# US 2024/0201191 A1 · Citrullinated proteins as biomarkers and therapy targets for cancer

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- Number: US 2024/0201191 A1
- Document ID: US20240201191A1
- Status: Published application
- Kind: A1
- Application number: 18/285,205
- Filed: 2022-03-30
- Published: 2024-06-20
- Earliest claimed: 2022-03-30
- Applicants: Board of Regents, the University of Texas System
- Inventors: Samir HANASH; Hiroyuki KATAYAMA
- Web page: https://patentgrep.com/patent/US20240201191A1

## CPC

- G01N 33/575: for cancer
- G01N 33/5758: {involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites}
- C07K 14/4748: {Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE}
- 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 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 2333/988: Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
- G01N 2440/18: citrullination
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Provided herein are methods of detecting cancer, diagnosing a subject with cancer, treating a subject with cancer, and reducing the risk of or preventing cancer in a subject. The methods are based in part on the discovery of dysregulated protein citrullination by protein arginine deaminase enzymes in cancer. Also provided are citrullinated peptides and kits that are expressed at a higher level in cancer cells than in corresponding normal cells. Kits and systems relating to these methods are also provided.

## Claims

### Claim 1

A method, comprising:
providing a plasma sample from a patient suffering from a cancer or suspected of suffering from the cancer;
incubating the plasma sample with at least one citrullinated protein selected from the group consisting of the proteins encoded by genes listed in Table 1, the proteins encoded by genes listed in Table 2, and the proteins encoded by genes listed in Table 10, under conditions sufficient for any autoantibodies against the citrullinated protein(s) that may be present in the plasma sample, to bind to the citrullinated protein(s);
incubating the citrullinated protein(s) and any bound autoantibodies against it/them with a detectable label, under conditions in which the detectable label will bind to the bound autoantibodies and will substantially not bind to other molecules;
detecting the detectable label bound to the bound autoantibodies;
classifying the patient as suffering from the cancer in response to the detected amount of the detectable label bound to the bound autoantibodies being equal to or greater than a threshold; and
classifying the patient as not suffering from the cancer in response to the detected amount of the detectable label bound to the bound autoantibodies being less than the threshold.

### Claim 2 (depends on 1)

The method of claim 1, wherein:
the cancer is selected from the group consisting of breast cancers, lung cancers, skin cancers, endometrial cancers, ovarian cancers, and colorectal cancers.

### Claim 3 (depends on 2)

The method of claim 2, wherein:
the citrullinated protein(s) is/are selected from the group consisting of citrullinated vimentin and citrullinated α-enolase.

### Claim 4 (depends on 1)

The method of claim 1, wherein the detectable label is an antibody against the autoantibody, wherein the antibody comprises a fluorescent moiety.

### Claim 5 (depends on 1)

The method of claim 1, further comprising administering, to the patient, a cancer treatment selected from the group consisting of surgical resection, chemotherapy, monoclonal antibody therapy, checkpoint inhibitor therapy, oncolytic virus therapy, thermal therapy, radiotherapy, a cancer vaccine comprising the citrullinated protein(s), a targeted therapy against the citrullinated protein(s),and two or more thereof, in response to classifying the patient as suffering from the cancer; and
testing the patient for at least one other medical condition, in response to classifying the patient as not suffering from the cancer.

### Claim 6

A kit, comprising:
a substrate;
at least one citrullinated protein selected from the group consisting of the proteins encoded by genes listed in Table 1, the proteins encoded by genes listed in Table 2, and the proteins encoded by genes listed in Table 10; and
instructions for performing a method, comprising instructions to:
provide a plasma sample from a patient suffering from a cancer or suspected of suffering from the cancer;
incubate the plasma sample with the citrullinated protein(s) under conditions sufficient for any autoantibodies against the citrullinated protein(s) that may be present in the plasma sample, to bind to the citrullinated protein(s);
incubate the citrullinated protein(s) and any bound autoantibodies against it/them with a detectable label, under conditions wherein the detectable label will bind to the bound autoantibodies and will substantially not bind to other molecules;
detect the label bound to the bound autoantibodies;
classify the patient as suffering from the cancer, in response to the detected amount of the label bound to the bound autoantibodies being equal to or greater than a threshold; and
classify the patient as not suffering from the cancer, in response to the detected amount of the label bound to the bound autoantibodies being less than the threshold.

