# US 2022/0050110 A1 · Use of mcm5 as a marker in semen for prostate cancer

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- Number: US 2022/0050110 A1
- Document ID: US20220050110A1
- Status: Published application
- Kind: A1
- Application number: 17/413,878
- Filed: 2019-12-19
- Published: 2022-02-17
- Earliest claimed: 2018-12-20
- Assignees: Arquer Diagnostics Limited
- Applicants: Arquer Diagnostics Limited
- Inventors: Jacqueline STOCKLEY; Cheryl NYBERG
- Web page: https://patentgrep.com/patent/US20220050110A1

## CPC

- G01N 33/57555: of the prostate
- 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)

## Abstract

The present invention relates to a method for detecting the presence or absence of a prostate cancer in a subject, the method comprising steps of: providing a semen sample obtained from the subject; and detecting at least one biomarker or determining the concentration of at least one biomarker. The present invention also relates to lysis buffers, monoclonal antibodies, and kits that can be used in such methods.

## Claims

### Claim 1

A method for detecting the presence or absence of a prostate cancer in a subject, the method comprising steps of:
providing a semen sample obtained from the subject; and
detecting at least one biomarker or determining the concentration of at least one biomarker.

### Claim 2 (depends on 1)

The method of claim 1, further comprising a step of treating the semen sample to release the at least one biomarker from cells in the semen sample.

### Claim 3 (depends on 1, 2)

The method of claim 1 or 2, wherein the at least one biomarker comprises or consists of an MCM protein.

### Claim 4 (depends on 3)

The method of claim 3, wherein the MCM protein is MCM5.

### Claim 5

The method of any one of the preceding claims, wherein the semen sample is obtained after a period of abstinence.

### Claim 6

The method of any one of the preceding claims, wherein:
detecting the at least one biomarker comprises determining whether the concentration of the biomarker is higher than a pre-defined cut-off.

### Claim 7 (depends on 6)

The method of claim 6, wherein:
(a) the pre-defined cut-off is between 40 pg/mL and 100 pg/mL, between 50 pg/mL and 75 pg/mL, between 60 pg/mL and 70 pg/mL, or around 65 pg/mL;
(b) prostate cancer is likely to be present if the concentration of the biomarker is higher than the pre-defined cut-off; and/or
(c) the method is a method for diagnosing prostate cancer, and the method further comprises a step of diagnosing the subject as having prostate cancer if the concentration of the biomarker is higher than the pre-defined cut-off.

### Claim 8

The method of any one of the preceding claims, wherein the concentration of the at least one biomarker is compared to a reference, and prostate cancer is likely to be present if the concentration of the at least one biomarker is abnormal compared to or higher than the reference, optionally wherein the method is a method for diagnosing prostate cancer, and the method further comprises a step of diagnosing the subject as having prostate cancer if the concentration of the at least one biomarker is higher than the reference.

### Claim 9

The method of any one of the preceding claims, wherein:
(a) determining the concentration of the at least one biomarker or determining whether the concentration of the at least one biomarker is higher than a pre-defined cut-off is carried out using an ELISA assay;
(b) the method has a sensitivity of greater than 40%, greater than 50%, greater than 60%, between 40% and 100%, between 50% and 100%, between 60% and 100%, or around 61%;
(c) the method has a sensitivity of greater than 40%, greater than 50%, greater than 60%, between 40% and 100%, between 50% and 100%, between 60% and 100%, or around 61% using a pre-defined cut-off of 65 pg/mL;
(d) the method has a specificity of greater than 70%, greater than 75%, between 70% and 100%, between 75% and 100%, or around 79%;
(e) the method has a specificity of greater than 70%, greater than 75%, between 70% and 100%, between 75% and 100%, or around 79% using a pre-defined cut-off of 65 pg/mL;
(f) the method has a positive predictive value (PPV) of greater than 60%, greater than 70%, greater than 75%, between 60% and 100%, between 70% and 100%, between 75% and 100%, or around 78%;
(g) the method has a positive predictive value (PPV) of greater than 60%, greater than 70%, greater than 75%, between 60% and 100%, between 70% and 100%, between 75% and 100%, or around 78% using a pre-defined cut-off of 65 pg/mL;
(h) the method has a negative predictive value (NPV) of greater than 55%, greater than 60%, between 55% and 100%, between 60% and 100%, or around 63%;
(i) the method has a negative predictive value (NPV) of greater than 55%, greater than 60%, between 55% and 100%, between 60% and 100%, or around 63% using a pre-defined cut-off of 65 pg/mL; and/or
(j) the method has greater sensitivity, specificity, NPV and/or PPV than an equivalent method carried out using a urine sample, optionally wherein the equivalent method carried out using a urine sample uses a cut-off of 3 pg/mL.

