Primer-probe compositions, kits, and detection methods

Abstract

The present invention relates to a primer-probe composition, a kit and a detection method, the primer-probe composition being selected from one of groups (i) to (ix), and the kit comprising the primer-probe composition. According to the present invention, the AKR1C3 RNA content in an ex vivo sample of a patient can be detected.

Description

[Technical Field]

[0001] The present invention relates to the field of molecular biological detection technology, and in particular to primer-probe compositions, kits, and detection methods. [Background technology]

[0002] Aldehyde-ketone reductase family 1 member C3 (AKR1C3) is an enzyme encoded by the AKR1C3 gene in humans. AKR1C3 protein levels are overexpressed in various types of cancer. Due to the high expression of AKR1C3 in tumors, compounds OBI-3424 (also known as AST-3424 and TH-3424), OBI-3423, and OBI-2870 are designed to be specifically activated in tumors, but are unable to activate in normal cells with low AKR1C3 expression, resulting in tumor-specific targeting. Compound OBI-3424 is currently being investigated in multiple Phase I clinical trials in the United States (NCT04315324 and NCT03592264) and China (CXHL1900137 and CXHL2000263) for the treatment of over 14 types of human cancer, including solid tumors and hematological tumors. [ka]

[0003] The compound OBI-3424 is a novel prodrug dialkylating agent activated by AKR1C3. Studies have confirmed that the activation of compound OBI-3424 is AKR1C3-dependent, and its cytotoxicity and antitumor effects are highly correlated with the expression level of the AKR1C3 enzyme. Compound OBI-3424 has demonstrated AKR1C3-dependent cytotoxicity ex vivo and antitumor activity in various types of human cancer, supporting its further development as an anticancer drug for the treatment of various types of cancer. AKR1C3 can be used as a biomarker to analyze the status of cancer patients and further guide the selection of compound OBI-3424 for treatment. Therefore, in practical applications, the content of AKR1C3 enzyme in ex vivo samples from patients can be detected, and the administration of compound OBI-3424 to patients for treatment can be determined according to the detection results.

[0004] In the prior art, Western blotting or immunohistochemistry (IHC) is usually used to detect the content of AKR1C3 enzyme in ex vivo patient samples. (Harvey DJ, Singleton RS, Dachs GU, et al., "Bioreductive prodrug PR-104A is activated by human aldo-keto reductase 1C3 under aerobic conditions" [J] Cancer Research, 2010, 70(4):1573) reported that 2,700 tumor tissue samples were counted and analyzed by IHC, and the expression level of AKR1C3 enzyme was high in liver cancer, gastric cancer, esophageal cancer, and bladder cancer, but low in small cell lung cancer, breast cancer, leukemia, and prostate cancer. Therefore, it is theoretically feasible to use IHC to detect the expression level of AKR1C3 enzyme in solid tumor tissue samples.

[0005] However, although there are many types of cancer or tumors, patients with blood cancer (leukemia) other than solid tumors cannot provide tissue samples, so it is not possible to directly use Western blotting or immunochemical staining to detect the expression level of the AKR1C3 enzyme in blood cancer (leukemia) samples. Summary of the Invention [Problem to be solved by the invention]

[0006] The applicant's research and development team has investigated the relationship between the expression level of AKR1C3 enzyme and AKR1C3 RNA content in different solid cancer cells and the IC value of AST-3424 inhibition on cancer cell proliferation. 50 The IC value of AST-3424 inhibition on cancer cell proliferation was significantly correlated with the expression level of AKR1C3 enzyme and AKR1C3 RNA content in different blood cancer cell lines. 50 Therefore, by detecting the ARKR1C3 RNA content, it is possible to predict or characterize the enzyme content, which can be used as a guide for drug administration.

[0007] However, there is no corresponding detection method for detecting AKR1C3 RNA content in the prior art.Assuming that corresponding detection method can be used to evaluate AKR1C3 RNA content, the patient with higher AKR1C3 RNA content and most likely to respond to prodrug can be selected to be administered with compound OBI-3424, so as to achieve better cancer treatment effect.

[0008] Therefore, the present invention provides a primer-probe composition, a kit and a detection method, and aims to use the primer-probe composition, the kit and the detection method to detect AKR1C3 RNA content in ex vivo samples from patients with high accuracy, high analytical specificity, precision and a low detection limit. [Means for solving the problem]

[0009] To achieve the above objectives, the first aspect of the present invention comprises: (i) an upstream primer AKR1C3-F1, a downstream primer AKR1C3-R1 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; (ii) an upstream primer AKR1C3-F2, a downstream primer AKR1C3-R2 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 3, respectively; (iii) an upstream primer AKR1C3-F2, a downstream primer AKR1C3-R6 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 3, respectively; (iv) an upstream primer AKR1C3-F6, a downstream primer AKR1C3-R2 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:3, respectively; (v) an upstream primer AKR1C3-F6, a downstream primer AKR1C3-R6 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:3, respectively; (vi) an upstream primer AKR1C3-F5, a downstream primer AKR1C3-R5 and a probe AKR1C3-P2, the nucleotide sequences of which are set forth in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; (vii) an upstream primer AKR1C3-F3, a downstream primer AKR1C3-R3 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 3, respectively; (viii) an upstream primer AKR1C3-F4, a downstream primer AKR1C3-R3 and a probe AKR1C3-P2, whose nucleotide sequences are set forth in SEQ ID NO: 13, SEQ ID NO: 12 and SEQ ID NO: 10, respectively; (ix) an upstream primer AKR1C3-F7, a downstream primer AKR1C3-R7, and a probe AKR1C3-P3, whose nucleotide sequences are set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; The present invention provides a primer-probe composition selected from any one of the following groups:

[0010] In a preferred embodiment of the invention, the primer-probe composition is selected from any one of groups (iii), (iv) and (v) above, More preferably, said primer-probe composition is selected from group (iv).

[0011] In a preferred embodiment of the present invention, the 5'-end reporter of said probes AKR1C3-P1, AKR1C3-P2 and AKR1C3-P3 is FAM, and the 3'-end quencher of said probes AKR1C3-P1, AKR1C3-P2 and AKR1C3-P3 is MGB.

[0012] Based on a similar inventive concept, a second aspect of the present invention provides a kit comprising the primer-probe composition.

[0013] In a preferred embodiment of the present invention, the kit further comprises a primer-probe composition for a reference gene, Preferably, the reference gene is ACTB.

[0014] In a preferred embodiment of the present invention, the primer-probe composition for the reference gene comprises an upstream primer ACTB-F1, a downstream primer ACTB-R1 and a probe ACTB-P1, whose nucleotide sequences are set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0015] In a preferred embodiment of the present invention, the 5'-end reporter of the probe ACTB-P1 is VIC, and the 3'-end quencher of the probe ACTB-P1 is BHQ1.

[0016] In a preferred embodiment of the present invention, the kit further comprises a polymerase mixture, the polymerase mixture mainly comprising a DNA polymerase, MgCl, a buffer, and dNTPs; Preferably, the polymerase mixture is KAPA PROBE FAST RT-PCR Master Mix (2x).

[0017] In a preferred embodiment of the invention, the kit further comprises a reverse transcriptase mixture, Preferably, the reverse transcriptase mixture is Superscript VILO MARSTER MIX.

[0018] In a preferred embodiment of the present invention, the kit further comprises a negative control and a positive control, Preferably, the negative control is nuclease-free water, and / or Preferably, the positive control is a reference of known copy number.

[0019] Based on the same inventive concept, a third aspect of the present invention provides the use of said primer-probe composition or said kit in the preparation of a cancer therapeutic drug.

[0020] In a preferred embodiment of the present invention, the AKR1C3 RNA content in a patient's ex vivo sample is determined using the primer-probe composition or the kit, and an AKR1C3-activating anticancer drug is administered to patients whose AKR1C3 RNA content is equal to or greater than a predetermined level.

[0021] In a preferred embodiment of the present invention, the AKR1C3 RNA content is determined according to the ratio of AKR1C3 copy number / reference gene copy number; Preferably, the predetermined content is 0.0001 to 1, More preferably, the predetermined content is 0.00011 to 0.5, More preferably, the predetermined content is 0.00013 to 0.05.

[0022] In a preferred embodiment of the invention, said ex vivo sample comprises a blood sample, a bone marrow sample, or a tissue sample.

[0023] In a preferred embodiment of the present invention, the AKR1C3-activating anticancer drug of the present invention includes the AKR1C3-activating anticancer prodrug of the present invention. That is, the prodrug-form compound is reduced to a cytotoxic toxin under the catalytic action of AKR1C3 in the biochemical environment of the cell, thereby exerting a toxic effect on cancer cells. See the following patent applications: PCT / US2016 / 021581 (corresponding to International Publication No. 2016 / 145092 (Chinese Patent Application No. 2016800150788 (Chinese Patent Application Publication No. 107530556)) PCT / US2016 / 025665 (International Publication No. 2016 / 161342 (Corresponding to Chinese Patent Application No. 2016800446081 (Chinese Patent Application Publication No. 108290911)) PCT / US2016 / 062114 (International Publication No. 2017 / 087428 (Chinese Patent Application No. 2016800200132 (Chinese Patent Application Publication No. 108136214)) PCT / NZ2019 / 050030 (International Publication No. 2019 / 190331 (Corresponding to Chinese Patent Application No. 201980023423.6 (Chinese Patent Application Publication No. 111918864))

[0024] All of the compounds of the general formula and specific compounds disclosed in the above patent applications belong to AKR1C3-activating anti-cancer prodrugs (compounds), and the disclosures of the above patent applications are incorporated herein in their entirety.

[0025] Generally, AKR1C3-activating anticancer drugs are said to meet the following conditions: AKR1C3 inhibitors (such as TH-3021 disclosed in the above three patents, or compound 36, i.e., Flanagan et al. Bioorganic and Medicinal Chemistry (2014), 962-977) [ka] The inhibitory effect of the compound on the proliferation of cancer cells in the presence of an AKR1C3 inhibitor is lower than that of cancer cells in the absence of an AKR1C3 inhibitor (such as TH-3021 disclosed in the above three patents). 50 When using ELISA to quantify the inhibitory effect on cancer cell proliferation, the detected IC of a compound against a specific cancer cell line in the presence of an AKR1C3 inhibitor is 50 is greater than that in the absence of an AKR1C3 inhibitor, the compound can be determined to be an AKR1C3-activating anticancer drug.

[0026] Preferably, the AKR1C3-activating anticancer agent has the following structure: [ka] The compound is selected from compounds having the formula:

[0027] In a preferred embodiment of the present invention, the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, breast cancer, gastric cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, bladder cancer, cervical cancer, hepatocellular carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, renal cell carcinoma, cystadenocarcinoma, cystic carcinoma, medullary carcinoma, bronchial carcinoma, osteocyte carcinoma, epithelial carcinoma, bile duct carcinoma, choriocarcinoma, embryonal carcinoma, seminoma, and Wilms' tumor. tumor, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hematoblastoma, vocal cord neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, fibrochondroma, fibrocyst, fibromyxoma, fibroosteosarcoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocyst, myxochondroma, myxochondrosarcoma, myxofibrosarcoma, myxoadenoma, myxoblastoma, liposarcoma, lipoma, lipoadenoma, lipoblastoma, lipochondroma, lipofibroma, lipoangioma, myxolipoma, cartilage Sarcoma, chondroma, chondroleiomyoma, chordoma, choriocarcinoma, chorioepithelioma, chorioblastoma, osteosarcoma, osteoblastoma, osteochondrofibroma, osteochondroma, osteocytoma, osteoodontoma, osteofibroma, fibrosarcoma of bone, angiosarcoma, hemangioma, angiolipoma, hemangiochondroma, hemangioblastoma, angiokeratoma, hemangioglioma, hemangioendothelioma, angiofibroma, angiomyoma, angiolipoma, angiolymphoma, angiolipoleiomyoma, angiomyolipoma, angiomyoneuroma, angiomyxoma, angioreticuloma, lymphangiosarcoma, lymphogranuloma, lymphangioma, lymphoma, lymphomyxoma, lymphoma lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, peripheral T-cell lymphoma, nodular NK / T-cell lymphoma, endothelioma, endoblastoma, synovial sarcoma, mesothelioma, connective tissue tumor, Ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoblastoma, leiomyofibroma, rhabdomyoma, rhabdomyosarcoma, rhabdomyomyxoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic disease cell, polycythemia vera, lymphoma, endometrial cancer, glioma, colorectal cancer, thyroid cancer, urothelial carcinoma or multiple myeloma, Preferably, the cancer comprises ovarian cancer, cervical cancer, pancreatic cancer, breast cancer, colorectal cancer, esophageal cancer, gastric cancer, stem cell cancer, non-small cell lung cancer, prostate cancer, renal cell carcinoma, peripheral T-cell lymphoma, nodular NK / T-cell lymphoma, acute lymphocytic leukemia or acute myeloid leukemia.

[0028] Based on a similar inventive concept, a fourth aspect of the present invention provides: (1) extracting RNA from an ex vivo sample to be detected, adding the extracted RNA to a reverse transcription system, and reverse transcribing the extracted RNA to synthesize cDNA; (2) performing qPCR or digital PCR amplification using the primer-probe composition or the kit with cDNA as a template; (3) determining the AKR1C3 RNA content of the ex vivo sample to be detected according to the qPCR or digital PCR amplification results; The present invention provides a method for detecting AKR1C3 RNA content, comprising:

[0029] In a preferred embodiment of the present invention, in step (1), the concentration of the extracted RNA is detected, preferably using a Qubit RNA HS Assay Kit, the reverse transcription system comprises a reverse transcriptase, and preferably the mass-to-volume ratio of the extracted RNA and the reverse transcriptase is (0.5-2):4 in μg / μL, more preferably the mass-to-volume ratio of the extracted RNA and the reverse transcriptase is (1-1.8):4 in μg / μL, and even more preferably the mass-to-volume ratio of the extracted RNA and the reverse transcriptase is 2:4 in μg / μL.

