Anti-human adam9 antibodies for in vitro diagnostics

Abstract

The disclosure provides binding agents (e.g., antibodies) against isoform 1 of human ADAM9, as well as kits and methods for using the same (e.g., immunoassays) as part of a companion diagnostic and for other applications. In some aspects, the binding agents described herein may be used in assays for detecting non-small cell lung cancer (NSCLC), pancreatic cancer, triple-negative breast cancer (TNBC), gastroesophageal cancer (GC), prostate cancer, and renal cancer.

Description

ANTI-HUMAN ADAM9 ANTIBODIES FOR IN VITRO DIAGNOSTICSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of priority to U. S. Provisional application number 63 / 540,047, filed September 23, 2023, the entire contents of which is incorporated herein by reference in its entirely.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (039062- 00164_SequenceListing.xml; Size: 26,988 bytes; and Date of Creation: September 19, 2024) are herein incorporated by reference in its entirety.TECHNICAL FIELD

[0003] This invention generally relates to binding agents (e.g., antibodies) against isoform 1 of human ADAM9, as well as kits and methods for using the same (e.g., immunoassays) as part of a companion diagnostic and for other applications.BACKGROUND

[0004] The ADAM family of proteins is known to be involved in various physiological and pathological processes. See Amendola, R. S. et al., “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2,” J. Leukocyte Biol. 97(5):951-962 (2015); Edwars, D. R. et al., “The ADAM MelalloproleasesC Molec. Aspects Med. 29:258-289 (2008). The ADAM family is believed to include at least 21 functional genes in humans. See Li, J. et al. “Overexpression of ADAM9 Promotes Colon Cancer Cells Invasion,” J. Invest. Surg. 26(3): 127-133 (2016); Duffy, M. J. et al. “The ADAMs Family Of Proteases: New Biomarkers And Therapeutic Targets For Cancer?,” Clin. Proteomics 8:9: 1-13 (2011); see also U.S. Pre-Grant Pub. No. 2013 / 0045244.

[0005] Members of the ADAM family share a highly conserved structure that typically includes eight domains, including a metalloprotease domain and an integrin-binding (disintegrin) domain. See Duffy, M. J. et al., “The Role Of ADAMs In Disease Pathophysiology f Clin. Chim. Acta 403:31-36 (2009). The ADAM metalloprotease domain acts as a sheddase, cleaving extracellular portions of transmembrane proteins, and thus releasing the soluble ectodomains from the cell surface, and has been reported to modulate a series of biological signaling pathways. See Amendola, R. S. et al. “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2,” J. Leukocyte Biol. 97(5):951-962 (2015); Ito, N. et al. “ADAMs, A Disintegrin And Metalloproteinases, Mediate Shedding Of Oxytocinase,” Biochem. Biophys. Res. Commun. 314 (2004) 1008-1013 (2004).

[0006] ADAM9 is a member of the ADAM family and assigned Uniprot Accession No. Q13443-1. An 819 amino acid isoform (“isoform 1,” SEQ ID NO: 2) is considered to represent the canonical sequence of this protein. ADAM9 is initially synthesized as an inactive form which is proteolytically cleaved to generate an active enzyme. ADAM9 is known to be expressed in fibroblasts (Zigrino, P. et al., “The Disintegrin-Like And Cysteine Rich Domains Of ADAM-9 Mediate Interactions Between Melanoma Cells And Fibroblasts,” J. Biol. Chem. 286:6801-6807 (2011)), activated vascular smooth muscle cells (Sun, C. et al., “ADAMI 5 Regulates Endothelial Permeability And Neutrophil Migration Via Src / ERKI / 2 Signalling,” Cardiovasc. Res. 87:348-355 (2010)), monocytes (Namba, K. et al., “Involvement Of ADAM9 In Multinucleated Giant Cell Formation Of Blood Monocytes,” Cell. Immunol. 213: 104-113 (2001)), and activated macrophages (Oksala, N. et al., “ADAM- 9, ADAM-15, And ADAM-17 Are Upregulated In Macrophages In Advanced Human Atherosclerotic Plaques In Aorta And Carotid And Femoral Arteries — Tampere Vascular Study,” Ann. Med. 41 :279-290 (2009)).

[0007] ADAM9’s metalloprotease activity plays a role in the degradation of matrix components, thereby allowing migration of tumor cells (Amendola, R. S. et al., “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2,” J. Leukocyte Biol. 97(5):951-962 (2015)). Its disintegrin domain meidates the interaction between ADAMS and integrins, and enables ADAM9 to modulate, positively or negatively, cell adhesion events (Zigrino, P. et al., “The Disintegrin- Like And Cysteine-Rich Domains Of ADAM-9 Mediate Interactions Between Melanoma Cells And Fibroblasts,” J. Biol. Chem. 286:6801-6807 (2011); Karadag, A. et al., “ADAM-9 (MDC- 9 / Meltringamma), A Member Of The A Disintegrin And Metalloproteinase Family, Regulates Myeloma-Cell-Induced Interleukin-6 Production In Osteoblasts By Direct Interaction With The Alpha(v)Beta5 Integrin,” Blood 107:3271-3278 (2006); Cominetti, M. R. et al., “Inhibition Of Platelets And Tumor Cell Adhesion By The Disintegrin Domain Of Human ADAM9 To Collagen I Under Dynamic Flow Conditions,” Biochimie 91 : 1045-1052 (2009)). Prior research has shown that the ADAM9 disintegrin domain interacts with the a6pi, a6p4, avP5 and a9pi integrins.

[0008] The expression of ADAM9 has been found to be relevant to multiple diseases, including cancer. For example, ADAM9 has been found to cleave and release a number of molecules with important roles in tumorigenesis and angiogenesis, such as TEK, KDR, EPHB4, CD40, VCAM1 and CDH5. ADAM9 is expressed by many types of tumor cells, including breast cancers, colon cancers, gastric cancers, gliomas, liver cancers, non-small cell lung cancers, melanomas, myelomas, pancreatic cancers and prostate cancers. See, e.g., Yoshimasu, T. et al., “Overexpression Of ADAM9 In Non-Small Cell Lung Cancer Correlates With Brain Metastasis,” Cancer Res. 64:4190-4196 (2004); Peduto, L. et al., “Critical Function For ADAM9 In Mouse Prostate Cancer,” Cancer Res. 65:9312-9319 (2005); Zigrino, P. et al., “ADAM-9 Expression And Regulation In Human Skin Melanoma And Melanoma Cell Lines,”Int. J. Cancer 116:853-859 (2005); Fritzsche, F. R. et al., “ADAM9 Is Highly Expressed In Renal Cell Cancer And Is Associated With Tumour Progression,” BMC Cancer 8: 179: 1-9 (2008); Fry, J. L. et al., “Secreted And Membrane-Bound Isoforms Of Protease ADAM9 Have Opposing Effects On Breast Cancer Cell Migration,” Cancer Res. 70, 8187-8198 (2010); Chang, L. et al., “Combined Rnai Targeting Human Stat3 And ADAM9 As Gene Therapy For Non-Small Cell Lung Cancer,” Oncology Letters 11 : 1242-1250 (2016); Fan, X. et al., “ADAM9 Expression Is Associate with Glioma Tumor Grade and Histological Type, and Acts as a Prognostic Factor in Lower-Grade Gliomas,” Int. J. Mol. Set. 17: 1276: 1-11 (2016).

[0009] Increased ADAM9 expression has been found to correlate positively with tumor malignancy and metastatic potential, highlighting the clinical significance of this gene. See, e.g., Amendola, R. S. et al., “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2,” J. Leukocyte Biol. 97(5):951- 962 (2015); Fan, X. et al., “ADAM9 Expression Is Associate with Glioma Tumor Grade and Histological Type, and Acts as a Prognostic Factor in Lower-Grade Gliomas,” Int. J. Mol. Sci. 17: 1276: 1-11 (2016); Li, J. et al. “Overexpression of ADAM9 Promotes Colon Cancer Cells Invasion,” J. Invest. Surg. 26(3): 127-133 (2016). Moreover, ADAM9 and its secreted soluble isoform appear to be crucial for cancer cells to disseminate (Amendola, R. S. et al., “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2,” J. Leukocyte Biol. 97(5):951-962 (2015); Fry, J. L. et al. “Secreted And Membrane -Bound Isoforms Of Protease ADAM9 Have Opposing Effects On Breast Cancer Cell Migration, ” Cancer Res. 70, 8187-8198 (2010); Mazzocca, A., “A Secreted Form Of ADAM9 Promotes Carcinoma Invasion Through Tumor-Stromal Interactions,” Cancer Res. 65:4728-4738 (2005); see also U.S. Patent Nos. 9,150,656; 7,585,634; 7,829,277; 8,101,361; 8,445,198; and 11,242,402; and U.S. Pre-Grant Pub. No.2009 / 0023149.

