WO2017156280A1 - Methods of treating cancer using monoclonal antibodies to agr2 and c4.4a - Google Patents

Abstract

Provided herein are monoclonal antibodies that recognize, bind to, and block interactions of AGR2 and C4.4A. Also, provided herein are methods of using said antibodies to treat cancer.

Description

METHODS OF TREATING CANCER USING MONOCLONAL ANTIBODIES TO AGR2

AND C4.4A

[0001] This application claims the benefit of United States Provisional Patent Application No. 62/306,043 filed March 9, 2016, the entirety of which is incorporated herein by reference.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates generally to the fields of cellular biology and oncology. More particularly, it concerns AGR2 and C4.4A antibodies and methods of their use in anticancer therapies.

BACKGROUND OF THE DISCLOSURE

[0003] Breast cancer is the most common women's malignancy, with growing incidence primarily in advanced countries. Despite improvements in treatment, 30^40% of women are diagnosed with metastatic cancer or develop metastases and die from their disease. The most important group of breast cancers is hormone sensitive tumors, characterized by expression of estrogen and progesterone receptors (ER and PgR, respectively). These tumors encompass approximately 70% of all breast cancers and are significantly clinicopathologically different from ER(-)and PgR(-) tumors. Thus, determination of ER and PgR status is an essential part of the diagnostic procedure in breast cancer patients. The presence of ER and PgR receptors indicates response to endocrine therapy and improved disease-free survival.

[0004] Tamoxifen is a selective estrogen receptor modulator (SERM), and is used for systemic treatment of all stages of ER-positive breast cancer during the past 30 years. Despite the undeniable benefit, approximately one third of patients with ER-positive breast cancer do not respond to tamoxifen or develop resistance, constituting a serious clinical problem.

[0005] AGR2 is expressed in a wide variety of tumors formed in different tissues with diverse patterns of genetic alterations including cancers of the breast, pancreas, prostate, lung, and colorectum. AGR2 has been shown to support aggressive growth and metastasis of a variety of cancer cells.

[0006] Recent findings suggest that AGR2 plays a prominent role in mediating pro-oncogenic signals of ER, and there is a correlation between increased AGR2 expression and poor outcome of therapy in patients with ERa-(+) breast cancers. Historically, AGR2 mRNA was discovered as selectively expressed in ER-positive breast cancer cell lines. AGR2 has been functionally characterized and shown to act as an inhibitor of the tumor suppressor p53 and a mediator of metastatic spread in rodent models. It has been shown that AGR2 mediates a pro-survival pathway in human breast cancer cells and is involved in pro-oncogenic signals of ER. In addition, AGR2 expression was elevated both in vitro and in vivo in response to tamoxifen adjuvant therapy, indicating that AGR2 mediates a partial agonist effect of this drug.

[0007] Although the mode of action of AGR2 after tamoxifen treatment remains to be defined, it is believed that AGR2 may mediate resistance to tamoxifen (and other SERMs), and potentially to an emerging class of selective estrogen receptor downregulators (SERDs) which target the ER for proteosomal degradation, thereby driving the progression and metastasis of ER(+) breast cancers.

[0008] Accordingly, monoclonal antibodies against AGR2 and/or its receptor, C4.4A, may have therapeutic potential in both SERM and SERD-resistant ER(+) breast cancers.

SUMMARY OF THE DISCLOSURE

[0009] Provided herein is a disclosure to monoclonal antibodies against AGR2 and C4.4A, as well as methods of their use in treating cancer.

[0010] In some embodiments, the present disclosure is directed towards an isolated or recombinant monoclonal antibody that specifically binds to an AGR2 polypeptide.

[0011] In some embodiments, the present disclosure is directed towards an isolated or recombinant monoclonal antibody that specifically binds to a C4.4A polypeptide.

[0012] In some aspects, an antibody of the embodiments may be an IgG (e.g., IgGl, IgG2,

IgG3 or IgG4), IgM, IgA, or an antigen binding fragment thereof. The antibody may be a Fab', a F(ab')2 a F(ab')3, a monovalent scFv, a bivalent scFv, or a single domain antibody. The antibody may be a human, humanized, or de-immunized antibody. In some aspects, the antibody may be conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

[0013] In some embodiments, there is provided an isolated polynucleotide molecule comprising nucleic acid sequence encoding an antibody or a polypeptide comprising an antibody VH or VL domain disclosed herein.

[0014] In further embodiments, a host cell is provided that produces a monoclonal antibody or recombinant polypeptide of the embodiments. In some aspects, the host cell is a mammalian cell, a yeast cell, a bacterial cell, a ciliate cell, or an insect cell. In certain aspects, the host cell is a hybridoma cell.

[0015] In still further embodiments, there is provided a method of manufacturing an antibody of the present disclosure comprising expressing one or more polynucleotide molecule(s) encoding a VL or VH chain of an antibody disclosed herein in a cell and purifying the antibody from the cell.

[0016] In additional embodiments, there are pharmaceutical compositions comprising an antibody or antibody fragment as discussed herein. Such a composition further comprises a pharmaceutically acceptable carrier and may or may not contain additional active ingredients.

[0017] In embodiments of the present disclosure, there is provided a method for treating a subject having a cancer comprising administering to the subject an effective amount of an agent that inhibits the AGR2/C4.4A autocrine signaling loop. In one aspect, the agent may be an agent that disrupts the AGR2/C4.4A interaction.

[0018] In embodiments of the present disclosure, there is provided a method for treating a subject having a cancer comprising administering an effective amount of an AGR2 or C4.4A antibody disclosed herein.

[0019] In certain aspects, the cancer may be a breast cancer, lung cancer, head & neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer. In one aspect, the cancer may be an endocrine-resistant form of breast cancer.

[0020] In one aspect, the antibody may be administered systemically. In additional aspects, the antibody may be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally. The method may further comprise administering at least a second anticancer therapy to the subject. Examples of the second anticancer therapy include, but are not limited to, surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, or cytokine therapy. In one aspect, the subject may be a human subject.

[0021] In further aspects, the method may further comprise administering a composition of the present disclosure more than one time to the subject, such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more times.

