EA045060B1 - PROTEIN MARKER FOR DIAGNOSTICS OF PROSTATE CANCER, METHOD FOR MEASURING THE QUANTITY OF MARKER AND ALGORITHM FOR INTERPRETING THE RESULT - Google Patents

Description

The invention relates to the field of biology and medicine, namely to oncology, and can be used for diagnosing prostate cancer.

State of the art

After skin cancer, prostate cancer (prostate cancer, PCa) is the most commonly diagnosed form of cancer. In many countries, prostate cancer is the second/third leading cause of death in men over 40 years of age. Currently, it is believed that the most important factor in the fight against prostate cancer is early diagnosis.

The traditional and most common method for detecting prostate cancer is a test for PSA (prostate specific antigen PSA) in blood serum (Stamey T.A. et al. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate // N. Engl. J. Med 1987 Oct 8;317(15):909-16). However, over 30 years of clinical practice, a large amount of statistical data has accumulated indicating the low specificity of PSA as a tumor marker, which manifests itself in a significant number of false-positive results. Therefore, the attention of many researchers is focused on finding new tumor markers.

Two different methods for diagnosing prostate cancer may be in demand in medical practice: the first is non-invasive, fast, easy, implemented on standard clinical laboratory equipment, which can be used for routine/serial tests during clinical examination, examination of the population, monitoring of risk groups, monitoring of relapses, monitoring course of the disease, monitoring of therapy, etc.; the second is the method required for final differential analysis, for which the only critical parameter is close to 100% accuracy.

Currently, a number of prostate cancer biomarkers are being actively studied (for example, PCA3, TMPRSS2:ERG, PSCA (prostate stem cell antigen), AMCAR), which are determined by non-invasive techniques by measuring their amount in urine (see, for example, Koo K.M. et al Colorimetric TMPRSS2ERG Gene Fusion Detection in Prostate Cancer Urinary Samples via Recombinase Polymerase Amplification // Theranostics., 2016 Jun 15; 6(9):1415-24; Deras I.L. et al., PCA3: a molecular molecular assay for predicting prostate biopsy outcome // J. Urol. 2008 Apr; 179(4):1587-92). However, these markers require the use of rather expensive methods based on polymerase chain reaction and/or immunohistochemistry for their detection, and these tests are difficult to perform in a standard clinical laboratory.

Recently, the possibility of diagnosing prostate cancer using urine exosome biomarkers has been actively studied. For example, US Pat. No. 7,897,356 B2 discloses a method for diagnosing prostate cancer by identifying a biosignal on exosomes, including capture and isolation of exosomes using common exosome surface proteins CD63, CD9 or CD81, and then qualitative and quantitative analysis of the level of prostate cancer-specific markers PSMA, PCSA, EpCam, B7H3 (CD276). Also known is a method and kit for the quantitative assessment and determination of exosomes for the diagnosis of prostate cancer and prostate hyperplasia (US 20130196355 A1), based on an enzyme-linked immunosorbent assay using anti-Rab5 as a capture antibody to isolate all exosomes, from which exosomes are subsequently isolated using an anti-PSA detection antibody and compare the level of such exosomes with a reference level. However, firstly, a biomarker suitable for non-invasive, rapid PCa diagnostics implemented on standard clinical laboratory equipment has not been found; and secondly, the isolation of exosomes, necessary for the implementation of the above methods, is a very non-trivial task that cannot be solved for most clinical laboratories. Thus, despite existing research on the diagnosis of prostate cancer, the medical practice of clinical examination of the population, as well as the tasks of monitoring risk groups, the course of the disease, its therapy and relapses require a routine method for analyzing prostate cancer, implemented on standard equipment.

Disclosure of the Invention

The objective and technical result of the invention is to find a new diagnostic marker for prostate cancer (PCa), allowing for non-invasive diagnosis of the disease, as well as to develop on its basis a simple, sensitive, reliable method for diagnosing prostate cancer, applicable in a standard clinical laboratory of a clinical or outpatient medical institution.

This problem is solved through the use of a new biomarker for the diagnosis of prostate cancer, which is alpha V integrin or its fragment, detected in the patient’s whole urine. According to the invention, integrin alpha V or a fragment thereof can be used as a biomarker by identifying them both as individual subunits and as part of integrin heterodimers, such as αγβ1, αγβ3, αγβ5, αγβ6 and/or αγβ8.

The problem is also solved by implementing a method for diagnosing prostate cancer, which includes identifying alpha V integrin or its fragment in the patient’s whole urine.

