Monoclonal antibody for diagnosis and early morbidity of prostate cancer

Abstract

The invention provides a monoclonal antibody designed for diagnosis and early detection of prostate cancer, and particularly relates to a specific monoclonal antibody developed to target soluble ADAM9 variants (sADAM9v2) secreted near prostate cancer.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application 63 / 377,801, filed on September 30, 2022. The entire content of the foregoing application is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application incorporates by reference in its entirety the content of an electronic sequence listing entitled 23P0419_final.xml (size 12,288 bytes), created on September 26, 2023. Technical Field

[0005] The present invention relates to a monoclonal antibody designed for the diagnosis and early detection of prostate cancer, and more particularly to the development of a monoclonal antibody with targeting specificity for a soluble ADAM9 variant (sADAM9v2) secreted near prostate cancer. Background Art

[0006] According to cancer registration data in Taiwan, China in 2021, the incidence of prostate cancer ranks sixth among men in Taiwan, China, and the mortality rate ranks fifth. It is worth noting that the early manifestations of prostate cancer are usually subtle and similar to those of prostate hyperplasia, making it possible to be overlooked. Concerns are only raised when prostate cancer infiltrates the seminal vesicles, presenting symptoms such as hematospermia, ejaculation pain, or metastasizes to other organs.

[0007] Therefore, an effective method for the early detection of prostate cancer is crucial. Summary of the Invention

[0008] ADAM9 has been shown to have increased expression in many cancer epithelial cells, such as prostate cancer, breast cancer, kidney cancer, and lung cancer. The enhanced expression is positively correlated with cancer progression and metastasis trends. Studies have shown that the main protein expression can be attributed to soluble ADAM9 (sADAM9).

[0009] Meanwhile, two different splicing variants of sADAM9 have been identified, including sADAM9v1 reported by Mazzocca et al., and a novel variant sADAM9v2 identified by the inventors of the present application. It is worth emphasizing that sADAM9v2 is not only expressed by malignant prostate cancer cells but also in the surrounding cells near prostate cancer.

[0010] In the following, the present invention provides an antibody or an antigen-binding fragment thereof that binds to the sADAM9v2 protein or a peptide fragment thereof. This antibody or antigen-binding fragment is characterized in that: a heavy chain that comprises a CDR1 having the amino acid sequence of SEQ ID NO:1, a CDR2 having the amino acid sequence of SEQ ID NO:2, and a CDR3 having the amino acid sequence of SEQ ID NO:3; and a concatenated light chain that comprises a CDR1 having the amino acid sequence of SEQ ID NO:4, a CDR2 having the amino acid sequence of SEQ ID NO:5, and a CDR3 having the amino acid sequence of SEQ ID NO:6.

[0011] Preferably, the antibody or antigen-binding fragment comprises: a heavy chain variable region that comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity with SEQ ID NO:7; and a light chain variable region that comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity with SEQ ID NO:8.

[0012] Preferably, the heavy chain comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity with SEQ ID NO:9.

[0013] Preferably, the light chain comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity with SEQ ID NO:10.

[0014] Preferably, the antibody or antigen-binding fragment is conjugated to a reagent such as a therapeutic compound, a fluorescent label, a chemiluminescent label, a colorimetric indicator, an enzyme tag, a radioisotope, and an affinity label.

[0015] For another aspect of the present invention, there is provided a polynucleotide encoding the antibody or antigen-binding fragment thereof as described above.

[0016] For another object of the present invention, it further provides a reagent for predicting or diagnosing sADAM9v2-related diseases, determining the drug efficacy after treatment with an sADAM9v2 inhibitor, or screening for subjects who are highly effective for treatment with an sADAM9v2 inhibitor, wherein the reagent comprises the antibody or antigen-binding fragment thereof as described above.

[0017] For another object of the present invention, it further provides a method for predicting or diagnosing an sADAM9v2-related disease or a tendency to develop an sADAM9v2-related disease in a subject, wherein the method comprises the following steps: (a) contacting a sample isolated from the subject with an antibody or an antigen-binding fragment thereof of any one of the above; (b) detecting the sADAM9v2 protein in the sample by detecting the binding between the antibody or its antigen-binding fragment and the sample; and (c) comparing the level of sADAM9v2 protein in the sample with a control, wherein an sADAM9v2 protein level higher than the control indicates that the subject has the disease or is at risk of developing the disease.

[0018] Preferably, the sADAM9v2-related disease may be a cancer expressing sADAM9v2; optionally, the sADAM9v2-related disease is a cancer overexpressing sADAM9v2.

[0019] Preferably, the cancer may be prostate cancer.

[0020] For another object of the present invention, it further provides the use of an antibody or an antigen-binding fragment thereof of any one of the above for preparing a pharmaceutical composition for treating an sADAM9v2-related disease.

[0021] For another object of the present invention, it further provides a pharmaceutical composition comprising an effective dose of an antibody or an antigen-binding fragment thereof of any one of the above as an active ingredient and a pharmaceutically acceptable carrier.

[0022] As disclosed above, the present invention provides a monoclonal antibody developed using the specificity of a soluble ADAM9 variant secreted around prostate cancer, which can detect the occurrence of prostate cancer at an early stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention, which can be better understood by referring to the drawings and in conjunction with the detailed description of a specific embodiment presented herein.

[0024] Figure 1Presents the results of PCR analysis, which shows the difference between the length of ADAM9 and the predetermined length. The cell lines involved in these studies include NC (representing the negative control, without ADAM9), PC (representing the positive control, which contains the ADAM9 gene), BPH-1 (representing the cell line of Benign Prostate Hyperplasia-1), LNCaP (representing the cancer cell line, which is derived from a prostate cancer patient with lymph node metastasis), C4-2 (a branch of the LNCaP cell line characterized by androgen insensitivity), C4-2B (a subset of C4-2 cells, which is extracted from the bone metastatic cancer of a mouse model), CWR22Rv1 (a prostate cancer cell line, which shows the ARV7 mutation of the androgen receptor mutation), DU145 (a prostate cancer cell line, which is derived from a prostate cancer patient with brain metastasis), PC3 (a prostate cancer cell line, which is obtained from a prostate cancer patient with bone metastasis), and PC3M (a variant of PC3 cells, which shows obvious bone metastasis in an animal model).

[0025] Figure 2 Shows the results of the comprehensive sequencing map of the ADAM9 gene, which shows the existence of alternative splicing variants. Although ADAM9 variant 1 has been previously documented, the data also indicate the expression of a novel ADAM9 variant 2, which has not been described in any previous reports.

[0026] Figure 3 Depicts that sADAM9 was identified only in tumor cells and the surrounding cells near the cancer. This figure provides the protein expression analysis of ADAM9 in LN (LNCaP), CWR (CWR22rv1), PC3, and DU145, and these proteins are from total cell lysates and protein isolates from the culture medium (conditioned medium). In addition, cells from the benign (WHN) and tumor (WHC) regions of prostate cancer patients were obtained to verify whether tumor-associated surrounding cells secrete sADAM9.

[0027] Figure 4 Shows that when using ELISA to detect the expression of ADAM9 in the blood of patients with benign prostate hyperplasia (BPH) and prostate cancer, the expression of sADAM9 in the blood of cancer patients increased significantly.

[0028] Figure 5 Shows the sADAM9v2 expression in cancer cells and benign cells isolated from 3 patients by laser capture microdissection (LCM).

[0029] Figure 6 Shows that according to the computational model, the alternative splicing site has high antigenicity.

[0030] Figure 7 Showed the results of the hybridoma assay for sADAM9v2.

[0031] Figure 8 Showed that sADAM9v2 improved the dose-dependent migration of prostate cancer cells in transwell, and the migration could be inhibited by sADAM9v2 mAb.

[0032] Figure 9 Showed the migration and metastasis of prostate cancer cells induced by the downstream signal of AKT activated by sADAM9. Specific embodiments

[0033] For the description in this article and the appended patent claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes more than one protein, and reference to "a compound" refers to more than one compound. "Comprise", "comprising", "include", "includes", and "including" are used interchangeably and are not intended to be limiting. It should be further understood that in cases where the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific cases, the language "consisting essentially of" or "consisting of" can be used alternatively to describe specific embodiments.

[0034] In the case of providing a range of numerical values, unless the context clearly dictates otherwise, it should be understood that each intermediate integer of the said numerical value, and each tenth of each intermediate integer of the said numerical value, between the upper and lower limits of the said range, and any other specified or intermediate value within the said specified range, are covered by the present invention. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges, or can be included in the present invention, subject to any specific exclusions within the said range. In the case where the said specified range includes one or both of the said limitations, the range is included in the present invention except for (i) any one or (ii) both of the included said limitations. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0035] All publications, patents, patent applications, and other documents cited in the present invention are incorporated herein by reference in their entirety for all purposes to the extent that each individual publication, patent, patent application, or other document is separately indicated to be incorporated herein by reference for all purposes.

[0036] It should be understood that the foregoing general description (including the accompanying drawings) and the following detailed description are merely exemplary and explanatory and do not limit the present invention.

[0037] Unless otherwise specifically defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs.

[0038] I.sADAM9v2 antibody or an antigen-binding fragment thereof

[0039] Provided is an antibody or an antigen-binding fragment thereof that binds to the sADAM9v2 protein or a partial peptide thereof, wherein the antibody or the antigen-binding fragment thereof comprises:

[0040] A heavy chain that comprises

[0041] CDR1 containing the amino acid sequence of SEQ ID NO:1,

[0042] CDR2 containing the amino acid sequence of SEQ ID NO:2, and

[0043] CDR3 containing the amino acid sequence of SEQ ID NO:3; and

[0044] A light chain that comprises

[0045] CDR1 containing the amino acid sequence of SEQ ID NO:4,

[0046] CDR2 containing the amino acid sequence of SEQ ID NO:5, and

[0047] CDR3 containing the amino acid sequence of SEQ ID NO:6.