[0022] In accordance with certain aspects of the present disclosure, there is provided a method for treating cancer comprising administering an amount of a AGR2-binding protein and/or an C4.4A-binding protein effective to treat the cancer of the patient. In some aspects, a method comprises treating a patient who either has previously been determined to have a cancer or is determined to have a cancer, such as an endocrine-resistant form of breast cancer.

[0023] In another aspect of the present disclosure, the breast cancer is an estrogen receptor positive (ER+) breast cancer.

[0024] In another aspect of the present disclosure, the endocrine-resistant form of breast cancer is tamoxifen-resistant ER(+) breast cancer.

[0025] In another aspect of the present disclosure, the breast cancer is a Her2(+), ER(-), and PR(+) breast cancer.

[0026] In another aspect of the present disclosure, wherein the breast cancer is Her2(+), ER(- ), and PR(+) breast cancer, it is resistant to anti-Her2 mAbs.In certain embodiments of the present disclosure, the AGR2-binding protein and/or C4.4A-binding protein may be in combination with a Selective Estrogen Receptor Modulators (SERMs) and / or an Oral Selective Estrogen Receptor Downregulators (SERDs). Exemplary examples of SERMs are tamoxifen, toremifene, lasofoxifene, and raloxifene.

[0027] In certain embodiments, the AGR2 -binding protein and/or C4.4A-binding protein may be an antibody, which may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, an affinity matured antibody, a humanized antibody, a human antibody, or an antigen-binding antibody fragment. Preferably, the antibody is a monoclonal antibody or a humanized antibody. In embodiments where the antibody is an antibody fragment, preferred fragments include Fab, Fab', Fab'-SH, F(ab')2, or scFv molecules.

[0028] For certain medical or clinical applications, the antibody may be attached to an agent to be targeted to a C4.4A-expressing cell. The agent may be a cytotoxic agent, a cytokine, an anti-angiogenic agent, a chemotherapeutic agent, a diagnostic agent, an imaging agent, a radioisotope, a pro-apoptosis agent, an enzyme, a hormone, a growth factor, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an antigen, a survival factor, an anti-apoptotic agent, a hormone antagonist, a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a nanoparticle, a magnetic bead, a microdevice, a cell, a nucleic acid, or an expression vector. Where the targeted molecule is a protein, the coding regions for the respective protein molecule and antibody may be aligned in frame to permit the production of a "fused" molecule where desired. In other embodiments, however, the antibody may be conjugated to the molecule using conventional conjugation techniques. [0029] Certain embodiments are directed to an antibody or recombinant polypeptide composition comprising an isolated and/or recombinant antibody or polypeptide that specifically binds AGR2 or C4.4A. In certain aspects the antibody or polypeptide has a sequence that is, is at least, or is at most 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical (or any range derivable therein) to all or part of any AGR2 or C4.4A monoclonal antibody . In still further aspects the isolated and/ or recombinant antibody or polypeptide has, has at least, or has at most 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, 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, 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, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more contiguous amino acids from any of the sequences provided herein or a combination of such sequences.

[0030] In still further aspects, an antibody or polypeptide of the embodiments comprises one or more amino acid segments of the any of the amino acid sequences disclosed herein. For example, the antibody or polypeptide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid segments comprising about, at least or at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 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, 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, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 amino acids in length, including all values and ranges there between, that are at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to any of the amino acid sequences disclosed herein. In certain aspects the amino segment(s) are selected from one of the amino acid sequences of a AGR2-binding antibody or C4.4A-binding antibody as provided in Table 1.

[0031] In still further aspects, an antibody or polypeptide of the embodiments comprises an amino acid segment of the any of the amino acid sequences disclosed herein, wherein the segment begins at amino acid position 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 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 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, 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, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 in any sequence provided herein and ends at amino acid position 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 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, 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, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 in the same provided sequence. In certain aspects the amino segment(s), or portions thereof, are selected from one of the amino acid sequences of a AGR2 -binding antibody or a C4.4A-binding antibody disclosed Table 1.

[0032] In yet further aspects, an antibody or polypeptide of the embodiments comprises an amino acid segment that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical (or any range derivable therein) to a V, VJ, VDJ, D, DJ, J or CDR domain of a AGR2 -binding antibody or C4.4A-binding antibody (as provided in Table 1). For example, a polypeptide may comprise 1, 2 or 3 amino acid segments that are at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical (or any range derivable therein) to CDRs 1, 2, and/or 3 of a AGR2-binding antibody or C4.4A- binding antibody disclosed in Table 1.

[0033] In one embodiment, a composition comprising a AGR2-binding antibody and/or C4.4A-binding antibody is provided for use in the treatment of cancer in a patient. In another embodiment, the use of a AGR2-binding antibody and/or C4.4A-binding antibody in the manufacture of a medicament for the treatment of a cancer is provided. Said AGR2 -binding antibody and/or C4.4A-binding antibody may be any AGR2-binding antibody and/or C4.4A- binding antibody of the embodiments. [0034] Embodiments discussed in the context of methods and/or compositions of the disclosure may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the disclosure as well.

[0035] As used herein the terms "encode" or "encoding" with reference to a nucleic acid are used to make the disclosure readily understandable by the skilled artisan; however, these terms may be used interchangeably with "comprise" or "comprising," respectively.

[0036] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0037] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0038] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0039] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein

[0041] FIG. 1. Anti-AGR2 and anti-C4.4A mAbs Inhibit the Growth of Orthotopic Tamoxifen-Resistant ER+ Breast Tumors. Antibodies and isotype control IgG are dosed intraperitoneally twice-weekly; fulvestrant is dosed subQ once-weekly for a period of 28 days. 1) Tamoxifen + vehicle + mouse isotype control IgG; 2) Tamoxifen + vehicle + anti-AGR2 mAb dosed at 15 mg/kg; 3) Tamoxifen + vehicle + anti-C4.4A mAb dosed at 15 mg/kg; 4) Tamoxifen + vehicle + anti-AGR2 mAb dosed at 7.5 mg/kg + anti-C4.4A mAb dosed at 7.5 mg/kg; 5) Tamoxifen + fulvestrant dosed at 200 mg/kg weekly + mouse isotype control IgG;

6) Tamoxifen + fulvestrant dosed at 200 mg/kg weekly + anti-AGR2 mAb dosed at 15 mg/kg;

7) Tamoxifen + fulvestrant dosed at 200 mg/kg weekly + anti-C4.4A mAb dosed at 15 mg/kg; and 8) Tamoxifen + fulvestrant dosed at 200 mg/kg weekly + anti-AGR2 mAb dosed at 7.5 mg/kg + anti-C4.4A mAb dosed at 7.5 mg/kg.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0042] The present disclosure is based, in part, on the finding that C4.4A (LYPD3) is the functional cell surface receptor for extracellular AGR2. Herein, C4.4A (LYPD3) was identified as the functional cell surface receptor for extracellular AGR2. To support the idea that an AGR2/C4.4A autocrine loop may be a therapeutic target against cancer, monoclonal antibodies against both AGR2 and C4.4A were developed. These results suggest that the AGR2/C4.4A interaction is a target with therapeutic potential for cancer therapy.

