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

Abstract

Disclosed herein is a method of diagnosing and treating prostate cancer using an antibody against zinc finger protein-like 1 (ZFPL1). The method may comprise: obtaining a serum sample from the patient; detecting whether zinc finger protein sample 1 (ZFPL1) exists in the serum sample or not; when it is detected that ZFPL1 exists in the serum sample, the patient is diagnosed as having prostate cancer; and then administering to the diagnosed patient an effective amount of an anti-ZFPLl antibody.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 371,902, filed on August 19, 2022, and titled "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 Serial No. 63 / 387,115, filed on December 13, 2022, and titled "Neuroendocrine Marker (ZFPL1) for Prostate Cancer Diagnosis and Monitoring and the Method of Measuring the Same".

[0003] Reference to Sequence Listing XML

[0004] 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 was created on August 2, 2023, has a name of 019976 - 187142 - 00_SL.xml, and is 17,392 bytes in size. Background of the Invention

[0005] Prostate cancer is the second most common cancer among men globally and the sixth leading cause of cancer death. The characteristics of prostate cancer show great diversity, ranging from slow - growing tumors with little clinical significance to aggressive metastatic disease. This presents a great opportunity for identifying multiple biomarkers that represent different stages of cancer progression. Unfortunately, prostate - specific antigen (PSA) is the only established blood biomarker used for multiple purposes, including prostate cancer detection, stratifying patients into prognostic risk groups, determining overall tumor burden, and tracking response to local or systemic treatment. In addition, the prognosis of this disease is still evaluated using routine pathological parameters such as the Gleason score, the number or percentage of positive nuclei, and the maximum percentage of tumor involvement in any nucleus.

[0006] PSA is a kallikrein protease that is mainly produced by prostate luminal cells, but a small amount is also secreted by the pancreas and uterus. PSA is not cancer-specific but is normally produced in the prostate; its level increases in prostate cancer as well as in several benign conditions such as benign prostatic hyperplasia (BPH) and prostatitis. Thus, the serum PSA test non-specifically detects many benign conditions as well as many low-grade and thus indolent prostate tumors. Therefore, PSA-based diagnosis requires confirmation by invasive, repetitive, and expensive procedures such as transrectal ultrasound-guided biopsy. On the other hand, approximately 15% of prostate cancer cases show low or normal serum PSA levels, most of which are highly aggressive and have neuroendocrine (NE) features, indicating that the PSA test may not detect all fatal prostate cancers that require aggressive treatment.

[0007] Calcitonin (CT) and its receptor (CTR) have been reported to be selectively expressed by the basal cells of benign prostatic epithelium, but not by secretory cells. However, all malignant prostatic epithelial cells express CT and CTR, and their expression increases with tumor progression. In addition, activation of the CT-CTR axis induces an invasive phenotype in benign prostatic cells. To date, non-invasive methods for accurately diagnosing prostate cancer in patients have remained elusive. Summary of the Invention

[0008] The invention disclosed herein relates to a method for diagnosing and treating prostate cancer by targeting zinc finger protein-like 1 (ZFPL1). Through a subtractive hybridization process, ZFPL1 has been identified as selectively expressed only in malignant prostatic cells, but not in benign prostatic cells. Further findings indicate that the ZFPL1 protein plays a role in tumor development. Therefore, treatment targeting ZFPL1 is effective in treating prostate cancer.

[0009] An immunosensor for detecting zinc finger protein-like 1 (ZFPL1) in a biological sample embodying the features of the present invention may include a monoclonal anti-ZFPL1 antibody immobilized on a substrate. 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.

[0010] In another embodiment of the immunosensor, the monoclonal antibody comprises: a heavy chain variable region (HCVR) comprising heavy chain complementarity determining regions (CDRs), the heavy chain CDRs comprising SEQ ID No: 1, 2, and 3; and a light chain variable region (LCVR) comprising light chain CDRs, the light chain CDRs comprising SEQ ID No: 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 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 (IC 50 50).

[0011] In another embodiment of the immunosensor, the light source generates white light, and the optical sensor is configured to detect light in the wavelength range of 500 nm to 900 nm (including the end values). In another embodiment, the immunosensor further comprises a processor that calculates the 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 a reading of less than 5% of the monoclonal antibody binding to ZFPL1, and the second sensor reading is a reading of greater than 5% of the monoclonal antibody binding to ZFPL1. In another embodiment, the frequency shift is detected when the concentration of ZFPL1 in the biological sample is less than 1 pg / ml.

[0012] The immunoassay method for detecting prostate cancer in a biological sample according to the present invention may 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. 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.

[0013] In another embodiment of the immunoassay method, the monoclonal antibody comprises: an HCVR comprising heavy chain CDRs, the heavy chain CDRs comprising SEQ ID NO: 1, 2, and 3; and an LCVR comprising light chain CDRs, the light chain CDRs comprising SEQ ID NO: 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 (IC 50 ).

[0014] In another embodiment of the immunoassay method, the presence of ZFPL1 in a biological sample is detected when the ZFPL1 concentration is less than 1 pg / ml. In another embodiment, the step of 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, the step of detecting the presence of ZFPL1 in the biological sample comprises performing at least one of the following: chemiluminescence assay, immunofluorescence assay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, Western blot assay, enzyme immunoassay, immunoprecipitation assay, immunohistochemical assay, immunochromatographic assay, dot blot assay, slot blot assay, lateral flow assay, optical immunoassay. In another embodiment, the step of 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 a substrate and obtaining a second reading after contacting the biological sample with the monoclonal antibody immobilized on a substrate, wherein the step of obtaining a reading comprises: collimating a white light source through a lens to irradiate the monoclonal antibody immobilized on the substrate, and detecting a transduction signal using an optical detector, including reflected optical interference fringes. In another embodiment, the step of detecting the presence of ZFPL1 in the biological sample comprises comparing the first reading with the second reading and measuring the frequency shift. In another embodiment, a data peak in the range of 550 nm to 750 nm is used to measure the frequency shift.

[0015] A method for diagnosing prostate cancer embodying the features of the present invention may include the following steps: First, obtaining a serum sample from a patient; Second, detecting the presence of ZFPL1 in the serum sample; and Third, diagnosing the patient as having prostate cancer when ZFPL1 is detected in the serum sample. More specifically, such a 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 ZFPL1; determining the ZFPL1 level in the patient sample; and identifying the patient as having a malignant prostate tumor when the ZFPL1 level is higher than the baseline ZFPL1 level observed in corresponding healthy subjects. 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: an HCVR comprising heavy chain CDRs, the heavy chain CDRs comprising SEQ ID NO:1, 2 and 3; and an LCVR comprising light chain CDRs, the light chain CDRs comprising SEQ ID NO: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 (IC 50 ) of 10 nM. In another embodiment, the ZFPL1 level is determined using a label-free optical immunoassay. In another embodiment, the baseline ZFPL1 level in the serum sample is 3.3 ng / mL.

[0017] A method for diagnosing and treating prostate cancer embodying the features of the present invention may include the following steps: First, obtaining a serum sample from a patient; Second, detecting the presence of zinc finger protein-like 1 (ZFPL1) in the serum sample; Third, diagnosing the patient as having prostate cancer when 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.

[0018] In another embodiment of the diagnostic method, the monoclonal antibody comprises: an HCVR comprising heavy chain CDRs, the heavy chain CDRs comprising SEQ ID NOs: 1, 2, and 3; and an LCVR comprising light chain CDRs, the light chain CDRs 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 (IC 50 ) value of 10 nM. In another embodiment, the ZFPL1 level is determined using a label-free optical immunoassay. In another embodiment, the baseline ZFPL1 level in the serum sample is 3.3 ng / mL.

[0019] 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 the number of tumor cells, delay in 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, increase in progression-free survival, increase in overall survival, complete remission, partial remission, and disease stabilization.

[0020] A method of treating cancer in a human subject embodying the features of the present invention may include administering to a patient with prostate cancer an effective amount of an anti-ZFPL1 antibody. 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.

[0021] In another embodiment of the treatment method, 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 within 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 within SEQ ID NO: 11. In another embodiment, the anti-ZFPL1 antibody is a chimeric antibody or a humanized antibody. In another embodiment, the anti-ZFPL1 antibody comprises a variant Fc domain.

[0022] 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 the number of tumor cells, delay in tumor growth, abscopal effect, inhibition of tumor metastasis, reduction in metastatic foci over time, reduction in the use of chemotherapeutic or cytotoxic agents, reduction in tumor burden, increase in progression-free survival, increase in overall survival, complete remission, partial remission, and disease stabilization.