[0030] In a preferred embodiment of the present invention, in the step (2), a primer-probe composition selected from any one of the groups (i) to (ix) is mixed with a primer-probe composition for a reference gene in a qPCR reaction system; Preferably, in the qPCR reaction system, the molar ratio of the AKR1C3 upstream primer, the AKR1C3 downstream primer, and the AKR1C3 probe is (2 to 10):(2 to 10):3; More preferably, in the qPCR reaction system, the molar ratio of the AKR1C3 upstream primer, the AKR1C3 downstream primer, and the AKR1C3 probe is (3 to 7):(3 to 7):3; Even more preferably, in the qPCR reaction system, the molar ratio of the AKR1C3 upstream primer, the AKR1C3 downstream primer and the AKR1C3 probe is 5:5:3; and / or In the qPCR reaction system, Preferably, the molar ratio of the upstream primer ACTB-F1, the downstream primer ACTB-R1, and the probe ACTB-P1 is (2 to 10):(2 to 10):3; More preferably, the molar ratio of the upstream primer ACTB-F1, the downstream primer ACTB-R1, and the probe ACTB-P1 is (3 to 7):(3 to 7):3; Even more preferably, the molar ratio of the upstream primer ACTB-F1, the downstream primer ACTB-R1 and the probe ACTB-P1 is 5:5:3; and / or In the qPCR reaction system, Preferably, the amounts of the AKR1C3 upstream primer, the AKR1C3 downstream primer, and the AKR1C3 probe are the same as the amounts of the upstream primer ACTB-F1, the downstream primer ACTB-R1, and the probe ACTB-P1, respectively.

[0031] In a preferred embodiment of the present invention, in the step (2), dUTP, UNG enzyme, cDNA template, and polymerase mixture are also added to the qPCR reaction system; Preferably, the volume of the polymerase mixture is 0.3 to 0.8 of the total volume of the qPCR reaction system; More preferably, the volume of the polymerase mixture is 0.5 times the total volume of the qPCR reaction system.

[0032] In a preferred embodiment of the present invention, in the step (2), AKR1C3 and the reference gene are amplified by an AKR1C3 digital PCR detection system and a reference gene digital PCR detection system, respectively; Preferably, in the AKR1C3 digital PCR detection system, the molar ratio of the AKR1C3 upstream primer, the AKR1C3 downstream primer, and the AKR1C3 probe is (5 to 15):(5 to 15):3; More preferably, the molar ratio of the AKR1C3 upstream primer, the AKR1C3 downstream primer, and the AKR1C3 probe is (8 to 14):(8 to 14):3; Even more preferably, the molar ratio of AKR1C3 upstream primer, AKR1C3 downstream primer and AKR1C3 probe is 12:12:3; and / or In the reference gene digital PCR detection system, Preferably, the molar ratio of the upstream primer ACTB-F1, the downstream primer ACTB-R1, and the probe ACTB-P1 is (5 to 15):(5 to 15):3; More preferably, the molar ratio of the upstream primer ACTB-F1, the downstream primer ACTB-R1, and the probe ACTB-P1 is (8 to 14):(8 to 14):3; Even more preferably, the molar ratio of the upstream primer ACTB-F1, the downstream primer ACTB-R1 and the probe ACTB-P1 is 12:12:3.

[0033] In a preferred embodiment of the present invention, in the step (3), the step of determining the AKR1C3 RNA content of the ex vivo sample to be detected according to the qPCR or digital PCR amplification result comprises: (A) determining the copy numbers of AKR1C3 and reference genes in the ex vivo sample to be detected according to the qPCR or digital PCR amplification results; (B) calculating the ratio of AKR1C3 copy number / reference gene copy number to determine the AKR1C3 RNA content of the ex vivo sample to be detected; Includes.

[0034] In a preferred embodiment of the present invention, when the qPCR method is used, the step (A) further comprises: (A1) plotting a standard curve of the Ct value and initial copy number lg value of AKR1C3 and a standard curve of the Ct value and initial copy number lg value of a reference gene; (A2) determining the copy numbers of AKR1C3 and the reference gene in the ex vivo sample to be detected based on the Ct values ​​of the detected AKR1C3 and the reference gene, respectively, according to the calibration curve; Includes.

[0035] In a preferred embodiment of the invention, the method is used to detect AKR1C3 RNA content in a blood, bone marrow or tissue sample.

[0036] Based on a similar inventive concept, a fifth aspect of the present invention provides a method for detecting the expression level of AKR1C3 enzyme, which uses the above method to detect the AKR1C3 RNA content of an ex vivo sample to be detected, and then determines the expression level of AKR1C3 enzyme in the ex vivo sample to be detected according to the AKR1C3 RNA content of the ex vivo sample to be detected.

[0037] In a preferred embodiment of the present invention, there is a linear correlation between the AKR1C3 RNA content in the ex vivo sample to be detected and the expression level of the AKR1C3 enzyme in the ex vivo sample to be detected. [Brief explanation of the drawings]

[0038] [Figure 1] Figure 1 shows the ex vivo AKR1C3-dependent cytotoxicity of compound OBI-3424. The left panel shows the correlation between the expression level of AKR1C3 protein in hepatocellular carcinoma cells and the IC50 of OBI-3424. The middle panel shows the correlation between the expression level of AKR1C3 RNA in hepatocellular carcinoma cells and the IC50 of OBI-3424. The right panel shows the correlation between the expression level of AKR1C3 RNA in non-small cell lung cancer cells and the IC50 of OBI-3424. [Figure 2]Figure 2 shows the cytotoxicity of compound OBI-3424 against leukemia cell lines. Figure 2a shows the cytotoxicity of compound OBI-3424 against six types of B-ALL cell lines. Figure 2b shows the cytotoxicity of compound OBI-3424 against seven types of T-ALL cell lines. Figure 2c shows the correlation between ex vivo AKR1C3 protein expression and cell viability in 18 types of ALL PDX at an OBI-3424 concentration of 10 nmol / L. Figure 2d shows the correlation between ex vivo AKR1C3 protein expression and cell viability in 18 types of ALL PDX at an OBI-3424 concentration of 100 nmol / L. [Figure 3] Figure 3 shows the correlation between AKR1C3 mRNA expression levels and protein expression levels in leukemia cell lines. Figure 3a shows the AKR1C3 mRNA expression levels in various ALL PDXs detected by RNA-Seq analysis. Figure 3b shows the AKR1C3 protein expression levels in various ALL PDXs detected by Western blotting. Figure 3c shows the correlation between AKR1C3 RNA expression levels and protein expression levels. Figure 3d shows the correlation between AKR1C3 RNA expression levels and the IC50 of OBI-3424. [Figure 4] FIG. 4 shows the location information of the AKR1C3 primer-probe in this application. [Figure 5] Figure 5 shows the amplification results of nine pairs of AKR1C3 primer-probes. Figure 5a shows the amplification results of the F1R1P1 primer-probe, Figure 5b shows the amplification results of the F2R2P1 primer-probe, Figure 5c shows the amplification results of the F2R6P1 primer-probe, Figure 5d shows the amplification results of the F6R2P1 primer-probe, Figure 5e shows the amplification results of the F6R6P1 primer-probe, Figure 5f shows the amplification results of the F5R5P2 primer-probe, Figure 5g shows the amplification results of the F3R3P1 primer-probe, Figure 5h shows the amplification results of the F4R3P2 primer-probe, and Figure 5i shows the amplification results of the F7R7P3 primer-probe. [Figure 6]Figure 6 shows the amplification results and standard curves of three pairs of AKR1C3 primer-probes. Figure 6a shows the amplification results of the F2R6P1 primer-probe. Figure 6b shows the standard curve of the F2R6P1 primer-probe (○ indicates the standard, E (amplification rate) = 82.5%, R2 = 0.987, Slope = -3.826, y-int (y-intercept) = 51.961). Figure 6c shows the standard curve of the F6R2P1 primer-probe. The amplification results are shown in Figure 6d, and the calibration curve for the F6R2P1 primer-probe is shown (○ indicates the standard, E (amplification rate) = 96.5%, R2 = 0.982, Slope = -3.408, y-int (y-intercept) = 49.591). Figure 6e shows the amplification results for the F6R6P1 primer-probe, and Figure 6f shows the calibration curve for the F6R6P1 primer-probe (○ indicates the standard, E (amplification rate) = 98.0%, R2 = 0.992, Slope = -3.371, y-int (y-intercept) = 49.385). [Figure 7] Figure 7 shows the amplification results and standard curve for the AKR1C3 plasmid. Figure 7a shows the amplification results for the AKR1C3 plasmid, and Figure 7b shows the standard curve for the AKR1C3 plasmid (○ indicates the standard, E (amplification rate) = 100.7%, R2 = 0.999, Slope = -3.305, y-int (y-intercept) = 42.336). [Figure 8] Figure 8 shows the amplification results of four types of reference gene primer-probes. Figure 8a shows the amplification results of the reference gene GAP, Figure 8b shows the amplification results of the reference gene GOLGA1, Figure 8c shows the amplification results of the reference gene ACTB, and Figure 8d shows the amplification results of the reference gene HPRT1. [Figure 9] FIG. 9 shows the Western blot results of AKR1C3 expression in different cell lines. [Figure 10]Figure 10 shows the results of detecting AKR1C3 expression in different cell lines using four types of reference genes. Figure 10a shows the results of detecting AKR1C3 expression in different cell lines using the reference gene HPRT1. Figure 10b shows the results of detecting AKR1C3 expression in different cell lines using the reference gene GAP. Figure 10c shows the results of detecting AKR1C3 expression in different cell lines using the reference gene ACTB. Figure 10d shows the results of detecting AKR1C3 expression in different cell lines using the reference gene GOLGA1. [Figure 11] FIG. 11 shows the results of different cell lines detected by the AKR1C3-ACTB system. [Figure 12] Figure 12 shows the results of the AKR1C3-ACTB system in the CCRF-CEM cell line. Figure 12a shows the amplification results for AKR1C3 in the CCRF-CEM cell line, and Figure 12b shows the calibration curve for AKR1C3 in the CCRF-CEM cell line (target: AKR1C3, Eff (amplification rate) % = 94.19%, R2 = 0.998, slope = -3.47, y-intercept = 34.595). Figure 12c shows the amplification results for ACTB in the CCRF-CEM cell line, and Figure 12d shows the calibration curve for ACTB in the CCRF-CEM cell line (target: ACTB, Eff (amplification rate) % = 95.302%, R2 = 0.991, slope = -3.44, y-intercept = 28.854). [Figure 13] Figure 13 shows the results of the plasmids detected with the AKR1C3-ACTB system. Figure 13a shows the amplification results for the AKR1C3 plasmid, and Figure 13b shows the calibration curve for the AKR1C3 plasmid (target: AKR1C3, Eff (amplification rate)% = 101.853%, R2 = 0.998, slope = -3.278, y-intercept = 38.048). Figure 13c shows the amplification results for the ACTB plasmid, and Figure 13d shows the calibration curve for the ACTB plasmid (target: ACTB, Eff (amplification rate)% = 94.134%, R2 = 1, slope = -3.471, y-intercept = 40.821). [Figure 14] FIG. 14 shows the qPCR detection results of the Jurkat cell line. [Figure 15] Figure 15 shows the results of digital PCR detection of cell lines (FAM and VIC were detected simultaneously). [Figure 16] Figure 16 shows the results of AKR1C3 expression in two types of cell lines detected by digital PCR. Figure 16a shows the results of AKR1C3 expression in the CCRF-CEM cell line detected by digital PCR, and Figure 16b shows the results of AKR1C3 expression in the MOLT-4 cell line detected by digital PCR. [Figure 17] Figure 17 shows the results of ACTB expression in two types of cell lines detected by digital PCR. Figure 17a shows the results of ACTB expression in the CCRF-CEM cell line detected by digital PCR, and Figure 17b shows the results of ACTB expression in the MOLT-4 cell line detected by digital PCR. [Figure 18] Figure 18 shows the results of plasmid amplification in real sample tests using the AKR1C3-ACTB system. Figure 18a shows the amplification results of the AKR1C3 plasmid, and Figure 18b shows the calibration curve for the AKR1C3 plasmid (target: AKR1C3, Eff (amplification rate)% = 99.108%, R2 = 0.996, slope = -3.344, y-intercept = 38.128). Figure 18c shows the amplification results of the ACTB plasmid, and Figure 18d shows the calibration curve for the ACTB plasmid (target: ACTB, Eff (amplification rate)% = 93.445%, R2 = 0.998, slope = -3.49, y-intercept = 40.636). [Figure 19] FIG. 19 is a histogram of FAM copy number / VIC copy number in the actual sample. [Figure 20] Figure 20 shows the results of copy numbers of 5-fold dilutions of cDNA from cell lines detected by digital PCR. DETAILED DESCRIPTION OF THE INVENTION

[0039] Unless otherwise defined, technical or scientific terms used in one or more examples herein have the ordinary meaning as understood by one of ordinary skill in the art to which this disclosure belongs.

[0040] Unless otherwise specified, all experimental methods in the following examples are conventional methods. All raw materials such as pharmaceuticals and reagents used in the following examples are commercially available products unless otherwise specified.

[0041] "Administering" a drug to a patient refers to direct administration (whether administered to the patient by a health care professional or self-administered) and / or indirect administration (such as writing a drug prescription). For example, a physician who instructs a patient to self-administer a drug or provides a patient with a drug prescription is administering the drug to the patient.

[0042] "Cancer" refers to leukemias, lymphomas, carcinomas, and other malignancies (including solid tumors) that can spread locally by invasion and potentially systemically by metastasis with unrestricted growth. Examples of cancer include, but are not limited to, cancers of the adrenal gland, bone, brain, breast, bronchus, colon and / or rectum, gallbladder, head and neck, kidney, throat, liver, lung, nervous tissue, pancreas, prostate, parathyroid gland, skin, stomach, and thyroid gland. Other specific cancer examples include acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, poorly differentiated cervical epithelial and intraepithelial carcinoma, Ewing's sarcoma, epidermoid carcinoma, giant cell tumor, glioblastoma multiforme, hairy cell tumor, intestinal ganglion cell tumor, proliferative corneal nerve tumor, pancreatic islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, mammary gland, and thyroid gland. These include ruffianoid habitus tumor, myeloid epithelial carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, granuloma mycosis, neuroblastoma, osteosarcoma, osteogenic sarcoma and other sarcomas, ovarian tumors, pheochromocytoma, polycythemia vera, primary brain tumors, small cell lung cancer, ulcerative and papillary squamous cell carcinoma, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, focal skin lesions, reticulum cell sarcoma, and Wilms' tumor.