In this case, when alpha V integrin or its fragment is detected in whole urine in an amount of 20 ng/ml, the absence of prostate cancer is diagnosed.

In some embodiments of the invention, when implementing a method for diagnosing prostate cancer, additional clinical, laboratory and/or instrumental examinations are performed.

- 1 045060

In some particular embodiments of the invention, additional clinical, laboratory and/or instrumental testing is performed if the amount of alpha V integrin or fragment thereof detected in the patient's urine is 20 ng/ml or less than this level.

The problem is also solved by developing a kit for diagnosing prostate cancer in whole patient urine, which includes at least one antibody or a functional fragment thereof that is specific to alpha V integrin or a fragment thereof and does not have cross-specificity to other urine peptides.

In some embodiments of the invention, the kit is a test system for performing an enzyme-linked immunosorbent assay.

Brief description of graphic materials

Fig. 1. Results of measurements of alpha V integrin in the morning urine of men over 50 years old: n - number of measurements. Horizontal line - 20 ng/ml biomarker.

Fig. 2. Results of mathematical processing of the data shown in Fig. 1. On the X axis - the concentration of alpha V integrin, ng/ml, on the Y axis - the probability of detecting alpha V integrin in the urine of different groups of subjects. Vertical lines are mathematical expectations.

Terms and Definitions

As used herein, the terms includes and including are interpreted to mean includes, among other things. These terms are not intended to be construed as consisting only of.

The term PCa stands for prostate cancer, BPH refers to benign formations of the prostate (adenoma, prostatitis), and the term normal describes samples obtained from individuals over 50 years of age without prostate pathologies.

The term whole urine refers to urine samples, both untreated and samples obtained after centrifugation, filtration, or other procedures that separate the soluble components of urine from the insoluble fraction.

By marker or biomarker, unless otherwise specified, in this description we mean integrin alpha V (UniProtKB identification number P06756-1) and its isoforms (P06756-2; P06756-3). Integrin alpha V (other names - aV, CD51, MSK8, vitronectin receptor a (VNRa)) is a membrane protein, a glycoprotein from the integrin superfamily, a product of the ITGaV gene, located on chromosome 2 at position 2q31-q32.

As used herein, the term antibody refers to a whole immunoglobulin or a fragment thereof, for example a Fab or (Fab') ₂ type, that retains antigen-binding activity. This may be a naturally occurring antibody or an antibody produced by immunization (including, for example, a chimeric antibody or a humanized antibody), or a recombinant antibody constructed using genetic engineering techniques.

In the present description, an antibody or a fragment thereof means an antibody that specifically binds to alpha V integrin or a fragment thereof and does not have cross-specificity to other proteins (peptides, fragments thereof) in urine, i.e. an antibody that has (significantly) greater affinity for alpha V integrin or a fragment thereof than for other proteins (peptides, fragments thereof) in urine.

The diagnostic kit of the present invention may use antibodies that can be obtained from suppliers or these antibodies can be obtained by the above or other methods.

Without further elaboration, it is assumed that, based on the above description, one skilled in the art is capable of practicing the present invention in its entirety. The following options are illustrative only. They do not limit the disclosure of the present invention in any way. All cited publications are included by reference.

The possibility of objective manifestation of a technical result when using the invention is confirmed by reliable data given in examples containing experimental information obtained in the process of conducting research using methods accepted in this field. The essence of the invention is illustrated by graphic materials.

The following examples are provided for the purpose of illustrating the method according to the present invention and should not be construed as limiting the scope of the invention in any way.

Detailed Disclosure of the Invention

Integrin alpha V (HGNC: 6150; Entrez Gene: 3685; Ensembl: ENSG00000138448; OMIM: 193210; UniProtKB: P06756) is a membrane protein, a glycoprotein from the integrin superfamily, the product of the ITGaV gene, located on the 2nd chromosome at position 2q31-q32. Integrin alpha V consists of 1048 amino acids. The mature alpha V integrin molecule is a glycoprotein with a length of 1018 amino acids (see SEQ ID NO: 1), the molecular weight of the protein part is 116.0 kDa.

The mature alpha V integrin molecule forms a heterodimeric complex with beta 1, beta 3, beta 5, beta 6 or beta 8 integrins. Alpha V integrins are receptors for a number of ligands recognizing the sequence arginine-glycine-aspartate (R-G-D). Alpha V integrins are involved in many processes of embryonic development, angiogenesis and osteoporosis.

