Novel methods and compounds for diagnosing prostate cancer

Abstract

The present disclosure relates to a human antibody or its antigen-binding fragment capable of binding to prostasomes, and the diagnosis and prognosis of prostate cancer. More specifically, the proposed technology relates to a method for diagnosing prostate cancer using a human antibody or its antigen-binding fragment for detecting prostasomes in body fluids. The present disclosure includes a human antibody capable of specifically and selectively detecting prostasomes in body fluids, and a method for diagnosing prostate cancer using the human antibody. The present disclosure further includes providing a prognosis for prostate cancer, assessing the severity, and determining the effectiveness of medical treatment for prostate cancer.

Description

[Technical Field]

[0001] The present disclosure relates to a human antibody or its antigen-binding fragment capable of binding to prostasomes, and the diagnosis and prognosis of prostate cancer. More specifically, the proposed technology relates to a method for diagnosing prostate cancer using a human antibody or its antigen-binding fragment for detecting prostasomes in body fluids. The present disclosure includes a human antibody capable of specifically and selectively detecting prostasomes in body fluids, and a method for diagnosing prostate cancer using the human antibody. The present disclosure further includes providing a prognosis for prostate cancer, assessing the severity, and determining the effectiveness of medical treatment for prostate cancer. [Background technology]

[0002] Cancer is one of the most common fatal diseases and, despite recent advances in diagnosis and treatment, still causes a significant number of deaths each year. As an example, prostate cancer is the most common cancer disease in men and presents with symptoms (such as urinary problems or bone pain) due to local tumors or metastatic spread of tumors. Moreover, the disease is often at an advanced stage at the time of diagnosis.

[0003] Prostate-specific antigen (PSA) measurement has changed the pattern of prostate cancer diagnosis, with more cases detected at early stages and fewer at advanced stages. However, serum PSA is not an ideal diagnostic marker because it is not a specific marker for prostate cancer in serum, and therefore is not suitable for prostate cancer screening. The PSA test cannot distinguish between benign prostatic hyperplasia and early-stage prostate cancer, and furthermore, it cannot distinguish between prostate cancer with a high metastatic potential (invasive prostate cancer) and prostate cancer that is not invasive or has a low risk of developing (indolent prostate cancer).

[0004] Cells have previously been shown to secrete vesicles called exosomes, which are released when multivesicular bodies fuse with the plasma membrane. These vesicles are primarily derived from membrane rafts in the source cell and possess a protein configuration specific to the cell of origin. Exosomes are present in many eukaryotic body fluids, including blood, urine, and cell culture medium. Exosomes contain various molecular components (including proteins and RNA) of their source cell. Although the protein composition of exosomes varies depending on the cell and tissue of origin, most exosomes contain a common, evolutionarily conserved set of protein molecules. The protein content of a single exosome is approximately 20,000 molecules, which may represent its cell of origin. Thus, by examining the protein content, it may be possible to determine the cell type that secreted the exosome. Exosomes have also been shown to contain double-stranded DNA. Evidence further suggests that exosomes have specialized functions and play important roles in processes such as coagulation, intercellular signaling, and waste management. As a result, there is growing interest in the clinical applications of exosomes, which could potentially be used as prognosticators for therapy and biomarkers for health and disease.

[0005] Submicron membrane vesicles have previously been found to be secreted by prostate acinar cells and enter semen. These submicron membrane vesicles are a type of exosome, called prostasomes. The altered tissue architecture in malignant tumors makes it easier for prostasomes to be released into the interstitial space rather than the acinar space of the prostate. Therefore, prostasomes also leak into the external bloodstream. It has been demonstrated that malignant prostate cells secrete prostasomes and that the presence of malignant prostate tumors increases the number of prostasomes present in peripheral blood (Tavoosidana et al. PNAS, May 24, 2011, vol. 108, no. 21, 8809-8814). Therefore, it is speculated that prostasomes may be a potential biomarker for prostate cancer. Summary of the Invention

[0006] It is an object of the present disclosure to provide a method and an antibody or fragment thereof that seeks to mitigate, alleviate, or eliminate the above-mentioned deficiencies and disadvantages in the prior art, singly or in any combination. This object is achieved by a novel antibody or antigen-binding fragment thereof (including synthetic fragments) for use in determining the level of prostasomes in a body fluid sample of the subject in a subject in need thereof.

[0007] In one aspect, a human monoclonal antibody or antigen-binding fragment thereof (including a synthetic fragment) that selectively binds to a prostasome is provided. In some aspects, the human monoclonal antibody or antigen-binding fragment thereof is an antigen-binding (Fab) fragment or an antigen-binding single-chain Fv (scFv) fragment (such as a human synthetic scFv fragment).

[0008] In a further aspect, the human monoclonal antibody or antigen-binding fragment thereof selectively binds to prostasomes by binding to one or more prostasome surface antigens selected from the group consisting of SEQ ID NOs: 60-104 as set forth in Table 3.

[0009] In some aspects, the human monoclonal antibody or antigen-binding fragment thereof selectively binds to prostasomes by binding to multiple prostasome surface antigens, ie, the human monoclonal antibody or antigen-binding fragment thereof selectively binds to prostasomes by binding to multiple prostasome surface antigens, where the prostasome surface antigens form clusters on the surface of the prostasome membrane, and the clusters are identified by the monoclonal antibody or antigen-binding fragment thereof and bound to by the monoclonal antibody or antigen-binding fragment thereof.

[0010] In one embodiment, the human monoclonal antibody or antigen-binding fragment thereof binds to a protein / antigen mass comprising at least five antigens selected from Table 11. For each of scFv fragments 1-12, at least five proteins / antigens are present in the bound mass, as defined for each fragment in Table 11.

[0011] In some aspects, the human monoclonal antibody or antigen-binding fragment thereof enables a sensitivity of at least 10 nanograms per milliliter (ng / mL) in an immunoassay using the antibody or antigen-binding fragment thereof as a capture antibody.

[0012] In some embodiments, the human monoclonal antibody or antigen-binding fragment thereof comprises at least six complementarity determining regions (CDRs) in any combination of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and the selection of the CDRs is determined by: CDR-H1 selected from SEQ ID NOs: 25, 27, and 28; CDR-H2 selected from SEQ ID NOs: 26, 29, and 30; a CDR-H3 selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; CDR-L1 selected from SEQ ID NOs: 56 and 57; CDR-L2 selected from SEQ ID NOs: 58 and 59; The CDR-L3 is selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24.

[0013] In a related embodiment, the human monoclonal antibody or antigen-binding fragment thereof may comprise at least six CDRs as described above, wherein CDR-H1, CDR-H2, and CDR-H3 are contained in a heavy chain variable region (VH) sequence selected from the group consisting of SEQ ID NOs: 32-43 and sequences that are 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95% or more identical) thereto, and CDR-L1, CDR-L2, and CDR-L3 are contained in a light chain variable region (VL) sequence selected from the group consisting of SEQ ID NOs: 44-55 and 56-59 and sequences that are 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95% or more identical) thereto.

[0014] In some embodiments, the human monoclonal antibody or antigen-binding fragment thereof comprises at least four complementarity determining regions (CDRs) in any combination of CDR-H1, CDR-H2, CDR-H3, and CDR-L3, and the selection of the CDRs is determined by: CDR-H1 selected from SEQ ID NOs: 25, 27, and 28; CDR-H2 selected from SEQ ID NOs: 26, 29, and 30; a CDR-H3 selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; The CDR-L3 is selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23, and CDR sequences that are 95% or more identical thereto (e.g., 96%, 97%, 98%, 99% or more).

[0015] In a related embodiment, the human antibody or antigen-binding fragment thereof may comprise at least four CDRs as described above, wherein CDR-H1, CDR-H2, and CDR-H3 are contained in a heavy chain variable region (VH) sequence selected from the group consisting of SEQ ID NOs: 32 to 43 and sequences that are 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95% or more identical) thereto, and CDR-L3 is contained in a light chain variable region (VL) sequence selected from the group consisting of SEQ ID NOs: 44 to 55 and sequences that are 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95% or more identical) thereto.

[0016] In one embodiment, the human monoclonal antibody or antigen-binding fragment thereof i) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 4, and CDR-L3 defined by SEQ ID NO: 16 an scFv fragment having ii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 7, and CDR-L3 defined by SEQ ID NO: 19 an scFv fragment having iii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 1, and CDR-L3 defined by SEQ ID NO: 13 an scFv fragment having iv) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO:2, and CDR-L3 defined by SEQ ID NO: 14 an scFv fragment having v) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 3, and CDR-L3 defined by SEQ ID NO: 15 an scFv fragment having vi) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 5, and CDR-L3 defined by SEQ ID NO: 17 an scFv fragment having vii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 6, and CDR-L3 defined by SEQ ID NO: 18 an scFv fragment having viii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 8, and CDR-L3 defined by SEQ ID NO: 20 an scFv fragment having ix) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 9, and CDR-L3 defined by SEQ ID NO: 21 an scFv fragment having x) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 10, and CDR-L3 defined by SEQ ID NO: 22 an scFv fragment having xi) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 11, and CDR-L3 defined by SEQ ID NO: 23 an scFv fragment having xii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 12, and CDR-L3 defined by SEQ ID NO: 24 The synthetic scFv fragment is selected from the group comprising the scFv fragment having the following structure:

[0017] In one embodiment, the human antibody or antigen-binding fragment thereof is a synthetic scFv fragment comprising the above CDRs, the fragment having a variable heavy chain VH and a variable light chain VL connected via a linker, the fragment comprising: i) a VH defined by SEQ ID NO: 35; VL defined by SEQ ID NO: 47 an scFv fragment having ii) a VH defined by SEQ ID NO: 38; VL defined by SEQ ID NO: 50, an scFv fragment having iii) a VH defined by SEQ ID NO: 32; VL defined by SEQ ID NO: 44, an scFv fragment having iv) a VH defined by SEQ ID NO: 33; VL defined by SEQ ID NO: 45, an scFv fragment having v) VH defined by SEQ ID NO: 34; VL defined by SEQ ID NO: 46, an scFv fragment having vi) VH defined by SEQ ID NO: 36; VL defined by SEQ ID NO: 48, an scFv fragment having vii) VH defined by SEQ ID NO: 37; VL defined by SEQ ID NO: 49, an scFv fragment having viii) VH defined by SEQ ID NO: 39; VL defined by SEQ ID NO: 51, an scFv fragment having ix) a VH defined by SEQ ID NO: 40; VL defined by SEQ ID NO: 52, an scFv fragment having x) a VH defined by SEQ ID NO: 41; VL defined by SEQ ID NO: 53, an scFv fragment having xi) a VH defined by SEQ ID NO: 42; VL defined by SEQ ID NO: 54, an scFv fragment having xii) VH defined by SEQ ID NO: 43; VL defined by SEQ ID NO: 55, and further selected from the group comprising an scFv fragment having

[0018] In one aspect, an in vitro method for determining whether prostate cancer is present in a subject is provided, the method comprising: providing a human monoclonal antibody or antigen-binding fragment thereof that selectively binds to prostasomes; reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from the subject; detecting any prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof to obtain a prostasome level; comparing the prostasome level with a predetermined threshold; and determining that prostate cancer is present in the subject if the detected prostasome level is higher than the predetermined threshold.

[0019] In a further aspect, detecting any prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof comprises detecting the prostasomes using an anti-prostasome detection antibody or antigen-binding fragment thereof.

[0020] In yet another aspect, the method is a sandwich immunoassay, and detecting prostasomes bound by the human monoclonal antibody or its antigen-binding fragment comprises detecting the anti-prostasome detection antibody using an additional detection antibody, wherein the human monoclonal antibody or its antigen-binding fragment is a capture antibody, the anti-prostasome detection antibody is a primary detection antibody, and the additional detection antibody is a secondary detection antibody.

[0021] In one aspect, an in vitro method for providing a prognosis for prostate cancer in a subject in need thereof is provided, the method comprising: providing a human monoclonal antibody or antigen-binding fragment thereof that selectively binds to prostasomes; reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from the subject; detecting the prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof to obtain a prostasome level; comparing the prostasome level with first and second predetermined thresholds; and providing the prognosis for prostate cancer, wherein a poor prognosis for prostate cancer is defined when the detected prostasome level is higher than the first predetermined threshold, and a good prognosis is defined when the detected prostasome level is lower than the second predetermined threshold.

[0022] In one aspect, an in vitro method for assessing the severity of prostate cancer in a subject in need thereof is provided, the method comprising: providing a human monoclonal antibody or antigen-binding fragment thereof that selectively binds to prostasomes; reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from the subject; detecting prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof to obtain a prostasome level; comparing the prostasome level with first and second predetermined thresholds; and assessing the severity of the prostate cancer, wherein the prostate cancer is assessed as severe if the detected prostasome level is higher than the first predetermined threshold, as moderate if the detected prostasome level is lower than the first predetermined threshold but higher than a second predetermined threshold, or as mild if the detected prostasome level is lower than the second predetermined threshold.

[0023] In one aspect, an in vitro method for evaluating the effectiveness of prostate cancer treatment in a subject in need thereof is provided, the method comprising: detecting the level of prostasomes in a sample from the subject before prostate cancer treatment; administering an anti-prostate cancer treatment to the subject; detecting the level of prostasomes in a sample from the subject after the prostate cancer treatment; comparing the prostasome level before the treatment with the level after the treatment; and determining the effectiveness of the treatment, wherein if the prostasome level after the treatment is reduced compared to the level before the treatment, the treatment is determined to be effective, and if the prostasome level remains the same or increases, the treatment is determined to be ineffective. In some aspects, detecting the level of prostasomes in a sample from the subject comprises providing a human monoclonal antibody or antigen-binding fragment thereof that selectively binds to prostasomes; reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from the subject; detecting the prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof to obtain the level of prostasomes.

[0024] In some embodiments, the sample from the subject is a bodily fluid sample and is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and a cell suspension.

[0025] In some embodiments, the predetermined threshold used in a diagnostic method is 10 ng of prostasomes per mL of bodily fluid sample, a first predetermined threshold is 10 ng of prostasomes per mL of bodily fluid sample, and a second predetermined threshold is 1 ng of prostasomes per mL of bodily fluid sample, and used in a method for providing a prognosis or severity of prostate cancer disease.

[0026] In some aspects, the human monoclonal antibody or antigen-binding fragment thereof used in the above methods is an antibody or antigen-binding fragment thereof described above.

[0027] In another aspect, there is provided an antibody or antigen-binding fragment thereof as described above for use in the above method.

[0028] It should be noted that the invention relates to all possible combinations of the features recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0029] Other objects and advantages will become apparent to those skilled in the art from a review of the following detailed description taken in conjunction with the following illustrative drawings and the appended claims.

