Anti-human ADAM9 antibodies for in vitro diagnostics

Abstract

The present disclosure provides binding agents (e.g., antibodies) directed against human ADAM9 allotrope 1, as well as kits and methods (e.g., immunoassays) that use them as part of companion diagnostics and for other applications. In some aspects, the binding agents described herein can be used in assays for the detection of non-small cell lung cancer (NSCLC), pancreatic cancer, triple negative breast cancer (TNBC), gastroesophageal cancer (GC), prostate cancer, and kidney cancer.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 540,047, filed September 23, 2023, the entire contents of which are incorporated herein by reference. Reference to electronic sequence listing

[0002] The contents of the electronic sequence list (039062-00164_SequenceListing.xml; size: 26,988 bytes; and creation date: September 19, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention generally relates to binders (e.g., antibodies) against human ADAM9 isoform 1, and kits and methods for using them as part of companion diagnostics and for other applications (e.g., immunoassays). Background Technology

[0004] The ADAM protein family is known to participate in various physiological and pathological processes. See Amendola, RS 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, DR et al., “The ADAM Metalloproteases,” 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, MJ 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-authorization Publication No. 2013 / 0045244.

[0005] Members of the ADAM family share a highly conserved structure, typically containing eight domains, including a metalloproteinase domain and an integrin-binding (unintegrin) domain. See Duffy, MJ et al., “The Role Of ADAMs In Disease Pathophysiology,” Clin. Chim. Acta 403:31-36 (2009). The ADAM metalloproteinase domain acts as an exfoliative enzyme, cleaving the extracellular portion of transmembrane proteins to release the soluble extracellular domain from the cell surface, and has been reported to regulate a range of biological signaling pathways. See Amendola, RS, et al., "ADAM9 Biophys. Res. Commun. 314 (2004) 1008-1013 (2004).

[0006] ADAM9 is a member of the ADAM family, and its Uniprot accession number is Q13443-1. An 819-amino acid isoform (“Isoform 1”, SEQ ID NO: 2) is considered to represent the typical sequence of this protein. ADAM9 is initially synthesized as an inactive form, which is cleaved by proteolysis to generate the active enzyme. ADAM9 is known to be expressed in the following cells: 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., “ADAM15 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 HumanAtherosclerotic Plaques In Aorta And Carotid And Femoral Arteries—Tampere Vascular Study," Ann. Med. 41:279-290 (2009)).

[0007] ADAM9’s metalloproteinase activity plays a role in the degradation of matrix components, thereby allowing tumor cell migration (Amendola, RS et al., “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins And CXCR2,” J. LeukocyteBiol. 97(5):951-962 (2015)). Its disintegrin domain mediates the interaction between ADAM9 and integrin, enabling ADAM9 to positively or negatively regulate 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, MR et al., “Inhibition Of Platelets And Tumor Cells”). Adhesion By The Disintegrin Domain Of Human ADAM9 ToCollagen I Under Dynamic Flow Conditions,” Biochimie 91:1045-1052 (2009)). Previous studies have shown that the ADAM9 disintegrin domain interacts with α6β1, α6β4, αvβ5, and α9β1 integrins.

[0008] ADAM9 expression has been found to be associated with a variety of diseases, including cancer. For example, ADAM9 has been found to cleave and release many molecules that play important roles in tumorigenesis and angiogenesis, such as TEK, KDR, EPHB4, CD40, VCAM1, and CDH5. ADAM9 is expressed by a variety of tumor cell types, including breast cancer, colon cancer, gastric cancer, glioma, liver cancer, non-small cell lung cancer, melanoma, myeloma, pancreatic cancer, and prostate cancer.See, for example, Yoshimasu, T. et al., “Overexpression Of ADAM9 In Non-Small Cell Lung Cancer Correlates With BrainMetastasis,” Cancer Res. 64:4190-4196 (2004); Peduto, L. et al., “CriticalFunction For ADAM9 In Mouse Prostate Cancer,” Cancer Res. 65:9312-9319(2005); Zigrino, P. et al., “ADAM-9 Expression And Regulation In Human SkinMelanoma 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 IsAssociated With Tumour Progression,” BMC Cancer 8:179:1-9 (2008); Fry, J. L. et al., “Secreted And Membrane-Bound Isoforms Of Protease ADAM9 Have OpposingEffects On Breast Cancer Cell Migration,” Cancer Res. 70, 8187-8198 (2010); Chang, L. et al., “Combined Rnai Targeting Human Stat3 And ADAM9 As Gene TherapyFor Non-Small Cell Lung Cancer,” Oncology Letters 11:1242-1250 (2016); Fan, X. et al., “ADAM9 Expression Is Associate with Glioma Tumor Grade and HistologicalType, and Acts as a Prognostic Factor in Lower-Grade Gliomas,” Int. J. Mol.Sci. 17:1276:1-11 (2016).

[0009] Increased ADAM9 expression has been found to be positively correlated with tumor malignancy and metastatic potential, highlighting the clinical significance of this gene. See, for example, Amendola, RS et al., “ADAM9 Disintegrin Domain Activates Human Neutrophils Through An Autocrine Circuit Involving Integrins AndCXCR2,” 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 CancerCells Invasion,” J. Invest. Surg. 26(3):127-133 (2016).Furthermore, ADAM9 and its secreted soluble isoforms have shown crucial role in cancer cell proliferation (Amendola, RS 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, JL 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 Publication No. 2009 / 0023149).

[0010] Given this clinical significance, ADAM9 has become 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, MJ et al., “Role Of ADAMs In Cancer Formation And Progression,” Clin. Cancer Res. 15:1140-1144 (2009); Duffy, MJ 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 ADAM9 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-authorization publications 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 The publication numbers are 2009 / 0285840, 2009 / 0203051, 2004 / 0092466, 2003 / 0091568, and 2002 / 0068062, as well as PCT publication numbers WO2016 / 077505, WO2014 / 205293, WO2014 / 186364, WO2014 / 124326, WO2014 / 108480, WO2013 / 119960, WO2013 / 098797, WO2013 / 049704, and WO2011 / 100362. Furthermore, ADAM9 expression has been found to be associated with lung disease and inflammation.See, for example, U.S. pre-authorization publication numbers 2016 / 0068909; 2012 / 0149595; 2009 / 0233300; 2006 / 0270618; and 2009 / 0142301. Antibodies binding to ADAM9 are commercially available from several suppliers, including Abcam, Thermofisher, Sigma-Aldrich, and others. Summary of the Invention

[0011] Antibodies and other binding agents specific to ADAM9 are commercially available. However, there remains a need in the art for additional high-affinity ADAM9 binding agents, particularly those exhibiting specificity for isoform 1 of ADAM9. As mentioned above, the human form of ADAM9 is known to have multiple isoforms. Existing antibodies against ADAM9 typically detect these multiple isoforms, interfering with efforts to accurately determine the location or level of isoform 1 of ADAM9. This resulting noise affects the accuracy of diagnostic assays and other applications of such assays.

[0012] This disclosure addresses these and other drawbacks of existing ADAM9 binders by providing a variety of binders capable of selectively binding to allotypes of ADAM9 (e.g., as a control for allotype 2, SEQ ID NO 13). In alternative embodiments, chimeric or recombinant antibodies (Abs) or their antigen (Ag)-binding fragments or monomeric or dimeric antigen-binding proteins capable of specifically binding to human ADAM9 peptides are provided, said human ADAM9 peptides including those expressed on the surface of tumor cells (e.g., tumor cells including breast cancer, colon cancer, gastric cancer, glioma, liver cancer, non-small cell lung cancer, melanoma, myeloma, pancreatic cancer, and prostate cancer) and tumor-infiltrating lymphocytes (such as activated T cells), or human ADAM9 peptides expressed on tumor-infiltrating lymphocytes (TILs). In some aspects, the binder (e.g., the Ab or its Ag-binding fragment) selectively binds to allotype 1 of human ADAM9 but not to allotype 2.

