Antibodies specific to scube3 and methods of use

Abstract

Disclosed herein are antibodies that bind to SCUBE3 useful in treating cancer, and treating or preventing metastatic cancer in a subject.

Description

[0001] ANTIBODIES SPECIFIC TO SCUBE3 AND METHODS OF USE

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 593,055, filed on October 25, 2023. The content of this earlier filed application is hereby incorporated by reference in its entirety.

[0004] REFERENCE TO A SEQUENCE LISTING

[0005] The Sequence Listing submitted herein as a text file named “21105_0086Pl_SL.xml,’' created on September 13, 2024, and having a size of 24.576 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0006] BACKGROUND

[0007] Triple negative breast cancers (TNBCs) represent aggressive malignancy with no targeted therapies. Despite a positive chemotherapy response rate in early-stages. more than 60% of patients with TNBCs develop chemoresistance, leading to early relapse and shorter survival (Gluz, O., et al., Ann Oncol, 2009. 20(12): p. 1913-27). Moreover, the quality of life for patients who survive is often substantially reduced due to the toxicity associated with the chemotherapy. Thus, new therapeutic targets that more efficacious and less toxic treatments for TNBC patients are needed.

[0008] SUMMARY

[0009] Disclosed herein are isolated antibodies comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a complementarity determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 1 ; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 2; and a complementarity determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 3; and wherein the heavy chain variable region comprises a complementarity determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 5; a complementarity determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 6; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 7.

[0010] Disclosed herein are isolated antibodies comprising a light chain variable region amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region amino acid sequence of SEQ ID NO: 8.

[0011] Disclosed herein are isolated antibodies comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a complementarity determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 9; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 10; and a complementarity determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 11; and wherein the heavy chain variable region comprises a complementarity determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 13; a complementarity7determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 14; and a complementarity7determining region heavy7chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 15.

[0012] Disclosed herein are isolated antibodies comprising a light chain variable region amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region amino acid sequence of SEQ ID NO: 16.

[0013] Disclosed herein are isolated antibodies comprising a light chain variable region and a heavy7chain variable region, wherein the light chain variable region comprises a complementarity determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 1; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 2; and a complementarity7determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 3; and wherein the heavy chain variable region comprises a complementarity determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 5; a complementarity determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 6; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 7, wherein one or more of the CDRL1. CDRL2, CDRL3, CDRH1, CDRH2. or CDRH3 comprise 1, 2, 3. 4, or 5 conservative amino acid substitutions.

[0014] Disclosed herein are isolated antibodies comprising a light chain variable region amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region amino acid sequence of SEQ ID NO: 8, wherein the isolated antibodies comprise 1, 2, 3, 4, or 5 conservative amino acid substitutions in the light or heavy chain variable region ammo acid sequences.

[0015] Disclosed herein are isolated antibodies comprising a light chain variable region and a heavy7chain variable region, wherein the light chain variable region comprises a complementarity determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 9; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 10; and a complementarity7determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 11; and wherein the heavy chain variable region comprises a complementarity7determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 13: a complementarity determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 14; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 15, wherein one or more of the CDRL1, CDRL2, CDRL3, CDREI1, CDRH2, or CDRH3 comprise 1, 2, 3, 4, or 5 conservative amino acid substitutions.

[0016] Disclosed herein are isolated antibodies comprising a light chain variable region amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region amino acid sequence of SEQ ID NO: 16, wherein the isolated antibodies comprise 1, 2, 3, 4, or 5 conservative amino acid substitutions in the light or heavy chain variable region amino acid sequences.

[0017] Disclosed herein are methods of reducing tumor volume in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more of the isolated antibodies or compositions disclosed herein.

[0018] Disclosed herein are methods of treating or reducing metastatic cancer in a subject or preventing metastasis in a subject having cancer at risk for metastasis, the method comprising administering to the subject a therapeutically effective amount of one or more of the isolated antibodies or compositions disclosed herein.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGS. 1A-I show Scube3 as a potent therapeutic adjuvant. FIG. 1A shows genome wide loss of function screen using Mission Lentiplex pooled shRNA library in the presence and absence of doxorubicin (DOX) in MDA-MB-231 cells. FIGS. IB and 1C show viability of MDA-MB-231 and MDA-MB-468 cells stably transfected with scrambled or SCUBE3 shRNA in the presence of increasing concentration of DOX. FIGS. ID and IE show time dependent growth inhibition curves of MDA-MB-231 and MDA-MB-468 cells upon SCUBE3 knockdown. FIG. IF depicts representative images showing reduction of long term cell viability, migration and invasion (Left) and its quantification (Right) upon SCUBE3 silencing. FIG. 1G depicts a graph showing tumor growth upon orthotropic injection of stably transfected MDA-MB-231 cells with scrambled or shSCUBE3. FIG. 1H depicts a graph showing differences in tumor growth of mammary fat pad tumors formed by injection of MDA-MB-231 cells stably transfected with scrambled or SCUBE3 overexpression (O / E) plasmid (0.25xl06cells). FIG. II depicts a graph showing tumor growth in MDA-MB-231 cells stably transfected with shscrambled or shSCUBE3 treated with or without DOX. For scrambled (solid line) and scrambled + DOX (2.5 mg / kg body weight) (squares with dotted line), animals were sacrificed at day 29 as control tumors were too big and DOX-treated mice were showing toxicity. DOX treatment (1 mg / kg bw) for shSCUBE3 group (circles with dotted line) was started when tumors reached 150-200 mm3.

[0021] FIGS. 2A-F show that SCUBE3 binds and activates EGFR / TGFBR signaling. FIG. 2A show immunoprecipitation (IP) using SCUBE3 antibody followed by Western blot using EGFR antibody in MDA-MB-231 and MDA-MB-468 cells showing interaction of SCUBE3 with EGFR. FIG. 2B shows IP using SCUBE3, EGFR1, TGFpRII and TGFpRI antibodies followed by Western blot using SCUBE3 and EGFR1 antibodies in MDA-MB-231 and MDA-MB-468 cells. FIG. 2C shows the results of a proximity' ligation assay with MDA-MB- 231 cells co-transfected with GFP-EGFR and Myc-EGFR constructs followed by treatment with PBS (control), erlotinib, EGF or recombinant SCUBE3. The bright signal indicates close proximity of PLA probes indicating dimerization of EGFR. FIGS. 2D-E show Western blot analysis on stably transfected TNBC cells with scrambled or SCUBE3 shRNA using antibodies against indicated protein. FIG. 2F shows Western blot showing the expression of indicated proteins upon SCUBE3 silencing (left) or SCUBE3 overexpression (right) in MDA- MB-231 and MDA-MB-468 cells.

[0022] FIGS. 3A-D show that SCUBE3 regulates expression of pro-growth / survival signals. FIG. 3A shoyvs IPA analysis showing specific up and down regulated pathways in MDA- MB-231 SCUBE3 KD cells compared to scrambled transfected cells. FIGS. 3B-C show gene enrichment score (Left) and western blots (right) of genes and proteins associated with (FIG. 3B) DNA repair and (FIG. 3C) Myc in MDA-MB-231 and MDA-MB-468 scrambled and SCUBE3 knockdoyvn cells. FIG. 3D show gene enrichment score (left) of G2M checkpoint genes and cell cycle analysis (right) in MDA-MB-231 and MDA-MB-468 scrambled and SCUBE3 knockdown cells.

[0023] FIGS. 4A-D show hybridoma culture to generate monoclonal anti-SCUBE3 antibody secreting clones. FIG. 4A is a schematic illustrating methods used to generate antibody secreting hybridomas. In brief, mice were immunized with human SCUBE3 protein. The mouse serum was tested using ELISA to detect antibodies against SCUBE3. B cells producing antibodies against SCUBE3 were fused with myeloma cells to form hybndomas. Hybridomas were tested for the presence of antibodies against SCUBE3 using ELISA and seeded to form single cell hybridoma clones. Antibodies collected from subcloned hybridomas were functionally tested using short-term and long-term viability assays in breast cancer cell lines. Top three clones were selected for next steps including sequencing to determine CDR antibody sequence. The absorbance values were obtained from 500 ng of IG10.3 and 9G5.12 antibodies. FIG. 4B shows long-term colony formation in MDA-MB-231 and MDA-MB-468 breast cancer cell lines, cells were treated with SCUBE3 commercial antibody, IgG control antibody and top ten hybridoma clone supernatants. FIG. 4C shows ELISA results showing the absorbance values of the top three hybridoma clones compared with a SCUBE3 commercial antibody as a positive control, and non-immunized mouse serum as a negative control. FIG. 4D shows representative images of long term colony formation (left) and its quantification (right) of different breast cancer cell lines after treatment with supernatant of expanded cultures from clone IG10.3, 7F2.4 and 9G5.12.

[0024] FIGS. 5A-J show purification of anti-SCUBE3 antibody from mouse ascites or HEK culture and its therapeutic efficacy in-vivo. FIG. 5A shows long-term colony formation in MDA-MB-231. Cells were treated in duplicate with IgG control, and anti-SCUBE3 antibodies purified from ascites fluid. FIG. 5B shows Western blot validating anti-SCUBE3 antibodies purified from ascites fluid can recognize human SCUBE3 protein present in MDA-MB-231 cell culture media. FIG. 5C shows Western blot showing downregulation of phospho-ERK when MDA-MB-231 cells are treated with antibody against SCUBE3. FIG. 5D shows anti-tumor effect of 9G.5 clone. Athymic NU / J mice were orthotopically injected with l*106MDA-MB-231 cells to form tumors. When tumor reached -100 mm3volume the mice were randomized and treated with either saline or 12 pg / mouse 9G5. 12 anti-SCUBE3 antibody intratumorally twice a week. FIG. 5E depicts a graph showing tumor growth curve for 28 days for experimental setup in FIG. 5D. FIG. 5F show bar graphs showing tumor volume in control and 9G.5 treated tumors. FIG. 5G depicts an image showing immunohistochemistry staining of mammary tumor with Ki67 (left) and number of Ki67+cells (right) from the tumors harvested at the experimental endpoint of FIG. 5E. FIG. 5H shows monoclonal IgG anti-SCUBE3 antibody that was purified from IL HEK cell culture and was used to treat MDA-MB-231 cells at various concentrations. Graphs shows comparison of growth curve and IC50 values of erlotinib (left), 9G5.12 and IG10.3 anti- SCUBE3 antibody (right) treatment on MDA-MB-231 cells for 72 hrs. FIG. 51 depicts a graph showing difference in tumor volume of mammary tumors of NU / J mice injected intraperitoneally with either saline, 9G5.12 (100 pg / mouse) or IG10.3 (150 pg / mouse) antibodies once a w eek. FIG. 5J shows line graphs depicting tumor grow th in NOD-SCID mice implanted with tumor derived from a TNBC patient treated with IgG (control) or 9G5.12 (i.p; 200 pg / mouse twice a week) (n=4).

[0025] DETAILED DESCRIPTION

[0026] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0027] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0028] Disclosed are matenals, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. If a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D. B-E. B-F, C- D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0029] DEFINITIONS

[0030] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0031] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.

[0032] “Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0033] The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

[0034] As used herein, the term “another” means at least a second or more.

[0035] Ranges can be expressed herein as from “about” or “approximately” one particular value, and / or to “about” or “approximately” another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about.” or “approximately,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0036] Throughout the description and claims of this specification, the word “comprise’' and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.

[0037] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0038] “Inhibit,” “inhibiting” and “inhibition” mean to diminish or decrease an activity’, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% inhibition or reduction in the activity7, response, condition, or disease as compared to the native or control level. Thus, in some aspects, the inhibition or reduction can be a 10, 20, 30, 40, 50, 60, 70, 80. 90. 100%, or any amount of reduction in between as compared to native or control levels. In some aspects, the inhibition or reduction is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared to native or control levels. In some aspects, the inhibition or reduction is 0-25, 25-50, 50-75, or 75- 100% as compared to native or control levels.

[0039] “Modulate”, “modulating” and “modulation” as used herein mean a change in activity or function or number. The change may be an increase or a decrease, an enhancement or an inhibition of the activity, function or number.

[0040] “Promote,” “promotion,” and “promoting” refer to an increase in an activity7, response, condition, disease, or other biological parameter. This can include but is not limited to the initiation of the activity, response, condition, or disease. This may also include, for example, a 10% increase in the activity7, response, condition, or disease as compared to the native or control level. Thus, in some aspects, the increase or promotion can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or more, or any amount of promotion in between compared to native or control levels. In some aspects, the increase or promotion is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared to native or control levels. In some aspects, the increase or promotion is 0-25, 25-50, 50-75, or 75-100%, or more, such as 200, 300, 500, or 1000% more as compared to native or control levels. In some aspects, the increase or promotion can be greater than 100 percent as compared to native or control levels, such as 100, 150, 200, 250, 300, 350, 400. 450, 500% or more as compared to the native or control levels.

[0041] As used herein, the term '‘treat,” “treatment,” or “treating” refers to administration or application of a therapeutic agent (e.g. one or more of the disclosed antibodies) to a subject in need thereof, or performance of a procedure or modality on a subject, for the purpose of obtaining at least one positive therapeutic effect or benefit, such as treating a disease or health-related condition. For example, a treatment can include administration of a pharmaceutically effective amount of an antibody, or a composition or formulation thereof that specifically binds to Scube3 for the purpose of treating various cancers.

[0042] As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting or slowing progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. For example, the disease, disorder, and / or condition can be a cancer or metastatic cancer.

[0043] As used herein, the term “subject” refers to the target of administration, e g., a human. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term '‘subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In some aspects, a subject is a mammal. In some aspects, a subject is a human. The term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. As used herein, the term “subject” refers to either a human or a non-human animal, such as primates, mammals, and vertebrates having a cancer. In some aspects, the subject in need will or is predicted to benefit from treatment with one or more of the disclosed antibodies.

[0044] As used herein, the term '‘patient” refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects. In some aspects of the disclosed methods, the “patient” has been diagnosed with a need for treatment for a cancer, such as, for example, prior to the administering step.

[0045] The term “isolated” can refer to a nucleic acid, polypeptide or antibody that is substantially free of cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (when chemically synthesized). Moreover, an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel. Moreover, an “isolated nucleic acid fragment” or “isolated peptide” or “isolated antibody” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and / or is not typically in the functional state.

[0046] The term “providing” is used according to its ordinary meaning “to supply or furnish for use.” In some embodiments, the isolated antibody is provided directly by administering the isolated antibody, while in other embodiments, the isolated antibody is effectively provided by administering a nucleic acid that encodes the isolated antibody. In certain aspects the invention contemplates compositions comprising various combinations of antibodies, nucleic acid, antigens, peptides, and / or epitopes.

[0047] As used herein, the term “sample” is meant a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), which is assayed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.

[0048] As used herein, the term “determining” can refer to measuring or ascertaining a quantity or an amount or a change in activity’. For example, determining the amount of a disclosed polypeptide, protein, gene or antibody in a sample as used herein can refer to the steps that the skilled person would take to measure or ascertain some quantifiable value of the polypeptide protein, gene or antibody in the sample. The art is familiar with the ways to measure an amount of the disclosed polypeptide, proteins, genes or antibodies in a sample.

[0049] As used herein, the terms “disease” or “disorder” or “condition” are used interchangeably referring to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and / or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease or disorder or condition can also related to a distemper, ailing, ailment, disorder, sickness, illness, complaint, affection. In some aspects, the disease or disorder or condition can be a cancer or metastatic cancer. In some aspects, the cancer can be breast cancer, brain cancer, colorectal cancer, lung cancer, skin cancer (e.g., melanoma), thyroid cancer, uterine cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancers, pancreatic cancer and stomach cancer.

[0050] As used herein, the term “SCUBE3” refers to a polypeptide (the terms “polypeptide’' and “protein” are used interchangeably herein) or any native SCUBE3 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkey (cyno)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated, and, in certain aspects, included various SCUBE3 isoforms, related SCUBE3 polypeptides, including SNP variants thereof. An exemplary amino acid sequence of human SCUBE3 is accession number NP_689966.2.

[0051] Abbreviations for the amino acid residues that comprise polypeptides and peptides described herein, and conservative substitutions for these amino acid residues are shown in Table 1 below. A polypeptide that contains one or more conservative amino acid substitutions or a conservatively modified variant of a polypeptide described herein refers to a polypeptide in which the original or naturally occurring amino acids are substituted with other amino acids having similar characteristics, for example, similar charge, hydrophobicity / hydrophilicity, side-chain size, backbone conformation, structure and rigidity, etc. Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity’ or its specificity for antigen. In general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity. Table 1. Amino Acid Residues and Examples of Conservative Amino Acid

[0052] Substitutions

[0053] As used herein, the term “polypeptide” or “peptide” refers to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds. As used herein, the term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. “Polypeptides” can be proteins, protein fragments, protein analogs, oligopeptides and the like. The amino acids that comprise the polypeptide may be naturally derived or synthetic. The polypeptide may be purified from a biological sample. For example, a SCUBE3 polypeptide or peptide may be composed of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous amino acids of human SCUBE3. In some aspects, the polypeptide has at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145. 150, 155, 160, 165. 170, 175, 180, 185, 190. 195, 200. 205, 210, 215, 220, 225. 230, 235, 240, 245,

[0054] 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335,

[0055] 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425,

[0056] 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515,

[0057] 520, 525. 530, 535, 540, 545, 550, 555, 560. 565, 570, 575, 580, 585, 590, 595, 600, 605,

[0058] 610, 615. 620, 625, 630, 635, 640. 645, 650. 655, 660, 665. 670, 675. 680, 685, 690, 695,

[0059] 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785,

[0060] 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875,

[0061] 880, 885, 890, 895, 900, 905, 910, 915, 920. 925, 930, 935, 940, 945, 950, 955, 960, 965,

[0062] 970, 975. 980, 985, 990, or 993, contiguous amino acids of human SCUBE3. In some aspects, the SCUBE3 polypeptide comprises at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least contiguous 100 amino acid residues, at least 150 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 250 contiguous amino acid residues, at least 300 contiguous amino acid residues, at least 350 contiguous amino acid residues, at least 400 contiguous amino acid residues, at least 450 contiguous amino acid residues, at least 500 contiguous amino acid residues, at least 550 contiguous amino acid residues, at least 600 contiguous amino acid residues, at least 650 contiguous amino acid residues, at least 700 contiguous amino acid residues, at least 750 contiguous amino acid residues, at least 800 contiguous amino acid residues, at least 850 contiguous amino acid residues, at least 900 contiguous amino acid residues, at least 950 contiguous amino acid residues, at least 960 contiguous amino acid residues, at least 970 contiguous amino acid residues, at least 980 contiguous amino acid residues, or at least 990 contiguous amino acid residues of the amino acid sequence of the SCUBE3 polypeptide.