[0030] A feature described with respect to one aspect may also be incorporated into other aspects, and the advantages of that feature may apply to any aspect in which it is incorporated.

[0031] Other objects, features, and advantages of the inventive concepts will become apparent from the following detailed disclosure, the appended claims, as well as the drawings.

[0032] In general, all terms used in the claims should be interpreted according to their ordinary meaning in the art, unless expressly stated otherwise herein. Moreover, the use of terms such as "first," "second," etc. herein does not denote any order, quantity, or importance, but rather is used to distinguish one element from another. Any reference to "one / one / the [element, apparatus, component, means, step, etc.]" should be interpreted broadly as a reference to at least one instance of that element, apparatus, component, means, step, etc., unless expressly stated otherwise. The steps of any method disclosed herein need not be performed in the exact order disclosed, unless expressly stated otherwise. [Brief explanation of the drawings]

[0033] The above objects, features, and advantages of the present invention, as well as additional objects, features, and advantages of the present invention, will be better understood through the illustrative and non-limiting description of various embodiments of the inventive concept with reference to the accompanying drawings.

[0034] [Figure 1] FIG. 1 shows one representative assay using the antibodies of the present invention. [Figure 2] FIG. 2 shows an scFv fragment of the present invention. [Figure 3a] FIG. 3 shows an example of the design of a HeIL-11 synthetic scFv library and a HeIL-13 synthetic scFv library, where FIG. 3a shows the CDRs from Example 1 and FIG. 3b shows the amino acid content of those CDRs. [Figure 3b] FIG. 3 shows an example of the design of a HeIL-11 synthetic scFv library and a HeIL-13 synthetic scFv library, where FIG. 3a shows the CDRs from Example 1 and FIG. 3b shows the amino acid content of those CDRs. [Figure 4a] Figure 4 shows the construction of the HeIL-11 and HeIL-13 libraries. Figure 4a shows the amino acid sequence of the HeIL library scaffold, and Figure 4b shows the specific positions that contribute diversity (X) in the design of VH and VL of the HeIL-11 and HeIL-13 antibody libraries. [Figure 4b] Figure 4 shows the construction of the HeIL-11 and HeIL-13 libraries. Figure 4a shows the amino acid sequence of the HeIL library scaffold, and Figure 4b shows the specific positions that contribute diversity (X) in the design of VH and VL of the HeIL-11 and HeIL-13 antibody libraries. [Figure 5] FIG. 5 shows a flowchart of the disclosed method for diagnosing prostate cancer in a subject. [Figure 6] FIG. 6 shows a flowchart of the disclosed method for predicting the prognosis of prostate cancer in a subject. [Figure 7] FIG. 7 shows a flowchart of the disclosed method for assessing the severity of prostate cancer in a subject. [Figure 8] FIG. 8 shows a flowchart of a method of the present disclosure for determining the efficacy of a prostate cancer treatment in a subject.

[0035] The drawings are not necessarily to scale and generally show only those parts necessary to clarify the concept of the invention, while other parts may be omitted or only suggested. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present disclosure relates to novel monoclonal antibodies or antigen-binding fragments thereof that selectively bind to prostasomes, and the diagnosis and prognosis of prostate cancer using these monoclonal antibodies or fragments specific for prostasomes. Aspects of the disclosure will be more fully described below with reference to the accompanying drawings. However, the antibodies and methods disclosed herein can be embodied in many different forms and should not be construed as limited to the aspects shown herein. Like numbers in the drawings refer to like elements throughout.

[0037] The terminology used herein is for the purpose of describing particular aspects of the present disclosure only and is not intended to be limiting of the present disclosure. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. First, some terms may be defined to clarify the following disclosure.

[0038] In some embodiments, the non-limiting terms "antibody" or "antigen-binding fragment thereof" are used. The term "antibody" is used herein in its broadest sense and includes both monoclonal and polyclonal antibodies. As is well known, an antibody is an immunoglobulin molecule that can specifically bind to a target (antigen) (protein, carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. The human monoclonal antibody, typically a phage-displayed antibody, herein can be any type of human antibody or human antigen-binding fragment of an antibody that can selectively bind to prostasomes.

[0039] As used herein, the term "antibody" or "antigen-binding fragment thereof" encompasses not only full-length or intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fab3, Fv, and variants thereof), fusion proteins containing one or more antibody moieties, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of immunoglobulin molecules that contain the required antigen recognition site with specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies. A full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). Antibodies are assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are six major classes of antibodies: IgA, IgD, IgE, IgG, IgM, and IgY, some of which can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgG1, and IgG2. The term "full-length antibody" as used herein refers to any class of antibody, such as IgD, IgE, IgG, IgA, IgM, or IgY (or any subclass thereof). The subunit structures and three-dimensional configurations of different classes of antibodies are well known. The term "antigen-binding fragment" refers to a portion or region of an antibody molecule or a derivative thereof that retains all or a significant portion of the antigen-binding ability of the corresponding full-length antibody. An antigen-binding fragment can include the heavy chain variable region (VH), the light chain variable region (VL), or both. Each of the VH and VL typically contains three complementarity determining regions, CDR1, CDR2, and CDR3, with the CDRs from the VH domain designated CDR-H1, CDR-H2, and CDR-H3, and the CDRs from the VL domain designated CDR-L1, CDR-L2, and CDR-L3.The three CDRs in a VH or VL are flanked by framework regions (FR1, FR2, FR3, and FR4). As briefly listed above, non-limiting examples of antigen-binding fragments include: (1) Fab fragments (monovalent fragments having VL-CL chains and VH-CH chains); (2) Fab' fragments (Fab fragments having heavy chain hinge regions); (3) F(ab')2 fragments (dimers of Fab' fragments, e.g., with joined heavy chain hinge regions linked by disulfide bridges at the hinge regions); (4) Fc fragments; (5) Fv fragments (the minimum antibody fragments having the VL and VH domains of a single arm of an antibody); (6) single-chain Fv (scFv) fragments (a single polypeptide chain in which the VH and VL domains of an scFv are linked by a peptide linker); (7) (scFv)2 (comprising two VH domains and two VL domains, with the two VH domains associated via disulfide bridges), and (8) domain antibodies (which can be single variable domain (VH or VL) polypeptides of an antibody that specifically binds to an antigen). Antigen-binding fragments can be prepared by conventional methods. For example, F(ab')2 fragments can be generated by pepsin digestion of full-length antibody molecules, and Fab fragments can be generated by reducing disulfide bridges in F(ab')2 fragments. Alternatively, fragments can be prepared by recombinantly expressing heavy and light chain fragments in suitable host cells (e.g., E. coli, yeast, mammalian, plant, or insect cells) and assembling them either in vivo or in vitro to form the desired antigen-binding fragment. Single-chain antibodies can be prepared by recombinantly linking nucleotide sequences encoding heavy and light chain variable regions. For example, a flexible linker can be incorporated between the two variable regions. In one embodiment, CDRs present in a library scaffold are linked via a linker to form a synthetic scFv fragment. Thus, the term "antibody or antigen-binding fragment thereof" as used herein also encompasses synthetic binding fragments (e.g., human synthetic scFv fragments). Accordingly, the general terms "antibody" or "human antibody" are used throughout this specification.These terms are used in their broadest sense and therefore encompass all variants and fragments described above and below. Thus, the term "human antibody" also encompasses human-binding fragments of antibodies.

[0040] The term "human monoclonal antibody" or "human antibody" refers to an antibody or related fragment derived entirely from human sequences and free of murine sequences, including antibody fragments and synthetic scFv fragments. Human antibodies have typically been generated through two sources: phage display technology and transgenic mice. The human antibodies of the present invention do not include, for example, humanized antibodies, in which murine monoclonal antibodies have been engineered to resemble more human sequences. Humanization involves the replacement of murine constant and variable (V) framework regions with human sequences, resulting in an antibody in which only the complementarity-determining regions (CDRs) of the variable (V) regions are of murine sequence origin. Conversely, human monoclonal antibodies are fully human and are contrasted with humanized antibodies, which still contain non-human binding epitopes.

[0041] As used herein, the phrase "capable of binding to X," where X is an antigen, refers to a property of an antibody or antigen-binding fragment thereof that can be tested, for example, by ELISA, by surface plasmon resonance (SPR) technology, by kinetic exclusion assays (KinExA®), or by biolayer interferometry (BLI). These and other methods are known to those skilled in the art.

[0042] In some embodiments, the general term "immunoassay" is used. The immunoassay can be any type of immunoassay, such as a lateral flow immunochromatography assay, an immunomagnetic separation and electrochemiluminescence (IMS-ECL) assay, a fluorescent immunoassay, a time-resolved fluorescence (TRF) assay, a radioimmunoassay (RIA), or an enzyme-linked immunosorbent (ELISA), which uses the monoclonal antibody of the present invention to detect the level or amount of prostasomes in a sample from a subject. However, the "sample" can be a body fluid sample from a subject, such as a blood sample, plasma, serum, urine, cerebrospinal fluid, or a cell suspension, and the cell suspension can be, for example, derived from a biopsy.

[0043] The term "cluster" can be used herein to refer to a substance comprising multiple prostasomal antigens that form a three-dimensional arrangement of antigens bound to the prostate membrane or prostate membrane fragment. The cluster of protein antigens is present on the surface of the prostate membrane. In one embodiment, an antibody or fragment thereof of the present invention binds to multiple prostasomal surface antigens, which form a cluster on the surface of the prostasomal membrane, and the cluster is identified by a monoclonal antibody or antigen-binding fragment thereof. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to multiple antigens selected from Table 11. In some embodiments, the terms "cluster," "structure," or "aggregate" can be used synonymously when referring to a substance comprising multiple prostasomal antigens that form a three-dimensional arrangement of antigens.

[0044] The term "sensitivity," for example, in the context of a diagnostic assay, refers to the rate of true positives (probability of detection) for a condition of interest and measures the rate at which actual positives are correctly identified (e.g., the rate at which diseased individuals are correctly identified as having the condition). High sensitivity reduces the number of false positives, i.e., subjects identified as not having the condition when in fact they do. "Sensitivity," in the context of an antibody, e.g., a "sensitive antibody," is one that can detect even small amounts of the antigen of interest. Sensitivity can therefore be defined in terms of the minimum concentration of antigen that can be detected in an assay using that antibody.

[0045] The term "specificity," sometimes referred to as "selectivity," refers to the rate of true negatives for a condition of interest and measures the rate at which actual negatives are correctly identified as such (e.g., the rate of healthy individuals correctly identified as not having the condition). High specificity in an assay reduces the number of false positives, i.e., subjects identified as having the condition when in fact they do not. A selective or specific antibody does not cross-react, or does so to a low extent, with targets other than the intended antigen. Thus, the prostasome-specific antibody or antigen-binding fragment thereof of the present invention specifically or selectively binds to prostasomes or prostasome antigens, but does not bind to other antigens (such as antigens present on the surface of other types of exosomes or cells).

[0046] The term "diagnosis" or "diagnosing" as used herein refers to the process of determining whether a disease or condition (in this case, prostate cancer) exists in a subject under investigation. Diagnosis, in the context of a diagnostic procedure, can be considered as an attempt to classify an individual's condition into separate, distinct categories that allow medical decisions regarding treatment and prognosis to be made. A diagnostic option is then often described for a disease or other condition. The first task is to detect medical signs to perform the diagnostic procedure, such as detecting any deviation from what is known to be normal (in this case, prostasome levels in peripheral blood). Typically, aggressive prostate cancer is detected and diagnosed in the assay of the present invention, compared to indolent prostate cancer.

[0047] The term "prognosis" or "prognosing" as used herein refers to a prediction or estimate of the likelihood of recovering or surviving a disease. Cancer prognosis may depend on several factors, such as the stage of the disease at the time of diagnosis, the type and subtype of cancer, the molecular profile of the tumor, and may even depend on gender. The prognosis of prostate cancer of the present invention may be correlated with the level of prostasome in the sample from the subject being examined, with a high level indicating a poor prognosis (i.e., a high likelihood that the disease will have a poor outcome), and a low level indicating a good prognosis (i.e., a high likelihood that the disease will have a good outcome).

[0048] "Prostasomes" refer to submicron membranous vesicles, also known as exosomes, secreted by prostate epithelial cells and transported into semen. Prostasomes are therefore a type of exosome. They are characterized by a size of 50–500 nm, a lipid plasma membrane bilayer with an exceptionally high cholesterol / phospholipid ratio, and characteristic proteins on their surface membrane, such as caveolin-1, prostate stem cell antigen, prostate-specific membrane antigen (PSMA), CD10, CD13, and CD26, while containing RNA and DNA (Beneficial Effects of Seminal Prostasomes on Sperm Functional Parameters, Amit Kumar, Sujata Pandita, Subha Ganguly, Simson Soren, and Nileshkumar Pagrut, J Entomology and Zoology Studies 2008;6(5):2464–2471). Based on this information and the information in the cited papers, a person skilled in the art would be able to distinguish prostasomes from microvesicles or from any other type of exosomes when encountered in an isolate or in peripheral blood.Due to the special characteristics of prostasomes, it is possible to obtain prostasome-specific antibodies that do not cross-react with other exosomes.Prostasomes typically present one or more protein antigens listed in Table 2 herein below.

[0049] The term "prostate cancer," in the broadest sense, in the context of a subject suffering from prostate cancer, refers to the presence of a single prostate cancer cell. Even if a single cell is not enough to exceed the threshold for detectable prostasome levels in bodily fluids outside of semen, early diagnosis of prostate cancer is possible. Detected or diagnosed prostate cancers of the present invention may typically be at a stage where a biopsy can be performed to recover cancer cells, and preferably are not metastatic and are present only in the prostate, making them surgically removable. Therefore, early detection of prostate cancer using the methods of the present invention has the technical effect of increasing the number of cancers that can be treated by surgery and may also be used as a decision support tool to determine which cancers can be treated by surgery. Typically, a detected prostasome level of 10 ng / mL or less in a subject's sample would be considered surgically treatable, while a level above 100 ng / mL would be considered surgically untreatable. A detected prostasome level of 10 ng / mL to 100 ng / mL may be used as an indication that further characterization of the prostate cancer is required before determining the treatment to be performed. Because prostate cancer must remain within the prostate gland and not metastasize in order for surgery to be possible, high levels detected by the assay of the present invention can predict that surgery is no longer possible.

[0050] PSA is currently the most commonly used marker for detecting prostate cancer, but unfortunately, this biomarker is not specific enough. Therefore, better markers for screening prostate cancer are needed. A preferred marker should be specific for both the prostate gland and the cancerous disease. One promising marker is prostasomes, vesicles released from acinar cells within the prostate.

