Monoclonal antibodies for prostate cancer diagnosis and early onset

Abstract

The present invention provides a monoclonal antibody designed for the diagnosis and early detection of prostate cancer, in particular regarding the development of a monoclonal antibody specific for targeting the soluble ADAM9 variant (sADAM9v2) secreted in the vicinity of prostate cancer.

Description

[0001] Cross-references to related applications

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

[0003] sequence list

[0004] This application contains the contents of an electronic sequence list named 23P0419_final.xml (12,288 bytes in size), created on September 26, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0005] This 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 target specificity against a soluble ADAM9 variant (sADAM9v2) secreted near prostate cancer. Background Technology

[0006] According to Taiwan's 2021 cancer registry data, prostate cancer ranks sixth in incidence and fifth in mortality among men in Taiwan. It is worth noting that early symptoms of prostate cancer are often subtle and similar to those of hyperplasia, making them easy to overlook. It only becomes a cause for concern when prostate cancer invades the seminal vesicles, causing symptoms such as hematospermia and painful ejaculation, or when it metastasizes to other organs.

[0007] Therefore, effective methods for early detection of prostate cancer are crucial. Summary of the Invention

[0008] ADAM9 has been shown to be increased in the epithelial cells of many cancers, such as prostate cancer, breast cancer, kidney cancer, and lung cancer. Increased expression is positively correlated with cancer progression and metastasis. Studies have shown that the major 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 this application. It is worth emphasizing that sADAM9v2 is expressed not only by malignant prostate cancer cells but also in peripheral cells near prostate cancer.

[0010] In the following, the present invention provides an antibody or antigen-binding fragment thereof that binds to the sADAM9v2 protein or a peptide fragment thereof. This antibody or antigen-binding fragment is characterized by: a heavy chain comprising CDR1 having the amino acid sequence of SEQ ID NO:1, CDR2 having the amino acid sequence of SEQ ID NO:2, and CDR3 having the amino acid sequence of SEQ ID NO:3; and a tandem light chain comprising CDR1 having the amino acid sequence of SEQ ID NO:4, CDR2 having the amino acid sequence of SEQ ID NO:5, and 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 containing 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 containing 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 contains an amino acid sequence that is at least 70%, preferably at least 80%, or more preferably at least 90% identical to 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 with reagents such as therapeutic compounds, fluorescent markers, chemiluminescent markers, colorimetric indicators, enzyme tags, radioisotopes, and affinity markers.

[0015] In another aspect of the invention, a polynucleotide encoding an antibody or an antigen-binding fragment thereof for any of the above is provided.

[0016] In view of another objective of the present invention, a reagent is further provided for predicting or diagnosing sADAM9v2-related diseases, determining the efficacy of drugs after sADAM9v2 inhibitor treatment, or screening subjects for whom sADAM9v2 inhibitor treatment is highly effective, wherein the reagent comprises the antibody or its antigen-binding fragment described above.

[0017] In view of another object of the present invention, it further provides a method for predicting or diagnosing sADAM9v2-related diseases or a predisposition to sADAM9v2-related diseases in a subject, wherein the method comprises the steps of: (a) contacting a sample separated from the subject with an antibody or antigen-binding fragment thereof of any one of the above; (b) detecting 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 a higher sADAM9v2 protein level than the control indicates that the subject has the disease or is at risk of developing the disease.

[0018] Preferably, sADAM9v2-related diseases can be cancers that express sADAM9v2; optionally, sADAM9v2-related diseases are cancers that overexpress sADAM9v2.

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

[0020] Another object of the present invention is to provide the use of any of the above-mentioned antibodies or antigen-binding fragments thereof for the preparation of pharmaceutical compositions for treating sADAM9v2-related diseases.

[0021] For another objective of the invention, it further provides a pharmaceutical composition comprising an effective dose of any of the above-mentioned antibodies or antigen-binding fragments thereof 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. Attached Figure Description

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

[0024] Figure 1The results of PCR analysis are presented, showing the difference between the length of ADAM9 and the predetermined length. The cell lines involved in these studies include NC (representing a negative control, without ADAM9), PC (representing a positive control, containing the ADAM9 gene), BPH-1 (representing a benign prostatic hyperplasia-1 cell line), LNCaP (representing a cancer cell line derived from prostate cancer patients exhibiting lymph node metastases), C4-2 (a branch of the androgen-insensitive LNCaP cell line), C4-2B (a subset of C4-2 cells extracted from bone metastases in mouse models), CWR22Rv1 (a prostate cancer cell line exhibiting the androgen receptor mutation ARV7), DU145 (a prostate cancer cell line derived from prostate cancer patients with brain metastases), PC3 (a prostate cancer cell line obtained from prostate cancer patients with bone metastases), and PC3M (a variant of PC3 cells exhibiting significant bone metastases in animal models).

[0025] Figure 2 The results of the comprehensive sequencing of the ADAM9 gene are shown, revealing the presence of an alternative splicing variant. 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 This figure depicts the identification of sADAM9 only in tumor cells and peripheral cells near cancer. The figure provides protein expression analysis of ADAM9 in LN (LNCaP), CWR (CWR22rv1), PC3, and DU145, derived from total cell lysates and protein isolates from culture medium (conditioned medium). Furthermore, cells from benign (WHN) and tumor (WHC) regions of prostate cancer patients were obtained to verify whether tumor-associated peripheral cells secrete sADAM9.

[0027] Figure 4 The results showed that when ADAM9 expression in the blood of patients with benign prostatic hyperplasia (BPH) and prostate cancer was detected using ELISA, sADAM9 expression in the blood of cancer patients was significantly increased.

[0028] Figure 5 The expression of sADAM9v2 in cancer cells and benign cells from three patients isolated by laser capture microdissection (LCM) is shown.

[0029] Figure 6 The results show that, according to the computational model, the selective splicing site has high antigenicity.

[0030] Figure 7 The results of hybridoma analysis for sADAM9v2 are shown.

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

[0032] Figure 9 This study demonstrates prostate cancer cell migration and metastasis induced by AKT downstream signaling activated by sADAM9. Detailed Implementation Plan

[0033] In connection with the description herein and the claims of the appended patent applications, the singular forms “a” and “an” include the plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. The terms “comprise,” “comprising,” “include,” “includes,” and “including” are used interchangeably and are not intended to be limiting. It should be further understood that where the term “comprising” is used in the description of various embodiments, those skilled in the art will appreciate that in certain specific cases, the language “substantially consisting of” or “consisting of” may be used instead to describe a particular embodiment.

[0034] When a range of numerical values ​​is provided, it should be understood that each intermediate integer of the value, and each tenth of each intermediate integer, between the upper and lower limits of the range, and any other specified or intermediate value within the specified range, are covered within this invention, unless the context explicitly specifies otherwise. The upper and lower limits of these smaller ranges may be included independently within the smaller range or within this invention, subject to any specific exclusions within the range. Where the specified range includes one or both of the limitations, the range is included in this invention except for (i) any one or (ii) both of the included 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 this 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 individually identified as being cited herein for all purposes.

[0036] It should be understood that the foregoing general description (including the accompanying drawings) and the following detailed description are for illustrative and explanatory purposes only and do not limit the invention.

[0037] Unless otherwise specifically defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention pertains.

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

[0039] An antibody or an antigen-binding fragment thereof is provided that binds to sADAM9v2 protein or a portion thereof, wherein the antibody or antigen-binding fragment comprises:

[0040] Heavy chains, which contain

[0041] CDR1 contains 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] Light chains, which contain

[0045] CDR1 contains the amino acid sequence of SEQ ID NO:4.

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

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

[0048] In one embodiment, the antibody or its antigen-binding fragment comprises: a heavy chain variable region containing 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 containing an amino acid sequence having at least 70%, preferably at least 80%, or more preferably at least 90% identity with 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 with 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 with SEQ ID NO:10.