[0013] In a first general aspect, this disclosure provides a binding agent capable of specifically binding to the human ADAM9 allotype 1 polypeptide (SEQ ID NO: 2), the binding agent comprising: a heavy chain variable domain comprising a sequence or fragment thereof having at least 90% sequence identity with SEQ ID NO: 9; and / or a light chain variable domain comprising a sequence or fragment thereof having at least 90% sequence identity with SEQ ID NO: 10. In some aspects, the heavy chain variable domain comprises a sequence or fragment thereof having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. In some aspects, the light chain variable domain comprises a sequence or fragment thereof having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. As used herein, the fragment may comprise any portion of the polypeptide less than the full-length sequence. For example, for a polypeptide of 100 amino acids in length, the fragment will constitute at least one and at most 99 consecutive amino acids of the full-length sequence. It should also be understood that, as used herein, the fragment may contain the minimum number of consecutive 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 of a given fragment).

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

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

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

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

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

[0021] In some aspects, the binding agent includes: a) an antigen-binding fragment (“F…”) ab b) Fab' fragment; c) F(ab')2 fragment; d) F(ab')2 fragment and pFc' fragment; e) single-chain variable fragment ("scFv"); f) (scFv)2, or two scFv or double scFv; g) microantibody; h) Bivariate, trivariate, or tetravariate antibodies; i) Single-domain antibodies (“dAb”); and / or j) Multiple CDR fragments, or multispecific antibodies formed by 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 conserved amino acid substitutions, wherein the heavy chain variable domain is specifically capable of binding to the human ADAM9 isoform 1 polypeptide; b) the light chain variable domain comprises SEQ ID NO: 10 having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, wherein the light chain variable domain is specifically capable of 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) When compared using distance matrix alignment, the heavy chain variable structural domain and SEQ ID NO: 9 have a Z score of about 2 to about 8, or at least 8; and / or f) when compared using distance matrix alignment, the heavy chain variable structural domain and SEQ ID NO: 10 have a Z score of about 2 to about 8, or at least 8.

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

[0024] In some respects, the binder does not specifically bind to the ADAM9 isoform 2 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, the heavy chain being operatively linked, paired, associated, 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 isomorph 1 polypeptide.

[0026] In some aspects, the binder further comprises a detectable agent or binding moiety. In some aspects, the detectable agent optionally comprises: an enzyme, biotin, a fluorescent or chemiluminescent label, a fluorophore, anthocyanin or sulfonylindocyanine, Nile red, rhodamine, perylene, fluorene, coumarin, 7-methoxycoumarin (“Mca”), dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (“NBD”), Nile blue, Tamra or tetramethylrhodamine (“TMR”), HRPMAGENTA™ chromogen (Dako Omnis, Agilent), boron-dipyrrole methylene (“BODIPY”), dye, radioisotope, quantum dot or photoluminescent aqueous nanocrystal, hapten or antibody binding epitope or domain. In some aspects, the enzyme is a peroxidase, alkaline phosphatase or β-galactosidase. In some aspects, the peroxidase is horseradish peroxidase (“HRP”). ​​In some aspects, the hapten includes biotin, theophylline, digoxigenin, carborane, fluorescein, or bromodeoxyuridine. In some aspects, the dye includes anthocyanin dyes; or Cy3 or Cy5. In some aspects, the fluorophore includes dansyl, fluorescein, or carboxyfluorescein (“FAM”) or 6-FAM.

[0027] In some aspects, the binding moiety includes: a glutathione S-transferase (“GST”) or ligand tag, a polyhistidine (polyhis) 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, this disclosure provides additional binding agents for the specific detection of allotype 1 of ADAM9. For example, in some aspects, a binding agent capable of specifically binding to the human ADAM9 allotype 1 polypeptide, the binding agent comprising: a heavy chain variable domain comprising a sequence or fragment thereof having at least 90% sequence identity with SEQ ID NO: 22; and / or a light chain variable domain comprising a sequence or fragment thereof having at least 90% sequence identity with SEQ ID NO: 23. In some aspects, the binding agent comprises a chimeric or recombinant antibody (“Ab”), 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 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.

[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 binder comprises a heavy chain variable domain containing a sequence having at least 90% sequence identity with SEQ ID NO: 9 or 22 or a corresponding fragment thereof; and / or a light chain variable domain containing a sequence having at least 90% sequence identity with SEQ ID NO: 10 or 23 or a corresponding fragment thereof.

[0031] In some aspects, a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 3 or 16 or having at least 90% sequence identity with SEQ ID NO: 3 or 16, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 4 or 17 or having at least 90% sequence identity with SEQ ID NO: 4 or 17, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 5 or 18 or having at least 90% sequence identity with SEQ ID NO: 5 or 18; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 6 or 19 or having at least 90% sequence identity with SEQ ID NO: 6 or 19, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 7 or 20 or having at least 90% sequence identity with SEQ ID NO: 7 or 20, and / or a polypeptide sequence represented by SEQ ID NO: 8 or 21 or having at least 90% sequence identity with SEQ ID NO: 6 or 19. NO: 8 or 21, with at least 90% sequence identity in CDR3 regions.

[0032] In some aspects, a) the heavy chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 3 or 16, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 4 or 17, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 5 or 18; and / or b) the light chain variable domain comprises a CDR1 region having a polypeptide sequence represented by SEQ ID NO: 6 or 19, a CDR2 region having a polypeptide sequence represented by SEQ ID NO: 7 or 20, and / or a CDR3 region having a polypeptide sequence represented by SEQ ID NO: 8 or 21.

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

[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 or constitutive promoter). In some aspects, the nucleic acid is a recombinant nucleic acid that further encodes additional protein or peptide portions or domains (e.g., purified portions or domains that aid in the purification or isolation of chimeric or recombinant antibodies or their antigen-binding fragments).

[0035] In a third general aspect, this disclosure provides methods for detecting the presence of human ADAM9 isoform 1 protein in or above cells, tissues, or organs or parts thereof. In some aspects, such methods include a) contacting a cell, tissue, or organ or part thereof with any of the aforementioned components with any ADAM9 binding agent described herein; and b) detecting specific binding of the ADAM9 binding agent to the human ADAM9 isoform 1 polypeptide in the cell, tissue, organ or part thereof; and c) detecting the presence of human ADAM9 isoform 1 protein in the cell, tissue, organ or part thereof based on the detection of specific binding in step b). In some aspects, the contact step (a) is performed using immunohistochemistry (IHC).

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

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

[0038] In some aspects, the antibody, the antigen-binding fragment, or the secondary antibody further comprises or is attached to or conjugated thereto with: biotin, fluorescent or chemiluminescent labeling, fluorophore, anthocyanin or sulfonylindocyanine, Nile red, rhodamine, perylene, fluorene, coumarin, McA, dabcyl, NBD, Nile blue, Tamra or TMR, BODIPY, HRP MAGENTA™ chromogen (DakoOmnis, Agilent), dye, radioisotope, quantum dot, photoluminescent aqueous nanocrystals, and / or hapten. In some aspects, the dye comprises anthocyanin dye or Cy3 or Cy5; and / or the hapten comprises biotin, theophylline, digoxigenin, carborane, fluorescein, or bromodeoxyuridine.

[0039] In a fourth general aspect, this disclosure provides a method for detecting cancer expressing the ADAM9 isoform 1 protein in a subject. In some aspects, such a method includes 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 the binding agent specifically binds to the human ADAM9 isoform 1 protein in the cell, tissue, or organ sample or a portion thereof, wherein the detection of specific binding indicates the expression or presence of the human ADAM9 isoform 1 protein in the cell, tissue, or organ sample or a portion thereof. In some aspects, such a method further includes c) diagnosing that the subject has or may have a cancer expressing the protein when the binding agent is found to specifically bind to the human ADAM9 isoform 1 protein in the cell, tissue, or organ sample or a portion thereof in step b).

[0040] In some aspects, the cancers expressing the ADAM9 isoform 1 protein include: non-small cell lung cancer (“NSCLC”), pancreatic cancer, triple-negative breast cancer (“TNBC”), gastroesophageal cancer (“GC”), prostate cancer, or kidney cancer. In some aspects, step b) is performed using immunohistochemistry (“IHC”) assay or flow cytometry. In some aspects, the flow cytometry includes fluorescence-activated cell sorting (“FACS”) or impedance flow cytometry. In some aspects, the cell, tissue, or organ sample, or a portion thereof, is obtained from a biopsy of the subject.