[0051] The inventors have previously demonstrated that malignant prostate cells secrete prostasomes and that the presence of malignant prostate tumors increases the number of prostasomes present in peripheral blood, indicating that prostasomes may be a potential biomarker for prostate cancer. However, developing a simple, robust, reliable, selective, and sensitive method for diagnosing prostate cancer using prostasomes has been challenging.

[0052] Although several methods using monoclonal antibodies against prostasomes to diagnose prostate cancer have previously been described, none of them have the sensitivity to detect low amounts of prostasomes in blood, serum, or plasma using a single capture antibody. Also, they generally appear to lack the specificity to detect prostasomes rather than exosomes. Therefore, there is a need for new, robust methods that can be used to diagnose prostate cancer and that have both sufficient sensitivity and specificity to avoid false negatives and false positives when performing diagnostic tests.

[0053] As shown in the present disclosure, the above-mentioned problems and shortcomings of the prior art have been overcome by using fully human monoclonal antibodies or binding fragments thereof for diagnosing prostate cancer. The fully human monoclonal antibodies or binding fragments of the present invention achieve the desired sensitivity and specificity / selectivity that allows for practical diagnosis of prostate cancer in serum and plasma samples from patients using a single capture antibody. Accordingly, the present disclosure relates to a method for providing a diagnosis or prognosis for a subject suspected of having prostate cancer, the method comprising detecting prostasomes in vitro in a peripheral blood sample from the subject, and comparing the detected level of prostasomes to a threshold or reference value derived from a healthy subject. The threshold and reference value can be predetermined or calibrated for a particular test.

[0054] Previous methods for detecting prostasomes for prostate cancer diagnosis typically used mouse monoclonal antibodies, or, at best, humanized monoclonal antibodies. These methods failed to provide the specificity and sensitivity required to effectively diagnose or prognose prostate cancer. The biggest problem was likely sensitivity, resulting in false negatives due to the inability of antibodies to detect low levels of prostasomes in samples. For example, in one previous study, an enzyme-linked immunosorbent assay (ELISA) was designed to determine the number of antibodies capable of functioning as appropriate capture or detection antibodies for prostasomes (E. Thermaenius et al., DiVA, id: diva2:544923). Two surface proteins specific to prostasomes were used as capture antigens in this study, and proteins that bind to the surface of prostasomes were used as secondary antibodies in the assay. In this experimental setup, the detection limit was 2500 ng / mL, which was deemed insufficient to detect prostasomes in cancer. In another previous study (Tavoosidana et al. PNAS, May 24, 2011, vol. 108, no. 21, 8809-8814), prostasome levels were detected in serum. This required the use of five antibodies in combination to achieve a sufficient level of sensitivity and specificity for diagnostic use. In contrast, the present invention provides a method that requires only one monoclonal antibody in a detection assay to specifically and selectively detect prostasomes for diagnostic purposes. Therefore, the use of the antibodies of the present invention provides a much simpler assay, which can be easily scaled up and is less expensive to manufacture.

[0055] False-positive assays can also be problematic if the antibodies used are not sufficiently specific. For example, antibodies used in assays may react with other exomes because all exomes share common epitopes. As shown by the Human Protein Atlas, a program aimed at mapping all human proteins in cells, tissues, and organs, random antibodies exhibit significant cross-reactivity with different tissues. Prostasome surface antigens have previously been shown to be the major autoantigens causing immune infertility and antisperm antibodies (see, for example, L. Carlsson et al. Journal of Andrology, Vol. 25, No. 5, September / October 2004 and A. Minelli et al. ANTICANCER RESEARCH 25: 4399-4402 (2005)). Thus, autoantibodies against prostasomes are common in men with clinical infertility. One defining feature of immune infertility is that affected men are infertile but generally lack any other underlying conditions. The inventors have interpreted this to be because these autoantibodies, which are antibodies produced by human subjects themselves, are specific to prostasomes and do not react with other types of cells or tissues. Therefore, using human antibodies from a human DNA library will result in better and more specific / selective prostasomes than using mouse monoclonal antibodies or humanized antibodies. Therefore, using human antibodies or antibody fragments based on human antigen-binding epitopes to detect prostasomes as biomarkers for cancer will achieve higher specificity and selectivity than antibodies suggested in the prior art.

[0056] Unlike the prior art, the present disclosure provides novel monoclonal antibodies or binding fragments that are fully human, specific enough to avoid false positives, and sensitive enough to detect small amounts of prostasomes in bodily fluids. These prostasome-specific antibodies can be used in different detection assays to detect prostasome levels in bodily fluids (such as blood, serum, or plasma). When performing a biopsy or prostate massage, cell suspensions from the biopsy or urine containing prostasomes resulting from the massage can also be used as sample bodily fluids. Prostasomes are secreted by prostate cells into semen, so in healthy subjects, prostasomes are generally present in semen, with only a small percentage leaking into the external bloodstream. However, prostasomes are also secreted by malignant prostate cells, increasing the number of prostasomes present in peripheral blood. It has been found that the reason prostasome levels in peripheral blood rapidly increase when cancer cells secrete prostasomes is not only because the number, and therefore leakage, increases, but also because prostate cells actually lose their polarity when they become cancerous, thereby secreting prostasomes into the blood. Therefore, even slightly elevated levels of prostasomes in peripheral blood are indicative of prostate cancer. Highly sensitive assays are needed to be able to effectively detect, diagnose, and predict the prognosis of prostate cancer, including early-stage prostate cancer, which may have only slightly elevated levels of prostasomes in peripheral blood.

[0057] The assay of the present invention uses a fully human monoclonal antibody or binding fragment thereof to detect the presence of elevated levels of prostasomes in the blood, and therefore the presence of malignant prostate cancer cells. A bodily fluid sample (e.g., a blood sample) is collected from a subject potentially in need of diagnosis (e.g., a patient in a hospital or clinic), and the sample is either directly screened for elevated levels of prostasomes or first processed to obtain a serum or plasma sample, which is then screened in the assay. The amount of prostasomes detected in the bodily fluid is compared to a predetermined threshold or reference value, and an increase in the detected value above the threshold or compared to the reference value indicates the presence of prostate cancer in the subject being examined. Typically, a prostasome level (concentration) of about 1-2 nanograms (ng) per milliliter (mL or ml) of bodily fluid sample indicates cancer, and levels above 10 ng / mL can be used for definitive diagnosis. The detected prostasome level compared to one or more thresholds or one or more reference values ​​can also be used to predict the prognosis of prostate cancer. For example, a detected value higher than the reference value indicates a later stage of prostate cancer and therefore a worse prognosis, while a detected value lower than the reference value indicates an earlier stage of prostate cancer and therefore a better prognosis. Prognosis can also be defined by several thresholds. In this case, levels above or below these thresholds indicate different prognoses. For example, if the prostasome level is above a certain threshold (a first predetermined threshold), a poor prognosis of prostate cancer is predicted, whereas if the level is below a certain threshold (such as a second predetermined threshold), a good prognosis of prostate cancer is predicted. The first threshold can be 10 ng of prostasome per 1 mL of sample, and the second threshold can be 1 ng of prostasome per 1 mL of sample. The prostasome level can also indicate the severity of prostate cancer and can be linked to possible treatments (such as surgery or no surgery). Cancer progression and treatment effectiveness can also be determined by measuring prostasome levels at different time points in the same subject.In this case, a decrease in the amount of prostasomes indicates tumor regression and a high treatment efficacy, whereas an increase in the amount indicates tumor progression and a low treatment efficacy. Thus, the effectiveness of treatment can be determined by measuring the prostasome levels before and after a treatment regimen, with a decrease in the prostasome levels indicating an effective treatment. The treatment can be, for example, surgery, administration of a medical substance to a subject, or radiation therapy. The medical substance can be, for example, an oral medication or a substance administered via injection or infusion.

[0058] The assay of the present invention can be any assay suitable for detecting an analyte in a body fluid sample using a monoclonal antibody. The assay can be an immunoassay, which relies on the ability of an antibody to recognize and bind to a specific macromolecule in a sample containing a complex mixture of macromolecules. In immunology, the specific macromolecule bound by an antibody is called an antigen, and the region on the antigen to which the antibody binds is called an epitope. In addition to the binding of an antibody to its corresponding antigen, another key feature of any immunoassay is a means of generating a measurable signal in response to binding. Most, if not all, immunoassays involve chemically coupling the antibody or antigen to some type of detectable label. Numerous labels exist in modern immunoassays, allowing for detection through different means. Many labels are detectable because they can be induced to emit radiation, produce a color change in solution, fluoresce under light, or emit light. The assay of the present invention can be, for example, a lateral flow immunochromatography assay, an immunomagnetic separation and electrochemiluminescence (IMS-ECL) assay, a time-resolved fluorescence (TRF) assay, or an enzyme-linked immunosorbent (ELISA) assay. In one embodiment, the assay is an immunoassay or immunocapture assay (e.g., a sandwich immunoassay), in which the monoclonal antibody of the present invention is used to capture and then detect prostasomes in a body fluid sample (a body fluid extracted from a subject potentially requiring diagnosis or prognosis). For example, the assay can be a sandwich ELISA, in which a surface is prepared, to which a known amount of a capture antibody (i.e., one of the monoclonal antibodies of the present invention) is bound, and a body fluid sample containing prostasomes is applied. The prostasomes are captured by the capture antibody, the surface is washed, and a primary detection antibody is applied and binds to the prostasomes. A secondary antibody conjugated to an enzyme is applied as a detection antibody, which also specifically binds to the Fc region of the antibody, thereby detecting the presence of the primary detection antibody and therefore the bound prostasomes. The surface is washed to remove any unbound antibody-enzyme conjugate, and a chemical is added that is enzymatically converted to a color, fluorescent, or electrochemical signal.The absorbance, fluorescence, or electrochemical signal (e.g., current) of the plate wells is measured to determine the presence and amount of analyte (i.e., prostasomes). The amount of prostasomes can then be compared to a reference value, and elevated levels are associated with the presence of malignant prostate cancer cells in the subject being diagnosed. The primary detection antibody can be, for example, a chicken anti-PSA or anti-prostasome antibody, and the secondary detection antibody can be an anti-chicken antibody, which can be labeled with HRP (horseradish peroxidase). A commonly used enzyme marker in ELISA assays is, for example, OPD (o-phenylenediamine dihydrochloride), which turns amber to detect HRP. HRP is often used as a conjugated protein. Alternatively, TMB (3,3',5,5'-tetramethylbenzidine) can be used, which turns blue when detecting HRP and turns yellow after the addition of sulfuric or phosphoric acid. Alternatively, ABTS (2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) can be used, which turns green when detecting HRP. Figure 1 shows an example of using a human monoclonal antibody or antigen-binding fragment thereof (101) of the present disclosure as a capture antibody for capturing prostasomes (200). After capture, the bound prostasomes (200) can be detected using a primary detection antibody (102) bound to the prostasomes, followed by detection with a secondary detection antibody (103). The primary detection antibody (102) can be an anti-prostasome or anti-PSA antibody and can be of chicken origin (chicken anti-prostasome or chicken anti-PSA), and the secondary detection antibody (103) can be an antibody labeled with horseradish peroxidase (HRP) and will be anti-species to the primary detection antibody, i.e., anti-chicken if the primary detection antibody is chicken (HRP-labeled anti-chicken antibody). Thus, the present invention provides an assay that is sensitive enough to use a single capture antibody and a single primary detection antibody together with a secondary detection antibody as an assay for detecting prostasome levels to diagnose prostate cancer, which was not possible in the prior art.Thus, in one embodiment, a single capture antibody is used in an immunoassay to detect prostasomes in a sample, while in another embodiment, multiple capture antibodies are used (e.g., two, three, or four, but less than five, capture antibodies of the invention).

[0059] The antibodies or fragments of the present invention are fully human monoclonal antibodies or fragments thereof that are prostasome-specific (selective for prostasomes over other cells or exosomes) and sufficiently sensitive to allow detection of low levels of prostasomes in a sample. The sensitivity required for an assay (such as an ELISA) in which an antibody functions as a single capture antibody is typically at least 10 nanograms (ng) of prostasomes per milliliter (mL). Thus, the assays of the present invention enable detection of prostasomes at levels of 10 ng / mL or less. This sensitivity can be achieved using the antibodies or antigen-binding fragments thereof of the present invention. Sensitivity can even be higher than 10 ng / mL, achieving a sensitivity range of 1-2 ng / mL. The absence of antibodies in the prior art has been shown to enable this sensitivity in similar assays, and therefore, the prior art does not provide an antibody that can be used as a single capture antibody in a detection assay to detect prostasomes for sufficient prostate cancer diagnosis.

[0060] The antibodies or fragments of the present invention bind to clusters of protein antigens present on the surface of the prostate membrane. The aggregated antigens form a three-dimensional structure, a mass of antigen and prostate membrane, which is identified by the antibodies or fragments of the present invention. Therefore, the antibodies or fragments of the present invention are ideal for measuring the presence of total prostasomes in a sample compared to prior art antibodies. Therefore, both the antibodies or fragments of the present invention have greater specificity and sensitivity than other antibodies or binding fragments. Therefore, using just one of these antibodies and fragments, it is possible to detect prostasomes in a sample (such as a blood or serum sample) at a sensitivity of 10 ng of prostasomes per mL, or even greater, e.g., 1-2 ng / mL. This is not possible with any prior art antibodies.

[0061] Therefore, unlike prior art antibodies used in diagnosing prostate cancer, the antibodies of the present invention are fully human and bind to several prostasome antigens (e.g., clusters of antigens). Furthermore, their specificity and sensitivity are higher than those of the prior art. In Tavoosidana et al., several antibodies (e.g., five or more) must be used in combination to achieve the sensitivity range achieved with a single antibody or fragment of the present invention in an assay, as shown, for example, in Figure 1. It is simply not correct to state that an anti-CD13 antibody achieves a detection sensitivity of 0.5 mg / mL. Because five antibodies and a DNA fragment are used in Tavoosidana et al. (see Figure 1), this method also requires an additional PCR step. Because several antibodies and a DNA fragment are required to achieve that sensitivity, it cannot be said that the antibody in Tavoosidana et al. enables a sensitivity of at least 10 ng / mL in an immunoassay by using it as the sole capture antibody in the immunoassay. Therefore, the individual antibodies of Tavoosidana et al. are not fully human, do not bind to several prostasome antigens or clusters of antigens on the surface of the prostasome membrane, and do not individually achieve the specificity of the antibodies / fragments of the present invention. The method of the present invention also has the additional advantage that it is simpler, cheaper, and more efficient than the method of Tavoosidana et al.

