interchangeably to refer to a subject that is a mammal, preferably a human. The subject may have lung cancer. The subject may be suspected of having lung cancer. The subject may have been tested positive for lung cancer using ultrasound or monitoring. The subject may have been previously diagnosed as having lung cancer and/or in partial or complete remission. The subject may be receiving treatment for lung cancer. The subject may be undergoing surgery, television assisted thoracoscopic surgery, radiation therapy, chemotherapy, immunotherapy, radiofrequency ablation, biological therapy, cryotherapy, and/or photodynamic therapy.
B.Method for detecting autoantibodies
In general, the invention provides immunoassays for the detection of autoantibodies immunologically specific for tumor marker proteins associated with lung cancer. Immunoassays can be used to detect or diagnose lung cancer.
According to a first aspect of the present invention there is provided a method of detecting lung cancer in a mammalian subject by detecting three or more autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein the three autoantibodies are immunologically specific for the tumour marker antigens p53, p62 and SSX1, and wherein the method comprises the steps of:
(a) contacting the test sample with a set of three or more tumor marker antigens, wherein the three tumor marker antigens are p53, p62, and SSX 1; and
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample,
wherein the presence of a complex comprising at least p53, p62, and SSX1 is indicative of the presence of lung cancer.
In certain embodiments, the methods of the present invention may further comprise the steps of:
(c) detecting the amount of specific binding between the tumor marker antigen and autoantibodies present in the test sample,
wherein the presence or absence of autoantibodies is based on a comparison between the amount of specific binding observed and a predetermined cut-off.
In this embodiment, the amount of specific binding between the tumor marker antigen and the autoantibodies present in the test sample can be the relative amount of binding or the absolute amount of binding.
Here, an autoantibody may be considered to be present if the amount of specific binding between the tumour marker antigen and the autoantibody present in the test sample is above or below a predetermined cut-off. However, if the amount of specific binding between the tumor marker antigen and the autoantibodies present in the test sample is above a predetermined cut-off, the autoantibodies are generally considered to be present. The predetermined cutoff can be determined by performing control assays on samples known to be negative (e.g., normal individuals) in case-control studies. A "normal" individual will preferably be an age-matched control without any diagnosis of lung cancer based on clinical imaging and/or biochemical criteria. In certain embodiments, a sample known to be negative may be derived from an individual with benign lung disease, i.e., those at high risk of lung cancer but who do not show any evidence of lung cancer. Preferably, the normal individual is free of any diagnosis of any cancer. Here, the amount of specific binding between the tumor marker antigen and autoantibodies present in test samples from normal patients can be detected and averaged to provide a predetermined cut-off. In certain embodiments, the predetermined cutoff can be determined by selecting the cutoff value that gives the largest you den's value that retains greater than 90% specificity.
The inventors have surprisingly found that a core set of three tumor marker antigens is particularly effective for the accurate detection and diagnosis of lung cancer. Within the scope of the present invention, it is contemplated that autoantibodies immunologically specific to a group of three or more tumor marker antigens are detectable, wherein the three tumor marker antigens are p53, p62 and SSX 1. In this embodiment, the diagnosis of lung cancer can be confirmed based on the presence of complexes of all three tumor marker antigens bound to their respective autoantibodies. The invention also contemplates the detection of autoantibodies immunologically specific for a set of three tumor marker antigens, wherein the three tumor marker antigens are p53, p62 and SSX1, and the detection of one or more additional autoantibodies immunologically specific for one or more additional tumor marker proteins.
In another embodiment, the invention contemplates that autoantibodies immunologically specific to a group of four or more tumor marker antigens can be detected, wherein the four tumor marker antigens are p53, p62, SSX1 and HuD. In this embodiment, the diagnosis of lung cancer can be confirmed based on the presence of complexes of all four tumor marker antigens bound to their respective autoantibodies. The invention also contemplates the detection of autoantibodies that are immunologically specific for a group of four tumor marker antigens, wherein the four tumor marker antigens are p53, p62, SSX1 and HuD, and the detection of one or more additional autoantibodies that are immunologically specific for one or more additional tumor marker proteins.
In another embodiment, the invention contemplates that autoantibodies immunologically specific for a group of five or more tumor marker antigens can be detected, wherein the five tumor marker antigens are p53, p62, SSX1, HuD and MAGE a 4. In this embodiment, the diagnosis of lung cancer can be confirmed based on the presence of complexes of all five tumor marker antigens bound to their respective autoantibodies. The invention also contemplates the detection of autoantibodies immunologically specific for a group of five tumour marker antigens, wherein the five tumour marker antigens are p53, p62, SSX1, HuD and MAGE a4, and the detection of one or more further autoantibodies immunologically specific for one or more further tumour marker proteins.
In certain embodiments, the mammalian subject may have lung cancer. The subject may have non-small cell lung cancer (NSCLC), such as adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, large cell carcinoma, or sarcomatoid carcinoma; or the subject may have Small Cell Lung Cancer (SCLC).
In other embodiments, the mammalian subject may be suspected of having lung cancer. The mammalian subject may have previously been tested positive in a lung cancer screen. Any lung cancer screening is contemplated herein. In other embodiments, the subject may have been previously tested positive for lung cancer using ultrasound monitoring or any other imaging method. In certain embodiments, the subject may have been previously diagnosed as having lung cancer and/or in partial or complete remission. The subject may be receiving treatment for lung cancer. The subject may be undergoing surgery, television assisted thoracoscopic surgery, radiation therapy, chemotherapy, immunotherapy, radiofrequency ablation, biological therapy, cryotherapy, and/or photodynamic therapy.
For purposes of the present invention, a subject who is undergoing lung cancer therapy or who has previously undergone lung cancer therapy may still be considered "suspected of having lung cancer". Herein, lung cancer treatment may be performed at any time, and the subject may or may not be subsequently tested for the presence of lung cancer.
Due to the presence of known lung cancer risk factors, the subject may be suspected of having lung cancer. In certain embodiments, the subject may be a smoker; the subject may have been exposed to second-hand smoke, radon, asbestos, arsenic, diesel exhaust (diesel exhaust), high air pollution, or other carcinogens; the subject may have received radiation therapy; and/or the subject may have a past or family history of lung cancer. Any method of determining these risk factors is contemplated, and the subject may or may not be receiving or has received treatment related to the risk factors.
Within the scope of the present invention, a subject may be tested as positive in a lung cancer screen at any time prior to performing the methods of the present invention. For example, a lung cancer screen can be conducted 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years, five years, six years, seven years, eight years, nine years, ten years, or more before the method of the invention is performed.
C.Set of tumor marker antigens
The present invention provides methods relating to the detection of three or more autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, wherein the three autoantibodies are immunologically specific for the tumour marker antigens p53, p62 and SSX 1.
In certain embodiments of the invention, the method can detect three or more autoantibodies, four or more autoantibodies, or five or more autoantibodies. For example, the method can detect three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty-eleven, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, or more autoantibodies.
It is generally believed that by testing for the presence of multiple autoantibodies, the sensitivity of the assay will be improved. Thus, in some embodiments, the methods of the invention contemplate the use of a set comprising a plurality of tumor marker antigens such as: three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty-eleven, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight or more tumor marker antigens.
For some embodiments involving the use of a set comprising a plurality of tumor marker antigens, the method may require the presence of an immune complex comprising three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty-eleven, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight or more antigens to obtain a positive assay result.
These methods may be referred to hereinafter as "group assays". Such assays are typically more sensitive than the detection of autoantibodies to a single tumour marker antigen and produce false negative results at a much lower frequency (see WO99/58978, WO2004/044590 and WO2006/126008, the contents of which are incorporated herein by reference).
The set of tumor marker antigens can be tailored to the particular ethnic background of the subject. The inventors have identified a core set of three tumor marker antigens that can be used to detect relevant autoantibodies for accurate diagnosis of lung cancer in the chinese population.
According to the core of the invention, the method comprises contacting the test sample with a set of three or more tumor marker antigens, wherein the three tumor marker antigens are p53, p62 and SSX 1.
In certain embodiments, the methods comprise contacting a test sample with a set of three or more tumor marker antigens, wherein the set comprises p53, p62, and SSX1 and one or more tumor marker antigens selected from the group consisting of HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H, and alpha-enolase-1. In this embodiment, the panel can comprise three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen of the enumerated tumor marker antigens.
In certain preferred embodiments, the methods can detect four or more autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, wherein the four autoantibodies are immunologically specific for tumor marker antigens p53, p62, SSX1 and HuD. In some particularly preferred embodiments, the method comprises contacting the test sample with a set of four or more tumor marker antigens, wherein the four tumor marker antigens are p53, p62, SSX1, and HuD. In certain embodiments, the presence of a complex comprising at least p53, p62, SSX1, and HuD is indicative of the presence of lung cancer.
In certain preferred embodiments, the methods can detect five or more autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, wherein the five autoantibodies are immunologically specific for the tumor marker antigens p53, p62, SSX1, HuD and MAGE a 4. In some particularly preferred embodiments, the method comprises contacting the test sample with a set of five or more tumor marker antigens, wherein the five tumor marker antigens are p53, p62, SSX1, HuD, and MAGE-a 4. In certain embodiments, the presence of a complex comprising at least p53, p62, SSX1, HuD, and MAGE a4 is indicative of the presence of lung cancer.
In certain embodiments, the set of five or more tumor marker antigens comprises p53, p62, SSX1, HuD, and MAGE A4, and one or more tumor marker antigens selected from SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, and KRAS-G13C/Q61H. In this embodiment, the panel may comprise five, six, seven, eight, nine, ten, eleven or twelve of the listed tumor marker antigens.
