Compounds - Diagnostic markers for kidney cancer, methods for detecting enzyme activity, methods for diagnosing kidney cancer, kits containing the compounds, uses of the compounds, and methods for treating kidney cancer

Abstract

The present invention relates to a novel compound, diagnostic marker, for use in medical treatment, more particularly in cancer diagnosis, particularly in kidney cancer diagnosis.The present invention also relates to an in vitro method for detecting enzyme activity present in subject's body fluid, particularly from kidney cancer cells, using the compound.The present invention further relates to an in vitro method for diagnosing kidney cancer using the compound, a kit containing the compound, and the use of the compound for detecting enzyme activity specific to kidney cancer, as well as the use of the compound for diagnosing kidney cancer.The present invention also relates to the compound for use as a diagnostic marker for kidney cancer, and to a method for treating kidney cancer, comprising the procedure of carrying out the above-mentioned method for diagnosing kidney cancer using the compound.

Description

[Technical field]

[0001] The present invention relates to a novel compound, diagnostic marker, for use in medical treatment, more particularly in cancer diagnosis, particularly for kidney cancer diagnosis.The present invention also relates to an in vitro detection method for detecting the enzyme activity present in subject's body fluid, particularly from kidney cancer cells, using the compound, an in vitro method for diagnosing kidney cancer using the compound, a kit comprising the compound, the use of the compound for detecting kidney cancer specific enzyme activity, the use of the compound for diagnosing kidney cancer, and the compound for use as a diagnostic marker for kidney cancer.The present invention also relates to a method for treating kidney cancer, comprising the procedure of carrying out the kidney cancer diagnosis method as described above. [Background technology]

[0002] In 2020, over 430,000 patients worldwide developed kidney cancer, making it the 14th most commonly diagnosed malignant neoplasm in the world. Kidney cancer is a relatively rare cancer, accounting for 2.4% of newly diagnosed solid tumors worldwide. Malignant kidney neoplasms are divided into primary and secondary, the most common of which is renal cell carcinoma (RCC), accounting for approximately 90% of all malignant kidney neoplasms. RCC represents a heterogeneous group of lesions that differ in both histological architecture and clinical manifestations and molecular alterations. The most common histological subtype is clear cell renal cell carcinoma (ccRCC), which accounts for 70-80% of all RCC cases and is also the most aggressive subtype, with the highest rates of local invasion, metastasis, and mortality. Early-stage kidney cancer is not associated with pain or other characteristic symptoms. More than 30% of patients with RCC have metastases at the time of diagnosis, and 30% of patients with organ-confined RCC will develop metastases after local treatment. RCC remains a challenge to diagnose and treat. In the majority of patients, the cancer is detected incidentally as a localized lesion during imaging studies (X-ray, CT, MRI) performed intentionally or for other reasons.

[0003] Prognosis depends on the stage of the neoplasm. Early diagnosis is crucial; 75% of patients diagnosed at an early stage survive for more than 5 years. There are no clinical tests, "cancer markers" or test sets that allow for early and reliable diagnosis of kidney cancer. Imaging modalities such as X-ray, CT scan, MRI, or positron-emission magnetic resonance imaging are used for effective diagnosis, followed by a tumor biopsy. A small patient population (5-10%) shows altered expression of certain genes, directing this population to genetic testing.

[0004] It is known that the processes of initiation, proliferation and dissemination of cancer cells involve many factors, including many enzymes, especially hydrolases, especially proteolytic enzymes. Such enzymes catalyze the process of enzymatic (hydrolytic or proteolytic) cleavage of proteins and peptides into their smaller fragments. This process allows cancer cells to colonize new tissues and to grow and spread by enhancing the process of blood vessel formation (angiogenesis) that allows effective nutrient delivery to the tumor. Moreover, these enzymes are present as a result of the death of healthy cells due to the tumor proliferation process. All these processes form a characteristic and specific profile of enzymatic (proteolytic) activity of cancer cells, which is characteristic of tumors.

[0005] Chromogenic peptide molecules are known in the art that change or increase the color of a test solution upon enzymatic degradation into smaller fragments, the coloring effect being the result of the release of a chromophore (e.g., 4-nitroanilide or 2-aminobenzoic acid) from the chromogenic peptide molecule.

[0006] Chromogenic molecules of this kind and their uses are known, for example, from the publications Erlanger BF, Kokowsky N, Cohen W. "The preparation and properties of two new chromogenic substrates of trypsin", Arch Biochem Biophys., November 1961; 95: 271-8 and Hojo K, Maeda M, Iguchi S, Smith T, Okamoto H, Kawasaki K. Amino acids and peptides. XXXV. "Facile preparation of p-nitroanilide analogs by the solid-phase method", Chem Pharm Bull (Tokyo), November 2000; 48 (11): 1740-4.

[0007] However, the use of this class of compounds for the diagnosis of kidney cancer has not been reported to date. Methods for obtaining chromogenic peptides are also known in the prior art, in which the individual components are coupled under suitable time and stoichiometric conditions. The coupling method consists of successive steps of coupling the individual building blocks (amino acid derivatives), washing off the residues, sequentially removing the protecting groups and washing again. This cycle is repeated for each amino acid residue. The resulting peptide is separated from the resin by reaction under acidic conditions. The solution is then separated from the resin in a filtration step, after which the peptide is precipitated from the solution by a non-polar solvent.

[0008] However, chromogenic peptide compounds suitable for specific and early diagnosis of kidney cancer and methods for obtaining them are not known in the prior art. Thus, there is an urgent need in the art for "cancer markers" for kidney cancer that allow for early, sensitive and specific diagnosis of kidney cancer in a non-invasive and reliable manner, and for diagnostic and therapeutic methods using such diagnostic markers.

[0009] The object of the present invention is to provide novel and specific diagnostic markers for kidney cancer and diagnostic methods for non-invasive, rapid, sensitive and specific early detection of kidney cancer using such markers (which may also be suitable as screening tests), as well as therapeutic methods using such markers.