[0051] An antibody (which may be used interchangeably in various forms) is an immunoglobulin molecule that specifically binds to a target antigen (e.g., sADAM9v2 in this invention) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term “antibody” encompasses not only complete (i.e., full-length) polyclonal or monoclonal antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins containing antibody moieties, humanized antibodies, chimeric antibodies, biantibodies, nanobodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of an immunoglobulin molecule including an antigen recognition site with desired specificity, including glycosylated 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 subclasses thereof), and antibodies need not be of any particular class. Immunoglobulins can be classified into different classes based on the antibody amino acid sequence of the antibody heavy chain constant domain. Immunoglobulins are classified into five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant heavy chain domains corresponding to different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well-known. As used herein, the term "isolated antibody" refers to an antibody that is substantially free of naturally bound molecules, i.e., naturally bound molecules constitute at most 20% of the dry weight of the formulation containing the antibody. Purity can be measured by appropriate methods, such as column chromatography, polyacrylamide gel electrophoresis, and HPLC.

[0052] Typical antibody molecules contain a heavy chain variable region (V) that is usually involved in antigen binding. H ) and light chain variable region (V L V H District and V LThe region can be further subdivided into hypervariable regions, also known as “complementarity-determining regions” (“CDRs”), which contain more conserved regions called “frame regions” (“FRs”). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The extent of the frame regions and CDRs can be precisely identified using methods known in the art, such as the Kabat definition, IMGT definition, Chothia definition, AbM definition, and / or contact definition, all of which are well known in the art. See, for example, Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, 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, Allazikani et al. (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). As used herein, a CDR may refer to a CDR defined by any method known in the art. Two antibodies with the same CDR refer to two antibodies that have the same amino acid sequence of CDR 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 at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, or higher) of sADAM9v2 activity. The apparent inhibition constant (Kiapp or Ki,app) provides a measure of inhibitory potency, relating to the concentration of inhibitor required to reduce enzyme activity, but independent of enzyme concentration. The inhibitory activity of the anti-sADAM9v2 antibody described herein can be determined using conventional methods known in the art.

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

[0055] In some embodiments, the anti-sADAM9v2 antibody described herein binds to the same epitope in the sADAM9v2 antigen as the reference antibody disclosed herein, or competes with the reference antibody for binding to the sADAM9v2 antigen. An epitope is a site on a target compound that binds to an antibody (such as a Fab or full-length antibody). Epitopes can be linear, typically 6-15 amino acids in length. Alternatively, epitopes can be conformational. An antibody that binds to the same epitope as the reference antibody described herein may serve as a reference antibody by binding to an identical or substantially overlapping epitope (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). Whether two antibodies compete with each other for binding to a homologous antigen can be determined by a competition assay, as is well known in the art. Such antibodies can be identified as is known to those skilled in the art, for example, antibodies with substantially similar structural features (e.g., complementarity-determining regions), and / or antibodies identified by assays known in the art. For example, a reference antibody can be used for competitive assays to determine whether a candidate antibody binds to the same epitope as the reference antibody or competitively binds to the sADAM9v2 antigen.

[0056] In one instance, the antibody used in the methods described herein may be a humanized antibody. A humanized antibody is a form of non-human (e.g., murine) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or antigen-binding fragment thereof, containing a minimal sequence derived from a non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody) in which residues derived from the receptor complementarity-determining region (CDR) are replaced by CDR residues derived from a non-human species (donor antibody) (e.g., mouse, rat, or rabbit) with the desired specificity, affinity, and capability. In some cases, Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may include residues not found in the receptor antibody or in the introduced CDR or frame sequence, but included to further refine and optimize antibody performance. Typically, the humanized antibody will contain at least one, and usually substantially all, of at least two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin common sequences. Humanized antibodies will preferably also contain at least a portion of an immunoglobulin constant region or structural domain (Fc), typically of human immunoglobulins. The antibody may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) altered relative to the original antibody, also referred to as one or more CDRs “derived” from the original antibody. Humanized antibodies may also involve affinity maturation.

[0057] In some embodiments, the anti-sADAM9v2 antibodies described herein specifically bind to the corresponding target antigen or its epitope. "Specifically binding" an antibody to an antigen or epitope is a well-known term in the art. A molecule exhibits "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity for a particular target antigen than with other target antigens. An antibody "specifically binds" a target antigen or epitope if its binding to a target antigen or epitope has a higher affinity, stronger affinity, easier binding, and / or 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 its antigenic epitope refers to an antibody target that binds to that target antigen with a longer affinity, stronger affinity, faster binding rate, and / or longer duration compared to binding to other antigens or other epitopes of the same antigen. It should also be understood from this definition 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. In this way, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. In some instances, antibodies that "specifically bind" to a target antigen or its epitope may not bind to other antigens or other epitopes of the same antigen (e.g., binding cannot be detected in routine assays).

[0058] In some embodiments, the antibodies described herein specifically bind to sADAM9v2 of a particular species (e.g., human sADAM9v2) relative to sADAM9v2 derived from other species. For example, the antibodies described herein can specifically bind to human sADAM9v2 relative to mouse sADAM9v2. In other embodiments, the antibodies described herein can cross-react with human sADAM9v2 and one or more sADAM9v2 derived from non-human species (e.g., non-human primates, such as rhesus monkeys). In some embodiments, the antibodies cross-react with humans and rhesus monkeys with similar binding affinity, but with significantly reduced binding affinity to mouse sADAM9v2. In some embodiments, the anti-sADAM9v2 antibodies as described herein have suitable binding affinity to the target antigen (e.g., human sADAM9v2) or its epitopes.

[0059] As used herein, “binding affinity” refers to the apparent binding constant, or KA, which is the ratio of the binding constant to the dissociation constant, K-on and K-off, respectively. KA is the reciprocal of the dissociation constant (KD). The anti-sADAM9v2 antibody described herein may have at least 10 binding affinity to the target antigen or epitope. -8 10 -9 10 -10 M, 10 -11M or lower binding affinity (KD). For example, anti-sADAM9v2 antibodies may have a binding affinity of 10 for sADAM9v2. -9 M, 10 -10 M or lower binding affinity. Increased binding affinity corresponds to a decrease in KD value. Higher affinity binding of an antibody to a first antigen (relative to a second antigen) can be expressed as a higher KA (or lower numerical KD) binding to the first antigen compared to the KA (or numerical KD) binding to the second antigen. In this case, the antibody is specific to the first antigen (e.g., the first protein in the first conformation or its mimicry) relative to the second antigen (e.g., the same first protein in the second conformation or its mimicry). In some embodiments, the anti-sADAM9v2 antibody described herein has a higher binding affinity to sADAM9v2 (higher KA or lower KD) compared to its binding affinity to another cytokine or chemokine. In some embodiments, the anti-sADAM9v2 antibody may have a higher binding affinity to a specific species (e.g., human sADAM9v2) compared to sADAM9v2 derived 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 may undergo further affinity maturation to increase the binding affinity of the antibody to the target antigen or its epitopes.

[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 spectroscopic methods (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 target protein concentration. The concentration of the binding protein ([bound]) is generally related to the concentration of the free target protein ([free]), as follows:

[0061] [Binding] = [Free] / (Kd + [Free])

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

[0063] In some cases, amino acid residue alterations can be conserved amino acid residue substitutions. As used herein, “conserved amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein to which the substitution is made. Variants can be prepared according to methods known to those skilled in the art for altering polypeptide sequences, as can be found, for example, in references compiling such methods, such as *Molecular Cloning: A Laboratory Manual*, J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or *Current Protocols in Molecular Biology*, F.M. Usubel 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 include a heavy chain constant region (5CH) or a portion thereof (e.g., CH1, CH2, CH3, or combinations thereof). The heavy chain constant region can be from any suitable source, such as human, mouse, rat, or rabbit. In one particular instance, the heavy chain constant region is derived from human IgG (γ heavy chain), such as IgG1, IgG2, or IgG4. In one instance, the heavy chain constant region is a subclass of IgG1.

[0065] The light chain of any anti-sADAM9v2 antibody described herein may also include a light chain constant region (CL), which may 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 chain and light chain constant region of the antibody are well known in the art and are provided, for example, 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, anti-sADAM9v2 antibodies can be any antibody form, including but not limited to, full-length (i.e., complete) antibodies, antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv), single-chain antibodies, bispecific antibodies, or nanobodies.