[0010] In view of this clinical significance, ADAMS has emerged as a potential target for anticancer therapy. See Peduto, L., “ADAM9 As A Potential Target Molecule In Cancer,” Curr. Pharm. Des. 15:2282-2287 (2009); Duffy, M. J. et al., “Role Of ADAMs In Cancer Formation And Progression,” Clin. Cancer Res. 15: 1140-1144 (2009); Duffy, M. J. et al., “The ADAMs Family Of Proteases: New Biomarkers And Therapeutic Targets For Cancer?” Clin. Proteomics 8:9: 1-13 (2011); Josson, S. et al., “Inhibition of AD AM9 Expression Induces Epithelial Phenotypic Alterations and Sensitizes Human Prostate Cancer Cells to Radiation and Chemotherapy,” Prostate 71(3):232-240 (2011); see also U.S. Pre-Grant Pub. Nos. 2016 / 0138113, 2016 / 0068909, 2016 / 0024582, 2015 / 0368352, 2015 / 0337356, 2015 / 0337048, 2015 / 0010575, 2014 / 0342946, 2012 / 0077694, 2011 / 0151536, 2011 / 0129450, 2010 / 0291063, 2010 / 0233079, 2010 / 0112713, 2009 / 0285840, 2009 / 0203051, 2004 / 0092466, 2003 / 0091568, and 2002 / 0068062, and PCT Publication Nos. WO2016 / 077505, WO2014 / 205293, WO2014 / 186364, WO2014 / 124326, W02014 / 108480, WO2013 / 119960, WO2013 / 098797, W02013 / 049704, and WO2011 / 100362). Additionally, the expression of ADAM9 has also been found to be relevant to pulmonary disease and inflammation. See, e.g., U.S. Pre-Grant Pub. Nos. 2016 / 0068909; 2012 / 0149595; 2009 / 0233300; 2006 / 0270618; and 2009 / 0142301. Antibodies that bind to ADAM9 are commercially available from multiple vendors, including Abeam, Thermofisher, Sigma-Aldrich, and other companies.BRIEF SUMMARY OF THE DISCLOSURE

[0011] Antibodies and other binding agents specific for ADAM9 are commercially available. However, there remains a need in the art for additional high affinity ADAM9- binding agents, and in particular ADAM9-binding agents that exhibit specificity for isoform 1 of ADAM9. As noted above, the human form of ADAM9 is known to have multiple isoforms. Prior antibodies for ADAM9 often detect multiple isoforms of ADAM9, interfering with effortsto accurately determine localization or levels of isoform 1 of ADAM9. The resulting noise impacts the accuracy of diagnostic assays and other applications of such assays.

[0012] The present disclosure addresses these and other shortcomings of prior ADAM9 binding agents by providing various binding agents capable of selectively binding to isoform of ADAM9 (e.g., as opposed to isoform 2, SEQ ID NO 13). In alternative embodiments, provided are chimeric or recombinant antibodies (Abs), or an antigen (Ag) binding fragments thereof, or a monomeric or dimeric antigen binding protein, capable of specifically binding to a human ADAM9 polypeptide, including human ADAM9 polypeptides expressed on the surface of tumor cells (e.g., tumor cells, including breast cancers, colon cancers, gastric cancers, gliomas, liver cancers, non-small cell lung cancers, melanomas, myelomas, pancreatic cancers and prostate cancers), and lymphocytes such as activated T cells that have infiltrated tumors, or human ADAMS polypeptides expressed on tumor infiltrating lymphocytes (TILs). In some aspects, the binding agent (e.g., an Ab, or Ag-binding fragment thereof) selectively binds to isoform 1 of human ADAM9 but not to isoform 2.

[0013] In a first general aspect, the disclosure provides a binding agent capable of specifically binding to a human ADAM9 isoform 1 polypeptide (SEQ ID NO: 2), comprising: a heavy chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NO: 9, or a fragment thereof; and / or a light chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NO: 10, or a fragment thereof. In some aspects, the heavy chain variable domain comprises a sequence having at least 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 9, or a fragment thereof. In some aspects, the light chain variable domain comprises a sequence having at least 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 10, or a fragment thereof. A fragment, as used herein, may comprise any portion of a polypeptide less than the full-length sequence. For example, for a polypeptide 100 amino acids in length, a fragment thereof wouldconstitute at least 1 and at most 99 contiguous amino acids of the full-length sequence. It is further understood that a fragment, as used herein, may comprise a minimum number of contiguous amino acids of the full-length sequence (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids of any such full-length sequence that forms the basis for a given fragment).

[0014] In some aspects, the binding agent comprises a chimeric or a recombinant antibody (“Ab”), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein.

[0015] In some aspects, a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 3, or having at least 90% sequence identity with SEQ ID NO: 3, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 4, or having at least 90% sequence identity with SEQ ID NO: 4, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 5, or having at least 90% sequence identity with SEQ ID NO: 5; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 6, or having at least 90% sequence identity with SEQ ID NO: 6, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 7, or having at least 90% sequence identity with SEQ ID NO: 7, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 8, or having at least 90% sequence identity with SEQ ID NO: 8.

[0016] In some aspects, a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 3, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 4, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 5; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 6, aCDR2 region having a polypeptide sequence represented by SEQ ID NO: 7, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 8.

[0017] In some aspects, the heavy chain variable domain comprises SEQ ID NO: 9; and / or the light chain variable domain comprises SEQ ID NO: 10.

[0018] In some aspects, the heavy chain variable domain comprises SEQ ID NO: 9; and the light chain variable domain comprises SEQ ID NO: 10.

[0019] In some aspects, the heavy chain variable domain comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions compared to SEQ ID NO: 9, or a fragment thereof; and / or the light chain variable domain comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions compared to SEQ ID NO: 10, or a fragment thereof.

[0020] In some aspects, the heavy chain variable domain comprises a CDR1 having a polypeptide sequence represented by SEQ ID NO: 3, a CDR2 having a polypeptide sequence represented by SEQ ID NO: 4, and a CDR3 having a polypeptide sequence represented by SEQ ID NO: 5; and / or the light chain variable domain comprises a CDR1 having a polypeptide sequence represented by SEQ ID NO: 6, a CDR2 having a polypeptide sequence represented by SEQ ID NO: 7, and a CDR3 having a polypeptide sequence represented by SEQ ID NO: 8.

[0021] In some aspects, the binding agent comprises: a) an antigen-binding fragment (“Fab”); b) an Fab' fragment; c) an F(ab')2 fragment; d) an F(ab')2 fragment and an pFc' fragment; e) a single-chain variable fragment (“scFv”); f) an (scFv)2, or a di-scFv or a bi-scFv; g) a minibody; h) a diabody, a triabody or a tetrabody; i) a single-domain antibody (“dAb”); and / or j) a plurality of CDR fragments, or a multi-specific antibody formed from two or more antibody fragments.

[0022] In some aspects, a) the heavy chain variable domain comprises SEQ ID NO: 9 having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, wherein theheavy chain variable domain is capable of specifically binding to the human AD AMP isoform1 polypeptide; b) the sequence of the light chain variable domain comprises SEQ ID NO: 10 having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, wherein the light chain variable domain is capable of specifically binding to the human ADAM9 isoform 1 polypeptide; c) the sequence of the heavy chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO:9; d) the sequence of the light chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 10; e) the heavy chain variable domain and SEQ ID NO: 9 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment; and / or f) the heavy chain variable domain and SEQ ID NO: 10 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment.

[0023] In some aspects, the binding agent comprises an IgM, IgG, IgA or IgE isotype antibody heavy chain, and / or a kappa or lambda isotype antibody light chain.

[0024] In some aspects, the binding agent does not specifically bind to an ADAM9 isoform2 polypeptide (SEQ ID NO: 13).

[0025] In some aspects, the binding agent is a chimeric or recombinant Ab comprising: a heavy chain having a polypeptide sequence represented by SEQ ID NO: 9 operatively linked to, paired with, associated with, or configured with a light chain having a polypeptide sequence represented by SEQ ID NO: 10, wherein the chimeric or recombinant Ab is capable of selectively binding to the human ADAM9 isoform 1 polypeptide.

[0026] In some aspects, the binding agent further comprises a detectable agent or a binding moiety. In some aspects, the detectable agent optionally comprises: an enzyme, a biotin, a fluorescent or chemiluminescent label, a fluorophore, a cyanine or sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, 7-methoxy coumarin (“Mca”), dabcyl, [2-(4-nitro-2,l,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (“NBD”), Nile blue, Tamra or tetramethylrhodamine (“TMR”), HRP MAGENTA™ chromogen (Dako Omnis, Agilent), boron-dipyrromethene (“BODIPY”), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten or an antibody binding epitope or domain. In some aspects, the enzyme is a peroxidase, an alkaline phosphatase, or a beta-galactosidase. In some aspects, the peroxidase is a horse radish peroxidase (“HRP”). In some aspects, the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine. In some aspects, the dye comprises a cyanine dye; or Cy3 or Cy5. In some aspects, the fluorophore comprises dansyl, fluorescein or carboxyfluorescein (“FAM”) or 6-FAM.

