Methods for diagnosing and treating prostate cancer using zinc finger protein-like 1 antibodies

Abstract

Disclosed herein is a method for diagnosing and treating prostate cancer using an antibody against zinc finger protein-like 1 (ZFPL1). The method can include obtaining a serum sample from a patient, detecting whether zinc finger protein-like 1 (ZFPL1) is present in the serum sample, diagnosing the patient with prostate cancer if the presence of ZFPL1 is detected in the serum sample, and then administering an effective amount of an anti-ZFPL1 antibody to the diagnosed patient.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 371,902, filed August 19, 2022, and entitled "Neuroendocrine Marker (ZFPL1) for Prostate Cancer Diagnosis and Monitoring and the Method of Measuring the Same." This application also claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 387,115, filed December 13, 2022, and entitled "Neuroendocrine Marker (ZFPL1) for Prostate Cancer Diagnosis and Monitoring and the Method of Measuring the Same."

[0002] Reference to sequence table XML This application contains a Sequence Listing that has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference. The XML file (created on August 2, 2023) is named 019976-187142-00_SL.xml and is 17,392 bytes in size. [Background technology]

[0003] background Prostate cancer is the second most common cancer worldwide and the sixth leading cause of cancer deaths in men. Prostate cancer exhibits high diversity in its characteristics, ranging from clinically insignificant, slow-growing tumors to aggressive, metastatic disease. This provides a unique opportunity to identify multiple biomarkers representing different stages of cancer progression. Unfortunately, prostate-specific antigen (PSA) is the only established blood biomarker for multiple purposes, including prostate cancer detection, patient stratification into prognostic risk groups, determination of overall tumor burden, and tracking response to local or systemic treatment. Furthermore, the prognosis of this disease is still assessed by conventional pathological parameters such as Gleason score, number or percentage of positive cores, and maximum percentage of tumor involvement in any core.

[0004] PSA is a kallikrein protease primarily produced by luminal cells of the prostate gland, but also secreted in small amounts by the pancreas and uterus. PSA is not cancer-specific but is normally produced in the prostate; its levels are elevated in prostate cancer and some benign conditions (e.g., benign prostatic hyperplasia (BPH) and prostatic inflammation). As a result, serum PSA testing nonspecifically detects many benign conditions and many low-grade, and therefore indolent, prostate tumors. Therefore, PSA-based diagnosis requires confirmation by invasive, repeated, and expensive procedures (e.g., transrectal ultrasound-guided biopsy). On the other hand, approximately 15% of prostate cancer cases show low or normal serum PSA levels, the majority of which are highly aggressive with neuroendocrine (NE) features. This suggests that PSA testing may not detect all lethal prostate cancers requiring aggressive treatment.

[0005] It has been reported that calcitonin (CT) and its receptor (CTR) are selectively expressed by basal cells but not by secretory cells in benign prostate epithelium. However, all cells in malignant prostate epithelium express CT and CTR, and their expression increases with tumor progression. Furthermore, activation of the CT-CTR axis induces an invasive phenotype in benign prostate cells. To date, a noninvasive method for accurately diagnosing prostate cancer in patients has yet to be discovered. Summary of the Invention [Means for solving the problem]

[0006] Abstract The invention disclosed herein relates to a method for diagnosing and treating prostate cancer by targeting zinc finger protein-like 1 (ZFPL1).Through subtraction hybridization, ZFPL1 has been identified as being selectively expressed only in malignant prostate cells, but not in benign prostate cells.It has also been discovered that ZFPL1 protein plays a role in tumorigenesis.Therefore, treatment that targets ZFPL1 is effective as a treatment for prostate cancer.

[0007] An immunosensor for detecting zinc finger protein-like 1 (ZFPL1) in a biological sample embodying features of the invention can include an anti-ZFPL1 monoclonal antibody immobilized on a substrate, wherein the monoclonal antibody specifically binds to a ZFPL1 protein comprising an epitope selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0008] In another embodiment of the immunosensor, the monoclonal antibody comprises a heavy chain complementarity determining region (CDR), wherein the heavy chain CDR comprises a heavy chain variable region (HCVR) comprising SEQ ID NOs: 1, 2, and 3; and a light chain CDR, wherein the light chain CDR comprises a light chain variable region (LCVR) comprising SEQ ID NOs: 4, 5, and 6. In another embodiment, the monoclonal antibody comprises an HCVR (SEQ ID NO: 7) and an LCVR (SEQ ID NO: 8). In another embodiment, the monoclonal antibody isotyped as immunoglobulin G (IgG). In another embodiment, the antibody has an equilibrium dissociation constant (KD) value of 100 nM and a half maximal inhibitory concentration (IC50) of 10 nM.

[0009] In another embodiment of the immunosensor, the light source generates white light and the optical sensor is configured to detect light within a wavelength range of 500 nm to 900 nm (inclusive). In another embodiment, the immunosensor further includes a processor that calculates a frequency shift between a first sensor reading of a substrate substantially free of ZFPL1 and a second sensor reading of ZFPL1 bound to the monoclonal antibody. In another embodiment, the first sensor reading is when less than 5% of the monoclonal antibody is bound to ZFPL1, and the second sensor reading is when more than 5% of the monoclonal antibody is bound to ZFPL1. In another embodiment, the frequency shift is detected at a ZFPL1 concentration in the biological sample of less than 1 pg / ml.

[0010] An immunoassay method for detecting prostate cancer in a biological sample according to the present invention can include a first step of contacting the biological sample with a monoclonal antibody immobilized on a substrate, and a second step of detecting the presence of ZFPL1 in the biological sample, wherein the monoclonal antibody specifically binds to a ZFPL1 protein comprising an epitope selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0011] In another embodiment of the immunoassay method, the monoclonal antibody comprises heavy chain CDRs, wherein the heavy chain CDRs comprise HCVRs comprising SEQ ID NOs: 1, 2, and 3; and light chain CDRs, wherein the light chain CDRs comprise LCVRs comprising SEQ ID NOs: 4, 5, and 6. In another embodiment, the monoclonal antibody comprises HCVR (SEQ ID NO: 7) and LCVR (SEQ ID NO: 8). In another embodiment, the monoclonal antibody isotyped as immunoglobulin G (IgG). In another embodiment, the antibody has an equilibrium dissociation constant (KD) value of 100 nM and a half maximal inhibitory concentration (IC50) of 10 nM.

[0012] In another embodiment of the immunoassay method, the presence of ZFPL1 in the biological sample is detected at a ZFPL1 concentration of less than 1 pg / ml. In another embodiment, contacting the biological sample with the monoclonal antibody immobilized on a substrate comprises an incubation period of at least 30, 45, 60, 90, 120, or 180 minutes. In another embodiment, detecting the presence of ZFPL1 in the biological sample comprises performing at least one of a chemiluminescence assay, an immunofluorescence assay, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay, a Western blot assay, an enzyme immunoassay, an immunoprecipitation assay, an immunohistochemical assay, an immunochromatography assay, a dot blot assay, a slot blot assay, a lateral flow assay, or an optical immunoassay. In another embodiment, detecting the presence of ZFPL1 in the biological sample comprises performing a label-free optical immunoassay. In another embodiment, the method further comprises obtaining a first reading before contacting the biological sample with the monoclonal antibody immobilized on the substrate, and obtaining a second reading after contacting the biological sample with the monoclonal antibody immobilized on the substrate, wherein obtaining the reading comprises illuminating the monoclonal antibody immobilized on the substrate with a collimated white light source through a lens and detecting a transmitted signal comprising reflected optical interference fringes using an optical detector. In another embodiment, detecting the presence of ZFPL1 in the biological sample comprises comparing the first reading with the second reading and measuring a frequency shift. In another embodiment, data peaks ranging from 550 nm to 750 nm are used to measure the frequency shift.

[0013] A method for diagnosing prostate cancer embodying the features of the present invention can include the following steps: first, obtaining a serum sample from a patient; second, detecting whether ZFPL1 is present in the serum sample; and third, diagnosing the patient with prostate cancer if the presence of ZFPL1 is detected in the serum sample.More specifically, this method can be achieved by contacting the serum sample with a monoclonal antibody specific for ZFPL1 immobilized on a substrate, detecting the specific binding between the monoclonal antibody and the ZFPL1, determining the level of ZFPL1 in the patient sample, and identifying the patient as having malignant prostate tumor if the level of ZFPL1 is higher than the baseline level of ZFPL1 observed in corresponding healthy subjects.The monoclonal antibody specifically binds to ZFPL1 protein comprising an epitope selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0014] In another embodiment of the diagnostic method, the monoclonal antibody comprises a heavy chain CDR, the heavy chain CDR comprising an HCVR comprising SEQ ID NOs: 1, 2, and 3; and a light chain CDR, the light chain CDR comprising an LCVR comprising SEQ ID NOs: 4, 5, and 6. In another embodiment, the monoclonal antibody comprises an HCVR (SEQ ID NO: 7) and an LCVR (SEQ ID NO: 8). In another embodiment, the monoclonal antibody isotype is immunoglobulin G (IgG). In another embodiment, the antibody has an equilibrium dissociation constant (KD) value of 100 nM and a half maximal inhibitory concentration (IC50) of 10 nM. In another embodiment, the level of ZFPL1 is determined using a label-free optical immunoassay. In another embodiment, the baseline level of ZFPL1 in the serum sample is 3.3 ng / mL.