[0067] II. Preparation of anti-sADAM9v2 antibody

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

[0069] In some embodiments, antibodies specific to a target antigen (e.g., sADAM9v2) can be prepared using conventional hybridoma techniques. Full-length target antigens or fragments thereof, optionally conjugated to a carrier protein (e.g., KLH), can be used to immunize host animals to generate antibodies that bind to said antigen. As further described herein, the immunization routes and protocols for host animals are generally consistent with established and conventional antibody stimulation and production techniques. General techniques for generating mouse, humanized, and human antibodies are known in the art and are described herein. Any mammalian subject, including human or antibody-producing cells, is expected to be manipulated as a basis for mammalian production, including human hybridoma cell lines. Typically, host animals are inoculated with an immunogen (including those described herein) via intraperitoneal, intramuscular, oral, subcutaneous, plantar, and / or intradermal administration.

[0070] If needed, the target antibody (monoclonal or polyclonal) (e.g., generated by hybridoma) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the target antibody can be held in a vector within host cells, which can then be amplified and frozen for future use. Alternatively, the polynucleotide sequence can be used for genetic manipulation to "humanize" the antibody or improve its affinity (affinity maturation) or other characteristics. For example, if the antibody is intended for clinical trials and treatment in humans, 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 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 while still maintaining its binding specificity to the target antigen.

[0071] In other implementations, fully human antibodies can be obtained using commercially available mice engineered to express specific human immunoglobulins. Transgenic animals designed to produce a more desirable (e.g., fully human antibody) or stronger immune response can also be used to generate humanized or human antibodies. An example of this type of technology is Xenomouse® from Amgen, Inc. (Fremont, CA). TM And HuMAb-MouseR, derived from Medarex, Inc. (Princeton, NJ) TM With TC Mouse TM Or H2L2 derived from Harbour Antibodies BV (Holland). Alternatively, antibodies can be prepared via phage display or yeast recombinant technology. See, for example, 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 gene library of immunoglobulin variable (V) domains from non-immunized donors.

[0072] Antigen-binding fragments of complete antibodies (full-length antibodies) can be prepared using conventional methods. For example, the F(ab')2 fragment can be produced by digesting antibody molecules with pepsin, while the Fab fragment can be produced by reducing the disulfide bridges of the F(ab')2 fragment. Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced using, for example, conventional recombinant techniques. In one instance, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional methods, such as by using oligonucleotide probes that specifically bind to the heavy and light chain genes encoding the monoclonal antibody. Hybridoma cells are a preferred source of this type of 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, which do not produce immunoglobulins, to obtain monoclonal antibody synthesis in recombinant host cells. See, for example, PCT Publication WO 87 / 04462. Then, DNA can be modified, for example, by replacing homologous mouse sequences with coding sequences of 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 an 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 using recombinant technology by linking nucleotide sequences encoding the variable region of the heavy chain with nucleotide sequences encoding the variable region of the light chain. Preferably, a flexible linker is incorporated between the two variable regions.

[0074] Antibodies known in the art and obtained by methods described herein can be characterized using methods well-known in the art. For example, methods may be used to identify antigen-binding epitopes or "epitope localization." Many methods known in the art are used to locate and confirm epitope localization on proteins, including resolving the crystal structure of antibody-antigen complexes, 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, NY, 1999. In one instance, epitope localization can be accomplished using H / D-Ex (hydrogen-deuterium exchange) combined with protein hydrolysis and mass spectrometry. In another instance, epitope localization can be used to determine the antibody-binding sequence. Epitopes can be linear epitopes, i.e., contained in a single amino acid fragment, or conformational epitopes (linear sequences of primary structure) formed by the three-dimensional interactions of amino acids that are not necessarily contained in a single fragment. Peptides of varying lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used for antibody binding assays. In another example, epitopes binding to antibodies can be determined in systematic screening by using overlapping peptides derived from target antigen sequences and by measuring antibody binding. Based on gene fragment expression assays, open reading frames encoding target antigens are randomly or through specific genetic constructs, and the reactivity of the expressed antigen fragments with the test antibody is determined. For example, gene fragments can be generated by PCR, then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. The binding of the antibody to the radiotagged antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Some epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, the binding of a defined library of overlapping peptide fragments to the test antibody can be tested in a simple binding assay. In another example, mutagenesis of the antigen-binding domain, domain exchange assays, and alanine scanning mutagenesis can be performed to identify the sufficient, and / or essential residues required for epitope binding. For example, a domain exchange assay can be performed using a target antigen mutant, in which fragments of the sADAM9v2 peptide have been replaced (exchanged) with sequences derived from closely related but antigenically different proteins (such as CD-28). By evaluating the binding of the antibody to the mutant sADAM9v2, the importance of a specific antigen fragment to antibody binding can be assessed. Alternatively, a competitive assay can be performed using other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as other antibodies. Competitive assays are well known to those skilled in the art.

[0075] In some instances, anti-sADAM9v2 antibodies are prepared using recombinant techniques as described below. The nucleic acids encoding the heavy and light chains of the anti-sADAM9v2 antibody, as described herein, can be cloned into an expression vector, with each nucleotide sequence operatively linked to a suitable promoter. In one instance, each nucleotide sequence encoding the heavy and light chains is operatively linked to a different promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operatively linked to a single promoter, such that both the heavy and light chains are expressed by the same promoter. If 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 an antibody are cloned into two vectors, which can then be introduced into the same or different cells. When the two chains are expressed in different cells, they can be isolated from the host cells that express them, and the separated heavy and light chains can be mixed and cultured under suitable conditions to form antibodies.

[0077] Typically, a nucleic acid sequence encoding one or all strands of an antibody can be cloned into a suitable expression vector and operatively ligated to a suitable promoter using methods known in the art. 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 be paired with each other and linked together by a ligase. Alternatively, synthetic nucleic acid adapters can be ligated to the ends of a gene. These synthetic adapters 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 to produce the antibody.

[0078] Adjustable promoters can be used. Such adjustable promoters include those that use the E. coli lac repressor as a transcriptional regulator to regulate transcription of lac operons derived from mammalian cell promoters (Brown, M. et al., Cell, 49:603-612 (1987)), and those that use the tetracycline repressor (tetR) [Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-555115 (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 tunable promoter containing a repressor carrying an operon can be used. In one embodiment, the lac repressor derived from *E. coli* can act as a transcription regulator to regulate transcription derived from mammalian cell promoters 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 a tetracycline repressor (tetR) with a transcription activator (VP16) to create the tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP16), using a minimal promoter carrying tetO derived from a human cytomegalovirus (hCMV) promoter to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operon is correctly located downstream of the TATA element of the CMVIE promoter, the tetracycline repressor alone (tetR), rather than the tetR-mammalian cell transcriptomic fusion derivative, can act as an effective transregulator to control gene expression in mammalian cells (Yao et al., Human Gene Therapy). A particular advantage of this tetracycline-inducible switch is that it does not require the use of a tetracycline repressor-mammalian cell transactivator or repressor fusion protein, which, under certain circumstances, can be cellularly toxic (Gossen 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 the following: a selectable marker gene, such as a neomycin gene for selecting stable or transient transfectants in mammalian cells; an enhancer / promoter sequence derived from the immediate early human CMV gene for high-level transcription; transcription termination and RNA processing signals derived from SV40 for mRNA stability; SV40 polyomavirus origin of replication and ColE1 for appropriate free replication; an internal ribosome binding site (IRESe), a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known in the art and are available. Examples of polyadenylation signals that can be used to carry out the methods described herein include, but are not limited to, human collagen I polyadenylation signals, human collagen II polyadenylation signals, and SV40 polyadenylation signals.