[0023] In another embodiment of the treatment method, an effective amount of the anti-ZFPL1 antibody is between 0.5 and 5.0 mg per kilogram of patient body weight. In another embodiment, the treatment is administered for 2 or 3 consecutive days and then the administration is stopped for at least three weeks. In another embodiment, after three weeks, it is tested whether the protein of SEQ ID NO:11 is present in human serum. In another embodiment, if the protein of SEQ ID NO:11 is present at a concentration higher than 3 ng / ml, the treatment is administered for a second consecutive 2 or 3 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] This patent or application document contains at least one drawing executed in color. After a request has been made and the necessary fee has been paid, the Office will provide a copy of the published patent or patent application containing one or more color drawings.

[0025] 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- denotes small interfering; and p- denotes phosphorylated.

[0026] Figure 1A Is a representative micrograph showing the presence of amplified ZFPL1 mRNA in PC3M, DU145, LNCaP, M1 (stably expressing inactive CTR), C4, PC3-CTR, PC3 prostate cancer cells after a qRT-PCR reaction. Figure 1B Is a bar graph showing the quantitative representation of the ZFPL1 gene expression band normalized with the GAPDH housekeeping gene. Figure 1C Is a representative micrograph depicting the ZFPL1 product of the 34.1 kDa size band on an immunoblot. Figure 1DBar graph showing the mean ± SEM (n = 3) of the relative abundance of ZFPL1 mRNA in LNCaP-C4 cells after treatment with CT at concentrations of 0, 5, 10, 50, and 100 nM. Figure 1E Bar graph showing the mean ± SEM (n = 3) of the relative abundance of ZFPL1 mRNA in PC3-CTR cells after treatment with CT at concentrations of 0, 5, 10, 50, and 100 nM. Figure 1F Bar graph showing the dose-dependent increase in the relative abundance of ZFPL1 mRNA (mean ± SEM, n = 3) in LNCaP-C4 cells in response to the synthetic androgen R1881.

[0027] Figure 2A Bar graph showing the mean ± SEM (n = 6) percentage of ZFPL1-immunopositive cell populations per field of view (magnification, x400) in various normal human organs. Figure 2B Representative micrographs of ZFPL1-immunopositive cells in sections of normal human organs showing ZFPL1-immunopositive cells and normal prostate showing ZFPL1-immunonegative. Figure 2C Bar graph presenting the relative abundance of ZFPL1 mRNA in normal, BPH, and prostate cancer specimens with different Gleason scores. Figure 2D Shows data extracted from the TCGA and Oncomine portals, which demonstrate upregulation of ZFPL1 gene expression in prostate cancer specimens.

[0028] Figure 3A Micrographs depicting in situ hybridization specificity after treatment of prostate cancer specimens with sense ZFPL1 siRNA probe (left) or antisense ZFPL1 siRNA probe (right). Figure 3B Micrographs depicting ZFPL1 mRNA expression in prostate sections of different cancer stages compared to non-cancer specimens. Figure 3C The left panel of is a representative micrograph showing the presence of ZFPL1-immunopositive cells (red) in prostate cancer tissue (left) relative to matched normal tissue (right) by immunofluorescence. The nuclear stain is DAPI (blue). Figure 3C The right panel of is a bar graph showing the mean percentage (n = 6) of ZFPL1-immunopositive cells per field of view (magnification, ×400) in prostate cancer tissue tested against matched normal tissue. In Figure 3D The representative micrograph on the left shows H&E staining of a human prostate cancer tissue sample (dark blue staining is nuclear hematoxylin and pink staining is eosin), while the micrograph on the right shows green immunofluorescent labeling of ZFPL1 and blue labeling of nuclear DAPI. White arrows depict the corresponding stained cancerous regions. Figure 3ERepresentative micrographs showing ZFPL1-immunopositive cells (red) and nuclear DAPI (blue) in different samples of a prostate cancer tissue microarray. Figure 3F The bar graphs in Figure 3E present the quantitative data of the prostate cancer tissue microarray of

[0029] In Figure 4A representative micrographs show the co-localization of ZFPL1 and chromogranin A (CgA) in PC3-CTR cells (upper panel) and human prostate cancer tissues (lower panel) evaluated by immunofluorescence technique. In Figure 4B representative micrographs show the co-localization of ZFPL1 and CD44 in PC3-CTR cells (upper panel) and human prostate cancer tissues (lower panel) evaluated by immunofluorescence technique.

[0030] Figure 5A Representative micrograph showing the co-localization of ZFPL1 (green) and exosomal CD81 (red) in PC3-CTR and LNCaP PC cells. Figure 5B Representative micrograph revealing the co-localization of ZFPL1 (green) and exosome / scretosome marker CD63 (red). Figure 5C Representative micrograph showing the co-localization of ZFPL1 (green) and Golgi marker GM130 (red). Figure 5D Representative immunoblot showing the co-precipitation of CD81 with ZFPL1 in exosome isolates from PC3-CTR and LNCaP-C4PC cells.

[0031] Figure 6A Immunoblots showing 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 demonstrate that transfection of ZFPL1 expression plasmid in PC3-CTR and LNCaP-C4 cells leads to an increase in ZFPL1 protein levels in both cell lines.

[0032] Figure 7A Bar graph showing the effect of ±10 nM CT on the proliferation of PC-3CTR cells receiving either nonsense siRNA or ZFPL1 siRNA. Figure 7B Representative micrographs in Figure 7CThe bar graph presents the pooled data from four independent experiments conducted using the LNCaP-C4 and PC3-CTR cell lines. 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 vector plasmid. The next four pairs of micrographs reveal the expression of cleaved caspase-3 in PC3-CTR and LNCaP-C4 cells overexpressing ZFPL1. Figure 7E The bar graph shows the pooled data from four independent experiments presenting Figure D. In Figure 7F , representative micrographs show the localization of cleaved caspase-3 staining in the nuclei of LNCaP-C4 cells.

[0033] Figure 8A The representative micrographs in show the effect of ±10 nM CT on the invasiveness of PC3-CTR cells receiving nonsense siRNA or ZFPL1 siRNA (1, 2, or 3). Figure 8B shows Figure 8A The pooled data from the experiment summarized Figure 8C The representative micrographs in show the effect of ±10 nM CT on the invasiveness of LNCaP-C4 and PC3-CTR cells expressing the vector pCMV5-XL4 plasmid or the plasmid with ZFPL1 expression plasmid. Figure 8D shows Figure 8C The pooled data from the experiment summarized Figure 8E Next, shows representative micrographs of a wound healing assay for cell migration in PC3-CTR cells transfected with ZDPL1 siRNA3 (siRNA - row 2) or ZFPL1 expression vector (OVER - row 4) and treated with ±CT (10 nM). Figure 8F shows Figure 8E The bar graph presenting the pooled data from the experiment summarized

[0034] Figure 9A The representative immunoblot shows the effect of ±10 nM CT on the p-Akt473 and p-Akt308 proteins in PC3-CTR cells receiving nonsense (control) siRNA or ZFPL1 siRNA1, ZFPL siRNA2, or ZFPL1 siRNA3. Figure 9A Also included in is a normalized bar graph of the densitometric quantification of the immunoblot (pAkt / total Akt). Figure 9BRepresentative immunoblots showing the effect of ±10 nM CT on p-Akt473 and p-Akt308 proteins in LNCaP-C4 cells transfected with nonsense (control) siRNA or ZFPL1 siRNA1, ZFPL siRNA2, or ZFPL1 siRNA3. Figure 9B Also included are normalized bar graphs of densitometric quantification of the immunoblots (p-Akt / total Akt).

[0035] Figure 9C Representative immunoblots showing the effect of ±10 nM CT on p-Akt473 and p-Akt308 proteins in PC3-CTR cells transfected with vector plasmid or ZFPL1 expression plasmid, respectively. Figure 9C Also included are normalized bar graphs of densitometric quantification of the immunoblots (p-Akt / total Akt). Figure 9D Representative immunoblots showing the effect of ±10 nM CT on p-Akt473 and p-Akt308 proteins in LNCaP-C4 cells transfected with vector plasmid or ZFPL1 expression plasmid, respectively. Figure 9D Also included are normalized bar graphs of densitometric quantification of the immunoblots (p-Akt / total Akt).

[0036] Figure 9E Representative micrographs showing the effect of ±10 nM CT on pAkt staining in LNCaP-C4 and PC3-CTR cells transfected with nonsense or ZFPL1 siRNA (1, 2, or 3). Scale bar = 50 μm. Figure 9F Are two bar graphs summarizing pooled data from four independent experiments of PC3-CTR and LNCaP-C4 cells transfected with nonsense or ZFPL1 siRNA. Figure 9G Representative micrographs showing the effect of ±10 nM CT on the number of p-Akt immunopositive cells per field (magnification, ×400; green) in PC3-CTR cells transfected with expression vector plasmid or ZFPL overexpression plasmid. Scale bar = 50 μm. Figure 9H Are two bar graphs summarizing pooled data from four independent experiments of PC3-CTR and LNCaP-C4 cells transfected with expression vector plasmid (C) or ZFPL1 overexpression plasmid (OV). In Figure 9I Representative micrographs at higher magnification (×1,000) showing nuclear localization of pAKT (green).

