# US 2019/0376975 A1 · Antibody assay

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- Number: US 2019/0376975 A1
- Document ID: US20190376975A1
- Status: Published application
- Kind: A1
- Application number: 16/463,549
- Filed: 2017-11-24
- Published: 2019-12-12
- Earliest claimed: 2016-11-25
- Assignees: OncImmune Ltd.
- Applicants: Oncimmune Limited
- Inventors: Jared ALLEN; Isabel MACDONALD; Andrea MURRAY; Christopher WELBERRY
- Web page: https://patentgrep.com/patent/US20190376975A1

## CPC

- G01N 33/57525: of the liver or pancreas
- G01N 33/564: for pre-existing immune complex or autoimmune disease {, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9}
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to a method of detecting liver cancer in a mammalian subject by detecting an antibody in a test sample comprising a bodily fluid from the mammalian subject, wherein the antibody is an autoantibody immunologically specific for a tumour marker protein selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B, which method comprises contacting the test sample with a tumour marker antigen selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B and determining the presence or absence of complexes of the tumour marker antigen bound to autoantibodies present in the test sample where the presence of said complexes is indicative of the presence of liver cancer. Also included within the invention are corresponding methods of diagnosing and treating liver cancer in a mammalian subject, corresponding methods of predicting response to an anti-liver cancer treatment, a corresponding method of detecting an antibody in a test sample comprising a bodily fluid from a mammalian subject and kits suitable for performing methods of the invention.

## Claims

### Claim 1

A method of detecting liver cancer in a mammalian subject by detecting an antibody in a test sample comprising a bodily fluid from the mammalian subject, wherein the antibody is an autoantibody immunologically specific for a tumour marker protein selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B, which method comprises the steps of:
(a) contacting the test sample with a tumour marker antigen selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B; and
(b) determining the presence or absence of complexes of the tumour marker antigen bound to autoantibodies present in the test sample;
whereby the presence of said complexes is indicative of the presence of liver cancer.

### Claim 2

A method of detecting an antibody in a test sample comprising a bodily fluid from a mammalian subject, wherein the antibody is an autoantibody immunologically specific for a tumour marker protein selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B, which method comprises the steps of:
(a) contacting the test sample with a tumour marker antigen selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B; and
(b) determining the presence or absence of complexes of the tumour marker antigen bound to autoantibodies present in the test sample.

### Claim 3 (depends on 2)

The method of claim 2, wherein the mammalian subject is suspected of having liver cancer.

### Claim 4 (depends on 1)

The method of claim 1, wherein the mammalian subject has tested positive for alpha-fetoprotein (AFP), des-gamma carboxyprothrombin (DCP) or lectin-reactive alpha-fetoprotein (AFP-L3).

### Claim 5 (depends on 1)

The method of claim 1, wherein the mammalian subject has tested positive for liver cancer using ultrasound surveillance.

### Claim 6 (depends on 1)

The method of claim 1, wherein the mammalian subject has liver cirrhosis, non-alcoholic fatty liver disease, alcoholic liver disease, Wilson's disease, hereditary hemochromatosis, autoimmune hepatitis, hepatitis B, hepatitis C, documented aflatoxin exposure, schistosomiasis or diabetes mellitus.

### Claim 7 (depends on 1)

The method of claim 1, wherein two or more autoantibodies are detected, and wherein the method comprises the step of:
(a) contacting the test sample with a panel of two or more tumour marker antigens comprising a tumour marker antigen selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B and one or more further tumour marker antigens immunologically specific for at least one of said autoantibodies.

### Claim 8 (depends on 7)

The method of claim 7, wherein the panel comprises two or more tumour marker antigens which are distinct antigens.

### Claim 9 (depends on 8)

The method of claim 8, wherein the panel comprises two or more antigen variants of one or more of the distinct antigens.

### Claim 10 (depends on 7)

The method of claim 7, wherein the panel of two or more tumour marker antigens comprises MMP9, AIF1, EpCAM and CDKN1B.

### Claim 11 (depends on 7)

The method of claim 7, wherein the panel of two or more tumour marker antigens comprises one or more tumour marker antigens chosen from NY-ESO-1, vimentin, HSPA4, transferrin, HNRNP-L, HSPD1, HNRNP-A2, SALL4, Cyclin B1, AFP, NPM1, YWHAZ, DDX3X, p62, CAGE, MAGE A4, RalA, GBU4-5, SOX2, AKR1B10, ApoA1, BCL2, CD44, CK18, CPS1, FUCA1, GLUL, HSPA2, IL-8, MDM2, PEBP1, prolactin, RGN, SPP1, SSX2 and TGFB1.