In the course of the studies, it was unexpectedly discovered that the quantitative content of alpha V integrin in whole urine correlates with prostate cancer, which made it possible to use it as a biomarker for the diagnosis of prostate cancer, and also made it possible to develop a new method for diagnosing prostate cancer based on a new biomarker. Quantitative determination of alpha V integrin in whole urine allows for non-invasive diagnosis of the disease, and a simple analysis technique allows the proposed method to be used in a standard clinical laboratory of clinical or outpatient medical institutions.

The invention also covers kits for performing diagnosis of prostate cancer according to the invention. Such kits include antibodies against integrin alpha V or its fragments, which can be detected either as individual subunits or as heterodimers, such as heterodimeric complexes with integrins beta 1, beta 3, beta 5, beta 6 and /or beta-8. According to the invention, any antibodies or fragments thereof can be used that specifically bind to alpha V integrin and do not have cross-specificity to other urinary peptides. Such antibodies may include, but are not limited to, any anti-integrin alpha V antibodies/antibody fragments known in the art (for example: Polyclonal Antibody to Integrin Alpha V (ITGaV):

http://www.elabscience.com/Manual/Antibody/EN/ENT2365.pdf, http://cloud-clone.com/products/PAB282Hu01.html, http://www.genetex.com/Integrin-alpha- 5-antibody-C-term-GTX81964.html;

Monoclonal Antibody to Integrin Alpha V (ITGaV):

http://www.abcam.com/integrin-alpha-5-antibody-epr7854-ab150361.html, http://www.sigmaaldrich.com/catalog/product/mm/mabt207?lang=en&region=RU, http: //cloud-clone.com/products/MAB282Hu22.html, http://www.antibodies-online.com/abstract/Integrin+alpha+V+(ITGAV)+Antibody/), and new, previously unknown antibodies/ antibody fragments, including antibodies (antibody fragments) modified to increase their affinity by known methods, which can be obtained using known methods, such as the immunization method (including, for example, chimeric antibodies or humanized antibodies) or a method for producing recombinant antibodies constructed using genetic engineering methods.

The diagnostic kit includes at least one antibody/antibody fragment that specifically binds to alpha V integrin (e.g., either immobilized on a support, or in lyophilized form, or as an aqueous solution), and one or more reagents useful for carrying out diagnostic analysis. When an anti-alpha V integrin antibody is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme (eg, a precursor substrate that provides a detectable chromophore or fluorophore). In addition, other additives such as stabilizers, buffers (eg, blocking buffer or lysis buffer), etc. may be included in the kit. In certain embodiments, an anti-alpha V integrin antibody included in a diagnostic kit is immobilized on a solid surface, or a solid surface (eg, glass slide or plastic plate, or diagnostic strip, etc.) on which the antibody can be immobilized is included in the kit. The relative amounts of different reagents can be varied widely to provide reagent concentrations in solution that significantly optimize the sensitivity of the assay. In a particular embodiment, the antibody and one or more reagents may be provided (separately or together) as dry powders, typically lyophilized, containing excipients that, when dissolved, will provide a reagent solution having a suitable concentration.

In some embodiments, the prostate cancer diagnostic kit may be a test system of any design, such as a diagnostic strip, array, chip, etc., or a set of reagents intended for use in manual or automated analysis, or with a partially/fully automatic/robotic analyzer; the reagent kit may also include a device, such as a plate, with immobilized antibodies, allowing for solid-phase ELISA or any other analysis with a sensitivity of at least 0.2 ng/ml.

In some embodiments of the invention, when implementing a method for diagnosing prostate cancer, any clinical, laboratory and/or instrumental examination methods known from the prior art are additionally carried out.

The original biomarker integrin alpha V is detected in whole or any fraction of patient urine, including but not limited to exosomes.

In some embodiments, the alpha V integrin biomarker can be used in the diagnosis of prostate cancer in combination with any other biomarkers that improve the quality (reliability, accuracy, and/or other) of prostate cancer diagnosis. Such biomarkers include, but are not limited to, such as, for example, prostate specific antigen, PCA3, TMPRSS2:ERG, PSCA (prostate

- 3 045060 stem cell antigen), AMCAR and/or others.

Examples of embodiments of the invention

Diagnosis of prostate cancer according to the invention can be carried out by any method of analysis capable of determining the content of alpha V integrin in the patient's urine in the range from 0 to 100 ng/ml. In a particular case, the diagnosis was carried out as follows.

30 ml (up to 50 ml) of the first portion of morning urine were taken from the subject.