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

[0082] In some embodiments, the methods for preparing the antibodies described herein involve a recombinant expression vector encoding both the heavy and light chains of an anti-sADAM9v2 antibody, as also described herein. The recombinant expression vector can be introduced into suitable host cells (e.g., dhfr-CHO cells) using conventional methods (e.g., calcium phosphate-mediated transfection). Positively transformed host cells can be selected and cultured under suitable conditions to allow expression of both polypeptide chains that form the antibody, which can then be recovered from the cells or 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 the anti-sADAM9v2 antibody and the other encoding the light chain of the anti-sADAM9v2 antibody. These two recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) using conventional methods (e.g., calcium phosphate-mediated transfection).

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

[0085] The process involves preparing recombinant expression vectors, transfecting host cells, selecting transformants, culturing host cells, and recovering antibodies from the culture medium using standard molecular biology techniques. For example, some antibodies can be separated using affinity chromatography with protein A, protein G, or protein L conjugated to the matrix.

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

[0087] III. Pharmaceutical Composition

[0088] Antibodies as described herein, along with vectors encoding nucleic acids or nucleic acid sequences, or host cells containing said vectors, can be mixed with pharmaceutically acceptable excipients to form pharmaceutical compositions for treating a target disease. "Acceptable" means that the vector must be compatible with (and preferably, stabilize the active ingredient) the composition and harmless to the subject being 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. KEHoover.

[0089] The pharmaceutical compositions containing anti-sADAM9v2 antibodies disclosed herein may also contain suitable buffers. The buffer is a weak acid or weak base used to maintain the pH of the solution near a selected value after the addition of other acids or bases. In some instances, the buffers disclosed herein may be buffers that maintain physiological pH despite changes in carbon dioxide concentration (produced through cellular respiration). Exemplary buffers include, but are not limited to, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer, Dulbecco 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 composition described herein may maintain a pH value of about 5-8. For example, the pH value of the pharmaceutical composition may 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 may 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 comprise one or more suitable salts. Salts are ionic compounds that can be formed by the neutralization reaction of an acid with a base (Skoog, DA; West, DM; Holler, JF; Crouch, SR (2004), Chapters 14-16. Fundamentals of Analytical Chemistry (8th ed.)). Salts consist of a corresponding number of cations (positively charged ions) and anions (negative ions), thus the product is electrically neutral (without net charge). As described herein, an ion is an atom or molecule that gains or loses one or more valence electrons, giving 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 in this article, cations ( + A cation is an ion with fewer electrons than a proton, 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 cation. A non-limiting example of a monovalent cation is hydrogen (H₂O). + ), sodium (Na + ), potassium (K) + ), ammonium (NH4) + Lithium (Li) +), copper(Cu) + ), silver (Ag) + Examples of polyvalent cations include magnesium (Mg). 2+ ), calcium (Ca 2+ ), barium (Ba 2+ ), Beryllium (Be) 2+ ), copper (Cu) 2+ ), Fe 2+ ), iron (Fe3) + ), lead(II)(Pb 2+ ), lead(IV)(Pb4) + ), Manganese(II)(Mn 2+ ), Strontium (Sr) 2+ ), Tin(IV)(Sn4) + ), Zinc (Zn) 2+ )wait.

[0093] As described herein, anions are ions that have more electrons than protons, giving them a net negative charge. A non-limiting example of anion is an azide (N3). - ), bromide (Br - ), chloride (Cl) - ), fluoride (F - ), hydride (H) - ), iodide (I - ), nitrides (N - ), oxides (O) 2- ), sulfides (S 2- ), carbonates (CO3) 2- ), bicarbonate (HCO3) - ), hydrogen sulfate (HSO4) - ), hydroxide (OH) - ), dihydrogen phosphate (H2PO4) - ), sulfate (SO4) 2- ), sulfites (SO3) 2- ), silicate (SiO3) 2- )wait.

[0094] Suitable salts used in the pharmaceutical compositions described herein may include monovalent cations and monovalent or polyvalent anions. Alternatively, salts used in the pharmaceutical compositions described herein may include monovalent or polyvalent 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 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, emulsifiers, foaming agents, and dispersants. Suitable surfactants particularly include nonionic agents, such as polyoxyethylene sorbitol (e.g., Tween). TM 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span) TM 20, 40, 60, 80, or 85). Compositions containing surfactants will conveniently contain between 0.05% and 5% surfactant, and may be between 0.1% and 2.5%. It should be understood that other ingredients, such as mannitol or other pharmaceutically acceptable carriers, may be added if necessary.

[0096] Pharmaceutical compositions containing the 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 contain one or more antioxidants. As used herein, an antioxidant is an agent that prevents or delays the oxidative degradation of the active ingredient contained in the composition. Antioxidants used herein may 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, blocking chain reactions. Reducing agents are compounds with lower redox potentials and are therefore more easily oxidized than the drug they protect. Reducing agents remove 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+ Chelating agents can shorten induction time and increase oxidation rate. Trace amounts of these metal ions are often introduced into pharmaceuticals during the preparation process. Therefore, chelating agents do not possess antioxidant activity, but they can enhance the effects of phenolic antioxidants by inactivating catalytic metal ions through reaction.

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

[0099] The pharmaceutical composition used in the method of this invention may comprise a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KEHoover). The acceptable carrier, excipient, or stabilizer is non-toxic to the receptor at the dose and concentration used and may comprise buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl benzoates, 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 containing antibodies (or encoding nucleic acids) 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 a specific cycle time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by reverse-phase evaporation from a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter membrane with a defined pore size to produce liposomes with a desired diameter.

[0101] Antibodies or one or more encoding nucleic acids can also be encapsulated in microcapsules, for example, by coagulation techniques or by interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., 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 into sustained-release forms. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being in the form of a molded article, such as a film or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methyl methacrylate) or poly(vinyl alcohol)), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and 7-L2O ethyl glutamate, non-degradable ethylene-vinyl acetate, and degradable lactic-glycolic acid copolymers, such as LUPRON DEPOT. TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose isobutyrate acetate and poly-D-(-)-3-hydroxybutyric acid.

[0103] In other instances, the pharmaceutical compositions described herein can be formulated into sustained-release forms to selectively influence binding to tissues or tumors by implementing specific protease biology techniques, for example, through peptide masking of the antigen-binding site of an antibody, to target one or more proteases (such as Probody) in the tumor microenvironment. TM or Conditionally ActiveBiologics TM Selective protease cleavage of ). This activation can be formulated as reversible in a normal microenvironment.

[0104] Pharmaceutical compositions intended for internal administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filter membrane. Therapeutic antibody compositions are typically placed in containers with sterile inlets, such as intravenous solution bags or vials with stoppers that can be punctured by a subcutaneous 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 for administration by inhalation or blowing.

[0106] To prepare solid compositions (such as tablets), the main active ingredient can be mixed with a pharmaceutical carrier, such as conventional tablet ingredients like corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium hydrogen phosphate, or gum, and other pharmaceutical diluents (e.g., water), to form a solid preformulation composition containing a homogeneous mixture of the composition of the present invention or a non-toxic, pharmaceutically acceptable salt. When these preformulation compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition, allowing for easy further fractionation into equivalent unit dosage forms, such as tablets, pills, and capsules. The solid preformulation composition can then be further fractionated into unit dosage forms of the aforementioned type, containing 0.1 to approximately 500 mg of the active ingredient of the present invention. Tablets or pills of the new composition can be coated or otherwise compounded to provide dosage forms with the advantage of prolonged action. For example, tablets or pills may contain an internal dose and an external dose component, the latter covering the former in the form of a film. These two components can be separated by an enteric coating, which resists breakdown in the stomach and allows the inner components to enter the duodenum intact or delays release. A variety of materials can be used for such enteric coatings or coatings, including various polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0107] Commercially available fat emulsions (such as Intralipid) can be used. TM Liposyn TM Infonutrol TM Lipofundin TM and Lipiphysan TM To prepare a suitable emulsion, the active ingredient can be dissolved in a premixed emulsion composition, or dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and mixed with phospholipids (e.g., egg yolk lecithin, soybean lecithin, or soybean lecithin) and water to form an emulsion. It should be understood that other ingredients, such as glycerol or glucose, can be added to adjust the emulsion's tension. A suitable emulsion will typically contain up to 20% oil, for example, between 5% and 20%. Fat emulsions may contain 0.1 to 1.0 μm, particularly between 0.1 and 0.5 μm, and have a pH ranging from 5.5 to 8.0. The emulsion composition can be prepared by mixing an antibody with an intralipid... TM Those prepared by mixing their components (soybean oil, lecithin, glycerin, and water).