[0063] By "‘isolated polypeptide’’ or “purified polypeptide” is meant a polypeptide (or a fragment thereof) that is substantially free from the materials with which the polypeptide is normally associated in nature. The polypeptides of the invention, or fragments thereof, can be obtained, for example, by extraction from a natural source (for example, a mammalian cell), by expression of a recombinant nucleic acid encoding the polypeptide (for example, in a cell or in a cell-free translation system), or by chemically synthesizing the polypeptide. In addition, polypeptide fragments may be obtained by any of these methods, or by cleaving full length polypeptides.

[0064] As used herein, the term “analog” refers to a polypeptide that possesses a similar or identical function as a reference polypeptide but does not necessarily comprise a similar or identical amino acid sequence of the reference polypeptide, or possess a similar or identical structure of the reference polypeptide. The reference polypeptide may be a SCUBE3 polypeptide, a fragment of a SCUBE3 polypeptide, or an anti SCUBE3 antibody. A polypeptide that has a similar amino acid sequence with a reference polypeptide refers to a polypeptide having an amino acid sequence that is at least 30%. at least 35%. at least 40%. at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the reference polypeptide, which can be a SCUBE3 polypeptide or antibody as described herein. A polypeptide with similar structure to a reference polypeptide refers to a polypeptide that has a secondary, tertiary, or quaternary structure similar to that of the reference polypeptide, which can be a SCUBE3 polypeptide or an antibody described herein. The structure of a polypeptide can determined by methods know n to those skilled in the art, including, but not limited to, X-ray crystallography, nuclear magnetic resonance (NMR). and crystallographic electron microscopy.

[0065] The term “fragment” can refer to a portion (e.g., at least 5, 10, 25, 50, 100, 125, 150, 200, 250, 300, 350, 400 or 500, etc. amino acids or nucleic acids) of a protein or nucleic acid molecule that is substantially identical to a reference protein or nucleic acid and retains the biological activity of the reference. In some aspects, the fragment or portion retains at least 50%. 75%. 80%. 85%. 90%. 95% or 99% of the biological activity of the reference protein or nucleic acid described herein. Further, a fragment of a referenced peptide can be a continuous or contiguous portion of the referenced polypeptide (e.g., a fragment of a peptide that is ten amino acids long can be any 2-9 contiguous residues within that peptide).

[0066] As used herein, the term “variant” when used in relation to a SCUBE3 polypeptide or to an antibody refers to a polypeptide or an antibody having one or more amino acid sequence substitutions, deletions, and / or additions as compared to a native or unmodified SCUBE3 sequence or antibody sequence. For example, a SCUBE3 polypeptide or an antibody refers to a polypeptide or an antibody having one or more amino acid sequence substitutions, deletions, and / or additions as compared to a native or unmodified SCUBE3 sequence or antibody sequence can have about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5 amino acid sequence substitutions, deletions, and / or additions as compared to a native or unmodified SCUBE3 sequence or antibody sequence. A SCUBE3 variant can result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15. about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native SCUBE3. Also by way of example, a variant of an antibody can result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5 changes to an amino acid sequence of a native or previously unmodified antibody that binds to SCUBE3. Variants can be naturally occurring, such as allelic or splice variants, or can be artificially constructed. Polypeptide variants can be prepared from the corresponding nucleic acid molecules encoding the variants.

[0067] A “variant’' can mean a difference in some way from the reference sequence other than just a simple deletion of an N- and / or C-terminal amino acid residue or residues. Where the variant includes a substitution of an ammo acid residue, the substitution can be considered conservative or non-conservative. Conservative substitutions can include those within the following groups: Ser, Thr, and Cys; Leu, He, and Vai; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp; and Gin, Asn. Glu, Asp, and His. Variants can include at least one substitution and / or at least one addition, there may also be at least one deletion. Variants can also include one or more non-naturally occurring residues. For example, a variant may include selenocysteine (e.g., seleno-L- cysteine) at any position, including in the place of cysteine. Many other “unnatural” amino acid substitutes are known in the art and are available from commercial sources. Examples of non-naturally occurring amino acids include D-amino acids, amino acid residues having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, and omega amino acids of the formula NH2(CH2)nCOOH wherein n is 2-6 neutral, nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr. or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties of proline. A “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g., Met, Ala. Vai, Leu. and He), among residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn and Gin), among residues with acidic side chains (e.g., Asp, Glu), among amino acids with basic side chains (e.g., His, Lys, and Arg), or among residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.

[0068] The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) must be addressed by a particular mathematical model or computer program (e.g., an “algorithm”). Methods that may be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Lesk. A. M.. ed.. 1988, Computational Molecular Biology, New York: Oxford University Press; Smith, D. W., ed., 1993, Biocomputing Informatics and Genome Projects , New York: Academic Press; Griffin, A. M., et al., 1994, Computer Analysis of Sequence Data, Part I , New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology. New York: Academic Press; Gribskov, M. et al., 1991, Sequence Analysis Primer, New York: M. Stockton Press; and Carillo et al., 1988, Applied Math., 48:1073.

[0069] In calculating percent identity, the sequences being compared can be aligned in a way that gives the largest match between the sequences. An example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res., 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI), which is a computer algorithm used to align the two polypeptides or polynucleotides to determine their percent sequence identity. The sequences can be aligned for optimal matching of their respective amino acid or nucleotide sequences (the “matched span” as determined by the algorithm). A gap opening penalty (which is calculated as 3 times the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used, and the “diagonal’’ is the score or number assigned to each perfect amino acid match by the particular comparison matrix; and a gap extension penalty (which is usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62, are used in conjunction with the algorithm. In some aspects, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm. Exemplary parameters for determining percent identity for polypeptides or nucleotide sequences using the GAP program include the following: (i) Algorithm: Needleman et al., 1970, J. Mol. Biol., 48:443-453; (ii) Comparison matrix: BLOSUM 62 from Henikoff et al., Id.; (iii) Gap Penalty7: 12 (but with no penalty7for end gaps); (iv) Gap Length Penalty: 4; and (v) Threshold of Similarity: 0.

[0070] Certain alignment schemes for aligning two amino acid sequences can result in matching only a short region of the two sequences, and this small aligned region can have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (e.g., the GAP program) can be adjusted if so desired to result in an alignment that spans a representative number of amino acids, for example, at least 50 contiguous amino acids, of the target polypeptide.

[0071] Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that is identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity7, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity7can be achieved in various ways that are within the skill of the practitioner in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0072] As used herein, the term “derivative’’ refers to a polypeptide or antibody that comprises an amino acid sequence of a reference polypeptide or antibody that has been altered by the introduction of amino acid residue substitutions, deletions or additions. The reference polypeptide can be a SCUBE3 polypeptide or an antibody disclosed herein. The term "‘derivative” as used herein also refers to a SCUBE3 polypeptide or an antibody disclosed herein that has been chemically modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, a SCUBE3 polypeptide or an antibody disclosed herein can be chemically modified, e.g.. by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand, linkage to a peptide or protein tag molecule, or other protein, etc. The derivatives are modified in a manner that is different from the naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives may further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide. A derivative of a SCUBE3 polypeptide or an antibody disclosed herein may be chemically modified by chemical modifications using techniques known to those of skill in the art. including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis by tunicamycin, etc. Further, a derivative of a SCUBE3 polypeptide or an antibody disclosed herein can contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as the reference polypeptide, which can be a SCUBE3 polypeptide or an antibody described herein, especially a monoclonal antibody.

[0073] The term “fusion protein” as used herein refers to a polypeptide that includes amino acid sequences of at least two heterologous polypeptides. The term “fusion” when used in relation to a SCUBE3 polypeptide or to an antibody disclosed herein refers to the joining, fusing, or coupling of a SCUBE3 polypeptide or an antibody, variant and / or derivative thereof as disclosed herein, with a heterologous peptide or polypeptide. In some aspects, the fusion protein retains the biological activity of the SCUBE3 polypeptide or the antibody disclosed herein. In some aspects, the fusion protein includes an antibody VH region, VL region, VH CDR (one, two or three VH CDRs), and / or VL CDR (one, two or three VL CDRs) coupled, fused, or joined to a heterologous peptide or polypeptide, wherein the fusion protein binds to an epitope on a SCUBE3 protein or peptide. Fusion proteins may be prepared via chemical coupling reactions as practiced in the art, or via molecular recombinant technology.

[0074] As used herein, the term “composition” refers to a product containing specified component ingredients (e.g.. a polypeptide or an antibody disclosed herein) in, optionally, specified or effective amounts, as well as any desired product which results, directly or indirectly, from the combination or interaction of the specific component ingredients in, optionally, the specified or effective amounts.

[0075] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products.

[0076] The terms “antibody,” “immunoglobulin,” and “Ig” are used interchangeably herein in a broad sense and specifically cover, for example, individual antibodies, such as the antibodies described herein, (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies, peptide fragments of antibodies that maintain antigen binding activity); antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments of antibodies, as described herein. An antibody can be human, humanized, chimeric and / or affinity matured. An antibody may be from other species, for example, mouse, rat, rabbit, etc. The term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen. An antibody is ty pically composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa); and wherein the aminoterminal portion of the heavy' and light chains includes a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain includes a constant region (See, Borrebaeck (ed.), 1995, Antibody Engineering, Second Ed., Oxford University Press.; Kuby, 1997 Immunology, Third Ed., W.H. Freeman and Company, New York). In some aspects, the specific molecular antigen bound by an antibody provided herein includes a SCUBE3 polypeptide, a SCUBE3 peptide fragment, or a SCUBE3 epitope. An antibody or a peptide fragment thereof that binds to a SCUBE3 antigen can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art. An antibody or a fragment thereof binds specifically to a SCUBE3 antigen when it binds to a SCUBE3 antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassay s (RIA) and enzyme linked immunosorbent assays (ELISAs). Typically, a specific or selective binding reaction will be at least twice background signal or noise, and more typically more than 5-10 times background signal or noise. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion regarding antibody specificity.

[0077] Antibodies provided herein include, but are not limited to. synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments such as SCUBE3 binding fragments) of any of the above. A binding fragment refers to a portion of an antibody heavy or light chain polypeptide, such as a peptide portion, that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments. F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabodies. triabodies, tetrabodies and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen binding domains or molecules that contain an antigenbinding site that binds to a SCUBE3 antigen, (e.g., one or more complementarity determining regions (CDRs) of an antibody disclosed herein). Description of such antibody fragments can be found in, for example, Harlow and Lane, 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., 1993. Cell Biophysics, 22: 189-224; Pliickthun and Skerra, 1989, Meth. Enzymol., 178:497-515 and in Day, E.D., 1990, Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY. The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. Antibodies disclosed herein can be agonistic antibodies or antagonistic antibodies. In some aspects, the antibodies can be fully human, such as fully human monoclonal antibodies. In some aspects, the antibodies can be humanized, such as humanized monoclonal antibodies. In some aspects, the antibodies provided herein can be IgG antibodies, or a class (e.g., human IgGl or IgG4) or subclass thereof, in particular. IgGl subclass antibodies.

[0078] By “Fv” or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody. By "‘framework” as used herein is meant the region of an antibody variable domain exclusive of those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1, FR2, FR3 and FR4).

[0079] A four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the molecular weight of the four-chain (unreduced) antibody unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. At the N- terminus, each H chain has a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and e isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its carboxy terminus. The VL domain is aligned with the VH domain, and the CL domain is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site, although certain VH and VL domains can bind antigen without pairing with a VL or VH domain, respectively. The basic structure of immunoglobulin molecules is understood by those having skill in the art. For example, the structure and properties of the different classes of antibodies may be found in Terr. Abba I. et al., 1994, Basic and Clinical Immunology, 8th edition, Appleton & Lange, Norwalk. CT, page 71 and Chapter 6.

[0080] A “single-chain variable fragment (scFv)” means a protein comprising the variable regions of the heavy and light chains of an antibody. A scFv can be a fusion protein comprising a variable heavy chain, a linker, and a variable light chain. In some aspects, the linker can be a short, flexible fragment that can be about 8 to 20 amino acids in length. For example. (G4S)ncan be used (n=l, 2, 3 or 4).

[0081] A “fragment antigen-binding fragment (Fab)” or “Fab region” is a region of an antibody that binds to antigen. A Fab comprises constant and variable regions from both heavy and light chains. For example, Fab or Fab region as used herein is meant the polypeptide that comprises the VH, CHI, VL. and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody, antibody fragment or Fab fusion protein, or any other antibody embodiments as outlined herein. As used herein, the term '‘antigen” or ‘'target antigen” is a predetermined molecule to which an antibody can selectively bind. A target antigen can be a polypeptide, peptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some aspects, a target antigen can be a small molecule. In some aspects, the target antigen can a polypeptide or peptide, e.g., SCUBE3.

[0082] As used herein, the term '‘antigen binding fragment,” “antigen binding domain,” “antigen binding region." and similar terms refer to that portion of an antibody which includes the amino acid residues that interact with an antigen and confer on the antibody as binding agent its specificity and affinity for the antigen (e.g., the CDRs of an antibody are antigen binding regions). The antigen binding region can be derived from any animal species, such as rodents (e.g., rabbit, rat, or hamster) and humans. In some aspects, the antigen binding region can be of human origin.

[0083] An “isolated” antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source and / or other contaminant components from which the antibody is derived, or is substantially free of chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of an antibody that have less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein"’). In some aspects, when the antibody is recombinantly produced, it is substantially free of culture medium, e.g., culture medium represents less than about 20%, 15%, 10%, 5%, or 1% of the volume of the protein preparation. In some aspects, when the antibody is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, for example, it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the antibody have less than about 30%. 25%. 20%. 15%. 10%, 5%, or 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest. Contaminant components can also include, but are not limited to, materials that would interfere with therapeutic uses for the antibody, and can include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some aspects, the antibody is purified (1) to greater than or equal to 95% by weight of the antibody, as determined by the Lowry method (Lowry et al., 1951, J. Bio. Chem., 193: 265-275), such as 95%, 96%, 97%, 98%, or 99%, by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity' by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or silver stain. Isolated antibody also includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. An isolated antibody is typically prepared by at least one purification step. In some aspects, the antibodies provided herein are isolated.

[0084] The term “monoclonal antibody'’ refers to an antibody, or population of like antibodies, obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method, including but not limited to, monoclonal antibodies can be made by the hybridoma method first described by Kohler and Milstein (Nature, 256: 495-497, 1975), or by recombinant DNA methods.

[0085] As used herein, the term “binds” or “binding” refers to an interaction between molecules including, for example, to form a complex. Illustratively, such interactions embrace non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site of an antibody and its epitope on a target (antigen) molecule, such as SCUBE3, is the affinity7of the antibody or functional fragment forthat epitope. The ratio of association (kon) to dissociation (kou) of an antibody to a monovalent antigen (kon / koff) is the association constant Ka, which is a measure of affinity. The value of K varies for different complexes of antibody and antigen and depends on both konand k„n. The association constant K> for an antibody provided herein may be determined using any method provided herein or any other method known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants come into contact with antibodies containing multiple binding sites, the interaction of antibody with antigen at one site will increase the probability' of an interaction at a second binding site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity. The avidity of an antibody can be a better measure of its binding capacity than is the affinity of its individual binding sites. For example, high avidity' can compensate for low affinity as is sometimes found for pentameric IgM antibodies, which can have a lower affinity' than IgG, but the high avidity of IgM, resulting from its multivalence, enables it to bind antigen effectively.

[0086] “Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (Ka). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer to antigen. A variety of methods for measuring binding affinity are known in the art. any of which may be used for purposes of the present disclosure. Specific illustrative aspects include the following: In some aspects, the “Ka” or “Ka value” is measured by assays known in the art, for example, by a binding assay. The Ka can be measured in a radiolabeled antigen binding assay (RIA), for example, performed with the Fab portion of an antibody of interest and its antigen (Chen, et al.. 1999, J. Mol. Biol., 293:865- 881). The Ka or Ka value may also be measured by using surface plasmon resonance (SPR) assays (by BIAcore) using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ), or by biolayer interferometry (BLI) using, for example, the OctetQK384 system (ForteBio, Menlo Park. CA), or by quartz cry stal microbalance (QCM) technology. An “on-rate” or “rate of association” or “association rate” or “kon” can also be determined with the same surface plasmon resonance or biolayer interferometry techniques described above, using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ), or the OctetQK384 system (ForteBio, Menlo Park, CA).