[0015] A method for diagnosing and treating prostate cancer embodying features of the present invention can include the following steps: first, obtaining a serum sample from a patient; second, detecting whether zinc finger protein-like 1 (ZFPL1) is present in the serum sample; third, diagnosing the patient with prostate cancer if the presence of ZFPL1 is detected in the serum sample; and fourth, administering an effective amount of an anti-ZFPL1 antibody to the diagnosed patient. The monoclonal antibody specifically binds to a ZFPL1 protein comprising an epitope selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0016] In another embodiment of the diagnostic method, the monoclonal antibody comprises a heavy chain CDR, wherein the heavy chain CDR comprises an HCVR comprising SEQ ID NOs: 1, 2, and 3; and a light chain CDR, wherein the light chain CDR comprises an LCVR comprising SEQ ID NOs: 4, 5, and 6. In another embodiment, the monoclonal antibody comprises an HCVR (SEQ ID NO: 7) and an LCVR (SEQ ID NO: 8). In another embodiment, the isotype of the monoclonal antibody is immunoglobulin G (IgG). In another embodiment, the antibody has an equilibrium dissociation constant (KD) value of 100 nM and a half maximal inhibitory concentration (IC50) of 10 nM. In another embodiment, the level of ZFPL1 is determined using a label-free optical immunoassay. In another embodiment, the baseline level of ZFPL1 in the serum sample is 3.3 ng / mL.

[0017] In another embodiment, the monoclonal antibody treatment is administered intravenously, subcutaneously, or intraperitoneally. In another embodiment, the monoclonal antibody treatment results in at least one effect selected from the group consisting of inhibition of tumor growth, tumor regression, reduction in tumor size, reduction in tumor cell count, slowing of tumor growth, abscopal effect, inhibition of tumor metastasis, reduction in metastatic lesions over time, reduction in the use of chemotherapeutic or cytotoxic agents, reduction in tumor burden, increased progression-free survival, increased overall survival, complete response, partial response, and stable disease.

[0018] A method of treating cancer in a human subject embodying features of the invention can include administering to a patient suffering from prostate cancer an effective amount of an anti-ZFPL1 antibody, wherein the antibody specifically binds to a protein comprising an epitope selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0019] In another embodiment of the method of treatment, the anti-ZFPL1 antibody is a monoclonal antibody, an antigen-binding fragment thereof, or a protein ligand. In another embodiment, the anti-ZFPL1 antibody binds to at least four nucleotides within nucleotide positions 62-77, 127-284, or 293-308 of SEQ ID NO: 11. In another embodiment, the anti-ZFPL1 antibody binds to at least seven nucleotides within nucleotide positions 62-77, 127-284, or 293-308 of SEQ ID NO: 11. In another embodiment, the anti-ZFPL1 antibody is a chimeric or humanized antibody. In another embodiment, the anti-ZFPL1 antibody comprises a variant Fc domain.

[0020] In another embodiment, the monoclonal antibody treatment is administered intravenously, subcutaneously, or intraperitoneally. In another embodiment, the monoclonal antibody treatment results in at least one effect selected from the group consisting of inhibition of tumor growth, tumor regression, reduction in tumor size, reduction in tumor cell count, slowing of tumor growth, abscopal effect, inhibition of tumor metastasis, reduction in metastatic lesions over time, reduction in the use of chemotherapeutic or cytotoxic agents, reduction in tumor burden, increased progression-free survival, increased overall survival, complete response, partial response, and stable disease.

[0021] In another embodiment of the treatment method, the effective amount of the anti-ZFPL1 antibody is between 0.5 and 5.0 mg / kg of patient body weight. In another embodiment, the treatment is administered for two or three consecutive days, and then the administration is discontinued for at least three weeks. In another embodiment, after three weeks, the human's serum is tested for the presence of a protein of sequence SEQ ID NO: 11. In another embodiment, the treatment is administered for a second time for two or three consecutive days if the protein of sequence SEQ ID NO: 11 is present at a concentration greater than 3 ng / ml. [Brief explanation of the drawings]

[0022] BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] In the figures below, ZFPL1 refers to zinc finger protein-like 1; PC refers to prostate cancer; CT refers to calcitonin; BPH refers to benign prostatic hyperplasia; IHC refers to immunohistochemistry; ICC refers to immunocytochemistry; RT-qPCR refers to reverse transcription quantitative PCR; OV refers to overexpression; DEX refers to dexamethasone; si- refers to small interfering molecule; and p- refers to phosphorylated.

[0024] [Figure 1-1]Figure 1A is a representative photomicrograph showing the presence of amplified ZFPL1 mRNA in PC3M, DU145, LNCaP, M1 (stably expressing inactive CTR), C4, PC3-CTR, and PC3 prostate cancer cells after qRT-PCR. Figure 1B is a bar graph showing the quantitative representation of ZFPL1 gene expression bands normalized to the GAPDH housekeeping gene. Figure 1C is a representative photomicrograph showing a 34.1 kDa ZFPL1 product-sized band on an immunoblot. Figure 1D is a bar graph showing the mean relative ZFPL1 mRNA abundance ± SEM (n = 3) in LNCaP-C4 cells after treatment with CT at concentrations of 0 nM, 5 nM, 10 nM, 50 nM, and 100 nM. Figure 1E is a bar graph showing the mean relative ZFPL1 mRNA abundance ± SEM (n = 3) in PC3-CTR cells after treatment with CT at 0, 5, 10, 50, and 100 nM concentrations. Figure 1F is a bar graph showing the dose-dependent increase in relative ZFPL1 mRNA abundance (n = 3 mean ± SEM) in LNCaP-C4 cells in response to the synthetic androgen, R1881. [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0025] [Figure 2-1] Figure 2A is a bar graph showing the mean ± SEM (n = 6) percentage of ZFPL1-immunopositive cell population per field (magnification, ×400) in various normal human organs. Figure 2B is a representative photomicrograph of ZFPL1-immunopositive cells in normal human organ sections, showing ZFPL1-immunopositive cells along with normal prostate (which is ZFPL1-immunonegative). Figure 2C is a bar graph showing relative ZFPL1 mRNA abundance in normal, BPH, and prostate cancer specimens with different Gleason scores. Figure 2D illustrates data extracted from TCGA and Oncomine portals showing upregulation of ZFPL1 gene expression in prostate cancer specimens. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above.

[0026] [Figure 3-1] Figure 3A shows photomicrographs demonstrating the specificity of in situ hybridization after treatment of prostate cancer specimens with a sense ZFPL1 siRNA probe (left) or an antisense ZFPL1 siRNA probe (right). Figure 3B shows photomicrographs of ZFPL1 mRNA expression in prostate sections from different cancer stages compared with non-cancer specimens. The left panel of Figure 3C is a representative photomicrograph demonstrating the presence of ZFPL1 immunopositive cells (red) in prostate cancer tissue (left) versus matched normal tissue (right) by immunofluorescence. Nuclear staining is DAPI (blue). The right panel of Figure 3C is a bar graph representing the average percentage (n=6) of ZFPL1 immunopositive cells per field (magnification, ×400) in prostate cancer tissue versus matched normal tissue. In Figure 3D, the representative photomicrograph on the left shows H&E staining of a human prostate cancer tissue sample (the dark bluish staining is from nuclear hematoxylin, and the pink staining is from eosin), while the photomicrograph on the right shows green immunofluorescent labeling of ZFPL1 and blue nuclear DAPI labeling. White arrows indicate cancerous areas of corresponding staining. Figure 3E shows representative photomicrographs revealing ZFPL1 immunopositive cells (red) and nuclear DAPI (blue) in different samples of a prostate cancer tissue microarray. The bar graph in Figure 3F shows quantified data for the prostate cancer tissue microarray in Figure 3E. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 3-5] Same as above. [Figure 3-6] Same as above.

[0027] [Figure 4-1]In Figure 4A, representative photomicrographs show the colocalization of ZFPL1 and chromogranin A (CgA) in PC3-CTR cells (upper panel) and human prostate cancer tissues (lower panel) as assessed by immunofluorescence. In Figure 4B, representative photomicrographs show the colocalization of ZFPL1 and CD44 in PC3-CTR cells (upper panel) and human prostate cancer tissues (lower panel) as assessed by immunofluorescence. [Figure 4-2] Same as above.

[0028] [Figure 5-1] Figure 5A is a representative micrograph showing the colocalization of ZFPL1 (green) and exosomal CD81 (red) in PC3-CTR and LNCaP PC cells. Figure 5B is a representative micrograph revealing the colocalization of ZFPL1 (green) and the exosome / secretosome marker CD63 (red). Figure 5C is a representative micrograph showing the colocalization of ZFPL1 (green) and the Golgi apparatus marker GM130 (red). Figure 5D is a representative immunoblot illustrating the coprecipitation of CD81 and ZFPL1 in exosome isolates from PC3-CTR and LNCaP-C4 PC cells. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0029] [Figure 6-1] Figure 6A shows immunoblots demonstrating the comparable efficacy of three siRNAs against ZFPL1 in suppressing ZFPL1 protein levels in PC3-CTR and LNCaP-C4 cells by Western blot analysis. In Figure 6B, immunoblots showed that transfection of a ZFPL1 expression plasmid in PC3-CTR and LNCaP-C4 cells resulted in increased ZFPL1 protein levels in both cell lines. [Figure 6-2] Same as above.

[0030] [Figure 7-1] Figure 7A is a bar graph showing the effect of ±10 nM CT on the proliferation of PC-3CTR cells that received either nonsense siRNA or ZFPL1 siRNA. Representative photomicrographs in Figure 7B show the effect of either nonsense (control) or ZFPL1 siRNA (1, 2, or 3) ± CT on cleaved caspase 3 expression in PC3-CTR cells (upper panel) and LNCaP-C4 cells (lower panel). The bar graph in Figure 7C shows pooled data from four separate experiments performed with LNCaP-C4 and PC3-CTR cell lines. In Figure 7D, the first four pairs of photomicrographs show the expression of cleaved caspase 3 (green) in PC3-CTR and LNCaP-C4 cells expressing a carrier plasmid. The next four pairs of photomicrographs reveal the expression of cleaved caspase 3 in PC3-CTR and LNCaP-C4 cells overexpressing ZFPL1. Figure 7E shows a bar graph depicting pooled data from four separate experiments in Figure D. In Figure 7F, a representative photomicrograph shows the localization of cleaved caspase-3 staining in the nuclei of LNCaP-C4 cells. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above.