In certain embodiments, the set of five or more tumor marker antigens comprises or consists of one of the sets of tumor marker antigens selected from the group consisting of:
(i)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE；
(ii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20；
(iii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE；
(iv)p53、p62、SSX1、HuD、MAGEA4、SOX2、CAGE、CK20；
(v)p53、p62、SSX1、HuD、MAGE A4、NY-ESO-1、CAGE、CK20；
(vi)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、CK20；
(vii)p53、p62、SSX1、HuD、MAGE A4、SOX2、CK20、CK8、KRAS-G13C/Q61H；
(viii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20、CK8、p53-95、KRAS-G13C/Q61H；
(ix) p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, KRAS-G13C/Q61H; and
(x)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、GBU4-5、CK8、KRAS-G13C/Q61H。
in certain embodiments, seven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of seven or more tumor marker antigens, wherein the seven tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE is indicative of the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, and CK20 is indicative of the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CAGE, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CAGE is indicative of the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the presence of the eight tumor marker antigens p53, p62, SSX1, HuD, MAGE a4, SOX2, CAGE, and CK20, and a complex comprising at least p53, p62, SSX1, HuD, MAGEA4, SOX2, CAGE, and CK20 is indicative of the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, NY-ESO-1, CAGE, and CK20 is indicative of the presence of lung cancer.
In certain embodiments, nine or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, CAGE, and CK20 indicates the presence of lung cancer.
In certain embodiments, nine or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H indicates the presence of lung cancer.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, CK20, CK8, p53-95, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CK20, CK8, p53-95, and KRAS-G13C/Q61H is indicative of the presence of lung cancer.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK220, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H is indicative of the presence of lung cancer.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H is indicative of the presence of lung cancer.
The invention also contemplates methods of using a panel of two or more antigen variants comprising one or more different antigens.
Also provided herein is a method of detecting lung cancer in a mammalian subject by detecting autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein the autoantibodies are immunologically specific for a tumor marker antigen selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95 and CK8, and wherein the method comprises the steps of:
(a) contacting a test sample with a tumor marker antigen selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK 8; and
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample,
wherein the presence of the complex is indicative of the presence of lung cancer.
In certain embodiments, two, three, four, five, six, seven or more autoantibodies are detected and the method comprises the steps of:
(a) contacting a test sample with a set of two or more, three or more, four or more, five or more, six or more, or seven or more tumor marker antigens, wherein at least two, at least three, at least four, at least five, at least six, or seven tumor marker antigens are selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8, wherein the presence of a complex comprising at least two, at least three, at least four, at least five, at least six, or seven tumor marker antigens selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8 is indicative of the presence of lung cancer.
In certain embodiments, seven or more autoantibodies are detected and the method comprises the steps of: (a) contacting the test sample with a set of seven or more tumor marker antigens, wherein the seven tumor marker antigens are p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8, wherein the presence of a complex comprising at least one, at least two, at least three, at least four, at least five, at least six tumor marker antigens selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8 is indicative of the presence of lung cancer.
In certain embodiments, the presence of a complex comprising at least p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8 is indicative of the presence of lung cancer.
D.Assay format
The actual step of detecting autoantibodies in a sample of body fluid may be performed according to immunoassay techniques known per se in the art.
General characteristics of immunoassays (e.g., ELISA, radioimmunoassay, etc.) are well known to those skilled in the art (see, e.g., immunolassay, e.diamandis and t.christopoulus, Academic Press, inc., San Diego, CA, 1996). Immunoassays for the detection of antibodies with a particular immunological specificity generally require the use of reagents (antigens) that exhibit a particular immunological reactivity to the antibodies tested. Depending on the format of the assay, the antigen may be immobilized on a solid support. The sample for which the test antibody is present is contacted with the antigen, and if an antibody having the desired immunological specificity is present in the sample, the antibody will immunologically react with the antigen to form an antibody-antigen complex, which can then be detected or quantitatively measured.
The method of the invention may be carried out in any suitable format which enables a test sample suspected of containing autoantibodies to be contacted with the antigen. Conveniently, the contact between the test sample and the antigen may be performed in separate reaction chambers (e.g. wells of a microtiter plate) which allow for the parallel determination of different antigens or different amounts of antigen (if required). In some embodiments where varying amounts of antigen are desired (see antigen titration methods below), they may be coated onto the wells of a microtiter plate by preparing serial dilutions from the antigen stock solution between the wells of the microtiter plate. Antigen stocks may have known or unknown concentrations. Aliquots of the test sample may then be added to the wells of the plate, with the volume and dilution of the test sample remaining constant in each well. As will be understood by those skilled in the art, the absolute amount of antigen added to the wells of a microtiter plate may vary depending on such factors as the nature of the target autoantibody, the nature of the test sample, the dilution of the test sample, and the like. Generally, the amount of antigen and the dilution of the test sample will be selected to produce a range of signal intensities that fall within the acceptable detection range of the selected readout for detecting antigen/autoantibody binding in the method. Conveniently, the amount of antigen tested may vary from 1.6nM to 160 mM.
In another embodiment of the invention, antigens may be immobilized at discrete locations or reaction sites on a solid support. In some embodiments where different amounts of antigen are required (see antigen titration methods below), they may each be immobilized at discrete locations or reaction sites on the solid support. The entire support can then be contacted with the test sample and the binding of autoantibodies to antigen detected or measured separately at each discrete or reaction site. Suitable solid supports include microarrays. Where different amounts of antigen are required, microarrays can be prepared by immobilizing different amounts of a particular antigen at discrete, distinguishable reaction sites on the array. In other embodiments, the actual amount of immobilized antigenic molecule can remain substantially constant, but the size of the sites or spots on the array is varied to vary the amount of available binding epitope, thereby providing a titration series of sites or spots having different amounts of available binding epitope. In such embodiments, the two-dimensional surface concentration of the binding epitope on the antigen is important in preparing the titration series, rather than the absolute amount of antigen. Techniques for the preparation and interrogation of protein/peptide microarrays are well known in the art.
Microarrays can be used to perform multiple assays for autoantibodies with different specificities in parallel on a single sample. This can be done using an array comprising a plurality of antigens or groups of antigens.
Certain antigens may comprise or be derived from proteins or polypeptides isolated from natural sources, including, but not limited to, proteins or polypeptides isolated from patient tissues or body fluids (e.g., plasma, serum, whole blood, urine, sweat, lymph, stool, cerebrospinal fluid, ascites, pleural effusion, semen, sputum, nipple aspirates, post-operative seromas, and wound drainage fluids). In such embodiments, the antigen may comprise substantially all of the naturally occurring protein, i.e., substantially the protein in its form isolated from a natural source, or the antigen may comprise a fragment of a naturally occurring protein. In order to be effective as an antigen in the methods of the invention, any such fragment must retain immunological reactivity with the autoantibodies to be tested. Suitable fragments may be prepared, for example, by chemical or enzymatic cleavage of the isolated protein.
In certain embodiments, and depending on the precise nature of the assay in which the antigen is to be used, the antigen may comprise a naturally occurring protein or fragment thereof linked to one or more additional molecules that confer some desired property not naturally present in the protein. For example, the protein or fragment may be conjugated to an revealing label, such as a fluorescent label, a colored label, a luminescent label, a radioactive label or a heavy metal, such as colloidal gold. In other embodiments, the protein or fragment may be expressed as a recombinantly produced fusion protein. For example, the fusion protein may comprise a tag peptide at the N-or C-terminus to aid in purification of the recombinantly expressed antigen.
Depending on the format of the assay in which the antigen is to be used, the antigen may be immobilized on a solid support, such as a chip, a glass slide, a well of a microtiter plate, a bead, a membrane or a nanoparticle. Immobilization may be achieved by non-covalent adsorption, covalent attachment or by a label.
Any suitable means of attachment may be used provided that it does not adversely affect to a significant extent the ability of the antigen to immunoreact with the target autoantibody.
The invention is not limited to solid phase assays but also encompasses assays that are performed wholly or partially in the liquid phase, such as solution phase bead assays or competitive assays.
In one embodiment, the antigen may be labeled with a ligand (e.g., biotin) that facilitates immobilization. The antigen can then be diluted to the appropriate titration range and allowed to react with the autoantibodies in the patient sample solution. The resulting immune complex can then be immobilized on a solid support via a ligand-receptor interaction (e.g., biotin-streptavidin), and the remainder of the assay performed as described below.
To facilitate the production of biotinylated antigens for use in the assay methods of the invention, the eDNA encoding the full-length antigen, truncated forms thereof, or antigenic fragments thereof, may be expressed as a fusion protein labeled with a protein or polypeptide tag that can be linked to a biotin cofactor, e.g., by an enzymatic reaction.
Vectors for the production of recombinant biotinylated antigens are commercially available from a number of sources. Alternatively, biotinylated antigens can be produced by covalently linking biotin to the antigen molecule after expression and purification.
As mentioned above, the immunoassay for detecting the autoantibodies according to the invention may be based on standard techniques known in the art. In a most preferred embodiment, the immunoassay may be an ELISA. ELISA is generally known in the art. In a typical indirect ELISA, an antigen specific for the autoantibody being tested is immobilized on a solid surface (e.g. the well of a standard microtiter assay plate, or the surface of a microbead or microarray) and a sample comprising a bodily fluid for testing for the presence of the autoantibody is contacted with the immobilized antigen. Any autoantibodies with the desired specificity present in the sample will bind to the immobilized antigen. The bound antigen/autoantibody complex can then be detected using any suitable method. In a preferred embodiment, a labeled second anti-human immunoglobulin antibody that specifically recognizes one or more human immunoglobulin consensus epitopes is used to detect the antigen/autoantibody complex. Typically, the second antibody will be anti-IgG or anti-IgM. The secondary antibody is typically labeled with a detectable marker, typically an enzymatic marker, such as peroxidase or alkaline phosphatase, which allows quantitative detection by addition of a substrate for the enzyme that produces a detectable product, such as a colored chemiluminescent or fluorescent product. Other types of detectable labels known in the art may be used with equivalent effect.
Antigen titration method
In WO2006/126008 (the contents of which are incorporated herein by reference), it was determined that: the performance, and more particularly the clinical utility and reliability, of assays based on the detection of autoantibodies as disease biomarkers can be significantly improved by including an antigen titration step.
By testing a sample suspected of containing antibodies against a series of different amounts of antigen (also referred to herein as a titration series) and constructing a titration curve, a true positive screening result can be reliably identified independent of the absolute amount of antibody present in the sample. The antigen titration method of WO2006/126008 provides greater specificity and sensitivity than methods in which autoantibody reactivity is measured at a single antigen concentration or in which a serum sample is titrated instead of antigen.