[0087] Disclosed herein are isolated antibodies. Disclosed herein are isolated antibodies that can specifically bind to SCUBE3, antibodies that are specific for SCUBE3. antibodies that can specifically bind to a SCUBE3 epitope, antibodies that can selectively bind to a SCUBE3 epitope, and antibodies that can preferentially bind to SCUBE3. As used herein, the terms “anti-SCUBE3 antibody.” “anti-SCUBE3 monoclonal antibody”, “monoclonal SCUBE3”, “an antibody that specifically binds to SCUBE3,” or “antibody that is specific for SCUBE3,” “antibodies that specifically bind to a SCUBE3 epitope,” “an antibody that selectively binds to SCUBE3,” “antibodies that selectively bind to a SCUBE3 epitope,” “an antibody that preferentially binds to SCUBE3”, and analogous terms refer to antibodies capable of binding SCUBE3, i.e., WT SCUBE3 (SEQ ID NO: 21 or Accession Number NP_689966.2 or GI: 31377568). with sufficient affinity and specificity, particularly compared with mutants of SCUBE3.

[0088] For example, the wild-type SCUBE3 amino acid sequence can be:

[0089] MGSGRVPGLCLLVLLVHARAAQYSKAAQDVDECVEGTDNCHIDAICQNTPRSYKCI CKSGYTGDGKHCKDVDECEREDNAGCVHDCVNIPGNYRCTCYDGFHLAHDGHNCL DVDECAEGNGGCQQSCVNMMGSYECHCREGFFLSDNQHTCIQRPEEGMNCMNKN HGCAHICRETPKGGIACECRPGFELTKNQRDCKLTCNYGNGGCQHTCDDTEQGPRC GCHIKFVLHTDGKTCIETCAVNNGGCDSKCHDAATGVHCTCPVGFMLQPDRKTCKD IDECRLNNGGCDHICRNTVGSFECSCKKGYKLLINERNCQDIDECSFDRTCDHICVNT PGSFQCLCHRGYLLYGITHCGDVDECSINRGGCRFGCINTPGSYQCTCPAGQGRLHW NGKDCTEPLKCQGSPGASKAMLSCNRSGKKDTCALTCPSRARFLPESENGFTVSCGT PSPRAAPARAGHNGNSTNSNHCHEAAVLSIKQRASFKIKDAKCRLHLRNKGKTEEA GRITGPGGAPCSECQVTFIHLKCDSSRKGKGRRARTPPGKEVTRLTLELEAEVRAEET TASCGLPCLRQRMERRLKGSLKMLRKSINQDRFLLRLAGLDYELAHKPGLVAGERA EPMESCRPGQHRAGTKCVSCPQGTYYHGQTEQCVPCPAGTFQEREGQLSCDLCPGS DAHGPLGATNVTTCAGQCPPGQHSVDGFKPCQPCPRGTYQPEAGRTLCFPCGGGLT TKHEGAISFQDCDTKVQCSPGHYYNTSIHRCIRCAMGSYQPDFRQNFCSRCPGNTST DFDGSTSVAQCKNRQCGGELGEFTGYIESPNYPGNYPAGVECIWNINPPPKRKILIVV PEIFLPSEDECGDVLVMRKNSSPSSITTYETCQTYERPIAFTARSRKLWINFKTSEANS ARGFQIPYVTYDEDYEQLVEDIVRDGRLYASENHQEILKDKKLIKAFFEVLAHPQNY FKYTEKHKEMLPKSFIKLLRSKVSSFLRPYK (SEQ ID NO: 21).

[0090] By ‘‘specifically binds” is meant that an antibody recognizes and physically interacts with its cognate antigen (for example, SCUBE3) and does not significantly recognize and interact with other antigens; such an antibody may be a polyclonal antibody or a monoclonal antibody, which are generated by techniques that are well known in the art.

[0091] “Preferential binding” of antibodies as provided herein may be determined or defined based on the quantification of fluorescence intensity of the antibodies’ binding to SCUBE3 or variant or fragment thereof, for example, molecularly engineered cells, cell lines or tumor cell isolates. Preferential binding of an antibody as described to a SCUBE3 WT-expressing cell is indicated by a measured fluorescent binding intensity (MFI) value, as assessed by cell flow cytometry, of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or greater, as compared with binding of the antibody to a mutant SCUBE3 polypeptide or a mutant SCUBE3 -expressing cell, wherein the antibody to be assayed is directly or indirectly detectable by a fluorescent label or marker, such as FITC. In some aspects, the antibody to be assayed is directly labeled with a fluorescent marker, such as FITC. In some aspects, an antibody that preferentially or selectively binds SCUBE3 exhibits an MFI value of from 1.5- fold to 25-fold, or from 2-fold to 20-fold, or from 3-fold to 15-fold, or from 4-fold to 8-fold, or from 2-fold to 10-fold, or from 2-fold to 5-fold or more greater than the MFI value of the same antibody for binding a SCUBE3 or a SCUBE3 variant. Fold-fluorescence intensityvalues between and equal to all of the foregoing are intended to be included. In some aspects, the antibodies specifically and preferentially bind to a SCUBE3 polypeptide, such as a SCUBE3 antigen, peptide fragment, or epitope (e.g., human SCUBE3 such as a human SCUBE3 polypeptide, antigen or epitope). An antibody that specifically binds to SCUBE3, (e.g., wild type human SCUBE3) can bind to the extracellular domain (ECD) or a peptide derived from the ECD of SCUBE3. An antibody that specifically binds to a SCUBE3 antigen (e.g., human SCUBE3) can be cross-reactive with related antigens (e.g., cynomolgus (cyno) SCUBE3). In some aspects, an antibody that specifically binds to a SCUBE3 antigen does not cross-react with other antigens. An antibody that specifically binds to a SCUBE3 antigen can be identified, for example, by western blot, immunofluorescence binding assays, immunohistochemistry assay methods, immunoassay methods, Biacore, or other techniques known to those of skill in the art.

[0092] In some aspects, an antibody binds to an epitope of SCUBE3 that is conserved among SCUBE3 proteins from different species (e.g., between human and mouse SCUBE3). An antibody binds specifically to a SCUBE3 antigen when it binds to a SCUBE3 antigen with higher affinity- than to any cross reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISAs). Typically a specific or selective reaction will be at least twice background signal or noise and can be more than 10 times background. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion regarding antibody specificity. In some aspects, the extent of binding of the antibody to a “non-targef’ protein will be less than about 10% of the binding of the antibody to its particular target protein, for example, as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). In some aspects, the antibodies disclosed herein can be determined by the absorbance value. The absorbance value measures the binding activity of the antibody to human SCUBE3. In some aspects, the antibodies disclosed herein can have an absorbance value of great than or equal to 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1. 1.2, 1.3, 1.4, 1.5. 1.6, 1.7, 1.8, 1.9. or 2.

[0093] As used herein, in reference to an antibody, the term “heavy (H) chain” refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable (V) region (also called V domain) of about 115 to 130 or more amino acids and a carboxy-terminal portion that includes a constant (C) region. The constant region (or constant domain) can be one of five distinct types, (e.g., isotypes) referred to as alpha (a), delta (5), epsilon (c), gamma (y) and mu (p), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: a, 8 and y contain approximately 450 amino acids, while p and E contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well know n classes (e.g., isotypes) of antibodies, namely, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3 and IgG4. An antibody heavy chain can be a human antibody heavy chain.

[0094] As used herein in reference to an antibody, the term “light (L) chain” refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable domain of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate length of a light chain (both the V and C domains) is 211 to 217 amino acids. There are two distinct types of light chains, referred to as kappa (K) and lambda ( ). based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. An antibody light chain can be a human antibody light chain.

[0095] As used herein, the term “epitope” is the site(s) or region(s) on the surface of an antigen molecule to which a single antibody molecule binds, such as a localized region on the surface of an antigen, e.g.. a SCUBE3 polypeptide that is capable of being bound by one or more antigen binding regions of an antibody disclosed herein. An epitope can be immunogenic and capable of eliciting an immune response in an animal. Epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. An epitope can be a linear epitope and a conformational epitope. A region of a polypeptide contributing to an epitope can be contiguous amino acids of the polypeptide, forming a linear epitope, or the epitope can be formed from two or more non-contiguous amino acids or regions of the polypeptide, typically called a conformational epitope. The epitope may or may not be a three-dimensional surface feature of the antigen. In some aspects, a SCUBE3 epitope is a three-dimensional surface feature of a SCUBE3 polypeptide. In some aspects, a SCUBE3 epitope is linear feature of a SCUBE3 polypeptide.

[0096] An antibody binds “an epitope” or “essentially the same epitope” or “the same epitope” as a reference antibody, when the two antibodies recognize identical, overlapping, or adjacent epitopes in a three-dimensional space. The most widely used and rapid methods for determining whether two antibodies bind to identical, overlapping, or adjacent epitopes in a three-dimensional space are competition assays, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate, or expressed on a cell surface, and the ability of unlabeled antibodies to block the binding of labeled antibodies to antigen is measured using a detectable signal, e.g., radioactive, fluorescent or enzyme labels.

[0097] The term “compete” when used in the context of antibodies that compete for the same epitope or binding site on a SCUBE3 target protein or peptide thereof means competition as determined by an assay in which the antibody under study, or binding fragment thereof, prevents, blocks, or inhibits the specific binding of a reference molecule (e.g., a reference ligand, or reference antigen binding protein, such as a reference antibody) to a common antigen (e.g., SCUBE3 or a fragment thereof). Numerous types of competitive binding assays can be used to determine if a test antibody competes with a reference antibody for binding to SCUBE3 (e.g., human SCUBE3). Examples of assays that can be employed include solid phase direct or indirect radioimmunoassay (RIA); solid phase direct or indirect enzy me immunoassay (EIA); sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al.. 1986, J. Immunol. 137:3614-3619): solid phase direct labeled assay; solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using labeled iodine (1125 label) (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al.. 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of a purified antigen (e.g., SCUBE3) bound to a solid surface, or cells bearing either of an unlabeled test antigen binding protein (e.g., test antibody) or a labeled reference antigen binding protein (e.g., reference antibody). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of a known amount of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and / or antibodies binding to an adj acent epitope sufficiently proximal to the epitope bound by the reference antibody causing steric hindrance to occur. Additional details regarding methods for determining competitive binding are described herein. Usually, when a competing antibody protein is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 15%, or at least 20%, for example, without limitation, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% or greater, as well as percent amounts betw een the amounts stated. In some aspects, binding can be inhibited by at least 80%, 85%, 90%, 95%, 96% or 97%, 98%, 99% or more.

[0098] As used herein, the term '‘blocking” antibody or an “antagonist” antibody refers to an antibody that prevents, inhibits, blocks, or reduces biological or functional activity of the antigen to which it binds. Blocking antibodies or antagonist antibodies can substantially or completely prevent, inhibit, block, or reduce the biological activity or function of the antigen. For example, a blocking antibody can prevent, inhibit, block, or reduce the binding interaction between SCUBE3 and epidermal growth factor receptor (e.g., EGFR1) and / or TGFbR2 or TGFbRl, thus preventing, blocking, inhibiting, or reducing the functions associated with the epidermal growth factor receptor (e.g., EGFR1) and / or TGFbR2 or TGFbRl and SCUBE3 interactions. The terms block, inhibit, and neutralize are used interchangeably herein and refer to the ability of the antibodies disclosed herein to prevent or otherwise disrupt or reduce the epidermal growth factor receptor (e.g., EGFR1) and / or TGFbR2 or TGFbRl SCUBE3 interactions.

[0099] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0100] Antibody -based therapy is one of the most successful strategies for treating patients with cancers. This is also true for breast cancers as trastuzumab (a monoclonal antibody against Her2) is used as the first line therapy for Her2+ breast cancer. However, triple negative breast cancers (TNBCs), which exhibit aggressive phenotype with poor prognosis, lack effective targeted antibody-based therapies. TNBCs occur more frequently in younger patients (<50 years old), and progress aggressively with a tendency to relapse as distant metastases (Amos, K.D., et al., Int J Breast Cancer. 2012: p. 385978; and Carey, L.A., Oncologist. 15 Suppl 5: p. 49-56).

[0101] Described herein is a new and more effective therapy for TNBCs. Using loss-of- function genomic screen, a secretory protein was identified that can render survival advantage to TNBCs and therefore, can be targeted by antibody-based therapeutics. The unbiased genomic screen of more than 70,000 shRNAs targeting 16,000 genes identified Signal peptide CUB domain EGF-like 3 (SCUBE3) that is important for TNBC cell survival. SCUBE3 is a secreted glycoprotein identified in umbilical vein (Wu, B.T., et al., J Biol Chem, 2004. 279(36): p. 37485-90). The results show that SCUBE3 is a secreted protein and is highly expressed in TNBC patient blood compared to blood from healthy individuals.

[0102] The results described herein show that SCUBE3 promotes TNBC growth and progression by interacting with EGFR and TGFbR2 / l and by activating ERK / MAPK- F0XM1 -associated signaling. This is a significant finding given that 47 out of 146 core genes that are found to be dysregulated (over-expressed) in >90% of TNBC were directly regulated by FOXM1 hub network (Radovich, M., et al.. Breast Cancer Res Treat, 2014. 143(1): p. 57- 68). The results described herein also demonstrate that SCUBE3 renders TNBC cells resistant to doxorubicin (DOX) by altering the levels and activity' of DNA repair associated proteins. Importantly, the results provide evidence that an antibody against SCUBE3 inhibits TNBC growth. These findings show' that the disclosed antibody blocking SCUBE3 interactions with TGFbR2 and EGFR1 can sen e as a potent therapeutic for treating TNBC. Not only does the data demonstrate that secreted SCUBE3 can mediate its oncogenic function by interacting with two discrete receptors: TGFbR2 / TGFbRl and EGFR1, but also consequently by activating ERK / MAPK-FOXM1 signaling. This is an interesting finding as FOXM1 is highly expressed in TNBC patients and w as identified as an important nodal factor that regulates a substantial number of genes that are defined as TNBC core genes (Radovich, M., et al., Breast Cancer Res Treat. 2014.143(1): p. 57-68). Notably, comprehensive analysis of TCGA dataset identified hyperactivation of F0XM1 as a signature of basal-like breast cancer (most TNBCs are basal-like) (Koboldt, D C , et al.. Nature, 2012, 490(7418): p. 61-70). Furthermore, the results show' that SCUBE3 induces the expression of RAD51 and EXO1, DNA repair-associated proteins that are highly expressed in TNBC patients and acts as a downstream effector of F0XM1.

[0103] SCUBE3 knockout mice are viable and fertile (Xavier, G.M., L. Panousopoulos, and M.T. Coboume, PLoS One, 2013. 8(1): p. e55274), demonstrating that SCUBE3 can be dispensable for normal cells and therefore, targeting SCUBE3 for treating TNBC will not result in overt life-threatening toxicity. The results additionally show that suppression of SCUBE3 attenuated TNBC growth and invasion, while SCUBE3 overexpression promoted tumor grow th in pre-clinical mouse models. These findings led to testing whether SCUBE3 is a player in supporting TNBC growth and progression to metastases, and in mediating cellular response to PARP inhibitor (PARPi) and chemotherapy by regulating ERK / MAPK-FOXM1 - dependent signaling cascade; and that antibody blocking the SCUBE3-TGFbR2 / EGFRl interactions can serve as a potent therapeutic for treating TNBCs.

[0104] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be practiced within the scope of the appended claims.

[0105] COMPOSITIONS

[0106] Antibodies. Disclosed herein are isolated antibodies, including, but not limited to, anti-SCUBE3 antibodies or binding fragments thereof. Disclosed herein are antibodies or binding fragments thereof that can specifically bind to SCUBE3. Disclosed herein are antibodies or binding fragments thereof that can specifically bind to SCUBE3 and block or inhibit the function of the SCUBE 3 and TGFbR2 or TGFbRl and EGFR1 interactions (e.g., block or inhibit the binding of SCUBE3 to TGFbR2 or TGFbRl and / or EGFR1). Disclosed herein are antibodies or binding fragments thereof useful in treating cancer. Also disclosed herein are antibodies or binding fragments thereof specifically bind to SCUBE3 useful in the treating cancer and inhibiting or preventing tumor or cancer metastases.

[0107] As used herein, the term “variable (V) region" or “variable (V) domain" refers to a portion of the light (L) or heavy (H) chains of an antibody polypeptide that is generally- located at the amino-terminus of the L or H chain. The H chain V domain has a length of about 115 to 130 amino acids, while the L chain V domain is about 100 to 110 amino acids in length. The H and L chain V domains are used in the binding and specificity of each particular antibody for its particular antigen. The V domain of the H chain can be referred to as “VH.” The V region of the L chain can be referred to as “VL.” The term “variable” refers to the fact that certain segments of the V domains differ extensively in sequence among different antibodies. While the V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen, the variability is not evenly distributed across the 110-amino acid span of antibody V domains. Instead, the V domains consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called ‘‘hypervariable regions” or “complementarity determining regions” (CDRs) that are each about 9-12 amino acids long or 3-17 amino acids long. The V domains of antibody H and L chains each comprise four FRs, largely adopting a P sheet configuration, connected by three hypervariable regions, called, which form loops connecting, and in some cases forming part of, the sheet structure. The hypervariable regions in each chain are held together in close proximity' by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD). The C domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The V domains differ extensively in sequence among different antibody classes or types. The variability in sequence is concentrated in the CDRs. which are primarily responsible for the interaction of the antibody w ith antigen. In some aspects, the variable domain of an antibody is a human or humanized variable domain.

[0108] As used herein, the terms “complementarity determining region,” “CDR,” “hypervariable region,” “HVR,” and “HV” are used interchangeably. A “CDR” or “complementarity determining region” is a region of hypervari ability interspersed within regions that are more conserved, termed “framework regions” (FR). A “CDR” refers to one of three hypervariable regions (Hl, H2 or H3) within the non-framework region of the antibody VH p-sheet framework, or to one of three hypervariable regions (LI, L2 or L3) within the non-framework region of the antibody VL P-sheet framework. The term, when used herein, refers to the regions of an antibody V domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions: three (Hl, H2, H3) in the VH domain and three (LI, L2. L3) in the VL domain. Accordingly. CDRs are typically highly variable sequences interspersed within the framew ork region sequences of the V domain. “Framework” or “FR” residues are those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies.