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

[0042] In a sixth general aspect, this disclosure provides cells comprising chimeric or recombinant antibodies, or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins, as provided herein, recombinant nucleic acids, or expression cassettes, vectors, recombinant viruses, artificial chromosomes, granules, or plasmids, as provided herein, and said cells may be bacterial, fungal, mammalian, yeast, insect, avian, or plant cells.

[0043] In a seventh general aspect, this disclosure provides a method for detecting or diagnosing cancer expressing the ADAM9 protein, or cancerous tissue containing lymphocytes expressing ADAM9, or tumor-infiltrating lymphocytes (such as tumor-infiltrating activated T cells) expressing ADAM9, the method comprising: contacting a cell, tissue, or organ sample with an ADAM9 binding agent (e.g., a chimeric or recombinant antibody) encoded by a nucleic acid as provided herein or provided herein; detecting the expression or presence of a human ADAM9 protein (e.g., an allotype 1 of ADAM9) in the cell, tissue, or organ sample or a portion thereof; and detecting whether the chimeric or recombinant antibody specifically binds to the human ADAM9 protein in the cell, tissue, or organ sample or a portion thereof, and the detection of specific binding indicating the expression or presence of the human ADAM9 protein in the cell, tissue, or organ sample or a portion thereof.

[0044] In an eighth general aspect, this disclosure provides methods for detecting or diagnosing cancer expressing the ADAM9 protein as provided herein, or cancerous tissue containing ADAM9-expressing lymphocytes or ADAM9-expressing tumor-infiltrating lymphocytes (such as tumor-infiltrating activated T cells). In some aspects, the cells are activated T cells, tumor-infiltrating activated T cells, or tumor-infiltrating lymphocytes (TILs). In some aspects, a specifically binding detection indicates the expression or presence of the human ADAM9 protein in the cells, tissue, or organ sample or a portion thereof, thereby diagnosing or detecting the cancer. In some aspects, the cancer is selected from non-small cell lung cancer (“NSCLC”), pancreatic cancer, triple-negative breast cancer (“TNBC”), gastroesophageal cancer (“GC”), prostate cancer, or kidney cancer. In some aspects, the detection includes using or performing immunohistochemistry (IHC) assays or flow cytometry. In some aspects, performing or using the flow cytometry includes using a fluorescence-activated cell sorter (FACS) or impedance flow cytometry. In some respects, the cell, tissue, or organ sample or a portion thereof is or is derived from a patient's biopsy.

[0045] In a ninth general aspect, this disclosure provides methods for treating, improving, or preventing cancer, said methods comprising first detecting or diagnosing cancer in an individual in need using methods as provided herein, and then treating said individual in need. In some aspects, said cancer is selected from: non-small cell lung cancer (“NSCLC”), pancreatic cancer, triple-negative breast cancer (“TNBC”), gastroesophageal cancer (“GC”), prostate cancer, and kidney cancer.

[0046] In a tenth general aspect, this disclosure provides for the use of the ADAM9 binding agents described herein (e.g., chimeric and / or recombinant antibodies encoded by recombinant nucleic acids as provided herein, or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins) for the detection or diagnosis of cancer, or cancerous tissue containing lymphocytes expressing ADAM9 or tumor-infiltrating lymphocytes expressing ADAM9 (such as tumor-infiltrating activated T cells), or for the treatment, improvement, or prevention of said cancer.

[0047] In an eleventh general aspect, this disclosure provides kits comprising chimeric or recombinant antibodies, or antigen-binding fragments thereof, or monomeric or dimer antigen-binding proteins encoded by recombinant nucleic acids as provided herein or as provided herein. In some aspects, the kits provided herein contain components required for immunohistochemical (IHC) assays; and / or instructions for practicing the methods provided herein. In alternative embodiments of the kits provided herein, the chimeric antibody or recombinant antibody, its antigen-binding fragment, or the monomeric or dimer antigen-binding protein is substantially purified or isolated.

[0048] In a twelfth general aspect, this disclosure provides articles comprising chimeric or recombinant antibodies, antigen-binding fragments thereof, or monomeric or dimer antigen-binding proteins encoded by recombinant nucleic acids as provided herein or as provided herein. In some aspects, the articles comprise or are manufactured as slides, grooves, chips, biochips, arrays, trays, dishes, or microtiter plates or dishes. In some aspects, the chimeric antibody or recombinant antibody, its antigen-binding fragment, or the monomeric or dimer antigen-binding protein is substantially purified or isolated, or is in the form of unpurified or partially purified culture supernatant. In some aspects, this disclosure provides bacteriophages or phage particles comprising or expressing on their surface chimeric or recombinant antibodies, antigen-binding fragments thereof, or monomeric or dimer antigen-binding proteins encoded by any nucleic acid as provided herein or as provided herein.

[0049] To achieve the foregoing and related objectives, the one or more aspects include the features fully described and specifically pointed out below in the claims. Certain illustrative features of the one or more aspects are set forth in detail in the following description and drawings. However, these features indicate several of the various ways in which the principles of the aspects may be employed, and this description is intended to include all such aspects and their equivalents. Attached Figure Description

[0050] This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, the official authority will provide a copy of this patent or patent application publication with the color drawing.

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

[0052] Figure 1 The images show Western blots detected using (A) Cell Signaling® anti-ADAM9 CST2099, (B) anti-ADAM9 clone 11H9, and (C) anti-ADAM9 clone 11C9, respectively. In blot (A), bands are observed at approximately 100 kDa (pre-ADAM9-L), 70 kDa (ADAM9-L), and 45 kDa (ADAM9-S), revealing that the anti-ADAM9 CST2099 antibody binds to all three isoforms of ADAM9. In the two blots on the right (B) and (C), only two upper bands are present, revealing that anti-ADAM9 antibody clones 11H9 and 11C9 bind only to ADAM9-L and pre-ADAM9-L, respectively.

[0053] Figure 2 This image shows kidney staining using ADAM9 clone 11H9 (T0525B, batch number H00976) on Dako Omnis. Strong apical and cytoplasmic staining is shown in both the proximal tubules (black arrows) and distal tubules. The glomeruli are almost unstained (hollow arrows).

[0054] Figure 3 This image shows pancreatic staining using ADAM9 clone 11H9 (T0525B, batch number H00976) on Dako Omnis. Cytoplasmic staining is shown in the Langerhans islands (black arrows). Tip staining and cytoplasmic staining are shown in the ducts (hollow arrows). The acinar structures are unstained (red arrows), but the small blood vessels surrounding the glands show weak staining.

[0055] Figure 4 This is an image showing prostate staining using ADAM9 clone 11H9 (T0525B, batch number H00976) on Dako Omnis. Cytoplasmic staining (black arrows) and membrane staining (hollow arrows) are shown in the prostate.

[0056] Figure 5 This is an image showing colon staining using ADAM9 clone 11H9 (T0525B, batch number H00976) on Dako Omnis. Cytoplasmic staining is shown in the epithelium (black arrows).

[0057] Figure 6This image shows liver staining using ADAM9 clone 11H9 (T0525B, batch number H00976) on Dako Omnis. It shows diffuse cytoplasmic staining of macrophages (Kuppfer cells) and blood sinuses (black arrows), as well as cytoplasmic and membrane staining in the bile ducts (hollow arrows).

[0058] Figure 7 This image shows tonsil staining using ADAM9 clone 11H9 (T0525B, batch number H00976) on Dako Omnis. Weak staining of keratinocytes in the crypts is shown (black arrows), with up-concentration at the top (hollow arrows). Additionally, weak diffuse staining is prevalent in the vascular structures (red arrows).

[0059] Figure 8 This image shows the staining of breast cancer cells with ADAM9 clone 11H9 (T0525B, batch number H00976) on Dako Omnis. Moderate membrane (apex) is shown in normal ducts (black arrow). Tumor cells are unstained (hollow arrow).

[0060] Figure 9 This image shows the staining of carcinoid cells with ADAM9 clone 11H9 (T0525B, batch number H00976) on Dako Omnis. It shows weak to moderate cytoplasmic staining in the tumor cells, with a tendency for stronger staining at the periphery of the tumor group (black arrows).