[0108] Pharmaceutical compositions for inhalation or inhalation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered orally or via nasal inhalation to produce local or systemic effects. Compositions preferably in sterile, pharmaceutically acceptable solvents can be nebulized using a gas nebulizer. The nebulized solution can be inhaled directly from a nebulizer, or the nebulizer can be connected to a mask, tent, or intermittent positive pressure ventilation machine. Solution, suspension, or powder compositions can preferably be administered orally or nasally by a device that delivers the formulation in a suitable manner.

[0109] IV. Therapeutic applications

[0110] Any antibodies described herein, as well as those encoding nucleic acids or nucleic acid sequences, vectors containing them, or host cells containing said vectors, may be used to treat sADAM9v2-mediated diseases. As used herein, sADAM9v2-mediated diseases refer to any medical condition associated with increased levels of sADAM9v2 or increased sensitivity to sADAM9v2. Non-limiting examples of sADAM9v2-mediated diseases include prostate cancer, etc.

[0111] To practice the methods disclosed herein, an effective amount of the pharmaceutical composition described herein may be administered to a subject requiring treatment (e.g., a human) via a suitable route, such as intravenous administration, for example by bolus or continuous infusion over a period of time, via intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or local routes. Commercially available nebulizers (including jet nebulizers and ultrasonic nebulizers) for use with liquid formulations may be used for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, the antibodies described herein may be nebulized using fluorocarbon formulations and metered-dose inhalers, or inhaled as lyophilized and ground powders.

[0112] Subjects treated using the methods described herein may be mammals, more preferably humans. Mammals include, but are not limited to, farm animals, sporting animals, pets, primates, horses, dogs, cats, mice, and rats. Human subjects requiring treatment may be human patients who have or are suspected of having an inflammatory disease, an autoimmune disease, cancer, an infectious disease, or other condition requiring modulation of the immune response, or who are at risk of having said disease. Subjects with a target disease or condition may be identified by routine medical examinations (e.g., laboratory tests, organ function tests, CT scans, or ultrasound). Subjects suspected of having any such target disease / condition may exhibit one or more symptoms of said disease / condition. Subjects at risk of having said disease / condition may be subjects who have one or more risk factors for said disease / condition.

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

[0114] As used in this article, "overexpression" refers to the expression of sADAM9v2 on the surface of cancer cells being significantly higher than that of normal cells.

[0115] Determining whether a given amount of antibody achieves a therapeutic effect will be obvious to those skilled in the art. As is recognized by those skilled in the art, variations in effective amounts depend on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, body size, sex, and weight, duration of treatment, the nature of any concomitant treatment, the specific route of administration, and factors within the knowledge and experience of the healthcare professional. These factors are well known to those skilled in the art and can be resolved through routine laboratory procedures alone.

[0116] Typically, the preferred dose is the maximum dose of a single component or combination thereof, i.e., the highest safe dose based on reasonable medical judgment.

[0117] Empirical factors (such as half-life) often help determine dosage. For example, antibodies compatible with the human immune system (such as humanized antibodies or whole-human antibodies) can be used to extend the antibody's half-life and prevent it from being attacked by the host immune system. Dosage frequency can be determined and adjusted during treatment and, in general (but not always), is based on the treatment and / or inhibition and / or improvement and / or delay of the target disease / symptom. Alternatively, a sustained-release formulation of the antibody may be suitable. Various formulations and devices for achieving sustained release are known in the art.

[0118] In one instance, in an individual who has been administered an antibody once or multiple times, the dosage of the antibody described herein can be determined empirically. The individual is then given an increased dose of the antagonist. To assess the effectiveness of the antagonist, indicators of the disease / symptom can be tracked.

[0119] In general, the initial candidate dose for administering any of the antibodies described herein may be about 2 mg / kg. For the purposes of this invention, typical daily, weekly, bi-weekly, or bi-weekly dose ranges may be 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 days, weeks, months, or longer, depending on the condition, treatment may continue until desired symptom suppression is achieved or until a therapeutic level sufficient to alleviate the target disease or condition or its symptoms is reached. An exemplary dosing regimen includes 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 desired by the physician. For example, combination therapy with a dose of 1 mg / kg every 3 weeks in conjunction with at least one additional immunotherapy is also covered in this invention. In some embodiments, doses in the range of about 3 μg / mg to about 3 mg / kg (e.g., 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) may be used. In some embodiments, the dosing frequency is once weekly, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, or once every 10 weeks; or once monthly, once every 2 months, once every 3 months, or for longer periods. Progression of the therapy can be easily monitored using conventional techniques and trials. The dosing regimen (including the antibody used) may vary over time.

[0120] In some implementations, for normal-weight adult patients, a dose ranging from about 0.1 to 5.0 mg / kg may be administered. In some instances, the dose of the anti-sADAM9v2 antibody described herein may be 10 mg / kg. Specific dosing regimens (i.e., dose, timing, and repetitions) will depend on the individual and their medical history, as well as the nature of the individual agents (the half-life of the agent, and other considerations well known in the art).

[0121] For the purposes of this invention, the appropriate dose of the antibody as described herein will depend on the specific antibody, antibody, and / or non-antibody peptide (or combination thereof) used, the type and severity of the disease / condition, whether the antibody is administered for prophylactic or therapeutic purposes, prior therapy, the patient's clinical history and response to antagonists, and the judgment of the attending physician. Typically, clinicians will administer antibodies up to a dose that yields the desired outcome. In some embodiments, the desired outcome is tumor size reduction, prolonged progression-free survival, and / or overall survival. Methods for determining whether a dose produces the desired outcome will be apparent to those skilled in the art. Administration of one or more antibodies may be continuous or intermittent, depending on factors such as the physiological condition of the receptor, whether the administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Antibody administration may be substantially continuous over a predetermined period of time, or may be a series of intervals of doses, for example, before, during, or after the 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 suffering from a target disease or condition, having symptoms of the disease / condition, or being susceptible to the disease / condition, with the aim of curing, healing, reducing, alleviating, altering, remedying, improving, or influencing the condition, symptoms of the disease, or susceptibility to said disease or condition. Reducing the target disease / condition includes delaying the onset or development of the disease, or reducing the severity of the disease.

[0123] Reducing disease does not necessarily require a cure. As used herein, “delaying” the progression of a target disease or symptom refers to delaying, hindering, slowing, stabilizing, and / or prolonging the progression of the disease. The delay can vary in length, depending on the disease history and / or the individual being treated. A method of “delaying” or reducing disease progression or delaying disease onset, when compared to not using the method, is a method of reducing the likelihood of developing one or more disease symptoms and / or reducing the degree of symptom relief within a given timeframe. Such comparisons are typically based on clinical studies using a sufficient number of subjects to provide statistically significant results.

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

[0125] Depending on the type or location of the disease to be treated, the pharmaceutical composition may be administered to the subject using conventional methods known to a person skilled in the medical field. The composition may also be administered via other conventional routes, such as parenteral, topical, oral, via inhalation spray, rectal, nasal, buccal, vaginal, or via an implantable cartridge. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intraperitoneal, intratumoral, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrasheathal, intralesional, and intracranial injection or infusion techniques. Additionally, it may be administered to the subject via injectable reservoir routes, such as using injectable or biodegradable materials and methods with 1, 3, or 6-month reservoirs.

[0126] Injectable compositions may 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 via infusion, thereby infusing a pharmaceutical formulation containing antibodies and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% glucose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular formulations (e.g., sterile formulations of antibodies suitable for soluble salt form) can be dissolved and administered in pharmaceutical excipients (e.g., water for injection, 0.9% saline, or 5% glucose solution).