[0109] A number of hypervariable region delineations are in use and are encompassed herein. CDR regions are well known to those skilled in the art and have been defined by. for example, Kabat as the regions of most hypervariability within the antibody V domains (Kabat et al., 1977, J. Biol. Chem., 252:6609-6616; Kabat, 1978, Adv. Prot. Chem., 32: 1-75). The Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda. MD). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved P-sheet framework, and thus are able to adopt different conformations (Chothia et al., 1987, J. Mol. Biol., 196:901-917). Chothia refers instead to the location of the structural loops. The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). Both numbering systems and terminologies are well recognized in the art.

[0110] Recently, a universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol., 27(1 ):55-77). IMGT is an integrated information system specializing in immunoglobulins (Ig), T cell receptors (TR) and the major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin V domain is conserved betw een species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features. CDR and framework residues and are readily identified. This information can be used in grafting and in the replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger et al., 2001, J. Mol. Biol., 309: 657-670. Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, Id; Chothia et al., Id.; Martin, 2010, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag; and Lefranc et al., 1999, Nuc. Acids Res., 27:209-212).

[0111] CDR region sequences have also been defined by AbM, Contact and IMGT. The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Martin, 2010, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are noted below.

[0112] Exemplary delineations of CDR region sequences are illustrated in Table 2. The positions of CDRs within a canonical antibody variable region have been determined by comparison of numerous structures (Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948); Morea et al., 2000, Methods. 20:267-279). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable region numbering scheme (Al-Lazikani et al., Id). Such nomenclature is similarly well known to those skilled in the art.

[0113] Table 2. Exemplary Delineations of CDR Region Sequences

[0114] An “affinity matured” antibody is one with one or more alterations (e.g., amino acid sequence variations, including changes, additions and / or deletions) in one or more HVRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In some aspects, affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen, such as SCUBE3. Affinity matured antibodies are produced by procedures known in the art. For reviews, see Hudson and Souriau, 2003, Nature Medicine, 9: 129-134; Hoogenboom, 2005, Nature Biotechnol., 23: 1105-1116; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia, 4: 39-51.

[0115] The CDRs disclosed herein may also include variants. Generally, the amino acid identity between individual variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% . Thus, a “variant CDR’' is one with the specified identity to the parent or reference CDR of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity' and / or activity of the parent CDR. For example, a “variant CDR"’ can be a sequence that contains 1, 2, 3, 4 or 5 amino acid changes as compared to the parent or reference CDR of the invention, and shares or improves biological function, specificity and / or activity of the parent CDR.

[0116] The antibodies disclosed herein can be of the IgG, IgM, IgA, IgD, and IgE Ig classes, as well as polypeptides comprising one or more antibody CDR domains that retain antigen binding activity. Illustratively, the antibodies can be chimeric, affinity matured, humanized, or human antibodies. In some aspects, the antibodies can be monoclonal antibodies. In some aspects, the monoclonal antibody can be a humanized antibody. By known means and as described herein, polyclonal or monoclonal antibodies, antibody fragments, binding domains and CDRs (including engineered forms of any of the foregoing) may be created that are specific for SCUBE3 antigen, one or more of its respective epitopes, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural protein.

[0117] Also disclosed herein are compositions comprising the disclosed isolated antibodies, including, but not limited to antibodies that specifically bind to SCUBE3. In some aspects, the antibodies disclosed herein can be isolated antibodies. Examples of the CDR sequences and heavy or light chain variable region sequences of antibodies disclosed herein are show n in Table 3.

[0118] Disclosed herein are isolated antibodies comprising a light chain variable region and a heavy chain variable region. In some aspects, the light chain variable region can comprise a complementarity determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 1; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 2; and a complementarity determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 3. In some aspects, the heavy chain variable region can comprise a complementarity determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 5; a complementarity determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 6; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 7.

[0119] Also disclosed herein, are isolated antibodies comprising a light chain variable region amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region amino acid sequence of SEQ ID NO: 8.

[0120] In some aspects, any of the antibodies disclosed herein can comprise a light chain variable region amino acid sequence comprising SEQ ID NO: 4. In some aspects, any of the antibodies disclosed herein can comprise a heavy chain variable region amino acid sequence comprising SEQ ID NO: 8. In some aspects, a light chain variable region has an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO: 4. In some aspects, a heavy chain variable region has an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO: 8.

[0121] Disclosed herein are isolated antibodies comprising a light chain variable region and a heavy chain vanable region. In some aspects, the light chain variable region can comprise a complementarity determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 9; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 10; and a complementarity determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 11. In some aspects, the heavy chain variable region can comprise a complementarity determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 13; a complementarity determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 14; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 15.

[0122] Also disclosed herein, are isolated antibodies comprising a light chain variable region amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region amino acid sequence of SEQ ID NO: 16.

[0123] In some aspects, any of the antibodies disclosed herein can comprise a light chain variable region amino acid sequence comprising SEQ ID NO: 12. In some aspects, any of the antibodies disclosed herein can comprise a heavy chain variable region amino acid sequence comprising SEQ ID NO: 16. In some aspects, a light chain variable region has an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO: 12. In some aspects, a heavy chain variable region has an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO: 16.

[0124] Disclosed herein are isolated antibodies comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a complementarity determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 1; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 2; and a complementarity determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 3; and wherein the heavy chain variable region comprises a complementarity determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 5; a complementarity determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 6; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 7. wherein one or more of the CDRL1. CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 comprise 1, 2, 3, 4, or 5 conservative amino acid substitutions.

[0125] Disclosed herein are isolated antibodies comprising a light chain variable region amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region amino acid sequence of SEQ ID NO: 8, wherein the isolated antibody comprises 1. 2, 3, 4. or 5 conservative amino acid substitutions in the light or heavy chain variable region amino acid sequences.

[0126] In some aspects, the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises one or more complementarity determining region (CDRHs) CDRH1, CDRH2 and CDRH3 with amino acid sequences that have 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions in 1, 2 or 3 CDRHs having the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; and / or wherein the antibody has a light chain variable region comprising one or more complementarity determining region (CDRLs) CDRL1. CDRL2 and CDRL3 with the amino acid sequences that have 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions in 1, 2 or 3 CDRLs having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0127] Disclosed herein are isolated antibodies comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a complementarity determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 9; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 10; and a complementarity determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 11; and wherein the heavy chain variable region comprises a complementarity determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 131 a complementarity determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 14; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 15, wherein one or more of the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 comprise 1, 2, 3, 4, or 5 conservative amino acid substitutions.

[0128] Disclosed herein are isolated antibodies comprising a light chain variable region amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region amino acid sequence of SEQ ID NO: 16, wherein the isolated antibody comprises 1, 2, 3, 4, or 5 conservative amino acid substitutions in the light or heavy chain variable region amino acid sequences.

[0129] Table 3: Exemplary Amino Acid Sequences of Antibodies

[0130] Disclosed herein are nucleic acid sequences that encode a light chain variable region of an antibody comprising the sequence of SEQ ID NO: 17. Disclosed herein are nucleic acid sequences that encode a heavy chain variable region of an antibody comprising the sequence of SEQ ID NO: 18.

[0131] Disclosed herein are nucleic acid sequences that encode a light chain variable region of an antibody comprising the sequence of SEQ ID NO: 19. Disclosed herein are nucleic acid sequences that encode a heavy chain variable region of an antibody comprising the sequence of SEQ ID NO: 20.

[0132] Disclosed herein are nucleic acid sequences encoding an antibody comprising a variable light chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 17. In some aspects, an antibody comprises a variable light chain comprising a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a sequence set forth in SEQ ID NO: 17.

[0133] Disclosed herein are nucleic acid sequences encoding an antibody comprising a variable heavy chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 18. In some aspects, an antibody comprises a variable heavy chain comprising a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a sequence set forth in SEQ ID NO: 18.

[0134] Disclosed herein are nucleic acid sequences encoding an antibody comprising a variable light chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 19. In some aspects, an antibody comprises a variable light chain comprising a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a sequence set forth in SEQ ID NO: 19.

[0135] Disclosed herein are nucleic acid sequences encoding an antibody comprising a variable heavy chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 20. In some aspects, an antibody comprises a variable heavy chain comprising a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity7to a sequence set forth in SEQ ID NO: 20. Table 4: Exemplary Nucleic Acid Sequences Encoding Antibodies

[0136] In some aspects, any of CDR sequences disclosed herein can include a single amino acid change as compared to the parent or reference CDR. In some aspects, any of the CDR sequences disclosed herein can include at least two amino acid changes as compared to the parent or reference CDR. In some aspects, the amino acid change can be a change from a cysteine residue to another amino acid. In some aspects, the amino acid change can be a change from a glycine residue to another amino acid. The amino acid identity between individual variant CDRs can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Thus, a '‘variant CDR” can be one with the specified identity to the parent CDR of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%. 96%. 97%. 98%. or 99% of the specificity and / or activity of the parent CDR. For example, the parent CDR sequence can be one or more of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 9, 10, 11, 13, 14, and / or 15. The variant CDR sequence can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 9, 10, 11, 13, 14, and / or 15. The variant CDR sequence can also share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent CDR.

[0137] As discussed herein, minor variations in the amino acid sequences of any of the antibodies disclosed herein are contemplated as being encompassed by the instant disclosure, providing that the variations in the amino acid sequence maintains at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% sequence identity to the parent sequence. In some aspects, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non- polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are known to one of ordinary skill in the art.

[0138] In some aspects, amino acid substitutions can be those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physiocochemical or functional properties of such analogs. In some aspects, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the non-CDR sequence of the heavy chain, the light chain or both. In some aspects, one or more amino acid substitutions can be made in one or more of the CDR sequences of the heavy chain, the light chain or both.

[0139] In some aspects, the at least one amino acid change can be to substitute an NG motif (amino acid asparagine followed by a glycine). In some aspects, the glycine residue can be substituted or replaced with hydrophobic amino acid residue. In some aspects, the glycine residue can be substituted or replaced with alanine, aspartic acid, glutamic acid, or valine. In some aspects, the glycine residue can be substituted or replaced with arginine, lysine, or glutamine.

[0140] Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’)2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’)2 fragments using mild reduction has been useful. In some aspects, cysteine and methionine can be susceptible to rapid oxidation, which can negatively influence the cleavage of protecting groups during synthesis and the subsequent peptide purification. In some instances, cysteine residues in peptides used for antibody production can affect the avidity of the antibody, because free cysteines are uncommon in vivo and therefore may not be recognized by the native peptide structure. In some aspects, the disclosed antibodies and fragments thereof comprise a sequence where a cysteine reside outside of the CDR (e.g. in the non-CDR sequence of the heavy chain, the light chain or both) is substituted. In some aspects, cysteine can be replaced with serine and methionine replaced with norleucine (Nle). Multiple cysteines on a peptide or in one of the disclosed antibodies or fragments thereof may be susceptible to forming disulfide linkages unless a reducing agent such as dithiothreitol (DTT) is added to the buffer or the cysteines can be replaced with serine residues.

[0141] While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed antigen binding protein CDR variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of antigen binding protein activities as described herein.

[0142] Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about one (1) to about twenty (20) amino acid residues, although considerably larger insertions may be tolerated. Deletions range from about one (1) to about twenty (20) amino acid residues, although in some cases deletions may be much larger.

[0143] Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative or variant. Generally these changes are done on a few amino acids to minimize the alteration of the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, larger changes may be tolerated in certain circumstances.

[0144] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are as described in Table 1, supra. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Nonconservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.

[0145] In some aspects, the CDRs can be defined according to the Kabut definition. In some aspects, the CDRs can be defined according to the 1MGT definition.

[0146] In some aspects, the antibodies disclosed herein can be recombinantly engineered, chimerized, or humanized. In some aspects, the antibodies disclosed herein can be affinity matured or human antibodies. In some aspects, the antibodies disclosed herein can be a Fab. an Fab’, an F(ab’)2, a Fv, a scFv, a diabody or fragments thereof. In some aspects, the antibody can be a monoclonal antibody. In some aspects, the monoclonal antibodies can be humanized or chimeric forms thereof. In some aspects, the monoclonal antibody can be a humanized antibody. By known means and as described herein, polyclonal or monoclonal antibodies, antibody fragments, binding domains and CDRs (including engineered forms of any of the foregoing) may be created that are specific for SCUBE3 antigen, one or more of its respective epitopes, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural protein.

[0147] A “chimeric’’ antibody is one in which a portion of the H and / or L chain, e.g., the V domain, is identical with or homologous to a corresponding amino acid sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s), e.g., the C domain, is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as a fragment of such an antibody, so long as it exhibits the desired biological activity (see, e.g., U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-6855).

[0148] The term '‘humanized antibody” refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. A humanized antibody can include conservative amino acid substitutions or non-natural residues from the same or different species that do not significantly alter its binding and / or biologic activity. Such antibodies are chimeric antibodies that contain minimal sequence derived from non- human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat. camel, bovine, goat, or rabbit having the desired properties. Furthermore, humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. Thus, in general, a humanized antibody can comprise all or substantially all of at least one, and in one aspect two. variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also can comprise at least a portion of an immunoglobulin constant region (Fc), or that of a human immunoglobulin (see, e.g., Cabilly et al., U.S. Pat. No. 4,816,567: Cabilly et al., European Patent No. 0,125,023 Bl; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 Bl; Neuberger, M. S. et al., WO 86 / 01533; Neuberger, M. S. et al., European Patent No. 0,194,276 Bl; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Padlan, E. A. et al., European Patent Application No. 0,519,596 Al; Queen et al. (1989) Proc. Natl. Acad. Sci. USA, Vol 86: 10029-10033). The terms “human antibody” and “fully human antibody” are used interchangeably herein and refer to an antibody that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as practiced by those skilled in the art. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom et al., 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581 and yeast display libraries (Chao et al., 2006, Nature Protocols, 1:755- 768). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., 1985 Monoclonal Antibodies and Cancer Therapy, Alan R. Liss. p. 77; Boemer et al., 1991, J. Immunol., 147(1 ): 86-95. See also van Dijk et al., 2001, Curr. Opin. Pharmacol., 5: 368-74. Human antibodies can be prepared by administering an antigen to a transgenic animal whose endogenous Ig loci have been disabled, e.g., a mouse, and that has been genetically modified to harbor human immunoglobulin genes which encode human antibodies, such that human antibodies are generated in response to antigenic challenge (see. e.g., Jakobovits, A., 1995, Curr. Opin. Biotechnol. 6(5):561 -566; Briiggemann et al., 1997 Curr. Opin. Biotechnol., 8(4):455-8; and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example. Li et al., 2006, Proc. Natl. Acad. Sci. USA. 103:3557-3562 regarding human antibodies generated via a human B-cell hybridoma technology. In some aspects, human antibodies comprise a variable region and constant region of human origin. “Fully human” antibodies, in some aspects, can also encompass antibodies which bind SCUBE3 polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence. In some aspects, the antibodies provided herein are fully human antibodies. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al., 1991, Sequences of Proteins of Immunological Interest. Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The phrase “recombinant human antibody” includes human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant, combinatorial human antibody library; antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and / or transchromosomal for human immunoglobulin genes (see e.g.. Taylor, L. D. et al., 1992, Nucl. Acids Res. 20:6287-6295); or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Sendees, NIH Publication No. 91-3242). In some aspects, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and, thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0149] A monoclonal antibody is a single, clonal species of antibody wherein every antibody molecule recognizes the same epitope because all antibody producing cells are derived from a single, antibody-producing B-lymphocyte (or other clonal cell, such as a cell that recombinantly expresses the antibody molecule). The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. In some aspects, rodents such as mice and rats are used in generating monoclonal antibodies. In some aspects, rabbit, sheep, or frog cells are used in generating monoclonal antibodies. The use of rats is well known and may provide certain advantages. Mice (e.g., BALB / c mice) are routinely used and generally give a high percentage of stable fusions. Hybridoma technology as used in monoclonal antibody production involves the fusion of a single, antibody-producing B lymphocyte isolated from a mouse previously immunized with a SCUBE3 protein or peptide with an immortalized cell, e.g., a mouse cell line. This technology provides a method to propagate a single antibody-producing cell for an indefinite number of generations, such that unlimited quantities of structurally identical antibodies having the same antigen or epitope specificity, i.e., monoclonal antibodies, may be produced.

[0150] Methods have been developed to replace light and heavy chain constant domains of the monoclonal antibody with analogous domains of human origin, leaving the variable regions of the foreign antibody intact. Alternatively, ‘Tully human’’ monoclonal antibodies are produced in mice or rats that are transgenic for human immunoglobulin genes. Methods have also been developed to convert variable domains of monoclonal antibodies to more human form by recombinantly constructing antibody variable domains having both rodent and human amino acid sequences. In “humanized” monoclonal antibodies, only the hypervariable CDRs are derived from non-human (e.g., mouse, rat, chicken, llama, etc.) monoclonal antibodies, and the framework regions are derived from human antibody amino acid sequences. The replacement of amino acid sequences in the antibody that are characteristic of rodents with amino acid sequences found in the corresponding positions of human antibodies reduces the likelihood of adverse immune reaction to foreign protein during therapeutic use in humans. A hybridoma or other cell producing an antibody may also be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced by the hybridoma.

[0151] Engineered antibodies may be created using monoclonal and other antibodies and recombinant DNA technology to produce other antibodies or chimeric molecules that retain the antigen or epitope binding specificity of the original antibody, i.e., the molecule has a specific binding domain. Such techniques may involve introducing DNA encoding the immunoglobulin variable region or the CDRs of an antibody into the genetic material for the framework regions, constant regions, or constant regions plus framework regions, of a different antibody. See, for instance, U.S. Patent Nos. 5,091,513 and 6,881,557, which are incorporated herein by reference.