[0043] The terms "patient" and "individual" can be used interchangeably herein and refer to a mammal in need of cancer treatment. Generally, a patient is a human. Generally, a patient is a human who has been diagnosed with cancer. In certain instances, a "patient" or "individual" may be a non-human mammal used to screen, characterize, and evaluate drugs and treatments, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat.

[0044] A "prodrug" refers to a compound that, after administration, is metabolized or converted into a biologically active or more active compound (or drug) with respect to at least one property. A prodrug is chemically modified to be less active or inactive compared to a drug, such that the corresponding drug is produced by metabolic or other biological processes after administration of the prodrug. A prodrug can have altered metabolic stability or transport properties, fewer side effects or toxicity, or improved flavor compared to an active drug (see, e.g., Nogrady, 1985, Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, pages 388-392, the disclosure of which is incorporated herein by reference). A prodrug can be synthesized using reactants other than the corresponding drug.

[0045] "Solid tumor" refers to solid tumors, including, but not limited to, metastatic tumors in bone, brain, liver, lung, lymph node, pancreas, prostate, skin, and soft tissue (sarcomas).

[0046] "Treating" a condition or patient refers to taking steps to obtain a beneficial or desired result, including a clinical result. For purposes of this invention, a beneficial or desired clinical result includes, but is not limited to, alleviation or amelioration of one or more symptoms of cancer, reduction in the extent of disease, delay or slowing of disease progression; palliation, remission, or stabilization of disease; or other beneficial result. Treatment of cancer can also be a partial response or stable disease.

[0047] "Tumor cell" refers to a tumor cell of any suitable species, for example, a mammal, such as a mouse, dog, cat, horse, or human.

[0048] A "therapeutically effective amount" of a drug refers to the amount of drug that, when administered to a cancer patient, exerts the intended therapeutic effect, such as alleviating, ameliorating, remission, or eliminating one or more symptoms of cancer in the patient. The therapeutic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

[0049] In describing an optically active compound, the prefixes R and S are used to denote the absolute configuration of the molecule about its chiral center. (+) and (-) are used to denote the optical activity of the compound, i.e., the direction in which a plane of polarized light rotates through the optically active compound. The prefix (-) denotes that the compound is levorotatory, i.e., the compound rotates the direction of a plane of polarized light to the left or counterclockwise. The prefix (+) denotes that the compound is dextrorotatory, i.e., the compound rotates the direction of a plane of polarized light to the right or clockwise. However, the optical designations (+) and (-) are independent of the absolute R and S configuration of the molecule.

[0050] The structures of compounds OBI-3424 (also referred to as AST-3424 and TH-3424), OBI-3423 and OBI-2870 are shown below. [ka] wherein OBI-3424 is the S-enantiomer, OBI-3423 is the R-enantiomer, and OBI-2870 is a racemic mixture of OBI-3424 and OBI-3423 in a 1:1 ratio.

[0051] Previous studies have shown that compounds OBI-3424, OBI-3423, and OBI-2870 have good therapeutic effects on various human cancers, especially solid tumors and hematological malignancies. The therapeutic effect of compound OBI-3424 on solid tumors (including liver cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer, melanoma, prostate cancer, breast cancer, esophageal cancer, kidney cancer, gastric cancer, colorectal cancer, brain tumor, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, sarcomatoid cancer, and rectal cancer) is described in Chinese Patent Application Publication No. 108290911. The therapeutic effect of compound OBI-3424 on hematological malignancies (including B-lineage acute lymphoblastic leukemia (B-ALL) and T-lineage acute lymphoblastic leukemia (T-ALL)) is described in International Publication No. 2019 / 062919.

[0052] AKR1C3 is overexpressed in many types of cancer, particularly in liver cancer, gastric cancer, renal cancer, CRPC, and non-small cell lung cancer. Because of the high expression of AKR1C3 in tumors, the compound OBI-3424 was designed to be specifically activated in tumors, but was unable to activate in normal cells with low AKR1C3 expression, resulting in tumor-specific targeting. OBI-3424 was developed as a highly potent DNA-alkylating prodrug selectively activated by AKR1C3. In the presence of NADPH, OBI-3424 is reduced to an intermediate that spontaneously hydrolyzes from AKR1C3 to OBI-2660. OBI-2660 is a DNA dialkylating agent similar to thioTEPA (N,N',N''-triethylenethiophosphoramide), which induces DNA crosslinking at the N7 (or O6) position of guanine, subsequently leading to cell death, as shown below. [ka]

[0053] Further studies conducted by the applicant have confirmed that the activation of compound OBI-3424 is AKR1C3-dependent, and its cytotoxicity and antitumor effects are highly correlated with the expression level of AKR1C3 protein. Compound OBI-3424 has demonstrated ex vivo AKR1C3-dependent cytotoxicity and in vivo antitumor activity in various types of human cancer, supporting the further development of compound OBI-3424 as an anticancer agent that can be used to treat different types of cancer. AKR1C3 can be used as a biomarker to analyze the status of cancer patients and further guide patients to select compound OBI-3424 for treatment.

[0054] In Example 1 of the present application, AKR1C3 enzyme and AKR1C3 RNA content in solid tumors were measured to assess the IC value of compound OBI-3424 on cancer cells. 50 It was confirmed that the IC value of OBI-3424 on cancer cells was linearly correlated with the amount of AKR1C3 enzyme and AKR1C3 RNA after mathematical transformation, and the linear correlation coefficient was sufficiently high. In Examples 2 and 3, the IC value of OBI-3424 on cancer cells was linearly correlated with the amount of AKR1C3 enzyme and AKR1C3 RNA in blood tumors. 50 The therapeutic effect of the drug on patients was confirmed by the IC. 50 It is well known to those skilled in the art that the value of IC 50 The lower the value, the better the efficacy of the drug for the patient. In all experimental data in Examples 1 to 3, the IC 50 The researchers then detected relevant biometric values ​​that could be correlated with the values, and then predicted the efficacy of drugs in specific cancer patients, achieving targeted drug delivery (in practice, tumor tissue from specific patients was directly collected and IC was measured under conditions of cancer cell survival). 50 While this is best and most intuitive and accurate, it is not feasible in practice, so it is best to only detect relevant biometric values ​​that can be objectively measured and then use IC 50After extensive detection, the present application demonstrates that AKR1C3 enzyme and AKR1C3 RNA content in solid tumors and hematological tumors correlate with the IC value of compound OBI-3424 on cancer cells. 50 We confirmed that the AKR1C3 enzyme and AKR1C3 RNA contents in solid tumors and blood tumors not only correlated linearly with the IC value, but also had a sufficiently high linear correlation coefficient, indicating that the IC value of the compound OBI-3424 on cancer cells was significantly correlated with the IC value. 50 It has been fully demonstrated that the AKR1C3 enzyme content and AKR1C3 RNA content are highly correlated with the value.Therefore, in practical application, the AKR1C3 enzyme content and AKR1C3 RNA content of the ex vivo sample of patient can be detected, and then according to the detection result, compound OBI-3424 can be administered to patient for treatment.

[0055] In the prior art, Western blotting or immunochemical staining is usually used to detect AKR1C3 enzyme content, but there is no corresponding detection method for detecting AKR1C3 RNA content in the prior art.Assuming that AKR1C3 RNA content can be evaluated using a corresponding assay, patients with higher AKR1C3 RNA content and who are most likely to respond to prodrugs can be selected to be administered with compound OBI-3424 to achieve better cancer treatment effects.

[0056] Based on the above objectives, the present invention is based on real-time fluorescent quantitative PCR technology (qPCR), adopting RNA reverse transcription reaction, polymerase chain reaction and TaqMan probe technology to detect the expression of AKR1C3 gene, and establishes a method and corresponding kit for human AKR1C3 gene expression that can be applied to the home-test (LDT) system, which can finally be used for drug detection in clinical trial sample companion diagnostics.

[0057] The basic principles for primer and probe design are as follows: (1) Primer-probe length: The length of each primer should be 15–30 bases. (2) The GC% of the primer-probe should be in the range of 30–80%, and the Tm value of the primer should be in the range of 55–60°C. (3) Base pairing between the primer-probe itself and the 3' end of the primer should be avoided as much as possible. (4) Six or more consecutive base pairs between the primer-probe itself and the primer should be avoided as much as possible. (5) The length of the target fragment amplified by the upstream and downstream primers should be in the range of 50–180 bp. The probe should be located between the upstream and downstream primers as close to the upstream primer as possible. The use of guanine at the 5' end of the probe should be avoided. If the probe annealing temperature is too low, consider designing an LNA or MGB probe. (6) The selected primers and probe should be designed in a conserved region of the AKR1C3 gene to specifically detect AKR1C3 gene expression.

[0058] Technical principle: RNA reverse transcription, polymerase chain reaction, and TaqMan® probe technology are used to design specific primers and probes targeting AKR1C3 gene expression. The target sequence is amplified by PCR using specific primers, and the TaqMan® probe bound to the template is cleaved by Taq enzyme (5' to 3' exonuclease activity), separating the reporter from the quencher and generating and accumulating a fluorescent signal. A real-time amplification curve can be obtained by tracking the relationship between the fluorescent signal and the number of amplification cycles, allowing the expression level of AKR1C3 RNA to be detected.

[0059] This invention designs nine pairs of primers and probes for three target regions of AKR1C3 (exons 2-3, 4-5, and 5-6, respectively). The primer-probe location information is shown in Figure 1. Beacon Designer 8.12 software is used to assist in the evaluation of primer quality, which is consistent with the basic principles of primer-probe design, and the blast tool in the NCBI database is used to ensure that each primer-probe pair specifically amplifies the human gene sequence without any common SNP sites.

[0060] A first aspect of the present invention is (i) an upstream primer AKR1C3-F1, a downstream primer AKR1C3-R1 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; (ii) an upstream primer AKR1C3-F2, a downstream primer AKR1C3-R2 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 3, respectively; (iii) an upstream primer AKR1C3-F2, a downstream primer AKR1C3-R6 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 3, respectively; (iv) an upstream primer AKR1C3-F6, a downstream primer AKR1C3-R2 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:3, respectively; (v) an upstream primer AKR1C3-F6, a downstream primer AKR1C3-R6 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:3, respectively; (vi) an upstream primer AKR1C3-F5, a downstream primer AKR1C3-R5 and a probe AKR1C3-P2, the nucleotide sequences of which are set forth in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; (vii) an upstream primer AKR1C3-F3, a downstream primer AKR1C3-R3 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 3, respectively; (viii) an upstream primer AKR1C3-F4, a downstream primer AKR1C3-R3 and a probe AKR1C3-P2, whose nucleotide sequences are set forth in SEQ ID NO: 13, SEQ ID NO: 12 and SEQ ID NO: 10, respectively; (ix) an upstream primer AKR1C3-F7, a downstream primer AKR1C3-R7, and a probe AKR1C3-P3, whose nucleotide sequences are set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; The present invention provides a primer-probe composition selected from any one of the following groups:

[0061] In the present invention, amplification of AKR1C3 was carried out by using the above nine groups of primer-probe compositions, as well as groups (iii), (iv), and (v) selected from the nine groups of primer-probe compositions, as candidate primer-probe compositions according to amplification rate and specificity. To determine the best primer-probe composition, further AKR1C3 amplification was carried out in different samples using primer-probe compositions from each of groups (iii), (iv), and (v). According to the amplification rate, the primer-probe composition of group (iv) (upstream primer AKR1C3-F6, downstream primer AKR1C3-R2, and probe AKR1C3-P1) was finally determined to be the best primer-probe composition of the present invention.

[0062] The nine primer-probe compositions of the present invention can be used not only to detect AKR1C3 RNA content by qPCR, but also to detect AKR1C3 RNA content by digital PCR.The steps of detecting AKR1C3 RNA content by qPCR and digital PCR are described in detail below.

[0063] In a preferred embodiment of the present invention, the 5'-end reporter of probes AKR1C3-P1, AKR1C3-P2 and AKR1C3-P3 is FAM, and the 3'-end quencher of probes AKR1C3-P1, AKR1C3-P2 and AKR1C3-P3 is MGB.

[0064] A preferred probe for practicing the present invention is a probe labeled according to the TaqMan system. The TaqMan system is available from Life Technologies. According to the TaqMan system, an oligonucleotide probe specifically designed to hybridize to the amplified target DNA is covalently linked to the 5' end of a reporter and the 3' end of a quencher. Suitable reporters for use in the TaqMan system include, for example, 6-carboxyfluorescein (FAM) or tetrachlorofluorescein (TET). A typical quencher is a minor groove binder (MGB). The principle of the TaqMan system is that as long as the quencher and reporter are positioned very close to each other on the probe, the quencher inhibits reporter fluorescence. When an oligonucleotide probe hybridizes to target DNA during qPCR amplification, it is degraded by Taq polymerase as the enzyme extends the oligonucleotide primer along the DNA corresponding to the target DNA. This degradation releases the reporter and quencher, and because the quencher is no longer in close proximity to the reporter, the reporter can emit fluorescence that can be detected and measured by appropriate tools that are typically integrated into qPCR instruments (i.e., thermal cyclers).

[0065] Based on a similar inventive concept, a second aspect of the present invention provides a kit comprising the above primer-probe composition.

[0066] In a preferred embodiment of the present invention, the kit further comprises a primer-probe composition for a reference gene, and preferably, the primer-probe composition for a reference gene comprises an upstream primer ACTB-F1, a downstream primer ACTB-R1 and a probe ACTB-P1, whose nucleotide sequences are set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0067] When measuring gene expression levels using qPCR technology, the absolute expression level of the target gene is rarely measured directly, but the expression levels of the target gene and reference gene are measured separately. Specifically, the expression level of a standard reference gene is collected to measure the relative expression level of the target gene, and the relative expression levels between samples are compared. When measuring gene expression levels using relative quantification, the Ct value obtained by qPCR is an exponential relationship, not a linear relationship. Therefore, Ct values ​​cannot be directly used in statistical analysis methods that require normal distribution of data, such as t-tests and analysis of variance (ANOVA). Therefore, before performing statistical analysis, the original Ct value must first be doubled. -Ct After obtaining the data using the relative quantification method, the comparative CT method (2 -ΔΔCT The PCR amplification method is widely used. This method is based on the following two assumptions: (1) the amplification rate is 100%, i.e., the amount of product doubles in each PCR cycle, but this can be resolved by verifying the amplification rate; (2) there is an appropriate reference gene to correct for errors in sample loading; fold change = 2 -ΔΔCt The formula for calculating ΔΔCt is (Ct of target gene - Ct of reference gene) in the treatment group - (Ct of target gene - Ct of reference gene) in the control group. According to this formula, in the relative quantification method, without the Ct value of the reference gene, it is impossible to calculate the relative expression of the target gene, suggesting that the reference gene plays a very important role.

