HK1244816A1 - Blocking monoclonal antibodies to agr2 and its receptor c4.4a - Google Patents

Abstract

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

Description

Blocking monoclonal antibodies against AGR2 and its receptor C4.4A

This application claims the benefit of U.S. provisional patent application No. 62/048,037, filed 9/2014, the entire contents of which are incorporated herein by reference.

Background

1. Field of the invention

The present invention relates generally to the fields of cell biology and oncology. More particularly, it relates to antibodies that bind to AGR2 and c4.4a and methods of their use in anti-cancer therapy.

2. Description of the related Art

Pre-gradin 2(AGR2[ also known as hGA-2 (Thompson and Weigel, 1998) or Gob-4 (palace et al, 1999) ]) is a human ortholog of Xenopus laevis XGA-2. XAG-2 is secreted and is involved in the ectoderm formation and amphibian limb regeneration of frog embryos by interacting with the Ly6 superfamily of receptors, Prod-1 (Aberger et al, 1998; Kuma (Kumar) et al, 2007; Daschirwal (da Silva) et al, 2002). However, there is no human homolog of Prod-1. It is unknown whether AGR2 functions in humans through receptors on the cell surface or within cells. Tissue distribution of AGR2 in healthy adults indicates that it is restricted to organs with mucin-producing cells. The mouse gene deletion model of AGR2 shows changes in mucin synthesis (Park et al, 2009). Other studies support the concept that AGR2 shares sequence similarity with the Protein Disulfide Isomerase (PDI) family (Zhao et al, 2010; atthul (Altschul) et al, 1997; person (Persson) et al, 2005; Gupta (Gupta) et al, 2012). Members of the PDI protein family can catalyze the formation, reduction and isomerization of disulfide bonds, thus stabilizing intermediate conformations during protein maturation in the ER (Persson et al, 2005). However, in normal cells, the role of AGR2 in protein synthesis is not as that in amphibians and also does not explain its observed role in cancer.

AGR2 has been reported to bind to dystrophin proteoglycan-1 (DAG-1) and C4.4A based on yeast two-hybrid results (Fletcher et al, 2003). However, no evidence is provided to support the interaction of these molecules in mammalian cells or to confirm the biological function of these interactions. AGR2 is expressed in a wide variety of tumors that develop in different tissues with different patterns of genetic changes, including Pancreatic Ductal Adenocarcinoma (PDAC) (Ramachandran et al, 2008) and breast cancer (Thompson and weiger (Weigel), 1998; Fletcher et al, 2003), prostate cancer (Zhang et al, 2005), lung cancer (Zhu) et al, 2007) and colorectal cancer (Smirnov, 2005). AGR2 supports invasive growth and metastasis of various cancer cells (Liu) et al, 2005; Innes (Innes) et al, 2006; Barlacraflugh (Barraclough) et al, 2009). accordingly, AGR2 and its receptors may be useful therapeutic targets.

Summary of The Invention

Here, c4.4a (LYPD3) was identified as a functional cell surface receptor for extracellular AGR 2. Provided herein are novel monoclonal blocking antibodies to both AGR2 and c4.4a and methods of their use for treating cancer.

In some embodiments, the present invention is directed to an isolated or recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to an AGR2 polypeptide. In certain aspects, the antibody specifically binds to an AGR2 polypeptide corresponding to amino acids 25-125 of SEQ ID NO. 26. In certain aspects, the antibody specifically binds to AGR2 polypeptide according to SEQ ID No. 28. SEQ ID NO:28 corresponds to amino acid residues 75-103 of human AGR2(SEQ ID NO: 26). The inventors have found that a monoclonal antibody or antigen-binding fragment thereof specific for an AGR2 polypeptide as defined above provides significant advantages in use compared to prior art antibodies directed to AGR2, in particular monoclonal antibody Ab 56703. In a further aspect, a monoclonal antibody or antigen-binding fragment thereof specific for an AGR2 polypeptide as defined above inhibits tumor cell pancreatic ductal adenocarcinoma migration and resistance to gemcitabine-induced apoptosis. In certain aspects, the antibody competes with the 163-28B-1 monoclonal antibody for binding to the AGR2 polypeptide. In certain aspects, the antibody may comprise all or part of the heavy chain variable region and/or the light chain variable region of the 163-28B-1 monoclonal antibody. In a further aspect, the antibody can comprise an amino acid sequence corresponding to the first, second, and/or third Complementarity Determining Regions (CDRs) of the light variable/heavy variable chain of the 163-28B-1 monoclonal antibody from an embodiment of the invention.