I. AGR2 and C4.4A

[0043] AGR2 is associated with poor outcomes in several tumor types (Brychtova et al. , 2011) but the mechanisms have previously been unknown. AGR2 has been reported to be involved in protein maturation and folding (Park et al, 2009; Zhao et al, 2010; Altschul et al , 1997; Higa et al, 2011), to regulate cathepsins (Dumartin et al, 2011), and to modulate MUC-1 levels (Park et al, 2009; Norris et al, 2013). However, these roles of AGR2 do not explain its ability to act as an oncogene (Wang et al, 2008) or the ability of AGR2 to increase the aggressiveness of several types of cancer. It is therefore likely that this protein has multiple intracellular and extracellular functions. Potentially, its physiologic and pathologic roles differ. In the present study, extracellular addition of rAGR2 stimulated the proliferation, migration, invasion, and chemoresistance of PDAC cells. These actions required the presence of cell surface receptors. Thus, based on these data, and without being bound by theory, the role of AGR2 in cancer is mechanistically similar to its roles in amphibians, where it is a secreted signaling molecule that interacts with a specific receptor.

[0044] In amphibians, AGR2 promotes limb growth by interacting with Prodi (Kumar et al, 2007; da Silva et al, 2002), a GPI-linked receptor related to the Ly6 family of receptors in humans (Galat, 2008; Chatterjee and Mayor, 2001). The Lys6 family includes uPAR, C4.4A, and CD59 (Galat, 2008; da Silva et al, 2002). The present study indicated that the Lys family receptors (uPAR, C4.4A, and CD59) were co-immunoprecipitated with AGR2, likely because of the structural homologies between these receptors (Galat, 2008). Nevertheless, only blocking the interaction of AGR2 with C4.4A by silencing or blocking antibodies reduced the endogenous (basal) and extracellular rAGR2-stimulated PDAC cell functions. Though it was reported that in a yeast-two-hybrid system Dystoglycan- 1 bound to AGR2, the present co- immunoprecipitation study could not verify this interaction. Surprisingly, it was observed that silencing of other two receptors, CD59 and uPAR, slightly increased the migration of PDAC cells. This observation was unexpected, as a previous report suggested that silencing uPAR inhibited PDAC cell migration (Xue et al. , 2009). It is unclear what accounts for this difference, but it may be due to the studies being conducted in different cell lines. Nevertheless, the data shown here support a model in which AGR2 and C4.4A participate in an autocrine loop that activates survival mechanisms.

[0045] In previous gene profiling studies, C4.4A was found to be highly expressed in pancreatic cancer but not in normal or chronic pancreatitis tissue (Logsdon et al. , 2003). C4.4A is an orphan receptor described previously as a regulator of cancer cell metastasis (Rosel et al. , 1998; Jacobsen and Ploug, 2008). C4.4A increases metastasis in melanoma (Rosel et al , 1998) and non-small cell lung cancer (Hansen et al. , 2007), and C4.4A protein levels correlate with poor prognosis in breast cancer (Hansen et al. , 2007) and colorectal cancer (Paret et al. , 2007; Konishi et al. , 2010). C4.4A is herein identified as a functional cell surface receptor for AGR2. Silencing or antibody-mediated blocking of C4.4A eliminated the effects of extracellular AGR2, thus supporting AGR2 as the ligand for C4.4A. However, the mechanism of action of C4.4A is had not previously been investigated. Hence, the signaling complex molecules that interact with C4.4A were examined in order to identify specific molecules.

[0046] Like other glycosylphosphatidylinositol (GPI)-linked plasma membrane receptors, C4.4A does not have an intracellular domain to mediate downstream signaling mechanisms. On the basis of homologies between C4.4A and uPAR, another member of the Ly6 family, these interactions likely include extracellular matrix proteins and specific integrin receptors. C4.4A is known to promote migration by associating with α6β4 (Ngora et al , 2012). C4.4A was also previously reported to bind laminins 1 and 5, although functional studies were not conducted (Paret et al. , 2005). Laminins 1 and 5 are thought to interact primarily with integrin α3β1 (Smith et al , 2001; Higa et al , 2011). Integrin α3β1 is expressed by pancreatic ductal cells (Jiang et al , 2002). Silencing of laminin 1, laminin 5, or integrin βΐ abolished the effects of AGR2 treatments, thus suggesting their involvement in the AGR2-mediated C4.4A receptor complex.

[0047] To examine the potential therapeutic benefits of blocking the AGR2/C4.4A autocrine loop, blocking mAbs against the ligand (AGR2) and the receptor (C4.4A) were developed. Both Abs blocked basal and AGR2-mediated functions. Pre-clinical studies using the blocking mAbs in three different types of preclinical models resulted in significant reductions in tumor weight and metastasis and improved survival. Treatment with mAbs had better benefits than treatment with Gemcitabine (Gem), the clinical standard of care for PDAC. Partial or complete regression of tumors was observed in several mice after treatment with individual mAb or with the combination of both mAbs. Even several weeks post treatment, no tumor recurrence was observed.

[0048] Thus, AGR2 has extracellular functions to increase the aggressiveness of cancer cells and C4.4A is the functional receptor of AGR2. The signaling complex of C4.4A likely includes laminin 1, laminin 5, and βΐ integrin. Blocking mAbs against AGR2 and/or C4.4A significantly reduce tumor growth and metastasis, and lead to tumor regression resulting in remarkably improved survival.