[0068] Reference gene refers to a known reference gene whose expression level is not affected by research conditions and can be consistently expressed among multiple samples, and the expression level of this gene can be used to quantify the amount of initial material used with high accuracy.The expression level of housekeeping genes is not easily affected by environmental factors and can be continuously expressed in almost all tissues and various growth stages of organisms, so housekeeping genes are usually used as reference genes in experiments.

[0069] According to the reference (DONG Enni, LIANG Qing, LI Li, et al., "Selection of Reference Genes in Real-Time Quantitative PCR" [J]. China Animal Industry, 49(11):92-96. DOI:10.3969 / J.issn.0258-7033.2013.11.025) and the reference (ZHAO Wen-Jing, XU Jie, BAO Qiuhua, et al., "Selection of Reference Genes for Real-Time Quantitative PCR" [J]. Microbiology China, 2010(12):1825-1829. DOI:CNKI:SUN:WSWT.0.2010-12-019), the reference genes used in q-PCR are as follows:

[0070] [Table A]

[0071] Regarding reference genes, four reference genes (HPRT1 (hypoxanthine phosphoribosyltransferase), GAP (glyceraldehyde-3-phosphate dehydrogenase), ACTB (also known as β-actin), and GOLGA1 (Golgi protein, specifically Golgi autoantigen A1)) were screened in the present invention. The primer-probe compositions corresponding to each of the four reference genes (listed in Table 1) were used to detect the CCRF-CEM cell line, and the fluorescence intensity and amplification were evaluated. Different samples (including healthy blood samples and bone marrow samples from leukemia patients) and cell lines with different expression levels (including cell lines with high and low AKR1C3 expression) were further detected to verify expression stability and whether samples and cell lines with high and low expression levels could be distinguished. The results showed that using ACTB as a reference gene made it easier to distinguish between the cell lines RPMI8226 and CCRF-CEM, which had high expression levels, and the cell line Jurkat, which had low expression levels, compared with using the other three reference genes. The expression levels of AKR1C3 were high in bone marrow RNA samples from leukemia patients, whereas those from healthy individuals remained relatively low. Based on the amplification curve results, ACTB was selected as the reference gene. Therefore, the primer-probe composition for the reference gene in the above kit includes the upstream primer ACTB-F1, the downstream primer ACTB-R1, and the probe ACTB-P1.

[0072] In a preferred embodiment of the present invention, the 5'-end reporter of probe ACTB-P1 is VIC (green fluorescent protein), and the 3'-end quencher of probe ACTB-P1 is BHQ1 (black hole quencher 1).

[0073] The best AKR1C3 primer-probe compositions and reference ACTB primer-probe compositions finally determined after screening are shown in Table 1.

[0074] [Table 1-1] [Table 1-2]

[0075] Reference 1: Wu, X., Blackburn, P., Tschumper, R. et al., "TALEN-mediated gene modulation as a tool to analyze the function of acquired mutations in multiple myeloma cells," Blood Cancer Journal 4, e210 (2014) https: / / doi.org / 10.1038 / bcj.2014.32 and corresponding nucleic acid sequences disclosed therein. Reference 2: HPRT1 nucleic acid sequence disclosed in WO 2018 / 123764. Reference 3: Werner Kempf, Marshall E. Kadin, Ann M. Dvorak, Carol C. Lord, Gunter Burg, Norman L. Letvin, Igor J. Koralnik, "Endogenous but not exogenous retroviral elements are detected in CD30-positive lymphoproliferative disorders of the skin," Carcinogenesis, Volume 24, Issue 2, February 2003, Pages 301-306, https: / / doi.org / 10.1093 / carcin / 24.2.301 and corresponding nucleic acid sequences disclosed therein. Reference 4: Stecker C, Johann A, Herzberg C, Averhoff B, Gottschalk G., "Complete nucleotide sequence and gene organization of a 210-kilobase linear plasmid of Rhodococcus erythropolis BD2." J Bacteriol. 2003;185(17):5269-5274. doi:10.1128 / jb.185.17.5269-5274.2003. Corresponding nucleic acid sequence disclosed in

[0076] All sequences not listed in the source were new sequences or sequences newly developed or selected for the kit.

[0077] In a preferred embodiment of the invention, the kit further comprises a polymerase mixture, the polymerase mixture essentially comprises a DNA polymerase, MgCl2, a buffer, and dNTPs; Preferably, the polymerase mixture is KAPA PROBE FAST RT-PCR Master Mix (2x), which has been used in laboratories for many years and has been repeatedly verified to have a good amplification rate.

[0078] In a preferred embodiment of the present invention, the kit further comprises a reverse transcriptase mixture, preferably wherein the reverse transcriptase mixture is Superscript VILO MARSTER MIX.

[0079] Each PCR reaction must be detected and analyzed with a positive control and a negative control (NC, nuclease-free water). In a preferred embodiment of the present invention, the kit further comprises a negative control and a positive control, preferably the negative control is nuclease-free water and / or preferably the positive control is a reference with a known copy number.

[0080] The kit may further include components necessary for performing qPCR, such as reagents known to those skilled in the art. In addition to primer pairs and probes, the kit may further include one or more enzymes (Taq polymerase) or reagents used in the qPCR reaction. The enzymes may be in lyophilized form or in an appropriate buffer. In addition, the kit may include all additional components necessary for performing qPCR, such as buffers, extraction reagents, enzymes, pipettes, plates, nucleic acids, filter paper, gel materials, transfer materials, autoradiography equipment, and instructions (recording related procedures).

[0081] Based on the same inventive concept, a third aspect of the present invention provides the use of the primer-probe composition or kit in the preparation of a cancer therapeutic drug.

[0082] In a preferred embodiment of the present invention, the AKR1C3 RNA content in a patient's ex vivo sample is determined using the primer-probe composition or the kit, and an AKR1C3-activating anticancer drug is administered to patients whose AKR1C3 RNA content is equal to or greater than a predetermined content.

[0083] Therefore, the present invention provides determining the AKR1C3 RNA content in an ex vivo sample from a patient using a primer-probe composition or kit as described above; administering an AKR1C3-activating anticancer drug to a patient whose AKR1C3 RNA content is equal to or greater than a predetermined content, preferably administering a therapeutically effective amount of compounds OBI-3424, OBI-3423, and OBI-2870 to the patient; The present invention provides a method for treating a cancer patient, comprising:

[0084] In a preferred embodiment of the present invention, the AKR1C3 RNA content is determined according to the ratio of AKR1C3 copy number / reference gene copy number, for example, the ratio of AKR1C3 copy number / ACTB copy number is calculated, and if the ratio of AKR1C3 copy number / ACTB copy number is greater than X, the expression of AKR1C3 is determined to be high, and the patient is given a therapeutically effective amount of compounds OBI-3424, OBI-3423 and OBI-2870; if the ratio of AKR1C3 copy number / ACTB copy number is less than X, the expression of AKR1C3 is determined to be low, and the patient is not given a therapeutically effective amount of compounds OBI-3424, OBI-3423 and OBI-2870, but can be given other cancer drugs.

[0085] The value of the predetermined content X needs to be determined by detecting a large number of samples. Using a large number of clinical test samples and statistical methods, the present inventors finally determined that the predetermined content X is 0.0001 to 1, more preferably 0.00011 to 0.5, even more preferably 0.00013 to 0.05, and particularly preferably 0.00014 to 0.015. For example, the predetermined content X may be 0.0001 to 0.0005, 0.0001 to 0.001, 0.0001 to 0.005, 0.0001 to 0.01, 0.0001 to 0.05, 0.0001 to 0.1, 0.0001 to 0.5, 0.0001 to 0.9, 0.00011 to 0.0005, 0.00011 to 0.001, 0.00011 to 0.005, 0.00011 to 0.01, 0.00011 to 0.05, 0.00011 to 0.1, 0.00011 to 0. 5, 0.00011~1, 0.00013~0.0005, 0.00013~0.001, 0.00013~0.01, 0.00013~0.05, 0.00013~0.1, 0.00013~0.5, 0.00013~1, 0.0005~0.001, 0.0005~0.005, 0.0005~0.01, 0.0005~0.05, 0.0005~0.1, 0.0005~0.5, 0.0005~1, 0.00091~0.001, 0.0009 1~0.05, 0.00091~0.1, 0.00091~0.5, 0.00091~1, 0.001~0.005, 0.001~0.01, 0.001~0.05, 0.001~0.1, 0.001~0.5, 0.001~1, 0.005~0.01, 0.005~0.05, 0.005~0.1, 0.005~0.5, 0.005~1, 0.01~0.05, 0.01~0.1, 0.01~0.5, 0.01~1, 0.05~0.1, 0.05~ 0.5 or 0.05 to 1, and more preferably the predetermined content X is 0.0001, 0.00011, 0.00012, 0.00013, 0.00014, 0.00015, 0.000156, 0.00016, 0.000163, 0.000165, 0.00017, 0.000177, 0.00018, 0.000181, 0.00019, 0.000195, 0.0002, 0.000201, 0.000216, 0.000221, 0.00023, 0.000239, 0.00025, 0.00029, 0.0003, 0.00035, 0.0004, 0.00045, 0.0005, 0.00054, 0.0006, 0.00065, 0.0007, 0.00075, 0.0008, 0.00085, 0.0009, 0.00095, 0.001, 0.00 15, 0.00177, 0.002, 0.00238, 0.0025, 0.00266, 0.00296, 0.003, 0.00315, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.00679, 0.007, 0.00710, 0.0075, 0.00778, 0 .00791, 0.008, 0.0085, 0.009, 0.00939, 0.0095, 0.01, 0.011, 0.012, 0.01207, 0.013, 0.01365, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.025, 0.03, 0.035, 0.04, 0.0 45, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1.

[0086] Based on a similar inventive concept, a fourth aspect of the present invention provides: (1) extracting RNA from an ex vivo sample to be detected, adding the extracted RNA to a reverse transcription system, and reverse transcribing the extracted RNA to synthesize cDNA; (2) performing qPCR or digital PCR amplification using the primer-probe composition or kit described above with cDNA as a template; (3) determining the AKR1C3 RNA content of the ex vivo sample to be detected according to the qPCR or digital PCR amplification results; The present invention provides a method for detecting AKR1C3 RNA content, comprising:

[0087] The methodological principle of the method for detecting AKR1C3 RNA content of the present invention is as follows: First, the sample RNA is reverse transcribed into cDNA, and then the cDNA is amplified using specific primers and probes. The AKR1C3 gene and the reference gene are represented by FAM and VIC signals, respectively, and the quantitative expression level of the AKR1C3 gene in peripheral blood RNA samples is calculated based on the sample and QC (quality control) products. At the same time, the VIC signal in the AKR1C3 reaction solution is used to monitor the quality of the sample.

[0088] In this invention, two methods, qPCR and digital PCR, are used to simultaneously carry out comparative experiments under sufficient sample conditions. The same primer-probe set is used for digital PCR and qPCR. First, the primer-probe is optimized using the temperature gradient of qPCR, and the most appropriate temperature and primer-probe composition are selected for the digital PCR platform and comparative method for quantitative reference material.

[0089] The detection process of digital PCR mainly involves two parts: PCR amplification and fluorescent signal analysis. Before the PCR reaction, the sample is divided into tens of thousands of units (reaction chambers) so that each unit contains only a single DNA molecule. The amplification procedure and system of digital PCR are essentially the same as those of conventional PCR. However, unlike conventional qPCR, digital PCR employs a direct counting method for quantitative analysis. That is, after PCR amplification is completed, the presence of a fluorescent signal (product) is marked as 1, and the absence of a fluorescent signal (product) is marked as 0. A reaction unit with a fluorescent signal contains at least one copy of the target molecule. Theoretically, if the concentration of target DNA in a sample is very low, the number of reaction units with a fluorescent signal is equal to the copy number of the target DNA molecule. However, under normal circumstances, a reaction unit in digital PCR may contain two or more target molecules, which can be calculated using the Poisson distribution.

number

[0090] In the above formula, λ is the average copy number (concentration) of the target DNA molecule contained in each reaction unit, and p is the probability of k copies of the target DNA molecule contained in each reaction unit under specific λ conditions. λ is determined by the dilution factor m of the sample solution, λ = cm, where c is the original copy number (concentration) of the sample. When k = 0 (no target DNA molecules), the above formula becomes p = e -λ =e -cm where p is the ratio of the number of reaction units without a fluorescent signal to the total number of reaction units, i.e., it can be expressed by the following formula:

number

number

[0091] The initial copy number (concentration) of a sample can be determined from the total number of reaction units and the number of units with a fluorescent signal in digital PCR, as well as the sample dilution factor. Because quantitative digital PCR does not rely on the circularity threshold of the amplification curve, it is not affected by the amplification rate and does not require the use of a standard curve. High accuracy and reproducibility enable absolute quantitative analysis.

[0092] In the present invention, the specific reaction system for digital PCR differs from the specific reaction system for qPCR in the following respects: In the qPCR reaction system, a group of primer-probe compositions selected from groups (i) to (ix) is mixed with a primer-probe composition for the reference gene ACTB. Meanwhile, in the digital PCR reaction system, the maximum Ct difference can reach 10 Ct values ​​due to the large difference in the expression levels of AKR1C3 and ACTB in samples and cell lines. Therefore, the copy number of VIC may be much greater than the copy number of FAM during digital PCR detection. Therefore, the group of primer-probe compositions selected from groups (i) to (ix) is not mixed with the primer-probe composition for the reference gene ACTB, but the AKR1C3 digital PCR detection system and the reference gene digital PCR detection system are used to amplify AKR1C3 and the reference gene, respectively.

[0093] PCR amplification results are affected by various factors in the reaction system (including primer concentration, probe concentration, etc.) and changes in the amplification procedure. To achieve the best amplification rate and minimize nonspecific products, this application relies on several years of research and development experience to determine the reaction system and amplification procedure for digital PCR and qPCR. In combination with the selection of probe-primer, the correlation coefficient of the standard curve must be 0.98 or higher, and the E value (amplification rate) must be 85% to 110%.