[0061] Figure 10 This image shows a tumor tissue sample from colon adenocarcinoma stained with ADAM9 clone 11H9 (T0525B, batch number H00976) on a Dako Omnis. Moderate cytoplasmic and membrane staining is observed in the tumor cells (black arrows).

[0062] Figure 11 This is an image showing cerebellar staining using ADAM9 clone 11H9 (T0525B, batch number H00976) on Dako Omnis. No staining or background staining was observed.

[0063] Figure 12 This image shows a tumor tissue sample from pancreatic adenocarcinoma stained with ADAM9 clone 11H9 (T0525B, batch H00976) on a Dako Omnis. Strong cytoplasmic and membrane staining is observed in the tumor cells (black arrows).

[0064] Figure 13This image shows a tumor tissue sample from pancreatic adenocarcinoma stained with ADAM9 clone 11H9 (T0525B, batch H00976) on a Dako Omnis. Moderate cytoplasmic and membrane staining is observed in the tumor cells (black arrows).

[0065] Figure 14 This image shows a tumor tissue sample from pancreatic adenocarcinoma stained with ADAM9 clone 11H9 (T0525B, batch H00976) on a Dako Omnis. Moderate cytoplasmic and membrane staining is observed in the tumor cells (black arrows). Note the strong apical staining (hollow arrow).

[0066] Figure 15 This image shows a tumor tissue sample from breast cancer stained with ADAM9 clone 11H9 (T0525B, batch number H00976) on a Dako Omnis. Moderate cytoplasmic staining and membrane staining are observed in the tumor cells (black arrows).

[0067] Figure 16 This image shows a tumor tissue sample from breast cancer stained with ADAM9 clone 11H9 (T0525B, batch number H00976) on a Dako Omnis. Moderate cytoplasmic staining and membrane staining are observed in the tumor cells (black arrows).

[0068] Figure 17 This image shows a lung adenocarcinoma tumor tissue sample stained with ADAM9 clone 11H9 (T0525B, batch H00976) on a Dako Omnis. Weak to moderate cytoplasmic staining and moderate membrane staining are observed in the tumor cells (black arrows).

[0069] Figure 18 This image shows a lung adenocarcinoma tumor tissue sample stained with ADAM9 clone 11H9 (T0525B, batch H00976) on a Dako Omnis. Weak cytoplasmic staining and membrane staining are observed in the tumor cells (black arrows).

[0070] Figure 19 This image shows a lung adenocarcinoma tumor tissue sample stained with ADAM9 clone 11H9 (T0525B, batch H00976) on a Dako Omnis. Moderate membrane staining is observed in some tumor cells (black arrows).

[0071] Figure 20 This image shows a tumor tissue sample from a gastric adenocarcinoma stained with ADAM9 clone 11H9 (T0525B, batch H00976) on a Dako Omnis. Weak membrane OG cyt staining was observed in some tumor cells (black arrows).

[0072] Figure 21 This is an image showing the staining of a gastric adenocarcinoma tumor tissue sample with ADAM9 clone 11H9 (T0525B, batch H00976) on a Dako Omnis. Moderate cytoplasmic and membrane staining is observed in the tumor cells (black arrows).

[0073] Figure 22 This is an image showing the staining of a gastric cancer tumor tissue sample with ADAM9 clone 11H9 (T0525B, batch H00976) on a Dako Omnis. Strong cytoplasmic and membrane staining is observed in the tumor cells (black arrows).

[0074] Figure 23 This image shows a tumor tissue sample from colon adenocarcinoma stained with ADAM9 clone 11H9 (T0525B, batch number H00976) on a Dako Omnis. Moderate cytoplasmic and membrane staining is observed in the tumor cells (black arrows).

[0075] Figure 24 It is an image showing the reaction pattern of clone 11C9 on selected tissues in a large multi-tissue block.

[0076] Figure 25 This image shows a tumor tissue sample from pancreatic adenocarcinoma stained with ADAM9 clone 11C9 (T0531B, batch H00971) on a Dako Omnis. Moderate cytoplasmic and membrane staining is observed in the tumor cells (black arrows).

[0077] Figure 26 This is an image showing the staining of a pancreatic adenocarcinoma tumor tissue sample with ADAM9 clone 11C9 (T0531B, batch H00971) on a Dako Omnis. Weak to moderate cytoplasmic and membrane staining is observed in the tumor cells (black arrows).

[0078] Figure 27 This is an image showing the staining of a pancreatic adenocarcinoma tumor tissue sample with ADAM9 clone 11C9 (T0531B, batch H00971) on a Dako Omnis. Weak cytoplasmic staining is observed in the tumor cells (black arrows).

[0079] Figure 28 This image shows a tumor tissue sample from pancreatic adenocarcinoma stained with ADAM9 clone 11C9 (T0531B, batch H00971) on a Dako Omnis. Weak cytoplasmic staining and moderate membrane staining are observed in the tumor cells (black arrows).

[0080] Figure 29This is an image showing the staining of a pancreatic adenocarcinoma tumor tissue sample with ADAM9 clone 11C9 (T0531B, batch H00971) on a Dako Omnis. Weak cytoplasmic and membrane staining is observed in the tumor cells (black arrows).

[0081] Figure 30 This is an image showing the staining of a gastric adenocarcinoma tumor tissue sample with ADAM9 clone 11C9 (T0407, batch H00740) on a Dako Omnis. Weak to moderate cytoplasmic and membrane staining is observed in the tumor cells (black arrows).

[0082] Figure 31 This image shows a tumor tissue sample from breast cancer stained with ADAM9 clone 11C9 (T0407, batch number H00740) on a Dako Omnis. Moderate to strong cytoplasmic and membrane staining is observed in the tumor cells (black arrows). Detailed Implementation

[0083] In alternative embodiments, chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimer antigen-binding proteins, are provided that can specifically bind to human ADAM9 polypeptides (e.g., ADAM9 isoform 1, i.e., SEQ ID NO: 2 or a portion thereof). In alternative embodiments, articles and kits comprising chimeric or recombinant Abs, or antigen-binding fragments thereof, or monomeric or dimer antigen-binding proteins, or nucleic acids encoding them, as provided herein, are provided, along with methods for preparing and using them. In alternative implementations, chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins, as provided herein, may be used in in vitro diagnostics, such as by immunohistochemistry (IHC), for the diagnosis, detection, and / or treatment of cancers, such as bladder cancer, urothelial carcinoma, breast cancer or ductal carcinoma in situ (DCIS), carcinoid tumors, Hodgkin's lymphoma, chronic lymphocytic leukemia, colorectal cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), renal cell carcinoma or renal cell carcinoma, clear cell renal cell carcinoma (RCC), mesothelioma or malignant pleural mesothelioma, squamous cell carcinoma, anal squamous cell carcinoma, pancreatic cancer, and melanoma or malignant melanoma. Therefore, chimeric or recombinant antibodies (Abs), or antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins, as provided herein, can be used as companion diagnostics for the diagnosis and treatment of cancer.

[0084] Expression of recombinant chimeric antibodies

[0085] In alternative embodiments, chimeric and / or recombinant antibodies (Abs), their antigen-binding fragments, or monomeric or dimeric antigen-binding proteins (including exemplary chimeric or recombinant anti-human ADAM9 Abs containing the heavy chain variable region of SEQ ID NO: 9 or 22 and the light chain variable region of SEQ ID NO: 10 or 23, with or without a signal peptide) may be expressed as recombinant Abs using, for example, plasmids encoding the corresponding heavy and light chains or any expression media, or the heavy and light chains may be encoded in separate expression media.

[0086] In some implementations, the heavy and light chains can be expressed from any plasmid, granule, recombinant virus, or equivalent vector (e.g., from one or more pTT5™ vectors (National Research Council of Canada, NRC-CNRC, Canada) or equivalents) (cis or trans).