The following procedures were used to measure alpha V integrin levels in urine:

the urine sample was clarified by centrifugation for 15 min at a speed of 2000 rpm (filtration through membranes with a pore size of 0.22-0.45 nm or any other available method can be used to separate the soluble fraction of urine proteins from the insoluble sediment);

Alpha V integrin content was determined on a case-by-case basis by ELISA using commercially available SEB282Hu 96 Tests. Enzyme-linked Immunosorbent Assay Kit For Integrin Alpha V (ITGaV) from Cloud-Clone Corp. (http://cloud-clone.com/products/SEB282Hu.html), strictly following the instructions (http://cloud-clone.com/manual/ELISA-Kit-for-Integrin-Alpha-V-(ITGaV)- SEB282Hu.pdf).

For this:

µl of urine was added to the well of a 96-well plate with immobilized antibodies;

incubated for 1.5 h at 37°C;

the liquid was removed and 100 μl of detection reagent A was added;

incubated for 1.5 h at 37°C;

the liquid was removed and the well was washed 3 times with washing solution. After each wash, the liquid was carefully removed by shaking onto filter paper;

100 μl of detection reagent B was added;

the plate was incubated for 0.5 h at 37°C;

the liquid was removed and the well was washed 5 times with the washing solution. After each wash, the liquid was carefully removed by shaking onto filter paper;

90 μl of TMB substrate was added;

incubated for 20 min at 37°C;

50 μl of stop reagent was added;

the result was measured immediately photometrically at 450 nm. To determine the level of ITGAV in each experimental urine sample, a calibration curve obtained from measuring various dilutions of pure (homogeneous) protein was used according to the manufacturer's instructions.

At the same time, increasing the accuracy of analysis and reproducibility of results is possible in two ways: by controlling the urine collection process: exactly 30-50 ml of the first portion of morning urine is needed;

collecting 30-50 ml of the first portion of urine after prostate massage.

Urine can be stored at 4°C for 24 hours and for 2-3 weeks at -18°C.

In some cases, it may also be advisable to repeat the analysis. The invention can also be used to monitor the effectiveness of treatment for prostate cancer by performing repeated tests to detect the level of alpha V integrin in the patient's whole urine during, as well as after completion of treatment.

In fig. Figure 1 shows the experimental results of measuring the level of alpha V integrin in the morning urine of healthy people, patients with benign tumors and patients with prostate cancer, obtained using the method described above.

On the graph of Fig. Figure 2 shows the probability curves of the distribution of values for three sets of data and the mathematical expectations of the corresponding measurements (vertical lines). As follows from the graph, the mathematical expectation of the biomarker level in healthy people and patients with benign tumors (between 24 and 32 ng/ml) differs significantly from the mathematical expectation of the biomarker level in patients with prostate cancer (~4 ng/ml). Processing was carried out under the assumption of a Gaussian distribution of the original data.

As follows from the data obtained, the level of alpha V integrin in the urine above 20 ng/ml indicates the absence of prostate cancer (100%), while the patient may be healthy or have prostate hyperplasia (prostatitis/adenoma). An alpha V integrin level of 20 ng/ml or less requires additional analysis, for example, through puncture and histological analysis.

The data presented indicate that alpha V integrin can be used as a new biomarker for the diagnosis of prostate cancer, and its determination in urine can be used for non-invasive diagnosis of prostate cancer, as well as for the development based on it of a simple, sensitive, reliable, applicable in standard clinical laboratory of clinical or outpatient medical institutions for the diagnosis of prostate cancer. The proposed diagnostic method can be used to survey the population, monitor risk groups, monitor relapses, monitor the course of the disease, and monitor therapy.

Although the invention has been described with reference to the disclosed embodiments, for

- 4 045060 specialists in the art will understand that the specific experiments described in detail are provided only for the purpose of illustrating the present invention, and they should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications can be made without departing from the spirit of the present invention.