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

[0128] Targeted delivery can also be achieved using therapeutic compositions containing antisense polynucleotides, expression vectors, or subgenomic polynucleotides. 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 (JA.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; and Wu et al., J. Biol. Chem. (1991) 266:338.

[0129] The therapeutic composition comprising polynucleotides (such as those encoding antibodies described herein) is administered in the range of about 100 ng to about 200 mg of DNA for topical application in a gene therapy regimen. In some embodiments, during the gene therapy regimen, DNA concentrations ranging from 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 may also be used.

[0130] The therapeutic polynucleotides and peptides 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). Expression of these coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of the coding sequences can be constitutive or modulotropic.

[0131] Virus-based vectors for delivering desired polynucleotides and expressing them in desired cells are well known in the art. Exemplary virus-based vectors include, but are not limited to, recombinant retroviruses (see, for example, PCT Publications WO90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Patents 5,219,740 and 4,777,127; GB Patent 2,200,651; and EP Patent 0 345242), alphavirus-based vectors (e.g., Sindbis virus vector, 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-1246). VR-532), and adeno-associated virus (AAV) vectors (see, for example, PCT Publications WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO95 / 00655). Inactivated adenovirus linked to DNA may also be used, as described in Curiel, Hum. Gene Ther. (1992) 3:147.

[0132] Non-viral delivery vectors and methods may also be used, including, but not limited to, polycationically condensed DNA with or without being linked to a single inactivated adenovirus (see, for example, Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, for example, Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vectors (see, for example, U.S. Patent No. 5,814,482; PCT Publications WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338); and nucleic acid charge neutralization or fusion with the cell membrane. Naked DNA may also be used. Exemplary methods for introducing naked DNA are described in PCT Publication 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 Publications 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 Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0133] The specific dosing regimen (i.e., dosage, timing, and repetitions) used in the methods described herein will depend on the specific subject and their medical history. In some embodiments, more than one antibody or a combination of an antibody and another suitable therapeutic agent may be administered to the subject requiring treatment. Antibodies may also be used with other agents to enhance and / or complement the effects of said agents. The therapeutic efficacy against the target disease / condition can be evaluated using methods well-known in the art.

[0134] Antibodies and treatments involving, for example, the anti-sADAM9v2 antibodies and treatments described herein 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" means that the antibody composition is administered simultaneously or sequentially with 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, radiation therapy, gene therapy, and anti-infective therapies. Such therapies can be administered simultaneously or sequentially (in any order) with the treatment according to the invention.

[0135] When the antibody composition described herein is used in combination with a second therapeutic agent, a subtherapeutic dose of the composition or the second agent, or a subtherapeutic dose of both, may be used to treat subjects who are at risk of having a disease or condition associated with sADAM9v2-mediated signaling or who are at risk of having a disease or condition associated with sADAM9v2-mediated signaling. As used herein, a “subtherapeutic dose” means a dose less than that which would produce a therapeutic outcome in a subject when administered to the subject in the absence of one or more other agents. Thus, a subtherapeutic dose of an agent is a dose that would not produce the desired therapeutic outcome in a subject without administration of the anti-sADAM9v2 antibody described herein. Therapeutic doses of many agents used clinically are well known in the medical field, and other therapeutic doses can be determined by those skilled in the art without extensive experimentation. Therapeutic doses have been extensively described in references such as Remington's Pharmaceutical Sciences, 18th ed., 1990; and in many other medical references relied upon by the medical community as guidelines for the treatment of diseases and disorders. Other useful medications can be found in Physician's Desk Reference, 59th edition, (2005), Thomson P DR, Montvale NJ; 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 NJ.

[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. Results obtained from such assays 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 applications (e.g., diagnostic applications), the anti-sADAM9v2 antibodies described herein can be bound to a detectable tag (e.g., an imaging agent, such as a contrast agent) to detect the presence of sADAM9v2 (e.g., secreted sADAM9v2) in vivo or in vitro. As used herein, “conjugated” or “attached” refers to two entities bound together, preferably with sufficient affinity to achieve the 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 polymer linker.

[0138] Joining or attachment can include covalent or non-covalent bonds and other forms of bonding, such as capture, for example, capture of one entity on or within another entity, or capture of one or two entities on or within a third entity, such as micelles.

[0139] In one example, an anti-sADAM9v2 antibody, as described herein, can be attached to a detectable tag, which is a compound that directly or indirectly releases a detectable signal, thereby enabling the detection, measurement, and / or identification of aptamers in vitro or in vivo. Examples of such "detectable tags" include, but are not limited to, fluorescent tags, chemiluminescent tags, colorimetric tags, enzyme markers, radioisotopes, and affinity tags (such as biotin). These tags can be directly or indirectly bound to aptamers using conventional methods.

[0140] In some implementations, the detectable label may be a reagent suitable for in vitro detection of sADAM9v2 secretory cells, which may 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 Inoxyquinoline, 131 Sodium iodide, 99 mTc methyl bromfenac and 99 mTc red blood cells, 123 Sodium iodide, 99 mTc esamine oxime, 99 mTc macropolymer albumin, 99 mTc methylene diphosphate, 99 mTc thiopeptide 99 mTc oxyphosphonate, 99 mTc triaminepentaacetic acid, 99 mTc pertechnetate, 99 mTc stapbital 99 mTc sulfur colloid 99 mTc tetrophosphine, thallium-201, or xenon-133.

[0141] The reporter agent can be a dye, such as a fluorophore, which can be used to detect diseases mediated by sADAM9v2 secretory cells in tissue samples.

[0142] For in vitro diagnostic assays, anti-sADAM9v2 antibodies can be contacted with samples suspected of containing sADAM9v2 (e.g., sADAM9v2-secreting cells in the disease microenvironment or soluble sADAM9v2). The antibody and sample can be cultured under suitable conditions for an appropriate time to allow the antibody to bind to the sADAM9v2 antigen. This interaction can then be detected using conventional methods such as ELISA, histological staining, or FACS.

[0143] For in vivo diagnostic assays, an appropriate amount of anti-sADAM9v2 antibody conjugated to a tag (e.g., an imaging agent or contrast agent) can be administered to the subject requiring the test. The presence of the labeled antibody can be detected using standard methods based on the signal released by the tag.

[0144] For scientific research purposes, anti-sADAM9v2 antibodies can be used to study the biological activity of sADAM9v2, detect the presence of sADAM9v2 within cells, and / or regulate the secretion of 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 not previously identified as producing sADAM9v2). Cells are permeabilized before contact with the anti-sADAM9v2 antibody. The antibody and sample can be cultured under suitable conditions for an appropriate time to allow the antibody to bind to the sADAM9v2 antigen. This interaction can then be detected using conventional methods (e.g., ELISA, histological staining, or FACS). VI. Kits for Therapeutic and Diagnostic Applications

[0145] The present invention also provides kits for therapeutic or diagnostic applications as disclosed herein. Such kits may comprise one or more containers containing anti-sADAM9v2 antibodies (such as any of those described herein).

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

[0147] Instructions for use related to the anti-sADAM9v2 antibody typically include information on the intended therapeutic dose, dosing regimen, and route of administration. Containers may be single-dose, bulk packaging (e.g., multi-dose packs), or subunit doses. Instructions provided with the kits of this invention are typically written instructions on a label or packaging insert (e.g., paper 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 intended to treat, delay the onset of, and / or alleviate diseases or conditions that can be treated by modulating the immune response, such as autoimmune diseases. Instructions for use in carrying out any of the methods described herein may be provided.

[0149] The kit of the present invention is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth bottles, flexible packaging (e.g., sealed Mylar or plastic bags), etc.

[0150] Packaging for use in conjunction with specific devices, such as inhalers, nasal delivery devices (e.g., nebulizers), or infusion devices (e.g., micropumps), is also considered. The kit may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). The container may also have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured 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 include other components, such as buffers and explanatory information. Typically, the kit includes a container and a label or instruction manual on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above.