[0152] By known means as described herein, polyclonal or monoclonal antibodies, antibody fragments having binding activity, binding domains and CDRs (including engineered forms of any of the foregoing), may be created that specifically bind to SCUBE3 protein, one or more of its respective epitopes, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural compounds.

[0153] Antibodies may be produced from any animal source, including birds and mammals. In some aspects, the antibodies can be ovine, murine (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken. In addition, newer technology permits the development of and screening for human antibodies from human combinatorial antibody libraries. For example, bacteriophage antibody expression technology allows specific antibodies to be produced in the absence of animal immunization, as described in U.S. Patent No. 6,946,546, which is incorporated herein by reference. These techniques are further described in Marks et al., 1992, Bio / Technol. , 10:779-783; Stemmer, 1994, Nature, 370:389-391; Gram et al., 1992, Proc. Natl. Acad. Sci. USA, 89:3576-3580; Barbas et al., 1994, Proc. Natl. Acad. Sci. USA, 91 :3809-3813; and Schier et al. , 1996. Gene, 169(2): 147-155.

[0154] Methods for producing polyclonal antibodies in various animal species, as well as for producing monoclonal antibodies of various types, including humanized, chimeric, and fully human, are w ell known in the art and are highly reproducible. For example, the following U.S. patents provide descriptions of such methods and are herein incorporated by reference: U.S. Patent Nos. 3.817,837; 3.850,752; 3,939.350; 3,996,345; 4,196,265; 4,275, 149; 4,277,437; 4,366,241; 4,469,797; 4,472,509; 4,606,855; 4,703,003; 4,742,159; 4,767,720; 4,816,567; 4,867,973; 4,938,948; 4,946,778; 5,021,236; 5,164,296; 5,196,066; 5,223,409; 5,403,484; 5,420,253; 5,565,332; 5,571,698; 5,627,052; 5,656,434; 5,770,376; 5,789,208; 5,821,337; 5,844,091; 5,858,657; 5,861,155; 5,871,907; 5,969,108; 6,054,297; 6,165,464; 6,365,157; 6,406,867; 6,709.659; 6,709,873; 6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; 6.891,024; 7,407.659; and 8,178,098.

[0155] In some aspects, the antibody can be a single chain antibody. In some aspects, the antibody can be linked to a detectable label. In some aspects, antibody can be a monovalent or a bivalent antibody.

[0156] In some aspects, the antibodies disclosed herein can be an IgG, an IgM, an IgA, an IgD, or an IgE antibody or antigen binding fragment thereof. In some aspects, the antibodies can be of the IgG, IgM, IgA, IgD, and IgE Ig classes or a genetically modified IgG class antibody, as well as polypeptides comprising one or more antibody CDR regions that retain antigen binding activity. In some aspects, the antibody can be an IgG class of antibody. In some aspects, the IgG class antibody can be an IgGl, IgG2. IgG3, or IgG4 class antibody.

[0157] In some aspects, the antibody can be a bispecific antibody. Unifying two antigen binding sites of different specificity into a single construct, bispecific antibodies have the ability to bring together two discreet antigens with exquisite specificity and therefore have great potential as therapeutic agents. Bispecific antibodies were originally made by fusing two hybridomas. each capable of producing a different immunoglobulin. Bispecific antibodies can also be produced by joining two scFv antibody fragments while omitting the Fc portion present in full immunoglobulins. Each scFv unit in such constructs can contain one variable domain from each of the heavy (VH) and light (VL) antibody chains, j oined with one another via a synthetic polypeptide linker, the latter often being genetically engineered so as to be minimally immunogenic while remaining maximally resistant to proteolysis. Respective scFv units may be joined by a number of known techniques, including incorporation of a short (usually less than 10 amino acids) polypeptide spacer bridging the two scFv units, thereby creating a bispecific single chain antibody. The resulting bispecific single chain antibody is therefore a species containing two VH / VL pairs of different specificity on a single polypeptide chain, in which the VH and VL domains in a respective scFv unit are separated by a polypeptide linker long enough to allow intramolecular association between these two domains, such that the so-formed scFv units are contiguously tethered to one another through a polypeptide spacer kept short enough to prevent unwanted association between, for example, the VH domain of one scFv unit and the VL of the other scFv unit. Examples of antibody fragments suitable for use include, without limitation: (i) the Fab fragment, consisting of VL, VH, CL, and CHI domains; (ii) the “Fd” fragment consisting of the VH and CHI domains; (iii) the “Fv” fragment consisting of the VL and VH domains of a single antibody; (iv) the ’dAb" fragment, which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules C‘scFv”), in which a VH domain and a VL domain are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bi-specific single chain Fv dimers (see U.S. Patent No. 5,091,513); and (ix) diabodies, multivalent, or multispecific fragments constructed by gene fusion (U.S. Patent Appln. Pub. No. 20050214860). Fv, scFv, or diabody molecules may be stabilized by the incorporation of disulfide bridges linking the VH and VL domains. Minibodies comprising a scFv joined to a CH3 domain (Hu et al., 1996, Cancer Res., 56:3055-3061) may also be useful. In addition, antibody-like binding peptidomimetics are also contemplated. "‘Antibody like binding peptidomimetics” (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods, have been reported by Liu et al., 2003, Cell Mol. Biol., 49:209-216.

[0158] Animals may be inoculated with an antigen, such as a SCUBE3 polypeptide or peptide to generate an immune response and produce antibodies specific for the SCUBE3 polypeptide. Frequently, an antigen is bound or conjugated to another molecule to enhance the immune response. As used herein, a conjugate can be any peptide, polypeptide, protein, or non-proteinaceous substance bound to an antigen that is used to elicit an immune response in an animal. Antibodies produced in an animal in response to antigen inoculation comprise a variety of non-identical molecules (polyclonal antibodies) made from a variety of individual antibody producing B lymphocytes. A polyclonal antibody is a mixed population of antibody species, each of which may recognize a different epitope on the same antigen. Given the correct conditions for polyclonal antibody production in an animal, most of the antibodies in the animal’s serum will recognize the collective epitopes on the antigenic compound to which the animal has been immunized. This specificity is further enhanced by affinity purification to select only those antibodies that recognize the antigen or epitope of interest.

[0159] The antibodies described herein specific to SCUBE3 will have the ability to neutralize, block, inhibit, or counteract the effects of SCUBE3 binding to TGFbR2 or TGFbRl and / or EGFRlregardless of the animal species, monoclonal cell line or other source of the antibody. Certain animal species may be less preferable for generating therapeutic antibodies because they may be more likely to cause an immune or allergic response due to activation of the complement system through the “Fc” portion of the antibody. However, whole antibodies may be enzymatically digested into the “Fc"’ (complement binding) fragment, and into peptide fragments having the binding domains or CDRs. Removal of the Fc portion reduces the likelihood that this antibody fragment will elicit an undesirable immunological response and, thus, antibodies without an Fc portion may be preferential for prophylactic or therapeutic treatments. As described above, antibodies may also be constructed so as to be chimeric, humanized, or partially or fully human, so as to reduce or eliminate potential adverse immunological effects resulting from administering to an animal an antibody that has been produced in, or has amino acid sequences from, another species.

[0160] In some aspects, the antibodies disclosed herein specifically bind to human SCUBE3. In some aspects, the antibody specifically binds to SCUBE3 and inhibits binding of SCUBE3 to TGFbR2 or TGFbRl and / or EGFR1. In some aspects, the antibody specifically binds to human SCUBE3 and inhibits binding of human SCUBE3 to human TGFbR2 or TGFbRl and / or EGFR1.

[0161] The term “specifically binds'’ (or “immunospecifically binds”) is not intended to indicate that an antibody binds exclusively to its intended target. Rather, an antibody “specifically binds” if its affinity for its intended target is about, for example. 5-fold greater when compared to its affinity for a non-target molecule. Suitably there is no significant crossreaction or cross-binding with undesired substances. The affinity of the antibody will, for example, be at least about 5-fold, such as 10-fold, such as 25-fold, especially 50-fold, and particularly 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In some aspects, specific binding between an antibody or other binding agent and an antigen means a binding affinity of at least 106M-1. Antibodies may, for example, bind with affinities of at least about 107M-1, such as between about 108M-1to about 109M-1, about 109M-1to about IO10M-1. or about 10-1°M-1to about 1011M-1. Antibodies may, for example, bind with an EC50 of 50 nM or less. 10 nM or less, 1 nM or less. 100 pM or less, or more preferably 10 pM or less. In some aspects, the antibodies can bind with an EC50 of about 60 pg / ml, 59 pg / ml, 58 pg / ml, 57 pg / ml, 56 pg / ml, 55 pg / ml, 54 pg / ml, 53 pg / ml, 52 pg / ml, 51 pg / ml, 50 pg / ml or less. In some aspects, the antibodies can bind with an EC50 of about 50 pg / ml, 49 pg / ml, 48 pg / ml, 47 pg / ml, 46 pg / ml. 45 pg / ml, 44 pg / ml, 43 pg / ml. 42 pg / ml, 41 pg / ml, 40 pg / ml or less. In some aspects, the antibodies can bind with an EC50 of about 40 pg / ml, 39 pg / ml, 38 pg / ml, 37 pg / ml, 36 pg / ml, 35 pg / ml, 34 pg / ml, 33 pg / ml, 32 pg / ml, 31 pg / ml, 30 pg / ml or less.

[0162] In some aspects, the antibodies described herein comprise a heavy chain variable region, wherein the heavy chain variable region comprises one or more complementarity determining region (CDRHs) CDRH1, CDRH2 and CDRH3 with amino acid sequences that have 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions in 1, 2 or 3 CDRHs having the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; and / or a light chain variable region comprising one or more complementarity determining region (CDRLs) CDRL1, CDRL2 and CDRL3 with the amino acid sequences that have 0, 1. 2, 3, 4, or 5 conservative amino acid substitutions in 1, 2 or 3 CDRLs having the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0163] In some aspects, the antibodies disclosed herein can prevent, inhibit or block SCUBE3 binding to TGFbR2 or TGFbRl and / or EGFR1. In some aspects, the antibodies disclosed herein can inhibit the binding of human SCUBE3 to human TGFbR2 or TGFbRl and / or EGFR1. In some aspects, the antibodies disclosed herein can neutralize SCUBE3. In some aspects, the antibodies disclosed herein can reduce tumor volume and prevent, reduce or inhibit tumor metastasis. In some aspects, the antibodies disclosed herein can prevent, inhibit or block tumor metastases generally and. in some aspects, specifically, for example, prevent, inhibit or block metastatic spread of cancers of the breast, brain, colon, pancreas, lung, skin including melanoma, thyroid, uterus, prostate, kidney, bladder, head and neck, and stomach.

[0164] Antibody proteins may be recombinant, or synthesized in vitro. It is contemplated that in antibody-containing compositions as described herein can comprise between about 0.001 mg and about 10 mg of total antibody polypeptide per ml. Thus, the concentration of antibody protein in a composition can be about, at least about or at most about or equal to 0.001, 0.010. 0.050, 0.1, 0.2. 0.3, 0.4, 0.5, 0.6. 0.7, 0.8, 0.9, 1.0. 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0. 6.5, 7.0, 7.5, 8.0. 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein). Of this, about, at least about, at most about, or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

[0165] 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49. 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

[0166] 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73. 74. 75. 76. 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,

[0167] 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% may be an antibody that binds

[0168] SCUBE3. Disclosed herein are compositions comprising any of the antibodies or isolated antibodies described herein. In some aspects, the compositions can further comprise at least one pharmaceutically acceptable carrier or diluent.

[0169] In some aspects, the compositions described herein can comprise a detectable label or reporter. An antibody or an immunological portion of an antibody that retains binding activity, can be chemically conjugated to, or recombinantly expressed as, a fusion protein with other proteins. For the purposes as described herein, all such fused proteins are included in the definition of antibodies or an immunological portion of an antibody. In some aspects, antibodies and antibody-like molecules generated against SCUBE3 or polypeptides that are linked to at least one agent to form an antibody conjugate or payload are encompassed. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety to the antibody. Such a linked molecule or moiety may be. but is not limited to, at least one effector, detectable label or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules that may be attached to antibodies include toxins, therapeutic enzy mes, antibiotics, radio-labeled nucleotides and the like. By contrast, a reporter molecule or detectable label is defined as any moiety that may be detected using an assay. Non-limiting examples of reporter molecules and detectable labels that can be conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin, and the like. Several methods are known in the art for attaching or conjugating an antibody to a conjugate molecule or moiety. Some attachment methods involve the use of a metal chelate complex, employing by way of nonlimiting example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3-6a- diphenylglycouril-3 attached to the antibody. Antibodies, particularly the monoclonal antibodies as described herein, may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are conventionally prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In some aspects, an antibody as described herein, particularly a binding fragment thereof, may be coupled or linked to a compound or substance, such as polyethylene glycol (PEG), to increase its in vivo half-life in plasma, serum, or blood following administration.

[0170] In some aspects, the antibodies described herein can be specifically bind to their intended target. In some aspects, the antibodies described herein have no off site binding.

[0171] METHODS

[0172] Disclosed herein are methods for treating cancer in a subject. The methods can comprise administering to the subject a therapeutically effective amount of one or more of the isolated antibodies described herein or any of the compositions described herein. In some aspects, the cancer can be breast cancer, brain cancer, colorectal cancer, lung cancer, melanoma, thyroid cancer, uterine cancer, prostate cancer, kidney cancer, bladder cancer, head or neck cancers, stomach cancer, or pancreatic cancer.

[0173] Also disclosed herein are methods of treating or reducing metastatic cancer in a subject or preventing metastasis in a subject having cancer at risk for metastasis. Also disclosed herein are methods of preventing reoccurrence of a tumor in a subject. In some aspects, the methods can comprise administering therapeutically effective amount of one or more of the isolated antibodies disclosed herein or any of the compositions disclosed herein. In some aspects, the cancer can be a cancer of breast, brain, colon, pancreas, lung, skin (including melanoma), thyroid, uterus, prostate, kidney, bladder, head and neck, or stomach. In some aspects, the subject has cancer. In some aspects, the subject has metastatic cancer. In some aspects, the subject can have cancer or be a cancer patient and is at risk for cancer metastasis. In some aspects, administration of any of the antibodies disclosed herein can reduce the number of metastases. In some aspects, administration of any of the antibodies disclosed herein can prevent the occurrence or reoccurrence of metastasis. In some aspects, administration of any of the antibodies disclosed herein can increase the subject’s or patient’s survival time. In some aspects, administration of any of the antibodies disclosed herein can prevent the reoccurrence of a tumor in the subject.

[0174] Disclosed herein are methods for reducing tumor volume in a subject. The methods can comprise administering to the subject a therapeutically effective amount of one or more of the isolated antibodies described herein or any of the compositions described herein. In some aspects, the tumor can be a cancer. In some aspects, the cancer can be breast cancer, brain cancer, colorectal cancer, lung cancer, melanoma, thyroid cancer, uterine cancer, prostate cancer, kidney cancer, bladder cancer, head or neck cancers, stomach cancer, or pancreatic cancer. In some aspect, the subject has a cancer. In some aspects, method can reduce the size of the tumor or the level of one or more tumor markers by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or more relative to the size of the tumor or the level of one or more tumor markers prior to administering the any of the isolated antibodies described herein or any of the compositions described herein to the subject. In some aspects, any of one of the tumor markers described herein can be detected using an antibody-based detection assay (e.g., ELISA), molecular amplification assay (e.g., PCR) or a suitable blood-based assay. In some aspects, the tumor marker can be Ki67, FOXM1, c-Myc, or a combination thereof.

[0175] In some aspects, the subject can be identified in need of treatment before the administering step. In some aspects, the antibody can be administered in a pharmaceutically acceptable composition. In some aspects, the antibody can be administered systemically, intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously or locally into inflamed tissues, organs or tumors.

[0176] In some aspects, the methods can further comprising administering one or more drugs or therapeutic agents to the subject. Examples of drugs or therapeutic agents that can be administered in combination with any of the antibodies described herein include but are not limited to oxorubicin, paclitaxel, docetaxel, 5-FU. gemcitabine, vincristine. Olaparib, Niraparib, Atezolizumab, Avelumab, Durvalumab; Pembrolizumab, Nivolumab, Cemiplimab, or a combination thereof.

[0177] The term “in combination” in the context of the administration of other therapies (e.g., other agents, cancer drugs, cancer therapies, immunosuppressants) includes the use of more than one therapy (e.g., drug therapy and / or cancer therapy and / or immunosuppressants). Administration “in combination with” one or more further therapeutic agents includes simultaneous (e.g., concurrent) and consecutive administration in any order. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. By way of nonlimiting example, a first therapy (e.g.. agent, such as an antibody disclosed herein that can bind SCUBE3) may be administered before (e.g.. 1 minute. 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks. 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (e.g.. 1 minute, 15 minutes, 30 minutes. 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer) the administration of a second therapy (e.g., agent) to a subject having or diagnosed with cancer.

[0178] In some aspects, the second therapy or agent that can be used in combination with drugs used to treat cancer include but are not limited to chemotherapy drugs, PARP inhibitors, and / or immunotherapy. In some aspects, the chemotherapy drugs can be doxorubicin, paclitaxel, docetaxel, 5-FU, gemcitabine, vincristine or a combination thereof. In some aspects, the PARP inhibitors can be Olaparib, Niraparib, or a combination thereof. In some aspects, the immunotherapy can be Atezolizumab, Avelumab, Durvalumab; Pembrolizumab, Nivolumab, Cemiplimab, or a combination thereof.

[0179] In some aspects, the combination of therapies (e.g., use of agents, including therapeutic agents) may be more effective than the additive effects of any two or more single therapy (e.g., have a synergistic effect). For example, a synergistic effect of a combination of therapeutic agents frequently permits the use of lower dosages of one or more of the agents and / or less frequent administration of the agents to a cancer patient. The ability to utilize lower dosages of therapeutics and cancer therapies and / or to administer the therapies less frequently reduces the potential for toxicity7associated with the administration of the therapies to a subject without reducing the effectiveness of the therapies. In addition, a synergistic effect may result in improved efficacy of therapies in the treatment or alleviation of a cancer. Also, a synergistic effect demonstrated by a combination of therapies (e g., therapeutic agents) may avoid or reduce adverse or unwanted side effects associated with the use of any single therapy.