In certain aspects, the isolated antibody comprises CDR sequences at least 80%, 90%, or 95% identical to the CDR regions of the 163-28B-1 heavy and light chain amino acid sequences. In another aspect, the antibody comprises a CDR region identical to 163-28B-1 except for one or two amino acid substitutions, deletions or insertions in one or more of the CDRs. For example, an antibody can include CDRs, wherein the CDR sequences include those at V relative to the CDRs of the 163-28B-1 monoclonal antibody_HCDR1、V_HCDR2、V_HCDR3、V_LCDR1、V_LCDR2 and/or V_L1 or 2 amino acid substitutions in CDR 3. Thus, in some particular aspects, the antibodies of the embodiments comprise (a) a first VH CDR that is at least 80% identical to VH CDR1 of 163-28B-1 (SEQ ID NO: 20); (b) a second VH CDR that is at least 80% identical to VHCDR2 of 163-28B-1 (SEQ ID NO: 21); (c) a third VH CDR that is at least 80% identical to VH CDR3 of 163-28B-1 (SEQ ID NO: 22); (d) a first VL CDR that is at least 80% identical to VL CDR1 of 163-28B-1 (SEQ ID NO: 23); (e) a second VL CDR that is at least 80% identical to VL CDR2 of 163-28B-1 (SEQ ID NO: 24); and (f) a third VL CDR that is at least 80% identical to VL CDR3 of 163-28B-1 (SEQ ID NO: 25).

In other aspects, the isolated antibody comprises a first V that is at least 80% identical to the corresponding CDR sequence of monoclonal antibody 163-28B-1 (represented by SEQ ID NOS: 20, 21, 22, 23, 24, and 25, respectively)_HSecond V_HThe third V_HFirst V_LSecond V_LAnd a third V_LA CDR sequence. In one aspect, the isolated antibody comprises CDR sequences identical to the CDR sequences of monoclonal antibody 163-28B-1.

In another aspect, the isolated antibody comprises 163-V of 28B-1_HV whose domain (SEQ ID NO:18) is at least about 80% identical_HDomain and V to 163-28B-1_LV whose domain (SEQ ID NO:19) is at least about 80% identical_LA domain. In one aspect, the isolated antibody comprises those V that are identical to monoclonal antibody 163-28B-1_HAnd V_LDomain-identical V_HAnd V_LA domain. In a further aspect, the isolated antibody is 163-28B-1 antibody.

In some embodiments, the invention is directed to an isolated or recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to a c4.4a polypeptide. In certain aspects, the antibody specifically binds to the C4.4A polypeptide corresponding to amino acids 240-340 of SEQ ID NO: 27. In certain aspects, the antibody specifically binds to a c4.4a epitope in a c4.4a polypeptide according to SEQ ID NO: 29. SEQ ID NO:29 corresponds to amino acid residue 262-296 of human C4.4A (SEQ ID NO: 28). The inventors have found that a monoclonal antibody or antigen-binding fragment thereof having the specificity as defined above provides significant advantages in use compared to prior art antibodies directed against c4.4a, in particular the monoclonal antibody AF 5428. In a further aspect, a monoclonal antibody or antigen-binding fragment thereof specific for a c4.4a polypeptide as defined above inhibits tumor cell pancreatic ductal adenocarcinoma migration and resistance to gemcitabine-induced apoptosis. In certain aspects, the antibody competes for binding to the C4.4A polypeptide with the 162-1A-1 monoclonal antibody. In certain aspects, the antibody may comprise all or part of the heavy chain variable region and/or the light chain variable region of the 162-1A-1 monoclonal antibody. In a further aspect, the antibody can comprise an amino acid sequence corresponding to the first, second, and/or third Complementarity Determining Regions (CDRs) of the light variable/heavy variable chain of the 162-1A-1 monoclonal antibody from embodiments of the invention.