[0037] Figure 10 Is a scatter plot presenting serum ZFPL1 and PSA profiles of healthy donors and patients with a definitive diagnosis of prostate cancer.

[0038] Figure 11It is an embodiment of the ZFPL1 immunosensor.

[0039] Figure 12A It is a graph showing Figure 11 the wavelength shift of ZFPL1 recorded by the immunosensor, and Figure 12B it is a graph showing Figure 11 the wavelength shift of BSA recorded by the immunosensor.

[0040] Figure 13A It is the calibration curve of ZFPL1 for the ELISA test, and Figure 13B it is Figure 11 the calibration curve of ZFPL1 for the immunosensor.

[0041] In Figure 14A , the left graph is the Receiver Operating Characteristic Curve for the predictability of ZFPL1 negative and positive for prostate cancer, and the right graph is the corresponding prediction curve for normal (0) versus prostate cancer (1). In Figure 14B , the left graph is the Receiver Operating Characteristic Curve for the predictability of PSA negative and positive for prostate cancer, and the right graph is the corresponding prediction curve for normal (0) versus prostate cancer (1). In Figure 14C , the left graph is the Receiver Operating Characteristic Curve for the predictability of ZFPL1+PSA negative and positive for prostate cancer, and the right graph is the corresponding prediction curve for normal (0) versus prostate cancer (1).

[0042] In Figure 15A , the left graph is the Receiver Operating Characteristic Curve for the predictability of ZFPL1 negative and positive for prostate cancer in the gray area, and the right graph is the corresponding prediction curve for normal (0) versus prostate cancer (1). In Figure 15B , the left graph is the Receiver Operating Characteristic Curve for the predictability of PSA negative and positive for prostate cancer in the gray area, and the right graph is the corresponding prediction curve for normal (0) versus prostate cancer (1).

[0043] Figure 16 It is a line graph showing the effect of the ZFPL1 monoclonal antibody on the PC3-CTR and DU145 cell lines.

[0044] Figure 17 It is a scatter plot of the tumor volume over time in the animal model treatment experiment.

[0045] Figure 18 It is a graph of representative images of tumors (untreated and treated) and their weights at autopsy. Detailed Implementation Modes

[0046] This document discloses detailed implementation modes of the present invention; however, it should be understood that the disclosed implementation modes are merely examples of the present invention, which can be embodied in various forms. Therefore, the specific structural and functional details disclosed herein should not be construed as restrictive, but only as a basis for the claims and a representative basis for teaching those skilled in the art to adopt the present invention differently in almost any appropriate detailed structure. Alternative implementation modes can be designed without departing from the spirit or scope of the present invention. In addition, the terms and phrases used herein are not intended to be restrictive; rather, they provide an understandable description of the present invention. Although this specification ends with claims that define the features of the present invention believed to be novel, it is believed that the present invention will be better understood by considering the following description in conjunction with the accompanying drawings, in which like reference numerals are used throughout.

[0047] As used herein, the term "a" is defined as one or more than one. As used herein, the term "plural" is defined as two or more than two. As used herein, the term "another" is defined as at least a second or more. The term "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or other elements inherent to such process, method, article, or apparatus. Without further limitation, an element followed by "comprising" does not exclude the presence of additional identical elements in the process, method, article, or apparatus that comprises the element. As used herein, the term "comprising", "having", or "characterized by" is defined as "including" (i.e., an open-ended term). As used herein, the term "coupled" is defined as connected, but not necessarily in a direct manner and not necessarily in a mechanical manner. As used herein, the term "about" or "approximately" applies to all numerical values, whether or not expressly indicated. These terms generally refer to a range of numbers that a person skilled in the art would consider equivalent to the recited value (i.e., having the same function or result). In many instances, these terms may include numbers rounded to the nearest significant digit. Relational terms such as first and second, top and bottom, right and left, etc. are only used to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0048] Discovery of Prostate Cancer Markers: Prostate cancer is the most common visceral cancer in men. The successful management of prostate cancer patients depends largely on the detection of cancer before metastasis. Although serum PSA screening has improved the detection of this disease at the early stage, it has been observed that this test is not reliable and expensive, repetitive, and invasive TRUS-guided biopsies must be used to confirm positive results. This is because PSA is a natural product of the normal prostate and is present in the serum of healthy individuals as well as cancer patients. Therefore, the inclusion of new markers that are only present in the prostate of cancer patients should enhance the specificity and accuracy of prostate cancer detection, thereby reducing the need for diagnostic biopsies.

[0049] The inventors have found that zinc finger protein-like 1 (ZFPL1) is a novel prostate tumor-specific protein that co-localizes with chromogranin A (a marker of neuroendocrine differentiation) and CD44 (a marker of cancer stem cells) in prostate cancer cells, indicating that ZFPL1 provides a measure of the neuroendocrine population of prostate tumors. Since the neuroendocrine as well as the stem cell phenotype is associated with castration-resistant metastatic cancer cells, the new marker should not only detect cancer at the early stage but also provide its future course, particularly the likelihood of its ability to grow rapidly and metastasize. In short, the data presented herein indicate that this new marker will help identify prostate cancer patients with an aggressive phenotype.

[0050] This new evidence demonstrates that the ZFPL1 immunoreactive cell population selectively localizes to the malignant portion of the prostate. The ZFPL1-positive cells increase with the increase in tumor grade and Gleason score. ZFPL1 is secreted into the blood via exosomes, and the serum ZFPL1 level in cancer patients is several-fold higher than that in age-matched normal individuals. Analysis of serum samples from more than 100 patients showed that ZFPL1 is more reliable than PSA in the detection of true prostate cancer and can distinguish cancer patients from those without cancer in the gray zone (patients showing serum PSA levels in the range of 4 - 10 ng / ml). These results demonstrate that, compared with PSA alone, the ZFPL1 test significantly increases the specificity, efficacy, and accuracy of prostate cancer detection.

[0051] Based on these findings, a ZFPL1-specific antibody was generated against the synthetic peptide GLGLPLIDEVVSPEPEPLNT (SEQ ID NO 10), which is one of the epitopes of ZFPL1. Then, a novel immunosensor-based assay for the diagnosis of prostate cancer was developed using this antibody. Finally, both in vitro and in vivo experiments were conducted to study the efficacy of the ZFPL1 antibody of the present invention as a treatment for prostate cancer. The research results showed that the antibody significantly slowed down the growth of tumors.

[0052] Discovery / Characterization of Neuroendocrine Markers: The study reported a significant upregulation of calcitonin (CT) and / or its receptor in malignant prostate. In addition, activation of the CT-CTR axis induced an invasive phenotype in benign prostate cells. In contrast, knockdown of CT / CTR induced loss of the invasive phenotype in invasive prostate cancer cells. To identify key factors associated with the increased tumorigenicity and metastatic capacity of prostate cancer cells induced by the CT-CTR axis, the present inventors identified nine CT-responsive genes from a prostate cancer cDNA library by subtractive hybridization technology (Table 1). Among them, the inventors further characterized a protein, ZFPL1, which was the most prevalent among the nine CT-responsive genes in the prostate cancer cDNA library:

[0053]

[0054] Table 1

[0055] Expression of ZFPL1 mRNA in Prostate Cancer Cell Lines: In Figure 1A the relative abundance of ZFPL1 mRNA was determined by RT-qPCR in multiple prostate cancer cell lines (LNCaP, PC3, PC-3M, and LNCaP-C4). Figure 1B Results normalized by GAPDH mRNA levels are shown. The abundance of ZFPL1 mRNA in PC cell lines was compared to that of PC3 (which was set to 1). Among the cell lines studied, the PC3-CTR, DU145, and PC3M cell lines showed comparable ZFPL1 mRNA levels, but they were higher than those of PC3 cells. In contrast, the abundance of ZFPL1 mRNA in LNCaP and LNCaP-C4 cells was significantly lower compared to that of PC3 cells. Note that the M1 cell line (which expresses a negative mutant CT receptor) showed the highest ZFPL1 mRNA abundance.

[0056] Now turning to Figure 1C to confirm the expression of ZFPL1 protein in prostate cancer cell lines and that the protein expressed in the prostate was the same size as in other organs, the presence of ZFPL1 protein in PC3-CTR cell lysates was studied. ZFPL1 immunoprecipitates were obtained and their molecular weight was determined by Western blot analysis. The ZFPL1 immunoprecipitate showed 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 the control, ordinary one-way ANOVA, and Tukey multiple comparison tests).