[0027] In some aspects, the binding moiety comprises: a glutathione S-transferase (“GST”) or ligandin tag, a polyhistidine (poly-his) tag, a chitin binding protein (“CBP”), a STREP- TAG™ II tag (SEQ ID NO: 14), a FLAG tag (SEQ ID NO: 15), or a maltose binding protein.

[0028] In some aspects, the disclosure provides additional binding agents for specifically detecting isoform 1 of ADAM9. For example, in some aspects, binding agent capable of specifically binding to a human ADAM9 isoform 1 polypeptide, comprising: a heavy chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NO: 22, or a fragment thereof; and / or a light chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NO:23, or a fragment thereof. In some aspects, the binding agent comprises a chimeric or a recombinant antibody (“Ab”), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein. In some aspects, a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 16, or having at least 90% sequence identity with SEQ ID NO: 16, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 17 or having at least 90% sequence identity with SEQ ID NO: 17, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 18, or having at least 90% sequence identitywith SEQ ID NO: 18; and / or ) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 19, or having at least 90% sequence identity with SEQ ID NO: 19, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 20 or having at least 90% sequence identity with SEQ ID NO: 20, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 21, or having at least 90% sequence identity with SEQ ID NO: 21.

[0029] In some aspects, a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 16, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 17, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 18; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 19, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 20, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 21.

[0030] In some aspects, the binding agent comprises a heavy chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NOs: 9 or 22, or a respective fragment thereof; and / or a light chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NOs: 10 or 23, or a respective fragment thereof.

[0031] In some aspects, a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NOs: 3 or 16, or having at least 90% sequence identity with SEQ ID NOs: 3 or 16, a CDR2 region having a polypeptide sequence represented by SEQ ID NOs: 4 or 17 or having at least 90% sequence identity with SEQ ID NOs: 4 or 17, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NOs: 5 or 18, or having at least 90% sequence identity with SEQ ID NOs: 5 or 18; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NOs: 6 or 19, orhaving at least 90% sequence identity with SEQ ID NOs: 6 or 19, a CDR2 region having a polypeptide sequence represented by SEQ ID NOs: 7 or 20 or having at least 90% sequence identity with SEQ ID NOs: 7 or 20, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NOs: 8 or 21, or having at least 90% sequence identity with SEQ ID NOs: 8 or 21.

[0032] In some aspects, a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NOs: 3 or 16, a CDR2 region having a polypeptide sequence represented by SEQ ID NOs: 4 or 17, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NOs: 5 or 18; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NOs: 6 or 19, a CDR2 region having a polypeptide sequence represented by SEQ ID NOs: 7 or 20, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NOs: 8 or 21.

[0033] In a second general aspect, the disclosure provides a nucleic acid encoding any of the binding agents (e.g., antibodies or antigen-binding fragments thereof) described herein.

[0034] In some aspects, the nucleic acid is a recombinant nucleic acid. In some aspects, the nucleic acid is operatively linked to a transcriptional regulatory element (e.g., an inducible promoter or a constitutive promoter). In some aspects, the nucleic acid is a recombinant nucleic acids that further encodes an additional protein or peptide moiety or domain (e.g., a purification moiety or domain to aid in the purification or isolation of the chimeric or recombinant antibody, or antigen-binding fragment thereof).

[0035] In a third general aspect, the disclosure provides methods for detecting the presence of a human AD AMP isoform 1 protein in or on a cell, tissue, or organ, or a portion thereof. In some aspects, such methods comprise a) contacting the cell, tissue, or organ, or the portion thereof of any of the foregoing with any ADAM9 binding agent described herein, and b) detecting specific binding of the ADAM9 binding agent, with the human ADAM9 isoform 1 polypeptide in the cell, tissue, organ, or portion thereof; and c) detecting the presence of ahuman AD AMP isoform 1 protein in the cell, tissue, or organ, or the portion thereof, based on the detection of specific binding in step b). In some aspects, the contacting step (a) is performed using an immunohistochemistry (IHC) assay.

[0036] In some aspects, the method further comprises contacting the binding agent with a detection agent capable of signaling the specific binding of the binding agent to the human ADAM9 isoform 1 protein. In some aspects, the detection agent comprises an antibody, an antigen-binding fragment, or a secondary antibody, that specifically binds to the binding agent; optionally, wherein the detection agent comprises an antibody, an antigen-binding fragment, or a secondary antibody that specifically binds to a hapten or tag attached or conjugated to the binding agent.

[0037] In some aspects, the antibody, the antigen-binding fragment, or the secondary antibody further comprises or has attached or conjugated thereto a second detection agent or an enzyme (e.g., an alkaline phosphatase, a beta-galactosidase or a peroxidase).

[0038] In some aspects, the antibody, the antigen-binding fragment, or the secondary antibody further comprises or has attached or conjugated thereto: a biotin, a fluorescent or chemiluminescent label, a fluorophore, a cyanine or sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, Mca, dabcyl, NBD, Nile blue, Tamra or TMR, BODIPY, HRP MAGENTA™ chromogen (Dako Omnis, Agilent), a dye, a radioisotope, a quantum dot, a photoluminescent aqueous nanocrystal, and / or a hapten. In some aspects, the the dye comprises a cyanine dye, or Cy3 or Cy5; and / or the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine.

[0039] In a fourth general aspect, the disclosure provides methods for detecting an ADAM9 isoform 1 protein-expressing cancer in a subject. In some aspects, such methods comprise a) contacting a cell, tissue, or organ sample, or a portion thereof, obtained from the subject, with any of the binding agents described herein; and b) detecting whether or not the binding agentspecifically binds to the human AD AMP isoform 1 protein in the cell, tissue or organ sample, or the portion thereof, wherein the detection of specific binding indicates an expression or presence of the human ADAM9 isoform 1 protein in the cell, tissue or organ sample, or the portion thereof. In some aspects, such methods further comprise c) diagnosing the subject as having, or being likely to have, a protein-expressing cancer when the binding agent is found to specifically bind to the human ADAM9 isoform 1 protein in the cell, tissue or organ sample, or the portion thereof in step b).

[0040] In some aspects, the ADAM9 isoform 1 protein-expressing cancer comprises: nonSmall Cell Lung Cancer (“NSCLC”), pancreatic cancer, triple-negative breast cancer (“TNBC”), gastroesophageal cancer (“GC”), prostate cancer, or renal cancer. In some aspects, the detecting step b) is performed using an immunohistochemistry (“IHC”) assay or a flow cytometry. In some aspects, the flow cytometry comprises a fluorescence-activated cell sorter (“FACS”) or an impedance flow cytometer. In some aspects, the cell, tissue or organ sample, or the portion thereof, was obtained from a biopsy from the subject.

[0041] In a fifth general aspect, the disclosure provides expression cassettes, vectors, recombinant viruses, artificial chromosomes, cosmids or plasmids comprising any of the nucleic acids provided herein.

[0042] In a sixth general aspect, the disclosure provides cells comprising a chimeric or a recombinant antibody, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein as provided herein, a recombinant nucleic acid as provided herein, or an expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid as provided herein, and the cell can be a bacterial, fungal, mammalian, yeast, insect, avian or plant cell.

[0043] In a seventh general aspect, the disclosure provides methods for detecting or diagnosing an ADAM9 protein-expressing cancer, or a cancer tissue having contained thereinan AD AMP expressing lymphocyte, or an ADAM9 expressing tumor infiltrating lymphocyte such as a tumor infiltrating activated T cell, comprising: detecting the expression or presence of a human ADAM9 protein (e.g., isoform 1 of ADAM9) in a cell, tissue or organ sample or portion thereof by contacting the cell, tissue or organ sample with an ADAM9 binding agent (e.g., a chimeric or recombinant antibody) as provided herein, or encoded by a nucleic acid as provided herein, and detecting whether or not the chimeric or recombinant antibody specifically binds to a human ADAM9 protein in the cell, tissue or organ sample or portion thereof, and the detecting of specific binding indicates the expression or presence of the human ADAM9 protein in the cell, tissue or organ sample, or portion thereof.

[0044] In an eighth general aspect, the disclosure provides methods for detecting or diagnosing an ADAM9 protein-expressing cancer, or a cancer tissue having contained therein a ADAM9 expressing lymphocyte, or a ADAM9 expressing tumor infiltrating lymphocyte such as a tumor infiltrating activated T cell, as provided herein. In some aspects, the cell is an activated T cell, an activated T cell that has infiltrated a tumor, or a tumor infiltrating lymphocyte (TIL). In some aspects, the detecting of specific binding indicates the expression or presence of the human ADAM9 protein in the cell, tissue or organ sample, or portion thereof, thereby diagnosing or detecting the cancer. In some aspects, the cancer is selected from nonsmall cell lung cancer (“NSCLC”), pancreatic cancer, triple-negative breast cancer (“TNBC”), gastroesophageal cancer (“GC”), prostate cancer, or renal cancer. In some aspects, the detection comprises using or conducting an immunohistochemistry (IHC) assay or a flow cytometry. In some aspects, the conducting or using of the flow cytometry comprises use of a fluorescence-activated cell sorter (FACS) or an impedance flow cytometer. In some aspects, the cell, tissue or organ sample or portion thereof is or is derived from a biopsy from a patient.