### Claim 7 (depends on 6)

The kit of claim 6, wherein:
the cancer is selected from the group consisting of breast cancers, lung cancers, skin cancers, endometrial cancers, ovarian cancers, and colorectal cancers.

### Claim 8 (depends on 7)

The kit of claim 7, wherein:
the citrullinated protein is selected from the group consisting of citrullinated vimentin and citrullinated α-enolase.

### Claim 9 (depends on 6)

The kit of claim 6, wherein the detectable label is an antibody against the autoantibody, wherein the antibody comprises a fluorescent moiety.

### Claim 10

A method, comprising:
providing a tissue sample from a patient suffering from a cancer or suspected of suffering from the cancer;
assaying the tissue sample for at least one citrullinated protein selected from the group consisting of the proteins encoded by genes listed in Table 1, the proteins encoded by genes listed in Table 2, and the proteins encoded by genes listed in Table 10;
classifying the patient as suffering from the cancer in response to the tissue sample containing an amount of the citrullinated protein(s) equal to or greater than a threshold; and
classifying the patient as not suffering from the cancer in response to the tissue sample containing an amount of the citrullinated protein(s) less than the threshold.

### Claim 11 (depends on 10)

The method of claim 10, wherein:
the cancer is selected from the group consisting of breast cancers, lung cancers, skin cancers, endometrial cancers, ovarian cancers, and colorectal cancers.

### Claim 12 (depends on 11)

The method of claim 11, wherein:
the citrullinated protein(s) is/are selected from the group consisting of citrullinated vimentin and citrullinated α-enolase.

### Claim 13 (depends on 10)

The method of claim 10, wherein the assaying comprises lysing cells of the tissue sample, to yield a tissue sample cell lysate, and isolating a protein fraction from the tissue sample cell lysate.

### Claim 14 (depends on 10)

The method of claim 10, wherein the assaying comprises liquid chromatography-mass spectrometry (LC-MS).

### Claim 15

A kit, comprising:
a cell lysis agent; and
instructions for performing a method, comprising instructions to:
provide a tissue sample from a patient suffering from a cancer or suspected of suffering from the cancer;
lyse cells of the tissue sample by exposing the tissue sample to the cell lysis agent;
assay the tissue sample for at least one citrullinated protein selected from the group consisting of the proteins encoded by genes listed in Table 1, the proteins encoded by genes listed in Table 2, and the proteins encoded by genes listed in Table 10;
classify the patient as suffering from the cancer, in response to the tissue sample containing an amount of the citrullinated protein(s) equal to or greater than a threshold; and
classify the patient as not suffering from the cancer, in response to the tissue sample containing an amount of the citrullinated protein(s) less than the threshold.

### Claim 16 (depends on 15)

The kit of claim 15, wherein:
the cancer is selected from the group consisting of breast cancers, lung cancers, skin cancers, endometrial cancers, ovarian cancers, and colorectal cancers.

### Claim 17 (depends on 16)

The kit of claim 16, wherein:
the citrullinated protein(s) is/are selected from the group consisting of citrullinated vimentin and citrullinated α-enolase.

### Claim 18 (depends on 15)

The kit of claim 15, wherein the assaying comprises liquid chromatography-mass spectrometry (LC-MS).

### Claim 19

A method, comprising:
providing a tumor sample from a patient suffering from a cancer;
assaying the tumor sample for at least one citrullinated amino acid sequence selected from the group consisting of sequences and corresponding modifications listed in Table 2, Table 9, and/or Table 10, and sequences having at least 70% identity to the sequences listed in Table 2, Table 9, and/or Table 10 and comprising at least one arginine residue; and
presenting to the immune system of the patient at least one peptide, wherein each peptide comprises at least one of the citrullinated amino acid sequences, in response to the tumor sample containing an amount of the citrullinated amino acid sequence(s) equal to or greater than a threshold.

### Claim 20 (depends on 19)

The method of claim 19, wherein:
the cancer is selected from the group consisting of breast cancers, lung cancers, skin cancers, endometrial cancers, ovarian cancers, and colorectal cancers.