Thus, in certain embodiments, the present invention contemplates providing tumor marker antigens in a plurality of different amounts, and wherein the method comprises the steps of:
(a) contacting a test sample with a plurality of different amounts of a tumor marker antigen;
(b) determining the presence or absence of a tumor marker antigen complex that binds to autoantibodies present in the test sample;
(c) detecting the amount of specific binding between the tumor marker antigen and the autoantibody;
(d) plotting or calculating a curve of the amount of specific binding versus the amount of tumor marker antigen for each amount of tumor marker antigen used in step (a); and
(e) the presence or absence of autoantibodies is determined based on the amount of specific binding between the tumor marker antigen and the autoantibody at each different amount of tumor marker antigen used.
In practice, different amounts of tumor marker antigen will typically be provided by varying the concentration of the tumor marker antigen utilized. Thus, the terms "different amounts" and "different concentrations" are used interchangeably. However, any method of altering the amount of tumor marker antigen is contemplated within the scope of the present invention. The skilled reader will appreciate that the amount of antigenic determinant or epitope available for binding to a target autoantibody in the methods of the invention is important for establishing a titration series (i.e. a set of antigens provided in different amounts). In many assay formats, the amount of antigenic determinant or epitope available for binding is directly related to the amount of antigenic molecule present. However, in other embodiments, such as certain solid phase assay systems, the amount of exposed antigenic determinant or epitope may not be directly related to the amount of antigen, but may depend on other factors, such as attachment to the solid phase surface and conformational presentation. In these embodiments, reference herein to "different amounts of antigen" in a titration series may be taken to refer to different amounts of antigenic determinants or epitopes. In some embodiments, the change in the amount of antigen can be achieved by altering the antigen or epitope density to which the test sample is directed, or by maintaining the antigen or epitope density but increasing the surface area on which the antigen is immobilized, or both.
In this embodiment, a "antigen set" refers to a single antigen that will be tested in varying amounts in the methods of the invention.
According to the invention, the method comprises contacting the test sample with a set of three or more tumor marker antigens, wherein three of these tumor marker antigens are p53, p62 and SSX 1. In such embodiments where multiple antigens are contemplated, "different antigen sets" refer to a single antigen to be tested in different amounts in the methods of the invention, wherein each antigen is a "different antigen" derived from a different protein or polypeptide (e.g., an antigen derived from an unrelated protein encoded by a different gene), as defined above.
A given microarray may contain only different antigen sets derived from different proteins or polypeptides, or only different antigen sets containing different peptide epitopes derived from a single protein or polypeptide, or a mixture of both in any ratio. It should be noted that in any embodiment of the invention, each individual antigen set in different amounts will typically comprise only one antigen rather than a mixture thereof.
A set of antigen variants refers to a single antigen variant that will be tested in varying amounts in the methods of the invention.
In certain embodiments, the presence or absence of autoantibodies can be determined based on a collective value of the amount of specific binding for all amounts of tumor marker antigen used. In the method of the invention, the relative or absolute amount of specific binding between the autoantibody and the antigen is determined for each different amount of antigen (antigenic determinant or epitope) tested and used to plot or calculate a curve of the (relative or absolute) amount of specific binding versus the amount of antigen for each amount of antigen tested. The presence of autoantibodies in the test sample that react with the antigen used in the test is determined based on the amount of specific binding observed at each antigen amount and is typically represented by a dose-response curve, which is typically S-shaped or S-shaped. Thus, in certain embodiments, the presence or absence of autoantibodies is determined by screening a graph for the presence of a dose response curve (e.g., generally an S-shape or sigmoidal curve). If there is no detectable change in binding to different amounts of the test antigen, it can be scored as the absence of a detectable amount of autoantibody.
In certain embodiments, the presence or absence of autoantibodies is determined based on a collective value of the amount of specific binding for all amounts of tumor marker antigen used.
In certain embodiments, the presence or absence of autoantibodies is determined by screening a graph of the presence of a dose response curve in step (d).
In certain embodiments, the dose response curve is generally S-shaped or sigmoidal.
In one embodiment, the presence or absence of autoantibodies is determined by comparing the amount of specific binding between the autoantibody and the antigen to a predetermined cut-off value. Here, a curve of the amount of specific binding versus the amount of antigen for each amount of antigen used in the titration series is plotted, and the level of binding in a known positive sample (e.g., a population of patients with disease) is compared to the level of binding observed in a known negative sample (e.g., normal individuals) in a case-control study. The cut-off for autoantibody binding at one or more points on the titration curve is chosen to maximize sensitivity (few false negatives) while maintaining high specificity (few false positives). Assuming that the curve of the amount of specific binding versus the amount of antigen for each amount of antigen used in the titration series is a dose response curve, a measurement is considered positive if the amount of specific binding determined at one or more points on the titration curve is above a predetermined cut-off value. In certain embodiments, the predetermined cutoff value may be determined by selecting a cutoff value that gives the most approximate dengue value (Youden's value) while maintaining a specificity greater than 90%.
It should be noted that antigen titration embodiments can be used with all methods of the present invention, including methods of detecting lung cancer, methods of diagnosing and treating lung cancer, methods of predicting response to anti-lung cancer therapy, and methods of determining antibody profiles. In addition, antigen titration may be used in some embodiments where only a single autoantibody is detected as well as in some embodiments where a set of antigens is used to detect multiple autoantibodies.
Double cut-off method
It is generally believed that the sensitivity of the assay can be improved by measuring autoantibodies against a variety of antigens. However, such increased sensitivity is often associated with a proportional decrease in specificity, and thus the assay method may be limited by the number of antigens it can use. In certain embodiments, the present methods may account for the decrease in specificity by using an antigen titration method that determines the level of specific binding between the autoantibody and the antigen and evaluates a secondary curve parameter, wherein a test result is considered positive only if it is classified as positive compared to the two cut-off points in these metrics. This method will be referred to herein as the "double-cut-off" method and is fully described in WO2015/193678 (the contents of which are incorporated herein by reference).
In certain embodiments, the methods of the present invention further comprise the steps of:
(d1) calculating a quadratic curve parameter from the curve drawn or calculated in step (d); and
(e) determining the presence or absence of autoantibodies based on the following combinations:
(i) the amount of specific binding between the autoantibody and the tumour marker antigen determined in step (b); and
(ii) the conic parameter determined in step (d 1).
The double cut-off method utilizes the antigen titration method described above. The amount of antigen/autoantibody binding is detected at each amount of antigen used in the titration series, and after plotting a curve of the amount of specific binding versus the amount of antigen for each amount of antigen used in the titration series, a quadratic curve parameter is calculated. The conic parameters may be calculated from linear or logarithmic regression curves. As used herein, a conic parameter is any calculated value that provides an indication of the nature of the curve. For example, the secondary curve parameter may be slope, intercept, AUC, maximum slope, or dissociation constant (Kd).
Thus, in certain embodiments, the conic parameter is selected from the group consisting of slope, intercept, AUC, maximum slope, and dissociation constant (Kd).
In certain embodiments, the quadratic curve parameters are calculated from a linear or logarithmic regression curve.
In certain embodiments, the secondary curve parameter can be determined by fitting a logistic curve (e.g., a 4-parameter logistic curve) to a curve of specific binding versus antigen amount per antigen amount used in the titration series. In this embodiment, the conic parameter may be a maximum asymptote, a minimum asymptote, a Hill slope (or slope factor), or an inflection point.
Thus, in certain embodiments, the quadratic curve parameter is the maximum asymptote, minimum asymptote, Hill slope (or slope factor) or inflection point fitted to the logistic curve for each curve plotted or calculated in step (c).
Once the secondary curve parameters are obtained, they are combined with antigen/autoantibody binding data to determine the presence or absence of autoantibodies. Here, the amount of specific binding between the autoantibody and the antigen is compared with the predetermined cut-off value.
Cutoff values for the secondary curve parameters were determined using known positive samples (e.g., a set of case-control sample sets consisting of a population of patients with disease) and known negative samples (e.g., a population of normal individuals in a case-control study). For each sample, a curve of the amount of specific binding versus the amount of antigen for each amount of antigen used in the titration series is plotted, and the secondary curve parameters observed in known positive samples (e.g., patients with disease) are compared to the secondary curve parameters observed in known negative samples (e.g., normal individuals). The cut-off for the secondary curve parameters was chosen such that it maximizes specificity (few false positives) when used in combination with the cut-off for antigen/autoantibody binding discussed above.
After calculating the cut-off value for the secondary curve parameter, the directionality required for a positive reading is also determined, i.e. whether a value above or below the cut-off value is considered positive. The directionality required for a positive reading will depend on the antigen and the secondary curve parameters. A measurement is considered to be ultimately positive if it is both above the cut-off for antigen/autoantibody binding and exhibits the directionality required for a positive reading compared to the cut-off for the secondary curve parameter, i.e. indicates the presence of autoantibodies in the test sample.
It should be noted that the double cutoff embodiment can be used with all methods of the invention, including methods of detecting lung cancer, methods of diagnosing and treating lung cancer, methods of predicting response to anti-lung cancer therapy, and methods of determining antibody profiles. In addition, the double cutoff method can be used in some embodiments where only a single autoantibody is detected as well as in some embodiments where multiple autoantibodies are detected using a panel of antigens. It should be noted in this set of embodiments that the quadratic curve parameters calculated for each antigen in the set need not necessarily be the same. However, in some embodiments, the quadratic curve parameters calculated for each antigen within a group may be the same.
E.Application of the method
The immunoassay method according to the present invention can be used in a variety of different clinical situations. According to the present invention, the method can be used for the detection of lung cancer. In particular, the methods may be used for the detection or diagnosis of lung cancer, screening asymptomatic human subject populations for the presence of lung cancer, detecting primary or secondary (metastatic) lung cancer or screening for early neoplastic or early oncogenic changes in symptomatic patients.
Diagnosis and treatment of lung cancer
In certain embodiments, there is provided a method of diagnosing and treating lung cancer in a mammalian subject by detecting three or more autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein the three autoantibodies are immunospecific for tumor marker antigens p53, p62 and SSX1, the method comprising the steps of:
(a) contacting the test sample with a set of three or more tumor marker antigens, wherein the three tumor marker antigens are p53, p62, and SSX 1;
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to autoantibodies present in the test sample;
(c) diagnosing a subject having lung cancer when a complex comprising at least the tumor marker antigens p53, p62 and SSX1 that bind to autoantibodies present in the test sample is detected; and
(d) and (3) treating the lung cancer of the diagnosed object.