[0048] In one embodiment, the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:7, and a light chain variable region that comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:8.

[0049] In one embodiment, the heavy chain comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:9.

[0050] In one embodiment, the light chain comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity to SEQ ID NO:10.

[0051] An antibody (used interchangeably in various forms) is an immunoglobulin molecule that can specifically bind to a target antigen (such as sADAM9v2 in the present invention) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab’, F(ab’)2, Fv), single-chain (scFv), mutants thereof, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single-chain antibodies, multispecific antibodies (such as bispecific antibodies), and any other modified configurations including immunoglobulin molecules having antigen recognition sites with the desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include any class of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and the antibody does not have to be of any specific class. Immunoglobulins can be classified into different classes according to the amino acid sequence of the antibody heavy chain constant domain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are respectively designated as α, δ, ε, γ, and μ. The subunit structures and three-dimensional configurations of different types of immunoglobulins are well known. The term "isolated antibody" as used herein refers to an antibody that is substantially free of naturally associated molecules, i.e., the naturally associated molecules account for at most 20% dry weight of the preparation containing the antibody. Purity can be measured by appropriate methods, for example, column chromatography, polyacrylamide gel electrophoresis, and HPLC.

[0052] A typical antibody molecule contains a heavy chain variable region (V H ) that is typically involved in antigen binding and a light chain variable region (V L ). The V H region and the V LThe regions can be further subdivided into hypervariable regions, also called "complementarity determining regions" ("CDRs"), within which are located more conserved regions, called "framework regions" ("FRs"). Each VH and VL is generally composed of three CDRs and four FRs, which are arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The extent of the framework regions and CDRs can be precisely identified using methods known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, for example, Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; the internationalImMunoGeneTics information http: / / www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); Ruiz, M. et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P., Nucleic Acids Res., 29:207-209 (2001); Lefranc, M.-P., Nucleic Acids Res., 31:307-310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [Epub], 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 33:D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 37:D1006-1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015); Chothia et al., (1989) Nature 342:877; Chothia, C. et al., (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). As used herein, CDR can refer to CDRs defined by any method known in the art. Two antibodies having the same CDR refer to two antibodies having the same amino acid sequence of CDRs determined by the same method (e.g., IMGT definition).

[0053] In some embodiments, the isolated anti-sADAM9v2 antibody as described herein can bind to sADAM9v2 and inhibit the activity of at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, or higher) of sADAM9v2. The apparent inhibition constant (Kiapp or Ki,app) provides a measure of inhibitor potency and is related to the concentration of inhibitor required to reduce enzyme activity, independent of enzyme concentration. The inhibitory activity of the anti-sADAM9v2 antibody described herein can be determined by conventional methods known in the art.

[0054] Any antibody described herein can be a monoclonal or polyclonal antibody. "Monoclonal antibody" refers to a population of homologous antibodies, and "polyclonal antibody" refers to a population of heterologous antibodies. These two terms do not limit the source or the manner of preparation of the antibody.

[0055] In some embodiments, the anti-sADAM9v2 antibodies described herein bind to the same epitope in the sADAM9v2 antigen as the reference antibodies disclosed herein, or compete with the reference antibodies for binding to the sADAM9v2 antigen. An "epitope" refers to the site on a target compound to which an antibody (such as a Fab or a full-length antibody) binds. An epitope can be linear, typically 6-15 amino acids in length. Alternatively, an epitope can be conformational. Antibodies that bind to the same epitope as the reference antibodies described herein can bind to the exact same epitope or an epitope that is substantially overlapping (e.g., containing fewer than 3 non-overlapping amino acid residues, fewer than 2 non-overlapping amino acid residues, or only 1 non-overlapping amino acid residue) as the reference antibody. Whether two antibodies compete with each other for binding to a homologous antigen can be determined by a competition assay, which is well known in the art. Such antibodies can be identified as known to those skilled in the art, for example, antibodies having substantially similar structural features (such as complementarity determining regions), and / or antibodies identified by assays known in the art. For example, a competition assay can be performed using a reference antibody to determine whether a candidate antibody binds to the same epitope as the reference antibody or competes for binding to the sADAM9v2 antigen.

[0056] In one example, the antibodies used in the methods described herein can be humanized antibodies. A humanized antibody is a form of a non-human (e.g., murine) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or antigen-binding fragment thereof that contains a minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues from the recipient complementarity determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, a humanized antibody can contain residues not found in the recipient antibody or in the introduced CDR or framework sequences, but which are included to further refine and optimize antibody performance. Ordinarily, a humanized antibody will contain substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are those of the human immunoglobulin consensus sequence. A humanized antibody optimally will also contain at least a portion of the immunoglobulin constant region or domain (Fc), usually that of a human immunoglobulin. The antibody can have a modified Fc region as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that are altered relative to the original antibody and are also referred to as one or more CDRs "derived from" one or more CDRs of the original antibody. Humanized antibodies can also relate to affinity maturation.

[0057] In some embodiments, the anti-sADAM9v2 antibodies described herein specifically bind to a corresponding target antigen or an epitope thereof. An antibody that "specifically binds" to an antigen or epitope is a term well known in the art. A molecule exhibits "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity with a particular target antigen than with other target antigens. An antibody "specifically binds" to a target antigen or epitope if it binds to the target antigen or epitope with a higher affinity, avidity, more readily, and / or for a longer duration than its binding to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (e.g., human sADAM9v2) or an antigenic epitope thereof refers to an antibody that binds to the target antigen with a longer affinity, avidity, binding rate, and / or duration than its binding to other antigens or other epitopes in the same antigen. By this definition, it should also be understood that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. In some instances, an antibody that "specifically binds" to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen (e.g., no binding is detected in a conventional assay).

[0058] In some embodiments, the antibodies described herein specifically bind to a sADAM9v2 of a particular species (e.g., human sADAM9v2) relative to sADAM9v2 from other species. For example, the antibodies described herein may specifically bind to human sADAM9v2 relative to mouse sADAM9v2. In other embodiments, the antibodies described herein may cross-react with human sADAM9v2 and one or more sADAM9v2s from non-human species (e.g., non-human primates such as macaques). In some embodiments, the antibody cross-reacts with human and rhesus macaque with a similar binding affinity, but with a significantly reduced binding affinity for mouse sADAM9v2. In some embodiments, the anti-sADAM9v2 antibody as described herein has a suitable binding affinity for the target antigen (e.g., human sADAM9v2) or an antigenic epitope thereof.

[0059] As used herein, "binding affinity" refers to the apparent binding constant or KA, which is the ratio of the binding constant and the dissociation constant, K-on and K-off, respectively. KA is the reciprocal of the dissociation constant (KD). The anti-sADAM9v2 antibodies described herein may have at least 10 -8 、10 -9 、10 -10 M、10 -11A binding affinity (KD) of M or lower. For example, an anti-sADAM9v2 antibody may have a binding affinity for sADAM9v2 of 10 -9 M, 10 -10 M or lower. An increase in binding affinity corresponds to a decrease in the KD value. The binding of an antibody to a first antigen with a higher affinity (relative to a second antigen) can be represented as a higher KA (or a smaller numerical KD) for binding the first antigen compared to the KA for binding the second antigen. In this case, the antibody is specific for the first antigen (e.g., a first protein in a first conformation or its mimetic) relative to the second antigen (e.g., the same first protein in a second conformation or its mimetic; or a second protein). In some embodiments, the anti-sADAM9v2 antibodies described herein have a higher binding affinity (higher KA or smaller KD) for sADAM9v2 compared to the binding affinity for another cytokine or chemokine. In some embodiments, the anti-sADAM9v2 antibodies may have a higher binding affinity for a specific species (e.g., human sADAM9v2) compared to sADAM9v2 from a different species (e.g., mouse). The difference in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 2.5, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1,000, 5,000, 10,000, or 105-fold. In some embodiments, any anti-sADAM9v2 antibody can be further affinity matured to increase the binding affinity of the antibody for the target antigen or its epitope.

[0060] Binding affinity (or binding specificity) can be determined by various methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance (SPR), fluorescence-activated cell sorting (FACS), or spectroscopy (e.g., using fluorescence assays). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20) and PBS buffer (10 mM PO4-3, 137 mM NaCl, and 2.7 mM KCl). These techniques can be used to measure the concentration of the binding protein as a function of the concentration of the target protein. The concentration of the binding protein ([bound]) is typically related to the concentration of the free target protein ([free]) by the following equation:

[0061] [bound] = [free] / (Kd + [free])

[0062] However, it is not always necessary to precisely determine KA, because sometimes a quantitative measure of affinity that is proportional to KA is obtained (e.g., determined using methods such as ELISA or FACS analysis), and can thus be used for comparison, e.g., to determine if a higher affinity is, for example, 2-fold higher, to obtain a quantitative measure of affinity, or to obtain an inferred affinity, e.g., by activity in a functional assay (e.g., in vitro or in vivo assay) is sufficient.

[0063] In some cases, the amino acid residue change can be a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods known to those skilled in the art for altering polypeptide sequences, e.g., as can be found in references that compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel et al., John Wiley & Sons, Inc., New York.

[0064] In some embodiments, the heavy chain of any anti-sADAM9v2 antibody as described herein may also comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can be from any suitable source, e.g., human, mouse, rat, or rabbit. In a particular instance, the heavy chain constant region is derived from human IgG (γ heavy chain), e.g., IgG1, IgG2, or IgG4. In one instance, the heavy chain constant region is subclass IgG1.