[0010] These objects are achieved by the invention as defined in the appended claims, while preferred variants thereof are defined in the dependent claims. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Erlanger BF, Kokowsky N, Cohen W., “The preparation and properties of two new chromogenic substrates of trypsin”, Arch Biochem Biophys., November 1961; 95:271-8 [Non-Patent Document 2] Hojo K, Maeda M, Iguchi S, Smith T, Okamoto H, Kawasaki K. Amino acids and peptides. XXXV. “Facile preparation of p-nitroanilide analogs by the solid-phase method”, Chem Pharm Bull (Tokyo), November 2000; 48(11):1740-4 Summary of the Invention

[0012] The present invention relates to a compound of formula 1: X1 1 -Leu 2 -Pro 3 -Gly 4 -Thr 5 -X2 6 (Formula 1) The present invention provides a compound having the formula:

[0013] In the formula, X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, The compound undergoes enzymatic cleavage into fragments X1-Leu-Pro-Gly-Thr-OH (fragment 1) and X2 (fragment 2) and generates a measurable light signal due to the spatial separation of molecules C1 and C2.

[0014] The compounds according to the invention are preferably subject to hydrolytic cleavage, more preferably proteolytic cleavage. Preferably, in the compound according to the invention, the pair of molecules C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2), more preferably the pair of C1 and C2 is (ABZ) / pNA or ABZ / ANB-NH2.

[0015] Preferably, the compound according to the present invention is a compound having formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 (formula 2) or a compound having formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (formula 3).

[0016] More preferably, the compound according to the invention undergoes hydrolytic cleavage to generate the following fragment 1: ABZ-Leu-Pro-Gly-Thr-OH and fragment 2: ANB-NH2.

[0017] The present invention further provides an in vitro method for detecting an enzymatic activity, particularly from renal cancer cells, present in a body fluid of a subject, the method comprising: a) A body fluid sample was subjected to a reaction with a solvent according to Equation 1: X1 1 -Leu 2 -Pro 3 -Gly 4 -Thr 5 -X2 6 (Formula 1) contacting the compound having the formula [wherein X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, the compound undergoes enzymatic cleavage to give the fragments X1-Leu-Pro-Gly-Thr-OH (fragment 1) and X2 (fragment 2); and b) Detecting a measurable light signal generated by the spatial separation of molecules C1 and C2 This includes:

[0018] In the method for detecting an enzymatic activity according to the present invention, the enzymatic activity is preferably a hydrolytic activity, more preferably a proteolytic activity. In the method for detecting enzyme activity according to the present invention, the compound preferably has the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 (formula 2) or the formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (formula 3).

[0019] In the method for detecting enzyme activity according to the present invention, the body fluid used is preferably urine, more preferably human urine. The present invention also relates to an in vitro method for diagnosing kidney cancer, which detects the presence or absence of kidney cancer in a subject by measuring kidney cancer-specific enzyme activity in a body fluid sample of the subject, the absence of said enzyme activity indicating the absence of kidney cancer, whereas the presence of said enzyme activity indicates the presence of kidney cancer.

[0020] In the method for detecting / diagnosing kidney cancer according to the present invention, the detection of enzyme activity is carried out by the method for detecting enzyme activity defined above. In the method for detecting / diagnosing kidney cancer according to the present invention, the measurement of the enzyme activity is carried out by measuring the enzyme activity according to Formula 1: X1 1 -Leu 2 -Pro 3 -Gly 4 -Thr 5 -X2 6 (Formula 1) The reaction is carried out using a compound having the formula:

[0021] In the above formula, X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, The compound undergoes enzymatic cleavage into fragments X1-Leu-Pro-Gly-Thr-OH (fragment 1) and X2 (fragment 2) and generates a measurable light signal due to the spatial separation of molecules C1 and C2.

[0022] In the kidney cancer detection / diagnosis method according to the present invention, the body fluid sample is preferably incubated with the compound in an assay buffer having a neutral or alkaline pH, preferably physiological pH, in a sample to assay buffer ratio ranging from 1:2 to 1:10, preferably 1:5.

[0023] In the method for detecting / diagnosing kidney cancer according to the present invention, the compound is preferably used at a concentration of 0.1 to 10 mg / mL, particularly 0.25 to 7.5 mg / mL. In the method for detecting / diagnosing kidney cancer according to the present invention, the compound preferably has the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 (formula 2) or the formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (formula 3).

[0024] In the method for detecting / diagnosing kidney cancer according to the present invention, preferably a urine sample, more preferably human urine, is used as the sample. In the method for detecting / diagnosing kidney cancer according to the present invention, the measurement of the enzyme activity preferably comprises measuring the absorbance intensity within the range of 300 to 500 nm, more preferably 380 to 430 nm, particularly 405 nm, for 40 to 60 minutes at a temperature within the range of 25 to 40°C, more preferably 36 to 38°C.

[0025] The present invention further provides a kit comprising any compound according to the invention as defined above and an assay buffer. In the kit according to the present invention, the compound is preferably a compound having the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 or a compound having the formula 3: ABZ-Leu-Pro-Gly-Thr-pNA.

[0026] The present invention also provides the use of any compound according to the invention as defined above for detecting an enzymatic activity specific for renal cancer. The present invention also provides the use of any compound according to the invention as defined above for the diagnosis of renal cancer.

[0027] Preferably, in such uses, diagnosing kidney cancer includes detecting primary kidney cancer, detecting minimal residual disease following surgical resection of kidney cancer, and / or detecting recurrence of kidney cancer. Preferably, the compound in the use according to the invention has the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 or the formula 3: ABZ-Leu-Pro-Gly-Thr-pNA.

[0028] The present invention further provides any of the compounds according to the invention as defined above for use as a diagnostic marker for the detection of kidney cancer. Preferably, the compound for use as a diagnostic marker according to the present invention has the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 or the formula 3: ABZ-Leu-Pro-Gly-Thr-pNA.

[0029] The present invention further comprises: a) detecting the presence of an enzyme activity specific for kidney cancer in a body fluid sample to be tested by any of the methods defined above; and b) administering to the subject a treatment for kidney cancer if the presence of said enzymatic activity is found in said sample. The Company also offers treatments for kidney cancer.

[0030] Preferably, in the method of treatment according to the invention, after the end of the treatment according to point b), said enzymatic activity specific for kidney cancer is monitored at defined time intervals. Preferably, in the therapeutic method according to the invention a urine sample, preferably human urine, is used as sample.