[0031] [Figure 8-1]Representative photomicrographs in Figure 8A show the effect of ±10 nM CT on the invasiveness of PC3-CTR cells receiving either nonsense siRNA or ZFPL1 siRNA (1, 2, or 3). Figure 8B shows two bar graphs summarizing the pooled data of the experiment in Figure 8A. Representative photomicrographs in Figure 8C show the effect of ±10 nM CT on the invasiveness of LNCaP-C4 and PC3-CTR cells expressing either the carrier pCMV5-XL4 plasmid or a ZFPL1 expression plasmid. Figure 8D shows two bar graphs summarizing the pooled data of the experiment in Figure 8C. Next, Figure 8E shows representative photomicrographs of a wound-healing assay for cell migration of PC3-CTR cells transfected with ZFPL1 siRNA3 (siRNA-Row 2) or a ZFPL1 expression vector (OVER-Row 4) and treated with ±CT (10 nM). Figure 8F shows a bar graph summarizing the pooled data of the experiment in Figure 8E. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above.

[0032] [Figure 9-1] A representative immunoblot in Figure 9A shows the effect of ±10 nM CT on p-Akt473 and p-Akt308 proteins in PC3-CTR cells receiving either nonsense (control) siRNA or ZFPL1 siRNA1, ZFPL siRNA2, or ZFPL1 siRNA3. A normalized bar graph (pAkt / total Akt) of densitometric quantification of the immunoblot is also included in Figure 9A. A representative immunoblot in Figure 9B shows the effect of ±10 nM CT on p-Akt473 and p-Akt308 proteins in LNCaP-C4 cells receiving either nonsense (control) siRNA or ZFPL1 siRNA1, ZFPL siRNA2, or ZFPL1 siRNA3. A normalized bar graph (pAkt / total Akt) of densitometric quantification of the immunoblot is also included in Figure 9B. [Figure 9-2] Same as above.

[0033] [Figure 9-3] Representative immunoblots in Figure 9C show the effect of ±10 nM CT on p-Akt473 and p-Akt308 proteins in PC3-CTR cells transfected with either a carrier plasmid or a ZFPL1 expression plasmid, respectively. A normalized bar graph (pAkt / total Akt) of densitometric quantification of the immunoblot is also included in Figure 9C. Representative immunoblots in Figure 9D show the effect of ±10 nM CT on p-Akt473 and p-Akt308 proteins in LNCaP-C4 cells transfected with either a carrier plasmid or a ZFPL1 expression plasmid, respectively. A normalized bar graph (pAkt / total Akt) of densitometric quantification of the immunoblot is also included in Figure 9D. [Figure 9-4] Same as above.

[0034] [Figure 9-5]Representative photomicrographs in Figure 9E show the effect of ±10 nM CT on pAkt staining in LNCaP-C4 and PC3-CTR cells receiving either nonsense or ZFPL1 siRNA (1, 2, or 3). Scale bar = 50 μm. Figure 9F shows two bar graphs summarizing pooled data from four separate experiments with PC3-CTR and LNCaP-C4 cells receiving nonsense or ZFPL1 siRNA. Representative photomicrographs in Figure 9G show the effect of ±10 nM CT on p-Akt-immunopositive cells per field (magnification, ×400; green) in PC3-CTR cells expressing either a carrier plasmid or a ZFPL1-overexpression plasmid. Scale bar = 50 μm. Figure 9H shows two bar graphs summarizing pooled data from four separate experiments with PC3-CTR and LNCaP-C4 cells expressing either a carrier plasmid (C) or a ZFPL1-overexpression plasmid (OV). In Figure 9I, a representative photomicrograph at higher magnification (×1,000) shows nuclear localization of pAKT (green). [Figure 9-6] Same as above. [Figure 9-7] Same as above. [Figure 9-8] Same as above. [Figure 9-9] Same as above.

[0035] [Figure 10] FIG. 10 is a scatter plot showing the serum profiles of ZFPL1 and PSA in healthy donors and confirmed positive patients with prostate cancer.

[0036] [Figure 11] FIG. 11 is one embodiment of a ZFPL1 immunosensor.

[0037] [Figure 12] 12A is a graph showing the ZFPL1 wavelength shift recorded by the immunosensor of FIG. 11, and FIG. 12B is a graph showing the BSA wavelength shift recorded by the immunosensor of FIG.

[0038] [Figure 13] FIG. 13A is a ZFPL1 calibration curve for the ELISA test, and FIG. 13B is a ZFPL1 calibration curve for the immunosensor of FIG.

[0039] [Figure 14-1] In Figure 14A, the graph on the left is the receiver operating characteristic curve for the negative predictive value and positive predictive value of ZFPL1 for prostate cancer, and the graph on the right is the corresponding prediction curve for normal (0) vs. prostate cancer (1). In Figure 14B, the graph on the left is the receiver operating characteristic curve for the negative predictive value and positive predictive value of PSA for prostate cancer, and the graph on the right is the corresponding prediction curve for normal (0) vs. prostate cancer (1). In Figure 14C, the graph on the left is the receiver operating characteristic curve for the negative predictive value and positive predictive value of ZFPL1 + PSA for prostate cancer, and the graph on the right is the corresponding prediction curve for normal (0) vs. prostate cancer (1). [Figure 14-2] Same as above.

[0040] [Figure 15-1] In Figure 15A, the graph on the left is the receiver operating characteristic curve for the negative and positive predictive values ​​of ZFPL1 for prostate cancer in the gray zone, and the graph on the right is the corresponding prediction curve for normal (0) vs. prostate cancer (1). In Figure 15B, the graph on the left is the receiver operating characteristic curve for the negative and positive predictive values ​​of PSA for prostate cancer in the gray zone, and the graph on the right is the corresponding prediction curve for normal (0) vs. prostate cancer (1). [Figure 15-2] Same as above.

[0041] [Figure 16] FIG. 16 is a line graph depicting the effect of ZFPL1 monoclonal antibody on PC3-CTR and DU145 cell lines.

[0042] [Figure 17] FIG. 17 is a scatter plot of tumor volume over time in an animal model treatment experiment.

[0043] [Figure 18] FIG. 18 is a graph of representative images of tumors (untreated and treated) and their weights at necropsy. DETAILED DESCRIPTION OF THE INVENTION

[0044] Detailed Description Detailed embodiments of the present invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Accordingly, the specific structural and functional details disclosed herein should not be construed as limiting, but merely as a basis for the claims and as a representative basis for teaching those skilled in the art to variously employ the invention in substantially any appropriately detailed structure. Alternate embodiments may be devised without departing from the spirit or scope of the present invention. Furthermore, the terms and phrases used herein are not intended to be limiting; rather, they are intended to provide an understandable description of the invention. While the specification concludes with claims defining features of the present invention that are believed to be novel, it is believed that the present invention will be better understood from a consideration of the following description in conjunction with the drawings in which like reference numerals carry forward.

[0045] As used herein, the terms "a" or "an" are defined as one or more than one. The term "plurality," as used herein, is defined as two or more than two. The term "another," as used herein, is defined as at least a second or more. The terms "comprises," "comprising," or any other variation thereof, are intended to cover non-exclusive inclusions, such that a process, method, article, or apparatus that comprises a list of elements not only includes those elements, but may also include other elements that are not expressly listed or inherent to such process, method, article, or apparatus. The use of "comprises," without more constraints, does not exclude the existence of additional identical elements in a process, method, article, or apparatus that comprises the elements. The terms "including," "having," or "featuring," as used herein, are defined as including or encompassing (i.e., open-ended language). The term "coupled," as used herein, is defined as connected, although not necessarily directly, and not necessarily mechanically. As used herein, the terms "about" or "approximately" apply to all numerical values, whether explicitly stated or not. These terms generally refer to a range of numbers that one of ordinary skill in the art would consider equivalent to the recited value (i.e., having the same function or result). In many cases, these terms may include numbers that are rounded to the nearest significant figure. Correlation terms, such as first and second, above and below, right and left, etc., may be used only to distinguish one entity or act from another and do not necessarily require or imply any actual relationship or order between such entities or acts.

[0046] Prostate Cancer Marker Discovery: Prostate cancer is the most common internal cancer diagnosed in men. Successful management of prostate cancer patients depends heavily on detecting the cancer before it metastasizes. While serum PSA screening has improved detection of the disease at early stages, it has been observed that this test is not reliable, and positive results must be confirmed with costly, repeated, and invasive TRUS-guided biopsies. This is because PSA is a natural product of the normal prostate and is found in the serum of healthy individuals and cancer patients. Therefore, the inclusion of new markers found only in the prostate of cancer patients should enhance the specificity and accuracy of prostate cancer detection and reduce the need for diagnostic biopsies.

[0047] The present inventors have discovered that zinc finger protein-like 1 (ZFPL1) is a novel prostate tumor-specific protein that co-localizes with chromogranin A (a marker for neuroendocrine differentiation) and CD44 (a marker for cancer stem cells) in prostate cancer cells. This suggests that ZFPL1 provides a measure of the neuroendocrine population of prostate tumors. Because neuroendocrine and stem cell phenotypes are associated with castration-resistant metastatic cancer cells, this new marker should not only detect cancer at an early stage, but may also provide insight into its future course, particularly its ability to grow rapidly and metastasize. In short, the data presented herein suggest that this new marker will help identify prostate cancer patients with aggressive phenotypes.

[0048] This new evidence demonstrates that ZFPL1-immunopositive cell populations are selectively localized in malignant areas of the prostate. ZFPL1-positive cells increase with increasing tumor grade and Gleason score. ZFPL1 is secreted into the blood via exosomes, and serum ZFPL1 levels in cancer patients are several-fold higher than those in age-matched normal individuals. Analysis of serum samples from over 100 patients suggests that ZFPL1 is more reliable than PSA in detecting true prostate cancer and can distinguish cancer patients from those without gray-zone cancer (those with serum PSA levels in the 4-10 ng / ml range). These results demonstrate that ZFPL1 testing significantly increases the specificity, efficacy, and accuracy of prostate cancer detection compared with PSA alone.