[0045] In an ninth general aspect, the disclosure provides methods for treating, ameliorating or preventing a cancer comprising first detecting or diagnosing the cancer in anindividual in need thereof using a method as provided herein, followed by treatment of the individual in need thereof. In some aspects, the cancer is selected from the group consisting of: non-small cell lung cancer (“NSCLC”), pancreatic cancer, triple-negative breast cancer (“TNBC”), gastroesophageal cancer (“GC”), prostate cancer, and renal cancer.

[0046] In a tenth general aspect, the disclosures provides uses of an ADAMS binding agent described herein (e.g., a chimeric and / or a recombinant antibody, or an antigen binding fragment thereof, or a monomeric or dimeric antigen binding protein as provided herein, or encoded by a recombinant nucleic acid as provided herein, for detecting or diagnosing a cancer, or a cancer tissue having contained therein a ADAM9 expressing lymphocyte, or a ADAM9 expressing tumor infiltrating lymphocyte such as a tumor infiltrating activated T cell, or treating, ameliorating or preventing the cancer.

[0047] In an eleventh general aspect, the disclosure provides kits comprising a chimeric or a recombinant antibody, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen binding protein as provided herein, or encoded by a recombinant nucleic acid as provided herein. In some aspects, the kits as provided herein comprise components needed for an immunohistochemistry (IHC) assay; and / or, instructions for practicing a method as provided herein. In alternative embodiments of the kits as provided herein the chimeric or the recombinant antibody, the antigen-binding fragment thereof, or the monomeric or dimeric antigen binding protein, is substantially purified or isolated.

[0048] In a twelfth general aspect, the disclosure provides products of manufacture comprising a chimeric or a recombinant antibody, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen binding protein as provided herein, or encoded by a recombinant nucleic acid as provided herein. In some aspects, the product of manufacture comprises or is fabricated as or manufactured as a slide, a well, a chip, a biochip, an array, a tray, a dish or a microtiter plate or dish. In some aspects, the chimeric or the recombinant antibody, the antigen-binding fragment thereof, or the monomeric or dimeric antigen-binding protein, is substantially purified or isolated, or is in the form of an unpurified or partially purified culture supernatant. In some aspects, the disclosure provides phages or phagemids comprising or expressing on its surface a chimeric or a recombinant antibody, or an antigen-binding fragment thereof, or a monomeric or dimeric antigen binding protein as provided herein, or encoded by any nucleic acid provided herein.

[0049] To the accomplishment of the foregoing and related ends, the one or more aspects comprise the features hereinafter fully described and particularly pointed out in the claims. The following description and the annexed drawings set forth in detail certain illustrative features of the one or more aspects. These features are indicative, however, of but a few of the various ways in which the principles of various aspects may be employed, and this description is intended to include all such aspects and their equivalents.DESCRIPTION OF DRAWINGS

[0050] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0051] The drawings set forth herein illustrate and describe exemplary aspects of the disclosure and are not meant to limit the scope of the invention as defined by the claims.

[0052] FIG. 1 shows Western blots detected with (A) Cell Signaling® anti-ADAM9 CST2099, (B) anti-ADAM9 clone 11H9 and (C) anti-ADAM9 clone 11C9, respectively. In blot (A), bands are seen around lOOkDa (pro-ADAM9-L), 70kDa (ADAM9-L) and 45kDa (ADAM9-S), reveling that the anti-ADAM9 CST2099 antibody binds all three isoforms of ADAM9. In the two blots to the right, (B) and (C), only the two upper bands are present, revealing that anti-ADAM9 antibody clones 11H9 and 11C9, respectively, binds only ADAM9-L and pro-ADAM9-L.

[0053] FIG. 2 is an image showing staining of a kidney using ADAM9 clone 11H9 (T0525B, lot H00976) on Dako Omnis. A strong apical staining but also a cytoplasmic staining is shown in proximal tubule (black arrow) but also in distal tubule. Almost no staining in glomeruli (open arrow)

[0054] FIG. 3 is an image showing staining of a pancreas using ADAM9 clone 11H9 (T0525B, lot H00976) on Dako Omnis. A cytoplasmic staining is shown in the islets of Langerhans (black arrow). A apical staining but also a cytoplasmic staining is shown in ducts (open arrow). No staining in the acinous structures (red arrow), however with a weak stainings of small vessels around the glands

[0055] FIG. 4 is an image showing staining of a prostate using AD AMP clone 11H9 (T0525B, lot H00976) on Dako Omnis. A cytoplasmic staining (black arrow) and membrane staining (open arrow) is shown in prostate glands.

[0056] FIG. 5 is an image showing staining of a colon using ADAM9 clone 11H9 (T0525B, lot H00976) on Dako Omnis. A cytoplasmic staining is shown in epithelium (black arrow).

[0057] FIG.6 is an image showing staining of a liver using ADAM9 clone 11H9 (T0525B, lot H00976) on Dako Omnis. A diffuse cytoplasmic staining of macrophages (Kuppfer cells) and sinusoids (black arrow) and a cytoplasmic and membrane staining is shown in bile ducts (open arrow).

[0058] FIG. 7 is an image showing staining of a tonsil using ADAM9 clone 11H9 (T0525B, lot H00976) on Dako Omnis. A weak staining of keratinocytes in the crypt (black arrow) with an up-concentration at top (open arrow) is shown. Furthermore, weak diffuse staining in vessel structures in general (red arrow).

[0059] FIG. 8 is an image showing staining of a breast carcinoma using ADAM9 clone 11H9 (T0525B, lot H00976) on Dako Omnis. A moderate membrane (apical) in normal ducts is shown (black arrow). No staining in tumor cells (open arrow).

[0060] FIG. 9 is an image showing staining of a carcinoid using ADAM9 clone 11H9 (T0525B, lot H00976) on Dako Omnis. A weak to moderate cytoplasmic staining in tumor cells with a tendency to a stronger staining in the peripheral of a tumor group is shown (black arrow).

[0061] FIG. 10 is an image showing staining of a tumor tissue sample of colon adenocarcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A moderate cytoplasmic and membrane staining is observed in tumor cells (black arrow).

[0062] FIG. 11 is an image showing staining of a cerebellum using AD AMP clone 11H9 (T0525B, lot H00976) on Dako Omnis. No staining and no background staining observed.

[0063] FIG. 12 is an image showing staining of a tumor tissue sample of pancreas adenocarcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A strong cytoplasmic and membrane staining is observed in tumor cells (black arrow).

[0064] FIG. 13 is an image showing staining of a tumor tissue sample of pancreas adenocarcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A moderate cytoplasmic and membrane staining is observed in tumor cells (black arrow).

[0065] FIG. 14 is an image showing staining of a tumor tissue sample of pancreas adenocarcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A moderate cytoplasmic and membrane staining is observed in tumor cells (black arrow). Note the strong apical staining (open arrow).

[0066] FIG. 15 is an image showing staining of a tumor tissue sample of breast carcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A moderate cytoplasmic staining and membrane staining is observed in tumor cells (black arrow).

[0067] FIG. 16 is an image showing staining of a tumor tissue sample of breast carcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A moderate cytoplasmic staining and membrane staining is observed in tumor cells (black arrow).

[0068] FIG. 17 is an image showing staining of a tumor tissue sample of lung adenocarcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A weak to moderate cytoplasmic staining and a moderate membrane staining is observed in tumor cells (black arrow).

[0069] FIG. 18 is an image showing staining of a tumor tissue sample of lung adenocarcinoma with AD AMP, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A weak cytoplasmic staining and membrane staining is observed in tumor cells (black arrow).

[0070] FIG. 19 is an image showing staining of a tumor tissue sample of lung adenocarcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A moderate membrane staining is observed in a fraction of tumor cells (black arrow).

[0071] FIG. 20 is an image showing staining of a tumor tissue sample of stomach adenocarcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A weak membrane og cyt staining is observed in a fraction of tumor cells (black arrow).

[0072] FIG. 21 is an image showing staining of a tumor tissue sample of stomach adenocarcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A moderate cytoplasmic and membrane staining is observed in tumor cells (black arrow).

[0073] FIG. 22 is an image showing staining of a tumor tissue sample of stomach carcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A strong cytoplasmic and membrane staining is observed in tumor cells (black arrow).

[0074] FIG. 23 is an image showing staining of a tumor tissue sample of colon adenocarcinoma with ADAM9, clone 11H9 (T0525B, lot H00976) on Dako Omnis. A moderate cytoplasmic and membrane staining is observed in tumor cells (black arrow).