[0031] Preferably, in the method of treatment according to the invention, a compound having the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 or a compound having the formula 3: ABZ-Leu-Pro-Gly-Thr-pNA is used as the compound. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 shows the results of chromatographic analysis of the substrate cleavage, i.e., ABZ-Leu-Pro-Gly-Thr-ANB-NH2, in urine samples from subjects with renal cancer. [Diagram 2] Figure 2 shows the hydrolysis rate of the substrate ABZ-Leu-Pro-Gly-Thr-ANB-NH2 in urine samples from subjects diagnosed with kidney cancer (samples N1–N20) and urine collected from healthy subjects (samples 21–40). Arabic numbers indicate the numbers of the selected urine samples. [Diagram 3]FIG. 3 shows the hydrolysis selectivity of the substrate ABZ1-Leu2-Pro3-Gly4-Thr5-ANB6-NH2 (i.e., compound of formula 2) in a urine sample from a subject diagnosed with renal cancer (sample 1) and urine samples taken from subjects diagnosed with different neoplastic diseases (cancer) (samples 2-9). Arabic numerals indicate the number of a given cancer type. Samples tested for each type of cancer were obtained from 20 different patients for each cancer tested. Results are average values ​​for a given cancer type. Results show the selectivity of substrate cleavage in the case of urine from renal cancer patients compared to urine samples from other cancer patients. [Figure 4] FIG. 4 shows the dependence of the hydrolysis level of the substrate ABZ1-Leu2-Pro3-Gly4-Thr5-ANB6-NH2 on pH conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] It should be understood that the present invention is defined in the appended claims. In this description, various non-limiting aspects and embodiments of the present invention are illustrated. The present invention is not limited to any particular methodology, protocol, or reagent used to carry it out, unless otherwise specified. The terms and scientific and technical expressions used herein have the meanings commonly known and used by those skilled in the art of the present invention. However, for the sake of clarity, the following expressions / terms and acronyms used in this patent shall be understood as follows:

[0034] Chromogenic compound or molecule refers to a compound that has chromogenic properties. Chromogenic properties refers to the ability of a compound to form a colored product. A fluorescent compound or molecule refers to a compound that has fluorescent properties, which refers to the ability of a compound to form products that fluoresce.

[0035] NMP stands for N-methylpyrrolidone; DMF stands for dimethylformamide; DCM stands for methylene chloride or dichloromethane; pNA stands for 4-nitroaniline or para-nitroaniline; ABZ stands for 2-aminobenzoic acid, ANB-NH2 stands for the amide of 5-amino-2-nitrobenzoic acid; Boc stands for the tert-butyloxycarbonyl group; Fmoc stands for the 9-fluorenylmethoxycarbonyl group; and TFA stands for trifluoroacetic acid.

[0036] In the context of the present invention, the term renal cancer shall be understood to mean primary renal cancer (malignant neoplasm) that originates from tissue in the kidney.The most frequent renal cancer is renal cell carcinoma (about 90%).The term renal cancer used herein therefore includes all malignant renal neoplasms that originate from tissue in the kidney.

[0037] In the context of the present invention, the term diagnosis of kidney cancer shall be understood to mean the identification of the disease, especially at an early stage when other diagnostic methods are not sufficiently sensitive and / or specific. As used herein, diagnosis of kidney cancer also includes the detection of minimal residual disease (MRD) after surgical resection of kidney cancer, and the detection of recurrence of kidney cancer after previously completed kidney cancer treatment.

[0038] In the context of the present invention, the term treatment of kidney cancer shall be understood to mean a treatment at an early stage of the progression of the disease, making it possible to significantly extend the survival time and improve the quality of life of affected individuals.

[0039] In the context of the present invention, the term monitoring shall be understood to mean diagnosing the presence of minimal residual disease (MRD), i.e. a small number of surviving cancer cells in the body (during treatment or remission) in amounts that cannot be detected by standard diagnostic methods.

[0040] In the context of the present invention, the term subject is to be understood to mean a human subject or mammal suspected of having kidney cancer or belonging to a group at high risk of kidney cancer, or after resection of kidney cancer or after completion of treatment for kidney cancer. The subject is preferably a human subject.

[0041] The compounds according to the invention have color-developing and fluorescent properties due to the presence of a chromophore, i.e. they contain a fluorescent donor and a fluorescent acceptor molecule. Due to their structure, which has been developed in such a way that, as a result of contact with a test body fluid sample of a subject having renal cancer, an increase in color is observed in the wavelength range of 380-440 nm, while no such effect is observed in reaction with a body fluid sample of a healthy subject or a subject diagnosed with another type of cancer, these compounds make it possible to detect an enzyme activity specific for renal cancer and in particular to diagnose renal cancer with specificity and high sensitivity even at an early stage of the progression of this cancer. The subject to be examined is preferably a human subject. The body fluid is preferably urine, more preferably human urine.

[0042] In a first aspect of the present invention, there is provided a novel compound having the formula 1: X1 1 -Leu 2 -Pro 3 -Gly 4 -Thr 5 -X2 6 (Formula 1) where X1 is an amino acid derivative or peptide fragment containing molecule C1 or X1 consists of such molecule C1, X2 is an amino acid derivative or peptide fragment containing molecule C2 or X2 consists of such molecule C2, and the pair of molecules C1 and C2 is a fluorescent donor and fluorescent acceptor pair. The superscripts indicate the position of the subsequent residues in the compound according to the invention and the order of attachment of the residues during synthesis. According to the invention, in this context, formula 1 can alternatively be written without showing the numbering of the residues. The core of all compounds according to the invention is a tetrapeptide with the indicated four amino acid sequence (Leu-Pro-Gly-Thr). This is also shown in the sequence listing as SEQ ID NO: 1.

[0043] The compound according to the invention undergoes enzymatic cleavage into fragments: X1-Leu-Pro-Gly-Thr-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal due to the spatial separation of molecules C1 and C2. The measurable light signal is measured by a method for measuring the change in absorbance / fluorescence after enzymatic cleavage of the compound. Preferably, molecules C1 and C2 are separated from each other by 10 or fewer amino acid residues to ensure efficient quenching of the fluorescent donor by the fluorescent acceptor. It is clear to those skilled in the art that the key factor is the distance between the fluorescent donor and the acceptor. Thus, the distance between molecules C1 and C2 may be more than 10 amino acid residues, if the amino acid sequence separating them is folded into a twisted or condensed secondary structure, resulting in the molecules C1 and C2 being closer together compared to the primary structure.

[0044] Due to its chromogenic properties and the presence of a reactive site at position 5 allowing enzymatic (preferably proteolytic) cleavage into smaller fragments, the compound is particularly suitable for use as a diagnostic marker, in particular a specific diagnostic biomarker for kidney cancer, in particular for the early diagnosis of kidney cancer.

[0045] In a preferred embodiment, the compounds according to the invention undergo hydrolytic cleavage, more preferably proteolytic cleavage. In a preferred embodiment, the pair of molecules C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2), more preferably, the pair of molecules C1 and C2 is ABZ / pNA or ABZ / ANB-NH2.