### Claim 10 (depends on 2, 3, 4, 5, 6, 7, 8, 9)

The method of any one of claims 2-9, wherein the step of treating the semen sample to release the at least one biomarker comprises exposing the semen sample to a lysis buffer capable of releasing the at least one biomarker from cells in the semen sample, optionally wherein:
(a) exposing the semen sample to a lysis buffer capable of releasing the at least one biomarker from cells in the semen sample is carried out on a pellet of the cells in the semen sample obtained by centrifuging the semen sample;
(b) exposing the semen sample to a lysis buffer capable of releasing the at least one biomarker from cells in the semen sample comprises:
passing the non-invasive sample through a filter for capturing cells, such that cells are captured in the filter;
passing a lysis buffer through the filter, such that the captured cells are exposed to the lysis buffer; and/or
incubating the filter for a period of time, such that the lysis buffer causes the cells to release the at least one biomarker;
(c) the lysis buffer is capable of releasing an MCM protein from cells in the semen sample, optionally wherein the lysis buffer is capable of releasing MCM5 from cells in the semen sample;
(d) the lysis buffer is capable of releasing an MCM protein from epithelial cells in the semen sample, optionally wherein the lysis buffer is capable of releasing MCM5 from epithelial cells in the semen sample;
(e) the lysis buffer is capable of releasing MCM5 from cells in the semen sample and does not denature the MCM5 protein;
(f) the lysis buffer is capable of releasing MCM5 from epithelial cells in the semen sample and does not denature the MCM5 protein; and/or
(g) the lysis buffer does not denature an antibody.

### Claim 11 (depends on 10)

The method of claim 10, wherein:
(a) the lysis buffer comprises a detergent;
(b) the lysis buffer comprises a detergent which comprises Triton X-100;
(c) the lysis buffer comprises a detergent which comprises Triton X-100 at a concentration of between 0.01% and 25%, between 0.01% and 10%, between 0.05% and 5%, between 0.1% and 2%, between 0.5% and 2%, between 0.75% and 1.25%, or about 1%;
(d) the lysis buffer comprises a detergent which comprises or consists of sodium deoxycholate;
(e) the lysis buffer comprises a detergent which comprises or consists of sodium deoxycholate at a concentration between 0.1% and 20%, between 0.1 and 10%, between 0.1 and 5%, between 0.5% and 5%, between 0.5% and 2.5%, between 0.75% and 2.5%, between 0.75% and 1.25%, or about 1%;
(f) the lysis buffer comprises a detergent which comprises or consists of sodium dodecyl sulphate (SDS);
(g) the lysis buffer comprises a detergent which comprises or consists of sodium dodecyl sulphate (SDS) at a concentration of between 0.001% and 10%, between 0.01% and 5%, between 0.05% and 5%, between 0.01% and 1%, between 0.05% and 1%, between 0.05% and 0.5%, between 0.075% and 0.25%, or about 0.1%;
(h) the lysis buffer comprises a lysis buffer which comprises a detergent which consists of Triton X-100, sodium deoxycholate, and sodium dodecyl sulphate (SDS);
(i) the lysis buffer comprises a detergent which consists of between 0.5% and 2% of Triton X-100, between 0.5% and 2% of sodium deoxycholate, and between 0.05% and 0.5% of sodium dodecyl sulphate (SDS);
(j) the lysis buffer comprises a buffer component;
(k) the lysis buffer comprises a buffer component which has a pH of between pH 4 and pH 9, between pH 5 and pH 8.5, between pH 6 and pH 8, between pH 6.5 and pH 8, between pH 7 and pH 8, between pH 7.3 and pH 7.9, between pH 7.4 and pH 7.8, between pH 7.5 and pH 7.7, or about pH 7.6; and/or maintains the pH of the lysis buffer at between pH 4 and pH 9, between pH 5 and pH 8.5, between pH 6 and pH 8, between pH 6.5 and pH 8, between pH 7 and pH 8, between pH 7.3 and pH 7.9, between pH 7.4 and pH 7.8, between pH 7.5 and pH 7.7, or about pH 7.6;
(l) the lysis buffer comprises a buffer component which comprises or consists of Tris;
(m) the lysis buffer comprises a buffer component which comprises or consists of Tris at a concentration greater than 1 mM, between 1 mM and 350 mM, between 5 and 200 mM, between 5 and 100 mM, between 5 and 50 mM, between 5 mM and 40 mM, between 5 mM and 35 mM, between 10 mM and 35 mM, between 15 mM and 35 mM, between 15 mM and 30 mM, between 20 mM and 30 mM, or about 25 mM;
(n) the lysis buffer comprises a buffer component which consists of Tris at a concentration of between 15 mM and 35 mM;
(o) the lysis buffer comprises a salt;
(p) the lysis buffer comprises sodium chloride;
(q) the lysis buffer comprises sodium chloride at a concentration between 10 mM and 350 mM, between 20 mM and 300 mM, between 50 mM and 250 mM, between 100 mM and 250, between 100 mM and 200 mM, between 125 mM and 175 mM, or about 150 mM;
(r) the lysis buffer comprises sodium chloride at a concentration of between 100 mM and 200 mM;
(s) the lysis buffer comprises:
(i) between 1 mM and 100 mM Tris;
(ii) between 50 mM and 300 mM sodium chloride;
(iii) between 0.1 and 5% sodium deoxycholate;
(iv) between 0.01 and 1% sodium dodecyl sulphate; and/or
(v) between 0.1 and 5% Triton-X100; and/or
(t) the lysis buffer comprises:
(i) between 10 mM and 40 mM Tris, optionally about 25 mM Tris;
(ii) between 100 mM and 200 mM sodium chloride, optionally about 150 mM sodium chloride;
(iii) between 0.5 and 2% sodium deoxycholate, optionally about 1% sodium deoxycholate;
(iv) between 0.05 and 0.5% sodium dodecyl sulphate, optionally about 0.1% sodium dodecyl sulphate; and/or
(v) between 0.5 and 2% Triton-X100, optionally about 1% Triton-X100.