II. Therapeutic Antibodies

[0049] In certain embodiments, an antibody or a fragment thereof that binds to at least a portion of AGR2 or C4.4A protein and inhibits AGR2/C4.4A binding and its associated use in treatment of diseases are contemplated. As used herein, the term "antibody" is intended to refer broadly to any immunologic binding agent, such as IgG, IgM, IgA, IgD, and IgE as well as polypeptides comprising antibody CDR domains that retain antigen binding activity. The antibody may be selected from the group consisting of a chimeric antibody, an affinity matured antibody, a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or an antigen-binding antibody fragment or a natural or synthetic ligand. Preferably, the anti-AGR2 or anti-C4.4A antibody is a monoclonal antibody or a humanized antibody. By known means and as described herein, polyclonal or monoclonal antibodies, antibody fragments, and binding domains and CDRs (including engineered forms of any of the foregoing) may be created that are specific to AGR2 protein or C4.4A 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. [0050] Examples of antibody fragments suitable for the present embodiments 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 ("scFv"), wherein 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. Pat. No. 5,091,513); and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (US Patent App. Pub. 20050214860). Fv, scFv, or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains. Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al., 1996).

[0051] Antibody-like binding peptidomimetics are also contemplated in embodiments. Liu et al. (2003) describe "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.

[0052] AGR2 and C4.4A mRNA sequences (SEQ ID NOs: 1 and 2, respectively) may be used to produce recombinant proteins and peptides as well known to people skilled in the art. For example, such mRNA sequences could be engineered into a suitable expression system, e.g. , yeast, insect cells, or mammalian cells, for production of a AGR2 or C4.4A protein or peptide.

[0053] Animals may be inoculated with an antigen, such as a soluble AGR2 or C4.4A protein, in order to produce antibodies specific for AGR2 or C4.4A protein. Frequently an antigen is bound or conjugated to another molecule to enhance the immune response. As used herein, a conjugate is 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. [0054] A monoclonal antibody is a single species of antibody wherein every antibody molecule recognizes the same epitope because all antibody producing cells are derived from a single B- lymphocyte cell line. The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. In some embodiments, rodents such as mice and rats are used in generating monoclonal antibodies. In some embodiments, 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.

[0055] Hybridoma technology involves the fusion of a single B lymphocyte from a mouse previously immunized with an AGR2 or C4.4A antigen with an immortal myeloma cell (usually mouse myeloma). 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 (monoclonal antibodies) may be produced.

[0056] In one embodiment, the antibody is a chimeric antibody, for example, an antibody comprising antigen binding sequences from a non-human donor grafted to a heterologous non- human, human, or humanized sequence (e.g. , framework and/or constant domain sequences). 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, "fully human" monoclonal antibodies are produced in mice 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, for example, mouse, and human amino acid sequences. In "humanized" monoclonal antibodies, only the hypervariable CDR is derived from mouse monoclonal antibodies, and the framework and constant regions are derived from human amino acid sequences (see U.S. Pat. Nos. 5,091 ,513 and 6,881 ,557). It is thought that replacing amino acid sequences in the antibody that are characteristic of rodents with amino acid sequences found in the corresponding position of human antibodies will reduce the likelihood of adverse immune reaction during therapeutic use. 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.

[0057] 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 well known in the art and highly predictable. For example, the following U.S. patents and patent applications provide enabling descriptions of such methods: U.S. Pat. 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. All patents, patent application publications, and other publications cited herein and therein are hereby incorporated by reference in the present application.

[0058] Antibodies may be produced from any animal source, including birds and mammals. Preferably, the antibodies are 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. Pat. No. 6,946,546, which is incorporated herein by reference. These techniques are further described in: Marks et al. (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).

[0059] It is fully expected that antibodies to AGR2 and/or C4.4A will have the ability to block AGR2/C4.4A binding regardless 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 allergic response due to activation of the complement system through the "Fc" portion of the antibody. However, whole antibodies may be enzymatically digested into "Fc" (complement binding) fragment, and into antibody fragments having the binding domain or CDR. Removal of the Fc portion reduces the likelihood that the antigen antibody fragment will elicit an undesirable immunological response, and thus, antibodies without Fc may be preferential for prophylactic or therapeutic treatments. As described above, antibodies may also be constructed so as to be chimeric or partially or fully human, so as to reduce or eliminate the adverse immunological consequences resulting from administering to an animal an antibody that has been produced in, or has sequences from, other species.

[0060] 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 well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Non- conservative 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.

[0061] Proteins may be recombinant, or synthesized in vitro. Alternatively, a non-recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that a bacterium containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.

[0062] It is contemplated that in compositions there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml. Thus, the concentration of protein in a composition can be about, at least about or at most about 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, or at most about 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, 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, 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, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% may be an antibody that binds AGR2 or C4.4A.

[0063] An antibody or preferably an immunological portion of an antibody, can be chemically conjugated to, or expressed as, a fusion protein with other proteins. For purposes of this specification and the accompanying claims, all such fused proteins are included in the definition of antibodies or an immunological portion of an antibody.

[0064] Embodiments provide antibodies and antibody-like molecules against AGR2 and C4.4A, polypeptides and peptides that are linked to at least one agent to form an antibody conjugate or payload. 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. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g. , cytotoxic activity. Non- limiting examples of effector molecules that have been attached to antibodies include toxins, therapeutic enzymes, antibiotics, radio-labeled nucleotides and the like. By contrast, a reporter molecule is defined as any moiety that may be detected using an assay. Non-limiting examples of reporter molecules that have been 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.

[0065] Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3-6-diphenylglycouril attached to the antibody. Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.

III. Treatment of Diseases

[0066] Certain aspects of the present embodiments can be used to prevent or treat a disease or disorder associated with an AGR2/C4.4A-mediated autocrine survival loop. Functioning of the AGR2/C4.4A autocrine loop may be reduced by any suitable drugs to prevent the AGR2/C4.4A interaction. Preferably, such substances would be an anti-AGR2 or anti-C4.4A antibody.

[0067] "Treatment" and "treating" refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a treatment may include administration of a pharmaceutically effective amount of an antibody that inhibits the AGR2/C4.4A-mediated autocrine survival loop.