[0046] In a preferred embodiment, the compound according to the invention is Formula 2: ABZ 1 -Leu 2 -Pro 3 -Gly 4 -Thr 5 -ANB 6 -NH2 (Formula 2) or Formula 3: ABZ 1 -Leu 2 -Pro 3 -Gly 4 -Thr 5 -pNA 6 (Formula 3) In the formula, ABZ represents 2-aminobenzoic acid, ANB-NH2 represents the amide of 5-amino-2-nitrobenzoic acid, and pNA represents 4-nitroaniline.

[0047] The compounds undergo hydrolytic cleavage to generate the following fragment 1: ABZ-Leu-Pro-Gly-Thr-OH and fragment 2: ANB-NH2 for compounds having formula 2, while the following fragment 1: ABZ-Leu-Pro-Gly-Thr-OH and fragment 2: pNA for compounds having formula 3. Thus, fragment 2 is the free chromophore.

[0048] Upon enzymatic cleavage of the compound according to the invention, the molecules C1 and C2 are spatially separated, resulting in the generation of a measurable optical signal, since the fluorescence emitted by the fluorescent donor is no longer quenched by the fluorescent acceptor. Such a measurable optical signal can be detected at a wavelength preferably between 300 and 500 nm, more preferably between 380 and 430 nm.

[0049] The compounds according to the invention can be obtained by known methods. For example, they can be obtained using a method for obtaining chromogenic peptides, which consists in carrying out the process on a solid support in the form of a resin bearing an Fmoc group (which is removed in the course of the reaction). For example, it can be an amide resin, such as Teenage S RAM or RinkAmide, but any other commercially available resin can also be used. The resin used to carry out the method should be appropriately prepared. The preparation of the resin consists in increasing its volume by repeated washing with a hydrophobic solvent. Preferably, a resin with a loading of 0.23 mmol / g is used. The Fmoc protecting group must be removed from the resin by washing with a 20% solvent solution.

[0050] The known methods for obtaining chromogenic peptides then involve coupling the individual components under suitable time and stoichiometric conditions. The coupling method consists of successive steps of coupling the individual building blocks (amino acid derivatives), washing off the residues, sequentially removing the protecting groups and washing again. This cycle is repeated for each amino acid residue. The resulting peptide is separated from the resin by reaction under acidic conditions. The peptide is then precipitated from the resulting solution by a non-polar solvent after the solution has been separated from the resin in a filtration step. The peptide precipitate thus obtained is centrifuged.

[0051] Exemplary detailed, but non-limiting, syntheses of compounds according to the invention are set forth below and in Example 1 below. The synthesis of the compounds according to the invention is such that the process is carried out on a solid support, preferably in the form of a resin bearing an Fmoc group. Before starting the process, the solid support is prepared by increasing its volume by repeated washing with a hydrophobic solvent, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and removing the Fmoc protecting group, preferably by washing with a 10-30% piperidine solution in a solvent such as dimethylformamide, methylene chloride or N-methylpyrrolidone.

[0052] The method is then carried out in the following steps. a) Prior to attachment of 5-amino-2-nitrobenzoic acid (ANB) (or another chromophore suitable for use according to the invention as defined in the claims) onto the resin, the solid support is washed with a 3-6% solution of N-methylmorpholine (NMM) in DMF, then with DMF, after which a solution of ANB in ​​DMF is prepared, to which is added in the following order: TBTU, DMAP and finally diisopropylethylamine (DIPEA) in the following excess relative to polymer attachment: ANB / TBTU / DMAP / DIPEA 3:3:2:6. The mixture thus prepared is added to the resin and mixed until homogeneous. The resin is then filtered under reduced pressure and washed with solvents such as DMF, DCM and isopropanol, after which the conjugation of ANB to the resin is continued with an excess of hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU). After completion, the solid support is washed successively with DMF, DCM and isopropanol and gently dried.

[0053] b) The attachment of the amino acid residue to the ANB is carried out by reaction with the amino acid derivative Fmoc-Thr(OtBu)-OH, where at least a 5-fold molar excess of the amino acid derivative relative to the resin is dissolved in anhydrous pyridine and brought into contact with the resin with the attached ANB. After this, the whole is cooled to a temperature not lower than -20°C, then POCl3 is added in a ratio of 1:1 relative to the amount of amino acid derivative used, the whole is mixed, then the mixing steps are carried out at room temperature and then at elevated temperature, once the reaction is complete the resin is filtered under reduced pressure, washed with DMF and MeOH and gently dried, after which the intermediate compound obtained is subjected to an acylation step followed by the attachment of the Leu-Pro-Gly fragment.

[0054] c) Acylation of the intermediate compounds obtained is carried out with amino acid derivatives, preferably with Fmoc-Gly-OH, then with Fmoc-Pro-OH, then with Fmoc-Leu-OH and, in the final stage of the synthesis, with Boc-Abz-OH. The acylation is carried out stepwise from residue 6 to 1, using diisopropylcarbodiimide as coupling agent (used in excess). After each step, the resin is washed with DMF and preferably subjected to the chloranil test (test for the presence of free amino groups) to monitor the coupling of the amino acid derivatives.

[0055] d) Removal of the Fmoc protecting group is carried out by washing with a 10-30% piperidine solution in DMF, followed by washing with each of the solvents, namely DMF, isopropanol and methylene chloride.

[0056] e) Separation of the peptide from the resin is carried out using a mixture, namely TFA:phenol:water:TIPS, maintaining the ratio of 88:5:5:2 v / v / v / v respectively. The mixture is stirred for at least 1 hour, preferably 3 hours, the resulting precipitate is filtered under reduced pressure, then washed with diethyl ether and the resulting peptide is centrifuged.

[0057] f) The preparation of the final product is carried out by dissolving the peptide in water by ultrasound and then subjecting it to lyophilization. In a second aspect of the present invention, there is provided an in vitro detection method for detecting an enzymatic activity, preferably a proteolytic activity, present in a body fluid of a subject, in particular from renal cancer cells, comprising a) contacting a body fluid sample with a compound according to the present invention, and b) detecting a measurable optical signal generated by the spatial separation of molecules C1 and C2 present in the compound according to the present invention. In a preferred embodiment of this aspect, the subject examined in this case is a human subject. In another preferred embodiment of this aspect, the body fluid is urine, in particular human urine.

[0058] In a preferred embodiment of this aspect, a compound having the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 or a compound having the formula 3: ABZ-Leu-Pro-Gly-Thr-pNA is used.