[0049] Based on these findings, a ZFPL1-specific antibody was generated against the synthetic peptide GLGLPLIDEV VSPEPEPLNT (SEQ ID NO: 10), one of the epitopes of ZFPL1. This antibody was then used to develop a novel immunosensor-based assay for prostate cancer diagnosis. Finally, both in vitro and in vivo experiments were conducted to study the effectiveness of the ZFPL1 antibody of the present invention as a treatment for prostate cancer. The results of this study showed that the antibody significantly delayed tumor growth.

[0050] Neuroendocrine Marker Discovery / Characterization: Studies have reported a significant upregulation of calcitonin (CT) and / or its receptor in malignant prostate gland. Furthermore, activation of the CT-CTR axis induces an invasive phenotype in benign prostate cells. In contrast, knockdown of CT / CTR induces loss of the invasive phenotype in aggressive prostate cancer cells. To identify key factors associated with the CT-CTR axis-induced increase in tumorigenicity and metastatic potential of prostate cancer cells, we identified nine CT-responsive genes from a prostate cancer cDNA library by subtraction hybridization (Table 1). Among them, we further characterized one protein, ZFPL1, which was the most prevalent among the nine CT-responsive genes in the prostate cancer cDNA library: [Table 1] Table 1

[0051] ZFPL1 mRNA expression in prostate cancer cell lines: In Figure 1A, the relative abundance of ZFPL1 mRNA in multiple prostate cancer cell lines (LNCaP, PC3, PC-3M, and LNCaP-C4) was determined by RT-qPCR. Figure 1B shows the results normalized by GAPDH mRNA levels. ZFPL1 mRNA abundance in PC cell lines was compared with that of PC3 (which was set to 1). Among the cell lines studied, PC3-CTR, DU145, and PC3M cell lines showed comparable ZFPL1 mRNA levels, but they were higher than those of PC3 cells. In contrast, ZFPL1 mRNA abundance was significantly lower in LNCaP and LNCaP-C4 cells compared with that of PC3 cells. Notably, the M1 cell line (which expressed a negative mutant CT receptor) showed the highest ZFPL1 mRNA abundance.

[0052] Turning now to Figure 1C, to confirm that ZFPL1 protein is expressed in prostate cancer cell lines and that the protein expressed in the prostate is the same size as that in other organs, we examined the presence of ZFPL1 protein in PC3-CTR cell lysates. ZFPL1 immunoprecipitates were obtained, and their molecular weights were determined by Western blot analysis. ZFPL1 immunoprecipitates exhibited a band of approximately 35 kDa, consistent with the reported size of 34.1 kDa. For all graphs in Figure 1, the symbol * indicates P<0.05, and the symbol ** indicates P<0.0001 (significantly different from control; conventional one-way ANOVA and Tukey's multiple comparison test).

[0053] Regulation of ZFPL1 mRNA expression by CT and testosterone: Turning now to Figures 1D and 1E, to confirm that ZFPL1 is a CT-inducible gene, the effect of CT on ZFPL1 mRNA abundance was examined in PC3-CTR and LNCaP-C4 cell lines. Cells were cultured overnight and treated with CT (1–100 nM) for 4 hours. RNA was extracted, reverse transcribed, and qRT-PCR for ZFPL1 was performed. Bar graphs represent the mean relative ZFPL1 mRNA abundance ± SEM (n = 3) in LNCaP-C4 cells (Figure 1D) and PC3-CTR cells (Figure 1E) after treatment with CT at concentrations of 0 nM, 5 nM, 10 nM, 50 nM, and 100 nM. The results show that CT induced a dose-dependent increase in ZFPL1 mRNA levels in both cell lines. The control was set at 1.0.

[0054] Because testosterone is a key hormone for the structural and functional integrity of the prostate, its effect on ZFPL1 expression was also examined. The same procedure as for the CT study was used, except that cells were treated overnight with the testosterone agonist R1881 (10 mM–10 nM). Figure 1F shows that the androgen receptor agonist R1881 induced a similar dose-dependent increase in ZFPL1 mRNA expression in the LNCaP-C4 cell line (mean ± SEM for n = 3). The same was not investigated in PC3-CTR cells, because they lack the androgen receptor. The control was set at 1.0. For all graphs in Figure 1, the symbol * indicates P < 0.05, and the symbol ** indicates P < 0.0001 (significantly different from the control; conventional one-way ANOVA and Tukey's multiple comparison test).

[0055] ZFPL1 Expression in Normal Human Tissues: ZFPL1 immunofluorescence was performed on TRP-1 microarrays containing sections of normal human tissues. The percentage of positive ZFPL1-immunopositive cells per field (magnification ×400) was counted. The mean ± SEM (n=6) percentage of ZFPL1-immunopositive cell population per field is graphed in Figure 2A. The * symbol indicates P<0.05 (significantly different from normal prostate; conventional one-way ANOVA and Tukey's multiple comparison test). The results show that ZFPL1 protein was expressed in cell populations of the cerebrum, cerebellum, pancreas, and endometrium. However, ZFPL1-immunopositive cells were not detected in normal human prostate and several other human organs. Figure 2B contains representative photomicrographs of ZFPL1-positive cell populations in various ZFPL1-positive organs from the experiment in Figure 2A.

[0056] ZFPL1 mRNA in normal and malignant prostate: To measure ZFPL1 mRNA abundance in normal and pathological prostate tissues, total RNA was extracted from frozen primary prostate specimens and used for RT-qPCR. Figure 2C is a bar graph showing the relative ZFPL1 mRNA abundance in normal, BPH, and prostate cancer specimens with different Gleason scores. The * symbol indicates P<0.05 (significantly different from normal prostate; conventional one-way ANOVA and Tukey's multiple comparison test). The results show that ZFPL1 mRNA was barely detectable in normal prostate, and its level was slightly increased in BPH. However, the increase in ZFPL1 mRNA level was significantly higher and statistically significant in prostate cancer specimens. Furthermore, the mRNA abundance in prostate cancer tissues increased with increasing Gleason score of prostate cancer tumor specimens. For example, ZFPL1 mRNA abundance in tumors with Gleason score 9 was more than 70-fold higher than that in normal prostate. These results suggest that tumor aggressiveness can be predicted by a patient's serum ZFPL1 levels. Because Gleason score assessment can only be performed by examining a patient's biopsy, ZFPL1 testing may provide a non-invasive alternative for assessing cancer aggressiveness. In Figure 2D, data from public portals such as TCGA and Oncomine also revealed increased expression of ZFPL1 in prostate cancer tissue compared with normal prostate tissue. The * symbol indicates P<0.05 (significantly different from normal prostate, conventional one-way ANOVA and Tukey's multiple comparison test).

[0057] ZFPL1 mRNA expression in clinical prostate specimens: ZFPL1 mRNA was also examined in several paraffin-embedded human prostate specimens by in situ hybridization (ISH) using digoxigenin 11-UTP-labeled ZFPL1 sense (nonspecific binding) and antisense (specific binding) riboprobes. The specificity of the ISH method is shown in the photomicrograph in Figure 3A (scale bar = 100 μm). Only the antisense ZFPL1 siRNA (Figure 3A, right panel) was shown to hybridize with endogenous ZFPL1 mRNA in prostate cancer specimens, but the sense ZFPL1 siRNA (Figure 3A, left panel) did not.

[0058] This technique was then applied to 78 prostate sections. These sections ranged from BPH, high-grade prostatic intraepithelial neoplasia (HGPIN), and prostate cancer with Gleason scores between 1-6 and 7-10. The processed sections were then viewed under a Nikon Optiphot microscope, and six or more digital photomicrographs were acquired per section. A representative photomicrograph from this experiment is shown in Figure 3B (scale bar = 50 μm). Staining in the digital photomicrographs (×400) was quantified by determining the area of ​​staining using the iImage Biovision image analysis program. Staining intensity was determined on a scale of 0 to 3 (0 for none, 1 for low, 2 for moderate, and 3 for high). The IHC index was calculated by multiplying the area of ​​staining by the scale of staining. As is evident from the images in Figure 3B, ZFPL1 transcripts were undetectable in benign specimens, detected in HGPIN specimens, and significantly increased with tumor progression. The quantification data shown in Table 2 show a significant increase in HGPIN, with minimal values ​​for benign acini, and an even more significant increase in prostate cancer specimens with higher Gleason scores. [Table 2] a P < 0.05 represents a group significantly different from the benign acinar group; b P<0.05 represents a group significantly different from the rest of the group. PC, prostate cancer. * p<0.05 (significantly different from benign acini; unpaired t-test). Table 2

[0059] Expression of ZFPL1 in prostate tumors: immunohistochemistry: Figure 3C compares ZFPL1 immunofluorescence in prostate tumors (left) with matched normal tissues (right). ZFPL1 protein expression (red) is cancer-specific; no staining was detected in matched normal tissues. Nuclear staining is DAPI (blue) (scale bar = 50 μm). Figure 3C also includes a bar graph depicting the mean percentage of ZFPL1 immunopositive cells (n = 6) per field (magnification, ×400) in prostate cancer tissues versus matched normal tissues. The * symbol indicates P < 0.0001 (paired t-test). In a total of approximately 12% of tumor cells, ZFPL1 protein was detected in cancer tissues, but not in matched normal tissues.

[0060] Localization of ZFPL1 in cancer tissue: To investigate whether ZFLP1 localized to histologically positive cancerous areas of the specimen, H&E and ZFPL1 immunofluorescence were performed on serial sections of the same biopsy specimen. In Figure 3D, the representative photomicrograph on the left shows H&E staining of a human prostate cancer tissue sample (the dark blue staining is due to nuclear hematoxylin, and the pink staining is due to eosin), while the photomicrograph on the right shows green immunofluorescent labeling of ZFPL1 and blue nuclear DAPI labeling (scale bar = 50 μm). As indicated by the white arrow in Figure 3D, ZFLP1 staining was selectively localized to the cancerous portion of the specimen (as indicated by the large hematoxylin-stained nuclei).