[0057] Regulation of ZFPL1 mRNA Expression by CT and Testosterone: Next turning to Figure 1D andFigure 1E , to confirm that ZFPL1 is a CT-induced gene, the effect of CT on ZFPL1 mRNA abundance was examined in the PC3-CTR and LNCaP-C4 cell lines. Cells were cultured overnight and treated with CT (1 - 100 nM) for 4 hours. RNA was extracted and reverse transcribed, and qRT-PCR was performed on ZFPL1. The bar graph represents the mean relative abundance ± SEM (n = 3) of ZFPL1 mRNA in LNCaP-C4 ( Figure 1D ) and PC3-CTR ( Figure 1E ) cells after treatment with CT at concentrations of 0, 5, 10, 50, and 100 nM. The results showed that CT induced a dose-dependent increase in ZFPL1 mRNA levels in both cell lines. The control was set to 1.0.

[0058] Since testosterone is the main hormone for the structural and functional integrity of the prostate, its effect on ZFPL1 expression was also examined. The same procedure as the CT test was used, except that cells were treated overnight with the testosterone agonist R1881 (10 mM - 10 nM). Figure 1F It was shown that the androgen receptor agonist R1881 induced a similar dose-dependent increase in ZFPL1 mRNA expression in the LNCaP-C4 cell line (mean ± SEM, n = 3). The same study was not performed in PC3-CTR cells because they lack androgen receptors. The control was set to 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, one-way ANOVA, and Tukey's multiple comparison test).

[0059] Expression of ZFPL1 in Normal Human Tissues: ZFPL1 immunofluorescence was performed on a TRP-1 microarray containing normal human tissue sections. The percentage of ZFLP1 immunopositive cells in each field of view (magnification ×400) was counted. The mean ± SEM (n = 6) percentage of the ZFPL1 immunopositive cell population in each field of view was plotted in Figure 2A . The symbol * indicates P < 0.05 (significantly different from normal prostate, one-way ANOVA, and Tukey's multiple comparison test). The results showed that ZFPL1 protein was expressed in cell populations of the brain, cerebellum, pancreas, and endometrium. However, ZFPL1 immunopositive cells were not detected in normal human prostate and several other human organs. Figure 2B Including Figure 2A Representative micrographs of ZFPL1-positive cell populations in various ZFPL1-positive organs of the experiment.

[0060] ZFPL1 mRNA in Normal and Malignant Prostates: To measure ZFPL1 mRNA abundance in normal and diseased prostate tissues, total RNA was extracted from frozen primary prostate specimens and used for RT-qPCR. Figure 2C is a bar graph presenting the relative abundance of ZFPL1 mRNA in normal, BPH, and prostate cancer specimens with different Gleason scores. The symbol * indicates P < 0.05 (significantly different from normal prostate, ordinary one-way ANOVA, and Tukey multiple comparison tests). The results showed that ZFPL1 mRNA was barely detectable in normal prostate and its level was slightly increased in BPH. However, in prostate cancer specimens, the increase in ZFPL1 mRNA level was significantly higher and statistically significant. In addition, the mRNA abundance in prostate cancer tissues increased with the increase in Gleason score of prostate cancer tumor specimens. For example, the ZFPL1 mRNA abundance in tumors with Gleason score 9 was more than 70-fold higher than that in normal prostate. These results suggest that the invasiveness of tumors can be predicted by the serum ZFPL1 level of patients. Since Gleason score assessment can be performed only by examining the patient's biopsy, the ZFPL1 test can provide a non-invasive alternative for evaluating the invasiveness of cancer. Figure 2D In, data from public portals such as TCGA and Oncomine also showed that the expression of ZFPL1 was increased in prostate cancer tissues compared with normal prostate tissues. The symbol * indicates P < 0.05 (significantly different from normal prostate, ordinary one-way ANOVA, and Tukey multiple comparison tests).

[0061] Expression of ZFPL1 mRNA in Clinical Prostate Specimens: ZFPL1 mRNA in several paraffin-embedded human prostate specimens was also examined by in situ hybridization (ISH) using digoxigenin 11-UTP-labeled ZFPL1 sense (non-specific binding) and antisense (specific binding) riboprobes. The specificity of the ISH method is shown in Figure 3A the micrographs of. (Scale bar = 100 μm). It was shown that in prostate cancer specimens, only the antisense ZFPL1 siRNA ( Figure 3A , right panel) hybridized with endogenous ZFPL1 mRNA, while the sense ZFPL1 siRNA ( Figure 3A , left panel) did not.

[0062] This technique was then applied to 78 prostate sections, which differed according to BPH, high-grade prostatic intraepithelial neoplasia (HGPIN), and prostate cancer with Gleason scores between 1 - 6 and 7 - 10. The processed sections were then observed under a Nikon Optiphot microscope and six or more digital micrographs were captured for each section. Representative micrographs of this experiment are shown inFigure 3B Medium. (Scale bar = 50 μm). The staining in digital micrographs (×400) was quantified by determining the stained area using the iImage Biovision image analysis program. The staining intensity was determined on a scale of 0 - 3 (0 = none, 1 = low, 2 = medium, and 3 = high). The IHC index was calculated by multiplying the stained area by the staining grade. From Figure 3B the images, it can be seen that the ZFPL1 transcript was undetectable in benign specimens, detectable in HGPIN specimens, and increased significantly with tumor progression. The quantitative data presented in Table 2 showed that the values were lowest in benign acini, increased significantly in HGPIN, and even more significantly in prostate cancer specimens with higher Gleason scores.

[0063]

[0064] a P < 0.05 indicates significant difference from the benign acini group;

[0065] b P < 0.05 indicates significant difference from the remaining groups. PC, prostate cancer.

[0066] * p < 0.05 (significantly different from benign acini, unpaired t - test).

[0067] Table 2

[0068] Expression of ZFPL1 in Prostate Tumors: Immunohistochemistry: Figure 3C ZFPL1 immunofluorescence in prostate tumors (left) and matched normal tissues (right) was compared. ZFPL1 protein expression (red) was cancer - specific and no staining was detected in the matched normal tissues. The nuclear stain was DAPI (blue). (Scale bar = 50 μm). Figure 3C Also included is a bar graph representing the mean percentage of ZFPL1 - immunopositive cells per field of view (magnification, ×400) in prostate cancer tissues tested against matched normal tissues (n = 6). The symbol * indicates P < 0.0001 (paired t - test). ZFPL1 protein was detected in cancer tissues in approximately 12% of total tumor cells, while none was positive in the matched normal tissues.

[0069] Localization of ZFPL1 in Cancer Tissues: To investigate whether ZFLP1 was localized to the histologically positive cancer regions of the specimens, H&E and ZFPL1 immunofluorescence were performed on consecutive sections of the same biopsy specimen. In Figure 3DAmong them, the representative micrograph on the left shows the H&E staining of a human prostate cancer tissue sample (dark blue staining is nuclear hematoxylin, and pink staining is eosin), while the micrograph on the right shows the green immunofluorescent labeling of ZFPL1 and the blue labeling of nuclear DAPI. (Scale bar = 50 μm). As Figure 3D indicated by the white arrow in

[0070] The ZFPL1-Immunoreactive Cell Population in Prostate Cancer Increases with Tumor Progression: The tumor stage-specific expression of ZFPL1 protein was examined by immunofluorescence of a US Biomax prostate cancer tissue microarray. The array contained sections of 80 specimens (73 PCs and 7 normals). Immunohistochemistry (IHC) was performed, and multiple fluorescent images of each specimen were captured. The number of ZFPL1 immunopositive cells (red - TRITC) and total cells (blue - DAPI) in each field of view (magnification, ×400) was counted, and the IHC index was determined as described above. Figure 3E is a representative micrograph revealing ZFPL1 immunopositive cells (red) and nuclear DAPI (blue) in different samples of a prostate cancer tissue microarray. (Scale bar = 50 μm). As Figure 3E can be seen in Figure 3F the ZFPL1 immunostaining is distributed in the cytoplasm of cells in the prostate tumor epithelium, but not in the normal prostate epithelium. In addition, with the increase of tumor stage, a significant increase in the number of immunopositive cells as well as the staining intensity was observed. Figure 3E The bar graph in

[0071] ZFLP1 Colocalizes with Chromogranin A (Neuroendocrine Marker) and CD44 (Cancer Stem Cell Marker): Fixed PC3 - CTR cells and sections of paraffin - embedded prostate cancer specimens were processed with primary antibody pairs against ZFLP1 + CgA or ZFPL1 + CD44 for double immunofluorescence. In Figure 4AAmong them, representative micrographs showed the co-localization of ZFPL1 and chromogranin A (CgA) in PC3-CTR cells (upper panel) and human prostate cancer tissues (lower panel). (Scale bar = 50 μm). The results showed that in cells and tissues, ZFPL1 (green) co-localized with CgA (red) in the same cells. Similarly, as Figure 4B shown in the representative immunofluorescence micrographs, ZFPL1 (green) co-localized with CD44 (red) in PC3-CTR cells (upper panel) and human prostate cancer tissues (lower panel). (Scale bar = 50 μm). The iVision image analysis program statistically evaluated the co-localization of the two fluorescent dyes in each digital image and calculated the Pearson's co-efficient (maximum value of 1.000). The co-localization data of CgA-ZFPL1 and CD44-ZFPL1 showed Pearson's co-efficient values of > 0.83 and > 0.8 (mean), respectively, indicating very strong co-localization of these three antigens in the same cells.