[0059] In a third aspect of the invention, there is provided an in vitro method for diagnosing kidney cancer, which detects the presence or absence of kidney cancer in a subject by measuring kidney cancer-specific enzyme activity in a body fluid sample of the subject, the absence of said enzyme activity indicating the absence of kidney cancer, whereas the presence of said enzyme activity indicates the presence of kidney cancer. The detection of such enzyme activity is preferably carried out using the method for detecting enzyme activity described above. In a preferred embodiment of this aspect, the subject is a human subject. In a preferred embodiment of this aspect, the body fluid is urine, in particular human urine. In a preferred embodiment of this aspect, the kidney cancer-specific enzyme activity is a proteolytic activity. In a preferred embodiment of this aspect, a compound having the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 or a compound having the formula 3: ABZ-Leu-Pro-Gly-Thr-pNA is used.

[0060] Furthermore, in a preferred embodiment of this aspect, the measurement of the enzyme activity in the method according to the present invention comprises measuring the absorbance intensity at 300 to 500 nm, preferably at 380 to 430 nm, particularly at 405 nm, for 40 to 60 minutes at a temperature in the range of 25 to 40° C., preferably at 36 to 38° C. This makes it possible to obtain the strongest possible measurable optical signal resulting from an increase in absorbance or fluorescence.

[0061] Furthermore, in a preferred embodiment of the method according to the invention, the measurement of the enzyme activity is carried out with a compound according to the invention at a concentration in the range of 0.1-10 mg / mL, more preferably at a concentration of 1 mg / mL. In a preferred embodiment of the method according to the invention, the test sample is incubated with the compound according to the invention in an assay buffer having a neutral or alkaline pH, preferably physiological pH. The body fluid sample is preferably human urine, the ratio of sample (e.g. urine sample) to assay buffer being in the range of 1:2-1:10, preferably 1:5. The sample is preferably taken from a subject referred for a diagnosis of kidney cancer. Preferably, the absorbance intensity is measured in the range of 300-500 nm, preferably 380-430 nm, in particular 405 nm, for 40-60 minutes, at a temperature in the range of 25-40° C., preferably 36-38° C. Under the above conditions, a maximally intense measurable light signal is obtained as a result of the increase in absorbance or fluorescence.

[0062] In a fourth aspect, the present invention provides a kit comprising any compound according to the present invention and an assay buffer. Assay buffers are known in the art, and suitable buffers for use in the kit according to the present invention include, but are not limited to, Tris-HCl buffer. In a preferred embodiment, in the kit according to the present invention, the compound is a compound having the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2, or a compound having the formula 3: ABZ-Leu-Pro-Gly-Thr-pNA.

[0063] In a fifth aspect, the present invention provides the use of a compound according to the present invention for detecting enzyme activity specific for kidney cancer. In a sixth aspect, the present invention provides the use of a compound according to the present invention for diagnosing kidney cancer. Preferably, diagnosing kidney cancer according to the present invention includes detecting primary kidney cancer, detecting minimal residual disease after surgical resection of kidney cancer, and / or detecting recurrence of kidney cancer after previously completed kidney cancer treatment.

[0064] In a seventh aspect, the present invention provides a compound according to the present invention for use as a diagnostic marker for detecting kidney cancer. In a preferred embodiment of this aspect, the compound has formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 or formula 3: ABZ-Leu-Pro-Gly-Thr-pNA.

[0065] In an eighth aspect, the present invention provides a method for producing a composition comprising: a) detecting the presence of an enzymatic activity specific for kidney cancer in a body fluid sample to be examined by any of the methods according to the invention as defined above, and b) administering to the subject a treatment for kidney cancer if said enzymatic activity is found to be present in said sample; Provides treatment for kidney cancer.

[0066] In a preferred embodiment of the method, after completing the treatment according to point b), the enzyme activity specific for kidney cancer is monitored at a certain time interval as known in the art, for example, every week, every few weeks, every month, every few months, every year, or any other interval that the skilled person considers appropriate, in order to detect minimal residual disease or recurrence after surgical resection of kidney cancer.Furthermore, in a preferred embodiment of the method, a urine sample, preferably human urine, is used as a test sample.In a preferred embodiment of the method, a compound having formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH2 (formula 2) or a compound having formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (formula 3) is used as said compound.

[0067] The advantage of the present invention is to provide novel compounds with suitable properties for use in the specific and sensitive detection of enzyme activity specific for kidney cancer, for use as diagnostic biomarkers for kidney cancer detection, for use in the rapid and non-invasive diagnosis of kidney cancer, while allowing kidney cancer to be detected at an early stage of its progression. Another advantage is that the diagnostic method according to the present invention can also be successfully used as a screening test. This allows a complete diagnosis at an early stage of the cancer's progression and, as a result, a more effective treatment. Early diagnosis allows for a surgical treatment that significantly extends the patient's survival time. It is also important when monitoring the effectiveness of the applied kidney cancer surgical treatment and / or chemotherapy, since it allows the detection of any minimal residual disease or recurrence.

[0068] The invention will now be described in the following figures and examples which are not intended in any way to limit the scope of the invention as defined in the claims. EXAMPLES

[0069] The present invention is illustrated by the following non-limiting examples. Unless otherwise indicated, the following examples use known and / or commercially available equipment, methods, reaction conditions, reactants and kits that are commonly used in the field to which the present invention pertains and that are recommended by the manufacturers of the respective reactants and kits.

[0070] Example 1: Synthesis of compounds according to the invention In this example, one representative compound according to the present invention, namely compound ABZ 1 -Leu 2 -Pro 3 -Gly 4 -Thr 5 -ANB 6The synthesis of -NH2 is provided. The remaining peptides according to the invention can be synthesized in a similar manner. The superscripts indicate the position of the subsequent residues in the compounds according to the invention and the order of attachment of the residues during synthesis. The compounds according to the invention can alternatively be represented by a similar formula without indicating the positions of the residues. This does not change the sequence of the residues in the compounds according to the invention, which remains unchanged.

[0071] 1. Obtaining Chromogenic Peptides a) The first step of the synthesis was to obtain the chromogenic peptide, which was obtained by solid phase synthesis on a solid support using Fmoc / tBu chemistry, i.e. with protection.