In this regard, autoantibodies are considered to be present if the amount of specific binding between the tumour marker antigen and the autoantibodies present in the test sample is above or below a predetermined cut-off value as described above.
In certain embodiments, the set of three or more tumor marker antigens comprises p53, p62, and SSX1, and the one or more tumor marker antigens are selected from the group consisting of HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H, and alpha-enolase-1.
In a particularly preferred embodiment, the method comprises detecting four or more autoantibodies and the method comprises the steps of: (a) contacting the test sample with a set of four or more tumor marker antigens, wherein the four tumor marker antigens are p53, p62, SSX1, and HuD, and wherein the presence of at least a complex comprising p53, p62, SSX1, and HuD is detected.
In a particularly preferred embodiment, the method comprises detecting five or more autoantibodies and the method comprises the steps of: (a) contacting the test sample with a set of five or more tumor marker antigens, wherein the five tumor marker antigens are p53, p62, SSX1, HuD, and MAGE a4, and wherein the presence of at least a complex comprising p53, p62, SSX1, HuD, and MAGE-a4 is detected.
In certain embodiments, a set of five or more tumor marker antigens comprises p53, p62, SSX1, HuD, and MAGE A4, and the one or more tumor marker antigens are selected from the group consisting of SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, and KRAS-G13C/Q61H.
In certain embodiments, a set of five or more tumor marker antigens comprises or consists of one of the sets of tumor marker antigens selected from:
(i)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE；
(ii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20；
(iii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE；
(iv)p53、p62、SSX1、HuD、MAGEA4、SOX2、CAGE、CK20；
(v)p53、p62、SSX1、HuD、MAGEA4、NY-ESO-1、CAGE、CK20；
(vi)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、CK20；
(vii)p53、p62、SSX1、HuD、MAGE A4、SOX2、CK20、CK8、KRAS-G13C/Q61H；
(viii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20、CK8、p53-95、KRAS-G13C/Q61H；
(ix) p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, KRAS-G13C/Q61H; and
(x)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、GBU4-5、CK8、KRAS-G13C/Q61H。
in certain embodiments, seven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of seven or more tumor marker antigens, wherein the seven tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE indicates the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, and CK20 indicates the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CAGE, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CAGE indicates the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, CAGE, and CK20 indicates the presence of lung cancer.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, NY-ESO-1, CAGE, and CK20 is indicative of the presence of lung cancer.
In certain embodiments, nine or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, CAGE, and CK20 indicates the presence of lung cancer.
In certain embodiments, nine or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H indicates the presence of lung cancer.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CK20, CK8, p53-95, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CK20, CK8, p53-95, and KRAS-G13C/Q61H indicates the presence of lung cancer.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H indicates the presence of lung cancer.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H indicates the presence of lung cancer.
It should be noted that the present invention is in no way limited to any particular lung cancer treatment. In certain embodiments, the lung cancer treatment may be selected from the group consisting of surgery, video assisted thoracoscopic surgery, radiation therapy, chemotherapy, immunotherapy, radiofrequency ablation, biological therapy, cryotherapy, and photodynamic therapy.
It is within the scope of the present invention that lung cancer treatment may be performed at any time after the diagnosis of lung cancer. For example, lung cancer treatment can be performed one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, ten hours, eleven hours, twelve hours, twenty-four hours, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, one year or more after lung cancer diagnosis. Multiple lung cancer treatments at any interval between treatment rounds are also contemplated.
Consider lung cancer treatment at a different geographical location than the geographical location where lung cancer diagnosis is performed. Furthermore, lung cancer treatment may be performed by a person who is different from the person making the diagnosis, regardless of whether the diagnosis and treatment are performed in the same or different geographic locations.
In this embodiment of the invention, all of the limitations discussed above with respect to the various methods of the invention are considered with respect to the methods of diagnosing and treating lung cancer.
Predicting response to lung cancer treatment
In one aspect, the autoantibody detection methods of the invention can be used for therapy stratification (stratification), i.e., to determine whether a particular subject or group of subjects can respond more or less to a particular lung cancer therapy. The methods can be used to predict a response of a lung cancer patient to a lung cancer treatment, select a lung cancer treatment for a particular patient, predict a response to a treatment, predict survival rate in response to a treatment, or predict a risk of an immune-related adverse event (irAE) in a patient undergoing immunotherapy (e.g., treatment with a checkpoint inhibitor). The lung cancer treatment or therapy can be, for example, surgery, video assisted thoracoscopic surgery, radiation therapy, chemotherapy, immunotherapy, radio frequency ablation, biological therapy, cryotherapy, and photodynamic therapy.
Accordingly, the present invention provides a method of predicting response to a lung cancer treatment, the method comprising detecting three or more autoantibodies in a test sample comprising bodily fluid from a mammalian subject, wherein the three autoantibodies are immunospecific for tumour marker antigens p53, p62 and SSX1, the method comprising the steps of:
(a) contacting the test sample with a set of three or more tumor marker antigens, wherein the three tumor marker antigens are p53, p62, and SSX 1;
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to autoantibodies present in the test sample;
(c) detecting the amount of specific binding between the tumor marker antigen and autoantibodies present in the test sample; and
(d) comparing the amount of specific binding between the tumor marker antigen and the autoantibody with a previously established relationship between the amount of binding and the likely outcome of the treatment,
wherein an alteration in the amount of specific binding is indicative that the patient will respond or will not respond to the lung cancer treatment when compared to a control.
Herein, a control may be a sample of bodily fluid derived from a subject known to have lung cancer and known to be non-responsive to the lung cancer treatment tested, i.e. a non-responsive control.
In certain embodiments, the set of three or more tumor marker antigens comprises p53, p62, and SSX1, and the one or more tumor marker antigens are selected from the group consisting of HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H, and alpha-enolase-1.
In a particularly preferred embodiment, the method comprises detecting four or more autoantibodies and the method comprises the steps of: (a) contacting the test sample with a set of four or more tumor marker antigens, wherein the four tumor marker antigens are p53, p62, SSX1, and HuD, and wherein the presence of at least a complex comprising p53, p62, SSX1, and HuD is detected.
In a particularly preferred embodiment, the method comprises detecting five or more autoantibodies and the method comprises the steps of: (a) contacting the test sample with a set of five or more tumor marker antigens, wherein the five tumor marker antigens are p53, p62, SSX1, HuD, and MAGE a4, and wherein the presence of at least a complex comprising p53, p62, SSX1, HuD, and MAGE a4 is detected.
In certain embodiments, a set of five or more tumor marker antigens comprises p53, p62, SSX1, HuD, and MAGE A4, and the one or more tumor marker antigens are selected from the group consisting of SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, and KRAS-G13C/Q61H.
In certain embodiments, a set of five or more tumor marker antigens comprises or consists of one of the sets of tumor marker antigens selected from:
(i)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE；
(ii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20；
(iii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE；
(iv)p53、p62、SSX1、HuD、MAGEA4、SOX2、CAGE、CK20；
(v)p53、p62、SSX1、HuD、MAGEA4、NY-ESO-1、CAGE、CK20；
(vi)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、CK20；
(vii)p53、p62、SSX1、HuD、MAGE A4、SOX2、CK20、CK8、KRAS-G13C/Q61H；
(viii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20、CK8、p53-95、KRAS-G13C/Q61H；
(ix) p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, KRAS-G13C/Q61H; and
(x)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、GBU4-5、CK8、KRAS-G13C/Q61H。
in certain embodiments, seven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of seven or more tumor marker antigens, wherein the seven tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE is detected.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1 and CK20 is detected.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CAGE, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CAGE is detected.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGEA4, SOX2, CAGE, and CK20 is detected.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGEA4, NY-ESO-1, CAGE, and CK20 is detected.
In certain embodiments, nine or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, and CK20 is detected.
In certain embodiments, nine or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H is detected.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CK20, CK8, p53-95 and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE-A4, SOX2, NY-ESO-1, CK20, CK8, p53-95 and KRAS-G13C/Q61H is detected.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H is detected.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H is detected.
It should be noted that the present invention is in no way limited to any particular lung cancer treatment. In certain embodiments, the lung cancer treatment may be selected from the group consisting of surgery, video assisted thoracoscopic surgery, radiation therapy, chemotherapy, immunotherapy, radiofrequency ablation, biological therapy, cryotherapy, and photodynamic therapy.
In this embodiment of the invention, all of the limitations discussed above with respect to the various methods of the invention are considered with respect to the method of predicting response to lung cancer treatment.
Determination of antibody profiles
The above-described aspects of the invention will generally be performed once. However, in vitro immunoassays are non-invasive and can be repeated as often as deemed necessary before the onset of lung cancer (as in the screening of "at risk" individuals) or throughout the course of the disease, to establish an autoantibody production profile in the subject. Thus, the methods can be used to determine an antibody profile in a subject having or suspected of having lung cancer.
In certain embodiments, there is provided an in vitro method of determining an autoantibody profile of an individual having lung cancer by detecting three or more autoantibodies in a test sample comprising a bodily fluid from a mammalian subject wherein the three autoantibodies are immunospecific for tumor marker antigens p53, p62 and SSX1, the method comprising the steps of:
a) contacting the test sample with a set of three or more tumor marker antigens, wherein the three tumor marker antigens are p53, p62, and SSX 1; and
b) determining the presence or absence of complexes of the tumor marker antigen that bind to autoantibodies present in the test sample, wherein the method is repeated to establish an autoantibody production profile.
In certain embodiments, the set of three or more tumor marker antigens comprises p53, p62, and SSX1, and the one or more tumor marker antigens are selected from the group consisting of HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H, and alpha-enolase-1.
In a particularly preferred embodiment, the method comprises detecting four or more autoantibodies and the method comprises the steps of: (a) contacting the test sample with a set of four or more tumor marker antigens, wherein the four tumor marker antigens are p53, p62, SSX1, and HuD, and wherein the presence of at least a complex comprising p53, p62, SSX1, and HuD is detected.