[0094] In a preferred embodiment of the present invention, in the step (3), the step of determining the AKR1C3 RNA content of the ex vivo sample to be detected based on the qPCR or digital PCR amplification results comprises: (A) determining the copy numbers of AKR1C3 and reference genes in the ex vivo sample to be detected based on the qPCR or digital PCR amplification results; (B) calculating the ratio of AKR1C3 copy number / reference gene copy number to determine the AKR1C3 RNA content of the ex vivo sample to be detected; Includes.

[0095] When the qPCR method is used, the step (A) further comprises: (A1) plotting a standard curve of the Ct value and initial copy number lg value of AKR1C3 and a standard curve of the Ct value and initial copy number lg value of a reference gene; (A2) determining the copy numbers of AKR1C3 and the reference gene in the ex vivo sample to be detected based on the Ct values ​​of the detected AKR1C3 and the reference gene, respectively, using the calibration curve (this can be calculated automatically by software); Includes.

[0096] Another difference between digital PCR and qPCR is that qPCR obtains the copy number of AKR1C3 and the reference gene in the ex vivo sample to be detected according to a standard curve, whereas digital PCR can directly obtain the copy number of AKR1C3 and the reference gene in the ex vivo sample to be detected according to the amplification results without drawing a standard curve.

[0097] The Ct value of the target gene AKR1C3 and the Ct value of the reference gene are explained below. The Ct value of the target gene AKR1C3 corresponds to the amplification signal (FAM signal) of the target gene AKR1C3. The Ct value of the reference gene corresponds to the amplification signal (VIC signal) of the reference gene. The thresholds for FAM and VIC are set during the exponential amplification phase.

[0098] In a preferred embodiment of the present invention, the method is used to detect AKR1C3 RNA content in blood samples, bone marrow samples, or tissue samples.Digital PCR and qPCR of the present invention can not only detect blood samples or bone marrow samples, but also detect solid tumor tissue samples, except that pretreatment processes (such as RNA extraction process) are different.For example, for blood samples, RNA extraction can be carried out directly using an extraction kit manufactured by Qiagen; for bone marrow samples or tissue samples, the mortar can be pre-cooled with liquid nitrogen, and the bone marrow samples or tissue samples are ground into powder in the mortar, and the dried tissue powder is placed in a 1.5ml EP tube, and then an RNA extraction kit is used for extraction.

[0099] In this application, performance tests were conducted on the above kit and detection method, and the test results are as follows. 1. Minimum detection limit: The minimum detection limit was determined by gradient dilution of a low-expressing cell line used as a sample to be detected. The detection results were 100% consistent when repeated 10 times. 2. Precision: Repeated detections at different times, with different instruments and by different experimenters yielded CV values ​​of less than 15%. 3. Positive concordance rate: When known positive samples and the standard curve were detected by qPCR, the detection rate was 100% concordant, and the E value of the standard curve was 90-110%, R 2 was above 0.98. 4. Negative concordance rate: When known negative samples were detected by qPCR, the consistent detection rate was 100%. 5. Different loading template amounts: 200 ng and 10 ng of RNA from known negative and positive samples, respectively, were selected as the initial amounts for reverse transcription, and a range of loading amounts was verified, with a consistent detection rate of 100%. 6. Analysis specificity: Two concentrations were selected as negative criteria, and each concentration was repeated three times, resulting in a 100% negative rate. 7. Real sample detection: Eight normal peripheral blood RNA samples were selected for real sample detection, and the concordance rate of the detection results was 100%.

[0100] The performance of the method was verified for minimum detection limit, accuracy, negative concordance rate, positive concordance rate, template amount, analytical specificity, and real sample detection. All seven performance criteria were met. The performance of the method met the requirements for clinical application, and it can be used for clinical sample detection.

[0101] Based on a similar inventive concept, a fifth aspect of the present invention uses the method to detect the AKR1C3 RNA content of the ex vivo sample to be detected, and then determines the expression level of the AKR1C3 enzyme in the ex vivo sample to be detected according to the AKR1C3 RNA content of the ex vivo sample to be detected.

[0102] In a preferred embodiment of the present invention, there is a linear correlation between the AKR1C3 RNA content in the ex vivo sample to be detected and the expression level of the AKR1C3 enzyme in the ex vivo sample to be detected.

[0103] In the prior art, the administration of compounds OBI-3424, OBI-3423, and OBI-2870 is usually determined based on whether the AKR1C3 reductase content is above a certain level, but Example 4 of the present invention demonstrates that the expression level of AKR1C3 enzyme is highly correlated with the expression level of AKR1C3 RNA. Therefore, in practical application, the AKR1C3 enzyme content is calculated based on the AKR1C3 RNA content measured by the above method, and then the administration of compounds OBI-3424, OBI-3423, and OBI-2870 is determined based on whether the AKR1C3 enzyme content reaches the predetermined level. [Example]

[0104] The technical embodiments provided by the present invention are further described in the following examples. The following examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0105] For the synthesis method of the AKR1C3-activating anticancer prodrug OBI-3424 (also known as AST-3424 or TH-3424) used in the following examples, please refer to International Publication No. 2017 / 087428 or Chinese Patent Application Publication No. 108290911.

[0106] [Example 1] AKR1C3 protein and RNA levels and IC in liver cancer and non-small cell lung cancer (NSCLC) cell lines 50 Correlation with values

[0107] 1. Experimental Materials and Methods

[0108] 1.1 Cell lines All human cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) or the JCRB Cell Bank (JCRB, Osaka, Japan) or Cobioer Biosciences (Nanjing, China).

[0109] 1.2 Ex vivo proliferation assay Exponentially growing cells were seeded. After 24 hours, the test compound OBI-3424 was added. After adding the test compound OBI-3424, the plates were incubated at 37°C in a standard tissue incubator for the indicated time. At the end of the experiment, viable cells were detected using the CellTiter Glo (CTG) assay kit or AlamarBlue. The drug concentration resulting in 50% growth inhibition compared to untreated controls (IC 50 ) were calculated using XLfit (IDBS, Boston, MA) or Prism 6 (GraphPad, San Diego, CA).

[0110] 1.3 Western blotting Human cell extracts were prepared and protein concentrations were determined. Proteins were detected using antibodies recognizing human AKR1C3 and tubulin or β-actin. Band densities of AKR1C3 and tubulin or β-actin were scanned and quantified using an Odyssey laser imaging system and software (LI-COR Biosciences, Lincoln, NE) or a UVP ChemStudio imaging system and VisionWorks software (Analytik Jena), and the ratios of AKR1C3 to tubulin or β-actin were calculated.

[0111] 2. Experimental Results Hepatocellular carcinoma cell lines were exposed to compound OBI-3424 for 96 hours, and NSCLC cell lines were exposed to compound OBI-3424 for 72 hours, after which IC was measured by ex vivo proliferation assay. 50The expression of AKR1C3 protein in hepatocellular carcinoma cell lines was determined by Western blotting (tubulin was used as a loading control). AKR1C3 RNA expression data in hepatocellular carcinoma and NSCLC cell lines was obtained from the CrownBio database (https: / / db.CrownBio.com / crownbio / OncoExpress_Login.aspx). The results are shown in Tables 2 and 3.

[0112] [Table 2]

[0113] [Table 3]

[0114] As shown in Tables 2 and 3, hepatocellular carcinoma cell lines with high AKR1C3 expression, both in protein and RNA, were more sensitive to OBI-3424 and showed IC 50 On the other hand, cells with low AKR1C3 expression were less sensitive to OBI-3424, with IC 50 was higher than 1000 nM. Similarly, after NSCLC cells were exposed to compound OBI-3424 for 72 hours, NSCLC cells also showed AKR1C3-dependent cytotoxicity (Table 3). 3424 IC in hepatocytes 50 was highly correlated with the amount of AKR1C3 protein (R 2 =0.71, Figure 1, left), 3424IC in hepatocytes 50 was highly correlated with the expression level of AKR1C3 RNA (R 2 =0.87, Figure 1, center), 3424IC in non-small cell lung cancer 50 was highly correlated with the expression level of AKR1C3 RNA (R 2 = 0.80, Figure 1, right). These results indicated that 3424-mediated cytotoxicity in hepatic and NSCLC cell lines was highly correlated with the expression level of AKR1C3.

[0115] [Example 2] AKR1C3 protein abundance and IC in leukemia cell lines 50 Correlation with values

[0116] 1. Experimental Materials and Methods

[0117] 1.1 Ex vivo testing of cell lines and PDX All cell lines were purchased from HD Biosciences. All experimental work was conducted under the approval of the respective institutional review boards and animal ethics committees, and the use of human-related tissue samples was carried out in accordance with the ethical and relevant legal regulations of the respective localities. We used 20-25g female non-obese / SCID (NOD.CB17-Prkdcscid / SzJ, NOD / SCID) or NOD / SCID / IL2 receptor gamma negative (NOD.Cg-Prkdcscid / SzJ, NOD / SCID) rats. scid Il2rg tm1Wjl Previously established serial PDXs in mice (SzJAusb, NSG) were used for experiments as described elsewhere (Lock RB, Liem N, Farnsworth ML, Milross CG, Xue C, Tajbakhsh M, et al., "A non-obese diabetic / severe combined immunodeficient (NOD / SCID) mouse model of pediatric acute lymphoblastic leukemia reveals intrinsic differences in biological features at diagnosis and relapse," Blood 2002;99:4100-8). The development of lentiviral-transduced ALL-11 PDX [empty vector (EV) and AKR1C3 overexpressing] has been previously described (Jamieson SM, Gu Y, Manesh DM, El-Hoss J, Jing D, Mackenzie KL, et al., "A novel fluorometric assay for aldo-ketoreductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukemia cells," Biochem Pharmacol 2014;88:36-45).

[0118] 1.2 Ex vivo cytotoxicity assay Leukemia cell lines were suspended in RPMI medium supplemented with FBS (Biosera), while ALL PDX cells were cultured in QBSF medium (Quality Biologicals) supplemented with Flt-3 ligand (20 ng / mL, BioNovus Life Sciences) or IL7 (10–20 ng / mL, Jomar Life Research). Cells were seeded at optimal cell density and incubated for 3 hours or overnight at 37°C and 5% CO2. PDX cells and leukemia cell lines were treated with OBI-3424 (10 mmol / L–1 pmol / L) or medium control for 48 or 72 hours, respectively. Viability was determined using the Alamar Blue reduction assay or the Cell Titer-Glo luminescent cell viability assay (Promega). The half-maximal inhibitory concentration (IC) was determined. 50 ) was used to calculate the interpolation of the nonlinear regression curves using GraphPad Prism 7 software.

[0119] 1.3 Western blotting Cryopreserved leukemia cells were thawed and lysed in RIPA lysis buffer, and protein concentration was quantified by BCA assay. Each sample was loaded with 20 µg of protein lysate in a 4–12% NuPAGE Bis-Tris protein gel, then electrophoresed at 120 V and transferred to a polyvinyl difluoride membrane at 30 V for 1 h. The membrane was probed using mouse anti-AKR1C3 (#A6229, Sigma-Aldrich, St. Louis, MO) or rabbit anti-actin primary antibody (#A2066, Sigma-Aldrich), followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody (GE Healthcare, Buckingham, UK), respectively. Immobilon Western chemiluminescent HRP substrate (Merck Millipore, Billerica, MA) was used to detect the conjugated secondary antibody, and the signal was quantified on a BioRad Chemidoc touch imaging system.

[0120] 2. Experimental Results The ex vivo cytotoxicity of AKR1C3-associated OBI-3424 was observed in 11 T-ALL cell lines, one B-ALL cell line transfected with granulocyte colony-stimulating factor, and one BCP-ALL cell line. AKR1C3 protein expression levels were determined by Western blot analysis. The ex vivo cytotoxicity of OBI-3424 was determined using the CellTiter-Glo assay and expressed as the 50% maximal inhibitory concentration (IC 50 ) was calculated as

[0121] OBI-3424 demonstrated ex vivo cytotoxicity, with IC 50 The IC ranged from 3.0 to 30.0 nM. In cell lines with moderate AKR1C3 expression, 50 ranged from 3.0 to 84.0 nM (Table 4).

[0122] [Table 4]

[0123] To evaluate the potential antileukemic activity of OBI-3424, ex vivo cytotoxicity assays were performed against various leukemia cell lines. OBI-3424 demonstrated its potential for treating T-ALL, B-ALL, acute myeloid leukemia, acute promyelocytic leukemia (APL), and erythroleukemia. OBI-3424 exhibited potent cytotoxicity against cell lines derived from T-ALL (T-lineage acute lymphoblastic leukemia), which have particularly high AKR1C3 expression, and IC 50 The IC between cell lines with high / medium AKR1C3 expression and cell lines with low AKR1C3 expression was in the low range (nmol / L) (see Table 5). 50 The difference was statistically significant (P = 0.0016).

[0124] [Table 5]

[0125] Consistent with the results obtained with leukemia cell lines, OBI-3424 exerted potent cell-killing effects against all leukemia cell lines. Figure 2a shows the cytotoxicity of OBI-3424 against six B-ALL cell lines, and Figure 2b shows the cytotoxicity of OBI-3424 against seven T-ALL cell lines. Figures 2a and 2b demonstrate the AKR1C3-dependent activity of OBI-3424 as a DNA alkylating agent. Furthermore, as shown in Figure 2c, at an OBI-3424 concentration of 10 nmol / L, AKR1C3 protein expression showed a significant negative correlation with ex vivo cell viability in 18 ALL PDXs (r = -0.53, P = 0.023). Similarly, as shown in Fig. 2d, at an OBI-3424 concentration of 100 nmol / L, AKR1C3 protein expression showed a significant negative correlation with ex vivo cell viability of 18 ALL PDXs (r = −0.56, P = 0.015).

[0126] [Example 3] AKR1C3 RNA levels and IC in leukemia cell lines 50 Valence correlation

[0127] Hematological cancer cell lines were exposed to the compound OBI-3424 for 72 hours, and then IC was measured by ex vivo proliferation assay. 50 The expression data of AKR1C3 RNA in hematological cancer cell lines was obtained from the CrownBio database (https: / / db.CrownBio.com / crownbio / OncoExpress_Login.aspx). The results are shown in Table 6.