[0087] In alternative embodiments, expression vectors (e.g., plasmids) containing one or more exemplary Ab-encoded nucleic acids as provided herein are expressed in an in vitro expression system or in cultured tissues, cells, or organoids, which may be bacterial, fungal, mammalian, yeast, insect, or plant cell expression systems, or hybrids or synthetic expression systems. For example, one or more exemplary Ab-encoded nucleic acids may be expressed in human embryonic kidney (HEK) cells (e.g., HEK293-6E cells). In alternative embodiments, one or more vectors expressing one or more exemplary Ab-encoded nucleic acids (e.g., exemplary heavy chains and / or light chains) are appendage-type or chromosome-integrated in stable cell lines, for example, capable of synthesizing, and optionally inducing, the synthesis of heavy chains and / or light chains.

[0088] In alternative embodiments, nucleic acids encoding chimeric or recombinant Abs as provided herein are provided. Nucleic acids as provided herein can be prepared, isolated, and / or manipulated, for example, by cloning and expressing cDNA libraries, via PCR amplification information, or genomic DNA. Nucleic acids (whether RNA, cDNA, genomic DNA, vectors, viruses, or hybrids thereof) for practicing the embodiments provided herein can be isolated, genetically engineered, amplified, and / or recombinantly expressed / produced from a variety of sources. Recombinant polypeptides derived from these nucleic acids can be isolated or cloned individually, and the desired activity of the recombinant polypeptides can be tested. 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 using well-known chemical synthesis techniques, such as those described below: 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; US Patent No. 4,458,066.

[0090] Techniques for manipulating nucleic acids (such as subcloning, labeled probes (e.g., random primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization, etc.) are well described in scientific and patent literature; see, for example, Sambrook (ed.), *MOLECULAR CLONING: A LABORATORY MANUAL* (2nd edition), Volumes 1–3, ColdSpring Harbor Laboratory, (1989); *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, Ausubel (ed.), John Wiley & Sons, Inc., New York (1997); *LABORATORY TECHNIQUESIN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACIDPROBES*, Part I. *Theory and Nucleic Acid Preparation*, Tijssen (ed.), Elsevier, NY (1993).

[0091] Another useful means of obtaining and manipulating nucleic acids for practicing embodiments as provided herein includes screening and recloning inserts isolated or amplified from, for example, genomic clones or cDNA clones. Sources of nucleic acids include recombinant nucleic acid sequences, genomes, or cDNA libraries contained in or expressed in, for example, vectors such as: 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 (YACs); bacterial artificial chromosomes (BACs); P1 artificial chromosomes, see, for example, Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see, for example, Kern (1997) Biotechniques 23:120-124; granules, recombinant viruses, bacteriophages, or plasmids.

[0092] In alternative implementations, nucleic acids, as provided herein, are operatively linked to transcriptional regulatory elements (including promoters), which may be constitutive or inducible transcriptional regulatory elements.

[0093] In an alternative, an "expression cassette" is provided, which contains a nucleotide sequence encoding, for example, a chimeric or recombinant antibody as provided herein. The expression cassette may include at least a transcriptional regulatory element (e.g., a promoter) operatively linked to the antibody-coding sequence, and optionally, a transcription termination signal. Additional factors (e.g., enhancers) necessary or helpful in achieving expression may also be used.

[0094] Alternatively, expression cassettes used in practicing embodiments as provided herein include plasmids, expression vectors, recombinant viruses, recombinant “naked DNA” vectors of any form. Alternatively, a “vector” used in practicing embodiments as provided herein may contain nucleic acids capable of infecting, transfecting, transiently or permanently transducing cells. Alternatively, a vector used in practicing embodiments as provided herein may be naked nucleic acid, or nucleic acid complexed with a protein or lipid. Alternatively, a vector used in practicing embodiments as provided herein may contain viral or bacterial nucleic acids and / or proteins, and / or membranes (e.g., cell membranes, viral lipid envelopes, etc.).

[0095] Alternatively, vectors used to practice embodiments as provided herein may include, but are not limited to, replicons (e.g., RNA replicons, bacteriophages), to which fragments of DNA may attach and be replicated. Thus, vectors include, but are not limited to, RNA, autonomously replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, for example, U.S. Patent No. 5,217,879), and may include both expression plasmids and non-expression plasmids. Alternatively, vectors used to practice embodiments as provided herein may be stably replicated by the cell as an autonomous structure during mitosis, or may be incorporated into the host genome.

[0096] In alternatives, the “promoter” used to practice embodiments as provided herein includes all sequences capable of driving transcription of coding sequences in cells (e.g., bacterial cells, yeast cells, fungal cells, plant cells, insect cells (e.g., baculoviruses), or mammalian cells). Therefore, promoters used in constructs include cis-acting transcriptional control elements and regulatory sequences involved in regulating or modulating the timing and / or rate of gene transcription. For example, promoters used to practice embodiments as provided herein can be cis-acting transcriptional control elements, including enhancers, promoters, transcription terminators, origins of replication, chromosomal integration sequences, 5' and 3' untranslated regions, or intron sequences involved in transcriptional regulation. These cis-acting sequences can interact with proteins or other biomolecules to perform (on / off, regulate, modulate, etc.) transcription.

[0097] "Constraint" promoters used in implementing the embodiments provided herein can be promoters that continuously drive expression under most environmental conditions and developmental or cell differentiation states. "Inducible" or "regulatory" promoters used in implementing the embodiments provided herein can direct the expression of nucleic acids as provided herein under the influence of environmental or developmental conditions. Examples of environmental conditions that may affect transcription via inducible promoters used in implementing the embodiments provided herein include the presence of inducing factors applied to the cell.

[0098] In alternative embodiments, antibodies used to practice embodiments as provided herein may include any “mimic” and / or “peptide mimic” form. In alternative embodiments, peptides and polypeptides used to practice embodiments as provided herein may include synthetic chemical compounds having substantially the same structural and / or functional characteristics as natural polypeptides, such as chimeric or recombinant antibodies as provided herein. Mimics used to practice embodiments as provided herein may consist entirely of synthetic, non-natural amino acid analogs, or chimeric molecules that are partly natural peptide amino acids and partly non-natural amino acid analogs. Mimics may also incorporate any amount of conserved substitutions of natural amino acids, provided that such substitutions do not substantially alter the structure and / or activity of the mimic. Routine experiments will determine whether the mimic is effective for practicing the invention, for example, whether the mimic composition is effective in specifically binding to the human ADAM9 protein. The methods detailed herein and other methods known to those skilled in the art can be used to select or guide the selection of mimics effective for practicing the compositions and / or methods provided herein.

[0099] Peptide mimic compositions used in practicing embodiments as provided herein can comprise any combination of non-natural structural components. Alternatively, mimic compositions used in practicing embodiments as provided herein can comprise one or all of three structural groups: a) residue-linking groups other than natural amide bonds (“peptide bonds”); b) non-natural residues replacing naturally occurring amino acid residues; or c) residues that induce secondary structure mimicry (i.e., induce or stabilize secondary structures, such as β-turns, γ-turns, β-sheets, α-helical conformations, etc.). For example, a polypeptide can be characterized as a mimic when all or some of its residues are linked by chemical means other than natural peptide bonds.

[0100] Purification and isolation of recombinant proteins

[0101] In alternative embodiments, the chimeric or recombinant antibody, its antigen-binding fragment or monomeric or dimer antigen-binding protein is substantially purified or isolated, and optionally, the substantially purified or isolated form is used in immunohistochemical methods and / or as a reagent, kit and / or article as provided herein.

[0102] In alternative embodiments, chimeric or recombinant antibodies, their antigen-binding fragments, or monomeric or dimer antigen-binding proteins are substantially purified or separated using: physicochemical fractionation, such as differential precipitation, size exclusion, or solid-state binding of immunoglobulins based on the size, charge, or other shared chemical properties of antibodies in a typical sample; class-specific affinity, such as solid-state binding of a specific antibody class (e.g., IgG or IgM) to immobilized biological ligands (e.g., proteins, lectins, etc.) with specific affinity for immunoglobulins, which can purify all antibodies of the target class without regard to antigen specificity; or antigen-specific affinity, such as affinity purification of only those antibodies in the sample that bind to a specific antigen molecule through their specific antigen-binding domains, where this purifies all antibodies bound to the antigen without regard to antibody class or isotype.