[0072] Subcellular Localization of ZFPL1 Protein in Cultured PC Cells: In cultured PC3-CTR and LNCaP-C4 cells, triple immunofluorescence was used to examine the subcellular localization of ZFPL1 (green) using markers for the Golgi GM130 (red), exosome CD81 (red), exosome-secretosome CD63 (red), and counterstaining of the nucleus with DAPI (blue). Figure 5A are representative micrographs showing the co-localization of ZFPL1 and exosome CD81 in PC3-CTR and LNCaP PC cells. Figure 5B are representative micrographs revealing the co-localization of ZFPL1 and the exosome / secretosome marker CD63. The cell boundaries were traced to show the localization of exosomes relative to the cells. The inset shows an enlarged image of the localization pointed by the arrow (magnification, ×1,000). Figure 5C are representative micrographs showing the co-localization of ZFPL1 and the Golgi marker GM130. (Scale bar = 25 μm). Figure 5A and Figure 5B The co-localization of ZFPL1 with CD81 and CD63 in Figure 5C suggests that ZFPL1 may be an exosomal protein. In addition,

[0073] In Figure 5DIn this study, the presence of ZFPL1 in exosomes was confirmed by separating the exosomal fractions of PC3-CTR cells and LNCaP-C4 cells and by Western blot analysis to verify its presence in the isolates. β-actin was used as a loading control. Co-precipitation of ZFPL1 with CD81 (an exosome marker) in the exosome isolates confirmed the presence of ZFPL1 in the exosomes of prostate cancer cell lines. Notably, the relative presence of ZFPL1 immunoreactivity was significantly higher in PC3-CTR cells than in LNCaP-C4 cells.

[0074] Function of ZFPL1 in Prostate Cancer Cells: To identify the potential role of ZFPL1 in prostate cancer progression, the effects of ZFPL1 knockdown and overexpression on prostate cancer cell characteristics such as cell proliferation, invasion, or apoptosis rates were examined. ZFPL1 overexpression was achieved by transfection with a constitutively active ZFPL1 expression plasmid. Knockdown was achieved by transfection with any one of three ZFPL1 siRNAs. β-actin was used as a housekeeping control. Knockdown ( Figure 6A ) and overexpression ( Figure 6B ) were verified by Western blot, and the protein bands were quantified by densitometry. The symbol * indicates P < 0.05. Figure 6A The results in Figure 6B showed that siRNA1 appeared to be the least effective in attenuating ZFPL1 expression, while siRNA3 appeared to be the most effective, and the latter was used in subsequent experiments unless otherwise specified.

[0075] 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-3 CTR cells receiving either nonsense siRNA or ZFPL1 siRNA. Data are presented as mean OD595 ± SEM (n = 4). The symbol * indicates P < 0.05 compared to the control receiving nonsense siRNA, 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 showed that knockdown of ZFPL1 in PC3-CTR cells led to a significant decrease in basal and CT-stimulated cell proliferation.

[0076] Effect of ZFPL1 Knockdown and Overexpression on Prostate Cancer Cell Apoptosis: Cell apoptosis in PC3-CTR and LNCaP cells was examined by immunofluorescence to analyze the presence of cleaved caspase-3 in the nucleus. Figure 7BRepresentative micrographs in Figure 7C demonstrate the effect of nonsense (control) or ZFPL1 siRNA (1, 2, or 3) ± CT on cleaved caspase-3 expression in PC3-CTR (upper panel) and LNCaP-C4 (lower panel) cells. DAPI staining in blue shows nuclei (scale bar = 100 μm). Results indicate that knockdown of ZFPL1 led to a marked increase in cleaved caspase-3 positive PC3-CTR cells. However, CT could significantly reverse or reduce this effect. The pooled data of these experiments are presented in Figure 7C . The graph presents the number of cleaved caspase-3 positive cells per field of view (magnification, ×400) for ± CT treatment. The symbol * indicates P < 0.05 compared to + CT of its own group, and the symbol ** indicates P < 0.001 compared to + CT of its own group. The symbol ^ indicates P < 0.05 compared to the corresponding nonsense siRNA control (one-way ANOVA and Tukey's multiple comparison test). Results show that knockdown of ZFPL1 by siRNA2 and 3 led to a significant increase in the number of cleaved caspase-3 positive cells in both cell lines, and CT could reverse / reduce this effect.

[0077] After treating the cells with / without DEX, the effect of ZFPL1 overexpression on DEX-induced apoptosis was examined. 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 vector plasmid. The next four pairs of micrographs reveal the expression of cleaved caspase-3 in cells overexpressing ZFPL1. The cells were also treated with vehicle, DEX (10 μM), CT (10 nM), or DEX + CT. DAPI staining is shown in blue. (Scale bar = 100 μm). Again, the results clearly reveal that treatment with CT and / or ZFPL1 overexpression significantly attenuated DEX-induced apoptosis in both cell lines. Figure 7E The pooled quantitative data of these experiments are shown. The mean number ± SEM of cleaved caspase-3 labeled cells per field of view (magnification, ×400) was plotted against treatment + CT ± DEX. Symbols The * indicates P < 0.05 compared to DEX + CT; the symbol x 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). Results show that ZFPL1 overexpression and / or treatment with CT significantly reduced the apoptotic population in both cell lines. In Figure 7FRepresentative micrographs show the localization of cleaved caspase-3 staining in the nuclei of LNCaP-C4 cells. (Scale bar = 25 μm). These results show that the cleaved caspase-3 staining in LNCaP-C4 cells is nuclear staining.

[0078] Effect of ZFPL1 Knockdown and Overexpression on Prostate Cancer Cell Invasion: Figure 8A Representative micrographs show the effect of ±10 nM CT on the invasiveness of PC3-CTR cells receiving nonsense siRNA or ZFPL1 siRNA (1, 2, or 3) (Scale bar = 50 μm). Knockdown of ZFPL1 significantly reduced the basal and CT-induced invasion of LNCaP-C4 and PC3-CTR cells. Figure 8B The bar graphs show the pooled data from these invasion assays, presented as the mean ± SEM of the number of invasive cells per field (magnification, ×400) in PC3-CTR and LNCaP-C4 cells receiving nonsense siRNA, siRNA1, siRNA2, or siRNA3. The symbol * indicates P < 0.05 when comparing -CT with +CT in each group, the symbol ** indicates P < 0.001, and the symbol *** indicates P < 0.0001. The symbol ^ indicates P < 0.01 when comparing nonsense siRNA with ZFPL1 siRNA, and the symbol ^^ indicates P < 0.001. All of the above P values were calculated using one-way ANOVA and Tukey's multiple comparison test.

[0079] In Figure 8C Representative micrographs show the effect of ±10 nM CT on the invasiveness of LNCaP-C4 and PC3-CTR cells expressing the vector pCMV5-XL4 plasmid or the plasmid with the ZFPL1 expression plasmid. The results show that overexpression of ZFPL1 in either cell line led to an increase in basal and CT-induced invasion. Figure 8D The bar graphs show the pooled data (mean ± SEM) from four independent invasion assays using PC3-CTR and LNCaP-C4 cells, respectively. The symbol * indicates P < 0.05 when comparing -CT with +CT in each group, the symbol ** indicates P < 0.001, and the symbol *** indicates P < 0.0001. The symbol ^ indicates P < 0.05 when comparing CT with OV+CT. All of the above P values were calculated using one-way ANOVA and Tukey's multiple comparison test.