[0128] [Table 6]

[0129] Table 6. AKR1C3 RNA (Log2 FPKM) expression levels and IC 50 (nM) was plotted as a curve graph as shown in Figure 3d. 50The correlation coefficient R was calculated by curve fitting between the log2 FPKM, an algebraically transformed value of the AKR1C3 expression level, and the corresponding curve fitting equation. 2 = 0.7889, and the expression level of AKR1C3 RNA was IC 50 It was shown that there is a significant linear correlation between

[0130] [Example 4] Correlation between AKR1C3 protein levels and AKR1C3 RNA levels in leukemia cell lines

[0131] 1. Experimental Materials and Methods

[0132] 1.1 Ex vivo testing of cell lines and PDX All cell lines were purchased from HD Biosciences. All experimental work was performed under the approval of the respective institutional review boards and animal ethics committees. We used 20-25g female non-obese / SCID (NOD.CB17-Prkdcscid / SzJ, NOD / SCID) or NOD / SCID / IL2 receptor gamma negative (NOD.Cg-Prkdcscid / SzJ, NOD / SCID) rats. scid Il2rg tm1Wjl Previously established serial PDXs in mice (SzJAusb, NSG) were used for experiments as described elsewhere (Lock RB, Liem N, Farnsworth ML, Milross CG, Xue C, Tajbakhsh M, et al., "A non-obese diabetic / severe combined immunodeficient (NOD / SCID) mouse model of pediatric acute lymphoblastic leukemia reveals intrinsic differences in biological features at diagnosis and relapse," Blood 2002;99:4100-8). The development of lentiviral-transduced ALL-11 PDX [empty vector (EV) and AKR1C3 overexpressing] has been previously described (Jamieson SM, Gu Y, Manesh DM, El-Hoss J, Jing D, Mackenzie KL, et al., "A novel fluorometric assay for aldo-ketoreductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukemia cells," Biochem Pharmacol 2014;88:36-45).

[0133] 1.2 Western blotting Cryopreserved leukemia cells were thawed and lysed in RIPA lysis buffer, and protein concentration was quantified by BCA assay. Each sample was loaded with 20 µg of protein lysate in a 4–12% NuPAGE Bis-Tris protein gel, then electrophoresed at 120 V and transferred to a polyvinyl difluoride membrane at 30 V for 1 h. The membrane was probed using mouse anti-AKR1C3 (#A6229, Sigma-Aldrich, St. Louis, MO) or rabbit anti-actin primary antibody (#A2066, Sigma-Aldrich), followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody (GE Healthcare, Buckingham, UK), respectively. Immobilon Western chemiluminescent HRP substrate (Merck Millipore, Billerica, MA) was used to detect the conjugated secondary antibody, and the signal was quantified on a BioRad Chemidoc touch imaging system.

[0134] 1.3 RNA-Seq analysis To analyze AKR1C3 expression in aspirates from primary patients, patients were divided into B-ALL and T-ALL and their associated subgroups. Paired-end reads were mapped to the GRCh37 human genome reference using the recommended round-trip mapping pipeline with default parameters using STAR (Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al., "STAR: An Ultrafast Universal RNA-seq Aligner," Bioinformatics 2013;29:15-21). Duplicate rates were marked using the Picard MarkDuplicates module. Gene annotation files were downloaded from Ensembl (http: / / www.ensembl.org / ) for STAR localization and subsequent assessment of gene expression levels. To assess gene expression profiles, read counts of annotated genes were retrieved by HTSeq (Anders S, Pyl PT, Huber W., "HTSeq - a Python framework for processing high-throughput sequencing data", Bioinformatics 2015;31:166-9.) and processed by the DESeq2 R program package (Anders S, Huber W., "Differential expression analysis of sequence count data", Genome Biol 2010;11:R106) to normalize gene expression to log2 values.

[0135] To analyze PDX samples, Illumina paired-end RNA-seq data were aligned to the human genome assembly (construct hg38) using STAR with the quantMode parameter set to TranscriptomeSAM, and the alignment was converted to transcript coordinates. Alignments were performed using the RSEM formula (version 1.2.31) to calculate approximate gene counts, TPM, FPKM, and isoform expression. All RNA-seq values ​​were expressed as fragments per million kilobases (FPKM). FPKM data were log-scaled.

[0136] 2. Experimental Results We detected the expression levels of AKR1C3 mRNA and protein in various types of ALL by RNA-Seq analysis (Figure 3a) and Western blot (Figure 3b). Analysis of the results confirmed that AKR1C3 expression was significantly higher in T-ALL (n = 25) than in B-ALL (n = 65, P < 0.0001). Furthermore, a significant correlation was observed between AKR1C3 mRNA and protein expression (r = 0.58, P = 0.0003, Figure 3c).

[0137] [Example 5] Primer and probe design and screening

[0138] 5.1 Experimental materials

[0139] 5.1.1 Sample Information The cell lines used in this example were purchased from Nanjing Cobioer Biosciences and the National Infrastructure of Cell Line Resources. Leukemia samples were obtained from clinically active acute lymphoblastic leukemia patients, and normal samples were obtained from healthy donors, who signed informed consent. Fresh blood samples were stored in PAXGENE tubes and kept in a refrigerator at -20°C.

[0140] The collection, use and subsequent processing of all human samples were carried out in accordance with relevant Chinese regulations for the experimental locations and relevant requirements of international ethics.

[0141] [Table 7-1]

[0142] [Table 7-2]

[0143] 5.1.2 Device information

[0144] [Table 8]

[0145] 5.1.3 Reagent Information

[0146] [Table 9]

[0147] 5.2 Nucleic acid extraction The RNA extraction kits used were the PAXGENE BloodRNA Kit (catalog number: 762174, used for blood stored in PAXGENE tubes) manufactured by Qiagen and the QIAamp RNA Blood Mini Kit (catalog number: 52304, used for blood stored in EDTA tubes) manufactured by Qiagen.

[0148] RNA was extracted from blood samples stored in PAXgene tubes using the PAXgene Blood RNA Kit-IVD (according to the manufacturer's instructions). RNA was extracted from blood samples stored in EDTA tubes using the QIAamp RNA Blood Mini Kit. The concentration of the extracted RNA was determined using the recommended RNA quantification kit (Qubit RNA HS Assay Kit, Thermo Fisher Scientific, Q32852).

[0149] 5.3 Reverse transcription Four microliters of AKR1C3 reverse transcriptase mixture (Thermo Fisher Superscript VILO MARSTER MIX (Cat. No. 11755050)) was absorbed and added to 16 μL of RNA sample (if the volume was insufficient, add water to 16 μL), vortexed briefly, mixed, and then briefly centrifuged. The PCR tube containing the reaction solution was placed in a PCR machine, and the PCR program was set for cDNA synthesis.

[0150] 5.4 AKR1C3 Primer Design and Selection Nine pairs of primers and probes were designed for a total of three target regions of AKR1C3. Primer-probe location information is shown in Figure 4. AKR1C3 gene primers were designed and synthesized by a Contract Research Organization (CRO).

[0151] 5.5 Choice of polymerase and reverse transcriptase The polymerase mixture is a key factor in the success or failure of PCR and affects the efficiency of amplification. The main components of the polymerase mixture include polymerase, MgCl2, buffer, and dNTPs. The above components in commercially available polymerase mixtures are optimized to their optimum ratios to create a concentrated mother solution. When used, the corresponding detection can be achieved as long as the polymerase buffer is added in proportion to the amplification agent. The polymerase KAPA PROBE FAST RT-PCR Master Mix (2x) (catalog number: KK4702), which has been repeatedly verified to have a good amplification rate, was used in this example.

[0152] In each PCR reaction, a positive standard and a negative control (NC, nuclease-free water) must be detected and analyzed. The qPCR detection process was as follows: (1) Remove the AKR1C3 polymerase mix, AKR1C3 reaction solution and standards, mix by shaking, and briefly centrifuge for future use. (2) Depending on the number of test samples and the total number of reaction tubes required for QC, 11.1 μL of AKR1C3 qPCR polymerase mix, 2.6 μL of AKR1C3 reaction mix, and 4.3 μL of nuclease-free water were added to each test, vortexed, briefly centrifuged, and then divided into 8 qPCR tubes, each containing 18 μL. (Note: Both test samples and QC samples must be replicated twice, and NTCs must be detected at least once.) (3) Add the cDNA of the test sample, QC, and standard to the corresponding 8-tube qPCR tubes, cap with the 8-tube cap, mix by vortexing, and briefly centrifuge. (4) Place the 8-tube tubes into the sample chamber of the qPCR instrument, record the arrangement order, and set the instrument amplification program according to the reaction conditions optimized for amplification. (5) After the experiment, wrap the PCR reaction strip in two layers of PE gloves and dispose of it as biological waste. The caps of the PCR tubes must not be opened to prevent contamination.

[0153] 5.6 Primer-probe selection

[0154] 5.6.1 AKR1C3 Primer-Probe Screening Test Using the above steps 5.2 to 5.5, first, nine primer-probe pairs were amplified, and the amplification results are shown in FIG.

[0155] Total RNA was extracted from peripheral blood of healthy volunteers and cDNA was synthesized by reverse transcription. The amplification results showed that F1R1P1, F2R2P1, F2R6P1, F6R2P1, F6R6P1, and F3R3P1 had higher fluorescence intensity values ​​than the other F5R5P2, F4R3P2, and F7R7P3.

[0156] Considering the specificity of the primer-probe, blasting was performed on nine pairs of primers, and F2R6P1, F6R2P1 and F6R6P1 showed better specificity and no non-specific products appeared.

[0157] 5.6.2 AKR1C3 Primer-Probe Real Sample Testing Using three sets of primer-probes (F2R6P1, F6R2P1, and F6R6P1), cDNA was obtained by reverse transcription using total RNA extracted from peripheral blood of healthy volunteers, and six samples were selected for testing. The results are shown in the table below.

[0158] [Table 10]

[0159] As can be seen from Table 10, the Ct value of the expression of the target gene AKR1C3 is relatively stable in the range of 27 to 30 in the healthy population.

[0160] 5.6.3 AKR1C3 Primer-Probe Amplification Rate Test cDNA was obtained by reverse transcription of RNA extracted from peripheral blood of one healthy volunteer using three primer-probe sets (F2R6P1, F6R2P1, and F6R6P1) for amplification rate testing. The cDNA concentration was 100 ng / µL and was diluted 5-fold, followed by further dilutions of 25, 125, and 625-fold to obtain a total of four concentration gradients from 5 to 625-fold. Two replicates were obtained for each concentration for qPCR testing. The calibration curve and amplification rate were determined based on the linear relationship between the Ct value and the logarithm of the initial concentration. The results are shown in Figure 6.

[0161] The results show that the amplification rates of F6R2P1 and F6R6P1 were higher than that of F2R6P1, exceeding 95%. Therefore, the F6R2P1 and F6R6P1 primer-probes were selected for further testing.

[0162] RNA samples extracted from the blood of four healthy volunteers were selected for amplification rate testing of F6R2P1 and F6R6P1, respectively. The results are shown in the table below.

[0163] [Table 11]

[0164] The results show that there was little difference in the amplification rate of the two primer-probe pairs, with slight variations in the different samples. However, the R 2 is better, and R 2is equal to 1 in sample 3. Therefore, in this kit, F6R2P1 was selected as the primer-probe for AKR1C3.

[0165] 5.6.4 AKR1C3 Plasmid Standard Curve Test After dissolving in 40 μL of nuclease-free water, the AKR1C3 plasmid dry powder was diluted 10-fold to a concentration of 5.78 ng / μL as measured by Qubit. 5 , 10 6 , 10 7 and 10 3 The AKR1C3 standard curve was measured using a 2x gradient, with four gradient concentrations for the qPCR test samples. Each gradient was repeated twice, and the standard curve was measured as shown in Figure 7.

[0166] As can be seen from the standard curve in Figure 7, the amplification rate of the AKR1C3 primer-probe reached 100%, and R 2 =0.999, suggesting a good amplification rate.

[0167] [Example 6] Screening of reference genes and design and screening of corresponding primers and probes

[0168] Regarding the selection of reference genes, references on leukemia gene expression ([1] Yanlan Wang, Yue Liu, Changhua Zhou et al., "AKR1C3-specific prodrugs with potent antitumor activity against T-ALL" [J], Leukemia & Lymphoma, 2020, Feb. DOI: 10.1080 / 10428194.2020.1728746, [2] Donya Moradi Manesh, Jad El-Hoss, Kathryn Evans et al., "The growth factor AKR1C3 is a biomarker for sensitivity to PR-104 in preclinical models of T-cell acute lymphoblastic leukemia therapeutic targets in clinical oncology" [J], BLOOD, 2015, 125:1193-1201, [3] Yuantong Tian, ​​Lijing Zhao, Haitao Zhang et al. Based on the previous study, four reference genes (HPRT1, GAP, ACTB, and GOLGA1) were selected for testing in the CCRF-CEM cell line, and their fluorescence intensity and amplification were evaluated. The results are shown in Figure 8.

[0169] In terms of fluorescence intensity, ACTB had the highest intensity, followed by HPRT1.

[0170] Regarding the amplification curves, the amplification curve of GOLGA1 is not a typical S-shaped curve, while the other three have typical S-shaped amplification curves.

[0171] Regarding Ct, the Ct values ​​of GAP and ACTB were low and their expression levels were relatively high, whereas the Ct values ​​of HPRT1 and GOLGA1 were high and their expression levels were relatively low.

[0172] Samples and cell lines were tested to verify their expression stability and whether high and low expressing samples could be distinguished.

[0173] In this experiment, we used RNAs 5, 6, and 7 from blood samples from healthy volunteers, RNAs BZQ and QQY from bone marrow samples from leukemia patients, and cell lines expressing AKR1C3 (RPMI8226, CCRF-CEM, and Jurkat). According to our data and previous results, RPMI8226 and CCRF-CEM cell lines were high expressers, while Jurkat was a low expresser (Figure 9).

[0174] The ΔΔCt calculation method was adopted for the experimental results, i.e., ΔΔCt = unknown sample FAM Ct - unknown sample VIC Ct - (QC FAM Ct - QC VIC Ct), and CCRF-CEM was the QC of each group. The results are shown in Figure 10.