[0180] In some aspects, any of the antibodies disclosed herein can improve the efficacy of one or more chemotherapy drugs, anti -cancer agents, or immunotherapy agents. In some aspects, the anti-cancer agent can be a PARP-inhibitor.

[0181] Treatment of diseases. Disclosed herein are antibodies or antigen binding fragments thereof, as described herein (e.g., an antibody that specifically and preferentially binds to SCUBE3 and blocks or inhibits binding of SCUBE3 to EGFR1 and / or TGFbR2 or TGFbRl) that can be used in treatment methods and administered to treat cancer, reduce tumor volume, or reduce or prevent metastatic cancer. Accordingly, provided herein are methods of treating cancer in a subject, reducing tumor volume in a subject, or treating or reducing metastatic cancer or preventing metastasis in a subject having cancer at risk for metastasis. In some aspects, the methods can comprise administering to a subject a therapeutically effective amount of one or more of the antibodies described herein or any of the compositions comprising at least one of antibodies as described herein. In some aspects, the drug or therapeutic agent can be an antibody or a composition comprising at least one antibody that bind to SCUBE3.

[0182] The compositions described herein can be administered to the subject (e.g., a human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of clinical disease. Accordingly, in some aspects, the patient can be a human patient. In therapeutic applications, compositions can be administered to a subject (e.g., a human patient) already with or diagnosed with a cancer in an amount sufficient to at least partially improve a sign or symptom or to inhibit the progression of (and preferably arrest) the symptoms of the disease or condition, its complications, and consequences. An amount adequate to accomplish this is defined as a “therapeutically effective amount.” A therapeutically effective amount of a composition (e g., a pharmaceutical composition) can be an amount that achieves a cure, but that outcome is only one among several that can be achieved. As noted, a therapeutically effective amount includes amounts that provide a treatment in which the onset or progression of the cancer is delayed, hindered, or prevented, or the cancer or a symptom of the cancer is ameliorated or its frequency can be reduced. One or more of the sy mptoms can be less severe. Recovery can be accelerated in an individual who has been treated. For example, treatment of cancer or metastatic cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the grow th rate of the cancer, or prevention of metastasis. Treatment of cancer or metastatic cancer may also refer to prolonging survival of a subject with cancer. In some aspects, the antibodies described herein can prolong the lifespan of a subject with cancer. In some aspects, the antibodies described herein can reduce or inhibit tumor cell growth.

[0183] Treatment of these subjects with an effective amount of at least one of the anti- SCRUBE3 antibodies as described herein can result in binding of one or more of the disclosed antibodies to SCUBE3, thereby preventing, blocking or inhibiting SCUBE3 from binding to its cognate receptor, TGFbR2 or TGFbRl and / or EGFR1, and thereby neutralizing SCUBE3. Accordingly, the methods as provided are advantageous for a subject who is in need of, capable of benefiting from, or who is desirous of receiving the benefit of, the anticancer results, the amelioration of one or more cancer symptoms results achieved by the practice of the present methods. A subject's seeking the therapeutic benefits of the methods involving administration of at least one antibody that binds to SCUBE3 in a therapeutically effective amount, or receiving such therapeutic benefits offer advantages to the art. In addition, the present methods offer the further advantages of eliminating or avoiding side effects, adverse outcomes, contraindications, and the like, or reducing the risk or potential for such issues to occur compared with other treatments and treatment modalities.

[0184] Cancers for which the present methods are useful include but are not limited to breast cancer, brain cancer, colorectal cancer, lung cancer, melanoma, thyroid cancer, uterine cancer, prostate cancer, kidney cancer, bladder cancer, head or neck cancers, stomach cancer, or pancreatic cancer.

[0185] Therapeutic compositions comprising antibodies are conventionally administered intravenously, such as by injection of a unit dose, for example. The term “unit dose” when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary7dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier, or vehicle. The compositions comprising any of the antibodies disclosed herein can be administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject’s system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimens for initial and booster administration are also contemplated and may typically involve an initial administration followed by repeated doses at one or more intervals (hours) by a subsequent injection or other administration. In some aspects, multiple administrations can be suitable for maintaining continuously high serum and tissue levels of antibody. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

[0186] It is contemplated that an antibody as described herein can be administered systemically or locally to treat disease (e.g.. cancer), such as to inhibit tumor cell growth, reduce tumor volume, or to kill cancer cells in cancer patients with cancer, locally advanced or metastatic cancers or at risk for metastatic cancers. The antibodies can be administered alone or in combination with anti-proliferative drugs or anticancer drugs. In some aspects, the antibodies can be administered to reduce the cancer load in the patient prior to surgery or other procedures. Alternatively, they can be administered at periodic intervals after surgery to ensure that any remaining cancer (e.g., cancer that the surgery failed to eliminate) is reduced in size or growth capacity and / or does not survive. As noted herein, a therapeutically effective amount of an antibody can be a predetermined amount calculated to achieve the desired effect. Thus, the dosage ranges for the administration of an antibody are those large enough to produce the desired effect in which the symptoms of tumor cell division and cell cycling are reduced. Optimally, the dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, neurological effects, and the like. Generally, the dosage will vary with age of, condition of, size and gender of, and extent of the disease in the subject or patient and can be determined by one of skill in the art such as a medical practitioner or clinician. Of course, the dosage may be adjusted by the individual physician in the event of any complication.

[0187] Treatment methods. In some aspects, the compositions and methods as described herein comprise the administration of an antibody as described herein, alone, or in combination with a second or additional drug or therapy. Such drug or therapy may be applied in the treatment of any disease that is associated with SCUBE3, and in some aspects, the interaction of human SCUBE3 or with human TGFbR2 or TGFbRl and / or EGFR1. For example, the disease can be cancer or metastatic cancer. The compositions and methods described herein can comprise at least one antibody that specifically binds to SCUBE protein and has a therapeutic or protective effect in the treatment of cancer or metastatic cancer, particularly by preventing, reducing, blocking, or inhibiting the SCUBE3 interaction with TGFbR2 or TGFbRl and / or EGFR1, thereby providing a therapeutic effect and treatment.

[0188] The compositions and methods, including combination therapies, have a therapeutic or protective effect and may enhance the therapeutic or protective effect, and / or increase the therapeutic effect of another drug, therapy or therapeutic agent (e.g., anti-cancer or anti- hyperproliferative therapy).

[0189] Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell, reducing tumor volume, and / or the inhibition of cellular hyperproliferation; reducing one or more symptoms of cancer or metastatic cancer). This process may involve administering an antibody or a binding fragment thereof that binds to SCUBE3and a second therapy. The second therapy may or may not have a direct cytotoxic effect. A tissue, tumor, and / or cell can be exposed to one or more compositions or pharmacological formulation(s) comprising one or more of the agents (e.g., an antibody or an anti-cancer agent), or by exposing the tissue, tumor, and / or cell with two or more distinct compositions or formulations, wherein one composition provides, for example, 1) an antibody, 2) an anti-cancer agent, 3) both an antibody and an anti-cancer agent, or 4) two or more antibodies. In some aspects, the second therapy can be also an antibody that binds to SCUBE3. Also, it is contemplated that such a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

[0190] By way of example, the terms “contacted” and “exposed,” when applied to a cell, are used herein to describe a process by which a therapeutic polypeptide, for example, an antibody as described herein, is delivered to a target cell or is placed in direct juxtaposition with the target cell, particularly to bind specifically to the target antigen, e.g.. SCUBE3. Such binding by a therapeutic antibody or binding fragment thereof prevents, blocks, inhibits, or reduces the interaction of SCUBE3 with TGFbR2 or TGFbRl and / or EGFR1, thereby neutralizing SCUBE3. In some aspects, a chemotherapeutic or radiotherapeutic agent can also be administered or delivered to the subject in conjunction with the antibody or binding fragment thereof that binds to SCUBE3. To achieve cell killing, for example, one or more agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[0191] Any of the antibodies disclosed herein may be administered before, during, after, or in various combinations relative to another treatment (e.g.. anti-cancer, or immunosuppressant agent). The administrations may be in intervals ranging from concurrently to minutes to days to weeks before or after one another. In some aspects, in which the antibody is provided to a patient separately from an anti-cancer agent or immunosuppressant agent, it would be generally ensured that a significant period of time did not expire between the time of each delivery, such that the administered compounds would still be able to exert an advantageously combined effect for the patient. Illustratively, in such instances, it is contemplated that one may provide a patient with the antibody and the anti-cancer therapy or immunosuppressant agent within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0192] In some aspects, a course of treatment or treatment cycle will last 1-90 days or more (this range includes intervening days and the last day). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days and the last day) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days and the last day) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there may be a period of time at which no second agent (e.g., anti-cancer treatment or immunosuppressant agent) is administered. This time period may last, for example, for 1-7 days, and / or 1-5 weeks, and / or 1-12 months or more (this such range includes intervening days and the upper time point), depending on the condition of the patient, such as prognosis, strength, health, etc. Treatment cycles would be repeated as necessary. Various combinations of treatments may be employed. In the representative examples of combination treatment regimens shown below, an antibody, such as an antibody or binding fragment thereof that binds to SCUBE3 is represented by “A” and an anti-cancer therapy is represented by “B”:

[0193] A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B

[0194] B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A

[0195] B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A.

[0196] Administration of any antibody or therapy as described herein to a patient will follow' general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some aspects there is a step of monitoring adverse events and toxicity, particularly those that may be attributable to combination therapy.

[0197] In some aspects, methods are disclosed comprising administering an antibody as described herein alone or in combination with another agent (e.g., anticancer agent or immunosuppressant agent) to a subject in need thereof, i.e., a subject with a cancer or a tumor). Prior to administration of the antibody, a sample of the subject’s tumor or cancer or one or more symptoms associated w ith the cancer may be evaluated for the presence or level of SCUBE3. If the results of such an evaluation reveals that the subject’s tumor or cancer or symptoms associated with the cancer is positive for SCUBE3 or the level of SCUBE3 is increased compared to a reference sample or prior sample from the same subject, the subject would be selected for treatment based on the likelihood that subject’s SCUBE3+ tumor or cancer or disease state or condition w'ould be more amenable to treatment with the antibody and treatment may proceed with a more likely beneficial outcome. A medical professional or physician may advise the subject to proceed with the antibody treatment method, and the subject may decide to proceed with treatment based on the advice of the medical professional or physician. In addition, during the course of treatment, the subject’s tumor or cancer cells or blood cells may be assayed for the presence of SCUBE3 as a way to monitor the progress or effectiveness of treatment. If the assay shows a change, loss, or decrease, for example, in SCUBE3 on the subject’s tumor or cancer cells or blood cells, a decision may be taken by the medical professional in conjunction with the subject as to whether the treatment should continue or be altered in some fashion, e.g., a higher dosage, the addition of another anticancer agent or therapy or immunosuppressant, and the like.

[0198] Chemotherapy. A wide variety of chemotherapeutic agents may be used in accordance with the treatment or therapeutic methods as described herein. The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” connotes a compound or composition that is administered in the treatment of cancer. Such agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle and cell growth and proliferation. Alternatively, a chemotherapeutic agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis in a cell.

[0199] Nonlimiting examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, cholophosphamide, estramustine. ifosfamide. mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin. and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids. such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'’-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ‘Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin. gemcitabine, navelbine, famesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Radiotherapy. Radiotherapy includes treatments with agents that cause DNA damage. Radiotherapy has been used extensively in cancer and disease treatments and embraces what are commonly known as y-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patent Nos. 5,760,395 and 4,870,287), and UV -irradiation. It is most likely that all of these factors affect a broad range of damage on DNA itself, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Exemplary dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, the uptake by the neoplastic cells, and tolerance of the subject undergoing treatment.

[0200] Immunotherapy. In some aspects of the methods, immunotherapies may be used in combination or in conjunction with administration of antibodies as described herein. In the context of cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. In some aspects, checkpoint inhibitors can also be administered in combination, including ipilimumab. The antibodies can be administered in combination with anti-PD-1 or anti-PD-Ll inhibitors, such as antibodies against PD-L1, which include atezolizumab, durvalumab, or avelumab, or antibodies against PD-1, including nivolumab, pembrolizumab, or pidilizumab. In addition, one or more of the antibodies as described herein can be administered in combination with each other. In some aspects, the antibodies can also be administered in combination with SCUBE3 inhibitors. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing or suppress the immune system. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target, e.g., the PD-1 on T-cells / PD-Ll on tumor cells interaction. Various effector cells include cytotoxic T cells and natural killer (NK) cells.

[0201] In some aspects of immunotherapy, the tumor cell must bear some marker (protein / receptor) that is amenable to targeting. Optimally, the tumor marker protein / receptor is not present on the majority of other cells, such as non-cancer cells or normal cells. Many tumor markers exist and any of these may be suitable for targeting by another drug or therapy administered with an antibody as disclosed herein. Common tumor markers include, for example, CD20, carcinoembryonic antigen (CEA), tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP. laminin receptor, erbB, and pl 55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist and include cytokines, such as IL -2, IL-4, IL-12, GM-CSF, gamma-IFN; chemokines, such as MIP-1, MCP-1, IL-8; and growth factors, such as FLT3 ligand.

[0202] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patent Nos. 5,801,005 and 5,739,169; Hui et al., 1998, Infection Immun., 66(l l):5329-5336; Christodoulides et al.. 1998, Microbiology, 144(Pt 11):3027- 3037); cytokine therapy, e.g., a. p, and y interferons; IL-1, GM-CSF. and TNF (Bukowski et al., 1998, Clinical Cancer Res., 4(10):2337-2347; Davidson et al., 1998, J. Immunother., 21(5):389-398; Hellstrand et al., 1998, Acta Oncologica, 37(4):347-353); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998, Proc. Natl. Acad. Sci. USA, 95(24): 14411-14416; Austin-Ward and Villaseca, 1998, Revista Medica de Chile. 126(7):838-845; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-pl 85 (Hollander, 2012, Front. Immun., 3:3; Hanibuchi et al., 1998, Int. J. Cancer, 78(4):480-485; U.S. Patent No. 5,824,311). It is contemplated that one or more anticancer therapies may be employed with the antibody therapies described herein.

[0203] Surgery. Approximately 60% of individuals with cancer undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery7includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conj unction with other therapies, such as antibody treatment as described herein, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies, as well as combinations thereof. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cry osurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery). Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by' perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every71, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of vary ing dosages as well.

[0204] Protein Purification. Protein, including antibody and, particularly, an antibody that binds to SCUBE3, purification techniques are well known to those of skill in the art. These techniques involve, at one level, the homogenization and crude fractionation of the cells, tissue, or organ into polypeptide and non-polypeptide fractions. The protein or polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity) unless otherwise specified. Analytical methods particularly suited to the preparation of a pure protein or peptide are ion-exchange chromatography, size-exclusion chromatography, reverse phase chromatography, hydroxyapatite chromatography, polyacry lamide gel electrophoresis, affinity chromatography, immunoaffinity' chromatography, and isoelectric focusing. A particularly efficient method of purifying peptides is fast-performance liquid chromatography (FPLC) or even high-performance liquid chromatography (HPLC). As is generally known in the art, the order of conducting the various purification steps may7be changed, and / or certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified polypeptide.

[0205] A purified polypeptide, such as an antibody as described herein, refers to a polypeptide which is isolatable or isolated from other components and purified to any degree relative to its naturally-obtainable state. An isolated or purified polypeptide, therefore, also refers to a polypeptide free from the environment in which it may naturally occur, e.g., cells, tissues, organs, biological samples, and the like. Generally, “purified’" will refer to a polypeptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. A “substantially purified’’ composition refers to one in which the polypeptide forms the major component of the composition, and as such, constitutes about 50%, about 60%. about 70%, about 80%. about 90%, about 95%, or more of the protein component of the composition.

[0206] Various methods for quantifying the degree of purification of polypeptides, such as antibody proteins, are know n to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS / PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity therein, assessed by a “fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification, and whether or not the expressed polypeptide exhibits a detectable activity.

[0207] There is no general requirement that the polypeptide will always be provided in its most purified state. Indeed, it is contemplated that less substantially purified products may have utility in some aspects. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “fold” purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

[0208] Affinity' chromatography is a chromatographic procedure that relies on the specific affinity between a substance (protein) to be isolated and a molecule to which it can specifically bind, e.g., a receptor-ligand type of interaction. The column material (resin) is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution that is passed over the column resin. Elution occurs by changing the conditions to those in which binding will be disrupted / will not occur (e.g., altered pH, ionic strength, temperature, etc.). The matrix should be a substance that does not adsorb molecules to any significant extent and that has a broad range of chemical, physical, and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding; however, elution of the bound substance should occur without destroying the sample protein desired or the ligand.

[0209] Size-exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated based on their size, or in more technical terms, their hydrodynamic volume. It is usually applied to large molecules or macromolecular complexes, such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates, resulting in the separation of a solution of particles based on size. Provided that all of the particles are loaded simultaneously or near simultaneously, particles of the same size should elute together.

[0210] High-performance (aka high-pressure) liquid chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used

[0211] Pharmaceutical Preparations. Where clinical application of a pharmaceutical composition comprising an antibody as disclosed herein is undertaken, it is generally beneficial to prepare a pharmaceutical or therapeutic composition appropriate for the intended application. In general, pharmaceutical compositions can comprise an effective amount of one or more polypeptides or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. In some aspects, pharmaceutical compositions may comprise, for example, at least about 0.1% of a polypeptide or antibody. In some aspects, a polypeptide or antibody may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable there between, including the upper and lower values. The amount of active compound(s) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose. Factors, such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations, are contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0212] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient (e.g.. an isolated antibody as described herein, including, but not limited to an antibody that binds to SCUBE3) to be effective, and which contains no additional components that would be are unacceptably toxic to a subject to whom the formulation would be administered. Such a formulation can be sterile, i.e.. aseptic or free from all living microorganisms and their spores, etc.