[0080] Similar studies were also conducted to examine the cell migration of PC3-CTR cells in a wound healing assay. Figure 8E Micrographs in the upper left quadrant show the wounds in the PC3-CTR cell layer at 0 h and 12 h in the absence or presence of 10 nM CT.Figure 8E The upper right quadrant of Figure 8F shows a similar experiment of PC3-CTR cells with ZFPL1 knocked down by siRNA3. The results are presented as the mean ± SEM of the number of migrating cells in the wound (magnification, ×100) in four independent wound healing assays shown in the bar graph of

[0081] This summary data shows that CT promoted cell migration of PC3-CTR cells. However, when ZFPL1 was knocked down, the baseline cell migration decreased and CT was also unable to promote cell migration. Figure 8E The micrograph in the lower left quadrant of Figure 8E again shows that CT promoted cell migration in PC3-CTR cells. However, as shown in the lower right quadrant of Figure 8F , overexpression of ZFPL1 increased cell migration in the absence and presence of CT. The summary data in

[0082] ZFPL1 and Akt Phosphorylation also demonstrated that overexpression of ZFPL1 increased cell migration of PC3-CTR cells and the addition of CT increased it further. These results are consistent with the effect of ZFPL1 on prostate cell invasion. The symbol * indicates P < 0.05 compared to the control of each group (i.e., siRNA or overexp (overexpression)), and the symbol ^ indicates P < 0.05 for overexp compared to overexp+CT (one-way ANOVA and Tukey's multiple comparison test). Figure 9A and Figure 9B summarize the immunoblot results of the effects of ±10 nM CT on p-Akt473 and p-Akt308 proteins in PC3-CTR ( Figure 9A ) and LNCaP-C4 ( Figure 9B ) cells treated with nonsense (control) siRNA or ZFPL1 siRNA 1, ZFPL siRNA2, or ZFPL1 siRNA3. Total Akt was used as a control protein and β-actin was used as a loading control. The normalized density bar graphs (p-Akt / total Akt) of the immunoblots are also included in Figure 9A and Figure 9B . The symbol * indicates P < 0.05 compared to siRNA+CT, and the symbol ^ indicates P < 0.05 (one-way ANOVA and Tukey's multiple comparison test). Figure 9A and9B The data in 9B revealed that knockdown of ZFPL1 led to a statistically significant decrease in the basal and CT-induced phosphorylation of Akt473 / Akt308 in both cell lines. CT increased Akt phosphorylation; however, knockdown of ZFPL1 significantly reduced CT-induced Akt phosphorylation. Consistent with earlier results, this experiment also revealed that siRNA3 was the most effective in downregulating Akt phosphorylation in both cell lines.

[0083] Figure 9C and Figure 9D summarize the immunoblot results of the effects of ±10 nM CT on p-Akt473 and p-Akt308 proteins in PC3-CTR ( Figure 9C ) and LNCaP-C4 ( Figure 9D ) cells transfected with either vector plasmid or ZFPL1 expression plasmid, respectively. Akt was used as a control protein, and β-actin was used as a loading control. Figure 9C and 9D also include the normalized densitometry bar graphs (p-Akt / total Akt) of the immunoblots. 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 demonstrated a minimal increase in Akt473 phosphorylation in CT-induced LNCaP cells overexpressing ZFPL1, further supporting the possibility that the activation of endogenous CT in prostate cancer cells has an indirect effect on PI3K pathway activation and that ZFPL1 may be a key mediator of this CT action.

[0084] Phosphorylation of Akt was also observed by immunofluorescence microscopy. Figure 9E Representative micrographs in Figure 9E show the changes in p-Akt staining (green) of phosphorylated (p)-Akt-immunopositive PC3-CTR and LNCaP-C4 cells receiving either nonsense or ZFPL1 siRNA (1, 2, or 3) when treated with ±10 nM CT. Blue is DAPI (scale bar = 50 μm). In cells treated with nonsense siRNA, a small fraction of cells were p-Akt positive (<20%). When treated with 10 nM CT for 30 min, the p-Akt positive population more than doubled. When treated with ZFPL1 siRNA, the p-Akt cell population was lower than that of cells treated with nonsense siRNA. However, treatment with CT increased the p-Akt positive cells but still significantly less than in the case of cells treated with nonsense sRNA. As Figure 9FAs shown, the results of the quantitative data of these experiments using PC-3CTR and LNCaP-C4 cells indicated that knockdown of ZFPL1 significantly attenuated / abolished basal and CT-induced Akt phosphorylation. Data are presented as the mean ± SEM of the number of p-Akt immunopositive cells per field (magnification, ×100) of PC3-CTR and LNCaP cells that sequentially received either nonsense siRNA (control) or ZFPL1 siRNA 1, 2, or 3. The symbol * indicates P < 0.05 when comparing control with CT-treated cells in each group. The symbol ^ indicates P < 0.05 when comparing control with siRNA-treated cells (one-way ANOVA and Tukey's multiple comparison test).

[0085] Similar experiments examined the effect of ZFPL1 overexpression on basal and CT-induced increases in P-Akt in the nuclei of PC-3CTR and LNCaP-C4 cells. Figure 9G Representative micrographs show the effect of ±10 nM CT on the number of p-Akt immunopositive cells per field (magnification, ×400; green) in PC3-CTR cells expressing either vector plasmid or ZFPL overexpression plasmid. Scale bar = 50 μm. When treated with 10 nM CT, the number of P-Akt positive LNCaP-C4 cells increased by nearly 70%. A similar increase was identified when LNCaP-C4 cells were transfected with the ZFPL1 overexpression vector. When these cells (ZFPL1ov) were treated with 10 nM CT, the nuclear co-localization of p-Akt further increased by approximately 35%. As Figure 9H shown, the pooled quantitative data of PC3-CTR and LNCaP-C4 cells indicated that ZFPL1 and CT may have an additive effect on Akt phosphorylation. Data are presented as the mean p-Akt ICC index ± SEM per field (magnification, ×100). The symbol * indicates P < 0.05 when comparing +CT with OV+CT, and the symbol ^ indicates P < 0.05 when comparing C with OV. (One-way ANOVA and Tukey's multiple comparison test).

[0086] Next, it was verified whether pAkt in these cells was localized to the nucleus. In Figure 9I , representative micrographs at a higher magnification (×1,000) show the nuclear localization of pAKT (green). Nuclear DAPI is blue (scale bar = 25 μm). It was revealed that P-Akt (green) co-localized with DAPI at a magnification of ×400.

[0087] Generation of Monoclonal Antibodies against ZFPL1: To generate antibodies that bind to ZFPL1, the ZFPL1 protein was sequenced (SEQ ID NO 11) and its epitopes were determined (see Table 3). To enable the antibodies or fragments thereof of the present invention to specifically bind to the ZFPL1 protein or its variant proteins, the antibodies specifically bind 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 range of amino acids 62 to 77 and 127 to 284.

[0088] Number Epitope Sequence Localization SEQ ID 1 LLLGLLGFLALLALMS 269-284 12 2 SRETTRLVCYDLFHWA 62-77 13 3 EPLTHAPRKVYDTRDD 205-220 14 4 RTPGLHGDCDDDKYRR 222-237 15 5 SDPNLDPLMNPHIRVG 293-308 16 6 ALGWLARLLRSRAGSR 240-255 17 7 GLGLPLIDEVVSPEPEPLNT 131-150 10

[0089] Table 3

[0090] 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 the broadest sense in the present invention. Specifically, 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 sites of the antibody that exhibit the desired biological activity (e.g., binding to ZFPL1)).

[0091] The antibodies of the present invention are antibodies in which the light and heavy chain CDRs contain specific amino acid sequences such that the antibodies can selectively bind to ZFPL1, and include both monoclonal and polyclonal antibodies, preferably monoclonal antibodies. In addition, the antibodies of the present invention include all of chimeric antibodies, humanized antibodies and human antibodies, and are preferably human antibodies.

[0092] In the present invention, the term'monoclonal' refers to the property of an antibody obtained from a substantially homogeneous population of antibodies, and does not necessarily mean that the antibody must be produced by any particular method. For example, the monoclonal antibodies of the present invention can be produced by the hybridoma method first described by Kohler et al. (1975, Nature 256:495) or by recombinant DNA methods (U.S. Patent No. 4,816,567). It can also be isolated from phage antibody libraries using, for example, the 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).

[0093] In the preferred embodiments 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 binding ability of the secreted PA1623 antibody was tested by ELISA. The cross-reactivity of the PA1623 antibody with prostate secretions was then tested and found to be specific for ZFPL1. These methods for generating antibodies are well known in the art and can be readily replicated by those skilled in the art to generate antibodies that bind to other ZFPL1 epitopes presented in Table 3.