[0061] The ZFPL1 immunoreactive cell population in prostate cancer increases with tumor progression:Tumor stage-specific expression of ZFPL1 protein was examined by immunofluorescence on a US Biomax prostate cancer tissue microarray. The array contained sections from 80 specimens (73 PC and 7 normal). Immunohistochemistry (IHC) was performed, and multiple fluorescent images were acquired for each specimen. The number of ZFPL1 immunopositive cells (red—TRITC) and total cells (blue—DAPI) per field of view (magnification, ×400) was counted, and the IHC index was determined as previously described. Figure 3E shows representative photomicrographs (scale bar = 50 μm) revealing ZFPL1 immunopositive cells (red) and nuclear DAPI (blue) in different samples of the prostate cancer tissue microarray. As evident in Figure 3E, ZFPL1 immunostaining was distributed in the cytoplasm of cells in the epithelium of prostate tumors but not in the epithelium of normal prostates. Furthermore, a clear increase in the number of immunopositive cells and staining intensity was observed with increasing tumor stage. The bar graph in Figure 3F shows the quantification data for the prostate cancer tissue microarray in Figure 3E. The mean ± SEM (n = 6) IHC index for each specimen in the microarray was calculated and plotted against the PC stage. The mean IHC index for each cancer group, except for T1N0M0, was significantly different from the control. The * symbol indicates P < 0.005 (one-way ANOVA and Tukey's multiple comparison test). These results suggest that the IHC index of PC specimens increased with increasing tumor stage and was highest in metastatic tumors at stage T4N1M1.

[0062] ZFLP1 colocalizes with chromogranin A (a neuroendocrine marker) and CD44 (a cancer stem cell marker):Sections of fixed PC3-CTR cells and paraffin-embedded prostate cancer specimens were processed for double immunofluorescence using primary antibody pairs against ZFPL1 + CgA or ZFPL1 + CD44. Representative photomicrographs in Figure 4A show the colocalization of ZFPL1 and chromogranin A (CgA) in PC3-CTR cells (upper panel) and human prostate cancer tissue (lower panel) (scale bar = 50 µm). The results illustrate that ZFPL1 (green) colocalizes with CgA (red) in the same cells and tissues. Similarly, as shown in the representative immunofluorescence photomicrographs in Figure 4B, ZFPL1 (green) colocalized with CD44 (red) in PC3-CTR cells (upper panel) and human prostate cancer tissue (lower panel) (scale bar = 50 µm). The iVision image analysis program statistically evaluated the colocalization of both fluorochromes in each digital image and calculated the Pearson coefficient (maximum of 1.000). CgA-ZFPL1 and CD44-ZFPL1 colocalization data showed mean Pearson coefficient values ​​of >0.83 and >0.8, respectively, suggesting very strong colocalization of these three antigens in the same cells.

[0063] Subcellular localization of ZFPL1 protein in cultured PC cells:In cultured PC3-CTR and LNCaP-C4 cells, the subcellular localization of ZFPL1 (green) was examined by triple immunofluorescence using the following markers: Golgi GM130 (red), exosomal CD81 (red), exosomal-secretome CD63 (red), and nuclear DAPI counterstaining (blue). Figure 5A is a representative micrograph showing the colocalization of ZFPL1 and exosomal CD81 in PC3-CTR and LNCaP-C4 cells. Figure 5B is a representative micrograph revealing the colocalization of ZFPL1 and the exosomal / secretome marker CD63. Cell boundaries were traced to indicate the location of exosomes relative to the cell. The inset shows a magnified image (magnification, ×1,000) of the location indicated by the arrow. Figure 5C is a representative micrograph showing the colocalization of ZFPL1 and the Golgi marker GM130 (scale bar = 25 µm). Colocalization of ZFPL1 with CD81 and CD63 in Figures 5A and 5B suggested that ZFPL1 might be an exosomal protein. Furthermore, colocalization of ZFPL1 with GM130 in Figure 5C suggested its presence in the Golgi.

[0064] In Figure 5D, the presence of ZFPL1 in exosomes was confirmed by isolating the exosome fractions of PC3-CTR and LNCaP-C4 cells and confirming its presence in the isolates by Western blot analysis. β-Actin is a loading control. Coprecipitation of ZFPL1 with CD81 (an exosome marker) in the exosome isolates confirms the presence of ZFPL1 in exosomes of prostate cancer cell lines. Notably, the relative abundance of ZFPL1 immunoreactivity in PC3-CTR cells was significantly higher than that in LNCaP-C4 cells.

[0065] Function of ZFPL1 in prostate cancer cells: To identify the potential role of ZFPL1 in prostate cancer progression, we examined the effects of ZFPL1 knockdown and overexpression on prostate cancer cell characteristics (e.g., cell proliferation, invasion, or apoptosis rate). ZFPL1 overexpression was achieved by transfecting a constitutively active ZFPL1 expression plasmid. Knockdown was achieved by transfecting one of three ZFPL1 siRNAs. β-actin was used as a housekeeping control. The knockdown (Figure 6A) and overexpression (Figure 6B) were verified using Western blot, and protein bands were quantified by densitometry. * indicates P<0.05. The results in Figure 6A indicate that siRNA1 appeared to be the least effective in attenuating ZFPL1 expression, whereas siRNA3 appeared to be the most potent and was used in subsequent experiments unless otherwise specified. The results in Figure 6B show that transfection of ZFPL1 expression plasmids in PC3-CTR and LNCaP-C4 cells resulted in increased ZFPL1 protein levels in both cell lines.

[0066] Effect of ZFPL1 knockdown on prostate cancer cell proliferation: Figure 7A is a bar graph showing the effect of ±10 nM CT on the proliferation of PC-3CTR cells receiving either nonsense siRNA or ZFPL1 siRNA. The data are presented as mean OD595 ± SEM (n=4). The symbol * indicates P<0.05, and the symbol *** indicates P<0.0001 compared to the control receiving nonsense siRNA (unpaired t-test). The symbol ^^^ indicates P<0.0001 compared to +CT receiving nonsense siRNA (unpaired t-test). The results show that knockdown of ZFPL1 in PC3-CTR cells resulted in a significant decrease in basal and CT-stimulated cell proliferation.

[0067] Effect of ZFPL1 knockdown and overexpression on apoptosis in prostate cancer cells:Apoptosis in PC3-CTR and LNCaP cells was examined by immunofluorescence analysis of the presence of cleaved caspase 3 in the nucleus. Representative photomicrographs in Figure 7B show the effect of either nonsense (control) or ZFPL1 siRNA (1, 2, or 3) ± CT on cleaved caspase 3 expression in PC3-CTR cells (upper panel) and LNCaP-C4 cells (lower panel). Blue DAPI staining indicates nuclei (scale bar = 100 μm). The results show that knockdown of ZFPL1 resulted in a clear increase in cleaved caspase 3-positive PC3-CTR cells. However, CT was able to significantly reverse / reduce this effect. Pooled data from these experiments are shown in Figure 7C. The graph shows the number of cleaved caspase 3-positive cells per field (magnification, ×400) for ± CT treatment. The symbol * indicates P<0.05, and the symbol ** indicates P<0.001 compared to +CT treatment alone. The symbol ^ indicates P<0.05 compared with the corresponding nonsense siRNA control (one-way ANOVA and Tukey's multiple comparison test). The results suggested that knockdown of ZFPL1 by siRNAs 2 and 3 resulted in a significant increase in the number of cleaved caspase-3-positive cells in both cell lines, and that CT could reverse / reduce this effect.

[0068] The effect of ZFPL1 overexpression on DEX-induced apoptosis was examined after treating cells with or without DEX. In Figure 7D, the first four pairs of micrographs show the expression of cleaved caspase 3 (green) in PC3-CTR and LNCaP-C4 cells expressing the carrier plasmid. The next four pairs of micrographs reveal the expression of cleaved caspase 3 in cells overexpressing ZFPL1. These cells also received either vehicle, DEX (10 μM), CT (10 nM), or DEX + CT. DAPI staining is shown in blue (scale bar = 100 μm). Again, the results clearly demonstrated that treatment with either CT and / or ZFPL1 overexpression significantly attenuated DEX-induced apoptosis in both cell lines. Figure 7E illustrates the pooled quantitative data from these experiments. The mean number ± SEM of cleaved caspase 3-labeled cells per field (magnification, ×400) was plotted against treatment with + CT ± DEX. The symbol * indicates P<0.05 compared with DEX+CT; X The symbol indicates P<0.001 compared to ZFPL1 overexpression (one-way ANOVA and Tukey's multiple comparison test). The symbol indicates P<0.05 compared to C (ordinary one-way ANOVA and Tukey's multiple comparison test). The results show that ZFPL1 overexpression and / or treatment with CT significantly reduced the apoptotic population in both cell lines. In Figure 7F, a representative photomicrograph shows the localization of cleaved caspase 3 staining in the nuclei of LNCaP-C4 cells (scale bar = 25 μm). These results indicate that cleaved caspase 3 staining in LNCaP-C4 cells was nuclear.

[0069] Effect of ZFPL1 knockdown and overexpression on prostate cancer cell invasion:Representative photomicrographs in Figure 8A show the effect of ±10 nM CT on the invasiveness of PC3-CTR cells receiving either nonsense siRNA or ZFPL1 siRNA (1, 2, or 3) (scale bar = 50 μm). Knockdown of ZFPL1 significantly reduced basal and CT-induced invasion of LNCaP-C4 and PC3-CTR cells. The bar graphs in Figure 8B reveal that pooled data from these invasion assays are expressed as the mean ± SEM number of invading cells per field (magnification, ×400) with PC3-CTR and LNCaP-C4 cells receiving either nonsense siRNA, siRNA1, siRNA2, or siRNA3. The symbol * indicates P<0.05, the symbol ** indicates P<0.001, and the symbol *** indicates P<0.0001 comparing −CT vs. +CT in each group. The symbol ^ indicates P<0.01 and the symbol ^^ indicates P<0.001 comparing nonsense siRNA vs. ZFPL1 siRNA. All P values ​​above were calculated using one-way ANOVA and Tukey's multiple comparison test.