[0062] The method in Yu et al.'s paper ("Electrical and Label-Free Quantification of Exosomes Using Reduced Graphene Oxide Field-Effect Transistor Biosensors," ANALYTICAL CHEMISTRY, vol. 91, no. 16, 23 July 2019 (2019-07-23), pages 10679-10686), like that of Tavoosidana et al., is based on the analysis of DNA (PNA), which is far more complex than a simple immunoassay and further demonstrates that DNA and immunoassays are fundamentally different. While DNA tests typically achieve high sensitivity, immunological (e.g., antibody-based) tests are simpler and less expensive. Furthermore, Yu et al.'s paper notes that the anti-CD63 antibody used has very low tissue specificity (see, for example, Protein Atlas, URL https: / / www.proteinatlas.org / ENSG00000135404-CD63). This means that it cannot distinguish prostasomes from other types of exosomes, nor can it distinguish prostasomes from whole cells (anti-CD63 antibodies would react with whole cells). The antibody of Yu et al. is used to detect exosomes in pure samples and to detect the nonspecific occurrence of cancer, simply based on the fact that cancer produces more exosomes. There is no specificity in a diagnosis based solely on the number of unspecified exosomes in blood. Furthermore, in a sample (such as the blood sample of the present invention), the antibody is thought to detect not only all exomes present (which may indicate the presence of a wide range of cancers or other disorders), but also whole cells. Therefore, the antibody is thought to be unable to distinguish between prostasomes and prostate cancer, nor to distinguish between exosomes in a blood sample. Therefore, the antibody of Yu et al. cannot be used in a method for detecting prostate cancer in a patient sample. For example, the antibody of Yu et al. is not fully human, does not selectively bind to prostasomes, does not bind to several prostasome antigens or clusters of antigens on the surface of the prostasome membrane, and does not achieve the sensitivity of the antibody / fragment of the present invention in a simple immunoassay.

[0063] The human monoclonal antibody or antigen-binding fragment thereof of the present invention is selected from the group consisting of a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fc fragment, an Fv fragment, a single-chain Fv fragment, an (scFv)2 fragment, and a domain antibody. In one embodiment, the antibody or antigen-binding fragment thereof is selected from a full-length antibody, a Fab fragment, and an scFv fragment. In one embodiment, the antibody comprises 12 complementarity-determining regions (CDRs). In another embodiment, the antibody or antigen-binding fragment thereof comprises six CDRs, i.e., CDR-H1 / CDR-H2 / CDR-H3 / CDRL1 / CDR-L2 / CDR-L3. In a more particular such embodiment, the antibody or antigen-binding fragment thereof comprises at least three complementarity-determining regions (CDRs), which can be, for example, from the heavy chain, i.e., CDR-H1, CDR-H2, and CDR-H3, or from the light chain, i.e., CDR-L1, CDR-L2, and CDR-L3.

[0064] In one embodiment, a human monoclonal antibody of the present invention is an antigen-binding fragment (Fab) of an antibody. The antigen-binding fragment (Fab) is a region on an antibody that binds to an antigen. It consists of one constant domain and one variable domain from each of the heavy and light chains. The variable domain contains a paratope (antigen-binding site) at the amino terminus of the monomer, which includes a set of complementarity-determining regions.

[0065] In one embodiment, the human monoclonal antibodies of the invention are single-chain variable fragments (scFv) of antibodies. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and are not actually antibody fragments, but instead are fusion proteins consisting of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins connected by a short peptide linker ranging from 10 to about 25 amino acids in length. These molecules retain the same binding function as full-length antibodies but have several advantageous features, such as rapid tissue penetration, ease of manipulation, rapid clearance of their immune complexes, and the possibility of production in simple expression systems (e.g., bacteria or yeast). Figure 2 shows a schematic diagram of an scFv fragment of the invention, which has a VH domain containing heavy chain CDRs (CDR-H1, CDR-H2, and / or CDR-H3) and a VL domain containing light chain CDRs (CDR-L1, CDR-L2, and / or CDR-L3) joined together using a linker.

[0066] In one embodiment, the human monoclonal antibodies of the present invention are human synthetic single-chain variable fragments (scFv) of antibodies. This scFv fragment has a molecular weight of approximately 30 kDa and can comprise heavy and light chains linked via a linker. The heavy and light chains can have constant and variable regions, and the loops therein can be referred to as CDRs, which can be variable. In some embodiments, there are at least four variable CDRs, of which CDR-H3 and CDR-L3 are typically the most variable because they form the most exposed portion of the binding molecule or fragment. CDR-L1 and CDR-L2 can be absent (allowing for constant sequences), while CDR-H1 and CDR-H2 can have slight variations. In some embodiments, the synthetic scFv fragment comprises short heavy chain sequences from CDR-H1, CDR-H2, and / or CDR-H3 on a VH scaffold in combination with short light chain sequences from CDR-L3 on a VL scaffold, which are linked using a linker to form the scFv fragment. Synthetic scFv fragments can be derived from human synthetic scFv libraries in which the CDRs reside on a library scaffold (e.g., the HeIL-11 or HeIL-13 scaffold libraries (Sall et al., "Generation and synthesis of human synthetic antibody libraries and their application to protein microarrays." Protein Eng Des Sel. 2016 Oct;29(10):427-437)) or the similar MAW-16 library. The CDR-H3 is typically 8-22 amino acids in length, and the CDR-L3 is 8-12 amino acids in length. The entire scFv fragment, including the library scaffold, the heavy and light chain CDR portions, and both the heavy and light chain CDRs, and the linker, is typically approximately 250-300 amino acids (e.g., 273 amino acids). The scFv fragment can be modified by attaching it to another structure, such as an antibody or antibody portion, a HIS tag, a FLAG, or biotin. The CDRs can also be attached to the surface of a full-length antibody using the constant regions of CDR-L1 and CDR-L2 as the corresponding antibody CDRs.

[0067] The human antibodies or binding fragments of the present invention can be generated using various methods, such as phage display and libraries. Monoclonal antibodies based on phage display technology are an attractive alternative to traditional hybridoma technology. Gene sequences encoding specific antibodies are incorporated into the DNA sequence of filamentous bacteriophages, which then express them on the surface of the bacteriophage capsid. This specificity establishes a link between genotype and phenotype. The phage infects E. coli and utilizes its internal replication system to continuously display new phages without killing the host cell. This allows for the rapid production of large numbers of antibodies. Libraries of naive or immune phage are thus constructed, which can be used to detect interesting antigen-antibody interactions through screening methods. Libraries can be generated from humans to access the human antibody repertoire.

[0068] The human genome offers distinct advantages for specific antibodies against prostasomes. Autoantibodies against prostasomes are frequently generated and, when present, cause immune-mediated infertility. These antibodies do not cause damage to other organs in the body beyond infertility. This is a strong indication that human antibodies against prostasomes are highly specific. High selectivity is clearly advantageous, for example, when detecting prostasomes in blood where other cell types are present. Therefore, one method for generating the antibodies or fragments of the present invention is to develop scFv antibodies against human prostasomes using phage display technology. Another advantage of using antibodies from the same species is immune tolerance, which should reduce the number of antibodies against antigens commonly presented in the body. Immune tolerance is a complex set of mechanisms that impair the immune system from mounting a response against self-antigens. Studies of scFv antibodies have also shown that these antibodies are better binders than mouse antibodies when used to detect prostasomes, allowing for the detection of much smaller amounts of prostasomes. This is surprising, because it contradicts the commonly held belief that phage-displayed antibodies often have lower affinity than hybridoma antibodies. One hypothesis for this finding is that it may be related to the fact that the antibodies are directed against prostasomes.

[0069] In one embodiment, the antibodies or binding fragments thereof originate from libraries screened for their ability to bind to prostasomes (full-sized whole prostasomes). Numerous approaches exist for generating antibody libraries from which binders can be selected, which can be divided into natural or synthetic depending on the source of genetic diversity. Natural antibody repertoires exploit the immune system's ability to generate diversity, whereas diversity in synthetic repertoires is artificially introduced into defined regions of the antibody sequence. Synthetic libraries generate large genetic diversity but are not selected for function. Thus, the trend in synthetic library design has been to attempt to mimic the diversity found in natural antibodies while still promoting genetic diversity to maximize function.

[0070] The following example describes the generation of a synthetic scFv library that can be used to screen the synthetic scFv fragments described herein. Two human synthetic antibody fragment libraries were previously developed and generated as part of a two-step process (see Sall et al., "Generation and synthesis of a human synthetic antibody library and its application to protein microarrays." Protein Eng Des Sel. 2016 Oct;29(10):427-437) that utilized initial experiments with a first-generation library (HelL-11) to design a second-generation library (HelL-13). While the CDR-H3 and CDR-L3 design (Figure 3) differs slightly, both template scFv genes used as scaffolds for the Hell-11 and Hell-13 libraries were constructed based on the human IGHV3-23 and IGKV1-39 genes, of which the kappa light chain IGKV1-39 and heavy chain IGHV3-23 were used as scaffolds to construct the libraries. The antibodies comprising these genes are the most frequently found in the natural antibody repertoire, and this particular VH / VL combination has been shown to be favorable. Instead of Hell-11 and Hell-13, the MAW-16 library may be used, in which the variable heavy chain is also based on IGHV3-23 and the variable light chain is also based on IGKV1-39.

[0071] Diversity was introduced at defined positions in four of the six CDR loops, namely, CDR-H1, CDR-H2, CDR-H3, and CDR-L3. Residues distributed within the CDR-H1, CDR-H2, CDR-H3, and CDR-L3 loops were selected for diversification. The scFv fragments from the different libraries are shown in Figure 4a, and the VH and VL regions are shown in Figure 4b. While one primer was used for CDR-H1 and CDR-H2, respectively, three or five primers and ten or fifteen primers (one primer for each of these loops of allowed length) were used to diversify CDR-L3 and CDR-H3 of HeIL-13 and HeIL-11, respectively. The introduced diversity was biased toward tyrosine, serine, and glycine because these amino acids are highly abundant in the antigen-binding site and their favorable role in antigen recognition has been demonstrated in several studies. The residues targeted by CDR-H1 and CDRH2 were restricted to this trimer code. While biased toward tyrosine, serine, and glycine, CDR-H3 and CDR-L3 allowed for much more complex chemical diversity, based on findings commonly found in natural antibodies. While solvent-accessible residues in CDR-H1 and CDR-H2 were restricted to tyrosine, serine, and glycine, more complex diversity schemes were possible in CDR-H3 and CDR-L3. The allowed lengths of CDR-H3 and CDR-L3 in HelL-11 were 8–22 and 8–12 residues, respectively. In HelL-13, the corresponding lengths were 8–17 and 8–10 residues. The variability introduced here was primarily limited to amino acids commonly found at these positions in natural antibodies. In HeIL-11, the lengths of CDR-H3 and CDR-L3 were allowed to vary from 8 to 22 and 8 to 12, respectively, thereby covering over 90% of the most frequently occurring lengths in natural antibodies. The HeIL-13 library was designed to contain CDR-H3 loop lengths of 8 to 17 and CDR-L3 loop lengths of 8 to 10, thereby covering approximately 90% of the functionally selected binders derived from HeIL-11.To further increase the proportion of functional binders in the HelL-13 natural repertoire, a CDR-L3 loop without a stop codon was included. The IGKV1-39 germline sequence was selected as the template for CDR-L3 (positions 105-115). At position 116, which is naturally derived from the J-segment, a tyrosine was included due to its generally favorable binding properties.

[0072] In one embodiment, the human monoclonal antibodies of the present invention are human synthetic single-chain variable fragments (scFv) of antibodies. Techniques for developing libraries and screening antibody fragments utilize focused single-chain antibody fragment (scFv) repertoires (Persson et al., (2006) Generation of focused antibody libraries for improved hapten recognition. J Mol Biol 357, 607-620. (Abstract) (GenBank)). The scFv genes used as templates for the HeIL-11 and HeIL-13 libraries were constructed based on the human immunoglobulin heavy chain variable 3-23 precursor (IGHV3-23) and immunoglobulin kappa variable 1-39 precursor (IGKV1-39) genes. The phagemid vector backbone was pFab5c. As shown in Figure 3a, residues distributed within CDR-H1, CDR-H2, CDR-H3, and CDR-L3 were cloned and the resulting sequences were introduced into the library scaffold gene. The lengths of CDR-H1 and CDR-H2 were 8 amino acid residues for both Hell-11 and Hell-13. The lengths of CDR-H3 and CDR-L3 for Hell-11 were 8–22 and 8–12 amino acid residues, respectively. For Hell-13, the corresponding lengths were 8–17 and 8–10 residues. (Sall et al., Generation and synthesis of a human synthetic antibody library and its application to protein microarrays. Protein Eng Des Sel. 2016 Oct;29(10):427–437)

[0073] Screening the library against a panel of different prostasomes using phage display yielded diverse and highly specific binders to prostasomes. Analysis showed that members selected from such populations were an excellent source for developing high-affinity binders after affinity maturation by random mutagenesis and stringent phage display selection. The mutations in the high-affinity variants were distributed throughout the scaffold and therefore would not have been easily predicted.

[0074] The synthetic scFv fragments of the invention may comprise one VH portion comprising CDRs H1, H2, and H3, and one VL portion comprising either CDR L3 or CDRs L1, L2, and L3. CDR-L1 and CDR-L2 may be considered CDRs present in the synthetic scFv fragment, and the positions of CDR-L1 and CDR-L2 are shown in Figure 4b. However, because they do not vary between different fragments of the invention, they may also be considered part of the scaffold. Thus, in one embodiment, only CDRs H1, H2, H3, and L3 are defined for each fragment. In other embodiments, such as when using CDRs within an antibody, a total of six CDRs may be defined, including CDRs L1 and L2.

[0075] Synthetic scFv fragments of the invention can comprise one VH domain comprising CDRs H1, H2, and H3, and one VL domain comprising CDR L3, with the VH and VL domains combined using a linker, which can be a gly-ser sequence of about 15 amino acids (such as GGGGSGGGSGGGGS as shown in SEQ ID NO: 31). In some embodiments, the VH fragment is 128 amino acids, the VL fragment is 128 amino acids, and the total scFv fragment is 273 amino acids. In some embodiments, the fragments can be shorter by dropping some amino acids, as indicated by the dotted circle in Figure 4b.

[0076] In one embodiment, the antibody or antigen-binding fragment can comprise a CDR of the VH portion (e.g., a heavy chain CDR, which can be referred to as CDR-H3) and a CDR of the VL portion (e.g., a light chain CDR, which can be referred to as CDR-L3), wherein CDR-H3 is selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; and CDR-L3 is selected from SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, with which the CDR sequences are at least 95% identical (96%, 97%, 98%, 99% or more).

[0077] The most important variable regions comprise the CDR-H3 and CDR-L3 regions. The CDR-H1, CDR-H2, CDR-L1, and CDR-L2 regions can be constant or variable, as shown in Table 1 below.