### Claim 12 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The method of any one of claims 2-11, wherein the cells which release the at least one biomarker are epithelial cells, optionally epithelial cells which have been shed from the lining of the prostate gland of the subject.

### Claim 13

The method of any one of the preceding claims, wherein the step of detecting the at least one biomarker or determining the concentration of the at least one biomarker comprises:
exposing the semen sample to a first monoclonal antibody and/or a second monoclonal antibody; and
detecting the at least one biomarker bound to the first monoclonal antibody and/or the second monoclonal antibody or determining the concentration of the at least one biomarker bound to the first monoclonal antibody and/or the second monoclonal antibody, optionally wherein the first monoclonal antibody and the second monoclonal antibody bind to MCM5.

### Claim 14 (depends on 13)

The method of claim 13, wherein the first monoclonal antibody is an antibody which:
(i) binds to a polypeptide having an amino acid sequence of SEQ ID NO: 1;
(ii) comprises at least one Complementary Determining Region (CDR) selected from the group consisting of:
(a) 12A7 CDRH1 which has a sequence of SEQ ID NO: 9 or a sequence that differs from SEQ ID NO:9 by a single amino acid substitution;
(b) 12A7 CDRH2 which has a sequence of SEQ ID NO: 11 or a sequence that differs from SEQ ID NO:11 by a single amino acid substitution;
(c) 12A7 CDRH3 which has a sequence of SEQ ID NO: 13 or a sequence that differs from SEQ ID NO:13 by a single amino acid substitution;
(d) 12A7 CDRL1 which has a sequence of SEQ ID NO: 3 or a sequence that differs from SEQ ID NO:3 by a single amino acid substitution;
(e) 12A7 CDRL2 which has a sequence of SEQ ID NO: 5 or a sequence that differs from SEQ ID NO:5 by a single amino acid substitution; and
(f) 12A7 CDRL3 which has a sequence of SEQ ID NO: 7 or a sequence that differs from SEQ ID NO:7 by a single amino acid substitution;
(iii) comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 29;
(iv) comprises a light chain variable region sequence having a sequence at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 27; or
(v) competes with the antibody of (i), (ii), (iii), or (iv); and/or
the second monoclonal antibody is an antibody which:
(i) binds to a polypeptide having an amino acid sequence of SEQ ID NO: 2;
(ii) comprises at least one Complementary Determining Region (CDR) selected from the group consisting of:
(a) 4B4 CDRH1 which has a sequence of SEQ ID NO: 21 or a sequence that differs from SEQ ID NO:21 by a single amino acid substitution;
(b) 4B4 CDRH2 which has a sequence of SEQ ID NO: 23 or a sequence that differs from SEQ ID NO:23 by a single amino acid substitution;
(c) 4B4 CDRH3 which has a sequence of SEQ ID NO: 25 or a sequence that differs from SEQ ID NO:25 by a single amino acid substitution;
(d) 4B4 CDRL1 which has a sequence of SEQ ID NO: 15 or a sequence that differs from SEQ ID NO:15 by a single amino acid substitution;
(e) 4B4 CDRL2 which has a sequence of SEQ ID NO: 17 or a sequence that differs from SEQ ID NO:17 by a single amino acid substitution; and
(f) 4B4 CDRL3 which has a sequence of SEQ ID NO: 19 or a sequence that differs from SEQ ID NO:19 by a single amino acid substitution;
(iii) comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 33;
(iv) comprises a light chain variable region sequence having a sequence at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 31; or
(v) competes with the antibody of (i), (ii), (iii), or (iv).