[0070] In Figure 8C, representative photomicrographs reveal the effect of ±10 nM CT on the invasiveness of LNCaP-C4 and PC3-CTR cells expressing either the carrier pCMV5-XL4 plasmid or the ZFPL1-expressing plasmid. The results show that overexpression of ZFPL1 in both cell lines resulted in increased basal and CT-induced invasion. The bar graphs in Figure 8D show pooled data (mean ± SEM) from four separate invasion assays in PC3-CTR and LNCaP-C4 cells, respectively. The * symbol indicates P<0.05, the ** symbol indicates P<0.001, and the *** symbol indicates P<0.0001 for the comparison of -CT vs. +CT in each group. The ^ symbol indicates P<0.05 for the comparison of CT vs. OV+CT. All P values ​​above were calculated using one-way ANOVA and Tukey's multiple comparison test.

[0071] Similar studies were also performed to examine cell migration of PC3-CTR cells in a wound-healing assay. The photomicrographs in the upper left quadrant of Figure 8E reveal wounds in the PC3-CTR cell layer after 0 and 12 hours in the absence or presence of 10 nM CT. The upper right quadrant of Figure 8E shows a similar experiment in PC3-CTR cells in which ZFPL1 was knocked down using siRNA3. The results are shown in the bar graph of Figure 8F, which is presented as the mean ± SEM of the number of migratory cells that migrated within the wound (magnification, ×100) in four separate wound-healing assays. The pooled data indicate that CT promoted cell migration of PC3-CTR cells. However, when ZFPL1 was knocked down, baseline cell migration was reduced, and CT also failed to promote cell migration.

[0072] The next experiment examined the effects of ZFPL1 overexpression in PC3-CTR cells. The photomicrograph in the lower left quadrant of Figure 8E again demonstrated that CT promoted cell migration in PC3-CTR cells. However, as shown in the lower right quadrant of Figure 8E, ZFPL1 overexpression increased cell migration in the absence of CT as well as in its presence. The pooled data in Figure 8F also show that ZFPL1 overexpression increased cell migration in PC3-CTR cells, and the addition of CT further enhanced it. These results were consistent with the effect of ZFPL1 on prostate cell invasion. The * symbol indicates P<0.05 compared to the control (i.e., either siRNA or overexpression) for each group. The ^ symbol indicates P<0.05 for overexpression compared to overexpression + CT (one-way ANOVA and Tukey's multiple comparison test).

[0073] ZFPL1 and Akt Phosphorylation: Because knockdown of ZFPL1 led to apoptosis of prostate cancer cells and its overexpression reduced DEX-induced apoptosis, the effect of ZFPL1 on the activation of the PI3K survival pathway was investigated by examining Akt phosphorylation in PC3-CTR and LNCaP-C4 cells. Figures 9A and 9B summarize the immunoblot results of the effect of ±10 nM CT on p-Akt473 and p-Akt308 proteins in PC3-CTR cells (Figure 9A) and LNCaP-C4 cells (Figure 9B) receiving either nonsense (control) siRNA or ZFPL1 siRNA1, ZFPL1 siRNA2, or ZFPL1 siRNA3. Total Akt was used as a control protein, and β-actin was used as a loading control. Normalized densitometry bar graphs (p-Akt / total Akt) of the immunoblots are also included in Figures 9A and 9B. The symbol * indicates P<0.05, and the symbol ^ indicates P<0.05 compared to siRNA+CT (one-way ANOVA and Tukey's multiple comparison test). The data in Figures 9A and 9B revealed that knockdown of ZFPL1 resulted in a statistically significant decrease in basal and CT-induced phosphorylation of Akt473 / Akt308 in both cell lines. CT increased Akt phosphorylation, whereas knockdown of ZFPL1 significantly reduced CT-induced Akt phosphorylation. Consistent with previous results, this experiment also revealed that siRNA3 was the most potent at downregulating Akt phosphorylation in both cell lines.

[0074] Figures 9C and 9D summarize the immunoblot results of the effects of ±10 nM CT on p-Akt473 and p-Akt308 proteins in PC3-CTR cells (Figure 9C) and LNCaP-C4 cells (Figure 9D), respectively, transfected with either a carrier plasmid or a ZFPL1 expression plasmid. Akt was used as a control protein, and β-actin was used as a loading control. Normalized densitometry bar graphs (p-Akt / total Akt) of the immunoblots are also included in Figures 9C and 9D. The * symbol indicates P < 0.05 compared to the control (one-way ANOVA and Tukey's multiple comparison test). As expected, overexpression of ZFPL1 in these cell lines produced the opposite effect, as indicated by a significant increase in basal and CT-induced Akt phosphorylation. The results of this experiment showed that CT induced a minimal increase in Akt473 phosphorylation in ZFPL1-overexpressing LNCaP cells, further supporting the possibility that the effect of endogenous CT activation on PI3K pathway activation in prostate cancer cells is indirect and that ZFPL1 may be an important mediator of this CT action.

[0075] Akt phosphorylation was also observed by immunofluorescence microscopy. Representative photomicrographs in Figure 9E show the change in pAkt staining (green) in phosphorylated (p)-Akt immunopositive PC3-CTR and LNCaP-C4 cells receiving either nonsense or ZFPL1 siRNA (1, 2, or 3) upon treatment with ±10 nM CT. Blue is for DAPI (scale bar = 50 μm). In nonsense siRNA-treated cells, a small population of cells was p-Akt positive (<20%). Treatment with 10 nM CT for 30 min increased the p-Akt-positive population by more than twofold. Treatment with ZFPL1 siRNA reduced the p-Akt cell population compared with nonsense siRNA treatment. However, treatment with CT increased the number of p-Akt-positive cells, although still significantly less than that in nonsense sRNA-treated cells. As shown in Figure 9F, the quantification results of these experiments in PC-3CTR and LNCaP-C4 cells suggested that knockdown of ZFPL1 significantly attenuated / abolished basal and CT-induced phosphorylation of Akt. The data are presented as the mean ± SEM number of p-Akt-immunopositive cells per field (magnification, ×100) for PC3-CTR and LNCaP cells receiving either nonsense siRNA (control) or ZFPL1 siRNA 1, 2, or 3, in that order. The * symbol indicates P<0.05, comparing the control with the CT-treated cells in each group. The ^ symbol indicates P<0.05, comparing the control with the siRNA-treated cells (one-way ANOVA and Tukey's multiple comparison test).

[0076] In a similar experiment, we examined the effect of ZFPL1 overexpression on the basal and CT-induced increase in p-Akt in the nuclei of PC-3CTR and LNCaP-C4 cells. Representative photomicrographs in Figure 9G show the effect of ±10 nM CT on p-Akt-immunopositive cells per field (magnification, ×400; green) in PC3-CTR cells expressing either a carrier plasmid or a ZFPL-overexpression plasmid. Scale bar = 50 μm. P-Akt-positive LNCaP-C4 cells increased by nearly 70% when treated with 10 nM CT. A similar increase was identified when LNCaP-C4 cells were transfected with a ZFPL1 overexpression vector. When these cells (ZFPL1ov) were treated with 10 nM CT, nuclear colocalization of p-Akt increased by an additional ∼35%. As shown in Figure 9H, pooled quantitative data from PC3-CTR and LNCaP-C4 cells suggest that ZFPL1 and CT may have additive effects on Akt phosphorylation. The data are presented as the mean p-Akt ICC index per field ± SEM (magnification, ×100). The * symbol indicates P<0.05 comparing +CT with OV +CT, and the ^ symbol indicates P<0.05 comparing C with OV (one-way ANOVA and Tukey's multiple comparison test).

[0077] We next examined whether pAkt in these cells localizes to the nucleus. In Figure 9I, a representative photomicrograph at higher magnification (×1,000) shows nuclear localization of pAKT (green). Nuclear DAPI is in blue (scale bar = 25 μm). p-Akt (green) was found to colocalize with DAPI at ×400 magnification.

[0078] Generation of monoclonal antibodies against ZFPL1:To generate antibodies that bind to ZFPL1, the ZFPL1 protein was sequenced (SEQ ID NO: 11) and its epitope was determined (see Table 3). In order for the antibody of the present invention or a fragment thereof to specifically bind to the ZFPL1 protein or its variant protein, the antibody specifically binds to a polypeptide within the sequence of amino acids 62 to 308 of the ZFPL1 protein represented by SEQ ID NO: 11, preferably within the ranges of amino acids 62 to 77 and 127 to 284. [Table 3] Table 3

[0079] The antibodies of the present invention may also be referred to as "anti-ZFPL1 antibodies," "humanized anti-ZFPL1 antibodies," or "modified humanized anti-ZFPL1 antibodies," and are used in their broadest sense herein. In particular, the antibodies include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., variable regions and other portions of antibodies that exhibit a desired biological activity (e.g., binding to ZFPL1)).

[0080] The antibodies of the present invention are antibodies containing specific amino acid sequences in the light and heavy chain CDRs so that the antibodies can selectively bind to ZFPL1, and include both monoclonal and polyclonal antibodies, preferably monoclonal antibodies. Furthermore, the antibodies of the present invention include all of chimeric antibodies, humanized antibodies, and human antibodies, preferably human antibodies.

[0081] As used herein, the term "monoclonal" refers to the property of an antibody being obtained from a population of substantially homogeneous antibodies and does not necessarily imply that the antibody must be produced by any particular method. For example, the monoclonal antibody of the present invention may be produced through the hybridoma method first described in Kohler et al. (1975, Nature 256: 495) or through recombinant DNA methods (U.S. Patent No. 4,816,567). It may also be isolated from phage antibody libraries using techniques described in the literature (Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597 and Presta (2005) J. Allergy Clin. Immunol. 116:731).