[0078] [Table 1]

[0079] The amino acid designated as X can be any naturally occurring amino acid, however in a preferred embodiment the amino acid X is selected from T, S or G.

[0080] In one embodiment, the antibody or antigen-binding fragment comprises at least three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3) and one light chain CDR (CDR-L3) in any combination thereof, CDR-H1 is selected from any variant of SEQ ID NOs: 25 and 27-28; CDR-H2 is selected from any variant of SEQ ID NOs: 26 and 28; a CDR-H3 selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; CDR-L3, CDR sequences selected from SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, are 95% or more identical thereto (96%, 97%, 98%, 99% or more).

[0081] In some embodiments, the antibody or antigen-binding fragment comprises at least two CDRs, i.e., CDR-H3 and CDR-L3, or at least four CDRs, i.e., CDR-H1, CDR-H2, CDR-H3, and CDR-L3, or at least six CDRs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, among which CDR-H1 is selected from any variant of SEQ ID NOs: 25, 27, and 28; CDR-H2 is selected from any variant of SEQ ID NOs: 26, 29, and 30; a CDR-H3 selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; CDR-L1 is selected from any variant of SEQ ID NOs: 56 and 57; CDR-L2 is selected from any variant of SEQ ID NOs: 58 and 59; The CDR-L3, CDR sequence selected from SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 is 95% or more identical thereto (96%, 97%, 98%, 99% or more). The CDRs can be grafted onto any suitable human antibody or antibody scaffold (e.g., constructed on the human IGHV3-23 and IGKV1-39 genes, using the kappa light chain IGKV1-39 and heavy chain IGHV3-23 as references).

[0082] In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) sequence selected from the group consisting of SEQ ID NOs: 32-43 and sequences that are 80% or more (85%, 90%, 95% or more) identical thereto. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) sequence selected from the group consisting of SEQ ID NOs: 44-55 and sequences that are 80% or more (85%, 90%, 95% or more) identical thereto.

[0083] In some embodiments, the antigen-binding fragment is an scFv fragment comprising a VH domain selected from SEQ ID NOs: 32-43 and a VL domain selected from SEQ ID NOs: 44-55 joined by a linker (such as the linker of SEQ ID NO: 31).

[0084] In one embodiment, the human monoclonal antibody or binding fragment thereof is a synthetic scFv fragment constructed, for example, on a HeIL-11 scaffold or a HeIL.13 scaffold, and comprises CDRs selected from the 12 scFv fragments of Table 2, wherein VH CDR-H1 for fragments scFv 1 to scFv 12 corresponds to SEQ ID NO: 25, VH CDR-H2 for fragments scFv 1 to scFv 12 corresponds to SEQ ID NO: 26, VH CDR-H3 for fragments scFv 1 to scFv 12 corresponds to SEQ ID NO: 1 to 12, VL CDR-L3 for fragments scFv 1 to scFv 12 corresponds to SEQ ID NO: 13 to 24, and the linker between the fragments corresponds to SEQ ID NO: 31.

[0085] [Table 2]

[0086] The human antibodies and binding fragments of the present invention can bind to prostasomes and thus be used as capture antibodies in assays to diagnose prostate cancer. These antibodies or binding fragments can recognize and bind to any possible epitope on prostasomes, which epitopes are known in the prior art. Some relevant epitopes are summarized in Table 3 below.

[0087] [Table 3]

[0088] A method according to the present concepts for diagnosing, prognosing, assessing the severity or effectiveness of treatment for prostate cancer will now be described with reference to Figures 5-8. For clarity and brevity, the method is described in "steps." The steps are not necessarily processes that are separated in time or isolated from one another; two or more "steps" may be performed simultaneously in a parallel manner.

[0089] The inventive concept has been primarily described above with reference to certain embodiments, however, those skilled in the art will readily recognize that embodiments other than those disclosed above are equally possible within the scope of the inventive concept, which is defined by the appended claims.

[0090] Example Operation

[0091] The proposed method will now be described in more detail with reference to Figures 5 to 8. It should be appreciated that the operations do not have to be performed in order. Furthermore, it should be appreciated that not all operations need to be performed.

[0092] 5 shows an in vitro method for determining whether a subject has prostate cancer, the method comprising the steps of (S1) providing a human monoclonal antibody or antigen-binding fragment thereof that selectively binds to prostasomes; (S2) reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from the subject; (S3) detecting any prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof to obtain a prostasome level; (S4) comparing the prostasome level with a predetermined threshold; and (S5) determining that the subject has prostate cancer if the detected prostasome level is higher than the predetermined threshold. In this regard, determining that prostate cancer is not present includes determining that clinically detectable prostate cancer is not present, i.e., that any cancer cells that may be present are too few to be detected or labeled as present prostate cancer. However, a few cancer cells may of course be present, and they will eventually develop into detectable prostate cancer. However, this small amount would be considered to be prostate cancer that is not present at the time of diagnosis. The predetermined threshold corresponds to a level of 10 ng of prostasomes per mL of sample. In another embodiment, the predetermined threshold is as low as 1-2 ng of prostasomes per mL of sample. Thresholds of 3, 4, 5, 6, 7, 8, or 9 ng / mL are also possible.

[0093] In one aspect, detecting any prostasomes bound by the human monoclonal antibody or its antigen-binding fragment (S3) comprises detecting prostasomes using an anti-prostasome detection antibody or its antigen-binding fragment. Typically, when the subject is male, a small amount of prostasomes is always present in the sample. In a further aspect, the method is a sandwich immunoassay, and detecting prostasomes (200) bound by the human monoclonal antibody or its antigen-binding fragment (101) (S3) comprises detecting the anti-prostasome detection antibody (102) using a further detection antibody (103), wherein the human monoclonal antibody or its antigen-binding fragment (101) is a capture antibody, the anti-prostasome detection antibody (102) is a primary detection antibody, and the further detection antibody (103) is a secondary detection antibody. In a further aspect, the primary detection antibody (102) is a chicken antibody, and the secondary detection antibody (103) is an anti-chicken antibody. In one aspect, the sample from the subject is a body fluid sample.In another aspect, the body fluid sample from the subject is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and cell suspension.Blood, serum, and plasma are obtained from peripheral blood samples collected from patients, while cell suspensions are typically obtained from biopsies.Urine can be used, for example, after prostate massage.In this case, prostasomes leak into the urine after prostate massage.In one aspect, the human monoclonal antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof of the present disclosure.

[0094] FIG. 6 shows an in vitro method for providing a prognosis of prostate cancer in a subject in need thereof, the method comprising the steps of: preparing a human monoclonal antibody or antigen-binding fragment thereof that selectively binds to prostasomes (S11); reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from the subject (S12); detecting the prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof to obtain a prostasome level (S13); comparing the prostasome level with first and second predetermined thresholds (S14); and providing a prognosis of the prostate cancer (S15), wherein if the detected prostasome level is higher than the first predetermined threshold, the prognosis of prostate cancer is defined as poor (S15a); and if the detected prostasome level is lower than the second predetermined threshold, the prognosis is defined as good (S15b). In one embodiment, the first threshold is 10 ng of prostasomes per mL of sample and the second threshold is 1 ng of prostasomes per mL of sample.

[0095] In one aspect, detecting prostasomes bound by the human monoclonal antibody or its antigen-binding fragment (S13) comprises detecting prostasomes using an anti-prostasome detection antibody or its antigen-binding fragment. In a further aspect, the method is a sandwich immunoassay, and detecting prostasomes (200) bound by the human monoclonal antibody or its antigen-binding fragment (101) (S13) comprises detecting the anti-prostasome detection antibody (102) using an additional detection antibody (103), wherein the human monoclonal antibody or its antigen-binding fragment (101) is a capture antibody, the anti-prostasome detection antibody (102) is a primary detection antibody, and the additional detection antibody (103) is a secondary detection antibody. In a further aspect, the primary detection antibody (102) is a chicken antibody, and the secondary detection antibody (103) is an anti-chicken antibody. In one aspect, the sample from the subject is a body fluid sample. In a further aspect, the bodily fluid sample from the subject is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and a cell suspension. In one aspect, the human monoclonal antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment of the present disclosure.

[0096] FIG. 7 shows an in vitro method for assessing the severity of prostate cancer in a subject in need thereof, the method comprising the steps of: providing a human monoclonal antibody or antigen-binding fragment thereof that selectively binds to prostasomes (S21); reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from the subject (S22); detecting the prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof to obtain a prostasome level (S23); and measuring the prostasome level using first and second ELISA assays. The method includes comparing the detected level of prostasomes with a predetermined threshold (S24), and assessing the severity of the prostate cancer (S25), wherein if the detected level of prostasomes is higher than a first predetermined threshold, the prostate cancer is assessed as severe (S25a), if the detected level of prostasomes is lower than a first predetermined threshold, the prostate cancer is assessed as moderate (S25b), and if the detected level of prostasomes is lower than a second predetermined threshold, the prostate cancer is assessed as mild (S25c). In one embodiment, the first threshold is 10 ng of prostasomes per mL of sample, and the second threshold is 1 ng of prostasomes per mL of sample.

[0097] In one aspect, detecting prostasomes bound by the human monoclonal antibody or its antigen-binding fragment (S23) comprises detecting prostasomes using an anti-prostasome detection antibody or its antigen-binding fragment. In a further aspect, the method is a sandwich immunoassay, and detecting prostasomes (200) bound by the human monoclonal antibody or its antigen-binding fragment (101) (S23) comprises detecting the anti-prostasome detection antibody (102) using an additional detection antibody (103), wherein the human monoclonal antibody or its antigen-binding fragment (101) is a capture antibody, the anti-prostasome detection antibody (102) is a primary detection antibody, and the additional detection antibody (103) is a secondary detection antibody. In a further aspect, the primary detection antibody (102) is a chicken antibody, and the secondary detection antibody (103) is an anti-chicken antibody. In one aspect, the sample from the subject is a body fluid sample. In a further aspect, the subject's bodily fluid sample is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and cell suspension. In one aspect, the human monoclonal antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment of the present disclosure. The severity of prostate cancer is typically described in the prior art in relation to the Gleason score, which may be linked to both aggressiveness and severity as well as prognosis. Men with biochemical recurrence, a PSA doubling time of less than 12 months, and / or a biopsy Gleason score of 8-10 are considered candidates for androgen deprivation therapy (ADT) according to European Association of Urology (EAU) guidelines. The prostasome assay of the present invention distinguished patients with high and intermediate Gleason scores (8 / 9 and 7, respectively) from patients with low scores (≤6), thus reflecting the aggressiveness of the disease. Therefore, a measured prostasome level greater than 1 ng / mL may correspond to a Gleason score greater than 6.

[0098] FIG. 8 shows an in vitro method for evaluating the effectiveness of a prostate cancer treatment in a subject in need thereof, the method comprising detecting a level of prostasomes in a sample from the subject before the prostate cancer treatment (S31), providing the subject with an anti-prostate cancer treatment (S32), detecting a level of prostasomes in a sample from the subject after the prostate cancer treatment (S33), comparing the level of prostasomes before the treatment with the level after the treatment (S34), and determining the effectiveness of the treatment (S35), wherein if the level of prostasomes after the treatment is reduced compared to the level before the treatment, the treatment is determined to be effective (S35a), and if the level of prostasomes is the same or increased, the treatment is determined to be ineffective (S35b).

[0099] In one aspect, detecting prostasomes bound by the human monoclonal antibody or its antigen-binding fragment (S33) comprises detecting prostasomes using an anti-prostasome detection antibody or its antigen-binding fragment. In a further aspect, the method is a sandwich immunoassay, and detecting prostasomes (200) bound by the human monoclonal antibody or its antigen-binding fragment (101) (S33) comprises detecting the anti-prostasome detection antibody (102) using an additional detection antibody (103), wherein the human monoclonal antibody or its antigen-binding fragment (101) is a capture antibody, the anti-prostasome detection antibody (102) is a primary detection antibody, and the additional detection antibody (103) is a secondary detection antibody. In a further aspect, the primary detection antibody (102) is a chicken antibody, and the secondary detection antibody (103) is an anti-chicken antibody. In one aspect, the sample from the subject is a body fluid sample. In a further aspect, the bodily fluid sample from the subject is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and a cell suspension. In one aspect, the human monoclonal antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment of the present disclosure.

[0100] Thus, the present disclosure makes it possible to diagnose prostate cancer, determine the prognosis of prostate cancer (which is associated with the stage and aggressiveness of the cancer), assess the severity of the cancer (which can be used as decision support for selecting a treatment (such as whether to perform surgery or not)), and evaluate a treatment plan for prostate cancer in a subject. The subject is typically a human male. The effectiveness of the treatment plan can be evaluated by measuring prostasome levels in samples before and after the treatment, which treatment includes both surgery, radiation therapy, and administration of pharmaceutical substances. Thus, the method can include measuring prostasome levels in accordance with the present invention, administering a therapeutic agent, and again measuring prostasome levels after the drug has been administered and has had a period of time to affect the cancer. The period of time will depend on the drug used, and will be apparent to those skilled in the art depending on the treatment used.

[0101] The drawings and specification disclose representative aspects of the present disclosure. However, many variations and modifications can be made to these aspects without substantially departing from the principles of the present disclosure. Accordingly, the present disclosure is to be regarded as illustrative rather than restrictive, and is not limited to the particular aspects discussed above. Accordingly, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0102] The descriptions of exemplary embodiments presented herein are provided for illustrative purposes. The description is not intended to be exhaustive or to limit the exemplary embodiments to the precise form disclosed; modifications and variations are possible in light of the above teachings or may arise from practicing various alternatives to the exemplary embodiments. The examples discussed herein are selected and described to explain the principles and properties of various exemplary embodiments and their practical applications, so that those skilled in the art can utilize the exemplary embodiments in various ways and with various modifications suited to the particular use contemplated. Features of the embodiments described herein can be combined in all possible combinations of methods, products, and systems. It should be recognized that the exemplary embodiments presented herein can be practiced in any combination with each other.

[0103] It should be noted that the word "comprising" does not necessarily exclude the presence of other elements or steps than those listed, and the word "a" preceding an element does not exclude the presence of a plurality of such elements. It should further be noted that any reference signs do not limit the scope of the claims, and that the example embodiments may be realized in the broadest sense of the claims.

[0104] While the present invention has been described with reference to various representative aspects and embodiments, those skilled in the art will recognize that various changes can be made without departing from the scope of the invention and that equivalents may be substituted for elements thereof. In addition, many modifications may be made to adapt a particular situation to the teachings of the invention without departing from its essential scope. Therefore, it is not intended that the present invention be limited to any particular embodiment; rather, the present invention is intended to include all embodiments falling within the scope of the appended claims. The present invention is further illustrated by the following non-limiting examples. [Example]

[0105] Example 1: Construction of a synthetic antibody fragment library

[0106] The synthetic scFv fragments scFv1-12 of the present invention are derived from the human synthetic antibody fragment libraries HeIL-11 and HeIL-13 generated by Sall et al. (Sall et al., Generation and synthesis of a human synthetic antibody library and its application to protein microarrays. Protein Eng Des Sel. 2016 Oct; 29(10): 427-437), aspects of which are summarized below.