[0065] The light chain of any anti-sADAM9v2 antibody as described herein may also comprise a light chain constant region (CL), which can be any CL known in the art. In some instances, the CL is a κ light chain. In other instances, the CL is a λ light chain. The heavy and light chain constant regions of antibodies are well known in the art, e.g., as provided in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0066] As used herein, an anti-sADAM9v2 antibody can be any antibody format, including but not limited to, a full (i.e., full-length) antibody, antigen-binding fragments thereof (such as Fab, Fab’, F(ab’)2, Fv), single-chain antibodies, bispecific antibodies, or nanobodies.

[0067] II. Preparation of Anti-sADAM9v2 Antibodies

[0068] Antibodies that bind sADAM9v2 as described herein can be prepared by any method known in the art. See, e.g., Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0069] In some embodiments, antibodies specific for a target antigen (e.g., sADAM9v2) can be prepared by conventional hybridoma techniques. The full-length target antigen or a fragment thereof, optionally conjugated to a carrier protein (such as KLH), can be used to immunize a host animal to generate antibodies that bind to the antigen. As further described herein, the immunization route and protocol for the host animal are generally consistent with established and conventional antibody stimulation and production techniques. General techniques for generating murine, humanized, and human antibodies are known in the art and are described herein. Any mammalian subject, including humans or cells that produce antibodies therefrom, can be manipulated to serve as a basis for production in mammals, including human hybridoma cell lines. Typically, a host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intradermally, and / or intratibially with an amount of immunogen, including as described herein.

[0070] If desired, the target antibody (monoclonal or polyclonal), e.g., produced by a hybridoma, can be sequenced and then the polynucleotide sequence can be cloned into a vector for expression or propagation. The sequence encoding the target antibody can be maintained in a vector in a host cell, and then the host cell can be amplified and frozen for future use. In an alternative, the polynucleotide sequence can be used for genetic manipulation to “humanize” the antibody or improve the antibody’s affinity (affinity maturation) or other characteristics. For example, if the antibody is to be used in human clinical trials and treatment, the constant region can be engineered to be more similar to the human constant region to avoid an immune response. It may be desirable to genetically manipulate the antibody sequence to obtain a higher affinity for the target antigen and greater effectiveness in inhibiting sADAM9v2 activity. It will be apparent to those skilled in the art that one or more polynucleotide alterations can be made to the antibody and still retain its binding specificity for the target antigen.

[0071] In other embodiments, fully human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or stronger immune response can also be used to generate humanized or human antibodies. Examples of such technologies are the XenomouseR from Amgen, Inc. (Fremont, CA) TM and the HuMAb-MouseR from Medarex, Inc. (Princeton, NJ) TM and the TC Mouse TM or the H2L2 from Harbour Antibodies BV (Holland). In another alternative, antibodies can be recombinantly prepared by phage display or yeast technology. See, e.g., U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to generate human antibodies or antibody fragments in vitro from a library of immunoglobulin variable (V) domain genes of non-immunized donors.

[0072] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by conventional methods. For example, F(ab’)2 fragments can be generated by pepsin digestion of antibody molecules, and Fab fragments can be generated by reduction of the disulfide bridges of F(ab’)2 fragments. Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced by, for example, conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific for a target antigen can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA can be placed in one or more expression vectors and then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, human HEK293 cells, or myeloma cells that do not produce immunoglobulins, to obtain synthesis of the monoclonal antibody in the recombinant host cells. See, e.g., PCT Publication No. WO 87 / 04462. Then, the DNA can be modified, for example, by replacing homologous murine sequences with the coding sequences of the human heavy and light chain constant domains, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, genetically engineered antibodies with target antigen-binding specificity, such as “chimeric” or “hybrid” antibodies, can be prepared.

[0073] Single-chain antibodies can be prepared by recombinant techniques by linking the nucleotide sequence encoding the heavy chain variable region to the nucleotide sequence encoding the light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions.

[0074] Antibodies obtained by the methods described herein and known in the art can be characterized using methods well known in the art. For example, a method is to identify the epitope that binds the antigen or "epitope mapping". There are many methods known in the art for mapping and confirming the location of epitopes on proteins, including resolving the crystal structure of the antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In one example, epitope mapping can be accomplished using H / D-Ex (hydrogen-deuterium exchange) combined with proteolysis and mass spectrometry. In another example, epitope mapping can be used to determine the sequence to which the antibody binds. The epitope can be a linear epitope, i.e., contained within a single amino acid fragment, or it can be a conformational epitope formed by the three-dimensional interaction of amino acids that are not necessarily contained within a single fragment (the primary structure linear sequence). Peptides of different lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used in binding assays with the antibody. In another example, the epitope that binds the antibody can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and by measuring antibody binding. According to the gene fragment expression assay, the open reading frame encoding the target antigen is fragmented randomly or by specific genetic constructs, and the reactivity of the expressed antigen fragments with the antibody to be tested is determined. For example, gene fragments can be generated by PCR and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. Then, the binding of the antibody to the antigen fragment with the radioactive label is determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using a large random peptide sequence library (phage library) displayed on the surface of phage particles. Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In another example, mutagenesis of the antigen-binding domain, domain exchange experiments, and alanine-scan mutagenesis can be performed to identify the residues required, sufficient, and / or essential for epitope binding. For example, domain exchange experiments can be performed using mutant target antigens in which individual fragments of the sADAM9v2 polypeptide have been replaced (exchanged) with sequences from closely related but antigenically different proteins (such as the CD-28 protein). By evaluating the binding of the antibody to the mutant sADAM9v2, the importance of specific antigen fragments for antibody binding can be evaluated. Alternatively, competition assays can be performed using other antibodies known to bind the same antigen to determine whether the antibody binds the same epitope as the other antibody. Competition assays are well known to those skilled in the art.

[0075] In some instances, anti-sADAM9v2 antibodies are prepared by the recombinant techniques exemplified below. Nucleic acids encoding the heavy and light chains of the anti-sADAM9v2 antibodies described herein can be cloned into an expression vector, with each nucleotide sequence operably linked to a suitable promoter. In one instance, each nucleotide sequence encoding the heavy and light chains is operably linked to a different promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter such that both the heavy and light chains are expressed from the same promoter. When necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0076] In some instances, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, they can be separately isolated from the host cells expressing them, and the isolated heavy and light chains are mixed and cultured under suitable conditions to form the antibody.

[0077] Generally, nucleic acid sequences encoding one or all of the chains of an antibody can be cloned into a suitable expression vector using methods known in the art and operably linked to a suitable promoter. For example, under suitable conditions, the nucleotide sequence and the vector can be contacted with a restriction enzyme to generate complementary ends on each molecule, which can pair with each other and be ligated together by a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell used in producing the antibody.

[0078] Regulatable promoters can be used. Such regulatable promoters include those that use the lac repressor of Escherichia coli as a transcriptional regulator to regulate the transcription of the lac operon derived from a mammalian cell promoter (Brown, M. et al., Cell, 49:603-612 (1987)), those that use the tetracycline repressor (tetR) (Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P. et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)). Other systems include FK506 dimer, VP16, or p65 (using estradiol, RU486, diphenolmurislerone, or rapamycin). Inducible systems are available from Invitrogen, Clontech, and Ariad, among others.

[0079] A regulatable promoter containing a suppressor of the lac operon can be used. In one embodiment, the lac suppressor derived from Escherichia coli can act as a transcriptional regulator to regulate the transcription of a mammalian cell promoter carrying the lac operon (M. Brown et al., Cell, 49: 603-612 (1987)); Gossen and Bujard (1992) [M. Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992)) combined the tetracycline suppressor (tetR) with a transcriptional activator (VP16) to create the tetR-mammalian cell transcriptional activator fusion protein tTa (tetR-VP16), and used a minimal promoter carrying tetO derived from the human cytomegalovirus (hCMV) promoter to create a tetR-tet operon system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operon is correctly positioned downstream of the TATA element of the CMVIE promoter, the individual tetracycline suppressor (tetR) rather than the tetR-mammalian cell transcriptional fusion derivative can act as an effective trans-regulator to regulate gene expression in mammalian cells (Yao et al., Human Gene Therapy). A particular advantage of the tetracycline-inducible switch is that it does not require the use of a tetracycline suppressor-mammalian cell trans-activator or suppressor fusion protein, which may be toxic to cells under certain circumstances (Gossen 5 et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92: 6522-6526 (1995)) to achieve its regulatory effect.

[0080] In addition, the vector may contain some or all of, for example, the following: selectable marker genes, such as the neomycin gene for selecting stable or transient transfectants in mammalian cells; enhancer / promoter sequences derived from the human CMV immediate early gene, which can achieve high levels of transcription; transcription termination and RNA processing signals derived from SV40 to achieve mRNA stability; the SV40 polyomavirus replication origin and ColE1 for appropriate episomal replication; internal ribosome entry sites (IRESes), a multi-purpose multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNAs. Suitable vectors and methods for generating vectors containing transgenes are well known in the art and are available. Examples of polyadenylation signals that can be used to practice the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0081] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any antibody can be introduced into a suitable host cell to produce the antibody. The host cell can be cultured under suitable conditions to express the antibody or any of its polypeptide chains. Such antibody or its polypeptide chains can be recovered from the cultured cells (e.g., from the cells or the culture supernatant) by conventional methods (e.g., affinity purification). If desired, the polypeptide chains of the antibody can be cultured for a suitable period of time under suitable conditions to produce the antibody.

[0082] In some embodiments, the method for preparing the antibodies described herein involves recombinant expression vectors encoding the heavy and light chains of an anti-sADAM9v2 antibody, as also described herein. The recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection). Positive transformed host cells can be selected and cultured under suitable conditions to allow expression of the two polypeptide chains of the antibody, which can be recovered from the cells or the culture medium. If necessary, the two chains recovered from the host cells can be cultured under suitable conditions to form the antibody.