[0152] This document also provides kits for detecting secreted sADAM9v2 in samples. Such kits may contain any of the anti-sADAM9v2 antibodies described herein. In some cases, anti-sADAM9v2 antibodies may be conjugated to detectable tags (such as those described herein). As used herein, “conjugated” or “attached” refers to two entities being bound together, preferably with sufficient affinity to achieve the therapeutic / diagnostic benefit of the binding between the two entities. The binding between the two entities can be direct or through a connector, such as a polymer connector. 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 micelles.

[0153] Alternatively or additionally, the kit may contain a secondary antibody that binds to the anti-sADAM9v2 antibody. The kit may also include instructions for using the anti-sADAM9v2 antibody to detect secreted sADAM9v2.

[0154] VII. General Techniques

[0155] Unless otherwise stated, the present invention will be practiced using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the scope of the art. MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, 1998) Academic Press; Animal Cell Culture (edited by RI Freshney, 1987); Introduction toCell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell andTissue Culture: Laboratory Procedures (edited by A.Doyle, JBGriffiths, and DGNewell, 1993-8) J.Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by DMWeir and CCBlackwell); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. C. Salos, eds., 1987); Current Protocols in Molecular Biology (F. M. Usubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Olgan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA. Janeway and P. Travers, 1997); Antibodies (P.Finch (1997); Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995). Without further elaboration, it is believed that those skilled in the art can utilize the invention to its fullest extent based on the foregoing description. Therefore, the following specific embodiments should be interpreted as illustrative only and not as limiting the remainder of the invention in any way. All publications cited herein are incorporated by reference for the purposes or subject matter of this citation.

[0156] Without further elaboration, it is believed that those skilled in the art can utilize the invention to its fullest extent based on the above description. Therefore, the following specific embodiments should be interpreted as illustrative only and do not limit the remainder of the invention in any way. All publications cited herein are incorporated by reference for the purposes or subject matter of this citation.

[0157] The discovery of sADAM9v2

[0158] In PCR analysis assessing ADAM9 expression, differences in the length of some ADAM9 sequences were observed compared to a predetermined baseline (see reference). Figure 1(Indicated by red arrows). This 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), PC (representing the ADAM9 gene as a positive control), BPH-1 (benign prostatic hyperplasia-1), LNCaP (prostate cancer cell line derived from patients with lymph node metastases), C4-2 (androgen-insensitive derivative of LNCaP), C4-2B (representing bone metastasis tendency of C4-2), CWR22Rv1 (representing an androgen-mutant prostate cancer cell line), DU145 (prostate cancer cell line derived from patients exhibiting brain metastases), PC3 (prostate cancer cell line derived from patients exhibiting bone metastases), and PC3M (a cell derivative of PC3 that exhibits significant metastatic behavior in mouse models). The main objective of this invention is to determine the expression of ADAM9 mRNA in the aforementioned prostate cancer cell lines. RNA was isolated using the Qiagen RNA isolation system (provided by Qiagen, Inc.) and then converted to cDNA using the MMLV reverse transcriptase system. The PCR procedure was facilitated by introducing cDNA into PCR reagents (provided by ThermoFisher, Inc.) and performing PCR analysis via agarose gel electrophoresis. The results showed that these cell lines contained not only the complete ADAM9 mRNA sequence but also different variants. This outcome highlights the potential of ADAM9 messenger RNA to exhibit unique alternative splicing iterations in prostate cancer cells, which may contribute to the malignant and metastatic progression of prostate cancer. Therefore, the inventors isolated these PCR bands for gene sequencing.

[0159] After sequencing the entire ADAM9 genome, an ADAM9 genome diagram was created, and the inventors discovered alternative splicing variants (see...). Figure 2 Note that ADAM9-splicing form 1 exhibits alternative splicing between exon 18 at position 2046 and exon 19 at position 2147. This results in out-of-frame splicing and early termination of ADAM9 translation. Consequently, both the transmembrane and cytoplasmic domains of the full-length ADAM9 are missing. Furthermore, the inventors discovered a novel alternative splicing event confirming a deletion between exon 18 at position 2046 and exon 19 at position 2236. This results in the omission of the transmembrane domain, but the cytoplasmic sequence remains within the reading frame.

[0160] Further analysis of culture media from tumors, tumor-associated peripheral cells, and normal tissues revealed that sADAM9 was detected only in culture media from tumors and tumor-associated peripheral cells (see [link to relevant documentation]). Figure 3Cells were cultured for three days before the culture medium was collected, followed by protein concentration. Simultaneously, the cultured cells were collected and proteins were extracted using a protein extraction system (RIPA buffer, comprising NaCl 150 mM, Triton-X-100 1%, sodium deoxycholate 0.5%, SDS 0.1%, and Tris-HCl pH 8.0 50 mM). This invention aims to verify the expression of the ADAM9 gene in various prostate cancer cell lines and corresponding control samples. This group included NC (control, representing samples without prostate cancer), PC (as a positive control for the ADAM9 gene), BPH-1 (benign prostatic hyperplasia-1), LNCaP (prostate cancer cell line derived from patients exhibiting lymph node metastasis), C4-2 (androgen-insensitive branch of LNCaP), C4-2B (showing bone metastasis tendency of C4-2), CWR22Rv1 (prostate cancer cell line exhibiting androgen mutation), DU145 (prostate cancer cell line derived from patients with brain metastases), PC3 (prostate cancer cell line derived from patients exhibiting significant bone metastasis), and PC3M (a cell line derived from PC3 that showed significant metastatic tendency in a mouse model). For protein analysis, Western blotting was performed using an 8% polyacrylamide gel. Proteins were transferred from the gel to nitrocellulose membranes by electrophoresis. Specific antibodies, particularly ADAM9 (MAB939), purchased from R&D Biosystems, were used for Western blotting.

[0161] In one particular implementation, an ELISA system is used to perform a blood test to assess sADAM9 expression in patients with benign prostatic hyperplasia (BPH) and prostate cancer. Figure 4 As shown, sADAM9 expression was significantly higher in prostate cancer patients than in BPH patients. Serum samples (anonymous) from both BPH and prostate cancer patients were obtained from the Taipei Medical University (TMU) Human Biobank, comprising 10 samples from each patient. 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] like Figure 5As shown, as determined using laser capture microdissection (LCM) from three patients, sADAM9v2 expression in cancer-associated peripheral cells exceeded that in the corresponding benign cells from the same patients. To determine that sADAM9v2 is primarily expressed in prostate tumor-associated peripheral cells, LCM was performed on tissue samples from three different patients. The tumor and peripheral regions of these tissue slides (derived from TMU Biobank) were validated by pathologists. The LCM procedure was performed according to a commercially available kit (…). The kit, for DNA and RNA extraction and purification, was followed according to the instructions of Invitrogen, Inc. LCM was performed using the MMI CellCut and Cell Ector systems provided by the TMU core facility. Then, [the kit was used]. RNA from these cells was isolated using a frozen RNA isolation kit, and cDNA was synthesized using the MMLV cDNA system (Thermo Fisher Scientific, Inc.). Quantitative PCR analysis was performed on a 480 system (Roche Diagnostics, Inc.). Fold-change differences were calculated using the 2^ΔΔCT analysis method, as outlined in the NIH overview (published in Biostat. Bioinforma Biomath. 2013 Aug; 3(3):71-85). The final results are expressed as fold-changes derived from comparisons between cancerous and benign lesions.

[0163] These tests confirmed the expression of sADAM9v2 in tumor cells and surrounding cells. This disclosure includes structural simulations of ADAM9v2 and reveals highly antigenic alternative splicing sites, as indicated by arrows (see [link]). Figure 6 ).