[0107] Residues distributed throughout CDR-H1, CDR-H2, CDR-H3, and CDR-L3 were selected for diversification. Oligonucleotides targeting these loops were generated using a custom-made trimeric phosphoramidite mixture (TriLink BioTechnologies, San Diego, CA, USA) encoding the amino acid compositions shown in Figure 3. While one primer was used for each of CDR-H1 and CDR-H2, three or five primers, and ten or fifteen primers (one primer for each of these loops of allowed length) were used to diversify CDR-L3 and CDR-H3 of Hell-13 and Hell-11, respectively. The sequences encoded by the primers were introduced into the library scaffold gene using an optimized Kunkel mutagenesis method, and the Kunkel DNA was then electroporated into E. coli cells. Finally, scFv-displaying phages were recovered. Phage selection based on specific antigens was then performed to obtain specific antigen-binding fragments.

[0108] For example, Figure 3 shows the design of the HeIL-11 and HeIL-13 libraries. These two libraries have a similar design, and as shown in Figure 3a, both are built on a human scFv scaffold based on the heavy chain variable gene IGHV3-23 and the kappa light chain variable gene IGKV1-39 (for their full sequences, see Sall et al., "Generation and synthesis of human synthetic antibody libraries and their application to protein microarrays." Protein Eng Des Sel. 2016 Oct; 29(10): 427-437). These libraries were configured to contain diversity, represented by standard single-letter codes, at selected positions within and immediately adjacent to CDR-H1, CDR-H2, CDR-H3, and CDR-L3. Solvent-accessible residues in CDR-H1 and CDR-H2 were limited to tyrosine, serine, and glycine, whereas more complex diversity schemes were possible in CDR-H3 and CDR-L3. X and J each represent a mixture of 13 amino acids introduced in the proportions listed in Figure 3b. The allowed lengths for CDR-H3 and CDR-L3 in HelL-11 were 8–22 and 8–12 residues, respectively. In HelL-13, the corresponding lengths were 8–17 and 8–10 residues. (CDR boundaries and residue numbering are as specified by the IMGT nomenclature.)

[0109] The library design and construction are further illustrated in Figure 4. Figure 4a shows the amino acid sequence of the HeIL library scaffold. HeIL-11 and HeIL-13 are synthetic scFv libraries based on human scaffolds consisting of the heavy chain variable gene IGHV3-23 and the kappa light chain variable gene IGKV1-39, connected by a 15-amino acid Gly-Ser linker (underlined). CDRs H1, H2, H3, and L3 are indicated by boxes, and randomized positions are highlighted in gray. Because the Kunkel cloning procedure is not 100% efficient, stop codons were introduced into CDRs H3 and L3 of the HeIL-11 template and into CDR H3 (indicated by an *) of the HeIL-13 template gene. This ensured that only clones mutated within these regions were displayed on the surface of the phage. No stop codons were introduced into any other CDR loops, so unmutated versions of these loops will be found in the presented library. Template sequences for the first and second CDRs of both the heavy and light chains were selected to mimic sequences found in germline genes. Similarly, the IGKV1-39 sequence was selected for CDR-L3 (positions 105-115) of the HelL-13 scaffold gene. At position 116 of the VL, which is naturally derived from the J-segment, a tyrosine was included due to its generally favorable binding properties.

[0110] Figure 4b shows a "string of pearls" defining the positions that carry diversity (X) in the VH and VL design of the HeIL-11 and HeIL-13 antibody libraries. The diagram in this disclosure demonstrates that the sequences have CDR3 lengths of 13 (VH) and 8 (VL) residues, but the length of these hypervariable loops can vary depending on the design of the library. Residues defined by the IMGT numbering scheme but not present in these VH and VL sequences are represented by dotted circles.

[0111] Examples 2 to 5 below demonstrate different prostasome detection assays. Human monoclonal synthetic scFv fragments were generated (see Example 1 above) and screened against prostasome-coated microtiter plates. 64 positive clones were sequenced. Based on DNA sequencing, these positively selected clones represented 60 different human monoclonal antibodies.

[0112] Example 2: Testing of human monoclonal antibodies as scFv fragments 1 to 12

[0113] Purified prostasomes (1 mg / mL) were diluted 1:250 in PBS (phosphate-buffered saline) and 100 μL was added per well in a microtiter plate. The plate was incubated for 2 hours at room temperature and then washed three times with PBS containing 0.05% Tween® 20. 120 μL of 1% bovine serum albumin (BSA) was added to the wells, which were then incubated overnight at room temperature. The wells were washed three times with PBS containing 0.05% Tween® 20. Biotinylated human synthetic scFv fragments were added 1:250 to row A, followed by 1:5 dilutions downward. The plate was then incubated for 2 hours at room temperature and then washed three times with PBS containing 0.05% Tween® 20. 100 μL of streptavidin-HRP was added to the wells and incubated for 2 hours at room temperature. The wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of TMB (tetramethylbenzidine) substrate was added to the wells and incubated for 20 minutes, after which 25 μL of 1 M H2SO4 was added to the plate. The plate was read at 450 nm.

[0114] Seminal plasma was collected from the Reproduction Centre at Akademiska Hospital (Uppsala, Sweden) according to a previously described method (Ronquist, GK, et al., Characterization of prostasomal DNA and its transfer into human sperm. Mol Reprod Dev, 2011. 78(7): pp. 467-76). Seminal plasma was thawed and centrifuged at 3,000 g for 12 min. The supernatant was collected and centrifuged at 10,000 g for 30 min. The supernatant was then transferred to a new tube and ultracentrifuged at 100,000 g for 2 h in a 90Ti rotor (Beckman Coulter, Brea, CA, USA). The pellet was resuspended overnight at 4°C in 0.02 M NaH2PO4, 0.15 M NaCl, pH 7.2 (PBS). The resuspended pellet was then loaded onto a chromatography column (XK 60 / 70, GE Healthcare, Uppsala, Sweden) packed with Superdex 200 gel. Fractions were collected at a flow rate of 5 mL / h. The fractions were then measured spectrophotometrically. Both peaks at 260 nm (nucleic acid) and 280 nm (protein) corresponded to prostasomes. These fractions were collected, pooled, and ultracentrifuged at 100,000 g. The resulting pellet was resuspended in PBS. Purified prostasomes (1 mg / ml) were diluted 1:250 in PBS and 100 μL was added per well of an F96 Polysorb NUNC Immunoplate (Thermo Fisher Scientific, Uppsala, Sweden). The plate was incubated for 2 hours at ambient temperature and then washed three times with PBS containing 0.05% Tween® 20 (P1379, Sigma-Aldrich). 120 μL of PBS containing 1% bovine serum albumin (BSA, Sigma-Aldrich) was added to the wells, and the plate was incubated overnight at ambient temperature. The plate was washed three times with PBS containing 0.05% Tween® 20. 100 μL of biotinylated monoclonal antibody diluted to 4 μg / mL in PBS was added to the wells.After incubating the plate at ambient temperature for 2 hours, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of streptavidin-HRP (43-8322, Thermo Fisher Scientific) diluted 1:2000 in PBS was added to the plate. After incubating the plate at ambient temperature for 2 hours, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of TMB substrate (EC-Blue Enhanced TMB Substrate, Medicago, Uppsala, Sweden) was added to the plate, and the plate was incubated in the dark at ambient temperature for 20 minutes. 25 μL of 1 M H2SO4 was added to each well, and the plate was read at 450 nm in a SpectraMax 250 ELISA reader.

[0115] The results for the individual human monoclonal scFv fragments 1-12 are shown in Table 4 below.

[0116] [Table 4]

[0117] One conclusion from this experiment is that all synthetic scFv fragments give a positive response as detection antibodies when tested against purified prostasomes.

[0118] Example 3: Testing of chicken polyclonal antibodies as detectors

[0119] Purified prostasomes (1 mg / mL) were diluted 1:250 in PBS and 100 μL was added per well of a microtiter plate. The plate was incubated for 2 hours at room temperature and then washed three times with PBS containing 0.05% Tween® 20. 120 μL of 1% bovine serum albumin (BSA) was added to the wells, which were then incubated overnight at room temperature. The wells were washed three times with PBS containing 0.05% Tween® 20. Chicken anti-prostasome antibody was added to rows 1 and 2, and chicken anti-PSA antibody was added to rows 3 and 4. An initial dilution of 1:125 was used in row A, followed by 1:5 dilutions downward (except dilutions were performed in PBS-Tween®). The plate was then incubated for 1 hour at room temperature and then washed three times with PBS containing 0.05% Tween® 20. 100 μL of anti-chicken IgY-HRP diluted in PBS-Tween® was added to the wells and incubated for 1 hour at room temperature. The wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of TMB substrate was added to the wells and incubated for 10 minutes, after which 25 μL of 1 M H2SO4 was added to the plate. The plate was read at 450 nm.

[0120] Purified prostasomes (1 mg / mL) were diluted 1:250 in PBS and 100 μL was added per well of an F96 Polysorb NUNC Immunoplate (Thermo Fisher Scientific, Uppsala, Sweden). The plate was incubated at ambient temperature for 2 hours and then washed three times with PBS containing 0.05% Tween® 20 (P1379, Sigma-Aldrich). 120 μL of PBS containing 1% bovine serum albumin (BSA, Sigma-Aldrich) was added to the wells, and the plate was incubated overnight at ambient temperature. The plate was washed three times with PBS containing 0.05% Tween® 20. 100 μL of chicken anti-prostasome and chicken anti-PSA antibodies (Immunsystem AB, Uppsala, Sweden) diluted 1:125 in PBS containing 0.05% Tween® 20 were added to the wells. After incubating the plate at ambient temperature for 1 hour, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of chicken anti-IgY-HRP (A16130, Novex, Frederick, MD, USA) diluted 1:2000 in PBS-Tween® was added to the plate. After incubating the plate at ambient temperature for 1 hour, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of TMB substrate (EC-Blue Enhanced TMB Substrate, Medicago, Uppsala, Sweden) was added to the plate, and the plate was incubated in the dark at ambient temperature for 20 minutes. 25 μL of 1 M H2SO4 was added to each well, and the plate was read at 450 nm in a SpectraMax 250 ELISA reader. The results for these antibodies are shown in Table 5 below.

[0121] [Table 5]

[0122] One conclusion from this experiment is that both chicken antibodies gave positive responses as detection antibodies when tested against purified prostasomes. The tested antibodies reacted strongly and gave strong signals due to the polyclonal nature of the antibodies, which have many epitopes. Therefore, the polyclonal chicken antibodies are well suited for use as primary detection antibodies to detect prostasomes bound to the capture antibodies of the present invention.

[0123] Example 4: Testing of purified prostasomes added to normal plasma

[0124] Human synthetic scFv fragment 4 (2 μg / mL) and human synthetic scFv fragment 7 (2 μg / mL) were coated in PBS. 100 μL was added per well and incubated at room temperature for 2 hours, followed by washing three times with PBS containing 0.05% Tween® 20. 120 μL of 1% bovine serum albumin (BSA) was added to the wells, which were then incubated overnight at room temperature. After incubation, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of prostasomes diluted to 1 ng / mL in plasma from a female blood donor was added. 100 μL was added to row 1 and incubated for 2 hours at room temperature. Both chicken anti-prostasome antibody and chicken anti-PSA antibody, diluted 1:1000 in PBS-Tween®, were added. The plates were incubated at room temperature for 2 hours and then washed three times with PBS containing 0.05% Tween® 20. 100 μL of anti-chicken IgY-HRP diluted in PBS-Tween® was added to the wells and incubated for 2 hours at room temperature, followed by washing three times with PBS containing 0.05% Tween® 20. 100 μL of TMB substrate was added to the wells and incubated for 10 minutes, after which 20 μL of 1 M H2SO4 was added to the plate. The plate was read at 450 nm.

[0125] 100 μL of human synthetic scFv fragment 4 (final concentration: 2 μg / mL in PBS) and scFv fragment 7 (final concentration: 2 μg / mL in PBS) were added separately to each well of an F96 Polysorb NUNC Immunoplate (Thermo Fisher Scientific, Uppsala, Sweden). The plate was incubated at ambient temperature for 2 hours and then washed three times with PBS containing 0.05% Tween® 20 (P1379, Sigma-Aldrich). 120 μL of PBS containing 1% bovine serum albumin (BSA, Sigma-Aldrich) was added to each well, and the plate was incubated overnight at ambient temperature. The plate was washed three times with PBS containing 0.05% Tween® 20. 1 ng / mL of purified prostasomes (final concentration) was diluted in plasma from a female human blood donor, and 100 μL was added to each well. After incubating the plate at ambient temperature for 2 hours, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of chicken anti-prostasome and chicken anti-PSA antibodies (Immunsystem AB, Uppsala, Sweden) diluted 1:1000 in PBS containing 0.05% Tween® 20 were added to the wells separately. After incubating the plate at ambient temperature for 2 hours, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of anti-IgY-HRP (A16130, Novex, Frederick, MD, USA) diluted 1:2000 in PBS-Tween® was added to the plate. After incubating the plate at ambient temperature for 2 hours, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of TMB substrate (EC-Blue Enhanced TMB Substrate, Medicago, Uppsala, Sweden) was added to the plate, and the plate was incubated in the dark at ambient temperature for 20 minutes. 25 μL of 1 M H2SO4 was added to each well, and the plate was read at 450 nm in a SpectraMax 250 ELISA reader.The results for the scFv fragments are shown in Table 6 below.

[0126] [Table 6]

[0127] In previous experiments, the use of chicken detection antibodies was investigated using purified prostasomes. In this example, we investigated how well the antibodies performed when the prostasomes were diluted in a sample (such as serum or plasma). One conclusion from this experiment was that both chicken antibodies tested gave a positive response even in the presence of human plasma, indicating that they can also be used to detect prostasomes in plasma.