### Claim 12 (depends on 11)

The method of claim 11, wherein the panel of two or more tumour marker antigens comprises:
(i) CAGE, NY-ESO-1, MMP9, transferrin, MAGE A4, RalA, HSPA4, SALL4, Cyclin B1, EpCAM, DDX3X, and AIF1; or
(ii) CAGE, NY-ESO-1, MMP9, transferrin, MAGE A4, RalA, HSPA4, SALL4, Cyclin B1, EpCAM, DDX3X, AIF1, SOX2 and AFP; or
(iii) MMP9, AIF1, EpCAM, NY-ESO-1, HSPA4, vimentin, HNRNP-L and transferrin.

### Claim at position 13

13-17. (canceled)

### Claim 18 (depends on 11)

The method of claim 11, wherein the panel of two or more tumour marker antigens comprises:
(i) AIF1, EpCAM, HSPA4 and CPS1; or .
(ii) AIF1, CAGE, HSPD1, SOX2, SALL4, HSPA4 and transferrin.

### Claim 19

(canceled)

### Claim 20 (depends on 18)

The method of claim 18, wherein the subject is female.

### Claim 21 (depends on 11)

The method of claim 11, wherein the panel of two or more tumour marker antigens comprises:
(i) EpCAM, NY-ESO-1, vimentin, HSPA2, HSPA4 and HNRNP-L; or
(ii) EpCAM, CAGE, SOX2, RalA, MAGE A4, DDX3X and NY-ESO-1.

### Claim 22

(canceled)

### Claim 23 (depends on 21)

The method of claim 21, wherein the subject is male.

### Claim 24 (depends on 11)

The method of claim 11, wherein the panel of two or more tumour marker antigens comprises MMP9, AIF1, EpCAM, DDX3X, SALL4, MAGE A4, NY-ESO-1, CAGE, RalA and SOX2.

### Claim at position 21

25-31. (canceled)

### Claim 32 (depends on 11)

The method of claim 11, wherein the panel of two or more tumour marker antigens comprises NY-ESO-1, vimentin, HSPA4, transferrin, HNRNP-L, HSPD1, HNRNP-A2, SALL4, Cyclin B1, AFP, NPM1, YWHAZ, DDX3X, p62, CAGE, MAGE A4, RalA, GBU4-5, SOX2, AKR1B10, ApoA1, BCL2, CD44, CK18, CPS1, FUCA1, GLUL, HSPA2, IL-8, MDM2, PEBP1, prolactin, RGN, SPP1, SSX2 and TGFB1.

### Claim 33 (depends on 32)

The method of claim 32, wherein the panel of two or more tumour marker antigens comprises MMP9, AIF1, EpCAM, CDKN1B, NY-ESO-1, vimentin, HSPA4, transferrin, HNRNP-L, HSPD1, HNRNP-A2, SALL4, Cyclin B1, AFP, NPM1, YWHAZ, DDX3X, p62, CAGE, MAGE A4, RalA, GBU4-5, SOX2, AKR1B10, ApoA1, BCL2, CD44, CK18, CPS1, FUCA1, GLUL, HSPA2, IL-8, MDM2, PEBP1, prolactin, RGN, SPP1, SSX2 and TGFB1.

### Claim at position 24

34-43. (canceled)

### Claim 44

An in vitro method of determining an antibody profile of an individual suffering from liver cancer in a test sample comprising a bodily fluid from the mammalian subject wherein the antibody is an autoantibody immunologically specific for a tumour marker protein selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B, which method comprises the steps of:
a) contacting the test sample with a tumour marker antigen selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B; and
b) determining the presence or absence of complexes of the tumour marker antigen bound to autoantibodies present in the test sample, wherein the method is repeated to build up a profile of antibody production.

### Claim 45

A method of diagnosing and treating liver cancer in a mammalian subject by detecting an antibody in a test sample comprising a bodily fluid from the mammalian subject, wherein the antibody is an autoantibody immunologically specific for a tumour marker protein selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B, which method comprises the steps of:
(a) contacting the test sample with a tumour marker antigen selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B;
(b) determining the presence or absence of complexes of the tumour marker antigen bound to autoantibodies present in the test sample;
(c) diagnosing the subject with liver cancer when complexes of the tumour marker antigen bound to autoantibodies present in the test sample are detected; and
(d) administering a liver cancer treatment to the diagnosed subject.

### Claim 46

A method of predicting response to an anti-liver cancer treatment, the method comprising detecting an antibody in a test sample comprising a bodily fluid from a mammalian subject, wherein the antibody is an autoantibody immunologically specific for a tumour marker protein selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B, which method comprises the steps of:
(a) contacting the test sample with a tumour marker antigen selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B;
(b) determining the presence or absence of complexes of the tumour marker antigen bound to autoantibodies present in the test sample;
(c) detecting the amount of specific binding between the tumour 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 treatment;
whereby a change in the amount of specific binding, when compared to controls, predicts that the patient will or will not respond to the anti-liver cancer treatment.