List of sequences <110> Limited Liability Company "Prostagnost" <120> Protein marker for diagnosing prostate cancer, measurement method and algorithm for interpreting the result <130> 416132 <150> PCT/RU2017/000386 <151> 2017-06-02 < 160> 1 <210> 1 <211>1018 <212> PRT <213> Homo sapiens <400> 1

Phe Asn Leu Asp Vai Asp Ser Pro Ala Glu Tyr Ser Gly Pro Glu Gly

1015

Ser Tyr Phe Gly Phe Ala Vai Asp Phe Phe Vai Pro Ser Ala Ser Ser

2530

Arg Met Phe Leu Leu Vai Gly Ala Pro Lys Ala Asn Thr Thr Gln Pro

4045

Gly He Vai Glu Gly Gly Gln Vai Leu Lys Cys Asp Trp Ser Ser Thr

5560

Arg Arg Cys Gln Pro He Glu Phe Asp Ala Thr Gly Asn Arg Asp Tyr

70 7580

Ala Lys Asp Asp Pro Leu Glu Phe Lys Ser His Gln Trp Phe Gly Ala

9095

Ser Vai Arg Ser Lys Gln Asp Lys He Leu Ala Cys Ala Pro Leu Tyr

100 105110

His Trp Arg Thr Glu Met Lys Gln Glu Arg Glu Pro Vai Gly Thr Cys

115 120125

Phe Leu Gln Asp Gly Thr Lys Thr Vai Glu Tyr Ala Pro Cys Arg Ser

130 135140

Gln Asp He Asp Ala Asp Gly Gln Gly Phe Cys Gln Gly Gly Phe Ser

145 150 155160

He Asp Phe Thr Lys Ala Asp Arg Vai Leu Leu Gly Gly Pro Gly Ser

165 170175

Phe Tyr Trp Gln Gly Gln Leu lie Ser Asp Gln Vai Ala Glu He Vai

180 185190

Ser Lys Tyr Asp Pro Asn Vai Tyr Ser He Lys Tyr Asn Asn Gln Leu

- 5 045060

195 200205

Ala Thr Arg Thr Ala Gln Ala lie Phe Asp Asp Ser Tyr Leu Gly Tyr

210 215220

Ser Val Ala Val Gly Asp Phe Asn Gly Asp Gly lie Asp Asp Phe Val

225 230 235240

Ser Gly Val Pro Arg Ala Ala Arg Thr Leu Gly Met Val Tyr lie Tyr

245 250255

Asp Gly Lys Asn Met Ser Ser Leu Tyr Asn Phe Thr Gly Glu Gln Met

260 265270

Ala Ala Tyr Phe Gly Phe Ser Val Ala Ala Thr Asp lie Asn Gly Asp

275 280285

Asp Tyr Ala Asp Val Phe lie Gly Ala Pro Leu Phe Met Asp Arg Gly

290 295300

Ser Asp Gly Lys Leu Gln Glu Val Gly Gln Val Ser Val Ser Leu Gln

305 310 315320

Arg Ala Ser Gly Asp Phe Gln Thr Thr Lys Leu Asn Gly Phe Glu Val

325 330335

Phe Ala Arg Phe Gly Ser Ala lie Ala Pro Leu Gly Asp Leu Asp Gln

340 345350

Asp Gly Phe Asn Asp lie Ala lie Ala Ala Pro Tyr Gly Gly Glu Asp

355 360365

Lys Lys Gly lie Val Tyr lie Phe Asn Gly Arg Ser Thr Gly Leu Asn

370 375380

Ala Val Pro Ser Gln lie Leu Glu Gly Gln Trp Ala Ala Arg Ser Met

385 390 395400

Pro Pro Ser Phe Gly Tyr Ser Met Lys Gly Ala Thr Asp lie Asp Lys

405 410415

Asn Gly Tyr Pro Asp Leu lie Val Gly Ala Phe Gly Val Asp Arg Ala

420 425430 lie Leu Tyr Arg Ala Arg Pro Val lie Thr Val Asn Ala Gly Leu Glu

435 440445

Val Tyr Pro Ser lie Leu Asn Gln Asp Asn Lys Thr Cys Ser Leu Pro

450 455460

Gly Thr Ala Leu Lys Val Ser Cys Phe Asn Val Arg Phe Cys Leu Lys

465 470 475480

Ala Asp Gly Lys Gly Val Leu Pro Arg Lys Leu Asn Phe Gln Val Glu

485 490495

Leu Leu Leu Asp Lys Leu Lys Gln Lys Gly Ala lie Arg Arg Ala Leu

- 6 045060

500 505510

Phe Leu Tyr Ser Arg Ser Pro Ser His Ser Lys Asn Met Thr lie Ser

515 520525

Arg Gly Gly Leu Met Gln Cys Glu Glu Leu lie Ala Tyr Leu Arg Asp

530 535540

Glu Ser Glu Phe Arg Asp Lys Leu Thr Pro lie Thr lie Phe Met Glu

545 550 555560

Tyr Arg Leu Asp Tyr Arg Thr Ala Ala Asp Thr Thr Gly Leu Gln Pro

565 570575 lie Leu Asn Gln Phe Thr Pro Ala Asn lie Ser Arg Gln Ala His lie