[0103] In alternative implementations, the chimeric or recombinant antibody, its antigen-binding fragment, or monomeric or dimeric antigen-binding protein is substantially purified or separated using standard separation methods 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 recombinant antibodies, their antigen-binding fragments, or monomeric or dimer antigen-binding proteins are generated by using sequential expression and purification methods in a bioreactor (e.g., a perfusion bioreactor), such as those described in Vogg et al. Methods Mol Biol. 2018; Vol. 1850: 147-178; or by using a stirred tank or swaying bioreactor system followed by purification.

[0105] Products and reagent kits

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

[0107] In alternative embodiments, the article is attached or immobilized (optionally covalently bound) to or to a chimeric or recombinant antibody (Ab) as provided herein, or an antigen-binding fragment thereof, or a monomeric or dimer antigen-binding protein, and optionally, the article as provided herein is or includes an array, biochip, slide, tray, dish (e.g., microtiter dish), phage, or phage particle.

[0108] Immunohistochemistry

[0109] In alternative embodiments, immunohistochemical methods and / or reagents used to practice the compositions, articles, kits, or methods provided herein may include or contain the following uses: any IHC protocol, IHC medical device, apparatus, and / or image or data analysis system used to practice IHC, or IHC reagents known in the art, such as those described in the following U.S. Patent Nos. (USPN): 10,565,479 (describing a method for identifying blurred regions in digital images of stained tissue); 10,564,076 (describing a system for preparing analytical (or IHC) samples); 10,551,395 (describing an automated histological staining 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 an immunohistochemical method for IHC). (Simultaneous multiplex detection and quantification of protein expression); 10,488,340 (Describes a method for extracting images of target fluorophores in biological materials); 10,453,195 (Describes a method for detecting target tissue regions using digital pathological imaging); 10,438,381 (Describes apparatus, systems, and methods for generating digital images of tissue sections); 10,416,176 (Describes a method for processing samples in an automated histological staining system); 10,393,633 (Describes a method for processing IHC samples and inhibiting their degradation); 10,217,011 (Describes the processing of IHC slides); 10,209,165 (Describes an automated or semi-automated method for assessing the staining quality of samples containing cells); 10,126,216 (Describes a method for fixing tissue samples for IHC); 9,423,322.

[0110] In alternative implementations, the chimeric or recombinant antibodies, their antigen-binding fragments, or monomeric or dimer antigen-binding proteins in the IHC protocols or kits provided herein are substantially purified or isolated, or are in the form of unpurified or partially purified culture supernatants.

[0111] In alternative implementations, the methods provided herein may use or include reagents for detecting or visualizing antibody-antigen interactions using, for example, any products or methods and IHC protocols or reagents known in the art.

[0112] In alternative embodiments, the methods provided herein include the use of chromogenic immunohistochemistry (CIH), wherein a primary antibody (e.g., a chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimer antigen-binding protein, as provided herein) or a secondary antibody (e.g., wherein the secondary antibody binds to, after the (primary antibody) chimeric or recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimer antigen-binding protein, as provided herein, specifically binds to, pairs with, associates with, or is configured with a LAG-3 epitope or polypeptide) is conjugated to an enzyme capable of catalyzing a chromogenic reaction, such as a peroxidase (or immunoperoxidase), for example, horseradish peroxidase (HRP).

[0113] In alternative embodiments, methods as provided herein include the use of immunofluorescence, wherein a primary or secondary antibody is tagged with a fluorophore, such as fluorescein or fluorescein isothiocyanate (FITC); a triarylmethane dye, such as rhodamine or a rhodamine derivative (e.g., tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulforhodamine 101); aminomethylcoumarin acetate (AMCA); or an ALEXA™ or DYLIGHT™ fluorescent agent. 3,3'-Diaminobenzidine (DAB) may also be used.

[0114] In alternative implementations, methods such as those provided herein include the use of direct or one-step staining methods, wherein a primary antibody (e.g., a chimeric or recombinant antibody (Ab) or its antigen-binding fragment or monomeric or dimer antigen-binding protein as provided herein) is labeled or reacted directly with an antigen, for example, in tissue sections. While this technique utilizes only one antibody and is therefore simple and rapid, its sensitivity may be low due to minimal signal amplification.

[0115] In alternative implementations, methods such as those provided herein include the use of an indirect method, wherein an unlabeled primary antibody (first layer) binds to a target antigen (ADAM9, or more specifically, ADAM9 isoform 1 in some cases), for example, in a tissue or organ, and then a labeled secondary antibody (second layer) reacts with the primary antibody. The secondary antibody may target an isotype of the animal species in which the primary antibody is derived (e.g., IgG). If the secondary antibody is conjugated to a detection agent (such as a fluorescent or enzyme reporter), the method may be more sensitive than a direct detection strategy due to signal amplification resulting from the binding of several secondary antibodies to each primary antibody.

[0116] In an alternative implementation, further amplification is achieved if the secondary antibody is conjugated to several detection molecules (e.g., biotin molecules) that are complexes capable of recruiting avidin-binding enzymes, streptavidin-binding enzymes, or NEUTRAVIDIN™ protein-binding enzymes.

[0117] In alternative embodiments, IHC is performed on tissue sections or tissue biopsies (e.g., paraformaldehyde (PFA)-fixed tissue or organs or formalin-fixed paraffin-embedded tissue). In alternative embodiments, the tissue is sliced ​​or used whole. Prior to slicing, the tissue sample may be embedded in a medium (e.g., paraffin or cryopreservation medium). Tissue sections can be sliced ​​on a variety of instruments, most commonly a microtome, cryostat, or vibratory microtome. Slices can be cut at approximately 3µm to 5µm. μm The sample is cut into thin slices. The slices can be placed on a glass slide, dehydrated using alcohol washes of progressively increasing concentrations (e.g., 50%, 75%, 90%, 95%, 100%), clarified using a detergent such as xylene, and then imaged under a microscope.

[0118] Depending on the fixation and tissue preservation methods, samples may require additional steps to make the ADAM9 epitope available for antibody binding. These additional steps include deparaffinization and antigen retrieval. For formalin-fixed paraffin-embedded tissues, antigen retrieval is often necessary and may include pretreatment of sections with heat or proteases.

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

[0120] How to use

[0121] In some respects, the ADAM9 antibody (or its antigen-binding fragment) described herein, as well as kits and products containing it, can be used for clinical or diagnostic applications. For example, the ADAM9 antibody or its antigen-binding fragment can be used to detect ADAM9 isoform 1 via in vitro IHC assay to determine whether a subject has cancer or other diseases. Potential applications include, for example, using it as a companion diagnostic to identify patients who may benefit from ADAM9-targeted antibody-drug conjugate (ADC) therapy (e.g., provided by ImmunoGen Inc.). Potential indications include, but are not limited to, NSCLC, pancreatic cancer, TNBC, and GC. Example

[0122] Unless otherwise stated in the examples, all recombinant DNA techniques were performed according to standard protocols, such as those described in Sambrook et al. (2012) *Molecular Cloning: A Laboratory Manual*, 4th edition, Cold Spring Harbor Laboratory Press, NY, and Ausubel et al. (1994) *Current Protocols in Molecular Biology*, *Current Protocols*, USA, Volumes 1 and 2. Other references to standard molecular biology techniques include Sambrook and Russell (2001) *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Laboratory Press, NY; and Brown (1998) *Molecular Biology LabFax*, 2nd edition, Volumes I and II, Academic Press (UK). Standard materials and methods for polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press and McPherson et 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 embodiment describes the development of two exemplary rabbit anti-human ADAM9 antibodies. Five antigens were selected for the study, including one antigen containing recombinant ADAM9 protein and four antigens containing ADAM9 peptide. Each antigen was used to immunize four rabbits.

[0125] Post-immunohemorrhage assays showed that one of the four ADAM9 peptides and recombinant ADAM9 exhibited good specificity for ADAM9 in IHC of pancreatic adenocarcinoma tissue. All five antigens were engineered with ADAM9 isoform 1-specific sequences, but these sequences did not fall within the sequence range shared with ADAM9 isoform 2. Based on this initial screening, four inoculated rabbits demonstrating a strong immune response to ADAM9 were selected for B-cell selection.