[0175] These results indicate that ACTB, when used as a reference gene, can better distinguish between the high-expressing cell lines RPMI8226 and CCRF-CEM and the low-expressing cell line Jurkat compared with the other three reference genes. While the values ​​remained relatively low in healthy subjects, high expression of AKR1C3 was detected in bone marrow RNA samples from leukemia patients. Based on the amplification curve results, ACTB was selected as a reference gene.

[0176] After determining ACTB as the reference gene, five cell lines were tested, and the test results are shown in Figure 11.

[0177] The cell line test results were found to be consistent with the Western blot validation results (Figure 9), demonstrating the accuracy of the AKR1C3-ACTB primer-probe test results.

[0178] [Example 7] Examination of qPCR reaction system and conditions The quality of the qPCR reaction system is a prerequisite for successful development and detection, and its key components must be optimized.

[0179] 7.1 Identifying reaction systems The reverse transcription system is shown below.

[0180] [Table 12]

[0181] The reverse transcription reaction conditions are as follows:

[0182] [Table 13]

[0183] The polymerase mixture was an important component affecting PCR amplification rates. The polymerase mixture used in this example, KAPA PROBE FAST RT-PCR Master Mix (2x), was optimized for optimal performance by specific ratios to create a concentrated mother solution. When used, the polymerase buffer was added in proportion to the amplification agent, allowing for corresponding detection. According to the instructions, the recommended volume was half the reaction volume; for a 20 μL reaction, the volume should be 10 μL. A specific system is shown in the table below.

[0184] [Table 14]

[0185] 7.2 Optimization of qPCR reaction conditions The reaction conditions used in this example are shown below in Table 15. The reaction conditions were tested as preferred reaction conditions for RNA reactions after long-term validation in the early stages.

[0186] [Table 15]

[0187] The number of cycles is one of the direct factors affecting PCR results, and it determines the degree of PCR amplification. The number of cycles used for qPCR was usually 30 to 45. The more cycles used, the more pronounced nonspecific amplification became, while the fewer cycles used, the more pronounced the effect on detection sensitivity became.

[0188] 7.3 Calibration curve test of the AKR1C3-ACTB system

[0189] 7.3.1 Cell line calibration curve testing In the optimized reaction system, the primer-probe pairs for AKR1C3 and ACTB were mixed, and a standard curve was performed to examine the amplification rate of the primer-probe pairs. CCRF-CEM cells were used as the reverse transcription system described in 7.1. After obtaining cDNA, a 5-fold gradient dilution was performed to obtain four gradient stock solutions (5-, 25-, and 125-fold dilutions) with two replicates for each concentration. Detection was performed using a qPCR reaction system. A standard curve was generated based on the linear relationship between the Ct value and the initial copy number (lg), as shown in Figure 12.

[0190] From Figure 12, it can be seen that the amplification rate of the AKR1C3 primer-probe was 94.2%, and the amplification rate of the ACTB primer-probe was 95.3%, both of which met the requirements. 2 is 0.99 or more, which indicates that the linear relationship is good and meets the necessary conditions.

[0191] 7.3.2 Plasmid calibration curve test The dry powders of the AKR1C3 and ACTB plasmids used were dissolved in 40 μL of nuclease-free water. After 10-fold dilution, the concentrations were measured using Qubit and found to be 1.67 ng / μL and 5.78 ng / μL, respectively. -5 Dilute ACTB plasmid to 10 -3 The plasmids were diluted to 100 μL. 100 μL of each plasmid was mixed and then diluted 10-fold, 100-fold, and 1000-fold gradients. Each gradient was replicated twice for qPCR testing. A calibration curve was constructed according to the linear relationship between the Ct value and the logarithmic value of the initial concentration. The results are shown in Figure 13.

[0192] From the results shown in the figure, the amplification rate of AKR1C3 reached 101.9%, the amplification rate of ACTB reached 94.1%, and R 2is greater than 0.99, which means that the requirement is met.

[0193] [Example 8] Digital PCR test of AKR1C3-ACTB primer-probe

[0194] 8.1 Digital PCR detection system and reaction conditions In this example, we attempted a digital PCR test using the AKR1C3-ACTB primer-probe. Because the expression levels of AKR1C3 and ACTB in the samples and cell lines were quite different, the maximum Ct difference could reach 10 Ct values. Figure 14 shows the qPCR detection pattern for the low-expressing Jurkat cell line.

[0195] Therefore, in digital PCR detection, the copy number of VIC is much larger than that of FAM, as shown in Figure 15.

[0196] Therefore, AKR1C3 and ACTB were detected separately. Two cell lines, CCRF-CEM and MOLT-4, were used as examples. After reverse transcription of 2 μg of RNA, the transcripts were diluted 5-fold and the copy number of AKR1C3 was detected by digital PCR using the following system.

[0197] [Table 16]

[0198] The reaction procedure for digital PCR is shown in the table below.

[0199] [Table 17]

[0200] The detection results are shown in FIG.

[0201] For ACTB detection, the cDNA was diluted 5-fold and then 200-fold, and then the copy number of ACTB was detected using the following system.

[0202] [Table 18]

[0203] The detection results are shown in Figure 17. As can be seen from the results, the FAM copy number / VIC copy number of the cell line CCRF-CEM was 0.0092, and the FAM copy number / VIC copy number of the cell line MOLT-4 was 0.00129.

[0204] 8.2 Physical sample testing and calculation method evaluation Sample number 9 from a healthy volunteer, eight cases from leukemia patients (numbers: PSS, GZJ, HXB, HCL, ZYQ, LBJ, JRZ, LSX), and five cell lines (TF-1, CCRF-CEM, RPMI8226, MOLT-4, Jurkat) were used. The plasmids and concentrations used in the calibration curve were as described in step 7.3.2 of Example 7. The preparation method was to add 10 AKR1C3 plasmids to the 100 μg / ml tubes. -4 Dilute ACTB plasmid to 10 -2 The plasmids were diluted to 100 μL, mixed, and then diluted 10x, 100x, and 1000x. Copy numbers were quantified by digital PCR. qPCR tests were performed jointly with two replicates for each sample. The copy numbers of AKR1C3 and ACTB in each unknown sample were determined using a standard curve, and the ratio of AKR1C3 copy number to ACTB copy number was used to measure AKR1C3 expression. The measured standard curve is shown in Figure 18.

[0205] From Figure 18, the amplification rate of AKR1C3 reached 99.1%, the amplification rate of ACTB reached 93.4%, and R 2 is greater than 0.99, which means that the requirement is met.

[0206] Sample test results are shown in the table below.

[0207] [Table 19]

[0208] The results of FAM copy number / VIC copy number are shown in FIG.

[0209] As can be seen from Figure 19, the detection results of the cell lines are consistent with the Western blot results of the cell lines, verifying the accuracy of the calculation method. At the same time, as indicated by LBJ in the figure, samples with high AKR1C3 expression were detected in samples from leukemia patients. All eight cases were from patients who had undergone bone marrow transplantation.

[0210] Based on the above-mentioned research on AKR1C3 primer-probe design, reaction system and reaction conditions, a method for detecting AKR1C3 expression in RNA quantity was established, and a certain sensitivity and accuracy were achieved.

[0211] [Example 9] Performance test of the method for detecting AKR1C3 RNA amount

[0212] The optimal primer-probe composition, reference gene and corresponding primer-probe composition, reaction system, and reaction conditions for the qPCR method and digital PCR method were determined according to Examples 5 to 8. Based on these conditions, in this example, the qPCR method and digital PCR method were combined to perform performance tests including sensitivity, specificity, reproducibility, and accuracy.

[0213] The qPCR reaction process included the following steps. (1) Sample preparation The concentration of RNA was assumed to be greater than 5 ng / μL, and 20–200 ng of RNA was taken and made up to the specified volume with nuclease-free water. (2) Reverse transcription The reverse transcription mixture was added sequentially to the PCR tubes as shown in Table 12, and the mixture was mixed by vortexing or blowing with a pipettor. After a brief centrifugation, the mixture was placed on a standard PCR instrument. Reverse transcription was performed as shown in Table 13, and the reverse-transcribed cDNA was used as a template for qPCR reactions and could be stored at -20°C. (3) Preparation of primer dilution standard solution The dry powders of primers and probes were dissolved in TE buffer at room temperature for 2 hours (or overnight at 4°C). The concentration of the primer dilution standard solution was 10 μM. After dissolution, the primers and probes were mixed according to the specific ratio and combination. After thorough mixing, the solution was stored in a refrigerator at -20°C. The primer dilution standard solution can be stably stored at -20°C for one year. Before each use, the centrifuge tube containing the DNA primer amplification mixture was shaken to mix, then briefly centrifuged. The labeled volume (20 μL / reaction) was aliquoted and stored as needed for a single use. (4) Preparation of reaction system A positive control (STD), negative control (Neg), and blank control (NTC) were used for QC every time. qPCR reactions were prepared according to Table 14, mixed, and briefly centrifuged. qPCR amplification was then performed under the amplification conditions shown in Table 15.

[0214] The corresponding meanings of the sample numbers involved in the following tests are as follows: W1: standard not expressing AKR1C3, P1: strongly positive cell line expressing AKR1C3, P2: gradient of AKR1C3 reference standards with different copy numbers used to detect the calibration curve, P3: weakly positive cell line expressing AKR1C3, S1: standard with the lowest detection limit of AKR1C3, R1: strongly positive cell line expressing AKR1C3, R2: standard not expressing AKR1C3.

[0215] 9.1 Minimum detection limit

[0216] 9.1.1 Detection Methods RNA extracted from a low-expressing cell line was selected for reverse transcription, with the amount of RNA set at 2 μg. The reverse-transcribed cDNA was diluted 25-fold to determine the lowest detection limit, and the copy number of the reference was determined by digital PCR. Each reference was replicated 10 times.

[0217] 9.1.2. Process and Results of Confirming Copy Numbers Based on Minimum Detection Limits (1) Preparation of PCR mixture: 7.5 μL of PCR master mixture (per assay), 3 μL of primer mixture, and the total required amount of water were collected, mixed by vortexing, centrifuged, and dispensed into PCR reaction tubes. (2) Addition of sample: 0.5 μL of the sample to be detected was added to each corresponding PCR reaction tube, mixed by vortexing, and then centrifuged. (3) Sample loading: The corresponding number of chips were prepared, and 14.5 μL of reaction solution was taken for loading. Note that during this process, the center of the chip and the outer cover of the chip were not touched. The chip was pressed for at least 20 seconds. Mineral oil was added slowly, taking care not to spill the oil. After adding the mineral oil, the chip was sealed. (4) Online testing: Place the chip on the shelf of the digital PCR instrument, with the two-dimensional code and numbers on the shelf facing you. (5) Chip reading: The chip was removed from the dedicated PCR instrument, placed at room temperature, and then placed in a chip scanner to scan the fluorescent signal. The computer calculated the mutation ratio according to the fluorescent signal. (6) Detection results: Copy number calculation: Target copy number = copies (target) x 14.5 / load

[0218] Figure 20 shows the results of a 5-fold dilution of cDNA in this cell line: 261.8 copies / μL, with a detection limit of 52.4 copies / μL.

[0219] 9.1.3 qPCR validation results

[0220] [Table 20]

[0221] As can be seen from Table 20, the minimum detection limit criteria were detected by qPCR with 10 replicates. The positive detection rate was 100%, and the minimum detection limit met the validation criteria.

[0222] 9.2 Accuracy Precision refers to the degree of closeness between the results of multiple parallel tests under the same conditions. The closer the test values ​​are to each other, the higher the precision. Precision reflects the reproducibility of the kit operation. In this example, a negative standard, a weakly expressed precision standard, and a highly expressed precision standard were selected to determine the intra-batch precision. The precision performance of the kit was evaluated by calculating the negative agreement rate for the negative precision standard and the coefficient of variation (CV) of the relative expression levels of AKR1C3 for the weakly expressed and highly expressed precision standards.

[0223] 9.2.1 Detection Methods In this experiment, two precision standards were used as detection samples, and 24 detections were performed for each standard using two instruments, two operators, and three validation days. All negative standards were negative, and the positive CV was less than 15%. CV value = (standard deviation SD / mean) x 100%

[0224] 9.2.2 Experimental results

[0225] [Table 21-1] [Table 21-2]

[0226] The CV value of the 24 positive precision criteria Ct of AKR1C3 was 0.78%, and the CV value of the ACTB CT was 1.73%. All 24 negative precision criteria were negative.

[0227] As can be seen from Table 21, the samples for precision and reproducibility detection were detected 24 times by qPCR, and the CV values ​​were all less than 5%, suggesting that the precision and reproducibility met the validation criteria.

[0228] 9.3 Negative concordance rate

[0229] 9.3.1 Detection Methods Negative standard (AKR1C3 homologous plasmid AKR1C4, 9.06 × 10 3copies) were selected and the detection by the kit was repeated three times to evaluate for false positives.

[0230] 9.3.2 Experimental results

[0231] [Table 22]

[0232] As can be seen from Table 22, all repeated detection results were negative, the negative agreement rate was 100%, and the validation criteria were met.

[0233] 9.4 Negative concordance rate

[0234] 9.4.1 Detection Methods Two positive standards (AKR1C3 cell line verified by Western blot) were selected and detected by the kit to evaluate the detection accuracy. AKR1C3 and ACTB plasmids (quantified by digital PCR) were used in gradient dilutions to generate a standard curve, which was then detected by the kit to evaluate the amplification rate and linear relationship.

[0235] 9.4.2 Experimental results

[0236] [Table 23-1]

[0237] [Table 23-2]

[0238] As can be seen from Table 23, the selected positive standards (cell lines verified by Western blot) were detected using the above detection method. P1 is a strong positive standard with a high FAM copy number / VIC copy number ratio, and P3 is a weak positive standard with a low FAM copy number / VIC copy number ratio, and the CV meets the requirements. The detection accuracy of this method meets the requirements.

[0239] 9.5 Specificity

[0240] 9.5.1 Detection Methods Two negative criteria (1.14 × 10 4 and 1.14 × 10 3 The AKR1C3 homologous plasmid (AKR1C2) was selected at a copy number of 1. Each concentration level was replicated three times to assess specificity.

[0241] 9.5.2 Detection Results

[0242] [Table 24]

[0243] The two gradients of the negative standard were repeated three times, and all the results were negative. The specificity of the kit test met the requirements.