[0164] In the following sections, this disclosure describes a specificity test for sADAM9v2 with hybridoma. Cell lysates and conditioned medium (CM) of sADAM9v1 and sADAM9v2 were also collected to test antibody specificity. Results are as follows... Figure 7As shown. The inventors selected mouse antibodies that produce ADAM9 monoclonal antibodies for the following assays. NMRI mouse strains were used to generate sADAM9v2 hybridomas and mAbs. All animal procedures were performed according to recommended general animal care and treatment standards. Immunoconjugates were first prepared using N-(-3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) after a routine conjugation 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. NMRI mice aged 6 to 12 weeks were selected for immunization, administered 200 μL of PBS containing 150 μg of the immunoconjugate and 100 μL of complete Freund's adjuvant. A booster injection containing the same amount of immunoconjugate was given 6 weeks later. Blood samples were collected 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, mice were euthanized, and a suspension of spleen cells was prepared. This suspension was then resuspended in balanced salt buffer (BSS) composed of 125 mM NaCl, 5 mM KCl, 4 mM CaCl2, 2.5 mM MgCl2, and 5 mM Tris-HCl (pH 7.4). The mixture was then combined with PEG8000, and electroporation was promoted by balancing the ratio of spleen cells to myeloma cells, followed by resuscitation in HAT medium for one week. After stabilizing the cultured cells, hybridoma cloning was performed. The inventors ultimately identified a monoclonal hybridoma and demonstrated that it produced mAbs with significant specificity for ADAM9v2.

[0165] Preparation of Recombinant Human sADAM9v2 mAb: After identifying different hybridomas suitable for generating sADAM9v2 mAb, the inventors performed isotype assessment and classification to determine the unique isotype of sADAM9v2 mAb present within the hybridomas. Isotype screening was facilitated using a hybridoma isotype ELISA system from Thermo Fisher. With appropriate isotype validation, specific antibody isotypes present in the hybridomas were sequenced. After validating the distinct sequences of the heavy and light chains, the inventors proceeded to isolate and clone specific sequences associated with sADAM9v2 mAb. This was achieved via PCR targeting specific assay sequences, which were then integrated into TGEX-heavy and TGEX-light chain expression vectors for subsequent applications. The TGEX vectors containing the heavy and light chains of sADAM9v2 mAb ensure consistent and preserved expression of the inventors' patented antibodies within their system.

[0166] Based on the migration of prostate cancer cells using sADAM9: [the method employed] A systematic Transwell cell migration assay was used to assess the migration of prostate cancer cells after the introduction of sADAM9. The inventors, from R&D... Recombinant ADAM9 (rADAM9) was acquired and added to the Transwell setup. Observed data showed that sADAM9 (in rADAM9 form) promoted prostate cancer cell migration in a dose-proportional manner (as shown on the left in the figure). Furthermore, the mAb targeting sADAM9v2 inhibited cell migration activity within the Transwell system, suggesting that the sADAM9v2 mAb functions as a neutralizing antibody (nAb).

[0167] The results are as follows Figure 8 As shown, the migration rate of prostate cancer cells is dose-dependent with sADAM9 concentration, and the developed sADAM9v2 mAb can reduce the migration rate of prostate cancer cells.

[0168] Previous findings by the inventors highlighted the specificity of sADAM9v2 mAb and its role in inhibiting prostate cancer cell motility. Ample evidence suggests that the migration and invasion behavior of prostate cancer cells is associated with downstream cellular regulation of AKT and Src. Therefore, the inventors investigated the AKT pathway (reference: Int. J. Mol. Sci., 2020 Jun; 21(12):4507) and the Src pathway (reference: Cancer and Metastasis Reviews 2014 Feb; 33:595-606). Therefore, following the above tests, this disclosure further attempts to identify the mechanism by which sADAM9 increases prostate cancer motility and migration, and finds that sADAM9 increases prostate cancer motility and migration by activating downstream AKT effectors. Protein assays showed elevated AKT phosphorylation levels. Prostate cancer cells were grown on culture plates coated with collagen-1 (considering the abundance of collagen in bone) or uncoated. Cells were treated with 5 μg / ml sADAM9 at 30 or 60-minute intervals before harvest. Cell lysis was performed using protein lysis buffer (RIPA buffer) supplemented with phosphatase inhibitors. Antibodies specifically targeting the phosphorylated forms of AKT and Src were obtained to assess the phosphorylation levels of AKT and Src, thereby verifying the activities of AKT and Src.

[0169] like Figure 9 As shown, sADAM9 has the ability to enhance downstream signaling of cell motility, such as AKT phosphorylation. Furthermore, sADAM9v2 mAb blocks cell motility, such as... Figure 8As shown. Therefore, the inventors hypothesize that sADAM9v2 mAb blocks prostate cancer motility by inhibiting AKT phosphorylation.

[0170] Other implementation plans

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

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

[0173] equivalent

[0174] Although some embodiments of the invention have been described and illustrated herein, those skilled in the art will readily conceive of a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, each of which is considered to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and constructions described herein are exemplary, and actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications using the teachings of the invention. Those skilled in the art will recognize or be able to identify many equivalents of the specific embodiments of the invention described herein using only conventional experimentation. Therefore, it should be understood that the foregoing embodiments are given by way of example only, and embodiments of the invention may be practiced in ways other than those specifically described and claimed, within the scope of the appended claims and their equivalents. The embodiments of the invention disclosed herein relate to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention, provided that such features, systems, articles, materials, kits, and / or methods are not contradictory.

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

[0176] All references, patents and patent applications disclosed herein are incorporated herein by reference in relation to the subject matter cited therein, and in certain circumstances, the subject matter may encompass the entirety of this document.

[0177] Unless explicitly stated to the contrary, the indefinite articles “a” and “an” used in this document, in the specification and in the claims, shall be understood to mean “at least one”.

[0178] As used herein, the phrase “and / or” in the specification and claims should be understood to mean “one or two” of such combined elements, i.e., elements that coexist in a particular case but are discontinuous in others. Multiple elements listed with “and / or” should be interpreted in the same way, i.e., “one or more” of the elements thus connected. In addition to the elements explicitly identified by the “and / or” clause, other elements may optionally be present, whether related to or unrelated to these specifically identified elements. Thus, as a non-limiting example, referring to “A and / or B”, when used in conjunction with open-ended language such as “comprising,” in one embodiment may refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements); and so on.

[0179] As used herein, in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when items in a list are separated, “or” or “and / or” should be interpreted as inclusive, i.e., including at least one, but also more than one or more elements in the list, and optionally, other unlisted items. Only explicitly indicating the opposite terms, such as “only one” or “exactly one”, or when used in the claims, “consisting of” will refer to including only one element in the list of one or more elements. Generally, as used herein, the term “or” should only be interpreted as an exclusive alternative when indicating an exclusive choice (i.e., “one or the other, but not both at the same time”), such as “any one,” “one of,” “only one,” or “exactly one.” When used in the claims, “consisting substantially of” should have the ordinary meaning used in the field of patent law.

[0180] As used herein, when referring to a list of one or more elements in the specification and in the claims, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each element explicitly listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements referred to by the phrase "at least one," whether related to 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 refer to at least one, optionally including more than one A, but without B (and optionally including elements other than B); in another embodiment, it may refer to at least one, optionally including more than one B, but without A (and optionally including elements other than A); in yet another embodiment, it may refer to 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 a clear indication to the contrary, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are listed.

[0182] The amino acid sequences are shown in Table 1 below.

[0183] Table 1

[0184]

Claims

1. An antibody or an antigen-binding fragment thereof, which binds to sADAM9v2 protein or a portion thereof, wherein said antibody or antigen-binding fragment comprises: Heavy chains, which include: CDR1, as shown in the amino acid sequence of SEQ ID NO: 1, CDR2 of the amino acid sequence shown in SEQ ID NO: 2, and For example, CDR3 of the amino acid sequence of SEQ ID NO: 3; and Light chains, which include: CDR1, as shown in the amino acid sequence of SEQ ID NO: 4, CDR2 of the amino acid sequence shown in SEQ ID NO: 5, and CDR3, as shown in the amino acid sequence of SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: The heavy chain variable region contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 7; and The light chain variable region contains an amino acid sequence that is at least 90% identical to 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 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 90% identity with SEQ ID NO:

10.

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

6. A polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

7. A reagent for diagnosing prostate cancer, wherein the reagent comprises an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 for the preparation of a reagent for diagnosing prostate cancer in a subject.

9. 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 prostate cancer.

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

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