[0126] Several B-cell clones producing antibodies that exhibit ADAM9-specific staining in IHC were identified. These B-cell clones produced functional antibody heavy and light chains, which were used for transient transfection in HEK cells, and the reactivity of the resulting antibodies against the ADAM9 antigen was tested in biolayer interferometry z. These antibodies were further tested by IHC on clinically relevant tissue samples. Two clones (11H9 and 11C9) showed promising staining in pancreatic adenocarcinoma and other tissues. Staining obtained with antibodies produced from clones 11H9 and 11C9 was similar to or superior to staining obtained with the ADAM9 reference antibody. Furthermore, staining obtained with the antibody from clone 11H9 showed similar performance at both high and low TRS pH. Very distinct membrane staining was observed in pancreatic adenocarcinoma and other tissues at both high and low TRS pH. Figures 1-23 This illustrates the staining of various tissues using antibodies produced via clone 11H9. Clone 11C9 was produced using a similar method, and images showing the use of this antibody as a staining agent for various tissues are provided. Figures 24-31 Provided. Exemplary IHC assays using these antibodies as staining agents for various tissue types are summarized in Table 1 below. sequence list

Claims

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

10.

2. The binding agent according to claim 1, wherein the binding agent comprises a chimeric or recombinant antibody ("Ab"), an antigen-binding fragment thereof, or a monomeric or dimer antigen-binding protein.

3. The binder according to claim 1 or 2, wherein: a) The heavy chain variable structural domain includes Having a polypeptide sequence represented by SEQ ID NO: 3 or a CDR1 region having at least 90% sequence identity with SEQ ID NO: 3, Having a polypeptide sequence represented by SEQ ID NO: 4 or a CDR2 region having at least 90% sequence identity with SEQ ID NO: 4, and / or Having a polypeptide sequence represented by SEQ ID NO: 5 or a CDR3 region having at least 90% sequence identity with SEQ ID NO: 5; and / or b) The light chain variable structural domain includes Having a polypeptide sequence represented by SEQ ID NO: 6 or a CDR1 region having at least 90% sequence identity with SEQ ID NO: 6, Having a polypeptide sequence represented by SEQ ID NO: 7 or a CDR2 region having at least 90% sequence identity with SEQ ID NO: 7, and / or It has a polypeptide sequence represented by SEQ ID NO: 8 or a CDR3 region having at least 90% sequence identity with SEQ ID NO:

8.

4. The binder according to any one of claims 1-3, wherein: a) The heavy chain variable structural domain includes The CDR1 region contains the polypeptide sequence represented by SEQ ID NO:

3. Having a CDR2 region containing the polypeptide sequence represented by SEQ ID NO: 4, and / or Having a CDR3 region containing the polypeptide sequence represented by SEQ ID NO: 5; and / or b) The light chain variable structural domain includes The CDR1 region contains the polypeptide sequence represented by SEQ ID NO:

6. Having a CDR2 region containing the polypeptide sequence represented by SEQ ID NO: 7, and / or It has a CDR3 region containing the polypeptide sequence represented by SEQ ID NO:

8.

5. The binder according to any one of claims 1-4, wherein: The heavy chain variable structural domain includes SEQ ID NO: 9; and / or The light chain variable structural domain includes SEQ ID NO:

10.

6. The binder according to any one of claims 1-5, wherein: The heavy chain variable structural domain includes SEQ ID NO: 9; and The light chain variable structural domain includes SEQ ID NO:

10.

7. The binder according to claim 1 or 2, wherein: The heavy chain variable domain comprises a sequence or fragment thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions compared to SEQ ID NO: 9; and / or The light chain variable structural domain contains a sequence or fragment thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions compared to SEQ ID NO:

10.

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

8.

9. The binder according to any one of claims 1-8, wherein the binder comprises: a) Antigen-binding fragment ("F") ab (”); b) Fab' fragment; c) F(ab′)2 segment; d) F(ab′)2 fragment and pFc' fragment; e) Single-chained variable fragments ("scFv"); f) (scFv)2, or two scFv or double scFv; g) Microantibodies; h) Bivalent, trivalent, or tetravalent antibodies; i) Single-domain antibodies ("dAb"); and / or j) Multiple CDR fragments, or multispecific antibodies formed by two or more antibody fragments.

10. The binder according to 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 conserved amino acid substitutions, wherein the heavy chain variable domain is capable of specifically binding to the human ADAM9 isomorph 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 conserved amino acid substitutions, wherein the light chain variable domain is capable of specifically binding to the human ADAM9 isomorph 1 polypeptide. c) The sequence of the heavy chain variable structural 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 variable structural domain of the light chain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 10; e) When compared using distance matrix alignment, the heavy chain variable structural domain and SEQ ID NO: 9 have a Z score of about 2 to about 8, or a Z score of at least 8; and / or f) When compared using distance matrix alignment, the heavy chain variable structural domain and SEQ ID NO: 10 have Z scores of about 2 to about 8, or at least 8.

11. The binder according to any one of claims 1-10, wherein the binder comprises IgM, IgG, IgA or IgE isotype antibody heavy chains, and / or κ or λ isotype antibody light chain.

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

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

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

23.

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

16. The binder according to claim 14 or 15, wherein: a) The heavy chain variable structural domain includes Having a polypeptide sequence represented by SEQ ID NO: 16 or a CDR1 region having at least 90% sequence identity with SEQ ID NO: 16, Having a polypeptide sequence represented by SEQ ID NO: 17 or a CDR2 region having at least 90% sequence identity with SEQ ID NO: 17, and / or Having a polypeptide sequence represented by SEQ ID NO: 18 or a CDR3 region having at least 90% sequence identity with SEQ ID NO: 18; and / or b) The light chain variable structural domain includes Having a polypeptide sequence represented by SEQ ID NO: 19 or a CDR1 region having at least 90% sequence identity with SEQ ID NO: 19, Having a polypeptide sequence represented by SEQ ID NO: 20 or a CDR2 region having at least 90% sequence identity with SEQ ID NO: 20, and / or It has a polypeptide sequence represented by SEQ ID NO: 21 or a CDR3 region having at least 90% sequence identity with SEQ ID NO:

21.

17. The binder according to any one of claims 14-16, wherein: a) The heavy chain variable structural domain includes It has a CDR1 region containing the polypeptide sequence represented by SEQ ID NO:

16. Having a CDR2 region containing the polypeptide sequence represented by SEQ ID NO: 17, and / or Having a CDR3 region of the polypeptide sequence represented by SEQ ID NO: 18; and / or b) The light chain variable structural domain includes It has a CDR1 region containing the polypeptide sequence represented by SEQ ID NO:

19. Having a CDR2 region of the polypeptide sequence represented by SEQ ID NO: 20, and / or It has a CDR3 region containing the polypeptide sequence represented by SEQ ID NO:

21.

18. The binder according to any one of claims 14-17, wherein: The heavy chain variable structural domain includes SEQ ID NO: 22; and / or The light chain variable structural domain includes SEQ ID NO:

23.

19. The binder according to any one of claims 14-18, wherein: The heavy chain variable structural domain includes SEQ ID NO: 22; and The light chain variable structural domain includes SEQ ID NO:

23.

20. The binder according to claim 14 or 15, wherein: The heavy chain variable domain comprises a sequence or fragment thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions compared to SEQ ID NO: 22; and / or The light chain variable structural domain contains a sequence or fragment thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions compared to SEQ ID NO:

23.

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

21.

22. The binder according to any one of claims 14-21, wherein the binder comprises: a) Antigen-binding fragment ("F") ab (”); b) Fab' fragment; c) F(ab′)2 segment; d) F(ab′)2 fragment and pFc' fragment; e) Single-chained variable fragments ("scFv"); f) (scFv)2, or two scFv or double scFv; g) Microantibodies; h) Bivalent, trivalent, or tetravalent antibodies; i) Single-domain antibodies ("dAb"); and / or j) Multiple CDR fragments, or multispecific antibodies formed by two or more antibody fragments.