[0083] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-sADAM9v2 antibody and the other encoding the light chain of an anti-sADAM9v2 antibody. These two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection).

[0084] Alternatively, each expression vector can be introduced into a suitable host cell. Positive transformants can be selected and cultured under appropriate conditions to allow expression of the polypeptide chains of the antibody. When two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or from the culture medium. If desired, the polypeptide chains can be recovered from the host cell or from the culture medium and then cultured under suitable conditions to form the antibody. When two expression vectors are introduced into different host cells, they can be recovered from their respective host cells or respective culture media. Then, the two polypeptide chains can be cultured under suitable conditions to form the antibody.

[0085] Recombinant expression vectors are prepared, host cells are transfected, transformants are selected, host cells are cultured, and antibodies are recovered from the culture medium using standard molecular biology techniques. For example, some antibodies can be isolated by affinity chromatography using Protein A, Protein G, or Protein L coupled to a matrix.

[0086] Any nucleic acid encoding the heavy chain, light chain, or both of the anti-sADAM9v2 antibody as described herein, vectors (such as expression vectors) containing these; and host cells containing the said vectors are encompassed within the scope of the present invention.

[0087] III. Pharmaceutical Compositions

[0088] The antibodies as described herein, as well as the encoding nucleic acids or nucleic acid sets, vectors containing these, or host cells containing the said vectors can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for treating the target disease. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably, can stabilize the active ingredient) and harmless to the subject to be treated. Pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover.

[0089] The pharmaceutical compositions disclosed herein that contain anti-sADAM9v2 antibodies may also contain a suitable buffer. A buffer is a weak acid or base that is used to keep the pH of a solution near a selected value after the addition of other acids or bases. In some instances, the buffers disclosed herein can be buffers that are able to maintain physiological pH despite changes in carbon dioxide concentration (produced by cellular respiration). Exemplary buffers include, but are not limited to, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Dulbecco's phosphate buffered saline (DPBS) buffer, or phosphate buffered saline (PBS) buffer. These buffers may include disodium hydrogen phosphate and sodium chloride, or potassium dihydrogen phosphate and potassium chloride.

[0090] In some embodiments, the buffer of the pharmaceutical compositions described herein can maintain a pH value of about 5 - 8. For example, the pH value of the pharmaceutical composition can be about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In other instances, the pharmaceutical composition can have a pH value below 7, for example, about 7, 6.8, 6.5, 6.3, 6, 5.8, 5.5, 5.3, or 5.

[0091] The pharmaceutical compositions described herein contain one or more suitable salts. A salt is an ionic compound that can be formed by the neutralization reaction of an acid and a base (Skoog, D.A; West, D.M.; Holler, J.F.; Crouch, S.R. (2004) “Chapters 14 - 16”. Fundamentals of Analytical Chemistry (8th ed.)). A salt consists of a corresponding number of cations (positively charged ions) and anions (negatively charged ions), so that the product is electrically neutral (has no net charge). As described herein, an ion is an atom or molecule that has gained or lost one or more valence electrons to give the ion a net positive or negative charge. If a chemical substance has more protons than electrons, it carries a net positive charge. If there are more electrons than protons, the substance has a negative charge.

[0092] As described herein, a cation ( + ) is an ion that has fewer electrons than protons, giving it a positive charge. (Douglas W. Haywick, (2007 - 2008). “Elemental Chemistry”). A cation with one positive charge can be called a monovalent cation; a cation with more than one positive charge can be called a polyvalent or multivalent cation. Non-limiting examples of monovalent cations are hydrogen (H + ), sodium (Na + ), potassium (K + ), ammonium (NH4 + ), lithium (Li +)、Copper(I) (Cu + )、Silver (Ag + ) and the like. Non-limiting examples of multivalent cations are Magnesium (Mg 2+ )、Calcium (Ca 2+ )、Barium (Ba 2+ )、Beryllium (Be 2+ )、Copper (Cu 2+ )、Iron(II) (Fe 2+ )、Iron(III) (Fe3 + )、Lead(II) (Pb 2+ )、Lead(IV) (Pb4 + )、Manganese(II) (Mn 2+ )、Strontium (Sr 2+ )、Tin(IV) (Sn4 + )、Zinc (Zn 2+ ) and the like.

[0093] As described herein, an anion is an ion that has more electrons than protons, giving it a net negative charge. Non-limiting examples of anions are Azide (N3 - )、Bromide (Br - )、Chloride (Cl - )、Fluoride (F - )、Hydride (H - )、Iodide (I - )、Nitride (N - )、Oxide (O 2- )、Sulfide (S 2- )、Carbonate (CO3 2- )、Bicarbonate (HCO3 - )、Bisulfate (HSO4 - )、Hydroxide (OH - )、Dihydrogen Phosphate (H2PO4 - )、Sulfate (SO4 2- )、Sulfite (SO3 2- )、Silicate (SiO3 2- ) and the like.

[0094] Suitable salts for use in the pharmaceutical compositions described herein may include monovalent cations and monovalent or multivalent anions. Alternatively, the salts used in the pharmaceutical compositions described herein may include monovalent or multivalent cations and monovalent anions. Exemplary salts include, but are not limited to, Potassium Chloride (KCl), Sodium Chloride (NaCl), Calcium Chloride (CaCl2), Magnesium Chloride (MgCl2), Magnesium Sulfate (MgSO4), Sodium Bicarbonate (NaHCO3), Ammonium Sulfate ((NH4)2SO4), Calcium Carbonate (Ca2CO3), or combinations thereof.

[0095] The pharmaceutical compositions described herein comprise one or more suitable surface-active agents, such as surfactants. Surfactants are compounds that reduce the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. Surfactants can be used as detergents, wetting agents, emulsifying agents, foaming agents, and dispersing agents. Suitable surfactants include, in particular, nonionic agents such as polyoxyethylene sorbitan (e.g., Tween TM 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span TM 20, 40, 60, 80, or 85). Compositions having a surface-active agent will conveniently contain between 0.05 and 5% of the surface-active agent, and can be between 0.1 and 2.5%. It should be understood that other ingredients, such as mannitol or other pharmaceutically acceptable carriers, can be added if desired.

[0096] The pharmaceutical composition containing anti-sADAM9v2 described herein may comprise one or more amino acids. Exemplary amino acids include, but are not limited to, glycine, histidine, or arginine.

[0097] The pharmaceutical composition may also comprise one or more antioxidants. As used herein, an antioxidant is a reagent that prevents or delays the oxidative degradation of the active ingredients contained in the composition. The antioxidants used herein can be phenolic antioxidants (sometimes referred to as true antioxidants), reducing agents, or chelating agents. Phenolic antioxidants are sterically hindered phenols that can react with free radicals and block chain reactions. Reducing agents are compounds having a lower redox potential, and thus, are more easily oxidized than the drugs they protect. Reducing agents scavenge oxygen from the medium, thereby delaying or preventing drug oxidation. Chelating agents are sometimes referred to as antioxidant synergists. Metal ions (such as Co 2+ 、Cu 2+ 、Fe 3+ 、Fe 2+ and Mn 2+ ) can shorten the induction time and increase the oxidation rate. Trace amounts of these metal ions are often introduced into drugs during the preparation process. Therefore, chelating agents do not have antioxidant activity, but can enhance the action of phenolic antioxidants by reacting with catalytic metal ions to inactivate them.

[0098] The pharmaceutical compositions described herein may also comprise sugar derivatives. As used herein, sugar derivatives comprise saccharides derived from sugars and organic compounds. In some instances, the sugar derivatives can be non-reducing sugars, sugar alcohols, polyols, disaccharides, or polysaccharides.

[0099] The pharmaceutical compositions used in the method of the present invention may comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized preparations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed and may include buffering agents, such as phosphoric acid, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants, such as TWEEN TM , PLURONICS TM , or polyethylene glycol (PEG).

[0100] In some instances, the pharmaceutical compositions described herein comprise liposomes that contain an antibody (or encoding nucleic acid) that can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation times are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter membrane with a defined pore size to produce liposomes with the desired diameter.

[0101] The antibody or one or more encoding nucleic acids may also be encapsulated in microcapsules, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxyethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, prepared in colloidal drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules, or in macroemulsions. Such techniques are known in the art, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0102] In other instances, the pharmaceutical compositions described herein may be formulated in a sustained release form. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrix being in the form of a shaped article, for example, a film or a microcapsule. Examples of sustained release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), poly(lactide) (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT TM (injectable microspheres composed of a lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0103] In other instances, the pharmaceutical compositions described herein may be formulated in a sustained release form by implementing specific protease biological techniques to selectively affect binding to tissues or tumors, for example, by peptide masking of the antigen binding site of the antibody to effect selective protease cleavage of one or more proteases (such as Probody TM or Conditionally Active Biologics TM ) in the tumor microenvironment. This activation can be formulated to be reversible in the normal microenvironment.

[0104] The pharmaceutical compositions for in vivo administration must be sterile. This can be readily accomplished, for example, by filtration through sterile filtration membranes. Typically, the therapeutic antibody composition is placed in a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a subcutaneous injection needle.

[0105] The pharmaceutical compositions described herein may be in unit dosage forms, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral, or rectal administration, or by inhalation or insufflation.