### Claim 47 (depends on 45)

The method of claim 45, wherein the liver cancer treatment is selected from the group consisting of chemotherapy, radiofrequency ablation, liver resection, liver transplant, vaccination, anti-growth factor or signal transduction therapies, endocrine therapy, human antibody therapy, transcatheter arterial chemoembolization, percutaneous ethanol injection, microwave ablation, sorafenib administration and radioembolisation.

### Claim at position 29

48-83. (canceled)

### Claim 84 (depends on 1)

The method of claim 1, wherein the tumour marker antigen is a naturally occurring protein or polypeptide, a recombinant protein or polypeptide, a synthetic protein or polypeptide, a synthetic peptide, a peptide mimetic, a polysaccharide or a nucleic acid.

### Claim 85 (depends on 1)

The method of claim 1, wherein the liver cancer is hepatocellular carcinoma (HCC).

### Claim 86 (depends on 1)

The method of claim 1, wherein the bodily fluid is chosen from plasma, serum, whole blood, urine, sweat, lymph, faeces, cerebrospinal fluid, ascites fluid, pleural effusion, seminal fluid, sputum, nipple aspirate, post-operative seroma, saliva, amniotic fluid, tears and wound drainage fluid.

### Claim 87 (depends on 1)

The method of claim 1, wherein the method further comprises detecting alpha-fetoprotein (AFP), des-gamma carboxyprothrombin (DCP) or lectin-reactive alpha-fetoprotein (AFP-L3) in a test sample comprising a bodily fluid from the mammalian subject.

### Claim 88 (depends on 87)

The method of claim 87, wherein the method comprises detecting alpha-fetoprotein (AFP) in a test sample comprising a bodily fluid from the mammalian subject.

### Claim 89 (depends on 88)

The method of claim 88, wherein autoantibodies immunologically specific for the tumour marker proteins CAGE, NY-ESO-1, MMP9, transferrin, MAGE A4, RalA, HSPA4, SALL4, Cyclin B1, EpCAM, DDX3X, and AIF1 are detected in a test sample comprising a bodily fluid from the mammalian subject.

### Claim 90 (depends on 88)

The method of claim 88, wherein autoantibodies immunologically specific for the tumour marker proteins CAGE, NY-ESO-1, MMP9, transferrin, MAGE A4, RalA, HSPA4, SALL4, Cyclin B1, EpCAM, DDX3X, AIF1, SOX2 and AFP are detected in a test sample comprising a bodily fluid from the mammalian subject.

### Claim 91 (depends on 87)

The method of claim 87, wherein the bodily fluid is blood.

### Claim 92

Use of a tumour marker antigen selected from the group consisting of MMP9 AIF1, EpCAM and CDKN1 B in a method of detecting liver cancer in a mammalian subject by detecting an autoantibody immunologically specific for MMP9, AIF1, EpCAM or CDKN1 B in a test sample comprising a bodily fluid from the mammalian subject, which method comprises the steps of:
(a) contacting the test sample with a tumour marker antigen selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1 B; and
(b) determining the presence or absence of complexes of the tumour marker antigen bound to autoantibodies present in the test sample;
whereby the presence of said complexes is indicative of the presence of liver cancer.

### Claim 93 (depends on 1)

A kit suitable for performing the method of claim 1, wherein the kit comprises:
(a) one or more tumour marker antigens; and
(b) a reagent capable of detecting complexes of the tumour marker antigen bound to autoantibodies present in the test sample.

### Claim 94

A kit for the detection of autoantibodies in a test sample comprising a bodily fluid from a mammalian subject comprising:
(a) a tumour marker antigen selected from the group consisting of MMP9, AIF1, EpCAM and CDKN1B; and
(b) a reagent capable of detecting complexes of the tumour marker antigen bound to autoantibodies present in the test sample.

### Claim 95 (depends on 93)

The kit of claim 93, further comprising:
(c) means for contacting the tumour marker antigen with a test sample comprising a bodily fluid from a mammalian subject.

### Claim 96 (depends on 95)

The kit of claim 95, wherein the means for contacting the tumour marker antigen with a test sample comprising a bodily fluid from a mammalian subject comprises the tumour marker antigen immobilised on a chip, slide, plate, wells of a microtitre plate, bead, membrane or nanoparticle.

### Claim 97 (depends on 93)

The kit of claim 93, wherein the tumour marker antigen is present within a panel of two or more tumour marker antigens.