23. The binder according to 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 conserved amino acid substitutions, wherein the heavy chain variable domain is capable of specifically binding to the human ADAM9 isomorph 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 conserved amino acid substitutions, wherein the light chain variable domain is capable of specifically binding to the human ADAM9 isomorph 1 polypeptide. c) The sequence of the heavy chain variable structural 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 variable structural domain of the light chain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 23; e) When compared using distance matrix alignment, the heavy chain variable structural domain and SEQ ID NO: 22 have Z scores of about 2 to about 8, or at least 8; and / or f) When compared using distance matrix alignment, the heavy chain variable structural domain and SEQ ID NO: 23 have Z scores of about 2 to about 8, or at least 8.

24. The binder according to any one of claims 14-23, wherein the binder comprises IgM, IgG, IgA or IgE isotype antibody heavy chains, and / or κ or λ isotype antibody light chain.

25. The binder according to any one of claims 14-24, wherein the binder does not specifically bind to the ADAM9 isomorph 2 polypeptide.

26. The binder according to any one of claims 14-25, wherein the binder is a chimeric or recombinant Ab, the chimeric or recombinant Ab comprising: A heavy chain having a polypeptide sequence represented by SEQ ID NO: 22, the heavy chain being operatively linked, paired, associated, or configured together 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 isomorph 1 polypeptide.

27. A binder capable of specifically binding to the human ADAM9 isoform 1 polypeptide, said binder comprising: Heavy chain variable structural domain, the heavy chain variable structural domain comprising a sequence having at least 90% sequence identity with SEQ ID NO: 9 or 22 or a fragment thereof; and / or A light chain variable structural domain comprising a sequence having at least 90% sequence identity with SEQ ID NO: 10 or 23, or fragments thereof.

28. The binder according to claim 27, wherein: a) The heavy chain variable structural domain includes Having a polypeptide sequence represented by SEQ ID NO: 3 or 16, or a CDR1 region having at least 90% sequence identity with SEQ ID NO: 3 or 16, Having a polypeptide sequence represented by SEQ ID NO: 4 or 17 or a CDR2 region having at least 90% sequence identity with SEQ ID NO: 4 or 17, and / or Having a polypeptide sequence represented by SEQ ID NO: 5 or 18 or a CDR3 region having at least 90% sequence identity with SEQ ID NO: 5 or 18; and / or b) The light chain variable structural domain includes Having a polypeptide sequence represented by SEQ ID NO: 6 or 19, or a CDR1 region having at least 90% sequence identity with SEQ ID NO: 6 or 19, Having a polypeptide sequence represented by SEQ ID NO: 7 or 20 or a CDR2 region having at least 90% sequence identity with SEQ ID NO: 7 or 20, and / or It has a polypeptide sequence represented by SEQ ID NO: 8 or 21 or a CDR3 region having at least 90% sequence identity with SEQ ID NO: 8 or 21.

29. The binder according to claim 27, wherein: a) The heavy chain variable structural domain includes The CDR1 region contains the polypeptide sequence represented by SEQ ID NO: 3 or 16. Having a CDR2 region containing the polypeptide sequence represented by SEQ ID NO: 4 or 17, and / or Having a CDR3 region containing the polypeptide sequence shown in SEQ ID NO: 5 or 18; and / or b) The light chain variable structural domain includes The CDR1 region contains the polypeptide sequence represented by SEQ ID NO: 6 or 19. Having a CDR2 region containing a polypeptide sequence represented by SEQ ID NO: 7 or 20, and / or It has a CDR3 region containing a polypeptide sequence represented by SEQ ID NO: 8 or 21.

30. The binder according to claim 13 or 26, wherein the binder further comprises a detectable agent or binding portion. The detectable agent optionally includes: Enzymes, biotin, fluorescent or chemiluminescent labels, fluorophores, anthocyanins or sulfonylindocyanines, Nile red, rhodamine, perylene, fluorene, coumarin, 7-methoxycoumarin ("Mca"), dabcyl, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium ("NBD"), Nile blue, Tamra or tetramethylrhodamine ("TMR"), HRP MAGENTA™ chromogen (Dako Omnis, Agilent), boron-dipyrrole methylene ("BODIPY"), dyes, radioactive isotopes, quantum dots or photoluminescent aqueous nanocrystals, haptens or antibody-binding epitopes or domains; and Optionally, the enzyme is a peroxidase, alkaline phosphatase, or β-galactosidase; Optionally, the peroxidase is horseradish peroxidase ("HRP"). Optionally, the hapten includes biotin, theophylline, digoxin, carborane, fluorescein, or bromodeoxyuridine; Optionally, the dye includes cyan dye; or Cy3 or Cy5; Optionally, the fluorophore includes dansyl, fluorescein, or carboxyfluorescein ("FAM") or 6-FAM; and Optionally, the binding portion includes: a glutathione S-transferase ("GST") or ligand tag, a polyhistidine (polyhis) 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 a binding agent according to any one of claims 1-30.

32. A method for detecting the presence of human ADAM9 isoform 1 protein in or on cells, tissues, or organs or parts thereof, the method comprising: a) Contacting the cells, tissues, or organs or portions thereof of any of the foregoing claims with the binder according to any one of claims 1-30, and b) Detecting the specific binding of the binding agent to the human ADAM9 isoform 1 polypeptide in the cells, tissues, organs, or parts thereof; and c) Detect the presence of human ADAM9 isoform 1 protein in the cells, tissues, or organs or parts thereof based on the specific binding detection in step b).

33. The method of claim 32, wherein the contact step (a) is performed using immunohistochemistry ("IHC") assay.

34. The method of claim 32 or 33, wherein the method further comprises contacting the binder with a detection agent, the detection agent being capable of emitting a signal indicating that the binder specifically binds to the human ADAM9 isoform 1 protein.

35. The method of claim 34, wherein the detection agent comprises Antibodies, antigen-binding fragments, or secondary antibodies that specifically bind to the binder; Optionally, the detection agent includes an antibody, antigen-binding fragment, or secondary antibody that specifically binds to a hapten or tag attached to or conjugated to the binder.

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

37. The method of claim 36, wherein the enzyme is alkaline phosphatase, β-galactosidase, or peroxidase.

38. The method of claim 36 or 37, wherein the antibody, the antigen-binding fragment, or the secondary antibody further comprises, is attached to, or is conjugated thereto: Biotin, fluorescent or chemiluminescent labels, fluorophores, anthocyanins or sulfonylindocyanines, Nile red, rhodamine, perylene, fluorene, coumarin, Mca, dabcyl, NBD, Nile blue, Tamra or TMR, BODIPY, HRP MAGENTA™ chromogen (DakoOmnis, Agilent), dyes, radioactive isotopes, quantum dots, photoluminescent aqueous nanocrystals and / or haptens.

39. The method of claim 38, wherein The dyes include cyanine dyes, or Cy3 or Cy5; and / or The hapten includes biotin, theophylline, digoxin, carborane, fluorescein, or bromodeoxyuridine.

40. A method for detecting cancer expressing ADAM9 isoform 1 protein in a subject, the method comprising: a) Contacting a sample of cells, tissues or organs or a portion thereof obtained from the subject with the binder according to any one of claims 1-30; as well as b) Detect whether the binding agent specifically binds to the human ADAM9 isoform 1 protein in the cell, tissue, or organ sample or a portion thereof, wherein the detection of specific binding indicates the expression or presence of the human ADAM9 isoform 1 protein in the cell, tissue, or organ sample or a portion thereof.

41. The method of claim 40, further comprising: c) When the binding agent is found to specifically bind to the human ADAM9 isoform 1 protein in the cell, tissue, or organ sample or a portion thereof in step b), the subject is diagnosed with or may have a cancer expressing the protein.

42. The method of claim 40 or 41, wherein the cancer expressing ADAM9 isoform 1 protein comprises: Non-small cell lung cancer ("NSCLC"), pancreatic cancer, triple-negative breast cancer ("TNBC"), gastroesophageal cancer ("GC"), prostate cancer, or kidney cancer.

43. The method according to any one of claims 40-42, wherein step b of the detection is performed using immunohistochemistry ("IHC") or 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 according to any one of claims 40-44, wherein the cell, tissue, or organ sample or a portion thereof is obtained from a biopsy of the subject.

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