In certain aspects, the isolated antibody comprises CDR sequences at least 80%, 90%, or 95% identical to the CDR regions of the 162-1A-1 heavy and light chain amino acid sequences. In another aspect, the antibody comprises a CDR region identical to 162-1A-1 except for one or two amino acid substitutions, deletions or deletions in one or more CDRsAnd (4) inserting. For example, an antibody can include CDRs, wherein the CDR sequences include those at V relative to the CDRs of the 162-1A-1 monoclonal antibody_HCDR1、V_HCDR2、V_HCDR3、V_LCDR1、V_LCDR2 and/or V_L1 or 2 amino acid substitutions in CDR 3. Thus, in some particular aspects, the antibodies of the embodiments comprise (a) a first VH CDR that is at least 80% identical to VH CDR1 of 162-1A-1 (SEQ ID NO: 12); (b) a second VH CDR that is at least 80% identical to VHCDR2 of 162-1A-1 (SEQ ID NO: 13); (c) a third VH CDR that is at least 80% identical to VH CDR3 of 162-1A-1 (SEQ ID NO: 14); (d) a first VL CDR that is at least 80% identical to VL CDR1 of 162-1A-1 (SEQ ID NO: 15); (e) a second VL CDR that is at least 80% identical to VL CDR2 of 162-1A-1 (SEQ ID NO: 16); and (f) a third VL CDR that is at least 80% identical to VL CDR3 of 162-1A-1 (SEQ ID NO: 17).

In other aspects, the isolated antibody comprises first VH, second VH, third VH, first VL, second VL, and third VL CDR sequences that are at least 80% identical to the corresponding CDR sequences of monoclonal antibody 162-1A-1 (represented by SEQ ID NOS: 12, 13, 14, 15, 16, and 17, respectively). In one aspect, the isolated antibody comprises CDR sequences identical to the CDR sequences of monoclonal antibody 162-1A-1.

In another aspect, the isolated antibody comprises a V that is identical to 162-1A-1_HV whose domain (SEQ ID NO:10) is at least about 80% identical_HDomains and V with 162-1A-1_LV whose domain (SEQ ID NO:11) is at least about 80% identical_LA domain. In one aspect, the isolated antibody comprises those V that are identical to monoclonal antibody 162-1A-1_HAnd V_LDomain-identical V_HAnd V_LA domain. In a further aspect, the isolated antibody is the 162-1A-1 antibody.

In some aspects, the antibody of the embodiments can be an IgG (e.g., IgG1, 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 antibody, a humanized antibody or a deimmunized antibody. In some aspects, the antibody may be conjugated to an imaging agent, chemotherapeutic agent, toxin, or radionuclide.

In one embodiment, a method of treating a subject comprising antibody V is provided_HRecombinant polypeptide of Domain, the antibody V_HThe domain comprises a V of 163-28B-1_HCDR1-3 of the Domain (SEQ ID NOS: 20, 21, and 22) or V of 162-1A-1_HCDR1-3 of Domain (SEQ ID NOS: 12, 13, and 14). In another embodiment, antibodies comprising V are provided_LRecombinant polypeptide of Domain, the antibody V_LThe domain comprises a V of 163-28B-1_LCDR1-3 of the Domain (SEQ ID NOS: 23, 24, and 25) or V of 162-1A-1_LCDR1-3 of Domain (SEQ ID NOS: 15, 16, and 17).

In some embodiments, isolated polynucleotide molecules are provided that comprise a polynucleotide encoding an antibody V, including those disclosed herein_HOr V_LA nucleic acid sequence of an antibody or polypeptide of a domain.

In further embodiments, host cells are provided that produce the monoclonal antibodies or recombinant polypeptides 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.

In still further embodiments, methods of making an antibody of the invention are provided, the methods comprising expressing a V encoding an antibody disclosed herein in a cell_LOr V_HOne or more polynucleotide molecules of the chain and purifying the antibody from the cell.

In further embodiments, there are pharmaceutical compositions comprising an antibody or antibody fragment as disclosed herein. Such compositions further comprise a pharmaceutically acceptable carrier and may or may not contain additional active ingredients.

In an embodiment of the invention, there is provided a method for treating a subject having cancer, the method 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 can be an agent that disrupts AGR2/C4.4A interaction.

In an embodiment of the invention, there is provided a method for treating a subject having cancer, the method comprising administering an effective amount of an antibody disclosed herein. In certain aspects, the antibody is a monoclonal antibody of the invention, such as 162-1A-1 or 163-28B-1, or a recombinant polypeptide comprising an antibody segment derived from an antibody.

In certain aspects, the cancer can be breast cancer, lung cancer, head and 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 pancreatic ductal adenocarcinoma.

In one aspect, the antibody may be administered systemically. In further aspects, the antibody can be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically. The method can further comprise administering at least one second anti-cancer therapy to the subject. Examples of second anticancer therapies 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.

In additional aspects, the method can further comprise administering a composition of the invention to the subject more than once, such as, e.g., 1,2, 3,4, 5,6, 7,8, 9,10, 15, 20, or more times.