[0068] "Subject" and "patient" refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human. [0069] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0070] An antibody that binds to AGR2 or C4.4A may be administered to treat a cancer. The cancer may be a solid tumor, metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.

[0071] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; small cell lung cancer; non-small cell lung cancer; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra- mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

A. Pharmaceutical Preparations

[0072] Where clinical application of a therapeutic composition containing an inhibitory antibody is undertaken, it will generally be beneficial to prepare a pharmaceutical or therapeutic composition appropriate for the intended application. This will typically entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One may also employ appropriate buffers to render the complex stable and allow for uptake by target cells. In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1 % of an active compound. In other embodiments, an active compound 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 therein.

[0073] The therapeutic compositions of the present embodiments are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.

[0074] The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. 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 FDA Office of Biological Standards.

[0075] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g. , water, alcoholic/aqueous solutions, 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, surfactants, antioxidants, preservatives (e.g. , antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, 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. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0076] The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e. , the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the effect desired.

[0077] The actual dosage amount of a composition of the present embodiments 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 patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, a dose may also comprise from about 1 μg/kg/body weight to about 1000 mg/kg/body weight (this such range includes intervening doses) 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 μg/kg/body weight to about 100 mg/kg/body weight, about 5 μg/kg/body weight to about 500 mg/kg/body weight, etc. , can be administered. 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.

[0078] The active compounds can be formulated for parenteral administration, e.g. , formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid 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.

[0079] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0080] The proteinaceous compositions may be formulated into a neutral or salt form.

Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0081] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0082] Solutions of therapeutic compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0083] The therapeutic compositions of the present disclosure are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0084] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0085] In particular embodiments, the compositions of the present disclosure are suitable for application to mammalian eyes. For example, the formulation may be a solution, a suspension, or a gel. In some embodiments, the composition is administered via a biodegradable implant, such as an intravitreal implant or an ocular insert, such as an ocular insert designed for placement against a conjunctival surface. In some embodiments, the therapeutic agent coats a medical device or implantable device. The formulation of the disclosure may be applied to the eye in aqueous solution in the form of drops. These drops may be delivered from a single dose ampoule, which may preferably be sterile and thus render bacteriostatic components of the formulation unnecessary. Alternatively, the drops may be delivered from a multi-dose bottle, which may preferably comprise a device that extracts preservative from the formulation as it is delivered, such devices being known in the art. In other aspects, components of the disclosure may be delivered to the eye as a concentrated gel or similar vehicle that forms dissolvable inserts that are placed beneath the eyelids.

[0086] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0087] The therapeutic compositions of the present disclosure may include classic pharmaceutical preparations. Administration of therapeutic compositions according to the present disclosure will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Topical administration may be particularly advantageous for the treatment of skin cancers, to prevent chemotherapy- induced alopecia or other dermal hyperproliferative disorder. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, or respiratory tract, aerosol delivery can be used. Volume of the aerosol is between about 0.01 mL and 0.5 mL.

[0088] An effective amount of the therapeutic composition is determined based on the intended goal. For example, one skilled in the art can readily determine an effective amount of an antibody of the disclosure to be administered to a given subject, by taking into account factors such as the size and weight of the subject; the extent of the neovascularization or disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e. , the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0089] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are particular to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g. , alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.

B. Combination Treatments

[0090] In certain embodiments, the compositions and methods of the present embodiments involve an antibody or an antibody fragment against AGR2 or C4.4A to inhibit the AGR2/C4.4A interaction, in combination with a second or additional therapy. Such therapy can be applied in the treatment of any disease that is associated with an AGR2/C4.4A-mediated autocrine survival loop. For example, the disease may be cancer.

[0091] The methods and compositions, including combination therapies, enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. 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 and/or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with both an antibody or antibody fragment and a second therapy. A tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents (i.e. , antibody or antibody fragment or an anti-cancer agent), or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) an antibody or antibody fragment, 2) an anti-cancer agent, or 3) both an antibody or antibody fragment and an anticancer agent. Also, it is contemplated that such a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

[0092] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing, for example, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[0093] An inhibitory antibody may be administered before, during, after, or in various combinations relative to an anti-cancer treatment. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the antibody or antibody fragment is provided to a patient separately from an anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the antibody therapy and the anti-cancer therapy 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.

[0094] In certain embodiments, a course of treatment will last 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) 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 is a period of time at which no anti-cancer treatment is administered. This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary. [0095] Administration of any compound or therapy of the present embodiments 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 embodiments there is a step of monitoring toxicity that is attributable to combination therapy.