### Claim 21 (depends on 20)

The method of claim 20, wherein:
the citrullinated amino acid sequences(s) is/are selected from the group consisting of
(SEQ ID NO: 43)
GVMVSHR*SGETEDTF,
(SEQ ID NO: 44)
LAQANGWGVMVSHR*SGETEDTF,
and
(SEQ ID NO: 45)
AVEKGVPLYR*HIADLAGNS, wherein R* is citrulline.

### Claim 22 (depends on 19)

The method of claim 19, wherein the assaying comprises lysing cells of the tumor sample, to yield a tumor sample cell lysate, and quantifying the amount of the citrullinated amino acid sequence(s) in the tumor sample cell lysate.

### Claim 23 (depends on 19)

The method of claim 19, further comprising:
administering, to the patient, an additional cancer therapy selected from the group consisting of surgical resection, chemotherapy, monoclonal antibody therapy, checkpoint inhibitor therapy, oncolytic virus therapy, thermal therapy, radiotherapy, and two or more thereof.

### Claim 24

A kit, comprising:
at least one peptide, wherein each peptide comprises at least one citrullinated amino acid sequence selected from the group consisting of sequences and corresponding modifications listed in Table 2, Table 9, and/or Table 10, and sequences having at least 70% identity to the sequences listed in Table 2, Table 9, and/or Table 10 and comprising at least one arginine residue; and
instructions for performing a method, comprising instructions to:
provide a tumor sample from a patient suffering from a cancer;
assay the tumor sample for the citrullinated amino acid sequence(s) contained in each of the peptide(s); and
present to the immune system of the patient one or more of the peptides, in response to the tumor sample containing an amount equal to or greater than a threshold of the citrullinated amino acid sequence contained in the peptide(s).

### Claim 25 (depends on 24)

The kit of claim 24, wherein:
the cancer is selected from the group consisting of breast cancers, lung cancers, skin cancers, endometrial cancers, ovarian cancers, and colorectal cancers.

### Claim 26 (depends on 25)

The kit of claim 25, wherein:
the citrullinated amino acid sequences(s) is/are selected from the group consisting of
(SEQ ID NO: 43)
GVMVSHR*SGETEDTF,
(SEQ ID NO: 44)
LAQANGWGVMVSHR*SGETEDTF,
and
(SEQ ID NO: 45)
AVEKGVPLYR*HIADLAGNS, wherein R* is citrulline.

### Claim 27 (depends on 24)

The kit of claim 24, further comprising a cell lysis agent, and wherein the instructions comprise instructions to assay the tumor sample by lysing cells of the tumor sample, to yield a tumor sample cell lysate, and quantifying the amount of the citrullinated amino acid sequence(s) in the tumor sample cell lysate.

### Claim 28 (depends on 24)

The kit of claim 24, wherein the instructions further comprise instructions to:
administer, to the patient, an additional cancer therapy selected from the group consisting of surgical resection, chemotherapy, monoclonal antibody therapy, checkpoint inhibitor therapy, oncolytic virus therapy, thermal therapy, radiotherapy, and two or more thereof.

### Claim 29 (depends on 28)

The kit of claim 28, wherein the additional cancer therapy is selected from the group consisting of chemotherapy, monoclonal antibody therapy, checkpoint inhibitor therapy, and oncolytic virus therapy, and the kit further comprises one or more of a chemotherapeutic agent, a monoclonal antibody, a checkpoint inhibitor, or an oncolytic virus.

### Claim 30

A method, comprising:
providing a tumor sample from a patient suffering from a cancer;
assaying the tumor sample for a citrullinated protein encoded by a gene selected from the group consisting of genes listed in Table 1 as having a plasma membrane location; and
administering to the patient an anti-cancer agent targeting the citrullinated protein, in response to the tumor sample containing an amount of the citrullinated protein equal to or greater than a threshold.

### Claim 31 (depends on 30)

The method of claim 30, wherein:
the cancer is selected from the group consisting of breast cancers, lung cancers, skin cancers, endometrial cancers, ovarian cancers, and colorectal cancers.

### Claim 32 (depends on 30)

The method of claim 30, wherein the assaying comprises lysing cells of the tumor sample, to yield a tumor sample cell lysate, and quantifying the amount of the citrullinated amino acid sequence(s) in the tumor sample cell lysate.