### Claim 15 (depends on 13, 14)

The method of claim 13 or 14, wherein:
(a) the first monoclonal antibody and/or second monoclonal antibody has an affinity for MCM5 in the range of 0.001-1 nM;
(b) the first monoclonal antibody and/or the second monoclonal antibody is a Fab′2, a F′(ab)2, an Fv, a single chain antibody or a diabody;
(c) the first monoclonal antibody comprises 12A7 CDRH1, 12A7 CDRH2, and 12A7 CDRH3;
(d) the first monoclonal antibody comprises 12A7 CDRL1, 12A7 CDRL2, and 12A7 CDRL3;
(e) the first monoclonal antibody comprises a 12A7 CDRH1 which has a sequence of SEQ ID NO: 9, a 12A7 CDRH2 which has a sequence of SEQ ID NO: 11, and a 12A7 CDRH3 which has a sequence of SEQ ID NO: 13;
(f) the first monoclonal antibody comprises a 12A7 CDRL1 which has a sequence of SEQ ID NO: 3, a 12A7 CDRL2 which has a sequence of SEQ ID NO: 5, and a 12A7 CDRL3 which has a sequence of SEQ ID NO: 7;
(g) the second monoclonal antibody comprises 4B4 CDRH1, 4B4 CDRH2, and 4B4 CDRH3;
(h) the second monoclonal antibody comprises 4B4 CDRL1, 4B4 CDRL2 and 4B4 CDRL3;
(i) the second monoclonal antibody comprises a 4B4 CDRH1 which has a sequence of SEQ ID NO: 21, a 4B4 CDRH2 which has a sequence of SEQ ID NO: 23, and a 4B4 CDRH3 which has a sequence of SEQ ID NO: 25;
(j) the second monoclonal antibody has a 4B4 CDRL1 which has a sequence of SEQ ID NO: 15, a 4B4 CDRL2 which has a sequence of SEQ ID NO: 17, and a 4B4 CDRL3 which has a sequence of SEQ ID NO: 19;
(k) the first monoclonal antibody comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 29;
(l) the first monoclonal antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 29;
(m) the first monoclonal antibody comprises a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 27;
(n) the first monoclonal antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 27;
(o) the second monoclonal antibody comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 33;
(p) the second monoclonal antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 33; the second monoclonal antibody comprises a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 31; and/or
(r) the second monoclonal antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 31.

### Claim 16

Use of a lysis buffer as defined in any one of the preceding claims in a method of detecting the presence of absence of prostate cancer in a subject.

### Claim 17

Use of a first monoclonal antibody as defined in any one of the preceding claims and/or a second monoclonal antibody as defined in any one of the preceding claims in a method of detecting the presence or absence of prostate cancer in a subject.

### Claim 18

Use of a kit in a method of detecting the presence or absence of prostate cancer in a subject, wherein the kit comprises:
(a) a lysis buffer as defined in any one of the preceding claims;
(b) a first monoclonal antibody as defined in any one of the preceding claims; and/or
(c) a second monoclonal antibody as defined in any one of the preceding claims.

### Claim 19

A kit comprising:
(a) a lysis buffer as defined in any one of the preceding claims;
(b) a first monoclonal antibody as defined in any one of the preceding claims; and/or
(c) a second monoclonal antibody as defined in any one of the preceding claims; and
(d) instructions for use of the lysis buffer and/or the first monoclonal antibody and/or the second monoclonal antibody in a method of detecting the presence or absence of prostate cancer in a subject.

### Claim 20

The method, use or kit of any one of the preceding claims, wherein the prostate cancer is grade 4/5.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

The method, use or kit of any one of the preceding claims 1-20, wherein the prostate cancer is grade 1-3.