[0082] In a preferred embodiment described below, the generated ZFPL1 antibody (also referred to as the "PA1623 antibody") specifically binds to amino acids 131 to 150 (SEQ ID NO: 10) of the ZFPL1 protein (SEQ ID NO: 11). The antibody was generated by immunizing mice with the synthetic peptide GLGLPLIDEV VSPEPEPLNT (SEQ ID NO: 10). Hybridomas were generated, and the secreted PA1623 antibody was tested for binding ability by ELISA. The PA1623 antibody was then tested for cross-reactivity with prostate secretions and found to be specific for ZFPL1. These methods for generating antibodies are well known in the art and can be easily reproduced by those skilled in the art to generate antibodies that bind to other ZFPL1 epitopes shown in Table 3.

[0083] The PA1623 antibody is monoclonal and of the immunoglobulin (Ig) G isotype. The antibody contains a variant Fc domain. While the entire sequence of PA1623 is disclosed in SEQ ID NO: 9, those skilled in the art will recognize that portions of the sequence (outside the paratope correlated with the ZFPL1 epitope) may vary and still be effective in the diagnostic and treatment methods disclosed below. Furthermore, the claimed diagnostic and treatment methods may also be performed with antibodies that bind to any of the other epitopes of ZFPL1 (see Table 3). Each of these antibodies is monoclonal and of the immunoglobulin (Ig) G isotype. Furthermore, to perform their claimed functions, these antibodies have an equilibrium dissociation constant (K D ) value and a half maximal inhibitory concentration (IC) of approximately 10 nM. 50 )

[0084] Development of immunosensors for prostate cancer diagnosisAn exemplary embodiment of an immunosensor incorporating a ZFPL1 antibody is photographed in FIG. 11 and described below. The immunosensor shown is constructed on a gold-coated nano-AAO chip. A self-assembled monolayer (SAM) was prepared by performing a series of chemical reactions followed by covalent attachment of the monoclonal antibody to the chip surface. First, the chip was incubated with a mixed alkanethiol solution containing 5 mM 11-mercaptoundecanoic acid and 50 mM 8-mercapto-l-octanol. The SAM was activated by incubating the chip in a phosphate buffer containing 2 mM NHS: N-hydroxysuccinimide and 8 mM EDC: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The chip was then incubated with a novel primary antibody against ZFPL1 disclosed herein. In the specific embodiment photographed in FIG. 11 and used in the experiments described below, the PA1623 antibody was used. However, it should be understood that any of the other novel antibodies described in this application may be used as well. Furthermore, those skilled in the art will recognize that the particular methods for constructing the ZFPL1 optical immunosensors of the present invention are illustrative only and can be easily modified to suit various environments.

[0085] Although an optical immunosensor is employed in the preferred embodiment disclosed herein, those skilled in the art will recognize that many other assay methods, including chemiluminescence assays, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, and dot / blot assays, can be used to determine ZFPL1 levels in a sample. Furthermore, the claimed immunosensor includes a substrate to which the antibody of the present invention is coupled. The antibody can be coupled to the substrate by, for example, passive adsorption, or can be chemically bound to the substrate by, for example, covalent bonding. Such covalent bonding generally requires the initial introduction of a chemically active compound covalently bound to the substrate surface prior to antibody addition. The antibody itself may also require the addition of a chemically activating group to achieve substrate binding. These requirements are well known in the art. The substrate can be any medium capable of adsorbing or binding to antibodies, such as beads or nanoparticles (optionally chemically activated), but is preferably a flat structure (optionally activated) such as a microtiter plate or biochip. Biochips are thin, wafer-like substrates with a flat surface, which can be made of any suitable material such as glass or plastic, but preferably ceramic. The biochip can be chemically activated prior to antibody binding or can undergo passive adsorption of antibodies.

[0086] Returning to the preferred immunosensor shown in Figure 11, the immunosensor was washed, blocked, and incubated with a known concentration of ZFPL1 peptide or an unknown serum sample for approximately 1 hour and 30 minutes or more. The chip was then thoroughly washed and dried. Readings were taken with an optical detector as follows: After functionalizing the biochip and binding the antibody to the surface, a white light source was collimated by a lens and shone onto the chip. The transmitted signal was a reflected optical interference pattern, which was detected by the optical detector. This reading served as a blank. After the sample was applied to the sensor, the transmitted signal shifted due to binding of the antibody and biomarker, resulting in a different reflected signal. Data from peaks ranging from 550 nm to 750 nm were used to measure the mean shift. Figure 12 shows the wavelength shift recorded during testing of the immunosensor of the presently disclosed invention. In Figure 12A, the solid line represents the reading of a blank containing only the antibody, and the dotted line represents the reading of a chip to which ZFPL1 was applied. In Figure 12B, the solid line represents the reading of a blank containing only the antibody, and the dotted line represents the reading of a chip to which bovine serum albumin (BSA) was applied as a control. As is clear from the graph in Figure 12, no optical shift is observed when BSA is used, but ZFPL1 causes a significant wavelength shift.

[0087] Turning now to Figure 13, the immunosensor was tested for its ultrasensitivity and high specificity for detecting ZFPL1 and PSA in patient serum. The immunosensor-based assay is label-free. Unlabeled synthetic peptides of partial sequences were used as references for both antigens. The assay was tested for accuracy, precision, recovery, and linearity. The dilution curve of human serum paralleled the ZFPL1 standard curve in the range of 0.1 to 2 μl serum. The test included negative samples (a serum pool from patients who underwent prostatectomy; serum PSA <0.003 ng / ml) and positive samples (a serum pool from patients with biopsy-confirmed prostate cancer). To date, the intra- and inter-assay variability of the assay is less than 5% and 9%, respectively. To compare the effectiveness of the immunosensor with ELISA, we compared ZFPL1 calibration curves from both systems. The immunosensor (Figure 13B) was approximately 50-fold more sensitive than the corresponding non-equilibrium ELISA (Figure 13A). The assay is linear over a range of 1 to 64 pg, with a sensitivity of 1 pg / 50 μl. Thus, serum ZFPL1 levels can be measured in volumes as low as 1 pg ZFPL1 / mL serum. Thus, when considered together with the simplicity of the method—a label-free assay with a short 90-minute incubation period—the immunosensor of the present disclosure offers significant advantages over ELISA for measuring ZFPL1.

[0088] Presence of ZFPL1 in the serum of patients with prostate cancerBecause ZFPL1 is secreted by prostate cancer cells, its presence in the serum of healthy volunteers and patients with prostate cancer was next examined. PSA was used as a reference biomarker in the same cohort. The serum profiles of ZFPL1 and PSA in healthy donors and confirmed prostate cancer patients are shown in Figure 10. The scatter plot shows that serum ZFPL1 levels in non-cancer individuals (control; mean ± SEM 3.6 ± 0.286 ng / ml, n = 36) were significantly lower than all patients with prostate cancer (cancer: 11.41 ± 0.6135, n = 75), with no overlap (P < 0.0001, unpaired t-test). In contrast, PSA levels showed significant overlap between non-cancer and cancer patients (control: 6.26 ± 0.9, n = 37 vs. cancer: 22.85 ± 2.96, n = 42, not significant by unpaired t-test). This clear separation of ZFPL1 levels in non-cancer and cancer patients suggested that ZFPL1 testing significantly improved the specificity of prostate cancer detection. Furthermore, the data suggest that ZFPL1 concentrations of approximately 3.3 ng / mL or greater correlate with a positive prostate cancer diagnosis.

[0089] Hit rate of ZFPL1 immunosensor Next, serum ZFPL1 and PSA levels in healthy donors (n=119) and prostate cancer patients (n=205) were measured using the immunosensor assay of the present invention, and the diagnostic ability of both markers was quantified using the area under the receiver operating characteristic curve (AUC). These were also reflected in the prediction curve for normal (0) vs. prostate cancer (1). As shown in Figure 14A, the AUC of ZFPL1 was 0.9788 (close to a perfect 1.0) with P<0.0001. Furthermore, the average ZFPL1 values ​​of normal individuals were clearly different from those of prostate cancer patients. In contrast, as shown in Figure 14B, the AUC value of PSA was 0.9055, and there was significant overlap between normal samples and prostate cancer samples. Finally, as shown in Figure 14C, when the data from the two markers were combined, the AUC was 0.9793.

[0090] Tables 4 and 5 summarize the accuracy of the immunoassays and diagnostic methods when testing for ZFPL1 and PSA. The results show that the ZFPL1 immunoassay accurately predicted whether the serum was positive for cancer 92.59% of the time, compared with only 84.88% accuracy for the PSA immunoassay. This suggests that the ZFPL1 immunoassay significantly reduces incorrect diagnoses. It is important to study the negative predictive value in addition to the positive predictive value because it may prevent unnecessary follow-up tests, such as biopsies. [Table 4] Table 4 [Table 5] Table 5

[0091] Table 6 provides the mean ± SD of ZFPL1 and PSA levels in stratified conditions from the same cohort, as determined by the ZFPL1 immunosensor disclosed herein. ZFPL1 clearly distinguishes prostate cancer from other prostate diseases, and shows very high levels in cases of metastatic disease. This test can be useful for monitoring patients after treatment to check tumor recurrence (biochemical recurrence or BCR). [Table 6] Table 6

[0092] Accuracy in the gray zone:One of the weaknesses of PSA-based diagnostics is that PSA has high diagnostic sensitivity but relatively low specificity, which can lead to overdiagnosis and treatment of indolent prostate lesions. In particular, when PSA levels were between 4 ng / mL and 10 ng / mL, also known as the "gray zone," only 22% of patients had a positive prostate biopsy. To investigate the effectiveness of ZFPL1 testing in this gray zone, we prepared receiver operating characteristic curves (ROC) for ZFPL1 and PSA levels in patients with serum levels in the gray zone. The results show that the predictive value of PSA in this sample population (Figure 15B) was very low, with an AUC value of 0.770, while the predictive value of ZFPL1 in the same population (Figure 15A) had an AUC value of 0.9672. The ZFPL1 immunosensor accurately predicted that 19 of 23 patients were cancer-free and did not require biopsy / treatment with an NPV of 82.6. The ZFPL1 study had a PPV of 95.5, correctly predicting that 87 of 88 patients had cancer.