[0128] Example 5: Testing of patient samples, patients with elevated PSA (over 50), female blood donors, and male blood donors (under 35 years old)

[0129] Human synthetic scFv fragment 4 (2 μg / mL), human synthetic scFv fragment 7 (2 μg / mL), and human synthetic scFv fragment 10 (2 μg / mL) were coated in PBS. 100 μL was added per well, incubated at room temperature for 2 hours, and washed three times with PBS containing 0.05% Tween® 20. 120 μL of 1% bovine serum albumin (BSA) was added to the wells, incubated overnight at room temperature, and washed three times with PBS containing 0.05% Tween® 20. 100 μL of patient sample was added, and the plate was incubated at room temperature for 2 hours. Chicken anti-PSA diluted 1:1000 in PBS-Tween® was added, incubated at room temperature for 2 hours, and washed three times with PBS containing 0.05% Tween® 20. 100 μL of anti-chicken IgY-HRP diluted 1:2000 in PBS-Tween® was added to the wells and incubated for 2 hours at room temperature before washing three times with PBS containing 0.05% Tween® 20. 100 μL of TMB substrate was added to the wells and incubated for 10 minutes before adding 20 μL of 1 M H2SO4 to the plate. The plate was read at 450 nm.

[0130] One hundred microliters of human synthetic scFv fragment 4 (final concentration: 2 μg / mL in PBS), human synthetic scFv fragment 7 (final concentration: 2 μg / mL in PBS), and human synthetic scFv fragment 10 (final concentration: 2 μg / mL in PBS) were added per well of an F96 Polysorb NUNC Immunoplate (Thermo Fisher Scientific, Uppsala, Sweden). After incubating the plate at ambient temperature for 2 hours, the wells were washed three times with PBS containing 0.05% Tween® 20 (P1379, Sigma-Aldrich). One hundred twenty microliters of PBS containing 1% bovine serum albumin (BSA, Sigma-Aldrich) was added to the wells, and the plate was incubated overnight at ambient temperature. The plate was washed three times with PBS containing 0.05% Tween® 20. 100 μL of samples from patients with elevated PSA (>50 μg / L, labeled PSA in the table), female blood donors (controls), and male blood donors under 35 years of age (served as negative controls because young male blood donors rarely have prostate cancer) were added to the wells. After incubating the plates for 2 hours at ambient temperature, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of chicken anti-prostasome and chicken anti-PSA antibodies (Immunsystem AB, Uppsala, Sweden) diluted 1:1000 in PBS containing 0.05% Tween® 20 were added to the wells. After incubating the plates for 2 hours at ambient temperature, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of chicken anti-IgY-HRP (A16130, Novex, Frederick, MD, USA) diluted 1:2000 in PBS-Tween® was added to the plate. After incubating the plate at ambient temperature for 2 hours, the wells were washed three times with PBS containing 0.05% Tween® 20.100 μL of TMB substrate (EC-Blue Enhanced TMB Substrate, Medicago, Uppsala, Sweden) was added to the plate, and the plate was incubated in the dark at ambient temperature for 20 minutes. 25 μL of 1 M H2SO4 was added to each well, and the plate was read at 450 nm in a SpectraMax 250 ELISA reader. The results for the scFv fragments are shown in Tables 7-9 below.

[0131] [Table 7]

[0132] [Table 8]

[0133] [Table 9]

[0134] One conclusion from this experiment is that PSA-positive plasma samples yielded greater values ​​compared to female and younger male blood donors, indicating it is a marker for prostate cancer.

[0135] Example 6: Testing of mouse monoclonal antibodies as detectors in prostasome ELISA

[0136] Seminal plasma was collected from the Reproduction Centre at Akademiska Hospital (Uppsala, Sweden) according to a previously described method (Ronquist, GK, et al., Characterization of prostasomal DNA and its transfer into human sperm. Mol Reprod Dev, 2011. 78(7): pp. 467-76). Seminal plasma was thawed and centrifuged at 3,000 g for 12 min. The supernatant was collected and centrifuged at 10,000 g for 30 min. The supernatant was then transferred to a new tube and ultracentrifuged at 100,000 g for 2 h in a 90Ti rotor (Beckman Coulter, Brea, CA, USA). The pellet was resuspended overnight at 4°C in 0.02 M NaH2PO4, 0.15 M NaCl, pH 7.2 (PBS).

[0137] The resuspended pellet was then loaded onto a chromatography column (XK 60 / 70, GE Healthcare, Uppsala, Sweden) packed with Superdex 200 gel. Fractions were collected at a flow rate of 5 mL / h. The fractions were then measured spectrophotometrically. Both peaks at 260 nm (nucleic acid) and 280 nm (protein) corresponded to prostasomes. These fractions were collected, pooled, and ultracentrifuged at 100,000 g. The resulting pellet was resuspended in PBS.

[0138] Monoclonal antibodies were added in duplicate to each well of an F96 Polysorb NUNC Immunoplate (Thermo Fisher Scientific, Uppsala, Sweden) at a final concentration of 1 μg / mL in PBS (100 μL). The mouse monoclonal antibodies used for coating were anti-CD10 (0112, Immunotech, Marseille, France), anti-CD46 (0846, Immunotech), anti-CD38 (Sanquin, Amsterdam, The Netherlands), anti-CD59 (Sanquin), anti-CD13 (C8589, Sigma-Aldrich, St. Louis, MO, USA), and anti-PSMA (Invitrogen, Carlsbad, CA). Plates were incubated at ambient temperature for 2 hours and then washed three times with PBS containing 0.05% Tween® 20 (P1379, Sigma-Aldrich). 120 μL of PBS containing 1% bovine serum albumin (BSA, Sigma-Aldrich) was added to each well, and the plate was incubated overnight at ambient temperature. The plate was washed three times with PBS containing 0.05% Tween® 20. 100 μL of purified prostasomes diluted to 100 ng / mL in PBS was added to each well. According to a previous study (Tavoosidana et al., PNAS, May 24, 2011, vol. 108, no. 21, 8809-8814), the sensitivity of detecting prostasomes in the blood of prostate cancer patients is approximately 1 ng / mL. Therefore, the concentrations examined are well above this level.

[0139] After incubating the plate for 2 hours at ambient temperature, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of chicken anti-PSA antibody (Immunsystem AB, Uppsala, Sweden) diluted 1:1000 in PBS containing 0.05% Tween® 20 was added to the wells. The plate was incubated for 2 hours at ambient temperature, and the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of anti-IgY-HRP (A16130, Novex, Frederick, MD, USA) diluted 1:2000 in PBS-Tween® was added to the plate. After incubating the plate for 2 hours at ambient temperature, the wells were washed three times with PBS containing 0.05% Tween® 20. 100 μL of TMB substrate (EC-Blue Enhanced TMB Substrate, Medicago, Uppsala, Sweden) was added to the plate, and the plate was incubated in the dark at ambient temperature for 20 minutes. 25 μL of 1 M H2SO4 was added to each well, and the plate was read at 450 nm in a SpectraMax 250 ELISA reader. None of the antibodies tested gave a positive response.

[0140] Thus, the examples contained herein demonstrate that the proposed human monoclonal antibodies or binding fragments thereof are effective in selectively detecting low concentrations of prostasomes in a sample, whereas their murine counterparts are unable to detect low levels of prostasomes and are therefore not suitable for use in diagnosing prostate cancer.

[0141] Example 7: Testing the prognostic efficacy of fragments 4 and 7

[0142] A study was conducted to evaluate the ability to predict the severity and outcome of a patient / subject based on prostasome levels measured according to one embodiment of the present disclosure. The efficacy of fragments 4 and 7 was evaluated.

[0143] Blood samples were collected from patients with prostate cancer before surgery. Samples were collected between 1991 and 2001. After centrifugation, the resulting serum samples were stored at -70°C. Follow-up data regarding death and cause of death were extracted from patient files in February 2021. The endpoint used was death at follow-up after excluding patients with undocumented tumor recurrence. Eighty-one samples were thawed and analyzed using ScFv fragment 4 (AK4) and ScFv fragment 7 (AK7) as capture antibodies (capture ScFv fragments) and chicken anti-prostasome antibody as a detector. Fifty-one of these samples were also analyzed using AK4 and AK7 as capture antibodies (capture ScFv fragments) and chicken anti-PSA antibody as a detector.

[0144] For both experiments using AK4 and AK7 in combination with anti-prostasome antibodies (Pros) as a detector, higher measured levels of prostasomes correlated with death, whereas lower levels correlated with survival after 20 years or more. AK7 in combination with anti-PSA antibodies (PSA) as a detector was significantly associated with expected death or survival after 20 years or more. These results are shown in Table 10 below.

[0145] [Table 10]

[0146] Example 8: Evaluation of fragment binding targets using immunoprecipitation mass spectrometry

[0147] Immunoprecipitation coupled with mass spectrometry (IP-MS) can be used to verify that antibodies specifically interact with their putative targets. To clarify the targets on prostasomes to which each of fragments 1–12 bind, the binding of these fragments to dissolved prostasomes was examined using liquid chromatography (LC)-IP-MS analysis.

[0148] Purified prostasomes were dissolved in a high-salt buffer using a freeze-thaw and sonication procedure. Debris was removed by centrifugation, and the prostasome solution was mixed with magnetic beads, each coated with one of the antibody fragments 1–12. After 2 hours of incubation at 4°C, the beads were washed three times with a buffer containing 10 mM Tris-HCl, pH 7.9, 100 mM NaCl, and 0.1% (v / v) Nonidet P-40, and twice with the same buffer without detergent. The sample was eluted from the beads using water containing 0.5 M NH4OH. The eluate was dried in a Speed-Vac and then reconstituted in 50 μL of 50 mM ammonium bicarbonate. The sample was then reduced with 100 mM TCEP-HCl, alkylated with 500 mM iodoacetamide, and digested with trypsin.

[0149] The samples were then subjected to liquid chromatography (LC)-MS analysis. The enzyme specificity was set to trypsin, and a search was performed against human proteins. The top five proteins (Homo sapiens) identified in the complexes bound to each antibody fragment (1–12) are listed in Table 11 below.

[0150] [Table 11-1] [Table 11-2]

[0151] These results indicated that each of the individual fragments 1 to 12 of the present invention binds to several targets, rather than a single target protein / lipid / carbohydrate. The top targets identified by IP-MS analysis revealed that the targets bound by these fragments were not individual targets but complexes formed by several individual proteins / lipids / carbohydrates. The binding strength to each individual target, the pure antigen, in the dissolved prostasome sample was much weaker than the binding to the complex (which consisted of several individual targets aggregated or bound to prostasome membrane fragments), indicating that the scFv fragments actually bind to the three-dimensional structure or three-dimensional arrangement of the target protein on the surface of the prostasome. Therefore, the antigens bound by the scFv fragments of the present invention are not individual, isolated antigens, but rather structures on the surface of the prostasome membrane, or of aggregates or complexes of antigens bound to prostasome membrane fragments, making them ideal for measuring total prostasomes in a sample. Therefore, the proteins in Table 11 should not be viewed as individual targets / antigens bound by each fragment, but as part of the complex to which each fragment binds.

[0152] Itemized List of Embodiments

[0153] 1. A human monoclonal antibody or antigen-binding fragment thereof that selectively binds to prostasomes.

[0154] 2. A human monoclonal antibody or antigen-binding fragment thereof according to paragraph 1, which is a full-length antibody, an antigen-binding (Fab) fragment, or an antigen-binding single-chain Fv (scFv) fragment.

[0155] 3. A human monoclonal antibody or antigen-binding fragment thereof according to paragraph 1 or 2, which is a human synthetic scFv fragment.

[0156] 4. A human monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the antibody or antigen-binding fragment thereof enables a sensitivity of at least 10 ng / mL in an immunoassay using the human monoclonal antibody or antigen-binding fragment thereof as a capture antibody.

[0157] 5. A human monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 4, which selectively binds to prostasomes by binding to one or more prostasome surface antigens selected from the group consisting of SEQ ID NOs: 60 to 104.

[0158] 6. A human monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 5, comprising a heavy chain complementarity-determining region (CDR) selected from SEQ ID NOs: 1 to 12 and a light chain CDR selected from SEQ ID NOs: 13 to 24, and a CDR sequence that is 95% or more identical to these (e.g., 96%, 97%, 98%, 99%, or more).

[0159] 7. At least four complementarity determining regions (CDRs) in any combination of CDR-H1, CDR-H2, CDR-H3, and CDR-L3, wherein the selection of the CDRs is: CDR-H1 selected from SEQ ID NOs: 25, 27, and 28; CDR-H2 selected from SEQ ID NOs: 26, 29, and 30; a CDR-H3 selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; A human monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 6, comprising a CDR-L3 selected from SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, and a CDR sequence that is 95% or more identical thereto (e.g., 96%, 97%, 98%, 99%, or more).

[0160] 8. The antibody comprises at least six complementarity determining regions (CDRs) in any combination of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein the selection of the CDRs is: CDR-H1 selected from SEQ ID NOs: 25, 27, and 28; CDR-H2 selected from SEQ ID NOs: 26, 29, and 30; a CDR-H3 selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; CDR-L1 selected from SEQ ID NOs: 56 and 57; CDR-L2 selected from SEQ ID NOs: 58 and 59; A human monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 7, comprising a CDR-L3 selected from SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, and a CDR sequence that is 95% or more identical thereto (e.g., 96%, 97%, 98%, 99%, or more).

[0161] 9. A human monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8, comprising a heavy chain variable region (VH) sequence selected from the group consisting of SEQ ID NOs: 32 to 43 and sequences that are 80% or more identical thereto (e.g., 85%, 90%, 95%, or more).

[0162] 10. A human monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8, comprising a light chain variable region (VL) sequence selected from the group consisting of SEQ ID NOs: 44 to 55 and sequences that are 80% or more identical thereto (e.g., 85%, 90%, 95%, or more).

[0163] 11. A human monoclonal antibody or antigen-binding fragment thereof according to paragraphs 9 and 10, which is a VH fragment according to paragraph 9, a VL fragment according to paragraph 10, and a synthetic scFv fragment comprising a linker.

[0164] 12. The human monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 11, which is a synthetic scFv fragment selected from the group consisting of scFv fragment 4, scFv fragment 1, scFv fragment 2, scFv fragment 3, scFv fragment 5, scFv fragment 6, scFv fragment 7, scFv fragment 8, scFv fragment 9, scFv fragment 10, scFv fragment 11, and scFv fragment 12.

[0165] 13. The human monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 12, which is scFv fragment 4.

[0166] 14. An in vitro method for determining the presence of prostate cancer in a subject, comprising: providing a human monoclonal antibody or an antigen-binding fragment thereof that selectively binds to prostasomes (S1); reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from a subject (S2); detecting any prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof to obtain the level of prostasomes (S3); comparing said level of prostasomes with a predetermined threshold (S4); and determining (S5) that prostate cancer is present in the subject if the detected level of prostasomes is higher than the predetermined threshold.

[0167] 15. The method according to paragraph 14, further comprising determining (S5) that the subject is free of prostate cancer if the detected level of prostasomes is lower than the predetermined threshold.

[0168] 16. The method according to item 14 or 15, wherein detecting any prostasomes bound by the human monoclonal antibody or its antigen-binding fragment (S3) comprises detecting the prostasomes using an anti-prostasome detection antibody or its antigen-binding fragment.