[0106] For the preparation of solid compositions (such as tablets), the main active ingredient can be mixed with a pharmaceutical carrier, for example, conventional tablet ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dibasic calcium phosphate or gums, and other pharmaceutical diluents (such as water) to form a solid pre - formulation composition which comprises a homogeneous mixture of the composition of the present invention or a non - toxic pharmaceutically acceptable salt. When these pre - formulation compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition so that the composition can be easily re - divided into equivalent unit dosage forms such as tablets, pills and capsules. Then, the solid pre - formulation composition is re - divided into unit dosage forms of the above types which contain from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the new composition can be coated or otherwise compounded to provide dosage forms having the advantage of extended action. For example, a tablet or pill can contain an internal dosage and an external dosage component, the latter covering the former in the form of a film. These two components can be separated by an enteric layer which is used to resist decomposition in the stomach and allow the internal component to enter the duodenum intact or with delayed release. A variety of materials can be used for such enteric layers or coatings, including a variety of polymeric acids and mixtures of such polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

[0107] Commercially available fat emulsions (such as Intralipid TM , Liposyn TM , Infonutrol TM , Lipofundin TM and Lipiphysan TM ) can be used to prepare suitable emulsions. The active ingredient can be dissolved in the pre - mixed emulsion composition or can be dissolved in an oil (such as soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion is formed after mixing with phospholipids (such as egg yolk lecithin, soybean phospholipid or soy lecithin) and water. It should be understood that other ingredients such as glycerol or glucose can be added to adjust the tonicity of the emulsion. Suitable emulsions will generally contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can contain fat droplets in the range of 0.1 to 1.0 µm, especially between 0.1 and 0.5 µm, and have a pH value in the range of 5.5 to 8.0. The emulsion composition can be those prepared by mixing an antibody with Intralipid TM or its components (soybean oil, lecithin, glycerol and water).

[0108] A pharmaceutical composition for inhalation or insufflation comprises solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as shown above. In some embodiments, the composition is administered by oral or nasal inhalation routes to produce local or systemic effects. The composition may be aerosolized using a gas, preferably in a sterile pharmaceutically acceptable solvent. The aerosolized solution may be breathed directly from the nebulizing device, or the nebulizing device may be connected to a face mask, tent, or intermittent positive pressure ventilator. The solution, suspension, or powder composition may preferably be administered orally or nasally by a device that delivers the formulation in an appropriate manner.

[0109] IV. Therapeutic Applications

[0110] Any of the antibodies described herein, as well as the encoding nucleic acids or nucleic acid sets, vectors containing them, or host cells containing the vectors can be used to treat sADAM9v2-mediated disorders. As used herein, an sADAM9v2-mediated disease refers to any medical disease associated with an increase in the level of sADAM9v2 or an increase in sensitivity to sADAM9v2. Non-limiting examples of sADAM9v2-mediated diseases are prostate cancer and the like.

[0111] To practice the methods disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment by a suitable route, such as intravenous administration, e.g., by bolus injection or continuous infusion over a period of time, by intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or topical routes. Commercially available nebulizers (including jet nebulizers and ultrasonic nebulizers) for liquid formulations can be used for administration. The liquid formulation can be nebulized directly and the lyophilized powder can be nebulized after reconstitution. Alternatively, the antibodies described herein can be nebulized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and micronized powder.

[0112] The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats. The human subject in need of treatment can be a human patient suffering from or suspected of suffering from an inflammatory disease, an autoimmune disease, cancer, an infectious disease, or other conditions requiring modulation of the immune response or at risk of developing such diseases. Subjects suffering from a target disease or disorder can be identified by conventional medical examinations (e.g., laboratory tests, organ function tests, CT scans, or ultrasounds). Subjects suspected of suffering from any such target disease / condition may exhibit one or more symptoms of the disease / condition. Subjects at risk of developing the disease / condition can be subjects having one or more risk factors for the disease / condition.

[0113] As used herein, "effective amount" means the amount of each active agent required to confer a therapeutic effect on a subject, alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is to reduce sADAM9v2 activity.

[0114] As used herein, "overexpression" means that the expression of sADAM9v2 on the surface of cancer cells is significantly higher than that of normal cells.

[0115] Determining whether a certain amount of an antibody achieves a therapeutic effect will be obvious to those skilled in the art. As recognized by those skilled in the art, the change in the effective amount depends on the specific condition being treated, the severity of the condition, the parameters of the individual patient, including age, physical condition, body size, gender and weight, the duration of treatment, the nature of concomitant treatments (if any), the specific route of administration, and factors within the knowledge and experience of healthcare personnel. These factors are well known to those skilled in the art and can be addressed by routine experimentation alone.

[0116] It is generally preferred to administer the maximum dose of a single component or a combination thereof, i.e., the highest safe dose based on sound medical judgment.

[0117] Empirical factors (such as half-life) will generally assist in determining the dose. For example, antibodies compatible with the human immune system (such as humanized antibodies or fully human antibodies) can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The frequency of administration can be determined and adjusted during the course of treatment, and typically (but not necessarily) based on the treatment and / or inhibition and / or improvement and / or delay of the target disease / condition. Alternatively, sustained-release formulations of the antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0118] In one example, in an individual who has received one or more doses of an antibody, the dose of the antibody described herein can be determined empirically. The individual is administered increasing doses of the antagonist. To evaluate the effectiveness of the antagonist, indicators of the disease / condition can be tracked.

[0119] Under normal circumstances, for the administration of any of the antibodies described herein, an initial candidate dose can be about 2 mg / kg. For the purposes of the present invention, typical daily, weekly, bi-weekly, or tri-weekly dose ranges can be in any range from about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 100 μg / kg to 300 μg / kg to 0.6 mg / kg, 1 mg / kg, 3 mg / kg, up to 10 mg / kg, up to 30 mg / kg to 100 mg / kg or higher, depending on the factors described above. For repeated administration over several days, weeks, months or longer, depending on the condition, treatment is continued until the desired symptom suppression occurs or until a treatment level sufficient to alleviate the target disease or condition or its symptoms is reached. Exemplary dosing regimens include administering an initial dose of about 3 mg / kg every 3 weeks, followed by a maintenance dose of about 1 mg / kg antibody every 6 weeks, and then a maintenance dose of about 1 mg / kg every 3 weeks. However, other dose ranges may also be useful, depending on the pharmacokinetic decay pattern the physician wishes to achieve. For example, combination therapy with a dose of 1 mg / kg every 3 weeks and at least one additional immunotherapy is also encompassed in the present invention. In some embodiments, doses in the range of about 3 μg / mg to about 3 mg / kg (such as about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg and about 3 mg / kg) can be used. In some embodiments, the dosing frequency is once a week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks or every 10 weeks; or once a month, every 2 months or every 3 months or longer. The progress of the therapy can be easily monitored by conventional techniques and trials. The dosing regimen (including the antibody used) can vary over time.

[0120] In some embodiments, for adult patients of normal body weight, doses in the range of about 0.1 to 5.0 mg / kg can be administered. In some instances, the dose of the anti-sADAM9v2 antibody described herein can be 10 mg / kg. The specific dosing regimen (i.e., dose, time and repeated doses) will depend on the particular individual and the individual's medical history, as well as the nature of each agent (the half-life of the agent, and other considerations well known in the art).

[0121] For the purposes of the present invention, a suitable dose of an antibody as described herein will depend on the specific antibody, antibody, and / or non-antibody peptide (or combination thereof) being used, the type and severity of the disease / condition, whether the antibody is being administered for prophylactic or therapeutic purposes, previous therapies, the patient's clinical history and response to the antagonist, and the judgment of the attending physician. Generally, the clinician will administer the antibody until a dose is reached that achieves the desired result. In some embodiments, the desired result is tumor size reduction, prolonged progression-free survival, and / or overall survival. Methods for determining whether a dose produces the desired result will be apparent to those of skill in the art. Administration of one or more antibodies can be continuous or intermittent, depending, for example, on the physiological condition of the receptor, whether the administration is for therapeutic or prophylactic purposes, and other factors known to those of skill in the art. Administration of the antibody can be substantially continuous over a predetermined period of time or can be a series of spaced doses, e.g., before, during, or after development of the target disease or condition.

[0122] As used herein, the term "treatment" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or condition, having the symptoms of a disease / condition, or being susceptible to a disease / condition, with the purpose to cure, heal, alleviate, relieve, alter, remedy, improve, or affect the condition, the symptoms of the disease, or the susceptibility to the disease or condition. Alleviating a target disease / condition includes delaying the occurrence or development of the disease, or reducing the severity of the disease.

[0123] Alleviating a disease does not necessarily require a cure. As used herein, "delaying" the progression of a target disease or condition refers to delaying, impeding, slowing, retarding, stabilizing, and / or postponing the progression of the disease. The delay can have different time lengths, depending on the history of the disease and / or the individual being treated. A method for "delaying" or alleviating disease progression or delaying the onset of a disease is a method that, compared to not using the method, reduces the likelihood of occurrence of one or more disease symptoms within a given time frame and / or reduces the degree of symptom alleviation within a given time frame. Such comparisons are typically based on clinical studies using a sufficient number of subjects to give statistically significant results.

[0124] In some embodiments, the antibodies described herein are administered to a subject in need of treatment in an amount sufficient to inhibit target antigen activity in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher). In another embodiment, the antibody is administered in an amount effective to reduce the target antigen activity level by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher).

[0125] Depending on the type of disease or the site of the disease to be treated, the pharmaceutical composition can be administered to the subject using conventional methods known to those of ordinary skill in the medical art. The composition can also be administered by other conventional routes, for example, parenterally, topically, orally, by inhalation spray, rectally, nasally, buccally, vaginally, or by an implantable cartridge. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intraperitoneal, intratumoral, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject by an injectable depot administration route, such as using 1, 3, or 6-month depot injectable or biodegradable materials and methods.

[0126] Injectable compositions can contain various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by infusion, whereby a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations (e.g., sterile preparations of the antibody in a suitable soluble salt form) can be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% dextrose solution.