[0213] Further in some aspects, the composition suitable for administration can be provided in a pharmaceutically acceptable carrier with or without an inert diluent. As used herein, the term '‘pharmaceutically acceptable” or “pharmacologically acceptable” refers to molecular entities, formulations and compositions that do not produce an adverse, allergic, or other untoward or unwanted reaction when administered, as appropriate, to an animal, such as a human. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient are known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, Id. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by a regulatory agency of the Federal or a state government, such as the FDA Office of Biological Standards or as listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly, in humans.

[0214] As used herein, the term “carrier” includes pharmaceutically acceptable carriers, excipients, diluents, vehicles, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, succinate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, sucrose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. The term “carrier” can also refer to a diluent, adjuvant (e.g., Freund’s adjuvant, complete or incomplete), excipient, or vehicle with which the therapeutic is administered. Such carriers, including pharmaceutical carriers, can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is an exemplary carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include, without limitation, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral compositions, including formulations, can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences. (1990) Mack Publishing Co., Easton, PA. Compositions, including pharmaceutical compounds, can contain a therapeutically effective amount of an antibody that binds to SCUBE3 in isolated or purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject (e.g., patient). The composition or formulation should suit the mode of administration.

[0215] As used herein, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc ), fatty7acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g.. SDS. polysorbate, nonionic surfactant, etc.), saccharides (e.g.. sucrose, maltose, trehalose, etc.) and polyols (e.g.. mannitol, sorbitol, etc.). See, also, for reference. Remington’s Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, PA, which is hereby incorporated by reference in its entirety'.

[0216] In some aspects, the carrier should be assimilable and include liquid, semi-solid, e.g.. gels or pastes, or solid carriers. Examples of carriers or diluents include but are not limited to fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, ethanol, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g.. propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, inert gases, parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal), isotonic agents (e.g., sugars, sodium chloride), absorption delaying agents (e.g., aluminum monostearate, gelatin), salts, drugs, drug stabilizers (e.g., buffers, amino acids, such as glycine and lysine, carbohydrates, such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.), gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary' skill in the art. Except insofar as any conventional media, agent, diluent, or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in an administrable composition for the practice of the methods is appropriate. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters. In some aspects, the composition can be combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption, grinding, and the like. Such procedures are routine for those skilled in the art.

[0217] In some aspects, the compositions may comprise different ty pes of carriers depending on whether they are to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. The compositions can be formulated for administration intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e g., liposomes), or by other methods or any combination of the forgoing as would be know n to one of ordinary' skill in the art. See, for example, Remington 's Pharmaceutical Sciences, 18th Ed.. 1990. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid or reconstitutable forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0218] The antibodies may be formulated into a composition in a free base, neutral, or salt form. Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, or mandelic acid. Salts formed with the free carboxyl groups may also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine, or procaine. In some aspects, a pharmaceutical lipid vehicle composition that includes polypeptides, one or more lipids, and an aqueous solvent may be used. As used herein, the term “lipid” refers to any of a broad range of substances that are characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well know n to those of skill in the art, and as the term “lipid” is used herein, it is not limited to any particular structure. Examples include compounds that contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether- and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods. One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the antibody may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

[0219] The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantify of the therapeutic antibody or composition containing the therapeutic antibody calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantify to be administered, both according to number of treatments and unit dose, depends on the effect desired. The actual dosage amount of a composition as described herein that can be administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the subject, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. In other nonlimiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 milligram / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 milligram / kg / body weight to about 100 milligram / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above. The foregoing doses include amounts between those indicated and are intended to also include the lower and upper values of the ranges. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0220] The particular nature of the therapeutic composition or preparation is not intended to be limiting. For example, suitable compositions may be provided in formulations together with physiologically tolerable liquid, gel, or solid carriers, diluents, and excipients. In some aspects, the therapeutic preparations may be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual subjects, as described supra.

[0221] SCUBE3 as a biomarker. Disclosed herein are methods comprising the use of at least one antibody as described herein. In some aspects, the methods can comprise detecting the amount of level of SCUBE3 gene or protein in a sample obtained from a subject who has a cancer or tumor or is exhibiting one or more symptoms of cancer. Such methods may be useful in biomarker evaluations of the level of SCUBE3 in a sample obtained from a subject who has a cancer or tumor or is exhibiting one or more symptoms of cancer. For example, if the subject's sample is tested and determined to comprise a higher level of SCUBE3 compared to a reference sample, then the subject is a candidate for treatment with an antibody as described herein, alone, or in combination with another agent, for example, would benefit from the treatment. Such methods comprise obtaining a sample from a subject having a cancer or tumor (or exhibiting one or more symptoms of cancer), testing the sample for the presence of SCUBE3 derived from the subject’s sample using binding methods known and used in the art and as described herein, for example, using an antibody as described herein, and administering to the subject an effective amount of an antibody alone, or in combination with another agent, if the subject’s sample is found to have a higher level of SCUBE3 when compared to a reference sample. Diagnosing the subj ect as having a cancer or tumor or metastatic cancer prior to treatment allows for more effective treatment and benefit to the subj ect, as the administered antibody is more likely to block or inhibit the interaction of the subject’s SCUBE3 with the subject’s TGFbR2 or TGFbRl and / or EGFR1, thereby neutralizing SCUBE3. In some aspects, the methods can involve first selecting a subject whose cancer or tumor or disease state or condition may be amenable to testing for the presence of SCUBE3 levels.

[0222] Similar methods may be used to monitor the presence of SCUBE3 levels during a course of treatment or therapy, including combination treatments with an antibody that binds to SCUBE3and another anticancer drug or treatment or another immunosuppressant, over time, as well as after treatment has ceased. Such methods may also be used in companion diagnostic methods in which a treatment regimen or combination treatment, involves testing or assaying a sample obtained from the subject for SCUBE3 levels, prior to treatment and during the course of treatment, e.g., monitoring, to determine a successful outcome or the likelihood thereof.

[0223] Other Agents. It is contemplated that other agents may be used in combination with certain aspects of the compositions and methods disclosed herein to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions may increase the anti -hyperproliferative effects on the neighboring hyperproliferative cell population. In some aspects, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiments to improve the anti- hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.

[0224] In some aspects, antibodies and polypeptides as described herein may be conjugated to a marker, such as a peptide, to facilitate purification. In some aspects, the marker can be a hexa-histidine peptide, i.e., the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I. A. et al.. Cell, 37:767-778 (1984)), or the “flag” tag (Knappik, A. et al., Biotechniques 17(4):754-761 (1994)).

[0225] In some aspects, a moiety conjugated to the antibodies and polypeptides as described herein can be an imaging agent that can be detected in an assay. Such imaging agents can be enzymes, prosthetic groups, radiolabels, nonradioactive paramagnetic metal ions, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, bioluminescent molecules, photoaffinity molecules, or colored particles or ligands, such as biotin. In some aspects, suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials include, but are not limited to, luminol; bioluminescent materials include, but are not limited to, luciferase, luciferin, and aequorin; radioactive materials include, but are not limited to, bismuth (213Bi), carbon (14C), chromium (51Cr), cobalt (57Co), fluorine (18F), gadolinium (153Gd,159Gd), gallium (S8Ga,67Ga). germanium (68Ge). holmium (166Ho), indium (115In,113In,112In,inIn), iodine (131I,125I,123I,121I), lanthanium (140La), lutetium (177Lu), manganese (54Mn), molybdenum ("Mo), palladium (103Pd), phosphorous (32P), praseodymium (142Pr), promethium (149Pm), rhenium (186Re,188Re), rhodium (105Rh), ruthemium (97Ru), samarium (153Sm), scandium (47Sc), selenium (75Se), strontium (85Sr), sulfur (35S), technetium ("Tc), thallium (201Ti), tin (113Sn.117Sn), tritium (3H), xenon (133Xe). ytterbium (169Yb,175Yb), yttrium (90Y), zinc (65Zn); positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.

[0226] The imaging agent can be conjugated to the antibodies or polypeptides described herein either directly or indirectly through an intermediate (such as. for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 which reports on metal ions that can be conjugated to antibodies and other molecules as described herein for use as diagnostics. Some conjugation methods involve the use of a metal chelate complex employing, for example, an organic chelating agent, such as diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and / or tetrachloro-3-6a-diphenylglycouril-3, attached to the antibody. Monoclonal antibodies can also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers can be prepared in the presence of these coupling agents or by reaction with an isothiocyanate.

[0227] In some aspects, the antibodies or polypeptides described herein can also be attached to solid supports, which can be useful for carrying out immunoassays or purification of the target antigen or of other molecules that are capable of binding to the target antigen that has been immobilized to the support via binding to an antibody or antigen binding fragment as described herein. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polysty rene, polyvinyl chloride or polypropylene.

[0228] KITS AND DIAGNOSTICS

[0229] Disclosed herein are kits comprising therapeutic agents and / or other therapeutic and delivery' agents. In some aspects, the kits can be used for preparing and / or administering a therapy involving the antibodies described herein. The kits can comprise one or more sealed vials containing any of the pharmaceutical compositions as described herein. The kits can include, for example, at least one antibody disclosed herein, as well as reagents to prepare, formulate, and / or administer one or more antibodies disclosed herein or to perform one or more steps of the described methods. In some aspects, the kits can also comprise a suitable container means, which is a container that will not react with components of the kit, such as an Eppendorf tube, an assay plate, a syringe, a bottle, or a tube. The container may be made from sterilizable materials, such as plastic or glass.

[0230] The kits can further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill. The instruction information may be in a computer readable medium containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of the therapeutic agent. EXAMPLES

[0231] Example 1: Identification, characterization and anti-tumor effects of isolated antibodies

[0232] Materials and Methods. Loss of Function Screening. Genome-wide loss of function screen was performed using Mission Lentiplex pooled shRNA (against the annotated genes; each gene is targeted by at least 3 or more unique barcoded shRNAs) library to identify genes that may give survival advantage and also sensitize doxorubicin (DOX) response in triple negative breast cancer (TNBC) cells. The pooled viral library was stably infected into MDA- MB-231 cells and subjected to vehicle or doxorubicin treatment for seven days. The DNA from viable cells were pooled and ultra-deep sequenced using barcode-specific primers. Candidate genes were prioritized for validation by functional and interaction assays using sample t-test (with pooled variance), using Benjamini -Hochberg method of controlling the False Discovery Rate (FDR < 0.05) and by using the magnitude of response (lowest 2.5 percentile of the distribution of the meandrug / meancaiTier ratios). The screen resulted in 133 genes that were targeted by more than 5 shRNAs and the shRNAs tested inhibited the viability7of TNBC cells. Of those 133 genes, genes that are reported to encode for secretory7proteins were the focus of this study. One of those gene was SCUBE3. The six shRNAs against SCUBE3 showed the highest magnitude of difference in cell viability of TNBC in DOX-treated TNBC cells compared to untreated cells.

[0233] Cell viability assays. Breast cancer cells were seeded in 96-well plates at a density7of 2500 cells / well. After 24 h, cells were treated with vehicle or vary ing concentrations of drugs / antibodies for 96 h. Cell viability was measured using CellTiter-Glo assays (Promega Inc.)

[0234] Colony formation assays. Cells were plated in a 6-well plate at very7low density (1000 or 2500 cells per well). For antibody studies, cells were treated with vehicle or antibodies for 7 days, replenishing the media every 3 days. The plates were fixed and stained with 0.5% Crystal Violet in 20% methanol. Colonies were imaged and the number of colonies were counted using ImageJ software.

[0235] IncuCyte ZOOM™ Assay. To measure cell confluency, cells were plated in a 96-well plate at a very low density of 1000 cells / well in 100 pl complete growth medium. The plate was then incubated into the IncuCyte ZOOM™ apparatus and images of the cells were recorded every74 hours for a total duration of one week. Invasion and migration assays. About 2.5,000 MDA-MB-231 Scrambled or ShScube3 cells were added to the top chamber of the transwell and plates were placed in a 5% CO2 incubator overnight. For invasion assay, cells were added to the top well coaled with the matrigel of the invasion chamber. Migrated / invaded cells were fixed with 4% paraformaldehyde, and stained with 0.5% crystal violet. Migrated / invaded cells were visualized and counted under the microscope.

[0236] Gene-expression profiling using RNA sequencing and data analysis. Total RNA was isolated from MDA-MB-231 Scrambled and ShScube3 cells. Six libraries were generated (3 replicates for each group) from total RNA using the TruSeq Stranded mRNA Library- Preparation kit according to the manufacturer's protocol (Illumina Inc.) by the GCCRI Genome Sequencing Facility. Samples were sequenced on the Illumina HiSeq 3000 platform (Illumina Inc.) using the 50 base-pair single-read (50SR) sequencing module.

[0237] Sequence reads were mapped to the University of California, Santa Cruz genome build hg!9 using TopHat2 aligner (v2.0.6) with default parameters for stranded RNA. Gene expression was measured by HTSeq, and differential analyses were performed using DESeq. Up-regulated and down-regulated genes were determined by the following criteria: (i) absolute log2 fold change >1, (ii) average reads per kilobase per million mapped reads >1, and (iii) multiple-test adjusted (Benjamini -Hochberg) P values < 0.05. Using the differential expression fold-change, Gene Set Enrichment Analysis (GSEA) was also performed with the stand-alone package from the Broad Institute (v4.0.3) or the implementation provided by ClusterProfiler package (R / Bioconductor). The top 15 pathways / gene sets were selected from mSigDB (v7. 1).

[0238] Further analyses used the Database for Annotation, Visualization and Integrated Discovery (DAVID) to determine changes in molecular functions using Hallmark analysis; results were visualized using Adobe Illustrator.

[0239] Protein analysis using western blots. Cells were harvested and lysed using RIPA buffer containing halt protease and phosphatase inhibitor (Pierce). Media supernatant from the cell culture was collected and concentrated using protein concentrator of 10K molecular weight cutoff. SDS-PAGE and Western blots were performed to quantity- the protein levels, using P-actin or transferrin as a loading control.

[0240] Propidium iodide-based cell cycle analyses. For cell cycle analysis, breast cancer cells were harvested and fixed with 70% ethanol for 24 h and stained with propidium iodide. Live cells were subjected to cell cycle distribution using a BD LSRFortessa™ X-20 instruments. Data were processed using BD FACSDiva™ or FlowJo vl 0.7.2 software and visualized using a GraphPad Prism program.

[0241] Proximity Ligation Assay. MDA-MB-231 cells were co-transfected with EGFR-Myc and EGFR-GFP expressing plasmids. After transfection cells were treated with either PBS. EGF (10 nM for 20 mins), recombinant Scube3 protein (1 pg for 1 hr) or erlotinib (10 pM for 20 mins). After treatment cells were fixed with 3.7% paraformaldehyde and blocked with the blocking solution supplied by the Duolink PLA kit (Sigma- Aldrich) according to the manufacturer's instructions. Briefly, the fixed cells were incubated with anti-MYC or anti- GFP antibody overnight. After multiple washings, cells were incubated successively with PLA probes, ligation solution and amplification solution at 37 °C. Cover-slips were mounted and the images were examined using a fluorescent microscope.

[0242] Enzyme-Linked Immunosorbent Assays. ELISA was performed to test the presence of anti-SCUBE3 antibodies in the cell culture supernatant. Briefly. ELISA plates were coated with 1 pg / ml of recombinant Scube3 protein and incubated overnight. Next day plate was washed 3X with wash buffer (0.05% Tween20 in PBS) and incubated for 1 hr at room temperature with reagent diluent (l%BSA in PBS). After blocking plate was washed again 3X and incubated with samples, negative and positive controls (1 : 12000 anti-Scube3 Ab, sc- 514696) for 2 hrs at room temperature after which the samples were probed with anti- mouse / biotinylated detection antibody for 1 hr. Samples were then treated with Streptavidin- HRP (1:5000) for 20 mins followed by incubation with substrate solution (A:B, 1 :1) for 20 mins at room temperature in dark. Plate was read in a spectrophotometer at 650 nm wavelength.

[0243] Hybridoma culture. Hybridoma culture w as performed at Hybridoma Core Facility of University of Michigan. Briefly, to generate clones secreting monoclonal Scube3 antibody, 3 BALB / c mice were immunized with recombinant human Scube3 protein and presence of antibodies against Scube3 was determined by performing ELISA on serum of those animals. Spleen was isolated from the mouse that showed presence of anti-Scube3 antibodies and B cells were fused with myeloma cells to form hybridoma. Secretion of anti-Scube3 antibodies in hybridoma clones was determined by ELISA and they were further subclones and expanded. Top 3 clones were picked based on secretion and functional assay of the anti- Scube3 antibodies.

[0244] Animal studies. Female athymic nude mice (4-6 weeks old) were obtained from Jackson Laboratories and housed under recommended conditions. For orthotopic tumor xenograft assays, 2 * 106MDA-MB-231 cells (Scrambled or ShSCUBE3 / SUCBE3 OE) were mixed with an equal volume of Matrigel® and implanted into the mammary fat pads of athymic nude mice.

[0245] Once the tumor reached 250 mm3mice were randomized into control and treatment groups (n = 7 each) and then treated with either vehicle (DMSO in PBS) or Doxorubicin (2.5 mg / kg for Scrambled and 1 mg / kg for ShScube3, injected intraperitoneally) every 3 days.

[0246] To study the therapeutic efficacy of anti-Scube3 antibodies, MDA-MB-231 cells were implanted to form tumors. Once the tumor reached 100 mm3antibodies were administered either intratumorally or intraperitoneally to the animals. Tumor volumes and body weight were measured twice a week.