[0094] The PA1623 antibody is monoclonal and is of the immunoglobulin (Ig) G isotype. The antibody contains a variant Fc domain. Although the entire sequence of PA 1623 is disclosed in SEQ ID NO 9, those skilled in the art will recognize that portions of the sequence (outside of the complementary positions related to the ZFPL1 epitope) can vary and still be effective in the diagnostic and therapeutic methods disclosed below. In addition, the claimed diagnostic and therapeutic methods can also be carried out with antibodies that bind to any other epitope of ZFPL1 (see Table 3). Each of these antibodies will be monoclonal and of the immunoglobulin (Ig) G isotype. In addition, in order to perform their claimed functions, these antibodies will have an equilibrium dissociation constant (K D ) value of approximately 100 nM and a half-maximal inhibitory concentration (IC 50 ) of approximately 10 nM.

[0095] Development of an Immunosensor for the Diagnosis of Prostate Cancer: Exemplary embodiments of immunosensors incorporating the ZFPL1 antibody are depicted in Figure 11 and described below. The immunosensor shown is based on a gold-coated nano-AAO chip. A self-assembled monolayer (SAM) was prepared by performing a series of chemical reactions and then covalently linking 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-1-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 the novel anti-ZFPL1 primary antibody disclosed herein. In Figure 11In a specific embodiment depicted and used in the experiments described below, the PA1623 antibody was used. However, it should be understood that any other novel antibody described in this application can also be used. In addition, those skilled in the art will recognize that the specific methods used to establish the ZFPL1 optical immunosensor of the present invention are merely illustrative and can be easily modified to adapt to various situations.

[0096] Although optical immunosensors are employed in the preferred embodiments disclosed herein, those skilled in the art will recognize that many other assay methods can be used to determine the ZFPL1 level in a sample, including chemiluminescent assays, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, dot / slot blot assays. In addition, the claimed immunosensor includes a substrate to which the antibody of the present invention binds. The antibody can be bound to the substrate, for example, by passive adsorption, or can be chemically bound to the substrate, for example, by means of a covalent bond. Such covalent bonding generally requires the initial introduction of a chemically reactive compound covalently linked to the substrate surface prior to the addition of the antibody. The antibody itself may also require the addition of a chemical activation group to effect substrate bonding. These requirements are well known in the art. The substrate can be any medium capable of adsorbing or bonding the antibody, such as beads or nanoparticles (optionally chemically activated), but is preferably in a planar conformation (optionally chemically activated), such as a microtiter plate or a biochip. A biochip is a thin, planar, wafer-like substrate that can be made of any suitable material, such as glass or plastic, but is preferably made of ceramic. The biochip can be chemically activated prior to antibody bonding or is suitable for passive adsorption of the antibody.

[0097] Returning to Figure 11 the preferred immunosensor shown, the immunosensor is washed, blocked, and incubated with a known concentration of ZFPL1 peptide or an unknown serum sample for approximately one hour, and not less than 30 minutes. The chip is then washed thoroughly and dried. The readings are taken on an optical detector as follows: After the biochip has been functionalized and the antibody has been bound to the surface, a white light source is collimated by a lens to illuminate the chip. The transduction signal is the reflected optical interference fringe, which is detected by the optical detector. This reading serves as a blank. After the sample is applied to the sensor, due to the binding of the antibody and the biomarker, the transduction signal shifts and a different signal is reflected. The data of the peak in the range of 550 nm to 750 nm is used to measure the average shift. Figure 12 shows the wavelength shift recorded during the testing of the immunosensor of the present invention disclosed herein. In Figure 12A it, the solid line represents the reading of the blank with only the antibody, and the dashed line represents the reading of the chip to which ZFPL1 has been applied. In Figure 12BAmong them, the solid line represents the reading of the blank with only the antibody, and the dashed line represents the reading of the chip with bovine serum albumin (BSA) applied as a control. As can be seen from the graph in Figure 12, when using BSA, no optical shift was observed, but ZFPL1 produced a significant wavelength shift.

[0098] Now turning to Figure 13, the immunosensor was tested for ultrasensitivity and high specificity in detecting ZFPL1 and PSA in patient sera. The immunosensor-based assay is label-free. Unlabeled synthetic peptides of partial sequences were used as references for the two antigens. The accuracy, precision, recovery, and linearity of the assay were tested. In the range of 0.1 - 2 μl of serum, the dilution curve of human serum was parallel to the ZFPL1 standard curve. The tests included a negative sample pool (serum pool of patients who had undergone prostatectomy; serum PSA < 0.003 ng / ml) and a positive sample pool (serum pool of patients with prostate cancer confirmed by biopsy). Currently, the within-assay and between-assay variations of the assay were less than 5% and 9% respectively. To compare the efficacy of the immunosensor with ELISA, we compared the ZFPL1 calibration curves of the two systems. The sensitivity of the immunosensor ( Figure 13B ) was approximately 50 times higher than that of the corresponding non-equilibrium ELISA ( Figure 13A ). The assay was linear in the range of 1 - 64 pg, with a sensitivity of 1 pg / 50 μl. Thus, serum ZFPL1 levels can be measured in the form of as little as 1 pg ZFPL1 / mL serum. Therefore, when considered together with the simplicity of the method (label-free assay, short incubation period of 90 minutes), the currently disclosed immunosensor offers significant advantages over ELISA for ZFPL1 measurement.

[0099] Presence of ZFPL1 in the serum of prostate cancer patients: Since ZFPL1 is secreted by prostate cancer cells, its presence in the sera of healthy volunteers and prostate cancer patients was then examined. In the same cohort, PSA was used as a reference biomarker. The serum ZFPL1 and PSA profiles of healthy donors and patients clearly confirmed to have prostate cancer are presented in Figure 10Among them, the scatter plot shows that the serum ZFPL1 level of non-cancer individuals (control group; mean ± SEM 3.6 ± 0.286 ng / ml, n = 36) is significantly lower than that of all prostate cancer patients (cancer group: 11.41 ± 0.6135, n = 75), without overlap (P < 0.0001, unpaired t-test). In contrast, between non-cancer patients and cancer patients, the PSA levels showed significant overlap (control group: 6.26 ± 0.9, n = 37 vs. cancer group: 22.85 ± 2.96, n = 42, not significant by unpaired t-test). This obvious separation of ZFPL1 levels between non-cancer patients and cancer patients indicates that the ZFPL1 test will significantly improve the specificity of prostate cancer detection. In addition, this data indicates that a ZFPL1 concentration of approximately 3.3 ng / mL or higher is associated with a positive prostate cancer diagnosis.

[0100] Predictability of the ZFPL1 immunosensor: Next, the serum ZFPL1 and PSA levels of healthy donors (n = 119) and prostate cancer patients (n = 205) were measured by the immunoassay of the present invention, and the diagnostic potential of the two markers was quantified using the area under the receiver operating characteristic curve (AUC). These are also reflected in the prediction curves of normal (0) vs. prostate cancer (1). As Figure 14A shown, the AUC of ZFPL1 is 0.9788 (very close to the perfect 1.0), P < 0.0001. In addition, the average ZFPL1 values of normal individuals are significantly different from the corresponding values of prostate cancer patients. In contrast, looking at Figure 14B , the AUC value of PSA is 0.9055, and there is significant overlap between normal samples and prostate cancer samples. Finally, as Figure 14C shown, when the data of the two markers are combined, the AUC is 0.9793.

[0101] Tables 4 and 5 summarize the accuracy of immunoassays and diagnostic methods when testing ZFPL1 and PSA. The results show that the ZFPL1 immunoassay correctly predicted whether the serum was cancer-positive in 92.59% of cases, compared with an accuracy rate of only 84.88% for the PSA immunoassay, indicating that the ZFPL1 immunoassay significantly reduced inaccurate diagnoses. In addition to positive predictability, negative predictability is also important for the study because it can avoid unnecessary follow-up tests such as biopsies.

[0102]

[0103]

[0104] Table 4

[0105]

[0106] Table 5

[0107] Table 6 provides the mean ± SD of ZFPL1 and PSA levels in a stratified situation from the same cohort, as determined by the ZFPL1 immunosensor disclosed herein. ZFPL1 clearly differentiates prostate cancer from other prostate diseases and shows very high levels in cases of metastatic disease. This test can be used to monitor patients after treatment to check for tumor recurrence (biochemical recurrence or BCR).