[0096] Antibody and small molecule drugs that increase the immune response to cancer by modulating co-stimulatory or inhibitory interactions that influence the T cell response to tumor antigens, including inhibitors of immune checkpoints and modulators of co- stimulatory molecules, are also of particular interest in the context of the combination therapeutic methods encompassed herein and include, but are not limited to antibodies or an antibody fragmentsagainst AGR2 or C4.4A to inhibit the AGR2/C4.4A interaction. These agents that are involved in the immune response include IL-10 (Interleukin-10, human cytokine synthesis inhibitory factor, CSIF) and Galectins. Administration of therapeutic agents that bind to the CD47 protein, for example, antibodies or small molecules that bind to CD47 and prevent interaction between CD47 and SIRPa, SIRPare administered to a patient, causing the clearance of cancer cells via phagocytosis. The therapeutic agent that binds to the CD47 protein is combined with a therapeutic agent such as an antibody, a chemical small molecule or biologic drug disclosed herein, directed against one or more additional cellular targets of CD70 (Cluster of Differentiation 70), CD200 (OX-2 membrane glycoprotein, Cluster of Differentiation 200), CD154 (Cluster of Differentiation 154, CD40L, CD40 ligand, Cluster of Differentiation 40 ligand), CD223 (Lymphocyte-activation gene 3, LAG3, Cluster of Differentiation 223), KIR (Killer-cell immunoglobulin-like receptors), GITR (TNFRSF18, glucocorticoid-induced TNFR-related protein, activation-inducible TNFR family receptor, AITR, Tumor necrosis factor receptor superfamily member 18), CD28 (Cluster of Differentiation 28), CD40 (Cluster of Differentiation 40, Bp50, CDW40, TNFRSF5, Tumor necrosis factor receptor superfamily member 5, p50), CD86 (B7-2, Cluster of Differentiation 86), CD160 (Cluster of Differentiation 160, BY55, NK1, NK28), CD258 (LIGHT, Cluster of Differentiation 258, Tumor necrosis factor ligand superfamily member 14, TNFSF14, HVEML, HVEM ligand, herpesvirus entry mediator ligand, LTg), CD270 (HVEM, Tumor necrosis factor receptor superfamily member 14, herpesvirus entry mediator, Cluster of Differentiation 270, LIGHTR, HVEA), CD275 (ICOSL, ICOS ligand, Inducible T-cell co-stimulator ligand, Cluster of Differentiation 275), CD276 (B7-H3, B7 homolog 3, Cluster of Differentiation 276), OX40L (OX40 Ligand), B7- H4 (B7 homolog 4, VTCN1, V-set domain-containing T-cell activation inhibitor 1), GITRL (Glucocorticoid-induced tumor necrosis factor receptor-ligand, glucocorticoid-induced TNFR- ligand), 4-1BBL (4-1BB ligand), CD3 (Cluster of Differentiation 3, T3D), CD25 (IL2Ra, Cluster of Differentiation 25, Interleukin-2 Receptor a chain, IL-2 Receptor a chain), CD48 (Cluster of Differentiation 48, B-lymphocyte activation marker, BLAST- 1, signaling lymphocytic activation molecule 2, SLAMF2), CD66a (Ceacam-1, Carcinoembryonic antigen- related cell adhesion molecule 1, biliary glycoprotein, BGP, BGP1, BGPI, Cluster of Differentiation 66a), CD80 (B7-1, Cluster of Differentiation 80), CD94 (Cluster of Differentiation 94), NKG2A (Natural killer group 2A, killer cell lectin-like receptor subfamily D member 1, KLRD1), CD96 (Cluster of Differentiation 96, TActlLE, T cell activation increased late expression), CD112 (PVRL2, nectin, Poliovirus receptor-related 2, herpesvirus entry mediator B, HVEB, nectin-2, Cluster of Differentiation 112), CD115 (CSF1R, Colony stimulating factor 1 receptor, macrophage colony- stimulating factor receptor, M-CSFR, Cluster of Differentiation 115), CD205 (DEC-205, LY75, Lymphocyte antigen 75, Cluster of Differentiation 205), CD226 (DNAM1, Cluster of Differentiation 226, DNAX Accessory Molecule- 1, PTA1, platelet and T cell activation antigen 1), CD244 (Cluster of Differentiation 244, Natural killer cell receptor 2B4), CD262 (DR5, TrailR2, TRAIL-R2, Tumor necrosis factor receptor superfamily member 10b, TNFRSF10B, Cluster of Differentiation 262, KILLER, TRICK2, TRICKB, ZTNFR9, TRICK2A, TRICK2B), CD284 (Toll-like Receptor- 4, TLR4, Cluster of Differentiation 284), CD288 (Toll-like Receptor-8, TLR8, Cluster of Differentiation 288), TNFSF15 (Tumor necrosis factor superfamily member 15, Vascular endothelial growth inhibitor, VEGI, TL1A), TD02 (Tryptophan 2,3-dioxygenase, TPH2, TRPO), IGF-1R (Type 1 Insulin-like Growth Factor), GD2 (Disialoganglioside 2), TMIGD2 (Transmembrane and immunoglobulin domain-containing protein 2), RGMB (RGM domain family, member B), VISTA (V-domain immunoglobulin-containing suppressor of T-cell activation, B7-H5, B7 homolog 5), BTNL2 (Butyrophilin-like protein 2), Btn (Butyrophilin family), TIGIT (T cell Immunoreceptor with Ig and ITIM domains, Vstm3, WUCAM), Siglecs (Sialic acid binding Ig-like lectins), Neurophilin, VEGFR (Vascular endothelial growth factor receptor), ILT family (LIRs, immunoglobulin-like transcript family, leukocyte immunoglobulin-like receptors), NKG families (Natural killer group families, C-type lectin transmembrane receptors), MICA (MHC class I polypeptide-related sequence A), TGF (Transforming growth factor β), STING pathway (Stimulator of interferon gene pathway), Arginase (Arginine amidinase, canavanase, L-arginase, arginine transamidinase), EGFRvIII (Epidermal growth factor receptor variant III), and HHLA2 (B7-H7, B7y, HERV-H LTR- associating protein 2, B7 homolog 7) , inhibitors of PD- 1 (Programmed cell death protein 1 , PD-1, CD279, Cluster of Differentiation 279), PD-Ll (B7-H1, B7 homolog 1, Programmed death-ligand 1, CD274, cluster of Differentiation 274), PD-L2 (B7-DC, Programmed cell death 1 ligand 2, PDCD1LG2, CD273, Cluster of Differentiation 273), CTLA-4 (Cytotoxic T- lymphocyte-associated protein 4, CD152, Cluster of Differentiation 152), BTLA (B- and T- lymphocyte attenuator, CD272, Cluster of Differentiation 272), Indoleamine 2,3-dioxygenase (IDO, IDOl), TIM3 (HAVCR2, Hepatitis A virus cellular receptor 2, T cell immunoglobulin mucin-3, KIM-3, Kidney injury molecule 3, TIMD-3, T cell immunoglobulin mucin-domain 3), A2A adenosine receptor (ADO receptor), CD39 (ectonucleoside triphosphate diphosphohydrolase-1, Cluster of Differentiation 39, ENTPD1), and CD73 (Ecto-5'- nucleotidase, 5 '-nucleotidase, 5 '-NT, Cluster of Differentiation 73), CD27 (Cluster of Differentiation 27), ICOS (CD278, Cluster of Differentiation 278, Inducible T-cell Co- stimulator), CD137 (4-lBB, Cluster of Differentiation 137, tumor necrosis factor receptor superfamily member 9, TNFRSF9), OX40 (CD134, Cluster of Differentiation 134), and TNFSF25 (Tumor necrosis factor receptor superfamily member 25), including antibodies, small molecules, and agonists, are also specifically contemplated herein.

[0097] YERVOY^® (ipilimumab; Bristol-Meyers Squibb) is an example of an approved anti- CTLA-4 antibody.