### Claim 33 (depends on 30)

The method of claim 30, further comprising:
administering, to the patient, an additional cancer therapy not targeting the citrullinated protein, selected from the group consisting of surgical resection, chemotherapy, monoclonal antibody therapy, checkpoint inhibitor therapy, oncolytic virus therapy, thermal therapy, radiotherapy, and two or more thereof.

### Claim 34

A kit, comprising:
an anti-cancer agent targeting a citrullinated protein encoded by a gene selected from the group consisting of genes listed in Table 1 as having a plasma membrane location; and
instructions for performing a method, comprising instructions to:
provide a tumor sample from a patient suffering from a cancer;
assay the tumor sample for the citrullinated protein; and
administer to the patient an anti-cancer agent targeting the citrullinated protein, in response to the tumor sample containing an amount of the citrullinated protein equal to or greater than a threshold.

### Claim 35 (depends on 34)

The kit of claim 34, wherein:
the cancer is selected from the group consisting of breast cancers, lung cancers, skin cancers, endometrial cancers, ovarian cancers, and colorectal cancers.

### Claim 36 (depends on 34)

The kit of claim 34, further comprising a cell lysis agent, and wherein the instructions comprise instructions to assay the tumor sample by lysing cells of the tumor sample, to yield a tumor sample cell lysate, and quantifying the amount of the citrullinated amino acid sequence(s) in the tumor sample cell lysate.

### Claim 37 (depends on 34)

The kit of claim 34, wherein the instructions further comprise instructions to:
administer, to the patient, an additional cancer therapy selected from the group consisting of surgical resection, chemotherapy, monoclonal antibody therapy, checkpoint inhibitor therapy, oncolytic virus therapy, thermal therapy, radiotherapy, and two or more thereof.

### Claim 38 (depends on 37)

The kit of claim 37, wherein the additional cancer therapy is selected from the group consisting of chemotherapy, monoclonal antibody therapy, checkpoint inhibitor therapy, and oncolytic virus therapy, and the kit further comprises one or more of a chemotherapeutic agent, a monoclonal antibody, a checkpoint inhibitor, or an oncolytic virus.

### Claim 39

An isolated peptide comprising at least 70% sequence identity to a peptide selected from the group consisting of peptides listed in Table 2, Table 9, and Table 10.

### Claim 40 (depends on 39)

The peptide of claim 39, wherein the peptide comprises at least 6 contiguous amino acids of the peptide selected from the group consisting of peptides listed in Table 2, Table 9, and Table 10.

### Claim 41 (depends on 39)

The peptide of claim 39, wherein the peptide is 15 amino acids or fewer in length.

### Claim 42 (depends on 39)

The peptide of claim 39, wherein the peptide is 9 amino acids in length.

### Claim 43 (depends on 39)

The peptide of claim 39, wherein the peptide is 15 amino acids in length.

### Claim 44 (depends on 39)

The peptide of claim 39, wherein the peptide is 20-25 amino acids.

### Claim 45 (depends on 39)

The peptide of claim 39, wherein the peptide is 22-24 amino acids.

### Claim 46 (depends on 39, 40, 41, 42, 43, 44, 45)

The peptide of any one of claims 39-45, wherein the peptide is immunogenic.

### Claim 47 (depends on 39, 40, 41, 42, 43, 44, 45)

The peptide of any one of claims 39-45, wherein the peptide is modified.

### Claim 48 (depends on 47)

The peptide of claim 47, wherein the modification comprises conjugation to a molecule.

### Claim 49 (depends on 48)

The peptide of claim 48, wherein the molecule comprises an antibody, a lipid, an adjuvant, or a detection moiety.

### Claim 50 (depends on 39, 40, 41, 42, 43, 44, 45)

The peptide of any of claims 39-45, wherein the peptide has at least 90% sequence identity to the peptide selected from the group consisting of peptides listed in Table 2, Table 9, and Table 10.

### Claim 51 (depends on 39, 40, 41, 42, 43, 44, 45)

The peptide of any of claims 39-45, wherein the peptide has 1, 2 or 3 substitutions relative to the peptide selected from the group consisting of peptides listed in Table 2, Table 9, and Table 10.

### Claim 52 (depends on 39, 40, 41, 42, 43, 44, 45)

The peptide of any one of claims 39-45, wherein the peptide comprises 100% sequence identity to the peptide selected from the group consisting of peptides listed in Table 2, Table 9, and Table 10.