In a particularly preferred embodiment, the method comprises detecting five or more autoantibodies and the method comprises the steps of: (a) contacting the test sample with a set of five or more tumor marker antigens, wherein the five tumor marker antigens are p53, p62, SSX1, HuD, and MAGE a4, and wherein the presence of at least a complex comprising p53, p62, SSX1, HuD, and MAGE a4 is detected.
In certain embodiments, a set of five or more tumor marker antigens comprises p53, p62, SSX1, HuD, and MAGE A4, and the one or more tumor marker antigens are selected from the group consisting of SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, and KRAS-G13C/Q61H.
In certain embodiments, a set of five or more tumor marker antigens comprises or consists of one of the sets of tumor marker antigens selected from:
(i)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE；
(ii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CK20；
(iii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE；
(iv)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE、CK20；
(v)p53、p62、SSX1、HuD、MAGE A4、NY-ESO-1、CAGE、CK20；
(vi)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE、CK20；
(vii)p53、p62、SSX1、HuD、MAGE A4、SOX2、CK20、CK8、KRAS-G13C/Q61H；
(viii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20、CK8、p53-95、KRAS-G13C/Q61H；
(ix) p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, KRAS-G13C/Q61H; and
(x)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、GBU4-5、CK8、KRAS-G13C/Q61H。
in certain embodiments, seven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of seven or more tumor marker antigens, wherein the seven tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE is detected.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1 and CK20 is detected.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CAGE, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CAGE is detected.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGEA4, SOX2, CAGE, and CK20 is detected.
In certain embodiments, eight or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGEA4, NY-ESO-1, CAGE, and CK20 is detected.
In certain embodiments, nine or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, and CK20 is detected.
In certain embodiments, nine or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a set of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H is detected.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CK20, CK8, p53-95 and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CK20, CK8, p53-95 and KRAS-G13C/Q61H is detected.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H is detected.
In certain embodiments, eleven or more autoantibodies are detected, the method comprising the steps of: (a) contacting the test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H, and a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H is present.
In this embodiment of the invention, all limitations discussed above with respect to the various methods of the invention are considered with respect to the in vitro method of determining an antibody profile.
Application of tumor marker antigens in detection of lung cancer
The present invention provides the use of a set of three or more tumour marker antigens for the detection of lung cancer in a mammalian subject by detecting autoantibodies immunospecific for p53, p62 and SSX1 in a test sample comprising a bodily fluid from a mammalian subject.
In certain embodiments, the set of three or more tumor marker antigens comprises p53, p62, and SSX1, and the one or more tumor marker antigens are selected from the group consisting of HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H, and alpha-enolase-1.
In a particularly preferred embodiment, four or more autoantibodies are detected and the use comprises contacting the test sample with a panel of four or more tumor marker antigens, wherein the four tumor marker antigens are p53, p62, SSX1 and HuD, and wherein the presence of at least a complex comprising p53, p62, SSX1 and HuD is detected.
In a particularly preferred embodiment, five or more autoantibodies are detected and the method comprises the steps of: (a) contacting the test sample with a set of five or more tumor marker antigens, wherein the five tumor marker antigens are p53, p62, SSX1, HuD, and MAGE a4, and wherein the presence of at least a complex comprising p53, p62, SSX1, HuD, and MAGE a4 is detected.
In certain embodiments, a set of five or more tumor marker antigens comprises p53, p62, SSX1, HuD, and MAGE A4, and the one or more tumor marker antigens are selected from the group consisting of SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, and KRAS-G13C/Q61H.
In certain embodiments, a set of five or more tumor marker antigens comprises or consists of one of the sets of tumor marker antigens selected from:
(i)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE；
(ii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CK20；
(iii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE；
(iv)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE、CK20；
(v)p53、p62、SSX1、HuD、MAGE A4、NY-ESO-1、CAGE、CK20；
(vi)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE、CK20；
(vii)p53、p62、SSX1、HuD、MAGE A4、SOX2、CK20、CK8、KRAS-G13C/Q61H；
(viii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CK20、CK8、p53-95、KRAS-G13C/Q61H；
(ix) p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, KRAS-G13C/Q61H; and
(x)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE、GBU4-5、CK8、KRAS-G13C/Q61H。
in certain embodiments, seven or more autoantibodies are detected, the use comprising contacting a test sample with a panel of seven or more tumor marker antigens, wherein the seven tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE, and detecting the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX-2 and CAGE.
In certain embodiments, eight or more autoantibodies are detected, the use comprising contacting a test sample with a panel of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, and CK20, and detecting the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, and CK 20.
In certain embodiments, eight or more autoantibodies are detected, the use comprising contacting a test sample with a panel of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CAGE, and detecting the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1 and CAGE.
In certain embodiments, eight or more autoantibodies are detected, the use comprising contacting a test sample with a panel of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CAGE, and CK20, and detecting the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, CAGE, and CK 20.
In certain embodiments, eight or more autoantibodies are detected, the use comprising contacting a test sample with a panel of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, NY-ESO-1, CAGE, and CK20, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, NY-ESO-1, CAGE, and CK20 is detected.
In certain embodiments, nine or more autoantibodies are detected, the use comprising contacting a test sample with a panel of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, CAGE, and CK20, and detecting the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, CAGE, and CK 20.
In certain embodiments, nine or more autoantibodies are detected, the use comprising contacting the test sample with a panel of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, CK20, CK8, and KRAS-G13C/Q61H is detected.
In certain embodiments, eleven or more autoantibodies are detected, the use comprising contacting a test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CK20, CK8, p53-95, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CK20, CK8, p53-95, and KRAS-G13C/Q61H is detected.
In certain embodiments, eleven or more autoantibodies are detected, the use comprising contacting a test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE a4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H is detected.
In certain embodiments, eleven or more autoantibodies are detected, the use comprising contacting a test sample with a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8 and KRAS-G13C/Q61H, and the presence of a complex comprising at least p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, GBU4-5, 8 and KRAS-G13C/Q61H is detected.
In this embodiment of the invention, all of the limitations discussed above with respect to the various methods of the invention are considered with respect to this use.
Also provided is the use of a set of two or more, three or more, four or more, five or more, six or more or seven or more tumor marker antigens in a mammalian subject for the detection of lung cancer by detecting autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, said autoantibodies being immunospecific for two or more, three or more, four or more, five or more, six or more or seven tumor marker antigens selected from: p53, SSX1, SOX2, GBU4-5, HuD, p53-95 and CK 8.
Further applications of the method
The methods are useful for identifying individuals at risk of developing lung cancer in a population of asymptomatic individuals.
The assay method can be repeated in many cases to provide continuous monitoring of disease recurrence. The methods are useful for detecting recurrent disease in patients previously diagnosed with lung cancer and who have undergone treatment for lung cancer to reduce the amount of lung cancer present.
The methods can be used to assess prognosis of a patient diagnosed with lung cancer, monitor the progression of lung cancer in a patient, or monitor the response of a lung cancer patient to a lung cancer therapy (e.g., surgery, video-assisted thoracoscopic surgery, radiation therapy, chemotherapy, immunotherapy, radiofrequency ablation, biological therapy, cryotherapy, and photodynamic therapy).
When an immunoassay is used to monitor the progression of lung cancer in a subject, the presence of elevated levels of autoantibodies, as compared to a "normal control", is considered an indication of the presence of cancer in the patient. A "normal control" can be the level of autoantibodies present in control individuals who are preferably age matched, free of any diagnosis of cancer based on clinical, imaging and/or biochemical criteria. Alternatively, a "normal control" can be a "baseline" level established for a particular subject. The "baseline" level may be, for example, the level of autoantibodies present when a first diagnosis of lung cancer or a diagnosis of recurrent lung cancer is made. Any increase above the baseline level will be considered an indication that the amount of cancer present in the patient has increased, while any decrease below the baseline level will be considered an indication that the amount of cancer present in the patient has decreased.
Immunoassay methods can complement existing screening, diagnostic and monitoring methods. For example, the methods of the invention can be used in combination with existing methods to confirm a lung cancer diagnosis. In certain embodiments, the methods of the invention are performed in combination with a CT scan, chest x-ray, PET-CT scan, bronchoscopy and biopsy, thoracoscopy, or any other suitable method of lung cancer diagnosis.
F.Reagent kit
The present invention also encompasses a kit for detecting autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:
(a) a set of three or more tumor marker antigens, wherein the three tumor marker antigens are p53, p62, and SSX 1; and
(b) a reagent capable of detecting a complex of a tumor marker antigen bound to an autoantibody present in a test sample.
In certain embodiments, the kit further comprises:
(c) a device for contacting a tumor marker antigen with a test sample comprising a bodily fluid from a mammalian subject.
Examples of means for contacting the tumor marker antigen with a test sample comprising a bodily fluid from a mammalian subject include immobilization of the tumor marker antigen on a chip, a glass slide, a well of a microtiter plate, a bead, a membrane or a nanoparticle.
In certain embodiments, the set of three or more tumor marker antigens comprises p53, p62, and SSX1 and one or more tumor marker antigens selected from the group consisting of HuD, MAGEA4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H, and alpha-enolase-1. In this embodiment, the panel may comprise three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen of said tumour marker antigens.
In a particularly preferred embodiment, the kit comprises a set of four or more tumor marker antigens, wherein the four tumor marker antigens are p53, p62, SSX1 and HuD.
In a particularly preferred embodiment, the kit comprises a set of five or more tumor marker antigens, wherein the five tumor marker antigens are p53, p62, SSX1, HuD and MAGEA 4.
In certain embodiments, the group of five or more tumor marker antigens comprises p53, p62, SSX1, HuD and MAGEA4, and one or more tumor marker antigens selected from SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8 and KRAS-G13C/Q61H.
In certain embodiments, the set of five or more tumor marker antigens comprises or consists of one of the sets of tumor marker antigens selected from the group consisting of:
(i)p53、p62、SSX1、HuD、MAGEA4、SOX2、CAGE；
(ii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CK20；
(iii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE；
(iv)p53、p62、SSX1、HuD、MAGEA4、SOX2、CAGE、CK20；
(v)p53、p62、SSX1、HuD、MAGEA4、NY-ESO-1、CAGE、CK20；
(vi)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE、CK20；
(vii)p53、p62、SSX1、HuD、MAGEA4、SOX2、CK20、CK8、KRAS-G13C/Q61H；
(viii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CK20、CK8、p53-95、KRAS-G13C/Q61H；
(ix) p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, CAGE, CK20, CK8, KRAS-G13C/Q61H; and
(x)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE、GBU4-5、CK8、KRAS-G13C/Q61H。
in certain embodiments, the kit comprises a set of seven or more tumor marker antigens, wherein the seven tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, and CAGE.