[0072] Array ABZ 1 -Leu 2 -Pro 3 -Gly 4 -Thr 5 -ANB 6 Compounds having -NH2, where ABZ is 2-aminobenzoic acid, ANB-NH2 is the amide of 5-amino-2-benzoic acid, and ANB is 5-amino-2-benzoic acid, were obtained by solid phase chemical synthesis using the following amino acid derivatives: Boc-ABZ, Fmoc-Leu, Fmoc-Pro, Fmoc-Gly, Fmoc-Thr(OtBu).

[0073] The synthesis of the compound according to the invention, i.e. the diagnostic marker for kidney cancer detection, was carried out on a solid support that allows the conversion of 5-amino-2-benzoic acid to ANB-NH2 amide, i.e. the amide resin TentaGel S RAM (with a loading of 0.23 mmol / g) from RAPP Polymere (Germany). However, it is also possible to use any other amide resin, e.g. Rink Amide (Germany).

[0074] The synthesis of the compounds was carried out manually on a laboratory shaker. For most steps, 25 mL sintered syringes for solid-phase synthesis were used as reactors. All the final compounds obtained contained a 2-aminobenzoic acid (ABZ) molecule at position 1, i.e. the N-terminus, and a 5-amino-2-nitrobenzoic acid (ANB) molecule at position 6, i.e. the C-terminus, of their sequence. ABZ acts here as a fluorescence donor, whereas ANB acts as a fluorescence quencher and at the same time as a chromophore. The peptides contained at least and preferably one reactive site in their sequence (located between the amino acid residues Thr-ANB-NH2, i.e. in position 5 of the compound). The synthesis for coupling the amino acid derivatives is carried out from residue 6 to 1, i.e. from the C-terminus to the N-terminus.

[0075] b) Attachment of ANB to TentaGel S RAM resin: The synthesis of peptides was carried out on TentaGel S RAM resin from Rapp Polymere with a loading of 0.23 mmol / g. In a first step, the resin was prepared, including loosening it by a washing cycle. Afterwards, the Fmoc amino group protection was removed from the solid support with a 20% piperidine solution in NMP. Solvent washing cycles were then performed. A chloranil test was performed to confirm the presence of free amino groups.

[0076] Solvent wash cycle: DMF 1x10min; IsOH 1x10min; DCM 1x10min Removal of Fmoc protecting group: DMF 1x5 min; 20% piperidine in NMP 1x3 min; 20% piperidine in NMP 1x8 min Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min c) Chloranil test: The chloranil test consisted in transferring a few grains of resin with a spatula from the reactor (syringe) to a glass ampoule, after which 100 μL of a saturated solution of p-chloranil in toluene and 50 μL of fresh acetaldehyde were added to it. After 10 min, a control of the color of the grains was performed.

[0077] At this stage (after the test run), green particles were obtained, which is evidence of the presence of free amino groups. After confirming the removal of the 9-fluorenylmethoxycarbonyl protecting group from the resin, it was possible to proceed to the next step, i.e. the coupling of the ANB derivative (5-amino-2-nitrobenzoic acid).

[0078] d) Attachment of 5-amino-2-nitrobenzoic acid to a solid support The first step in the synthesis of a peptide library (mixture of peptides) was the attachment of ANB onto 1 g of resin. Prior to the attachment of the chromophore, the resin used in the reaction was washed with the following solvents: DMF, DCM, and again with DMF, after which the Fmoc protecting group was removed from the functional groups of the solid support. One cycle of removing the Fmoc protecting group included the following steps:

[0079] Removal of Fmoc protecting group: 20% piperidine in NMP 1x3 min; 20% piperidine in NMP 1x8 min e) Washing: DMF 3x2min; IsOH 3x2min; DCM 3x2min f) Chloranil test: The resin with free amino groups was washed with a 5% solution of N-methylmorpholine (NMM) in DMF and then with DMF. The procedure for removal of the Fmoc protecting group and the washing cycles were carried out in a Merrifield vessel. In a separate flask, ANB was dissolved in DMF, followed by TBTU, DMAP and finally diisopropylethylamine (DIPEA) in the following excess amounts relative to the polymer attachment: ANB / TBTU / DMAP / DIPEA 3:3:2:6 v / v / v / v. The mixture thus prepared was added to the resin and stirred for 3 hours. The resin was filtered under reduced pressure, washed with DMF, DCM and isopropanol and the whole acylation procedure was repeated twice. To carry out the next reaction of coupling ANB to the resin, hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) was used, followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU). In the last step, the resin was washed successively with DMF, DCM and isopropanol and air-dried.

[0080] g) Coupling of the C-terminal amino acid residue (Fmoc-Thr(OtBu)-OH) to ANB: The corresponding amino acid derivative (9-fold molar excess relative to the resin attachment) was dissolved in pyridine and transferred to the flask containing the ANB-attached resin. The whole was cooled (ice bath: 1 part by weight NH4Cl, 1 part by weight NaNO3, 1 part by weight ice) until the temperature reached -15°C. Once the desired temperature was reached, POCl3 was added (1:1 ratio relative to the amount of amino acid derivative used) and the whole was stirred on a magnetic stirrer for 20 minutes at -15°C, 30 minutes at room temperature, and 6 hours at 40°C (oil bath). Once the reaction was complete, the resin was filtered under reduced pressure, washed with DMF and MeOH, and left to dry.

[0081] In the next step, a residue was attached at the P2 position (Fmoc-Gly). After each coupling of amino acid residues, the resin was first washed with DMF for 5 min. Diisopropylcarbodiimide was used as a coupling agent for the subsequent couplings. The procedure was repeated twice.

[0082] After each acylation, a resin washing cycle was initiated and then a chloranil test was performed to monitor the binding of the amino acid derivatives to the free amino acid groups of the resin. Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min Chloranil Test: After the first two coupling steps, tests were performed showing that the color of the particles was initially green and then grey, so another acylation had to be performed, which resulted in the resin particles becoming colorless when tested with the chloranil test, which was evidence of the binding of ANB to the TentaGel S RAM resin, and therefore it was possible to proceed to the next peptide synthesis step.

[0083] h) Coupling of subsequent protected amino acid residues: After washing both the resin and the binding fragment ANB-Thr(OtBu) in the reaction vessel with DMF, the amino group was deprotected with Fmoc in order to couple the protected amino acid derivative Gly.

[0084] Removal of Fmoc protecting group: DMF 1x5 min; 20% piperidine in NMP 1x3 min; 20% piperidine in NMP 1x8 min Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min Chloranil Test: The chloranil test was successful, as evidenced by the green colour of the resin particles, making it possible to proceed to the next step, namely the coupling of the amino acid residue Fmoc-Pro-OH.