According to certain aspects of the present invention, there is provided a method of treating cancer, the method comprising administering an amount of AGR2 binding protein and/or C4.4 binding protein effective to treat cancer in a patient. In some aspects, the method includes treating a patient who has been previously determined to have cancer or is determined to have cancer, such as pancreatic ductal adenocarcinoma.

In certain embodiments, AGR2 binding proteins and/or c4.4a junctionsThe 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 '-SH, F (ab')₂Or scFv molecules.

For certain medical or clinical applications, the antibody may be attached to an agent to be targeted to c4.4a-expressing cells. The agent can be a cytotoxic agent, cytokine, anti-angiogenic agent, chemotherapeutic agent, diagnostic agent, imaging agent, radioisotope, pro-apoptotic agent, enzyme, hormone, growth factor, peptide, protein, antibiotic, antibody, Fab fragment of antibody, antigen, survival factor, anti-apoptotic agent, hormone antagonist, virus, bacteriophage, bacterium, liposome, microparticle, nanoparticle, magnetic bead, microdevice, cell, nucleic acid, or expression vector. If the molecules being targeted are proteins, the coding regions of the respective protein molecules and antibodies may be aligned in frame to allow production of the desired "fusion" molecule. However, in other embodiments, the antibody may be conjugated to the molecule by using conventional conjugation techniques.

Certain embodiments are directed to antibody or recombinant polypeptide compositions comprising isolated and/or recombinant antibodies or polypeptides that specifically bind to AGR2 or c4.4a. In certain aspects, the antibody or polypeptide has a sequence that is at least or at most 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) identical to all or part of any of the monoclonal antibodies provided herein. In still further aspects, the isolated and/or recombinant antibody or polypeptide 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 sequence or combination of these sequences provided herein.

In still further aspects, the antibody or polypeptide of the embodiments comprises one or more amino acid segments of any of the amino acid sequences disclosed herein. For example, an antibody or polypeptide can include a1, 2, 3,4, 5,6, 7,8, 9,10, or more amino acid segment that is 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, 96, 97, 98, 99, 98, 99, 100, 98, 100, or more amino acid segments in length, 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, 194, 195, 196, 197, 198, 199, or 200 amino acids, including all values and ranges therebetween, and any amino acid fragment thereof disclosed herein is at least 80%, 95%, 96%, 97%, 95%, 85%, 96%, 97%, or 200 amino acid sequence 98%, 99%, or 100% identical. In some aspects, the one or more amino acid segments are selected from one of the amino acid sequences of an AGR 2-binding antibody or a c4.4a-binding antibody as provided in table 1.

In still further aspects, the antibodies or polypeptides of the embodiments include an amino acid segment of 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, 94, 95, 93, 96, 97, 95, 96, 97, 95, 93, 97, 96, 97, 95, 96, 97, 95, 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 and terminating at amino acid position 4, 5,6, 7, 11, 17, 16, 11, 17, 16, 11, 17, 18,20, 138, 20, 172, 175, 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, 137, 118, 119, 120, 121, 122, 123, 124, 125, 127, 126, 131, 130, 135, 145, 142, 136, 143, 142, 136, 145, 142, 145, 142, 146, 145, 142, 136, 146, 145, 142, 145, 23, 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 certain aspects, the one or more amino acid segments, or portions thereof, are selected from one of the amino acid sequences of an AGR 2-binding antibody or a c4.4a-binding antibody as provided in table 1.

In yet further aspects, the antibody or polypeptide of embodiments includes an amino acid segment that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) identical to the V, VJ, VDJ, D, DJ, J, or CDR domains of an AGR 2-binding antibody or a C4.4-binding antibody (as provided in table 1). For example, the polypeptide may comprise 1,2, or 3 amino acid segments that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) identical to CDR1, 2, and/or 3 of the AGR 2-binding antibody or the c4.4a-binding antibody provided in table 1.

In one embodiment, compositions comprising AGR 2-binding antibodies and/or c4.4a-binding antibodies are provided for use in treating cancer in a patient. In another embodiment, there is provided the use of an AGR 2-binding antibody and/or a c4.4a-binding antibody in the manufacture of a medicament for the treatment of cancer. The AGR 2-binding antibody and/or C4.4-binding antibody can be any AGR 2-binding antibody and/or c4.4a-binding antibody of the examples.

The embodiments discussed in the context of the methods and/or compositions of the present invention may be used with respect to any other method or composition described herein. Thus, embodiments relating to one method or composition may also be applicable to other methods and compositions of the present invention.