In certain embodiments, the kit comprises a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, and CK 20.
In certain embodiments, the kit comprises a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, and CAGE.
In certain embodiments, the kit comprises a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, CAGE, and CK 20.
In certain embodiments, the kit comprises a set of eight or more tumor marker antigens, wherein the eight tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, NY-ESO-1, CAGE, and CK 20.
In certain embodiments, the kit comprises a panel of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, CAGE, and CK 20.
In certain embodiments, the kit comprises a panel of nine or more tumor marker antigens, wherein the nine tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, CK20, CK8, and KRAS-G13C/Q61H.
In certain embodiments, the kit comprises a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, CK20, CK8, p53-95, and KRAS-G13C/Q61H.
In certain embodiments, the kit comprises a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, CAGE, CK20, CK8, and KRAS-G13C/Q61H.
In certain embodiments, the kit comprises a panel of eleven or more tumor marker antigens, wherein the eleven tumor marker antigens are p53, p62, SSX1, HuD, MAGEA4, SOX2, NY-ESO-1, CAGE, GBU4-5, CK8, and KRAS-G13C/Q61H.
In the kits of the invention, the tumor marker antigen is a naturally occurring protein or polypeptide, a recombinant protein or polypeptide, a synthetic peptide, a peptidomimetic, a polysaccharide, or a nucleic acid.
In the kit of the present invention, the body fluid may be selected from the group consisting of plasma, serum, whole blood, urine, sweat, lymph, feces, cerebrospinal fluid, ascites, pleural effusion, semen, sputum, nipple aspirates, post-operative seroma, saliva, amniotic fluid, tears, and wound drainage fluid.
The kit of the present invention is suitable for carrying out any of the methods of the present invention described above. In particular, the kit of the present invention is suitable for detecting lung cancer. Thus, in certain embodiments, the kit is for detecting lung cancer.
Also provided herein is a kit for detecting autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:
(a) a set of two or more, three or more, four or more, five or more, six or more, seven or more tumor marker antigens, wherein at least two, at least three, at least four, at least five, at least six or seven tumor marker antigens are selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK 8; and
(b) a reagent capable of detecting a complex of a tumor marker antigen bound to an autoantibody present in a test sample.
Also provided herein is a kit for detecting autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:
(a) a set of seven or more tumor marker antigens, wherein the seven tumor marker antigens are p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK 8; and
(b) a reagent capable of detecting a complex of a tumor marker antigen bound to an autoantibody present in a test sample.
The invention will now be further understood with reference to the following non-limiting examples.
Examples
Example 1: method for measuring autoantibodies against tumor associated proteins (antigens) during the development of EarlyCDT lung test against chinese population
Samples of tumour marker antigens may be prepared by recombinant expression in a manner analogous to that described in WO99/58978, the contents of which are incorporated herein by reference. Briefly, cDNA encoding the marker antigen of interest (table 1) was cloned into pET21 or pET45 vector (Invitrogen) modified to encode a biotin tag and a 6 × histidine tag (His tag) to aid in purification of the expressed protein. The resulting clones were grown in BL21(DE3) e.coli (e.coli) and subsequently the bacteria were lysed. The expressed antigen was recovered by a nickel chelate affinity column (HiTrap, commercially available from GE Healthcare) according to the manufacturer's protocol. The purity, specificity and yield of the expressed proteins were assessed by SDS-PAGE, Western blotting and protein assay, followed by storage.
The negative control protein VOL was generated by transforming BL21(DE3) e.coli with the empty pET21 vector (i.e. cDNA not encoding a tumor associated antigen). The expressed and purified protein included the same His and biotin tag sequences found on recombinant tumor-associated antigens and allowed correction of non-specific autoantibody binding to residual bacterial contaminants.
Table 1: details of antigens and accession numbers
Figure BDA0002583997550000451
Figure BDA0002583997550000461
Figure BDA0002583997550000471
GeneID and protein accession numbers can be found on the NCBI website (www.ncbi.nlm.nih.gov).
Antigen and VOL (negative control) were diluted to appropriate concentrations (160 and/or 50nM) in borate-coated buffer (pH 8.5) and dispensed into wells of a microtiter plate at 100 ul/well using an automated liquid handling system according to the plate layout (fig. 1A). Plates were capped and stored at +18 to +22 ℃ for 18 to 24 hours, after which all wells were washed with PBS + 0.1% tween 20 using an automatic plate washer. The plates were tapped dry on absorbent paper and blocking buffer (PBS + 0.1% casein +300mM D (+) -trehalose dihydrate) was added at 200 ul/well.
The plates were then stored at +18 to +22 ℃ for 2 hours, then the contents of the wells were aspirated and the plates were allowed to air dry overnight.
Serum samples were thawed at +18 to +22 ℃, pooled and diluted at 1/110 in a specimen antibody diluent (PBS-1% BSA + 0.1% tween 80+ 0.01% Pluronic F-127 or PBS + 0.1% casein). According to the plate layout in fig. 1B, each diluted serum sample was dispensed into a microtiter plate at 100 ul/well.
The on-plate calibrator, high control and low control, all using chimeric human-rabbit anti-His tag monoclonal antibody (Sigma), were diluted in sample antibody diluent and dispensed into microtiter plates at 100 μ Ι/well according to the plate layout in fig. 1B. The plates were capped and incubated at room temperature for 1.5 hours with shaking.
Plates were washed as above and horseradish peroxidase conjugated rabbit anti-human immunoglobulin diluted in sample antibody diluent was dispensed at 100 μ Ι/well into all wells of microtiter plates. The plates were then incubated at room temperature for 1 hour with shaking. The plate was washed as described above.
A pre-prepared 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB) substrate was added to each plate at 100 μ l/well and incubated on the bench for 15 minutes. The plate was tapped to mix. After 15 minutes, stop solution (1M HCl) was added at 100. mu.l/well. The optical density of each well was determined using a standard spectrophotometer plate reader at 450 nm.
Example 2: detection of autoantibodies in Chinese Lung cancer patients Using a commercially available EarlyCDT Lung test kit
EarlyCDT lung kit assays (oncommume Limited, Nottingham, UK) were performed according to Instructions (Instructions for Use, IFU) and using the manufacturer's suggested cut-off values. Serum samples were collected in china from chinese population and the clinical and demographic status (demographics status) of this group (group 1) is given in table 2.
Table 2: demographic status of cohort 1 consisting of lung cancer cases and of control cohort of individuals with benign lung disease or no evidence of malignant tumors (healthy normal persons)
Figure BDA0002583997550000481
Briefly, the EarlyCDT lung test detects autoantibodies (AAb) against a panel of 7 antigens (table 3). The results (table 3) show that for this cohort, using the established cut-off values, the EarlyCDT lung test had a sensitivity of 32.1% for lung cancer and a specificity of 79.1% and 76.8% for healthy and benign control cohorts, respectively. It is therefore clear that both the sensitivity and specificity of this group of samples from chinese patients are lower than the performance requirements (sensitivity 41% and specificity 90%).
These results indicate that developing and validating an EarlyCDT lung test group for early detection of lung cancer in western patients may not be optimal for achieving the same goal in chinese patients, and that other cut-off values or autoantibodies may need to be measured in order to account for ethnic differences between two regional populations.
Table 3: single autoantibodies (AAb) per patient group (lung cancer cases, benign lung disease controls and healthy normal controls) using EarlyCDT pulmonary test kit and the positive rate of the group
Figure BDA0002583997550000491
Example 3: detection of autoantibodies in chinese lung cancer patients using commercially available cancer probe test
An autoantibody Test (English name: Seven Kinds of autoantibody detection kits (Seven Kinds of autoantibody Test kits) (ELISA), "Cancer Probe") for early lung Cancer detection is sold in China (manufactured by Handzhou Cancer Probe Biotechnology Company, Handzhou, China). The test also measured autoantibodies against a panel of 7 antigens, 4 of which were also present in the EarlyCDT lung test (p53, SOX2, CAGE and GBU4-5), but three others (GAGE-7, MAGEA1 and PGP 9.5).
Autoantibodies were measured in china from a panel of samples collected from chinese populations (n 62; subset of panel 1, table 2) using the cancer probe test according to instructions for use (IFU).
The performance of the cancer probe test (table 4) was compared to the performance of the EarlyCDT lung test (table 5) for the same subset of samples. Autoantibody levels were measured from each tested IFU.
Table 4: for the cancer Probe test, the positive rate of individual autoantibodies (AAb) and the entire group per patient cohort (lung cancer cases, benign lung disease controls and healthy normal controls)
Figure BDA0002583997550000501
Table 5: for the EarlyCDT lung test, the positive rate of individual autoantibodies (AAb) and the whole group per patient cohort (lung cancer cases, benign lung disease controls and healthy normal controls)
Figure BDA0002583997550000511
The results show that the sensitivity of the cancer probe test was 42.9% for this cohort and the specificity was 61.9% and 80.0% for benign and healthy controls, respectively. The results also show that EarlyCDT lung test has a sensitivity of 52.4% for the same group of patients and a specificity of 47.6% and 75.0% for benign and healthy control groups, respectively.
Example 4: identification of an antigen group optimized for early detection of lung cancer in the Chinese population
The following data were obtained from studies to explore the sensitivity and specificity of development assays (methods detailed in example 1) for an independent patient cohort, studying a panel of up to 14 markers selected from p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H and alpha-enolase. All antigens were coated at 50 nM. Following the IFU, the cancer Probe test was also performed on the same cohort.
The clinical and demographic status of the subjects (cohort 2) included in the study are given in table 6. It is a group of patients completely independent of the patients studied in examples 2 and 3 (cohort 1 and a subset of cohort 1, respectively).