[0075] FIG. 24 is an image showing the reaction pattern of clone 11C9 on selected tissues in large multi block.

[0076] FIG. 25 is an image showing staining of a tumor tissue sample of pancreas adenocarcinoma with ADAM9, clone 11C9 (T0531B, lot H00971) on Dako Omnis. A moderate cytoplasmic and membrane staining is observed in tumor cells (black arrow).

[0077] FIG. 26 is an image showing staining of a tumor tissue sample of pancreas adenocarcinoma with AD AMP, clone 11C9 (T053 IB, lot H00971) on Dako Omnis. A weak to moderate cytoplasmic and membrane staining is observed in tumor cells (black arrow).

[0078] FIG. 27 is an image showing staining of a tumor tissue sample of pancreas adenocarcinoma with ADAM9, clone 11C9 (T0531B, lot H00971) on Dako Omnis. A weak cytoplasmic staining is observed in tumor cells (black arrow)

[0079] FIG. 28 is an image showing staining of a tumor tissue sample of pancreas adenocarcinoma with ADAM9, clone 11C9 (T0531B, lot H00971) on Dako Omnis. A weak cytoplasmic staining and moderate membrane staning is observed in tumor cells (black arrow).

[0080] FIG. 29 is an image showing staining of a tumor tissue sample of pancreas adenocarcinoma with ADAM9, clone 11C9 (T0531B, lot H00971) on Dako Omnis. A weak cytoplasmic and membrane staining and is observed in tumor cells (black arrow).

[0081] FIG. 30 is an image showing staining of a tumor tissue sample of stomach adenocarcinoma with ADAM9, clone 11C9 (T0407, lot H00740) on Dako Omnis. A weak to moderate cytoplasmic and membrane staining and is observed in tumor cells (black arrow).

[0082] FIG. 31 is an image showing staining of a tumor tissue sample of breast carcinoma with ADAM9, clone 11C9 (T0407, lot H00740) on Dako Omnis. A moderate to strong cytoplasmic and membrane staining and is observed in tumor cells (black arrow).DETAILED DESCRIPTION

[0083] In alternative embodiments, provided are chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins, that can specifically bind to human ADAM9 polypeptides (e.g., isoform 1 of ADAM9, SEQ ID NO: 2, or a portion thereof). In alternative embodiments, provided are products of manufacture and kits comprising the chimeric or a recombinant Abs, or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, as provided herein, or nucleic acids encoding them, and methods for making and using them. In alternative embodiments, chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein are used for in vitro diagnostics, for example, by immuno-histochemistry (IHC), for example, in IHC protocols to diagnose, detect and / or treat a cancer, for example bladder cancer, urothelial carcinoma, a breast cancer or a mammary carcinoma or a ductal carcinoma in situ (DCIS), a carcinoid, Hodgkin’s Lymphoma, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, a lung cancer, a Non-Small Cell Lung Cancer (NSCLC), a kidney cancer or renal cell carcinoma or a renal carcinoma, a Renal Clear cell Carcinoma (RCC), mesothelioma or a malignant pleural mesothelioma, a squamous cell carcinoma, an anal squamous cell carcinoma, pancreatic cancer, and a melanoma or a malignant melanoma. Thus, the chimeric or recombinant antibodies (Abs), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein can be used as a companion diagnostic for the diagnosis and treatment of cancer.

[0084] Expression of Recombinant Chimeric Antibodies

[0085] In alternative embodiments, chimeric and / or recombinant antibodies (Abs), the antigen binding fragments thereof, or the monomeric or dimeric antigen binding proteins as provided herein, including the exemplary chimeric or recombinant anti-human ADAM9 Abscomprising the heavy chain variable region of SEQ ID NOs: 9 or 22, and light chain variable region of SEQ ID NOs: 10 or 23, with or without a signal peptide, can be expressed as a recombinant Ab using, for example, a plasmid or any expression vehicle encoding the respective heavy and light chains, or the heavy chain and the light chain can be encoded in separate expression vehicles.

[0086] In some embodiments, the heavy and light chains can be (cis- or trans-) expressed from any plasmid, cosmid, recombinant virus or equivalent vector, for example, from a pTT5™ vector(s) (National Research Council Canada, NRC-CNRC, Canada) or equivalents.

[0087] In alternative embodiments, the expression vehicles (such as a plasmid) containing exemplary Ab-encoding nucleic acid(s) as provided herein are expressed in in vitro expression systems or are expressed in cultured tissues, cells or organoids, which can be a bacterial, fungal, mammalian, yeast, insect or plant cell expression systems, or hybrid or synthetic expression system. For example, exemplary Ab-encoding nucleic acid(s) can be expressed in a human embryonic kidney (HEK) cell such as an HEK293- 6E cell. In alternative embodiment, the vector or vectors expressing exemplary Ab- encoding nucleic acid(s), for example, exemplary heavy and / or light chains, are episomal or are chromosomally integrated, for example, in a stable cell line capable of synthesizing, optionally inducibly synthesizing, the heavy and / or light chains.

[0088] In alternative embodiments, provided are nucleic acids encoding chimeric or recombinant Abs as provided herein. Nucleic acids as provided herein can be made, isolated and / or manipulated by, for example, cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. Nucleic acids used to practice embodiments as provided herein, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and / or expressed / generated recombinantly. Recombinant polypeptides generated from these nucleic acids can beindividually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, fungal, mammalian, yeast, insect or plant cell expression systems, or hybrid or synthetic expression systems.

[0089] Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, for example, Martin et al, ACS Synth. Biol. (2017) 6, 7, 1370-1379; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373- 380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Patent No. 4,458,066.

[0090] Techniques for the manipulation of nucleic acids, such as, for example, subcloning, labeling probes (for example, random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, for example, Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

[0091] Another useful means of obtaining and manipulating nucleic acids used to practice embodiments as provided herein comprises screening and re-cloning inserts isolated or amplified from, for example, genomic clones or cDNA clones. Sources of nucleic acids include recombinant nucleic acid sequences, genomic or cDNA libraries contained and / or expressed in, for example, mammalian artificial chromosomes (MACs), see, for example, U.S. Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, for example, Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes(BAC); Pl artificial chromosomes, see, for example, Woon (1998) Genomics 50:306-316; Pl- derived vectors (PACs), see, for example, Kern (1997) Biotechniques 23: 120-124; cosmids, recombinant viruses, phages or plasmids.

[0092] In alternative embodiments, nucleic acids as provided herein are operably linked to transcriptional regulatory elements, including promoters, with can be constitutive or inducible transcriptional regulatory elements.

[0093] In alternative aspects, provided are “expression cassettes” comprising a nucleotide sequence as provided herein, for example encoding a chimeric or recombinant antibody as provided herein. Expression cassettes can include at least a transcriptional regulatory element, for example, a promoter, operably linked with an antibody coding sequence, and optionally can also include transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, for example, enhancers.

[0094] In alternative aspects, expression cassettes used to practice embodiments as provided herein include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like. In alternative aspects, a “vector” used to practice embodiments as provided herein can comprise a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. In alternative aspects, a vector used to practice embodiments as provided herein can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. In alternative aspects, vectors used to practice embodiments as provided herein can comprise viral or bacterial nucleic acids and / or proteins, and / or membranes (for example, a cell membrane, a viral lipid envelope, etc.).

[0095] In alternative aspects, vectors used to practice embodiments as provided herein can include, but are not limited to replicons (for example, RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (for example,plasmids, viruses, and the like, see, for example, U.S. Patent No. 5,217,879), and can include both the expression and non-expression plasmids. In alternative aspects, the vector used to practice embodiments as provided herein can be stably replicated by the cells during mitosis as an autonomous structure, or can be incorporated within the host’s genome.

[0096] In alternative aspects, “promoters” used to practice embodiments as provided herein include all sequences capable of driving transcription of a coding sequence in a cell, for example, a bacterial, yeast, fungal, plant, insect (for example, baculovirus) or mammalian cell. Thus, promoters used in the constructs include cv.s-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and / or rate of transcription of a gene. For example, a promoter used to practice embodiments as provided herein can be a cv.s-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5’ and 3’ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences can interact with proteins or other biomolecules to carry out (turn on / off, regulate, modulate, etc.) transcription.

[0097] Constitutive” promoters used to practice embodiments as provided herein can be those that drive expression continuously under most environmental conditions and states of development or cell differentiation. “Inducible” or “regulatable” promoters used to practice embodiments as provided herein can direct expression of a nucleic acid as provided herein under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters used to practice embodiments as provided herein include the presence of an inducing factor administered to a cell.