### Claim 98 (depends on 97)

The kit of claim 97, wherein the panel comprises two or more tumour marker antigens which are distinct antigens.

### Claim 99 (depends on 97)

The kit of claim 97, wherein the panel of two or more tumour marker antigens comprises MMP9, AIF1, EpCAM and CDKN1B.

### Claim 100 (depends on 97)

The kit of claim 97, wherein the panel of two or more tumour marker antigens comprises one or more tumour marker antigens selected from the group consisting of NY-ESO-1, vimentin, HSPA4, transferrin, HNRNP-L, HSPD1, HNRNP-A2, SALL4, Cyclin B1, AFP, NPM1, YWHAZ, DDX3X, p62, CAGE, MAGE A4, RalA, GBU4-5, SOX2, AKR1B10, ApoA1, BCL2, CD44, CK18, CPS1, FUCA1, GLUL, HSPA2, IL-8, MDM2, PEBP1, prolactin, RGN, SPP1, SSX2 and TGFB1.

### Claim 101 (depends on 100)

The kit of claim 100, wherein the panel of two or more tumour marker antigens comprises:
(i) CAGE, NY-ESO-1, MMP9, transferrin, MAGE A4, RalA, HSPA4, SALL4, Cyclin B1, EpCAM, DDX3X, and AIF1; or
(ii) CAGE, NY-ESO-1, MMP9, transferrin, MAGE A4, RalA, HSPA4, SALL4, Cyclin B1, EpCAM, DDX3X, AIF1, SOX2 and AFP; or
(iii) MMP9, AIF1, EpCAM, NY-ESO-1, HSPA4, vimentin, HNRNP-L and transferrin.

### Claim at position 48

102-106. (canceled)

### Claim 107 (depends on 100)

The kit of claim 100, wherein the panel of two or more tumour marker antigens comprises:
(i) AIF1, EpCAM, HSPA4 and CPS1; or
(ii) AIF1, CAGE, HSPD1, SOX2, SALL4, HSPA4 and transferrin.

### Claim 108

(canceled)

### Claim 109 (depends on 107)

The kit of claim 107, wherein the subject is female.

### Claim 110 (depends on 100)

The kit of claim 100, wherein the panel of two or more tumour marker antigens comprises:
(i) EpCAM, NY-ESO-1, vimentin, HSPA2, HSPA4 and HNRNP-L; or
(ii) EpCAM, CAGE, SOX2, RalA, MAGE A4, DDX3X and NY-ESO-1.

### Claim 111

(canceled)

### Claim 112 (depends on 110)

The kit of claim 110, wherein the subject is male.

### Claim 113 (depends on 100)

The kit of claim 100, wherein the panel of two or more tumour marker antigens comprises MMP9, AIF1, EpCAM, DDX3X, SALL4, MAGE A4, NY-ESO-1, CAGE, RalA and SOX2.

### Claim at position 56

114-120. (canceled)

### Claim 121 (depends on 100)

The kit of claim 100, wherein the panel of two or more tumour marker antigens comprises NY-ESO-1, vimentin, HSPA4, transferrin, HNRNP-L, HSPD1, HNRNP-A2, SALL4, Cyclin B1, AFP, NPM1, YWHAZ, DDX3X, p62, CAGE, MAGE A4, RalA, GBU4-5, SOX2, AKR1B10, ApoA1, BCL2, CD44, CK18, CPS1, FUCA1, GLUL, HSPA2, IL-8, MDM2, PEBP1, prolactin, RGN, SPP1, SSX2 and TGFB1.

### Claim 122 (depends on 121)

The kit of claim 121, wherein the panel of two or more tumour marker antigens comprises MMP9, AIF1, EpCAM, CDKN1B, NY-ESO-1, vimentin, HSPA4, transferrin, HNRNP-L, HSPD1, HNRNP-A2, SALL4, Cyclin B1, AFP, NPM1, YWHAZ, DDX3X, p62, CAGE, MAGE A4, RalA, GBU4-5, SOX2, AKR1B10, ApoA1, BCL2, CD44, CK18, CPS1, FUCA1, GLUL, HSPA2, IL-8, MDM2, PEBP1, prolactin, RGN, SPP1, SSX2 and TGFB1.

### Claim 123

(canceled)

### Claim 124 (depends on 93)

The kit of claim 93 for the detection of liver cancer.

### Claim 125 (depends on 93)

The kit of claim 93, wherein the bodily fluids is selected from the group consisting of plasma, serum, whole blood, urine, sweat, lymph, faeces, cerebrospinal fluid, ascites fluid, pleural effusion, seminal fluid, sputum, nipple aspirate, post-operative seroma, saliva, amniotic fluid, tears and wound drainage fluid.