Table 6: demographic status of cohort 2 consisting of lung cancer cases and of control cohort of individuals with benign lung disease
Demographics Lung cancer Benign
Number of 98 55
Mean age 58.6 51.7
Age range 30 to 82 15 to 77
Male sex% 49.0％ 66.7％
Unknown sex and age 0 1
(i)Seven antigens in EarlyCDT Lung assay
The optimal cut-off (in RU) was determined using a multiple cut-off optimization algorithm based on simulated annealing.
The results (table 7) show that when the optimal set of cut-off values for this chinese cohort was applied to the assay results, this group corresponded to the EarlyCDT lung test group with a sensitivity of 31.6% for lung cancer and a specificity of 90.9% for the benign control cohort. It is evident that both sensitivity and specificity are lower than the EarlyCDT lung test performance requirements for this chinese cohort (sensitivity 41% and specificity 91%). This suggests that developing and validating a group for early diagnosis of lung cancer in western patients may not be optimal for achieving the same goal in chinese patients, and that other autoantibodies may need to be measured to account for ethnic differences between two regional populations.
Table 7: positive rates for individual autoantibodies (AAb) and EarlyCDT lung test groups in each patient cohort (lung cancer cases and benign lung disease controls) for the indicated cut-off values
Figure BDA0002583997550000531
(ii)Cancer Probe test group
Table 8 shows the results and performance of the cancer probe test for the same patient cohort with an overall sensitivity of 26.5% and a specificity of 96.4% for the benign control group.
Table 8: positive rates for the entire group of single autoantibody (AAb) and cancer rprobe tests in each patient cohort (lung cancer cases, benign lung disease controls and healthy normal controls)
Figure BDA0002583997550000541
To determine the sensitivity that may be accompanied by reduced specificity, simulated annealing optimization was performed based on the population examined. The set of cut-off values found by simulated annealing optimization indicated a maximum sensitivity of 40.8% and a specificity of 90.9%, however this optimization had a high probability of overfitting due to the small size of the control cluster.
(iii)Alternative test set of 3 to 14 markers
The optimal cut-off (in RU) for the assay results for this group of 14, 9, 5 and 3 markers was determined using a simulated annealing-based multivariate cut-off optimization algorithm. This approach identifies a number of different groups of different sizes and performance (tables 9 to 12, figures 2 to 5) which can be directly compared to the cancer probe test performance since they are identified using the exact same group of patients. For the group identified below with a specificity of 90.9%, the sensitivity of the group was 37.8% to 48.0%, and thus all groups showed better performance than the cancer probe test for the same chinese cohort.
Table 9: FIG. 2 Performance characteristics of the top-ranked group
Figure BDA0002583997550000551
Figure BDA0002583997550000561
Table 10: FIG. 3 Performance characteristics of the top-ranked group
Figure BDA0002583997550000562
Table 11: FIG. 4 Performance characteristics of the top-ranked group
Figure BDA0002583997550000571
Table 12: performance characteristics of the cut-off value sets A to D of FIG. 5
Group of Sensitivity (%) Degree of specificity (%)
Set A: p53.HuD, SSX1 37.8 90.9
And a set B: p62, HuD、SSX1 n/a* n/a*
And a set C: p53, p62, HuD 37.8 90.9
And (4) set D: p53, p62, SSX1 40.8 90.9
*Without p53, the optimization cannot find a group whose performance satisfies the search constraints
These results indicate that the groups of 3 to 14 markers (each incorporating at least p53, SSX1, and p62) performed the same or better than the cancer probe test for the same cohort. Even if the results are comparable to the cancer probe test results (e.g. three marker sets of p53, p62, SSX1), it is advantageous to simply use only three tumor marker antigens.
Example 5: detection of autoantibodies to an expanded antigen pool in chinese lung cancer patients using a developmental assay
The following data were obtained from feasibility studies to evaluate the sensitivity and specificity of development assays (methods detailed in example 1) of up to 21 markers (table 1), including those used in the EarlyCDT lung kit (example 2). This was done to evaluate the performance of a larger independent group of EarlyCDT lung groups. This study was aimed at determining whether optimization of the marker cutoff values and/or replacement of certain markers for the chinese population could improve test performance.
The antigens were coated at 50nM (p53, MAGE A4, SOX2, HuD and NY-ESO-1), 160nM (CAGE and GBU4-5) or two concentrations (CK8, CK20, EGFR1-ECD, EGFR1-EP, EGFR1-KD, EGFR2, EGFR-L858R, EGFR-VIII, KRAS, p16, p53-95, p62, alpha-enolase and SSX1), respectively.
The clinical and demographic status of the subjects (cohort 3) included in the study are given in table 13. Table 13: demographic status of cohort 3 consisting of lung cancer cases and control cohort of individuals without history of malignancy (healthy normal persons) for development of assays
Figure BDA0002583997550000581
Figure BDA0002583997550000591
Optimal cut-off values in Reference Units (RU) were determined for the results of the group analysis of the seven marker panels using a simulated annealing-based multivariate cut-off optimization algorithm. This method identifies many different groups, and the ROC scattergram (figure 6) shows the range of sensitivity and specificity combinations that can be obtained by combining various cut-off values. Table 14 details the seven markers that performed best, with a sensitivity of 41.2% and a specificity of 94.4%.
Table 14: positive rates for the entire set of individual autoantibody (AAb) markers and seven markers in each patient cohort (lung cancer cases and healthy normal controls) for the assigned cut-off values to develop the assay cohorts
Figure BDA0002583997550000601
a) The cut-off for autoantibodies against antigen coated at 50nM or b) the cut-off for autoantibodies against antigen coated at 160 nM.
These analyses show that by swapping some markers and optimizing the cut-off values for the chinese population, the performance of the set of seven markers can be improved to a level comparable to that described for the EarlyCDT lung test in the western population.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention (in addition to those described herein) will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered broadly applicable and can be combined with any and all other consistent embodiments, including those embodiments appropriately selected from other aspects of the invention (including alone).
Various publications and patent applications are cited herein, the disclosures of which are incorporated by reference in their entirety.

Claims (37)

1. A method of detecting lung cancer in a mammalian subject by detecting three or more autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein three of the autoantibodies are immunologically specific for the tumour marker antigens p53, p62 and SSX1, and wherein the method comprises the steps of:
(a) contacting the test sample with a set of three or more tumor marker antigens, wherein three of the tumor marker antigens are p53, p62, and SSX 1; and
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample,
wherein the presence of a complex comprising at least p53, p62, and SSX1 is indicative of the presence of lung cancer.
2. The method of claim 1, wherein the set of three or more tumor marker antigens comprises p53, p62, and SSX1, and one or more tumor marker antigens selected from the group consisting of: HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H and alpha-enolase-1.
3. The method of claim 1 or claim 2, wherein four or more autoantibodies are detected, wherein the method comprises the steps of: (a) contacting the test sample with a set of four or more tumor marker antigens, wherein four of the tumor marker antigens are p53, p62, SSX1, and HuD, and wherein the presence of a complex comprising at least p53, p62, SSX1, and HuD indicates the presence of lung cancer.
4. The method of claim 1 or claim 2, wherein five or more autoantibodies are detected, wherein the method comprises the steps of: (a) contacting the test sample with a set of five or more tumor marker antigens, wherein five of the tumor marker antigens are p53, p62, SSX1, HuD, and MAGE a4, and wherein the presence of a complex comprising at least p53, p62, SSX1, HuD, and MAGE a4 is indicative of the presence of lung cancer.
5. The method of claim 4, wherein the set of five or more tumor marker antigens comprises p53, p62, SSX1, HuD, and MAGE A4, and one or more tumor marker antigens selected from the group consisting of: SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8 and KRAS-G13C/Q61H.
6. The method of claim 5, wherein the set of five or more tumor marker antigens comprises or consists of one of the tumor marker antigen sets selected from the group consisting of:
(i)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE；
(ii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20；
(iii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE；
(iv)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE、CK20；
(v)p53、p62、SSX1、HuD、MAGEA4、NY-ESO-1、CAGE、CK20；
(vi)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、CK20；
(vii)p53、p62、SSX1、HuD、MAGE A4、SOX2、CK20、CK8、KRAS-G13C/Q61H；
(viii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20、CK8、P53-95、KRAS-G13C/Q61H；
(ix) p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, KRAS-G13C/Q61H; and
(x)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、GBU4-5、CK8、KRAS-G13C/Q61H。
7. the method of any one of the preceding claims, further comprising the steps of:
(c) detecting the amount of specific binding between the tumor marker antigen and autoantibodies present in the test sample,
wherein the presence or absence of the autoantibody is based on a comparison between the amount of specific binding observed and a predetermined cut-off value.
8. The method of any one of the preceding claims, wherein the tumor marker antigen is provided in a plurality of different amounts, and wherein the method comprises the steps of:
(a) contacting the test sample with a plurality of different amounts of the tumor marker antigen;
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample;
(c) detecting the amount of specific binding between said tumor marker antigen and said autoantibody;
(d) plotting or calculating a curve of the amount of specific binding versus the amount of tumor marker antigen for each amount of tumor marker antigen used in step (a); and
(e) determining the presence or absence of the autoantibody based on the amount of specific binding between the tumor marker antigen and the autoantibody at each different amount of tumor marker antigen used.
9. The method of claim 8, wherein the method further comprises the steps of:
(d1) calculating a secondary curve parameter from the curve plotted or calculated in step (d); and
(e) determining the presence or absence of the autoantibody based on a combination of:
(i) the amount of specific binding between said autoantibody and said tumor marker antigen determined in step (b); and
(ii) the secondary curve parameter determined in step (d 1).
10. An in vitro method for determining the autoantibody profile of an individual having lung cancer by detecting three or more autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, wherein three of said autoantibodies are immunologically specific for the tumor marker antigens p53, p62 and SSX1, the method comprising the steps of:
a) contacting the test sample with a set of three or more tumor marker antigens, wherein three of the tumor marker antigens are p53, p62, and SSX 1; and
b) determining the presence or absence of a complex of a tumor marker antigen that binds to autoantibodies present in the test sample, wherein the method is repeated to establish an autoantibody production profile.