[0098] In alternative embodiments, antibodies used to practice embodiments as provided herein can comprise any “mimetic” and / or “peptidomimetic” form. In alternativeembodiments, peptides and polypeptides used to practice embodiments as provided herein can comprise synthetic chemical compounds which have substantially the same structural and / or functional characteristics of the natural polypeptide, for example, a chimeric or recombinant antibody as provided herein. The mimetic used to practice embodiments as provided herein can be either entirely composed of synthetic, non- natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic’s structure and / or activity. Routine experimentation will determine whether a mimetic is effective for practicing the invention, for example, if a mimetic composition is effective in specifically binding a human ADAM9 protein. Methodologies detailed herein and others known to persons skilled in the art may be used to select or guide one to choose effective mimetic for practicing the compositions and / or methods as provided herein.

[0099] Polypeptide mimetic compositions for practicing embodiments as provided herein can comprise any combination of non-natural structural components. In alternative aspects, mimetic compositions for practicing embodiments as provided herein can comprise one or all of the following three structural groups: a) residue linkage groups other than the natural amide bond (“peptide bond”) linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, for example, a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. For example, a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.

[0100] Purification and Isolation of Recombinant Proteins

[0101] In alternative embodiments, chimeric or the recombinant antibodies, antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, are substantiallypurified or isolated, and optionally the substantially purified or isolated forms are the forms used in immunohistochemistry methodologies and / or as reagents, kits and / or products of manufacture as provided herein.

[0102] In alternative embodiments, chimeric or the recombinant antibodies, antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, are substantially purified or isolated using: physicochemical fractionation, for example, using differential precipitation, size-exclusion or solid-phase binding of immunoglobulins based on size, charge or other shared chemical characteristics of antibodies in typical samples; class-specific affinity, for example, solid-phase binding of particular antibody classes (for example, IgG or IgM) by immobilized biological ligands (for example, proteins, lectins, and the like) that have specific affinity to immunoglobulins, and this can purify all antibodies of the target class without regard to antigen specificity; or antigen- specific affinity, for example, affinity purification of only those antibodies in a sample that bind to a particular antigen molecule through their specific antigen-binding domains, where this purifies all antibodies that bind the antigen without regard to antibody class or isotype.

[0103] In alternative embodiments, chimeric or the recombinant antibodies, antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, are substantially purified or isolated using standard isolation methodologies such as chromatography, for example, Ion Exchange (IEX) Chromatography, Hydrophobic Interaction Chromatography (HIC), countercurrent chromatography, immunoaffinity and / or size exclusion chromatography.

[0104] In alternative embodiments, chimeric or the recombinant antibodies, antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, are generated in bioreactors, for example, a perfusion bioreactor, using continuous expression and purification processes, for example, as described by Vogg et al Methods Mol Biol. 2018; vol 1850: 147- 178, or using stirred-tank or rocking bioreactor systems, followed by purification.

[0105] Products of Manufacture and Kits

[0106] In some aspects, products of manufacture and kits comprising at least one antihuman ADAM9 Ab, or an antigen-binding fragment thereof, are provided, as well as methods of using the same; optionally, the products of manufacture and kits can further comprise some or all reagents needed to perform an IHC, and optionally can comprise instructions for practicing any of the methods described herein. Such products of manufacture and kits may comprise one or more anti-human ADAM9 Abs disclosed herein (e.g., recombinant or chimeric Abs), or antigen-binding fragments thereof. In some aspects, the Ab(s), or antigen-binding fragment(s) thereof, will comprise at least one Ab, or antigen-binding fragment thereof, specific for ADAM9, isoform 1. In some aspects, an Ab or antigen-binding fragment specific for ADAM9, isoform 1 is one that does not bind to ADAM9, isoform 2 under identical assay conditions.

[0107] In alternative embodiments, products of manufacture have attached thereto or affixed (optionally covalently bound) on or onto chimeric or a recombinant antibodies (Abs), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein, and optionally products of manufacture as provided herein are or comprise arrays, biochips, slides, trays, dishes (for example, microtiter dishes), phages or phagemids.

[0108] Immunohi stochemi stry

[0109] In alternative embodiments, immunohistochemistry methodologies and / or reagents used to practice compositions, products of manufacture, kits or methods as provided herein can include or comprise or comprise use of any IHC protocol, IHC armamentarium, device and / or image or data analysis system, for practicing IHC or IHC reagents known in the art, for example, as described in U.S. patent nos. (USPNs) 10,565,479 (describing methods for identifying blurred areas in digital images of stained tissue); 10,564,076 (describing systems for analytical ( or IHC) sample preparation); 10,551,395 (describing an automated histologicalstaining system); 10,551,378 (describing a tissue staining method); 10,504,224 (describing a digital tissue image analysis system for IHC); 10,501,777 (describing simultaneous, multiplexed detection and quantification of protein expression in IHC); 10,488,340 (describing method for extracting an image of a target fluorophore in a biological material); 10,453,195 (describing methods of detecting tissue areas of interest using digital pathology imaging); 10,438,381 (describing devices, systems and methods for generating a digital image of a tissue section); 10,416,176 (describing methods for processing specimens in an automated histological staining system); 10,393,633 (describing methods for processing and inhibiting the degradation of an IHC sample); 10,217,011 (describing handling of IHC slides); 10,209,165 (describing automated or semi-automated methods for assessing the quality of staining of a specimen containing cells); 10,126,216 (describing methods for fixing tissue samples for IHC); 9,423,322.

[0110] In alternative embodiments, chimeric or the recombinant antibodies, antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, in IHC protocols, or kits, as provided herein are substantially purified or isolated or are in the form of an unpurified or partially purified culture supernatant.[OHl] In alternative embodiments, methods as provided herein can use or comprise reagents for detecting or visualizing an antibody-antigen interaction using any products or methods know in the art, for example, and IHC protocol or reagents.

[0112] In alternative embodiments, methods as provided herein comprise use of chromogenic immunohistochemistry (CIH), wherein a primary antibody (for example, chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein) or secondary antibody (for example, where the secondary antibody binds to (the primary antibody) chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigenbinding proteins as provided herein after they have specifically bound to, paired with, associated with, or configured with a LAG-3 epitope or polypeptide) is conjugated to an enzyme, such as peroxidase (or immunoperoxidase), for example, a horseradish peroxidase (HRP), that can catalyze a color-producing reaction.

[0113] In alternative embodiments, methods as provided herein comprise use of immunofluorescence, where a primary or a secondary antibody is tagged to a fluorophore, such as fluorescein or fluorescein isothiocyanate (FITC), a triarylmethane dye such as rhodamine or rhodamine derivatives (for example, tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulforhodamine 101), aminomethylcoumarin acetate (AMCA), ALEXA™ or DYLIGHT™ fluors. 3,3’-Diaminobenzidine (DAB) also can be used.

[0114] In alternative embodiments, methods as provided herein comprise use of a direct method or one-step staining method where a primary antibody (for example, chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein) is labeled and reacts directly with an antigen, for example, in a tissue sections. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity may be lower due to little signal amplification.

[0115] In alternative embodiments, methods as provided herein comprise use of an indirect method where an unlabeled primary antibody (first layer) binds to a target antigen (ADAM9, or in some cases more specifically ADAMS, isoform 1), for example, in a tissue or organ, and a labeled secondary antibody (second layer) then is reacted with the primary antibody. The secondary antibody can be against the isotype, for example, IgG, of the animal species in which the primary antibody is derived. This method can be more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies toeach primary antibody if the secondary antibody is conjugated to a detecting agent such as a fluorescent or enzyme reporter.

[0116] In alternative embodiments, further amplification is achieved if the secondary antibody is conjugated to several detecting molecules, for example, biotin molecules, which can recruit complexes of avidin-, streptavidin- or NEUTRA VIDIN™ protein- bound enzyme.

[0117] In alternative embodiments, the IHC is performed on tissue sections or tissue biopsies, for example, paraformaldehyde (PF A) fixed tissues or organs, or formalin-fixed paraffin-embedded tissues. In alternative embodiments, a tissue is sliced or used whole. Before sectioning, the tissue sample can be embedded in a medium, for example, paraffin wax or cryomedia. Tissue sections can be sliced on a variety of instruments, most commonly using a microtome, cryostat, or vibratome. Specimens can be sliced at a range of about 3 pm to 5 am. The slices can be mounted on slides, dehydrated using alcohol washes of increasing concentrations (for example, 50%, 75%, 90%, 95%, 100%), and cleared using a detergent like xylene before being imaged under a microscope.

[0118] Depending on the method of fixation and tissue preservation, the sample may require additional steps to make the ADAM9 epitopes available for antibody binding, including deparaffinization and antigen retrieval. For formalin-fixed paraffin-embedded tissues, antigenretrieval is often necessary, and can comprise pre-treating the sections with heat or proteases.

[0119] In alternative embodiments, the IHC is performed using an ENVISION DUOFLEX DOUBLESTAIN SYSTEM™ (EnVision DuoFLEX Doublestain System) (Agilent, San Jose, CA), which allows for staining of two or more markers on a single slide. In alternative embodiments, the IHC is performed using an EnVision FLEX HRP Magenta, High pH (Dako Omnis) system, and binding can be visualized by EnVision FLEX HRP Magenta Chromogen. In alternative embodiments, the IHC is performed using EnVision FLEX Mini Kit, High pH, which is a high-sensitivity visualization system intended for use in IHC together with DakoAUTO STAINER™ instruments; this dual link system detects primary mouse and rabbit antibodies and the reaction is visualized by 3,3’-Diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized, for example, by a peroxidase).