580 585590

Leu Leu Asp Cys Gly Glu Asp Asn Val Cys Lys Pro Lys Leu Glu Val

595 600605

Ser Val Asp Ser Asp Gln Lys Lys lie Tyr lie Gly Asp Asp Asn Pro

610 615620

Leu Thr Leu lie Val Lys Ala Gln Asn Gln Gly Glu Gly Ala Tyr Glu

625 630 635640

Ala Glu Leu lie Val Ser lie Pro Leu Gln Ala Asp Phe lie Gly Val

645 650655

Val Arg Asn Asn Glu Ala Leu Ala Arg Leu Ser Cys Ala Phe Lys Thr

660 665670

Glu Asn Gln Thr Arg Gln Val Val Cys Asp Leu Gly Asn Pro Met Lys

675 680685

Ala Gly Thr Gln Leu Leu Ala Gly Leu Arg Phe Ser Val His Gln Gln

690 695700

Ser Glu Met Asp Thr Ser Val Lys Phe Asp Leu Gln lie Gln Ser Ser

705 710 715720

Asn Leu Phe Asp Lys Val Ser Pro Val Val Ser His Lys Val Asp Leu

725 730735

Ala Val Leu Ala Ala Val Glu lie Arg Gly Val Ser Ser Pro Asp His

740 745750

Val Phe Leu Pro lie Pro Asn Trp Glu His Lys Glu Asn Pro Glu Thr

755 760765

Glu Glu Asp Val Gly Pro Val Val Gln His lie Tyr Glu Leu Arg Asn

770 775780

Asn Gly Pro Ser Ser Phe Ser Lys Ala Met Leu His Leu Gln Trp Pro

785 790 795800

Tyr Lys Tyr Asn Asn Asn Thr Leu Leu Tyr lie Leu His Tyr Asp lie

- 7 045060

805 810815

Asp Gly Pro Met Asn Cys Thr Ser Asp Met Glu He Asn Pro Leu Arg

820 825830

He Lys He Ser Ser Leu Gin Thr Thr Glu Lys Asn Asp Thr Vai Ala

835 840845

Gly Gin Gly Glu Arg Asp His Leu He Thr Lys Arg Asp Leu Ala Leu

850 855860

Ser Glu Gly Asp He His Thr Leu Gly Cys Gly Vai Ala Gin Cys Leu

865 870 875880

Lys He Vai Cys Gin Vai Gly Arg Leu Asp Arg Gly Lys Ser Ala lie

885 890895

Leu Tyr Vai Lys Ser Leu Leu Trp Thr Glu Thr Phe Met Asn Lys Glu

900 905910

Asn Gin Asn His Ser Tyr Ser Leu Lys Ser Ser Ala Ser Phe Asn Vai

915 920925

He Glu Phe Pro Tyr Lys Asn Leu Pro He Glu Asp He Thr Asn Ser

930 935940

Thr Leu Vai Thr Thr Asn Vai Thr Trp Gly He Gin Pro Ala Pro Met

945 950 955960

Pro Vai Pro Vai Trp Vai He He Leu Ala Vai Leu Ala Gly Leu Leu

965 970975

Leu Leu Ala Vai Leu Vai Phe Vai Met Tyr Arg Met Gly Phe Phe Lys

980 985990

Arg Vai Arg Pro Pro Gin Glu Glu Gin Glu Arg Glu Gin Leu Gin Pro

995 10001005

His Glu Asn Gly Glu Gly Asn Ser Glu Thr

10101015

Claims (6)

1. Use of integrin alpha V or its fragment as a marker detected in whole urine for the diagnosis of prostate cancer. 2. A method for diagnosing prostate cancer, including detecting alpha V integrin or a fragment thereof in the patient’s whole urine. 3. The method according to claim 2, in which integrin alpha V or a fragment thereof is detected in urine in the form of individual subunits or as part of heterodimers of integrins ave1, av33, av35, av36 and/or ave8. 4. The method according to any one of claims 2 or 3, in which the absence of prostate cancer is diagnosed when alpha V integrin or its fragment is detected in the urine in an amount of more than 20 ng/ml. 5. The method according to claim 2, including additional clinical, laboratory and/or instrumental examination methods. 6. The method according to claim 5, in which additional clinical, laboratory and/or instrumental examination methods are carried out if the amount of alpha V integrin detected in the patient’s urine is no more than 20 ng/ml.