11. A method of diagnosing and treating lung cancer in a mammalian subject by detecting three or more autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein three of the autoantibodies are immunologically specific for the tumour marker antigens p53, p62 and SSX1, the method comprising the steps of:
(a) contacting the test sample with a set of three or more tumor marker antigens, wherein three of the tumor marker antigens are p53, p62, and SSX 1;
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample;
(c) diagnosing the subject as having cancer when a complex comprising at least tumor marker antigens p53, p62, and SSX1 that bind to autoantibodies present in the test sample is detected; and
(d) administering a lung cancer treatment to the diagnosed subject.
12. A method of predicting response to a lung cancer treatment, the method comprising detecting three or more autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, wherein three of the autoantibodies are immunologically specific for the tumour marker antigens p53, p62 and SSX1, the method comprising the steps of:
(a) contacting the test sample with a set of three or more tumor marker antigens, wherein three of the tumor marker antigens are p53, p62, and SSX 1;
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample;
(c) detecting the amount of specific binding between the tumor marker antigen and autoantibodies present in the test sample; and
(d) comparing the amount of specific binding between the tumor marker antigen and the autoantibody to a previously established relationship between the amount of binding and the likely outcome of treatment,
wherein an alteration in the amount of specific binding when compared to a control is predictive that the patient will or will not respond to the lung cancer treatment.
13. The method of claim 11 or claim 12, wherein the lung cancer treatment is selected from surgery, video assisted thoracoscopic surgery, radiation therapy, chemotherapy, immunotherapy, radiofrequency ablation, biologic therapy, cryotherapy, and photodynamic therapy.
14. Use of a set of three or more tumor marker antigens for detecting lung cancer in a mammalian subject by detecting autoantibodies immunologically specific to p53, p62 and SSX1 in a test sample comprising a bodily fluid from said mammalian subject.
15. The method of any one of claims 10 to 13 or the use of claim 14, wherein the set of three or more tumor marker antigens comprises p53, p62 and SSX1, and one or more tumor marker antigens selected from the group consisting of: HuD, MAGEA4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H and alpha-enolase-1.
16. The method of any one of claims 10 to 13 or the use of claim 14, wherein four or more autoantibodies are detected, wherein the method or use comprises contacting the test sample with a set of four or more tumor marker antigens, wherein four of the tumor marker antigens are p53, p62, SSX1 and HuD, and wherein the presence of a complex comprising at least p53, p62, SSX1 and HuD is detected.
17. The method of any one of claims 10 to 13 or the use of claim 14, wherein five or more autoantibodies are detected, wherein the method or use comprises contacting the test sample with a set of five or more tumor marker antigens, wherein five of the tumor marker antigens are p53, p62, SSX1, HuD and MAGE a4, and wherein the presence of a complex comprising at least p53, p62, SSX1, HuD and MAGE a4 is detected.
18. The method or use of claim 17, wherein the set of five or more tumor marker antigens comprises p53, p62, SSX1, HuD and MAGE a4, and one or more tumor marker antigens selected from the group consisting of: SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8 and KRAS-G13C/Q61H.
19. The method or use of claim 18, wherein the set of five or more tumor marker antigens comprises or consists of one of the tumor marker antigen sets selected from:
(i)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE；
(ii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20；
(iii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE；
(iv)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE、CK20；
(v)p53、p62、SSX1、HuD、MAGEA4、NY-ESO-1、CAGE、CK20；
(vi)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CAGE、CK20；
(vii)p53、p62、SSX1、HuD、MAGE A4、SOX2、CK20、CK8、KRAS-G13C/Q61H；
(viii)p53、p62、SSX1、HuD、MAGEA4、SOX2、NY-ESO-1、CK20、CK8、p53-95、KRAS-G13C/Q61H；
(ix) p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, KRAS-G13C/Q61H; and
(x)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、GBU4-5、CK8、KRAS-G13C/Q61H。
20. a kit for detecting autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:
(a) a set of three or more tumor marker antigens, wherein three of the tumor marker antigens are p53, p62, and SSX 1; and
(b) a reagent capable of detecting a complex of a tumor marker antigen that binds to an autoantibody present in the test sample.
21. The kit of claim 20, further comprising:
(c) means for contacting the tumor marker antigen with a test sample comprising a bodily fluid from a mammalian subject.
22. The kit of claim 21, wherein the means for contacting the tumor marker antigen with a test sample comprising a bodily fluid from a mammalian subject comprises the tumor marker antigen immobilized on a chip, slide, plate, well of a microtiter plate, bead, membrane, or nanoparticle.
23. The kit of any one of claims 20 to 22, wherein the set of three or more tumor marker antigens comprises p53, p62, and SSX1, and one or more tumor marker antigens selected from the group consisting of: HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8, KRAS-G13C/Q61H and alpha-enolase-1.
24. The kit of any one of claims 20 to 22, comprising a set of four or more tumor marker antigens, wherein four of the tumor marker antigens are p53, p62, SSX1, and HuD.
25. The kit of any one of claims 20 to 22, comprising a set of five or more tumor marker antigens, wherein five of the tumor marker antigens are p53, p62, SSX1, HuD, and MAGE a 4.
26. The kit of claim 25, wherein the set of five or more tumor marker antigens comprises p53, p62, SSX1, HuD, and MAGE a4, and one or more tumor marker antigens selected from the group consisting of: SOX2, NY-ESO-1, CAGE, CK20, GBU4-5, p53-95, CK8 and KRAS-G13C/Q61H.
27. The kit of claim 26, wherein the set of five or more tumor marker antigens comprises or consists of one of the tumor marker antigen sets selected from:
(i)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE；
(ii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20；
(iii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE；
(iv)p53、p62、SSX1、HuD、MAGE A4、SOX2、CAGE、CK20；
(v)p53、p62、SSX1、HuD、MAGE A4、NY-ESO-1、CAGE、CK20；
(vi)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、CK20；
(vii)p53、p62、SSX1、HuD、MAGE A4、SOX2、CK20、CK8、KRAS-G13C/Q61H；
(viii)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CK20、CK8、p53-95、KRAS-G13C/Q61H；
(ix) p53, p62, SSX1, HuD, MAGE A4, SOX2, NY-ESO-1, CAGE, CK20, CK8, KRAS-G13C/Q61H; and
(x)p53、p62、SSX1、HuD、MAGE A4、SOX2、NY-ESO-1、CAGE、GBU4-5、CK8、KRAS-G13C/Q61H。
28. the kit of any one of claims 20 to 27 for use in detecting lung cancer.
29. The method, use or kit of any preceding claim, wherein the tumor marker antigen is a naturally occurring protein or polypeptide, a recombinant protein or polypeptide, a synthetic peptide, a peptidomimetic, a polysaccharide or a nucleic acid.
30. The method, use or kit of any preceding claim, wherein the bodily fluid is selected from the group consisting of plasma, serum, whole blood, urine, sweat, lymph, stool, cerebrospinal fluid, ascites, pleural effusion, semen, sputum, nipple aspirates, post-operative seromas, saliva, amniotic fluid, tears and wound drainage fluid.
31. A method of detecting lung cancer in a mammalian subject by detecting autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein the autoantibodies are immunologically specific for a tumor marker antigen selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95 and CK8, and wherein the method comprises the steps of:
(a) contacting the test sample with a tumor marker antigen selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK 8; and
(b) determining the presence or absence of a complex of a tumor marker antigen that binds to an autoantibody present in the test sample,
wherein the presence of the complex is indicative of the presence of lung cancer.
32. The method of claim 31, wherein two, three, four, five, six, seven or more autoantibodies are detected and the method comprises the steps of:
(a) contacting the test sample with a set of two or more, three or more, four or more, five or more, six or more, or seven or more tumor marker antigens, wherein the presence of a complex comprising at least two, at least three, at least four, at least five, at least six, or seven tumor marker antigens selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8 selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8 is indicative of the presence of lung cancer.
33. The method of claim 31, wherein seven or more autoantibodies are detected and the method comprises the steps of: (a) contacting the test sample with a set of seven or more tumor marker antigens, wherein seven of the tumor marker antigens are p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8,
wherein the presence of a complex comprising at least one, at least two, at least three, at least four, at least five, at least six tumor marker antigens selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8 is indicative of the presence of lung cancer.
34. The method of claim 33, wherein the presence of a complex comprising at least p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8 is indicative of the presence of lung cancer.
35. A kit for detecting autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:
(a) a set of two or more, three or more, four or more, five or more, six or more, seven or more tumor marker antigens, wherein at least two, at least three, at least four, at least five, at least six or seven of the tumor marker antigens are selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK8, and
(b) a reagent capable of detecting a complex of a tumor marker antigen that binds to an autoantibody present in the test sample.
36. A kit for detecting autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:
(a) a set of seven or more tumor marker antigens, wherein seven of the tumor marker antigens are p53, SSX1, SOX2, GBU4-5, HuD, p53-95, and CK 8; and
(b) a reagent capable of detecting a complex of a tumor marker antigen that binds to an autoantibody present in the test sample.
37. Use of a set of two or more, three or more, four or more, five or more, six or more or seven or more tumor marker antigens for detecting lung cancer in a mammalian subject by detecting autoantibodies immunologically specific for two or more, three or more, four or more, five or more, six or more or seven tumor marker antigens selected from the group consisting of p53, SSX1, SOX2, GBU4-5, HuD, p53-95 and CK8 in a test sample comprising a bodily fluid from the mammalian subject.
CN202010677754.6A 2020-07-14 2020-07-14 Antibody assay Pending CN113933509A (en)

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CN202180062774.5A CN116324412A (en) 2020-07-14 2021-07-14 Use of antigen combinations for detection of autoantibodies in lung cancer
US18/005,568 US20230266331A1 (en) 2020-07-14 2021-07-14 Use of antigen combination for detecting autoantibodies in lung cancer
JP2023502699A JP2023533815A (en) 2020-07-14 2021-07-14 Use of antigen combinations to detect autoantibodies in lung cancer
EP21743191.5A EP4182693A2 (en) 2020-07-14 2021-07-14 Use of antigen combination for detecting autoantibodies in lung cancer
PCT/EP2021/069668 WO2022013321A2 (en) 2020-07-14 2021-07-14 Antibody assay
KR1020237004771A KR20230068378A (en) 2020-07-14 2021-07-14 Use of Antigen Combinations to Detect Autoantibodies in Lung Cancer

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