[0085] Binding of amino acid derivatives The resin was washed with DMF prior to the coupling step. The composition of the coupling mixture remained unchanged upon attachment of the protected serine residue.

[0086] After each acylation, a solvent washing cycle was performed according to a specific procedure, followed by a chloranil test to check for the presence of free amino acid groups in the solution. Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min Chloranil Test: During the test carried out after the second acylation, the resin particles were colorless, so it was possible to proceed to the next synthetic step, i.e. the introduction of another protected amino acid derivative, Fmoc-Leu, and a 2-aminobenzoic acid molecule. The coupling step was carried out according to the procedure discussed previously.

[0087] Tests carried out after coupling of the aforementioned residues showed good results, i.e. the resin particles were colourless. 2. Removal of peptides from solid support After synthesis, the amide of ABZ-Leu-Pro-Gly-Thr-ANB-NH2 peptide was removed from the solid support and simultaneously the side chain protecting groups were removed using a mixture, namely TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v), in a round bottom flask on a magnetic stirrer.

[0088] After 3 hours, the contents of the flask were filtered under reduced pressure through a Schott funnel and washed with diethyl ether. The precipitate obtained was centrifuged for 20 minutes in a SIGMA 2K30 Laboratory Centrifuges. The precipitate obtained after centrifugation was dissolved in water by ultrasound and then subjected to freeze-drying. The remaining compounds according to the invention can be obtained in the same manner.

[0089] The identity / characterization of the novel compounds according to the present invention was confirmed using HPLC analysis. The conditions of the HPLC analysis were as follows: RP Bio Wide Pore Supelco C8 column, 250 mm x 4 mm, phase system A: 0.1% TFA in water, B: 80% acetonitrile in A, flow rate 1 mL / min, UV detection: 226 nm. The analyses carried out confirm that the compound according to the invention was obtained.

[0090] Example 2: Testing the properties of the peptides according to the invention as cancer markers The activity of the novel compounds according to the invention was investigated in a group of 20 subjects diagnosed with renal cancer using a representative compound according to the invention. The mechanism of action of the compounds according to the invention (including the representative compound having formula 2) is a specific enzymatic cleavage, more specifically enzymatic hydrolysis, occurring at a position that leads to the release of a free molecule of the respective chromophore, ANB-NH2 (amide of 5-amino-2-nitrobenzoic acid) in the case of the compound having formula 2 or pNA (para-nitroanilide) in the case of the compound having formula 3, which exhibits an absorbance at a wavelength of 320-480 nm, in particular 380-430 nm, in particular 405 nm. The remaining compounds according to the invention are also characterized by a similar mechanism of action. For this purpose, the representative compound according to the invention, ABZ 1 -Leu 2 -Pro 3 -Gly 4 -Thr 5 -ANB 6 -NH2 was dissolved in dimethyl sulfoxide (at a concentration of 0.5 mg / mL) and 50 μL of this solution was mixed with 120 μL of buffer (200 mM Tris-HCl, pH 8.0) and 80 μL of urine from a subject suffering from renal cancer. The measurements were carried out on a 96-well plate designed for the measurement of absorbance, each sample was analyzed in triplicate at a temperature of 37 °C. The measurement time was 60 min. During the measurements, the wavelength characteristic of the released chromophore (ANB-NH2) was monitored at a wavelength of 405 nm (range 380-430 nm).

[0091] As shown in FIG. 1, RP HPLC analysis of a randomly selected series containing urine collected from individuals diagnosed with kidney cancer showed that the compound according to the present invention was cleaved into the peptide fragment ABZ-Leu-Pro-Gly-Thr-OH and the chromophore group of the compound (ANB-NH2).

[0092] Measurements showed that the color intensity of the solution increased over time in all urine samples taken from people diagnosed with kidney cancer. The magnitude of the observed increase in absorbance over time varied for each tested sample. For the 20 samples taken from healthy subjects (samples 21-40), different results were obtained, since no increase in absorbance within the diagnostic range was observed in any of the 20 tested urine samples.

[0093] The tests carried out show that all samples N1-N20 from kidney cancer patients underwent cleavage, but in the case of samples N3 and N6, the cleavage of the substrate, i.e., ABZ-Leu-Pro-Gly-Thr-ANB-NH2, proceeded less efficiently than in the case of samples N5 or N20 (Figure 2, Table 1). Such results may be due to differences in the activity and amount of the enzymes responsible for the enzymatic cleavage (proteolysis). Furthermore, the results shown in Table 1 below show that incubation of the substrate solution (compound according to the invention) with urine samples taken from healthy individuals (not diagnosed with cancer, marked with Arabic numerals 21-40 in order) does not result in an increase in absorbance, and therefore no hydrolysis of the test compound occurs. This result indicates the absence of proteolytic enzymes specific / characteristic of kidney cancer.

[0094] Table 1. Results of absorbance analysis

[0095] [Table 1-1]

[0096] [Table 1-2]

[0097] Furthermore, the dependence of the substrate, i.e., the cleavage selectivity of the compound according to the invention on the type of cancer examined was also investigated. The results of the carried out tests are shown in Figure 3. According to it, the test substrate, i.e., ABZ, incubated with samples taken from patients diagnosed with the following cancers, i.e., testicular cancer, prostate cancer, pancreatic cancer, bile duct cancer, lung cancer, ovarian cancer, and rectal cancer, was 1 -Leu 2 -Pro 3 -Gly 4 -Thr 5 -ANB 6 It has been shown that -NH2 does not undergo cleavage and does not cause an increase in absorbance within a specific range. The test samples are in each case a mixture of 20 samples obtained from each cancer investigated. This shows the cleavage selectivity of the compound according to the present invention, making it suitable for the specific detection of renal cancer-specific enzyme activity and the specific diagnosis of renal cancer.

[0098] Table 2 below shows the results of three measurements taken for each sample. Table 2. Analysis results of cleavage selectivity

[0099] [Table 2]

[0100] Furthermore, the dependence of the proteolytic activity of representative compounds according to the invention on reaction pH was also determined. Experiments showed that the investigated materials possess at least one enzyme that exhibits maximum activity at alkaline pH (Figure 4).

[0101] The analyses carried out confirmed that the compounds according to the invention are suitable for the sensitive and specific detection of enzyme activity specific for kidney cancer, as well as for the specific diagnosis of kidney cancer and as diagnostic markers for kidney cancer. The mechanism of action of the compounds according to the invention is that they are specifically enzymatically cleaved at a position which leads to the release of a free chromophore molecule, which generates a measurable light signal which can be used for diagnostic purposes, in particular for the diagnosis of kidney cancer according to the invention.