[0093] Effect of ZFPL1 monoclonal antibody on prostate cancer cell growth: To investigate the potential of ZFPL1 antibodies as druggable targets, two prostate cancer cell lines, PC3-CTR and DU-145, were cultured and incubated with 0 μl, 0.25 μl, 0.5 μl, or 1 μl of the PA1623 ZFPL1 antibody. PC3-CTR and DU-145 were selected for their ability to grow rapidly under androgen-independent conditions. Cell growth was monitored by measuring the optical density at 600 nm (OD 600 ) The results are summarized in Figure 16. Both cell lines showed reduced cell growth at all treatment doses compared to cells that did not receive ZFPL1 antibody treatment. The results demonstrate the potent, dose-related inhibitory effect of ZFPL1 antibodies on the growth of PC3-CTR and DU-145 cells. The antibody's inhibition of growth of these highly aggressive cell lines indicates promising potential for using the antibody as an effective therapeutic agent for castration-resistant prostate cancer.

[0094] Efficacy of ZFPL1 monoclonal antibodies as treatment in animal models:Next, a follow-up experiment was conducted to test the efficacy of ZFPL1 antibodies as a treatment in an animal model. In this study, adult nude mice weighing approximately 30 g were injected with a PC3-CTR cell suspension in Matrigel. TM The mixture (1:1 v / v) was injected into the flanks of the mice. The mice were then observed daily. Tumors became visible 11 to 14 days after cell implantation. Observation, treatment with the PA1623 ZFPL1 antibody, and caliper tumor volume measurement began approximately two weeks after implantation. The infected mice received either no treatment (the "untreated" group), saline (the "control" group), 1 μl of antibody solution (containing 4.5 μg of ZFPL1 antibody), or 2 μl of antibody solution (containing 9 μg of ZFPL1 antibody). The ZFPL1 antibody was injected intratumorally every Monday and Thursday until the end of the study. The "untreated" group was left untreated after PC3-CTR cell implantation, while the "control" group was injected with saline on the same schedule as the ZFPL1 antibody injections in the other groups.

[0095] Figure 17 is a scatter plot of tumor volume from day 15 after cell implantation. The results show that treatment with 4.5 μg ZFPL1 antibody significantly slowed tumor growth, and tumor growth inhibition was even greater at the higher dose of 9 μg. Figure 18 includes representative images of tumors (untreated and treated) and a graph of their body weights at necropsy. Significant differences were observed in the appearance and texture of the tumors. For example, tumors in untreated mice were extremely hard, as expected. In contrast, tumors in treated mice were very soft and had a strong odor of narcotic tissue. These results demonstrate the effectiveness of ZFPL1 antibody in slowing tumor growth and, therefore, treating prostate cancer.

[0096] Based on the overall results presented above, it is anticipated that a preferred embodiment of human treatment will involve administration of 0.5-5.0 mg of antibody per kg of body weight. The treatment is preferably administered intraperitoneally, but in other embodiments may be administered intratumorally, intravenously, or subcutaneously. Because the half-life of antibodies in humans is typically 3-4 weeks, the treatment is preferably administered approximately every 2-3 days for 1 week, followed by approximately 3 weeks without treatment. This process can be repeated until the patient's ZFPL1 and / or PSA levels reach normal or until the patient's tumor has decreased in size to such an extent that it can be surgically removed.

[0097] Administration of monoclonal antibodies as described herein can result in tumor growth inhibition, tumor regression, reduction in tumor size, reduction in tumor cell count, delay in tumor growth, abscopal effect, inhibition of tumor metastasis, reduction in metastatic lesions over time, reduction in the use of chemotherapeutic or cytotoxic drugs, reduction in tumor burden, increased progression-free survival, increased overall survival, complete response, partial response, and / or stable disease.This treatment method can be combined with one or more of surgery, radiation, chemotherapy, cancer vaccine, antibody-drug conjugate, anti-inflammatory drug, nutritional supplement, or any other treatment for prostate cancer and / or its symptoms that is currently known or may be developed in the future.

[0098] The foregoing description and accompanying drawings illustrate the principles, exemplary embodiments, and modes of operation of the present invention. However, the present invention should not be construed as limited to the particular embodiments discussed above. Many modifications of the embodiments described herein will come to mind to those skilled in the art having the benefit of the teachings presented in the foregoing description and the associated drawings. It should thus be recognized that variations to those embodiments may be made by those skilled in the art without departing from the scope of the present invention.

Claims

1. A composition for use in a method for diagnosing and treating prostate cancer in mammalian patients, comprising an anti-ZFPL1 antibody, wherein the method is a. The process of obtaining a serum sample from the patient; b. A step of detecting whether or not zinc finger protein-like 1 (ZFPL1) is present in the serum sample; c. The step of diagnosing the patient with prostate cancer if the presence of ZFPL1 is detected in the serum sample; and d. The step of administering the composition to the diagnosed patient. A composition that includes the following:

2. The composition according to claim 1, wherein the level of ZFPL1 is determined using an unlabeled optical immunoassay comprising a monoclonal antibody capable of specifically binding to ZFPL1, wherein the monoclonal antibody is immobilized on a substrate.

3. The composition according to claim 1, wherein the monoclonal antibody specifically binds to a protein containing an epitope selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:

17.

4. The aforementioned monoclonal antibody is a. A heavy chain complementarity determination region (CDR) comprising a heavy chain variable region (HCVR) including SEQ ID NOs: 1, 2, and 3; and b. A light chain CDR comprising a light chain variable region (LCVR) including sequence numbers 4, 5, and 6. The composition according to claim 1, comprising:

5. The composition according to claim 1, wherein the monoclonal antibody comprises HCVR (SEQ ID NO: 7) and LCVR (SEQ ID NO: 8).

6. The composition according to claim 1, wherein the isotype of the monoclonal antibody is immunoglobulin G.

7. The antibody has an equilibrium dissociation constant of 100 nM (K Ｄ ) value and 10 nM half-inhibitory inhibitory concentration (IC) ５０ The composition according to claim 1, having )

8. The composition according to claim 1, characterized in that the patient is diagnosed with prostate cancer if the serum sample has a ZFPL1 level higher than or equal to 3.3 ng / mL.

9. The composition according to claim 1, characterized in that the monoclonal antibody treatment is administered intravenously, subcutaneously, or intraperitoneally.

10. The composition according to claim 9, characterized in that the monoclonal antibody treatment is administered intraperitoneally.

11. The composition according to claim 1, wherein the monoclonal antibody treatment produces at least one effect selected from the group consisting of inhibition of tumor growth, tumor regression, reduction of tumor size, reduction of tumor cell count, delay of tumor growth, abscopal effect, inhibition of tumor metastasis, reduction of metastatic lesions over time, reduction of the use of chemotherapeutic agents or cytotoxic agents, reduction of tumor burden, increase in progression-free survival, increase in overall survival, complete response, partial response, and stable disease state.

12. The composition according to claim 1, wherein the method further comprises the step of administering a further therapeutic agent or treatment to the patient, wherein the further therapeutic agent or treatment is selected from the group consisting of surgical procedures, radiation, chemotherapeutic agents, cancer vaccines, antibody-drug conjugates, anti-inflammatory drugs, and nutritional supplements.

13. A composition for treating prostate cancer in a patient, comprising an anti-ZFPL1 antibody.

14. The composition according to claim 13, wherein the anti-ZFPL1 antibody is a monoclonal antibody, an antigen-binding fragment thereof, or a protein ligand.

15. The anti-ZFPL1 antibody is found in SEQ ID NO: 11 a. 269-284; b. 62-77; c. 205-220; d. 222-237; e. 293-308: f. 240-255; or g. 131-150; The composition according to claim 13, wherein at least four nucleotides are bound to the nucleotide position of the nucleotide.

16. The composition according to claim 13, wherein the anti-ZFPL1 antibody is bound to at least four nucleotides at nucleotide positions 62-77, 127-284, or 293-308 in SEQ ID NO:

11.

17. The composition according to claim 13, wherein the anti-ZFPL1 antibody is bound to at least seven nucleotides at nucleotide positions 62-77, 127-284, or 293-308 in SEQ ID NO:

11.

18. The composition according to claim 13, wherein the anti-ZFPL1 antibody is a chimeric antibody or a humanized antibody.

19. The composition according to claim 13, wherein the anti-ZFPL1 antibody comprises a variant Fc domain.

20. The anti-ZFPL1 antibody is an isolated monoclonal antibody or its antigen-binding moiety, as follows: a. HCDR1 containing an amino acid sequence having an amino acid substitution in the second amino acid, the fourth amino acid, or both the second and fourth amino acids of SEQ ID NO: 1; b. HCDR2 containing the amino acid sequence of SEQ ID NO: 2; c. HCDR3 containing an amino acid sequence having an amino acid substitution at the eighth amino acid of SEQ ID NO: 3; d. LCDR1 containing an amino acid sequence having an amino acid substitution in the 8th, 11th, or 13th amino acid of SEQ ID NO: 4, or in all of these amino acids; e. LCDR2 containing an amino acid sequence having an amino acid substitution at position 4 of SEQ ID NO: 5; and f. LCDR3 containing an amino acid sequence having an amino acid substitution in the fourth amino acid, the seventh amino acid, or both the fourth and seventh amino acids of SEQ ID NO: 6; including and g. The antibody or its antigen-binding portion contains 100 nM or less K in the polypeptide of SEQ ID NO:

11. Ｄ Join them together. The composition according to claim 13.

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