[0169] 17. The method according to item 16, which is a sandwich immunoassay, and wherein detecting (S3) the prostasomes (200) bound by the human monoclonal antibody or its antigen-binding fragment (101) comprises detecting the anti-prostasome detection antibody (102) using a further detection antibody (103), wherein the human monoclonal antibody or its antigen-binding fragment (101) is a capture antibody, the anti-prostasome detection antibody (102) is a primary detection antibody, and the further detection antibody (103) is a secondary detection antibody.

[0170] 18. The method according to paragraph 17, wherein the primary detection antibody (102) is a chicken antibody and the secondary detection antibody (103) is an anti-chicken antibody.

[0171] 19. The method according to any one of paragraphs 14 to 18, wherein the sample from the subject is a body fluid sample.

[0172] 20. The method according to paragraph 19, wherein the body fluid sample from the subject is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and a cell suspension.

[0173] 21. The method according to any one of paragraphs 19 or 20, wherein the predetermined threshold is 10 ng of prostasomes per mL of body fluid sample.

[0174] 22. The method according to any one of items 14 to 21, wherein the human monoclonal antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof according to any one of items 1 to 13.

[0175] 23. An in vitro method for providing a prognosis for prostate cancer in a subject in need thereof, comprising: Providing a human monoclonal antibody or an antigen-binding fragment thereof that selectively binds to prostasomes (S11); Reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from a subject (S12); detecting prostasomes bound to the human monoclonal antibody or its antigen-binding fragment to obtain the level of prostasomes (S13); Comparing the level of prostasomes with first and second predetermined thresholds (S14); and and providing a prognosis for the prostate cancer (S15), wherein the prognosis for the prostate cancer is defined as poor if the detected level of prostasomes is higher than a first predetermined threshold (S15a), and the prognosis is defined as good if the detected level of prostasomes is lower than a second predetermined threshold (S15b).

[0176] 24. The method according to paragraph 23, wherein the sample from the subject is a body fluid sample.

[0177] 25. The method according to paragraph 24, wherein the body fluid sample from the subject is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and a cell suspension.

[0178] 26. The method according to any one of paragraphs 24 to 25, wherein the first predetermined threshold is 10 ng of prostasomes per mL of body fluid sample and the second predetermined threshold is 1 ng of prostasomes per mL of body fluid sample.

[0179] 27. The method according to any one of items 23 to 26, wherein the human monoclonal antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof according to any one of items 1 to 13.

[0180] 28. An in vitro method for assessing the severity of prostate cancer in a subject in need thereof, comprising: providing a human monoclonal antibody or an antigen-binding fragment thereof that selectively binds to prostasomes (S21); reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from the subject (S22); detecting prostasomes bound by the human monoclonal antibody or its antigen-binding fragment to obtain the level of prostasomes (S23); comparing the level of prostasomes with first and second predetermined thresholds (S24); and The method includes assessing the severity of the prostate cancer (S25), wherein the prostate cancer is assessed as severe if the detected level of prostasomes is higher than a first predetermined threshold (S25a), as moderate if the detected level of prostasomes is lower than the first predetermined threshold but higher than a second predetermined threshold (S25b), or as mild if the detected level of prostasomes is lower than the second predetermined threshold (S25c).

[0181] 29. The method according to paragraph 28, wherein the sample from the subject is a body fluid sample.

[0182] 30. The method according to paragraph 29, wherein the body fluid sample from the subject is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and a cell suspension.

[0183] 31. The method according to any one of paragraphs 29 to 30, wherein the first predetermined threshold is 10 ng of prostasomes per mL of body fluid sample and the second predetermined threshold is 1 ng of prostasomes per mL of body fluid sample.

[0184] 32. The method according to any one of items 28 to 31, wherein the human monoclonal antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof according to any one of items 1 to 14.

[0185] 33. An in vitro method for assessing the efficacy of a prostate cancer treatment in a subject in need thereof, comprising: detecting a level of prostasomes in a sample from a subject prior to prostate cancer treatment (S31); providing an anti-prostate cancer treatment to the subject (S32); Detecting the level of prostasomes in a sample from the subject after the prostate cancer treatment (S33); Comparing the pre-treatment prostasome level with the post-treatment level (S34), and The method includes determining the effectiveness of the treatment (S35), wherein if the level of prostasomes after the treatment is decreased compared to the level before the treatment, the treatment is determined to be effective (S35a), and if the level of prostasomes remains the same or increases, the treatment is determined to be ineffective (S35b).

[0186] 34. Detecting the level of prostasomes in a sample from a subject (S31, S33), Providing a human monoclonal antibody or an antigen-binding fragment thereof that selectively binds to prostasomes (S31a, S33a); reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from the subject (S31b, S33b); Item 34. The method according to Item 33, comprising detecting prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof to obtain the level of prostasomes (S31c, S33c).

[0187] 35. The method according to any one of paragraphs 33 or 34, wherein the sample from the subject is a body fluid sample.

[0188] 36. The method according to paragraph 35, wherein the body fluid sample from the subject is selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and a cell suspension.

[0189] 37. The method according to any one of items 34 to 36, wherein the human monoclonal antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof according to any one of items 1 to 13.

[0190] 38. An antibody or antigen-binding fragment thereof according to any one of paragraphs 1 to 13 for use in a method according to any one of paragraphs 14 to 37.

[0191] 39. A method of treating a subject having prostate cancer, comprising: Providing a human monoclonal antibody or an antigen-binding fragment thereof that selectively binds to human prostasomes; reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample containing prostasomes from a subject; detecting prostasomes bound by the human monoclonal antibody or its antigen-binding fragment to obtain the level of prostasomes; comparing said level of prostasomes to a predetermined first threshold; determining that prostate cancer is present in the subject if the detected level of prostasomes is greater than the first predetermined threshold; determining that the detected level of prostasomes is lower than the second predetermined threshold; treating said subject in need thereof by performing surgery for said prostate cancer.

[0192] All references cited herein are incorporated by reference to the extent permitted.

Claims

1. A human monoclonal antibody or antigen-binding fragment thereof that selectively binds to prostasomes, comprising at least six complementarity-determining regions (CDRs): CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; The human monoclonal antibody or antigen-binding fragment thereof i) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 4, CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 16 an scFv fragment having ii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 7 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 19 an scFv fragment having iii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 1 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 13 an scFv fragment having iv) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 2 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 14 an scFv fragment having v) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 3 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 15 an scFv fragment having vi) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 5 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 17 an scFv fragment having vii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 6 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 18 an scFv fragment having viii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 8, CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 20 an scFv fragment having ix) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 9 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 21 an scFv fragment having x) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 10 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 22 an scFv fragment having xi) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 11 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 23 an scFv fragment having xii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 12 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 24 an antigen-binding single-chain Fv (scFv) fragment selected from the group consisting of: The human monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof selectively binds to prostasomes by binding to multiple prostasome surface antigens, and the prostasome surface antigens are selected from the group consisting of SEQ ID NOs: 60 to 104.

2. The human monoclonal antibody or its antigen-binding fragment described in claim 1, wherein the monoclonal antibody or its antigen-binding fragment selectively binds to prostasomes by binding to multiple prostasome surface antigens, wherein the prostasome surface antigens form clusters on the surface of the prostasome membrane, and the clusters are identified by the monoclonal antibody or its antigen-binding fragment, to which the monoclonal antibody or its antigen-binding fragment binds.

3. the mass comprises at least five antigens selected from Table 11; The human monoclonal antibody or its antigen-binding fragment described in claim 2, wherein the antibody or its antigen-binding fragment enables a sensitivity of at least 10 ng / mL in an immunoassay using the human antibody or its antigen-binding fragment as a capture antibody in the immunoassay. Table 1-1 Table 1-2

4. The human monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is an antigen-binding single-chain Fv (scFv) fragment.

5. The human monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) sequence selected from the group consisting of SEQ ID NOs: 32 to 43 and sequences that are 90% or more identical thereto, and a light chain variable region (VL) sequence selected from the group consisting of SEQ ID NOs: 44 to 55 and sequences that are 90% or more identical thereto.

6. the antibody or antigen-binding fragment thereof i) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 4 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 16 an scFv fragment having ii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 7 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 19 an scFv fragment having iii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 1 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 13 an scFv fragment having iv) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 2 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 14 an scFv fragment having v) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 3 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 15 an scFv fragment having vi) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 5 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 17 an scFv fragment having vii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 6 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 18 an scFv fragment having viii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO:8 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 20 an scFv fragment having ix) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 9 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 21 an scFv fragment having x) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 10 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 22 an scFv fragment having xi) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 11 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 23 an scFv fragment having xii) CDR-H1 defined by SEQ ID NO: 25 CDR-H2 defined by SEQ ID NO: 26 CDR-H3 defined by SEQ ID NO: 12 CDR-L1 defined by SEQ ID NO: 56 CDR-L2 defined by SEQ ID NO: 58, and CDR-L3 defined by SEQ ID NO: 24 2. The human monoclonal antibody or antigen-binding fragment thereof of claim 1, which is a synthetic scFv fragment selected from the group consisting of:

7. the antibody or antigen-binding fragment thereof is a synthetic scFv fragment having a variable heavy chain VH and a variable light chain VL connected via a linker, and selecting the fragment comprises: i) a VH defined by SEQ ID NO: 35, and VL defined by SEQ ID NO: 47 an scFv fragment having ii) a VH defined by SEQ ID NO: 38, and VL defined by SEQ ID NO: 50, an scFv fragment having iii) a VH defined by SEQ ID NO: 32, and VL defined by SEQ ID NO: 44, an scFv fragment having iv) a VH defined by SEQ ID NO: 33, and VL defined by SEQ ID NO: 45, an scFv fragment having v) a VH defined by SEQ ID NO: 34, and VL defined by SEQ ID NO: 46, an scFv fragment having vi) a VH defined by SEQ ID NO: 36, and VL defined by SEQ ID NO: 48, an scFv fragment having vii) VH defined by SEQ ID NO: 37, and VL defined by SEQ ID NO: 49, an scFv fragment having viii) VH defined by SEQ ID NO: 39, and VL defined by SEQ ID NO: 51, an scFv fragment having ix) a VH defined by SEQ ID NO: 40, and VL defined by SEQ ID NO: 52, an scFv fragment having x) a VH defined by SEQ ID NO: 41, and VL defined by SEQ ID NO: 53, an scFv fragment having xi) a VH defined by SEQ ID NO: 42, and VL defined by SEQ ID NO: 54, an scFv fragment having xii) VH defined by SEQ ID NO: 43, and VL defined by SEQ ID NO: 55, The human monoclonal antibody or antigen-binding fragment thereof according to claim 6, which is made from the group including an scFv fragment having the following structure:

8. The human monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is a synthetic scFv fragment, and the scFv fragment binds to a cluster containing titin isoform 12, myosin-1, myosin-2, myosin-4, and myosin-8.

9. 1. An in vitro method for determining the level of prostasomes in a sample from a subject, said method comprising: Providing a human monoclonal antibody or an antigen-binding fragment thereof that selectively binds to human prostasomes (S1, S11, S21); Reacting the human monoclonal antibody or antigen-binding fragment thereof with a sample from a subject potentially containing prostasomes (S2, S12, S22); detecting the prostasomes using an anti-prostasome detection antibody or an antigen-binding fragment thereof, thereby detecting any prostasomes bound by the human monoclonal antibody or an antigen-binding fragment thereof, thereby obtaining the level of prostasomes (S3, S13, S23, S31-33); Including, The method, wherein the human monoclonal antibody or antigen-binding fragment thereof is the human monoclonal antibody or antigen-binding fragment thereof of claim 1.

10. comparing the detected level of prostasomes with a predetermined threshold (S4); and determining that prostate cancer is present in the subject if the detected level of prostasomes is higher than the predetermined threshold (S5A); determining that the subject does not have clinically detectable prostate cancer if the detected level of prostasomes is lower than the predetermined threshold (S5B); further comprising wherein the sample from the subject is a bodily fluid sample selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and a cell suspension; and 10. The method of claim 9, wherein the predetermined threshold is 10 ng of prostasomes per mL of body fluid sample.

11. the method is a sandwich immunoassay, and detecting (S3) the prostasomes (200) bound to the human monoclonal antibody or its antigen-binding fragment (101) comprises detecting the anti-prostasome detection antibody (102) using a further detection antibody (103); The human monoclonal antibody or antigen-binding fragment thereof (101) is a capture antibody, the anti-prostasome detection antibody (102) is a primary detection antibody, and the further detection antibody (103) is a secondary detection antibody; and 11. The method of claim 10, wherein the primary detection antibody (102) is a chicken antibody and the secondary detection antibody (103) is an anti-chicken antibody.

12. comparing the detected level of prostasomes with first and second predetermined thresholds (S14); and Providing a prognosis for the prostate cancer (S15). further comprising 10. The method of claim 9, wherein a poor prognosis for prostate cancer is defined when the detected level of prostasomes is higher than a first predetermined threshold (S15a), and a good prognosis is defined when the detected level of prostasomes is lower than a second predetermined threshold (S15b), wherein the sample from the subject is a body fluid sample, the first predetermined threshold is 10 ng of prostasomes per mL of body fluid sample, and the second predetermined threshold is 1 ng of prostasomes per mL of body fluid sample.

13. comparing the detected level of prostasomes with first and second predetermined thresholds (S24); and further comprising assessing the severity of the prostate cancer (S25); 10. The method of claim 9, wherein the prostate cancer is assessed as severe if the detected level of prostasomes is higher than a first predetermined threshold (S25a), as moderate if the detected level of prostasomes is lower than the first threshold but higher than a second threshold (S25b), or as mild if the detected level of prostasomes is lower than a second threshold (S25c), wherein the sample from the subject is a body fluid sample, the first predetermined threshold is 10 ng of prostasomes per mL of body fluid sample, and the second predetermined threshold is 1 ng of prostasomes per mL of body fluid sample.

14. detecting any prostasomes bound by the human monoclonal antibody or antigen-binding fragment thereof; detecting a level of prostasomes in a sample from a subject prior to prostate cancer treatment (S31); providing an anti-prostate cancer treatment to the subject (S32); detecting a level of prostasomes in a sample from the subject after the prostate cancer treatment (S33); The method comprises: Comparing the pre-treatment prostasome levels with the post-treatment levels (S34); and determining the efficacy of said treatment (S35); If the level of prostasomes after the treatment is lower than the level before the treatment, the treatment is judged to be effective (S35a), and if the level of prostasomes remains the same or has increased, the treatment is judged to be ineffective (S35b).

10. The method of claim 9, wherein the sample from the subject is a bodily fluid sample selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, and a cell suspension.

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