[0244] 9.6 Real sample detection

[0245] 9.6.1 Detection Methods Eight normal blood samples were selected for detection, and the loading amount was 40 ng of cDNA from reverse-transcribed RNA. Each sample was detected twice, and the accuracy of the FAM copy number / VIC copy number ratio was calculated.

[0246] 9.6.2 Detection Results The detection results are shown in the table below.

[0247] [Table 25-1]

[0248] [Table 25-2]

[0249] Eight normal blood samples were detected twice, and the ratio of FAM copy number to VIC copy number was less than 0.001, with a detection CV of less than 5%. The accuracy of the kit met the requirements.

[0250] 9.7 Template amount verification 200 ng and 10 ng of RNA were used as the initial amount for reverse transcription, and a range of loading amounts was tested for bone marrow samples from three clinical leukemia patients.

[0251] [Table 26]

[0252] Conclusion: The detection results of 10ng and 200ng loaded samples were consistent.

[0253] 9.8 Verification Conclusion

[0254] [Table 27]

[0255] Conclusion: The performance of this method was verified for the minimum detection limit, accuracy, negative agreement rate, positive agreement rate, template amount, analytical specificity, and real sample detection. All seven performance parameters met the validation criteria. The performance of this detection method met the requirements for clinical application and can be used for clinical sample detection.

[0256] [Example 10] AKR1C3 RNA content detection kit (qPCR method)

[0257] In this example, the AKR1C3 RNA content detection kit comprises: (1) a qPCR polymerase mixture (a qPCR polymerase mixture mainly containing DNA polymerase, MgCl, buffer, and dNTPs (plus UNG enzyme and dUTP), preferably, the polymerase mixture is KAPA PROBE FAST RT-PCR Master Mix (2x)); (2) a reverse transcriptase mixture (preferably Superscript VILO MARSTER MIX); (3) an AKR1C3 reaction mixture (the AKR1C3 reaction mixture comprises a primer-probe composition for detecting the AKR1C3 gene and a primer-probe composition for detecting a reference gene, and the primer-probe composition for detecting the AKR1C3 gene is selected from any one of groups (i) to (ix) as described above, more preferably selected from one of groups (iii), (iv) and (v), and even more preferably selected from group (iv). In the probe for detecting the AKR1C3 gene, the 5'-end reporter was FAM and the 3'-end quencher was MGB. The primer-probe composition for detecting the reference gene comprises an upstream primer ACTB-F1, a downstream primer ACTB-R1 and a probe ACTB-P1, which are represented by SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively. The 5'-end reporter of the probe for the reference gene was VIC and the 3'-end quencher was BHQ1), and (4) a negative control (NC) (e.g., nuclease-free water); (5) a positive control (e.g., a reference of known copy number); (6) Instructions recording the relevant operating procedures; Includes.

[0258] [Example 11] AKR1C3 RNA content detection kit (digital PCR method)

[0259] As described in Example 8, separate detection of AKR1C3 and ACTB was required in the digital PCR method. Therefore, the kit of this example differs from the kit of Example 10 in that the AKR1C3 reaction mixture contains only a primer-probe composition for detecting the AKR1C3 gene, but does not contain a primer-probe composition for detecting the reference gene ACTB. The kit in this example contains both an AKR1C3 reaction mixture (containing a primer-probe composition for detecting the AKR1C3 gene) and a reference gene ACTB reaction mixture (containing a primer-probe composition for detecting the reference gene ACTB), and the remaining components are the same.

[0260] As described in Example 10 or 11, the AKR1C3 RNA content detection kit is used together with AKR1C3 activating anticancer drugs to carry out routine patient screening.By using the kit, medical personnel can easily carry out detection in different laboratories according to the uniform detection kit standard operating procedure (SOP) before deciding to administer the drug to patients.In this way, the AKR1C3 detection results obtained by the same reagent and the same procedure can meet the recommended detection results for specific cancers in the drug prescription of AKR1C3 activating cancer drugs.

[0261] The specific operating method of the kit is recorded in the instruction manual, i.e., the specific operating conditions in the instruction manual. Optionally, or as a preferred embodiment, the instruction manual also provides a predetermined amount X of the AKR1C3-activating anticancer drug for different types of cancer (tumor). For example, the predetermined X value for B-lineage acute lymphoblastic leukemia (B-ALL) and T-lineage acute lymphoblastic leukemia (T-ALL) is 0.00014 to 0.015, and the AKR1C3 copy number / ACTB copy number ratio of ex vivo peripheral blood samples from patients detected using the kit was 0.0092. Statistics show that the AKR1C3 copy number / ACTB copy number ratio of B-ALL patients treated with an AKR1C3-activating anticancer drug should not be lower than 0.00014. Therefore, doctors can prescribe the AKR1C3-activating anticancer drug to this patient. As another example, for T-lineage acute lymphoblastic leukemia (T-ALL), the AKR1C3 copy number / ACTB copy number ratio of ex vivo peripheral blood samples from patients with T-ALL detected by the above kit was 0.00012. Statistics show that the AKR1C3 copy number / ACTB copy number ratio of T-ALL patients treated with AKR1C3-activating anticancer drugs should not be lower than 0.00014. Therefore, doctors should not prescribe AKR1C3-activating anticancer drugs to these patients.

[0262] [Example 12] Method for detecting AKR1C3 RNA content and use of the kit for detecting AKR1C3 RNA content in the preparation of cancer therapeutic drugs

[0263] The ratio of AKR1C3 copy number / ACTB copy number in ex vivo peripheral blood samples from B-ALL patients detected by the AKR1C3 RNA content detection method established in Examples 5 to 9 or the AKR1C3 RNA content detection kit established in Examples 10 or 11 was 0.0092, which was greater than the predetermined content of 0.00014.

[0264] B-ALL patients were administered AKR1C3-activating anticancer drugs.

[0265] According to existing experimental verification, AKR1C3-activating anticancer drugs selected from the following structures have better therapeutic effects: [ka]

Claims

1. (iv) an upstream primer AKR1C3-F6, a downstream primer AKR1C3-R2, and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO:7, SEQ ID NO:5, and SEQ ID NO:3, respectively; (v) an upstream primer AKR1C3-F6, a downstream primer AKR1C3-R6 and a probe AKR1C3-P1, whose nucleotide sequences are set forth in SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:3, respectively; A primer-probe composition selected from any one of:

2. The primer-probe composition of claim 1, wherein the primer-probe composition is selected from group (iv).

3. The primer-probe composition according to claim 1, wherein the 5'-end reporter of the probe AKR1C3-P1 is FAM, and the 3'-end quencher of the probe AKR1C3-P1 is MGB.

4. A kit comprising the primer-probe composition of any one of claims 1 to 3.

5. further comprising a primer-probe composition for a reference gene; The kit according to claim 4, wherein the reference gene is ACTB.

6. 6. The kit of claim 5, wherein the primer-probe composition for the reference gene comprises an upstream primer ACTB-F1, a downstream primer ACTB-R1, and a probe ACTB-P1, whose nucleotide sequences are set forth in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively.

7. The kit of claim 6, wherein the 5'-end reporter of the probe ACTB-P1 is VIC and the 3'-end quencher of the probe ACTB-P1 is BHQ1.

8. Further comprising a polymerase mixture, The kit of any one of claims 4 to 7, wherein the polymerase mixture mainly comprises a DNA polymerase, MgCl2, a buffer, and dNTPs.

9. The kit according to any one of claims 4 to 8, further comprising a reverse transcriptase mixture.

10. Additionally, negative and positive controls are included. the negative control is nuclease-free water, and / or The kit of any one of claims 4 to 9, wherein the positive control is a reference of known copy number.

11. A primer-probe composition according to any one of claims 1 to 3 or a kit according to any one of claims 4 to 10 for use in cancer treatment.

12. determining the AKR1C3 RNA content in an ex vivo sample from a patient using the primer-probe composition according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 10; administering an AKR1C3-activating anticancer drug to a patient whose AKR1C3 RNA content is equal to or greater than a predetermined content; The primer-probe composition or kit for use in cancer therapy of claim 11 , wherein the ex vivo sample comprises a blood sample, a bone marrow sample, or a tissue sample.

13. The AKR1C3 RNA content is determined according to the ratio of AKR1C3 copy number / reference gene copy number; The primer-probe composition or kit for use in cancer treatment according to claim 12, wherein the predetermined content is 0.0001 to 1.

14. The AKR1C3-activating anticancer drug has the following structure: 【Chemistry 1】 The primer-probe composition or kit for use in cancer treatment according to claim 12 or 13, wherein the compound is selected from compounds having the formula:

15. The cancer is lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, breast cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colorectal cancer, ovarian cancer, bladder cancer, cervical cancer, hepatocellular carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, renal cell carcinoma, cystadenocarcinoma, cystic carcinoma, medullary carcinoma, bronchial carcinoma, bone cell carcinoma, epithelial carcinoma, bile duct carcinoma, choriocarcinoma, embryonal carcinoma, seminoma, Wilms' tumor, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma , ependymoma, pineal tumor, hematoblastoma, vocal cord neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, fibrochondroma, fibrocyst, fibromyxoma, fibroosteosarcoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocyst, myxochondroma, myxochondrosarcoma, myxofibrosarcoma, myxoadenoma, myxoblastoma, liposarcoma, lipoma, lipoadenoma, lipoblastoma, lipochondroma, lipofibroma, lipoangioma, myxolipoma, chondrosarcoma, chondroma, chondroleoma, chondroleoma, chordoma, choriocarcinoma, chorioepithelioma, chorioblastoma, Osteosarcoma, osteoblastoma, osteochondrofibroma, osteochondroma, osteocytoma, osteoodontoma, osteofibroma, fibrosarcoma of bone, angiosarcoma, hemangioma, angiolipoma, hemangiochondroma, hemangioblastoma, hemangiokeratoma, hemangioglioma, hemangioendothelioma, angiofibroma, angiomyoma, angiolipoma, hemangiolymphoma, angiolipoleiomyoma, angiomyolipoma, angiomyoneuroma, angiomyxoma, hemangioreticuloma, lymphangiosarcoma, lymphogranuloma, lymphangioma, lymphoma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, peripheral T-cell lymphoma 15. The primer-probe composition or kit for use in cancer treatment of any one of claims 11 to 14, comprising a tumor selected from the group consisting of: lymphoma, thyroid cancer ...

16. A primer-probe composition or kit for use in cancer treatment according to claim 15, wherein the cancer comprises ovarian cancer, cervical cancer, pancreatic cancer, breast cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, prostate cancer, renal cell carcinoma, peripheral T-cell lymphoma, nodular NK / T-cell lymphoma, acute lymphocytic leukemia or acute myeloid leukemia.

17. (1) extracting RNA from an ex vivo sample to be detected, adding the extracted RNA to a reverse transcription system, and synthesizing cDNA by reverse transcribing the extracted RNA; (2) performing qPCR or digital PCR amplification using cDNA as a template using the primer-probe composition according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 10; (3) determining the AKR1C3 RNA content of the ex vivo sample to be detected according to the qPCR or digital PCR amplification results; Used to detect AKR1C3 RNA content in blood samples, bone marrow samples or tissue samples, Methods for detecting AKR1C3 RNA content.

18. In the step (1), the concentration of the extracted RNA is detected, and / or the reverse transcription system comprises a reverse transcriptase; 18. The method of claim 17, wherein the mass-to-volume ratio of the extracted RNA and reverse transcriptase is (0.5-2):4 in μg / μL.

19. In the step (2), a primer-probe composition selected from any one of the groups (iv) and (v) is mixed with a primer-probe composition for a reference gene in a qPCR reaction system; In the qPCR reaction system, the molar ratio of the AKR1C3 upstream primer, the AKR1C3 downstream primer, and the AKR1C3 probe is (2-10):(2-10):3; and / or In the qPCR reaction system, the molar ratio of the upstream primer ACTB-F1, the downstream primer ACTB-R1 and the probe ACTB-P1 is (2-10):(2-10):3; and / or In the qPCR reaction system, The method according to claim 17 or 18, wherein the amounts of the AKR1C3 upstream primer, the AKR1C3 downstream primer and the AKR1C3 probe are the same as the amounts of the upstream primer ACTB-F1, the downstream primer ACTB-R1 and the probe ACTB-P1, respectively.

20. In the step (2), dUTP, UNG enzyme, cDNA template, and polymerase mixture are also added to the qPCR reaction system; 20. The method of claim 19, wherein the volume of the polymerase mixture is 0.3 to 0.8 of the total volume of the qPCR reaction system.

21. In the step (2), AKR1C3 and the reference gene are amplified by an AKR1C3 digital PCR detection system and a reference gene digital PCR detection system, respectively; In the AKR1C3 digital PCR detection system, the molar ratio of the AKR1C3 upstream primer, the AKR1C3 downstream primer and the AKR1C3 probe is (5-15):(5-15):3; and / or In the reference gene digital PCR detection system, The method according to any one of claims 17 to 20, wherein the molar ratio of the upstream primer ACTB-F1, the downstream primer ACTB-R1 and the probe ACTB-P1 is (5-15):(5-15):

3.

22. In the step (3), the step of determining the AKR1C3 RNA content of the ex vivo sample to be detected according to the qPCR or digital PCR amplification result, (A) determining the copy numbers of AKR1C3 and reference genes in the ex vivo sample to be detected according to the qPCR or digital PCR amplification results; (B) calculating the ratio of AKR1C3 copy number / reference gene copy number to determine the AKR1C3 RNA content of the ex vivo sample to be detected; The method according to any one of claims 17 to 21, comprising:

23. When the qPCR method is used, the step (A) further comprises: (A1) plotting a standard curve of the Ct value and initial copy number lg value of AKR1C3, and a standard curve of the Ct value and initial copy number lg value of a reference gene; (A2) determining the copy numbers of AKR1C3 and the reference gene in the ex vivo sample to be detected based on the Ct values ​​of the detected AKR1C3 and the reference gene according to the calibration curve; The method according to any one of claims 17 to 22, comprising:

24. The AKR1C3 RNA content of an ex vivo sample to be detected is detected using the method according to any one of claims 17 to 23, and then the expression level of the AKR1C3 enzyme in the ex vivo sample to be detected is determined according to the AKR1C3 RNA content of the ex vivo sample to be detected; A method for detecting the expression level of AKR1C3 enzyme, wherein there is a linear correlation between the AKR1C3 RNA content in the ex vivo sample to be detected and the expression level of AKR1C3 enzyme in the ex vivo sample to be detected.

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