[0127] In one embodiment, the antibody is administered by site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the antibody or local delivery catheters, such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, adventitia, shunts, and stents, or other implantable devices, site-specific carriers, direct injection, or direct application. See, for example, PCT Publication No. WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0128] Targeted delivery of therapeutic compositions comprising antisense polynucleotides, expression vectors or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods and Applications of Direct Gene Transfer (J.A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

[0129] Therapeutic compositions comprising polynucleotides (such as those encoding the antibodies described herein) are administered in the range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. In some embodiments, during a gene therapy protocol, DNA concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg or more can also be used.

[0130] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vectors. Gene delivery vectors can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Endogenous mammalian or heterologous promoters and / or enhancers can be used to induce the expression of such coding sequences. Expression of the coding sequences can be constitutive or regulatable.

[0131] Virus-based vectors for delivering a desired polynucleotide and expressing it in a desired cell are well known in the art. Exemplary virus-based vectors include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Patent Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655). Inactivated adenoviruses linked to DNA as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be used.

[0132] Non-viral delivery vectors and methods can also be used, including but not limited to, polycation condensed DNA linked or not linked to a separate inactivated adenovirus (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vector cells (see, e.g., U.S. Patent No. 5,814,482; PCT Publication No. WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338) and nucleic acid charge neutralization or fusion with cell membranes. Naked DNA can also be used. Methods for introducing exemplary naked DNA are described in PCT Publication No. WO 90 / 11092 and U.S. Patent No. 5,580,859. Liposomes capable of serving as gene delivery vectors are described in U.S. Patent No. 5,422,120; PCT Publication No. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and EP Patent No. 0524968. Other methods are described in Philip, Mol. Cell. Biol. (1994) 14:2411 and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0133] The specific dosing regimens (i.e., dose, timing, and repeated doses) used in the methods described herein will depend on the particular subject and the medical history of the subject. In some embodiments, more than one antibody or a combination of an antibody and another suitable therapeutic agent can be administered to a subject in need of treatment. Antibodies can also be used in combination with other agents that are used to enhance and / or supplement the action of the agent. The therapeutic effectiveness against the target disease / condition can be evaluated by methods well known in the art.

[0134] The anti-sADAM9v2 antibodies and treatment methods described, for example, in the present disclosure can be used in combination with other types of therapies for the target diseases or conditions disclosed herein. In this context, the term "in combination with" refers to the simultaneous or sequential administration of the antibody composition and the therapeutic agent. Examples include chemotherapy, immunotherapy (e.g., therapies involving anti-inflammatory drugs, immunosuppressants, therapeutic antibodies, antibodies, CAR T cells, or cancer vaccines), surgery, radiotherapy, gene therapy, etc. or anti-infective therapy. Such therapies can be administered simultaneously or sequentially (in any order) with the treatment according to the invention.

[0135] When the antibody compositions described herein are used in combination with a second therapeutic agent, a sub-therapeutic dose of the composition or the second agent, or a sub-therapeutic dose of both, can be used for the treatment of a subject having a disease or disorder associated with sADAM9v2-mediated signaling or at risk of having a disease or disorder associated with sADAM9v2-mediated signaling. As used herein, "sub-therapeutic dose" means less than the dose that produces a therapeutic outcome in a subject when administered to the subject in the absence of one or more other agents. Thus, a sub-therapeutic dose of an agent is a dose that would not produce the desired therapeutic outcome in a subject in the absence of administration of an anti-sADAM9v2 antibody as described herein. The therapeutic doses of many agents used in clinical practice are well known in the medical art, and other therapeutic doses can be determined by those of ordinary skill in the art without undue experimentation. Therapeutic doses have been widely described, for example, in references such as Remington’s Pharmaceutical Sciences, 18th ed., 1990; and many other medical references relied upon by the medical community as guides for treating diseases and disorders. See also Physician’s Desk Reference, 59.sup.th edition, (2005), Thomson P DR, Montvale N.J.; Gennaro et al., Remington’s The Science and Practice of Pharmacy, 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Harrison’s Principles of Internal Medicine, 15th supplement, (2001), McGraw Hill, NY; Berkow et al., The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0136] V. Diagnostic Applications

[0137] Any anti-sADAM9v2 antibody disclosed herein can also be used to detect the presence of sADAM9v2 (e.g., secreted sADAM9v2) in vitro or in vivo. The results obtained from such detection methods can be used for diagnostic purposes (e.g., diagnosing diseases associated with secreted sADAM9v2) or for scientific research purposes (e.g., identifying novel sADAM9v2-secreting cells, studying the biological activity and / or regulation of secreted sADAM9v2). For assay uses (such as diagnostic uses), the anti-sADAM9v2 antibodies described herein can be conjugated to a detectable label (e.g., an imaging agent, such as a contrast agent) to detect the presence of sADAM9v2 (e.g., secreted sADAM9v2) in vitro or in vivo. As used herein, "conjugated" or "attached" means that two entities are joined together, preferably with sufficient affinity to achieve a therapeutic / diagnostic benefit from the binding between the two entities. The binding between the two entities can be direct or through a linker, such as a polymeric linker.

[0138] Conjugation or attachment can include covalent or non-covalent bonds and other forms of binding, such as capture, e.g., capture of one entity on or within another entity, or capture of one or both entities on or within a third entity, such as a micelle.

[0139] In one example, an anti-sADAM9v2 antibody as described herein can be attached to a detectable label, which is a compound capable of directly or indirectly releasing a detectable signal, thereby enabling the detection, measurement, and / or identification of aptamers in vitro or in vivo. Examples of such "detectable labels" are intended to include, but are not limited to, fluorescent labels, chemiluminescent labels, colorimetric labels, enzyme labels, radioisotopes, and affinity labels (such as biotin). Such labels can be directly or indirectly conjugated to aptamers by conventional methods.

[0140] In some embodiments, the detectable label is a reagent suitable for in vitro detection of sADAM9v2-secreting cells, which can be a radioactive molecule, a radiopharmaceutical, or iron oxide particles. Radioactive molecules suitable for in vivo imaging include, but are not limited to, 122 I, 123 I, 124 I, 1 25 I, 131 I, 18 F, 75 Br, 76 Br, 77 Br, 211 At, 225 Ac, 177 Lu, 153 Sm, 186 Re, 188 Re, 67 Cu, 213 Bi,212 Bi, 212 Pb, and 67 Ga. Exemplary radiopharmaceuticals suitable for in vivo imaging include 111 In oxyquinoline, 131 I sodium iodide, 99 mTc mebrofenin, and 99 mTc red blood cells, 123 I sodium iodide, 99 mTc exametazime, 99 mTc macroaggregated albumin, 99 mTc methylene diphosphonate, 99 mTc mercaptoacetyltriglycine, 99 mTc phosphonates, 99 mTc diethylenetriaminepentaacetic acid, 99 mTc pertechnetate, 99 mTc sestamibi, 99 mTc sulfur colloid, 99 mTc teboroxime, thallium-201, or xenon-133.

[0141] The reporter agent can be a dye, e.g., a fluorophore, which can be used to detect diseases mediated by sADAM9v2-secreting cells in a tissue sample.

[0142] For in vitro diagnostic assays, an anti-sADAM9v2 antibody can be contacted with a sample suspected of containing sADAM9v2 (e.g., sADAM9v2-secreting cells or soluble sADAM9v2 in a disease microenvironment). The antibody and the sample can be incubated under suitable conditions for a suitable time to allow the antibody to bind to the sADAM9v2 antigen. Then, such interactions can be detected by conventional methods (e.g., ELISA, histological staining, or FACS).

[0143] For in vivo diagnostic assays, a suitable amount of an anti-sADAM9v2 antibody conjugated to a label (e.g., an imaging agent or a contrast agent) can be administered to a subject in need of examination. The presence of the labeled antibody can be detected by conventional methods based on the signal released by the label.

[0144] For scientific research assays, anti-sADAM9v2 antibodies can be used to study the biological activity of sADAM9v2, to detect the presence of sADAM9v2 intracellularly and / or to modulate the action of secreted sADAM9v2. For example, an appropriate amount of anti-sADAM9v2 can be contacted with a sample suspected of producing sADAM9v2 (e.g., a new cell type that has not previously been identified as producing sADAM9v2). The cells are permeabilized prior to contacting with the anti-sADAM9v2 antibody. The antibody and sample can be incubated for an appropriate time under suitable conditions to allow the antibody to bind to the sADAM9v2 antigen. Then, such interactions can be detected by conventional methods (e.g., ELISA, histological staining, or FACS). VI. Kits for Therapeutic and Diagnostic Applications

[0145] The invention also provides kits for therapeutic or diagnostic applications as disclosed herein. Such kits can include one or more containers comprising an anti-sADAM9v2 antibody (e.g., any of those described herein).

[0146] In some embodiments, the kit can include instructions for use according to any of the methods described herein. The included instructions can include instructions for administering the anti-sADAM9v2 antibody to treat, delay the onset, or alleviate the target diseases as described herein. The kit can also include a description for selecting a suitable individual for treatment based on identifying whether the individual has the target disease. In yet another embodiment, the instructions include instructions for administering the antibody to an individual at risk of having the target disease.

[0147] Instructions related to the use of the anti-sADAM9v2 antibody generally include information about the dosage, dosing regimen, and route of administration of the intended treatment. The container can be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kits of the invention are generally written instructions on a label or package insert (e.g., a sheet included in the kit), but machine-readable instructions (e.g., instructions recorded on a magnetic or optical storage disk) are also acceptable.

[0148] The label or package insert indicates that the composition is for treating, delaying the onset, and / or alleviating a disease or disorder treatable by modulating the immune response, such as an autoimmune disease. Instructions for performing any of the methods described herein can be provided.