[0108] Condition n PSA (ng / mL) ZFPL1 (ng / mL) Normal 61 1.795±2.141 1.204±2.143 BPH 10 5.396±3.629 3.096±2.244 Prostate cancer 79 15.30±38.18 59.09±6.912 BCR 11 3.885±7.253 5.615±6.912 Metastasis / death 3 1871±721.7 1661±742.4 Other cancers 44 8.295±4.858 10.72±5.712

[0109] Table 6

[0110] Predictability in the grey area: One of the drawbacks of using PSA for diagnosis is that PSA has high diagnostic sensitivity but relatively low specificity, which may lead to overdiagnosis and overtreatment of indolent prostate lesions. Specifically, when the PSA value ranges between 4 ng / mL and 10 ng / mL (also known as the "grey zone"), only 22% of patients have a positive prostate biopsy. To study the effectiveness of the ZFPL1 test in the grey zone, ROC curves of ZFPL1 and PSA levels in patients with serum levels within the grey zone were prepared. The results showed that the predictability of PSA ( Figure 15B ) in this sample population was very low, with an AUC value of 0.770, while the AUC value of the predictability of ZFPL1 ( Figure 15A ) in the same population was 0.9672. The ZFPL1 immunosensor correctly predicted that 19 out of 23 patients did not have cancer and they did not need to undergo biopsy / treatment, with an NPV of 82.6. The PPV of the ZFPL1 test was 95.5, correctly predicting that 87 out of 88 patients did have cancer.

[0111] Effect of the ZFPL1 monoclonal antibody on the growth of prostate cancer cells: To examine the potential of the ZFPL1 antibody as a druggable target, 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 PA1623 ZFPL1 antibody. PC3-CTR and DU-145 were selected because they are able to grow rapidly in androgen-independent conditions. Cell growth was measured by the optical density (OD 600 ) at 600 nm, and the results are summarized in Figure 16Among them. Compared with the cells not treated with the ZFPL1 antibody, both cell lines showed reduced cell growth at all treatment doses. The results indicate that the ZFPL1 antibody has a potent dose-related inhibitory effect on the growth of PC3-CTR and DU-145 cells. The inhibition of the growth of these highly invasive cell lines by the antibody shows promising potential for using the antibody as an effective therapeutic agent for castration-resistant prostate cancer.

[0112] Effect of the ZFPL1 monoclonal antibody as a treatment in an animal model: Next, a follow-up experiment was conducted to test the effectiveness of the ZFPL1 antibody as a treatment in an animal model. In this study, a suspension of PC3-CTR cells - Matrigel TM mixture (1:1 v / v) was injected into the flanks of adult nude mice weighing approximately 30 g. Then the mice were observed daily. Tumors could be observed 11 - 14 days after cell implantation. Approximately two weeks after this implantation, observations, treatment with the PA1623 ZFPL1 antibody, and measurement of tumor volume with calipers were started. Infected mice were not treated ("untreated" group), received a saline solution ("control" group), received 1 μl of the antibody solution (containing 4.5 μg of the ZFPL1 antibody), or received 2 μl of the antibody solution (containing 9 μg of the ZFPL1 antibody). The ZFPL1 antibody was injected intratumorally every Monday and Thursday until the end of the study. The "untreated" group remained unchanged after implantation of PC3-CTR cells, while the "control" group was injected with saline according to the same schedule as the ZFPL1 antibody injection in the other groups.

[0113] Figure 17 is a scatter plot of the tumor volume on the 15th day after cell implantation. The results show that treatment with 4.5 μg of the ZFPL1 antibody significantly slowed tumor growth, and at the higher dose of 9 μg, tumor growth inhibition was even more significant. Figure 18 A figure including representative images of the tumors (untreated and treated) and their weights at autopsy. Significant differences in the appearance and texture of the tumors were observed. For example, the tumors of untreated mice were extremely hard, as expected. In contrast, the tumors of treated mice were very soft and had a strong odor of anesthetized tissue. These results demonstrate the effectiveness of the ZFPL1 antibody in slowing tumor growth and thus treating prostate cancer.

[0114] Based on all of the results presented above, a preferred embodiment for human treatment is expected to involve administration of 0.5 - 5.0 mg antibody / kg body weight. The treatment is preferably administered intraperitoneally, but in other embodiments, it can be administered intratumorally, intravenously, or subcutaneously. Since the half-life of the antibody in humans is typically 3 - 4 weeks, the treatment is preferably administered approximately every 2 - 3 days for one week, followed by no treatment for approximately three weeks. This process can be repeated until the ZFPL1 and / or PSA levels of the patient reach normal levels, or until the size of the patient's tumor has been reduced to the extent that it can be surgically removed.

[0115] Administration of the monoclonal antibody as described herein may result in inhibition of tumor growth, tumor regression, reduction in tumor size, reduction in the number of tumor cells, delay in 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, increase in progression-free survival, increase in overall survival, complete remission, partial remission, and / or disease stabilization. This treatment method can be combined with one or more of surgery, radiation, chemotherapeutic agents, cancer vaccines, antibody-drug conjugates, anti-inflammatory drugs, dietary supplements, or any other treatment for prostate cancer and / or its symptoms that is currently known or may be developed in the future.

[0116] The foregoing description and the drawings illustrate the principles, exemplary embodiments, and modes of operation of the invention. However, the invention should not be construed as limited to the specific embodiments discussed above. Many modifications of the embodiments described herein will occur to those skilled in the art in light of the foregoing description and the teachings presented in the related drawings. Accordingly, it is to be understood that those skilled in the art can make changes to those embodiments without departing from the scope of the invention.

Claims

1. A method for diagnosing and treating prostate cancer in a mammalian patient, the method comprising: a. obtaining a serum sample from the patient; b. detecting the presence of zinc finger protein-like 1 (ZFPL1) in the serum sample; c. diagnosing the patient as having prostate cancer when ZFPL1 is detected in the serum sample; and d. administering to the diagnosed patient an effective amount of an anti-ZFPL1 antibody.

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

3. The method according to claim 1, wherein the monoclonal 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.

4. The method according to claim 1, wherein the monoclonal antibody comprises: a. a heavy chain variable region (HCVR) comprising heavy chain complementarity determining regions (CDRs), the heavy chain CDRs comprising SEQ ID No:1, SEQ ID No:2 and SEQ ID No:3; and b. a light chain variable region (LCVR) comprising light chain CDRs, the light chain CDRs comprising SEQ ID No:4, SEQ ID No:5 and SEQ ID No:

6.

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

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

7. The method according to claim 1, wherein the antibody has an equilibrium dissociation constant (K D ) value of 100 nM and a half maximal inhibitory concentration (IC 50 ) of 10 nM.

8. The method according to claim 1, wherein the patient is diagnosed as having prostate cancer when the serum sample has a ZFPL1 level of greater than or equal to 3.3 ng / mL.

9. The method according to claim 1, wherein the monoclonal antibody treatment is administered intravenously, subcutaneously or intraperitoneally.

10. The method according to claim 9, wherein the monoclonal antibody treatment is administered intraperitoneally.

11. The method according to claim 1, wherein 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 the number of tumor cells, delay in 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, increase in progression-free survival, increase in overall survival, complete remission, partial remission and disease stabilization.

12. The method according to claim 1, further comprising administering an additional therapeutic agent or therapy to the patient, wherein the additional therapeutic agent or therapy is selected from the group consisting of surgery, radiation, chemotherapeutic agents, cancer vaccines, antibody-drug conjugates, anti-inflammatory drugs, and dietary supplements.

13. A method of treating prostate cancer in a patient, the method comprising administering an effective amount of an anti-ZFPL1 antibody to a patient having prostate cancer.

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

15. The method according to claim 13, wherein the anti-ZFPL1 antibody binds to at least four nucleotides within the following nucleotide positions 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。 16. The method according to claim 13, wherein the anti-ZFPL1 antibody binds to at least four nucleotides within nucleotide positions 62-77, 127-284, or 293-308 in SEQ ID NO:

11.

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

11.

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

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

20. The method according to claim 13, wherein the anti-ZFPL1 antibody is an isolated monoclonal antibody or an antigen-binding portion thereof, comprising: a. an HCDR1 comprising an amino acid sequence having an amino acid substitution at the 2nd amino acid, the 4th amino acid, or both the 2nd amino acid and the 4th amino acid of SEQ ID NO: 1; b. an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; c. an HCDR3 comprising an amino acid sequence having an amino acid substitution at the 8th amino acid of SEQ ID NO: 3; d. an LCDR1 comprising an amino acid sequence having an amino acid substitution at the 8th amino acid, the 11th amino acid, the 13th amino acid, or all of these amino acids of SEQ ID NO: 4; e. an LCDR2 comprising an amino acid sequence having an amino acid substitution at the 4th amino acid of SEQ ID NO: 5; and f. an LCDR3 comprising an amino acid sequence having an amino acid substitution at the 4th amino acid, the 7th amino acid, or both the 4th amino acid and the 7th amino acid of SEQ ID NO:

6. g. and wherein said antibody or antigen-binding portion thereof binds to the polypeptide of SEQ ID NO: 11 with a K of 100 nM or less D binds to the polypeptide of SEQ ID NO: 11.

Images