[0098] KEYTRUDA^® (pembrolizumab; Merck) and OPDIVO^® (nivolumab; Bristol-Meyers Squibb Company) are examples of approved anti-PD-1 antibodies.

1. Chemotherapy

[0099] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer.

[0100] These 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. Alternatively, an 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.

[0101] 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 CB l-TMl); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, 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 antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, 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 deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, 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; PS Kpoly saccharide 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-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine,plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

2. Radiotherapy

[0102] Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-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. Patents 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, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), 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, and the uptake by the neoplastic cells.

3. Immunotherapy

[0103] The skilled artisan will understand that additional immunotherapies may be used in combination or in conjunction with methods of the embodiments. 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. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemo therapeutic, 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. Various effector cells include cytotoxic T cells and NK cells. [0104] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pl55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: 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.

[0105] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g. , Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al , 1998); cytokine therapy, e.g., interferons α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al, 1998; Davidson et al., 1998; Hellstrand et al, 1998); gene therapy, e.g. , TNF, IL-1, IL-2, and p53 (Qin etal, 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-pl85 (Hollander, 2012; Hanibuchi et al, 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.

4. Surgery

[0106] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).

[0107] 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, every 1, 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 varying dosages as well. 5. Hormone Therapy

[0108] Hormonal therapy for hormone receptor-positive breast cancer includes but is not limited to Selective Estrogen Receptor Modulators (SERMs), which are a class of drugs that act on the estrogen receptor (ER). A characteristic that distinguishes these substances from pure ER agonists and antagonists (that is, full agonists and silent antagonists) is that their action is different in various tissues, thereby granting the possibility to selectively inhibit or stimulate estrogen-like action in various tissues.

[0109] There are several SERMs for treatment of hormone receptor-positive breast cancer patients. Tamoxifen is a first- line hormonal treatment of ER-positive metastatic breast cancer. It is used for breast cancer risk reduction in women at high risk, and as adjuvant treatment of axillary node-negative and node -positive, ductal carcinoma in situ. Tamoxifen treatment is also useful in the treatment of bone density and blood lipids in postmenopausal women. Adverse effects include hot flushes and more serious is two to three times higher relative risk of developing endometrial cancer compared to women of an age-matched population.

[0110] Toremifene, a chlorinated tamoxifen derivative, causes fewer DNA adducts in liver than seen with tamoxifen in preclinical studies and was developed to avoid hepatic carcinomas. It is used as endocrine therapy in women with ER/PR-positive stage 4 or recurrent metastatic breast cancer and has demonstrated similar efficacy compared to tamoxifen as adjuvant treatment of breast cancer and in the treatment of metastatic breast cancer.

[0111] Raloxifene is used for prevention and treatment of postmenopausal osteoporosis and breast cancer prevention in high-risk postmenopausal women with osteoporosis. Preclinical and clinical reports suggest that it is considerably less potent than estrogen for the treatment of osteoporosis. It is associated with an acceptable endometrial profile and has not demonstrated tamoxifen-like effects in the uterus but has been associated with adverse effects such as venous thromboembolism and vasomotor symptoms, including hot flushes.

[0112] Oral Selective Estrogen Receptor Downregulators (SERDs) are drugs that eliminate estrogen receptor alpha (ERa) expression by targeting the receptor for proteasome-dependent degradation. Preclinical studies suggest that SERDs may have particular utility in the treatment of breast cancers that have progressed on tamoxifen and/or aromatase inhibitors. The discovery and development of orally bioavailable SERDs provide the opportunity to evaluate the utility of eliminating ERa expression in advanced metastatic breast cancers. 6. Other Agents

[0113] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments 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 would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can 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.

7. Kits and Diagnostics

[0114] In various aspects of the embodiments, a kit is envisioned containing therapeutic agents and/or other therapeutic and delivery agents. In some embodiments, a kit is contemplated for preparing and/or administering a therapy of the embodiments. The kit may comprise one or more sealed vials containing any of the pharmaceutical compositions of the present embodiments. The kit may include, for example, at least one AGR2 or C4.4A antibody as well as reagents to prepare, formulate, and/or administer the components of the embodiments or perform one or more steps of the inventive methods. In some embodiments, the kit may also comprise a suitable container, 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.

[0115] The kit may 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 in the art. The instruction information may be in a computer readable media 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 a therapeutic agentA [0116] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

EXAMPLES

Example 1

Antibody sequences

[0117] Sequences of VH and VL genes. Amino acid sequences of the VH and VL regions of C4.4A and anti-AGR2 monoclonal antibodies are shown in Table 1. The sequences of CDRs 1, 2 and 3 according to the definition of Kabat et al. (1991) are underlined with a solid line. Table 1. Antibody sequences.

NYGMN WINTDTG VTADSMD RSSOSLV KVSNRFS SOSTHVP (SEO ID KPTYTEE Y (SEO ID HSNGNIY (SEO ID LT (SEO

AGR2

NO:9) FKG (SEO NO: l l) LH (SEO NO:13) ID NO: 14)

ID NO: 10) ID NO: 12)

MDWLWNLLFLMAAAOSIOAOIOLV MKLPVRLLVLMFWIP AS S SD V VMT

OSGPELKKPGETVKISCKASGYTFTN OTPLSLPVSLGDOASISCRSSOSLVH

YGMNWVKOAPGKGLKWMGWINT SNGNIYLHWFLOKPGOSPKLLIYKV

DTGKPTYTEEFKGRFAFSLATSASTA SNRFSGVPDRFSGSGSGTDFTLKISR

YLOINNLRNEDTATYFCGRVTADSM VEAEDLGVYFCSOSTHVPLTFGAGT

DYWGOGTSVTVSS (SEO ID NO: 15) KLELK (SEO ID NO: 16)

Example 2

Anti-AGR2 and anti-C4.4A mAbs will be Tested in a Mouse Model of Tamoxif en-Sensitive

ER+ breast cancer

[0118] Anti-AGR2 and/or anti-C4.4A mAbs will be assessed for activity in an industry- standard mouse model of tamoxifen- sensitive human xenograft ER+ breast cancer. An exemplary human xenograft model of tamoxifen-sensitive ER+ breast cancer could be generated as follows: Time release pellets containing 0.72 mg 17-β estradiol are subcutaneously implanted into nu/nu mice. MCF-7 cells are grown in RPMI containing 10% FBS at 5% C02, 37 °C. Trypsinized cells are pelleted and resuspended in 50% RPMI (serum free) and 50% Matrigel at 1 x 107 cells/mL. MCF-7 cells are subcutaneously injected (100 μΕ ηίηωΙ) on the right flank 2-3 days post pellet implantation. Tumor volume (length x width²/2) is monitored biweekly. When tumors reach an average volume of ~200 mm³, animals are randomized into the following treatment groups: 1) human isotype control IgG; 2) anti-AGR2 mAb dosed at 25 mg/kg; and 3) anti-C4.4A mAb dosed at 25 mg/kg. Dosing is intraperitoneal or intravenous and is biweekly for 4 weeks. Key study endpoints include tumor volume and body weight, which are measured biweekly throughout the study.