[0149] The kits of the invention are in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), etc.

[0150] Packaging for use in combination with specific devices such as inhalers, nasal administration devices (such as atomizers) or infusion devices (such as micropumps) is also contemplated. The kit may have a sterile inlet (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile inlet (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an anti-sADAM9v2 antibody as described herein.

[0151] The kit may optionally provide other components such as buffers and explanatory information. Generally, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article comprising the contents of the kit described above.

[0152] Also provided herein are kits for detecting secreted sADAM9v2 in a sample. Such kits may contain any anti-sADAM9v2 antibody described herein. In some cases, the anti-sADAM9v2 antibody may be conjugated to a detectable label (such as those described herein). As used herein, "conjugated" or "attached" means that two entities are bound together, preferably with sufficient affinity to achieve a therapeutic / diagnostic benefit of the binding between the two entities. The binding between the two entities can be direct or through a linker, such as a polymeric linker. Conjugation or attachment can include covalent or non-covalent bonds and other forms of binding, such as capture, for example, capture of one entity on or within another entity, or capture of one or both entities on or within a third entity, such as a micelle.

[0153] Alternatively or additionally, the kit may contain a second antibody that can bind to the anti-sADAM9v2 antibody. The kit may also contain instructions for detecting secreted sADAM9v2 using the anti-sADAM9v2 antibody.

[0154] VII. General Techniques

[0155] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (ed. M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. J.E. Cellis, 1998) Academic Press; Animal Cell Culture (ed. R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (ed. A. Doyle, J.B. Griffiths, and D.G. Newell, 1993-8) J.Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (ed. D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (ed. J.M. Miller and M.P. Calos, 1987); Current Protocols in Molecular Biology (ed. F.M. Ausubel et al., 1987); PCR: The Polymerase Chain Reaction, (ed. Mullis et al., 1994); Current Protocols in Immunology (ed. J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley & Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practical approach (edited by D. Catty, IRL Press, 1988 - 1989); Monoclonal antibodies: a practical approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (by E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and J.D. Capra, Harwood Academic Publishers, 1995). Without further elaboration, it is believed that those skilled in the art can make the most of the present invention based on the above description. Therefore, the following specific embodiments should be construed as merely illustrative and not in any way limiting the remainder of the present invention. All publications cited herein are incorporated by reference for the purposes or subject matter cited herein.

[0156] Without further elaboration, it is believed that those skilled in the art can make the most of the present invention based on the above description. Therefore, the following specific embodiments should be construed as merely illustrative and not in any way limiting the remainder of the present invention. All publications cited herein are incorporated by reference for the purposes or subject matter cited herein.

[0157] Discovery of sADAM9v2

[0158] In a PCR assay evaluating ADAM9 expression, differences in the length of some ADAM9 sequences were observed when compared to a predetermined baseline (see Figure 1, indicated by the red arrow). The present invention aims to verify the expression of the ADAM9 gene in various prostate cancer cell lines and control samples. These include NC (negative control, indicating the absence of prostate cancer samples), PC (representing the ADAM9 gene as a positive control), BPH-1 (benign prostatic hyperplasia-1), LNCaP (a prostate cancer cell line derived from a patient with lymph node metastasis), C4-2 (an androgen-insensitive derivative of LNCaP), C4-2B (indicating the bone metastasis tendency of C4-2), CWR22Rv1 (representing a prostate cancer cell line with androgen mutations), DU145 (a prostate cancer cell line derived from a patient showing brain metastasis), PC3 (a prostate cancer cell line obtained from a patient showing bone metastasis), and PC3M (a cell derivative from PC3 that shows significant metastatic behavior in a mouse model). The main objective of the present invention is to determine the expression of ADAM9 mRNA in the above-mentioned prostate cancer cell lines. RNA was isolated using the Qiagen RNA isolation system (provided by Qiagen, Inc.), and then it was converted into cDNA by the MMLV reverse transcriptase system. The PCR procedure was facilitated by introducing the cDNA into PCR reagents (provided by ThermoFisher, Inc.) and performing PCR analysis by agarose gel electrophoresis. The results showed that these cell lines not only contained the complete ADAM9 mRNA sequence but also contained different variants. This result emphasizes the potential of ADAM9 messenger RNA to exhibit unique alternative splicing iterations in prostate cancer cells, which contribute to the malignant and metastatic progression of prostate cancer. Therefore, the inventors isolated these PCR bands for gene sequencing.

[0159] After mapping the ADAM9 gene diagram following whole-gene sequencing of ADAM9, the inventors discovered alternative splicing variants (see Figure 2 ). Note that ADAM9-splice form 1 exhibits alternative splicing between position 2046 of exon 18 and position 2147 of exon 19. This results in out-of-frame splicing and premature termination of ADAM9 translation. Therefore, there are deletions in the transmembrane and cytoplasmic domains of full-length ADAM9. In addition, the inventors discovered a novel alternative splicing event that confirmed the deletion between position 2046 of exon 18 and position 2236 of exon 19. This results in the omission of the transmembrane domain, but the cytoplasmic sequence remains in-frame.

[0160] Further detection of the culture media from tumors, tumor-associated peripheral cells, and normal tissues showed that sADAM9 was detected only in the culture media from tumors and tumor-associated peripheral cells (see Figure 3)。The cells were cultured for three days before collecting the culture medium, and then the proteins in the culture medium were concentrated. Meanwhile, the cultured cells were collected and protein extraction was performed using a protein extraction system (RIPA buffer, which includes 150 mM NaCl, 1% Triton-X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-HCL pH 8.0). The present invention aims to verify the expression of the ADAM9 gene in various prostate cancer cell lines and corresponding control samples. This group includes NC (control, which indicates the absence of prostate cancer samples), PC (as a positive control for the ADAM9 gene), BPH-1 (benign prostatic hyperplasia-1), LNCaP (a prostate cancer cell line derived from a patient showing lymph node metastasis), C4-2 (an androgen-insensitive LNCaP branch), C4-2B (showing a tendency for bone metastasis of C4-2), CWR22Rv1 (a prostate cancer cell line showing androgen mutation), DU145 (a prostate cancer cell line derived from a patient with brain metastasis), PC3 (a prostate cancer cell line obtained from a patient showing significant bone metastasis), and PC3M (a cell line from PC3 showing a significant tendency for metastasis in a mouse model). For protein analysis, Western Blotting using an 8% polyacrylamide gel was performed. By electrophoresis, the proteins were transferred from the gel to a nitrocellulose membrane. For Western Blotting, a specific antibody purchased from R&D Biosystem was used, particularly ADAM9 (MAB939).

[0161] In a specific embodiment, an ELISA system was utilized to perform a blood test to evaluate the expression of sADAM9 in patients with benign prostatic hyperplasia (BPH) and prostate cancer. As Figure 4 shown, the expression of sADAM9 in prostate cancer patients was significantly higher than that in BPH patients. Serum samples (with anonymized identities) from BPH and prostate cancer patients were obtained from the Human Bio-Resource Bank of Taipei Medical University (TMU), which included 10 samples each from BPH and prostate cancer patients. ELISA analysis was performed according to the standard protocol provided by R&D Biosystems, Inc., and the corresponding ELISA kit (DY939) was also purchased from the same company.

[0162] As Figure 5As shown, as measured by laser capture microdissection (LCM) from 3 patients, the expression of sADAM9v2 in cancer-associated peripheral cells exceeded that in benign corresponding cells of the same patients. To determine that sADAM9v2 is mainly expressed in prostate tumor-associated peripheral cells, tissue samples from three different patients were subjected to LCM. Both the tumor and peripheral regions of these tissue slides (derived from the TMU Biobank) have been verified by pathologists. The LCM procedure was carried out according to the instructions of a commercial kit ( Kit for DNA and RNA extraction and purification, Invitrogen, Inc.). The MMI CellCut and Cell Ector systems provided by the TMU Core Facility were used to perform the LCM operation. Then, the frozen RNA isolation kit was used to isolate the RNA of these cells, and cDNA was synthesized using the MMLV cDNA system (Thermo Fisher Scientific, Inc.). Quantitative PCR analysis was performed on the 480 system (Roche Diagnostics, Inc.). The 2^ΔΔCT analysis method was used to calculate the fold difference, as outlined by the NIH (published in Biostat. Bioinforma Biomath. 2013 Aug; 3(3): 71-85). The final results were expressed as fold changes, which were derived from the comparison results between cancerous and benign.

[0163] Based on these tests, it was confirmed that sADAM9v2 was expressed in tumor cells and peripheral cells. The present disclosure performed structural simulations on ADAM9v2 and found that the alternative splicing sites had high antigenicity, as indicated by the arrows (see Figure 6 ).

[0164] Hereinafter, the present disclosure performed specific tests of sADAM9v2 with hybridomas. Cell lysates and conditioned media (CM) of sADAM9v1 and sADAM9v2 were also collected to test the specificity of antibodies. The results are as Figure 7As shown. The inventors selected murine antibodies that produce ADAM9 monoclonal antibodies for the following assays. To generate sADAM9v2 hybridomas and mAbs, the NMRI mouse strain was used. All animal procedures were carried out in accordance with the recommended general animal care and handling standards. First, N-(-3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) was used to prepare immunoconjugates after a conventional coupling process. The peptide and its carrier (bovine serum albumin) were mixed in equal proportions and suspended in phosphate-buffered saline (PBS) supplemented with 50 mM NaHCO3 buffer. The inventors selected NMRI mice aged between 6 and 12 weeks for immunization, which were administered 200 μL of PBS containing 150 micrograms of the immunoconjugate, combined with 100 μL of complete Freund's adjuvant. A booster injection containing the same amount of the immunoconjugate was given after 6 weeks. Blood samples were taken 7 weeks after the first injection. For hybridoma development, murine SP2 / 0 cells were used. These cells were cultured in a solution containing 10 mL of RPMI-1640, 10% FBS, and 2 mM L-glutamine (5 mL). Seven weeks after immunization, the mice were euthanized and spleen cell suspensions were prepared. Then, they were suspended in balanced salt buffer (BSS), which consisted of 125 mM NaCl, 5 mM KCl, 4 mM CaCl2, 2.5 mM MgCl2, and 5 mM Tris-HCl (pH 7.4). Then, the mixture was mixed with PEG8000, and electroporation was facilitated by balancing the ratio of spleen cells to myeloma cells, and then suspended in HAT medium for one week. After stabilizing the cultured cells, hybridoma cloning was performed. The inventors finally identified a monoclonal hybridoma and confirmed that it could produce mAbs with significant specificity for ADAM9v2.