[0247] Ki-67 staining. Formalin-fixed and paraffin-embedded orthotopic xenograft tumor tissues were deparaffinized and stained with an antibody against Ki-67 (1:400 dilution, Cat # CRM325C, Biocare Medical). Slides were visualized using DAB and counterstained with hematoxylin, and then imaged using a MoticEasyScan digital slide scanner (Motic) at 20x magnification. The average number of Ki-67 -positive cells was determined from ten arbitrarily selected fields and then calculated as percent of Ki-67-positive cells after normalization to the control group.

[0248] Results. Scube3 promotes tumor progression and is associated with doxorubicin resistance in TNBC. To identify and characterize a more effective, and less toxic therapeutic targets for treating TNBCs, genome-wide loss of function screens were performed using Mission Lentiplex pooled shRNA (against the annotated genes; each gene is targeted by at least 3 or more unique barcoded shRNAs) library. The pooled viral library was stably infected into MDA-MB-231 cells and subjected to vehicle or doxorubicin treatment for seven days. The DNA from viable cells were pooled and ultra-deep sequenced using barcodespecific primers. Candidate genes were prioritized for validation by functional and interaction assays using sample t-test (with pooled variance), using Benjamini -Hochberg method of controlling the False Discovery Rate (FDR < 0.05) and by using the magnitude of response (lowest 2.5 percentile of the distribution of the meandrug / rneancamer ratios). The screen resulted in 133 genes that were targeted by more than 5 shRNAs and shRNAs tested inhibited the viability of TNBC cells. Of those 133 genes, that genes that were reported to encode for secretory proteins were the focus. One of those gene was SCUBE3. The six shRNAs against SCUBE3 showed the highest magnitude of difference in cell viability of TNBC in DOX- treated TNBC cells compared to untreated cells (FIG. 1 A). These results were further validated in secondary screen showing DOX sensitization (~10-fold) of TNBC cells with SCUBE3 shRNA (FIGS. IB, C). To examine the functional significance of SCUBE3 in TNBC, SCUBE3 was silenced and the rate of proliferation, long-term viability, migration and invasion ability of TNBC cells (MDA-MB-231 and MDA-MB-468) were tested. Depletion of SCUBE3 resulted in significantly reduced proliferation, long-term viability and migratory and invasive capabilities of TNBC cells (FIGS. 1D-F). To test SCUBE3’s tumor promoting activity, stably transfected MDA-MB-231 cells were injected with scrambled and SCUBE3 shRNA into mammary fat pad. As shown in FIGS. 1G, H, SCUBE3 shRNA expressing tumors were markedly smaller than control scrambled-ShRNA tumors, while SCUBE3 overexpression increased the tumor growth. Further, it was observed that SCUBE3 inhibition improves the efficacy of DOX in orthotopic xenograft model (FIG. II).

[0249] ScubeS Interacts with EGFR1 and TGFftR and activates EGFR dimerization as well as EGFRGGFbR activity and levels. The results show pro-tumorigenic activity of SCUBE3; and since SCUBE3 is a secreted protein, its receptor on TNBC cells was identified. For example, a close inspection of SCUBE3 structure revealed multiple EGF-like domains suggesting its potential interaction with EGFR. Indeed, immunoprecipitation analysis revealed that SCUBE3 interacts with EGFR1 (FIG. 2A). This is a significant finding as SCUBE3 will be a ligand of EGFR1. In addition, a previous study reported SCUBE3’s interaction with TGFpRII in lung cancer cells. To test that, immunoprecipitation was performed. Interestingly. SCUBE3 interacted with TGFpRII but also with TGFpRI (Fig. 2B). Since EGFR ligands are known to dimerize and activate EGFR, i it was tested whether SCUBE3 may also dimerize and activate EGFR. For dimerization, proximity ligation assay in TNBC cells transfected with Myc-tagged and GFP-tagged EGFR expression constructs was performed. Cells were then probed with antibodies against GFP and Myc followed by corresponding secondary antibodies coupled to PLA probes. Annealing of the PLA probes occurs when two EGFR monomers are in close proximity, and repeat sequences in the annealed oligonucleotide complexes are amplified and then recognized by a fluorescently labeled oligonucleotide probe. As shown in FIG. 2C, recombinant SCUBE3 treatment resulted in increased annealing of PLA probes resulting in intense yellow signal compared to PBS treated cell indicating EGFR dimerization. EGF served as the positive control, while erlotinib (EGFR inhibitor) served as the negative control. Next, it was tested whether SCUBE-induced EGFR dimerization affects its activity . Indeed, silencing of SCUBE3 resulted in significantly reduced levels of phospho-EGFR in TNBC cells (FIG. 2D). Furthermore, SCUBE3 silencing reduced the levels of phospho SMAD suggesting inhibition of TGF signaling (FIG. 2E). Activation of EGFR results in the induction of MAPK / ERK signaling. Similarly, TGFpRII / TGFpRI interaction induces PI3K / AKT-ERK pathway. Consistent with that, SCUBE3 silencing inhibited phospho ERK (pERK), while SCUBE3 overexpression induced pERK level in TNBC cells (FIGS. 2F,G). Interestingly, SCUBE3 knockdown resulted in reduced expression of both EGFR and TGFbR. Furthermore, EGFR ligand (like EGF) is known to induce the expression of EGFR by inducing the expression / recruitment of specific transcription factors to EGFR promoter. These observations further attest that SCUBE3 can induce EGFR / TGFbR-dependent signaling by promoting their expression and activity.

[0250] SCUBE3 regulates expression of pro-growth / survival genes. To understand the mechanism by which SCUBE3 may regulate TNBC growth, gene expression analysis was performed on TNBC cells stably silenced for SCUBE3. Ingenuity Pathway Analysis (IP A) revealed that G2M checkpoint and DNA repair-associated genes were downregulated. while IFNy, inflammatory response and immunity-associated pathways were highly upregulated in SCUBE3-silenced TNBC cells (FIG. 3A). Genes associated with DNA damage repair pathways showed highly decreased levels in SCUBE3-silenced TNBC cells (compared to scrambled control) which included F0XM1, c-Myc, RAD51 and Exol (FIGS. 3B, C). SCUBE3 depletion also showed cell cycle arrest at G2 / M checkpoint in TNBC cells (FIG. 3D).

[0251] Generation and testing the potency of SCUBES-specific monoclonal antibody. High expression of secreted SCUBE3 protein in drug resistant cells and in TNBC patients as well as secreted SCUBE3-mediated activation of EGFR / TGF0R signaling prompted led to testing whether blocking the SCUBE3 activity by SCUBE3 -specific antibody may have anti-tumor effects. As shown in FIG. 4 A, two anti-SCUBE3 monoclonal antibody clones (IG10.3 and 9G5. 12) were identified after screening of 375 hybridoma clones, followed by validation using ELISA using recombinant SCUBE3 protein and long term colony formation assay (FIG. 4B,C). The reduction in colony formation ability of TNBC cells by the antibodies treatment was observed even after the clones were expanded (FIG. 4D).

[0252] Anti-SCUBE3 antibody inhibits tumor growth in vivo. To test the therapeutic efficacy of the anti-SCUBE3 antibody in animal models, large volume of antibodies was generated from mouse ascites by inoculating hybridoma clone 9G5. 12 in mouse peritoneal cavity. The results demonstrate that antibody isolated from mouse ascites retained the ability to suppress the long-term colony forming ability’ of MDA-MB-231 cells (FIG. 5 A). It was also observed that the antibody can bind to cleaved SCUBE3 in the cell culture media (FIG. 5B). Next, the effects of the antibody on EGFR and TGFpR signaling was assessed. The results show that reduced levels of phosphorylated ERK (dow nstream target of EGFR and TGFPR signaling) with antibody treatment (FIG. 5C). To evaluate the in vivo therapeutic efficacy of antibody, tumor bearing nude mice were injected intratumorally with antibody. A remarkable reduction in tumor growth (FIGS. 5D-F) and reduction of expression of Ki67 proliferation markers in tumors (FIG. 5G) was observed with antibody treatment. Next, monoclonal IgG antibodies were isolated from HEK cell culture expressing 9G5. 12 and IG10.3 hybridoma clones. It was observed that both the antibodies had better effect in inhibiting cell growth compared to Erlotinib, which is a FDA approved EGFR inhibitor (FIG. 5H). Importantly, a significant reduction in tumor growth and progression in mice was observed upon systemic injections of both 9G5.12 and IG10.3 hybridoma clones (FIG. 51). To further support the therapeutic potential of 9G5.12, TNBC patient-derived xenograft model was used as a second preclinical model. Sections from TNBC patient derived xenograft were implanted into NOD-SCID mice. Once the tumor started growing, mice were treated with 200 pg / mouse of 9G5. 12 antibody twice a w eek. The results shown in FIG. 5J demonstrate that 9G5. 12 treatment resulted in significant inhibition of PDX tumors compared to IgG control providing evidence that the efficacy of 9G5. 12 can be used as a therapeutic.

[0253] Collectively, these data identify SCUBE3 as a ligand for EGFR. Furthermore, the data demonstrating SCUBE3 interactions with EFGRl / TGFpRII supporting TNBC growth provide evidence that an antibody blocking these interactions can have better clinical outcomes for treating TNBC patients than currently available therapeutic agents. Given that SCUBE3 knockout mice are viable, SCUBE3 antibody-based therapy will likely be safe and will have better clinical outcomes for treating cancers with overactive EGFR1 and / or TGFpR-associated signaling in general and TNBCs in particular. Furthermore, secreted SCUBE3 can serve as a biomarker for predicting therapy response in TNBC.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An isolated antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a complementarity determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 1; a complementarity7determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 2; and a complementarity determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 3; and wherein the heavy chain variable region comprises a complementarity7determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 5; a complementarity7determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 6; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 7.

2. The isolated antibody of claim 1, comprising a light chain variable region amino acid sequence of SEQ ID NO: 4.

3. The isolated antibody of claim 1. comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 8.

4. An isolated antibody comprising a light chain variable region amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region amino acid sequence of SEQ ID NO: 8.

5. The isolated antibody of claim 1, wherein the light chain variable region has an amino acid sequence that is at least 90% identical to amino acid sequence of SEQ ID NO: 4.

6. The isolated antibody of claim 1, wherein the heavy chain variable region has an amino acid sequence that is at least 90% identical to amino acid sequence of SEQ ID NO: 8.

7. An isolated antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a complementarity7determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 9; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 10; and a complementarity determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 11; and wherein the heavy chain variable region comprises a complementarity determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 13; a complementarity determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 14; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 15.

8. The isolated antibody of claim 7, comprising a light chain variable region amino acid sequence of SEQ ID NO: 12.

9. The isolated antibody’ of claim 7, comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 16.

10. An isolated antibody comprising a light chain variable region amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region amino acid sequence of SEQ ID NO: 16.

11. The isolated antibody of claim 10, wherein the light chain variable region has an amino acid sequence that is at least 90% identical to amino acid sequence of SEQ ID NO: 12.

12. The isolated antibody of claim 10, wherein the heavy chain variable region has an amino acid sequence that is at least 90% identical to amino acid sequence of SEQ ID NO: 16.

13. An isolated antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a complementarity' determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 1; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 2; and a complementarity determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 3; and wherein the heavy chain variable regioncomprises a complementarity determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 5; a complementarity determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 6; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO:

7. wherein one or more of the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 comprise 1, 2, 3, 4, or 5 conservative amino acid substitutions.

14. An isolated antibody comprising a light chain variable region amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region amino acid sequence of SEQ ID NO: 8, wherein the isolated antibody comprises 1, 2, 3, 4, or 5 conservative amino acid substitutions in the light or heavy chain variable region amino acid sequences.

15. The isolated antibody of claim 1, 4, 5, or 6, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises one or more complementarity determining region (CDRHs) CDRH1. CDRH2 and CDRH3 with amino acid sequences that have 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions in 1, 2 or 3 CDRHs having the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; and / or wherein the antibody has a light chain variable region comprising one or more complementarity determining region (CDRLs) CDRL1. CDRL2 and CDRL3 with the amino acid sequences that have 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions in 1, 2 or 3 CDRLs having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

16. An isolated antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a complementarity determining region light chain 1 (CDRL1) amino acid sequence of SEQ ID NO: 9; a complementarity determining region light chain 2 (CDRL2) amino acid sequence of SEQ ID NO: 10; and a complementarity determining region light chain 3 (CDRL3) amino acid sequence of SEQ ID NO: 11 ; and wherein the heavy7chain variable region comprises a complementarity determining region heavy chain 1 (CDRH1) amino acid sequence of SEQ ID NO: 13; a complementarity determining region heavy chain 2 (CDRH2) amino acid sequence of SEQ ID NO: 14; and a complementarity determining region heavy chain 3 (CDRH3) amino acid sequence of SEQ ID NO: 15,wherein one or more of the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 comprise 1, 2, 3, 4, or 5 conservative amino acid substitutions.

17. An isolated antibody comprising a light chain variable region amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region amino acid sequence of SEQ ID NO: 16, wherein the isolated antibody comprises 1, 2, 3, 4, or 5 conservative amino acid substitutions in the light or heavy chain variable region amino acid sequences.

18. The isolated antibody of claim 7, 10, or 16, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises one or more complementarity determining region (CDRHs) CDRH1. CDRH2 and CDRH3 with amino acid sequences that have 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions in 1, 2 or 3 CDRHs having the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; and / or wherein the antibody has a light chain variable region comprising one or more complementarity determining region (CDRLs) CDRL1, CDRL2 and CDRL3 with the amino acid sequences that have 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions in 1, 2 or 3 CDRLs having the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

19. The isolated antibody of claim 1 , 4, 7, 10, 13, or 16, wherein the antibody blocks Scube3 binding to epidermal growth factor receptor 1 and / or transforming growth factor receptor 2 (TGFPR2) or TGFpRl.

20. The isolated antibody of any of the preceding claims, wherein the antibody neutralizes Scube3.

21. The isolated antibody of any of the preceding claims, wherein the antibody inhibits binding of human Scub3 to human epidermal growth factor receptor and / or transforming growth factor receptor 2 (TGFPR2) or TGFpRl.

22. The isolated antibody of any one of the preceding claims, wherein the antibody is recombinantly engineered, chimerized, or humanized.

23. The isolated antibody of any one of the preceding claims, wherein the antibody is a Fab, an Fab’, an F(ab’)2, a Fv, a scFv, a diabody or fragments thereof.

24. The isolated antibody of any one of the preceding claims, wherein the antibody selectively binds to human SCUBE3 and inhibits binding of human SCUBE3 to human epidermal growth factor receptor, and / or transforming growth factor receptor 2 (TGF R2) or TGF0R1.

25. The isolated antibody of any one of the preceding claims, wherein the antibody is a monoclonal antibody.

26. The isolated antibody of any one of the preceding claims, wherein the antibody is a single chain antibody.

27. The isolated antibody of any one of the preceding claims, wherein the antibody is linked to a detectable label.

28. The isolated antibody of any one of the preceding claims, wherein the antibody is a monovalent or a bivalent antibody.

29. The isolated antibody of any one of the preceding claims, wherein the antibody is an IgG, an IgM. an IgA antibody or antigen binding fragment thereof.

30. The isolated antibody of any one of the preceding claims, wherein the complementarity determining regions are defined according to the Kabat definition.31 . The isolated antibody of any one of the preceding claims, wherein any one of the heavy chain CDR1, CDR2 or CDR3 or the light chain CDR1, CDR2, or CDR3 comprises at least one amino acid substitution as compared to the parent CDR.

32. The isolated antibody of claim 31, wherein the at least one amino acid substitution is a glycine to another amino acid.

33. The isolated antibody of claim 31, wherein the at least one amino acid substitution decreases deamidation.

34. A composition comprising an isolated antibody of any of the preceding claims, and at least one pharmaceutically acceptable carrier or diluent.

35. The composition of claim 34, wherein the composition is lyophilized.

36. The composition of claim 34, further comprising a detectable label or reporter.

37. A method of treating cancer in a subject, the method comprising administering to the subj ect a therapeutically effective amount of an isolated antibody of any of claims 1- 33 or the composition of any of claims 34-36.

38. The method of claim 37, wherein the cancer is breast cancer, brain cancer, colorectal cancer, lung cancer, melanoma, thyroid cancer, uterine cancer, prostate cancer, kidney cancer, bladder cancer, head or neck cancers, stomach cancer, or pancreatic cancer.

39. A method of reducing tumor volume in a subject, the method comprising administering to the subject a therapeutically effective amount of an isolated antibody of any of claims 1-33 or the composition of any of claims 34-36.

40. A method of treating or reducing metastatic cancer in a subject or preventing metastasis in a subject having cancer at risk for metastasis, the method comprising administering to the subject a therapeutically effective amount of an isolated antibody of any of claims 1-33 or the composition of any of claims 34-36.

41. The method of claim 40, wherein the cancer is breast cancer, brain cancer, colorectal cancer, lung cancer, melanoma, thyroid cancer, uterine cancer, prostate cancer, kidney cancer, bladder cancer, head or neck cancers, stomach cancer, or pancreatic cancer.

42. The method of any of claims 37-41, wherein the subject is identified in need of treatment before the administering step.

43. The method of any of claims 37-42, wherein the antibody is administered in a pharmaceutically acceptable composition.

44. The method of any of claims 37-43, wherein the antibody is administered systemically, intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously or locally into inflamed tissues, organs or tumors.

45. The method of any of claims 37-44, further comprising administering one or more of doxorubicin, paclitaxel, docetaxel, 5-FU, gemcitabine, vincristine, Olaparib,Niraparib, Atezolizumab, Avelumab, Durvalumab; Pembrolizumab, Nivolumab, or Cemiplimab to the subject.

46. The method of any of claims 37-45, wherein the subject is a human.

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