Example 3

Anti-AGR2 and anti-C4.4A mAbs Inhibit the Growth of Orthotopic Tamoxif en-Resistant ER+

Breast Tumors

[0119] Anti-AGR2 and/or anti-C4.4A mAbs will be assessed for activity in an industry- standard mouse model of tamoxifen-resistant human xenograft ER+ breast cancer. An exemplary human xenograft model of tamoxifen-resistant ER+ breast cancer could be generated as follows: Female nu/nu mice (with supplemental 17-β estradiol pellets; 0.72 mg; 60-day slow release) bearing MCF-7 tumors (mean tumor volume 200 mm3) are treated with Tamoxifen (citrate) by oral gavage. Tumor volume (length x width²/2) and body weight are monitored twice weekly. Following a significant anti-tumor response in which tumor volume remains static, at the first evidence of tumor growth the tamoxifen dose is increased. At this point, rapidly growing tumors are deemed tamoxifen-resistant and are selected for in vivo passage into new host animals. Tumor fragments from the tamoxifen-resistant tumors are subcutaneously implanted orthotopically into the mammary fat pad of female nu/nu mice (with 17-β estradiol pellets [0.72 mg; 60-day slow release]). Passaged tumors are maintained under constant tamoxifen selection, and tumor volume (length x width²/2) is monitored weekly. When tumor volume reaches ~ 100 mm³, animals are randomized into the following treatment groups under continued treatment with tamoxifen: Antibodies and isotype control IgG are dosed intraperitoneally twice-weekly; fulvestrant is dosed subQ once-weekly for a period of 28 days. 1) Tamoxifen + vehicle + mouse isotype control IgG; 2) Tamoxifen + vehicle + anti- AGR2 mAb dosed at 15 mg/kg; 3) Tamoxifen + vehicle + anti-C4.4A mAb dosed at 15 mg/kg; 4) Tamoxifen + vehicle + anti-AGR2 mAb dosed at 7.5 mg/kg + anti-C4.4A mAb dosed at 7.5 mg/kg; 5) Tamoxifen + fulvestrant dosed at 200 mg/kg weekly + mouse isotype control IgG;

6) Tamoxifen + fulvestrant dosed at 200 mg/kg weekly + anti-AGR2 mAb dosed at 15 mg/kg;

7) Tamoxifen + fulvestrant dosed at 200 mg/kg weekly + anti-C4.4A mAb dosed at 15 mg/kg; and 8) Tamoxifen + fulvestrant dosed at 200 mg/kg weekly + anti-AGR2 mAb dosed at 7.5 mg/kg + anti-C4.4A mAb dosed at 7.5 mg/kg (FIG. 1). The above treatments are conducted in the continued presence of tamoxifen, as the tamoxifen-resistant ER+ breast tumors proliferate in response to tamoxifen. Key study endpoints include tumor volume and body weight which are monitored twice or three times weekly for the duration of the study.

[0120] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the disclosure as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating cancer in a patient comprising administering to the patient an agent that disrupts AGR2 activity, C4.4A activity, and / or the AGR2/C4.4A interaction in an amount effective to treat the cancer.

2. The method of claim 1, wherein the cancer is a breast cancer, lung cancer, head & neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer

3. The method of claim 2, wherein the breast cancer is an estrogen receptor positive (ER+) breast cancer.

4. The method of claim 2, wherein the breast cancer is a Her2+ and ER- and PR- breast cancer.

5. The method of claim 3 wherein the ER+ breast cancer is resistant to one or more of the following:

a. Selective Estrogen Receptor Modulators (SERMs),

b. Selective Estrogen Receptor Downregulators (SERDs), and

c. Aromatase inhibitors.

6. The method of claim 4 wherein the Her2+ and ER- and PR- breast cancer is resistant to anti-Her2 mAbs.

7. The method of claim 1, wherein the antibody is administered systemically.

8. The method of claim 1, wherein the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.

9. The method of claim 5 or 6, further comprising administering at least a second anticancer therapy in combination to the subject.

10. The method of claim 7, wherein the second anticancer therapy in combination is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy or cytokine therapy.

11. The method of claim 8, wherein the is second anticancer therapy is a hormonal therapy selected from a Selective Estrogen Receptor Modulator (SERM), a Selective Estrogen Receptor Downregulator (SERDs) or an aromatase inhibitor.

12. The method of claim 9, wherein the Selective Estrogen Receptor Modulators (SERMs) is selected from Tamoxifen, Toremifene, and Raloxifene.

13. The method of claim 9, wherein the aromatase inhibitor is letrozole.

14. A method for treating cancer in a patient comprising administering to the patient an agent that disrupts AGR2 activity, C4.4A activity, or the AGR2/C4.4A interaction wherein the agent prevents or reverses ER+ breast cancer resistance to one or more of Selective Estrogen Receptor Modulators (SERMs), Selective Estrogen Receptor Down regulators (SERDs) or aromatase inhibitors.

15. A method for treating cancer in a patient comprising administering to the patient an agent that disrupts AGR2 activity, C4.4A activity, and / or the AGR2/C4.4A interaction wherein the agent prevents or reverses Her2+ breast cancer resistance to one or more of anti-Her2 therapies.

16. A method of treating a patent with estrogen receptor positive (ER+) breast cancer by administering:

a. tamoxifen in combination with anti-ARG2 antibody; or

b. tamoxifen in combination with an anti-C4.4A antibody.