[0165] Preparation of recombinant human sADAM9v2 mAb: After identifying different hybridomas suitable for producing sADAM9v2 mAbs, the inventors performed isotype evaluation and classification to determine the unique isotype of sADAM9v2 mAbs present within the hybridomas. The isotype screening was facilitated using the Hybridoma Isotyping ELISA System from Thermo Fisher. Since the isotype was properly verified, sequencing analysis was performed on the specific antibody isotype present in the hybridomas. After verifying the different sequences of the heavy and light chains, the inventors continued to isolate and clone the specific sequences related to sADAM9v2 mAbs. This was achieved by PCR targeting the specific sequences to be tested, which were then integrated into the TGEX-heavy chain and TGEX-light chain expression vectors for subsequent applications. The TGEX vectors containing the heavy and light chains of sADAM9v2 mAbs ensured consistent expression and retention of the inventors' patented antibodies within their system.

[0166] Prostate cancer cell migration according to sADAM9: The Transwell cell migration assay was used to evaluate the motility of prostate cancer cells after the introduction of sADAM9. The inventors obtained recombinant ADAM9 (rADAM9) from R&D and added it to the Transwell setup. The data observed showed that sADAM9 (in the form of rADAM9) promoted prostate cancer cell migration in a dose-proportional manner (as shown on the left side of the figure). In addition, the mAb against sADAM9v2 inhibited the cell migration activity within the Transwell system, indicating that the sADAM9v2 mAb functions as a neutralizing antibody (nAb). As shown

[0167] The results showed that the prostate cancer cell migration rate was dose-dependent on the sADAM9 concentration, and the developed sADAM9v2 mAb could reduce the prostate cancer cell migration rate. Figure 8

[0168] The inventors' previous findings emphasized the specificity of the sADAM9v2 mAb and its role in inhibiting prostate cancer cell motility. Sufficient evidence indicates that the migratory and invasive behaviors of prostate cancer cells are related to the downstream cell regulation of AKT and Src. Therefore, the inventors studied the AKT pathway (citation: Int. J. Mol. Sci., 2020 Jun; 21(12): 4507) and the Src pathway (citation: Cancer and Metastasis Reviews 2014 Feb; 33: 595 - 606). Therefore, after the above tests, the present disclosure further attempted to find out the mechanism by which sADAM9 increases the motility and migration of prostate cancer, and found that sADAM9 increases the motility and migration of prostate cancer by activating the downstream effector of AKT. Protein assays showed an increased level of AKT phosphorylation. Prostate cancer cells were grown on culture dishes coated with collagen-1 (considering that bone is rich in collagen) or uncoated. Before harvesting, the cells were treated with 5 μg / ml sADAM9 at intervals of 30 or 60 minutes. Cell lysis was performed using a protein lysis buffer (RIPA buffer) supplemented with phosphatase inhibitors. The phosphorylation levels of AKT and Src were evaluated by using antibodies obtained from Cell specifically targeting the phosphorylated forms of AKT and Src to verify the activities of AKT and Src.

[0169] As Figure 9 shown, sADAM9 has the ability to enhance the downstream signals of cell motility, such as AKT phosphorylation. In addition, the sADAM9v2 mAb blocks cell motility, as Figure 8 ​As shown. Therefore, the inventors hypothesized that the sADAM9v2 mAb blocks prostate cancer motility by inhibiting AKT phosphorylation.

[0170] Other embodiments

[0171] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by alternative features having the same, equivalent or similar purposes. Therefore, unless otherwise clearly stated, each disclosed feature is only an example of a series of equivalent or similar features.

[0172] Based on the above description, those skilled in the art can easily determine the basic features of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, another embodiment is also within the scope of the patent application.

[0173] Equivalents

[0174] Although some embodiments of the invention have been described and illustrated herein, those skilled in the art will readily conceive of many other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein. Each of these variations and / or modifications is considered to be within the scope of the embodiments of the invention described herein. In a more general sense, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and the actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications of the teachings of the present invention. Using only routine experimentation, those skilled in the art will recognize or be able to ascertain many equivalents of the specific embodiments of the invention described herein. Therefore, it should be understood that the foregoing embodiments are given by way of example only, and that the embodiments of the invention may be practiced in a manner different from that specifically described and claimed within the scope of the appended patent application and its equivalents. The embodiments of the invention disclosed herein relate to each and every separate feature, system, article, material, kit, and / or method described herein. Moreover, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present invention if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0175] As defined and used herein, all definitions should be understood to control dictionary definitions, definitions in the documents incorporated by reference, and / or the ordinary meaning of the defined terms.

[0176] All references, patents, and patent applications disclosed herein are incorporated herein by reference with respect to the subject matter to which they refer, and in particular cases, such subject matter may cover the entirety of this article.

[0177] Unless expressly stated to the contrary, the indefinite articles "a" and "an" as used in this specification and in the claims of the patent application shall be understood to mean "at least one".

[0178] As used herein, the phrase "and / or" as used in the specification and claims shall be understood to mean "one or both" of such conjoint elements, i.e., elements that coexist in a particular case and are discrete in other cases. Multiple elements listed with "and / or" shall be construed in the same manner, i.e., "one or more" of such connected elements. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, with respect to "A and / or B", when used in conjunction with open-ended language such as "comprising", in one embodiment, it may refer only to A (optionally including elements other than B); in another embodiment, it may refer only to B (optionally including elements other than A); in yet another embodiment, it may refer to both A and B (optionally including other elements); and so on.

[0179] As used herein, in the specification and in the claims of the patent application, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one, but also including a list of more than one or more elements, and optionally, other unlisted items. Only terms that expressly state the contrary, such as "only one" or "exactly one", or when used in the claims of the patent application, "consisting of" shall refer to including only one of the elements in a list of one or more elements. Generally, as used herein, the term "or" shall be interpreted as an exclusive alternative only when indicating an exclusive choice (i.e., "one or the other, but not both"), such as "any one", "one of", "only one" or "exactly one". When used in the claims of the patent application, "consisting essentially of" shall have the ordinary meaning used in the field of patent law.

[0180] As used herein, in the specification and in the claims, when referring to a list of one or more elements, the phrase "at least one" shall be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element expressly listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows elements other than those specifically identified in the list of elements referred to by the phrase "at least one" to optionally be present, whether related or unrelated to those specifically defined elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "and / or at least one of B"). In one embodiment, it may mean at least one, optionally including more than one A, but no B (and optionally including elements other than B); in another embodiment, it may mean at least one, optionally including more than one B, but no A (and optionally including elements other than A); in yet another embodiment, it may mean at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0181] It should also be understood that, unless there is an express contrary indication, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method need not be limited to the order in which the steps or acts of the method are recited.

Claims

1. An antibody or antigen-binding fragment thereof that binds to sADAM9v2 protein or a partial peptide thereof, wherein the antibody or antigen-binding fragment thereof comprises: A heavy chain, which comprises: CDR1 containing the amino acid sequence of SEQ ID NO:1, CDR2 containing the amino acid sequence of SEQ ID NO:2, and CDR3 containing the amino acid sequence of SEQ ID NO:3; and A light chain, which comprises: CDR1 containing the amino acid sequence of SEQ ID NO:4, CDR2 containing the amino acid sequence of SEQ ID NO:5, and CDR3 containing the amino acid sequence of SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, which comprises: A heavy chain variable region comprising an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity with SEQ ID NO:7; and A light chain variable region comprising an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity with SEQ ID NO:

8.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity with SEQ ID NO:

9.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain comprises an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity with SEQ ID NO:

10.

5. The antibody or antigen-binding fragment thereof according to claim 1, which is conjugated to a therapeutic agent, a fluorescent label, a chemiluminescent label, a colorimetric label, an enzyme label, a radioisotope, and an affinity label.

6. A polynucleotide that encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. A reagent for predicting or diagnosing sADAM9v2-related diseases, measuring the drug efficacy after treatment with an sADAM9v2 inhibitor, or screening for subjects who are highly effective for treatment with an sADAM9v2 inhibitor, wherein the reagent comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

8. A method for predicting or diagnosing sADAM9v2-related diseases or the tendency to develop sADAM9v2-related diseases in a subject, wherein the method comprises the following steps: (a) Contacting a sample isolated from the subject with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; (b) Detecting the sADAM9v2 protein in the sample by detecting the binding between the antibody or antigen-binding fragment thereof and the sample; and (c) Comparing the sADAM9v2 protein level in the sample with a control, wherein an sADAM9v2 protein level higher than the control indicates that the subject has the disease or is at risk of developing the disease.

9. The method according to claim 8, wherein the sADAM9v2-related disease is a cancer expressing sADAM9v2.

10. The method according to claim 9, wherein the cancer is prostate cancer.

11. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 for the preparation of a pharmaceutical composition for treating sADAM9v2-related diseases.

12. A pharmaceutical composition comprising an effective dose of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

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