[0120] Methods of Use

[0121] In some aspects, the ADAM9 antibodies (or antigen-binding fragments thereof), as well as the kits and products of manufacture comprising the same, described herein, may be used in clinical or diagnostic applications. For example, an ADAM9 antibody, or antigenbinding fragment thereof, may be used to detect ADAM9 isoform 1 via an in vitro IHC assay in order to determine whether a subject has a cancer or other disease. Potential applications included, e.g., use as a companion diagnostic to identify patients that could benefit from an ADAM9 targeting antibody-drug conjugate (ADC) therapy (e.g., provided by ImmunoGen Inc.). Potential indications, include without limitation NSCLC, Pancreatic Cancer, TNBC, and GC.EXAMPLES

[0122] Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0123] Example 1 : Development of two exemplary anti-ADAM9 antibodies

[0124] This example describes the development of two exemplary rabbit anti-human ADAM9 antibodies. Five antigens were selected for study, including one comprising a recombinant ADAM9 protein and four comprising AD AMP peptides. Each antigen was used to immunize four rabbits.

[0125] Post-immunization bleed testing showed that one of the four ADAM9 peptides, and the recombinant ADAM9, showed good specificity for ADAM9, in IHC on pancreas adenocarcinoma tissue. All 5 antigens were designed with ADAM9 isoform 1 specific sequences, but which were outside the sequence range shared with ADAM9 isoform 2. Four of the inoculated rabbits which displayed a strong immune response against ADAM9 were chosen for B-cell selection based on this initial screen.

[0126] Several B-cell clones were identified which produces antibodies showing ADAM9 specific staining in IHC. Cloning of these B-cells resulted in functional antibody heavy and light chainsthat were used for transient transfection in HEK cells and the resulting antibodies were tested for reactivity against the ADAM9 antigen in Bio-layer interferometry z. These antibodies were further tested by IHC on clinically relevant tissue samples. Two clones (11H9 and 11C9) showed promising staining on pancreas adenocarcinomas among other tissues. The staining obtained with antibodies produced from clone 11H9 and 11C9 were similar or superior to the staining obtained with an ADAM9 reference antibody. In addition, the staining obtained from antibodies from clone 11H9 was similar in performance in both TRS High pH and TRS Low pH. A very distinct membrane staining was observed with on pancreas adenocarcinoma and other tissues both with TRS High pH and TRS Low pH. FIGs. 1-23 illustrate the staining of various tissues using antibodies produced by clone 11H9. A similar process was used to generate clone 11C9 and images showing the use of this antibody as a stain for various tissuesare provided as FIGs. 24-31. The results of exemplary IHC assays using these antibodies as a stain with various tissue types, are summarized below in Table 1.SEQUENCE LISTING

Claims

CLAIMSWe claim:

1. A binding agent capable of specifically binding to a human ADAM9 isoform 1 polypeptide, comprising: a heavy chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NO: 9, or a fragment thereof; and / or a light chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NO: 10, or a fragment thereof.

2. The binding agent of claim 1, wherein the binding agent comprises a chimeric or a recombinant antibody (“Ab”), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein.

3. The binding agent of claims 1 or 2, wherein: a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 3, or having at least 90% sequence identity with SEQ ID NO: 3, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 4, or having at least 90% sequence identity with SEQ ID NO: 4, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 5, or having at least 90% sequence identity with SEQ ID NO: 5; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 6, or having at least 90% sequence identity with SEQ ID NO: 6, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 7, or having at least 90% sequence identity with SEQ ID NO: 7, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 8, or having at least 90% sequence identity with SEQ ID NO: 8.

4. The binding agent of any one of claims 1-3, wherein: a) the heavy chain variable domain comprisesa CDR1 region having a polypeptide sequence represented by SEQ ID NO: 3, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 4, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 5; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 6, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 7, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 8.

5. The binding agent of any one of claims 1-4, wherein: the heavy chain variable domain comprises SEQ ID NO: 9; and / or the light chain variable domain comprises SEQ ID NO: 10.

6. The binding agent of any one of claims 1-5, wherein: the heavy chain variable domain comprises SEQ ID NO: 9; and the light chain variable domain comprises SEQ ID NO: 10.

7. The binding agent of claims 1 or 2, wherein: the heavy chain variable domain comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions compared to SEQ ID NO: 9, or a fragment thereof; and / or the light chain variable domain comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions compared to SEQ ID NO: 10, or a fragment thereof.

8. The binding agent of any one of claims 1-7, wherein the heavy chain variable domain comprises a CDR1 having a polypeptide sequence represented by SEQ ID NO: 3, a CDR2 having a polypeptide sequence represented by SEQ ID NO: 4, and a CDR3 having a polypeptide sequence represented by SEQ ID NO: 5; and / or the light chain variable domain comprises a CDR1 having a polypeptide sequence represented by SEQ ID NO: 6, a CDR2 having a polypeptide sequence represented by SEQ ID NO: 7, and a CDR3 having a polypeptide sequence represented by SEQ ID NO: 8.

9. The binding agent of any one of claims 1-8, wherein the binding agent comprises: a) an antigen-binding fragment (“Fab”); b) an Fab' fragment; c) an F(ab')2 fragment; d) an F(ab')2 fragment and an pFc' fragment; e) a single-chain variable fragment (“scFv”); f) an (scFv)2, or a di-scFv or a bi-scFv; g) a minibody; h) a diabody, a triabody or a tetrabody; i) a single-domain antibody (“dAb”); and / or j) a plurality of CDR fragments, or a multi-specific antibody formed from two or more antibody fragments.

10. The binding agent of any one of claims 1-9, wherein: a) the heavy chain variable domain comprises SEQ ID NO: 9 having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, wherein the heavy chain variable domain is capable of specifically binding to the human ADAM9 isoform 1 polypeptide; b) the sequence of the light chain variable domain comprises SEQ ID NO: 10 having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, wherein the light chain variable domain is capable of specifically binding to the human ADAM9 isoform 1 polypeptide; c) the sequence of the heavy chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO:9; d) the sequence of the light chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 10; e) the heavy chain variable domain and SEQ ID NO:9 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment; and / or f) the heavy chain variable domain and SEQ ID NO: 10 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment.

11. The binding agent of any one of claims 1-10, wherein the binding agent comprises an IgM, IgG, IgA or IgE isotype antibody heavy chain, and / or a kappa or lambda isotype antibody light chain.

12. The binding agent of any one of claims 1-11, wherein the binding agent does not specifically bind to an ADAM9 isoform 2 polypeptide (SEQ ID NO: 13).

13. The binding agent of any one of claims 1-12, wherein the binding agent is a chimeric or recombinant Ab comprising: a heavy chain having a polypeptide sequence represented by SEQ ID NO: 9 operatively linked to, paired with, associated with, or configured with a light chain having a polypeptide sequence represented by SEQ ID NO: 10, wherein the chimeric or recombinant Ab is capable of selectively binding to the human ADAM9 isoform 1 polypeptide.

14. A binding agent capable of specifically binding to a human ADAM9 isoform 1 polypeptide, comprising: a heavy chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NO: 22, or a fragment thereof; and / or a light chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NO:23, or a fragment thereof.

15. The binding agent of claim 14, wherein the binding agent comprises a chimeric or a recombinant antibody (“Ab”), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein.

16. The binding agent of claims 14 or 15, wherein: a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 16, or having at least 90% sequence identity with SEQ ID NO: 16, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 17 or having at least 90% sequence identity with SEQ ID NO: 17, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 18, or having at least 90% sequence identity with SEQ ID NO: 18; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 19, or having at least 90% sequence identity with SEQ ID NO: 19,a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 20 or having at least 90% sequence identity with SEQ ID NO: 20, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 21, or having at least 90% sequence identity with SEQ ID NO: 21.

17. The binding agent of any one of claims 14-16, wherein: a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 16, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 17, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 18; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 19, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 20, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 21.

18. The binding agent of any one of claims 14-17, wherein: the heavy chain variable domain comprises SEQ ID NO: 22; and / or the light chain variable domain comprises SEQ ID NO: 23.

19. The binding agent of any one of claims 14-18, wherein: the heavy chain variable domain comprises SEQ ID NO: 22; and the light chain variable domain comprises SEQ ID NO: 23.

20. The binding agent of claims 14 or 15, wherein: the heavy chain variable domain comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions compared to SEQ ID NO: 22, or a fragment thereof; and / or the light chain variable domain comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions compared to SEQ ID NO: 23, or a fragment thereof.