Claims

1. Formula 1: X1 １ -Leu ２ -Pro ３ -Gly ４ -Thr ５ -X2 ６ (Equation 1) A compound having, In the formula, X1 contains or consists of molecule C1, and X2 contains or consists of molecule C2. Here, the pair of molecules C1 and C2 is a pair of a fluorescence donor and a fluorescence receptor. The compound undergoes enzymatic cleavage to form fragments X1-Leu-Pro-Gly-Thr-OH (fragment 1) and X2 (fragment 2), and the spatial separation of molecules C1 and C2 generates a measurable optical signal. compound.

2. The compound according to claim 1, wherein the compound undergoes hydrolytic cleavage or proteolytic cleavage.

3. The molecular pairs C1 and C2 are 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, and ABZ / ANB-NH. ２ , ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr (3-NO ２ A compound according to claim 1 or 2, selected from the group consisting of ).

4. The compound according to claim 3, wherein the pair of C1 and C2 is ABZ / pNA or ABZ / ANB-NH2.

5. The compound is a compound having the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH ２ The compound according to claim 1 or 2, which is a compound having the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH (Formula 2), or a compound having the formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (Formula 3).

6. The aforementioned compound undergoes hydrolytic cleavage to obtain the following fragment 1: ABZ-Leu-Pro-Gly-Thr-OH and fragment 2: ANB-NH ２ The compound according to claim 5, which produces [the product].

7. An in vitro method for detecting enzyme activity, particularly that derived from kidney cancer cells, present in the body fluids of a target, a) Prepare a body fluid sample using formula 1: X1 １ -Leu ２ -Pro ３ -Gly ４ -Thr ５ -X2 ６ (Equation 1) By contacting a compound having the following properties, [In the above formula, X1 contains or consists of molecule C1, and X2 contains or consists of molecule C2, Here, the pair of molecules C1 and C2 is a pair of a fluorescence donor and a fluorescence receptor. The compound undergoes enzymatic cleavage to form fragments X1-Leu-Pro-Gly-Thr-OH (fragment 1) and X2 (fragment 2), and b) Detect a measurable optical signal generated by the spatial separation of molecules C1 and C2. In vitro methods including the following.

8. The in vitro method according to claim 7, wherein the enzyme activity is hydrolytic activity, preferably proteolytic activity.

9. As the aforementioned compound, Formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH ２ The in vitro method according to claim 7 or 8, wherein a compound having (Formula 2) or a compound having Formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (Formula 3) is used.

10. The method according to claim 6 or 7, wherein urine or human urine is used as the bodily fluid.

11. An in vitro method for diagnosing kidney cancer in which the presence or absence of kidney cancer in a subject is detected by measuring the enzyme activity specific to kidney cancer in a body fluid sample of the subject, the absence of said enzyme activity indicates the absence of kidney cancer, and the presence of said enzyme activity indicates the presence of kidney cancer, wherein the measurement of said enzyme activity is given by Equation 1: X1 １ -Leu ２ -Pro ３ -Gly ４ -Thr ５ -X2 ６ (Equation 1) This is carried out using a compound having the following properties: In the above formula, X1 contains or consists of molecule C1, and X2 contains or consists of molecule C2. Here, the pair of molecules C1 and C2 is a pair of a fluorescence donor and a fluorescence receptor. The compound undergoes enzymatic cleavage to form fragments X1-Leu-Pro-Gly-Thr-OH (fragment 1) and X2 (fragment 2), and the spatial separation of molecules C1 and C2 generates a measurable optical signal. In vitro method.

12. The method according to claim 11, wherein the detection of enzyme activity is carried out by the method defined in claim 7.

13. The method according to claim 11 or 12, wherein the body fluid sample is incubated with the compound in a measurement buffer having a neutral, alkaline, or physiological pH, in a sample-to-measurement buffer ratio within the range of 1:2 to 1:10, or 1:

5.

14. The method according to claim 11 or 12, wherein the compound is used at a concentration of 0.1 to 10 mg / mL or 0.25 to 7.5 mg / mL.

15. As the aforementioned compound, Formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH ２ The method according to claim 11 or 12, wherein a compound having (Formula 2) or a compound having Formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (Formula 3) is used.

16. The method according to claim 11 or 12, wherein a urine sample or human urine is used as the aforementioned sample.

17. The method according to claim 11 or 12, wherein the measurement of the enzyme activity includes measuring the absorbance intensity in the range of 300 to 500 nm, 380 to 430 nm, or 405 nm for 40 to 60 minutes at a temperature in the range of 25 to 40°C, or 36 to 38°C.

18. A kit comprising the compound defined in claim 1 or 2 and a measurement buffer.

19. The compound has the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH ２ The kit according to claim 18, wherein the compound is having (Formula 2) or is having Formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (Formula 3).

20. Use of a compound as defined in claim 1 or 2 for detecting enzyme activity specific to kidney cancer.

21. A kit as defined in claim 18 for use in the diagnosis of kidney cancer.

22. The kit for use according to claim 21, wherein the diagnosis of kidney cancer includes the detection of primary kidney cancer, the detection of minimal residual disease after surgical resection of kidney cancer, and / or the detection of recurrence of kidney cancer.

23. The compound has the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH ２ A kit for use according to claim 21, wherein the compound is having (Formula 2) or is a compound having Formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (Formula 3).

24. A diagnostic marker for detecting kidney cancer, comprising the compound defined in claim 1 or 2.

25. The compound has the formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH ２ A diagnostic marker for use according to claim 24, which is a compound having (Formula 2) or a compound having Formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (Formula 3).

26. a) The presence of enzyme activity specific to kidney cancer is detected in the body fluid sample to be tested by the method defined in claim 7, and b) If the presence of the enzyme activity is found in the sample, it shall be applied to the treatment of kidney cancer. The kit according to claim 18 for use in the treatment of kidney cancer.

27. The kit for use according to claim 26, wherein, after the completion of treatment in accordance with point b), the enzyme activity specific to kidney cancer is monitored at predetermined time intervals.

28. The kit for use according to claim 26, characterized in that a urine sample, preferably human urine, is used as the sample.

29. As the aforementioned compound, Formula 2: ABZ-Leu-Pro-Gly-Thr-ANB-NH ２ A kit for use according to claim 26, wherein a compound having (Formula 2) or a compound having Formula 3: ABZ-Leu-Pro-Gly-Thr-pNA (Formula 3) is used.

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