21. The binding agent of any one of claims 14-20, whereinthe heavy chain variable domain comprises a CDR1 having a polypeptide sequence represented by SEQ ID NO: 16 a CDR2 having a polypeptide sequence represented by SEQ ID NO: 17, and a CDR3 having a polypeptide sequence represented by SEQ ID NO: 18; and / or the light chain variable domain comprises a CDR1 having a polypeptide sequence represented by SEQ ID NO: 19, a CDR2 having a polypeptide sequence represented by SEQ ID NO: 20, and a CDR3 having a polypeptide sequence represented by SEQ ID NO: 21.

22. The binding agent of any one of claims 14-21, wherein the binding agent comprises: a) an antigen-binding fragment (“Fab”); b) an Fab' fragment; c) an F(ab')2 fragment; d) an F(ab')2 fragment and an pFc' fragment; e) a single-chain variable fragment (“scFv”); f) an (scFv)2, or a di-scFv or a bi-scFv; g) a minibody; h) a diabody, a triabody or a tetrabody; i) a single-domain antibody (“dAb”); and / or j) a plurality of CDR fragments, or a multi-specific antibody formed from two or more antibody fragments.

23. The binding agent of any one of claims 14-22, wherein: a) the heavy chain variable domain comprises SEQ ID NO: 22 having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, wherein the heavy chain variable domain is capable of specifically binding to the human AD AMP isoform 1 polypeptide; b) the sequence of the light chain variable domain comprises SEQ ID NO: 23 having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, wherein the light chain variable domain is capable of specifically binding to the human ADAM9 isoform 1 polypeptide; c) the sequence of the heavy chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 22; d) the sequence of the light chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 23;e) the heavy chain variable domain and SEQ ID NO: 22 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment; and / or f) the heavy chain variable domain and SEQ ID NO:23 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment.

24. The binding agent of any one of claims 14-23, wherein the binding agent comprises an IgM, IgG, IgA or IgE isotype antibody heavy chain, and / or a kappa or lambda isotype antibody light chain.

25. The binding agent of any one of claims 14-24, wherein the binding agent does not specifically bind to an ADAM9 isoform 2 polypeptide.

26. The binding agent of any one of claims 14-25, wherein the binding agent is a chimeric or recombinant Ab comprising: a heavy chain having a polypeptide sequence represented by SEQ ID NO: 22 operatively linked to, paired with, associated with, or configured with a light chain having a polypeptide sequence represented by SEQ ID NO:23, wherein the chimeric or recombinant Ab is capable of selectively binding to the human ADAM9 isoform 1 polypeptide.

27. A binding agent capable of specifically binding to a human ADAM9 isoform 1 polypeptide, comprising: a heavy chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NOs: 9 or 22, or a respective fragment thereof; and / or a light chain variable domain comprising a sequence having at least 90% sequence identity with SEQ ID NOs: 10 or 23, or a respective fragment thereof.

28. The binding agent of claim 27, wherein: a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NOs: 3 or16, or having at least 90% sequence identity with SEQ ID NOs: 3 or 16, a CDR2 region having a polypeptide sequence represented by SEQ ID NOs: 4 or17 or having at least 90% sequence identity with SEQ ID NOs: 4 or 17, and / ora CDR3 region having a polypeptide sequence represented by SEQ ID NOs: 5 or18, or having at least 90% sequence identity with SEQ ID NOs: 5 or 18; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NOs: 6 or19, or having at least 90% sequence identity with SEQ ID NOs: 6 or 19, a CDR2 region having a polypeptide sequence represented by SEQ ID NOs: 7 or20 or having at least 90% sequence identity with SEQ ID NOs: 7 or 20, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NOs: 8 or21, or having at least 90% sequence identity with SEQ ID NOs: 8 or 21.

29. The binding agent of claim 27, wherein: a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NOs: 3 or16, a CDR2 region having a polypeptide sequence represented by SEQ ID NOs: 4 or17, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NOs: 5 or 18; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NOs: 6 or19, a CDR2 region having a polypeptide sequence represented by SEQ ID NOs: 7 or20, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NOs: 8 or21,30. The binding agent of claims 13 or 26, further comprising a detectable agent or a binding moiety, wherein the detectable agent optionally comprises: an enzyme, a biotin, a fluorescent or chemiluminescent label, a fluorophore, a cyanine or sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, 7-methoxy coumarin (“Mca”), dabcyl, [2-(4-nitro- 2,l,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (“NBD”), Nile blue, Tamra or tetramethylrhodamine (“TMR”), HRP MAGENTA™ chromogen (Dako Omnis, Agilent),boron-dipyrromethene (“BODIPY”), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten or an antibody binding epitope or domain; and optionally, the enzyme is a peroxidase, an alkaline phosphatase, or a betagalactosidase; optionally, the peroxidase is a horse radish peroxidase (“HRP”); optionally, the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine; optionally, the dye comprises a cyanine dye; or Cy3 or Cy5; optionally, the fluorophore comprises dansyl, fluorescein or carboxyfluorescein (“FAM”) or 6-FAM; and optionally the binding moiety comprises: a glutathione S-transferase (“GST”) or ligandin tag, a polyhistidine (poly-his) tag, a chitin binding protein (“CBP”), a STREP- TAG™ II tag (SEQ ID NO: 14), a FLAG tag (SEQ ID NO: 15), or a maltose binding protein.

31. A recombinant nucleic acid encoding the binding agent of any one of claims 1-30.

32. A method for detecting the presence of a human ADAM9 isoform 1 protein in or on a cell, tissue, or organ, or a portion thereof, comprising: a) contacting the cell, tissue, or organ, or the portion thereof of any of the foregoing with the binding agent of any one of claims 1-30, and b) detecting specific binding of the binding agent, with the human ADAM9 isoform 1 polypeptide in the cell, tissue, organ, or portion thereof; and c) detecting the presence of a human ADAM9 isoform 1 protein in the cell, tissue, or organ, or the portion thereof, based on the detection of specific binding in step b).

33. The method of claim 32, wherein the contacting step (a) is performed using an immunohistochemistry (“IHC”) assay.

34. The method of claims 32 or 33, wherein the method further comprises contacting the binding agent with a detection agent capable of signaling the specific binding of the binding agent to the human ADAM9 isoform 1 protein.

35. the method of clam 34, wherein the detection agent comprises an antibody, an antigen-binding fragment, or a secondary antibody, that specifically binds to the binding agent; optionally, wherein the detection agent comprises an antibody, an antigen-binding fragment, or a secondary antibody that specifically binds to a hapten or tag attached or conjugated to the binding agent.

36. The method of claim 35, wherein the antibody, the antigen-binding fragment, or the secondary antibody further comprises or has attached or conjugated thereto a second detection agent or an enzyme.

37. The method of claim 36, wherein the enzyme is an alkaline phosphatase, a betagalactosidase or a peroxidase.

38. The method of claims 36 or 37, wherein the antibody, the antigen-binding fragment, or the secondary antibody further comprises or has attached or conjugated thereto: a biotin, a fluorescent or chemiluminescent label, a fluorophore, a cyanine or sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, Mca, dabcyl, NBD, Nile blue, Tamra or TMR, BODIPY, HRP MAGENTA™ chromogen (Dako Omnis, Agilent), a dye, a radioisotope, a quantum dot, a photoluminescent aqueous nanocrystal, and / or a hapten.

39. The method of claim 38, wherein the dye comprises a cyanine dye, or Cy3 or Cy5; and / or the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine.

40. A method for detecting an ADAM9 isoform 1 protein-expressing cancer in a subject, comprising: a) contacting a cell, tissue, or organ sample, or a portion thereof, obtained from the subject, with the binding agent of any one of claims 1-30; and b) detecting whether or not the binding agent specifically binds to the human ADAM9 isoform 1 protein in the cell, tissue or organ sample, or the portion thereof, wherein thedetection of specific binding indicates an expression or presence of the human AD AMP isoform 1 protein in the cell, tissue or organ sample, or the portion thereof.

41. The method of claim 40, further comprising: c) diagnosing the subject as having, or being likely to have, a protein-expressing cancer when the binding agent is found to specifically bind to the human ADAM9 isoform 1 protein in the cell, tissue or organ sample, or the portion thereof in step b).

42. The method of claim 40 or 41, wherein the ADAM9 isoform 1 protein-expressing cancer comprises: non-Small Cell Lung Cancer (“NSCLC”), pancreatic cancer, triple-negative breast cancer (“TNBC”), gastroesophageal cancer (“GC”), prostate cancer, or renal cancer.

43. The method of any one of claims 40-42, wherein the detecting step b) is performed using an immunohistochemistry (“IHC”) assay or a flow cytometry.

44. The method of claim 43, wherein the flow cytometry comprises a fluorescence-activated cell sorter (“FACS”) or an impedance flow cytometer.

45. The method of any one of claims 40-44, wherein the cell, tissue or organ sample, or the portion thereof, was obtained from a biopsy from the subject.

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