As used herein, the terms "encode" or "encoding" with respect to a nucleic acid are used so that the invention may be readily understood by a skilled artisan; however, these terms may be used interchangeably with "comprising" or "comprising", respectively.

As used in the specification, "a" or "an" may mean one or more than one. As used in the claims, the words "a" or "an" when used in conjunction with the word "comprising" may mean one or more than one.

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

Throughout this application, the term "about" is used to indicate that a numerical value includes variations in error inherent to the apparatus, the method used to determine the value, or variations present in the subject.

Other objects, features and advantages of the present invention 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 invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief description of the drawings

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

FIGS. 1A-D. extracellular AGR2 stimulates PDCA cell invasiveness. Extracellular addition of rAGR2(0-500nM) to AsPC-1 cells resulted in dose-dependent increases in (A) cell proliferation, (B) migration, and (C) invasion. (D) Gemcitabine (Gem) addition resulted in increased apoptosis. Proliferation is shown as the percentage of viable cells relative to the control. However, extracellular AGR2 significantly reduced the level of Gem-induced apoptosis. Data shown are mean ± SEM of 3 experiments (./p < 0.05).

Figure 2A-c.c4.4a interacts with other LY6 family receptors and AGR 2. (A) Several candidate receptors (uPAR, C4.4A, and CD59) were co-immunoprecipitated with AGR2, however DAG-1 was not. (B) Purified recombinant AGR2 and c4.4a were also co-immunoprecipitated from their suspensions, supporting the direct interaction of AGR2 and c4.4a. (C) Silencing of c4.4a, CD59, and uPAR was achieved using siRNA at two different concentrations. Significant silencing was shown by western blotting with the respective antibodies, and the same membranes were blotted against β -actin as a loading control. Two concentrations of siRNA showed significant silencing. The micrographs shown represent three independent experiments.

FIG. 3A-E.C4.4A is required for rAGR 2-mediated function. AsPC-1 cells were transfected with siRNA to silence either c4.4a, CD59, or uPAR, and then treated daily with no (basal, left panel) and with AGR2(100nM, right panel). Only c4.4a silencing reduced both basal and rAGR 2-stimulated (a) proliferation, (B) migration, and (C) invasion. Proliferation is shown by the percentage of viable cells relative to the Si control (substrate) and Si control + AGR2 (treated). (D) In Si control cells, Gem addition stimulated apoptosis and the effect was improved by AGR 2. Silencing of c4.4a itself induced apoptosis, promoted Gem-mediated apoptosis, and abolished the survival effect of AGR2, showing a significant increase in apoptosis. Data shown are mean ± SEM of 3 experiments (./p < 0.05). (E) To determine the effect of specific c4.4a sirnas, four sirnas (sic4.4a 1-4) were used for apoptosis studies and had comparable results.

The effect of agr2 is mediated by the interaction of c4.4a with integrin beta1 and laminin 1 or laminin 5. (A) In Si control AsPC-1 cells, Gem addition stimulated apoptosis and addition of rAGR2 inhibited this effect. AspC-1 cells were also transfected with siRNAs against ITG- β 1, ITG- β 2, ITG- β 4, ITG- α 6, laminin 1 and laminin 5. Only silencing of laminin 1 and 5 and integrin beta1 increased Gem-induced apoptosis and abrogated the survival effect of AGR 2. (B) Silencing of laminin 1 and 5 and integrin beta1 by siRNA significantly abolished AGR 2-mediated proliferation of AsPC-1 cells. (C) Commercially available blocking antibodies directed against ITG- β 1, ITG- β 2, ITG- β 4, ITG- α 6, laminin 1, and laminin 5 showed similar results to siRNA treatment, with only antibodies directed against laminin 1 and 5 and integrin β 1 blocking the AGR 2-mediated survival effect. Data shown are mean ± SEM of 3 experiments (./p < 0.05).

FIGS. 5A-D.A highly specific monoclonal antibody was developed that blocks AGR2/C4.4A binding and biological effects. (A) From endogenous PDAC cell lysates (SU86.86), AGR2(18kD) and c4.4a (50kD) were identified by their respective newly developed mabs. Additional bands identified with endogenous proteins were also observed with recombinant proteins and may represent cleavage products. Commercially available antibodies also recognize these molecules. However, the commercially available Ab showed several nonspecific bands. Blocking mab (b) reduced AGR 2-stimulated PDAC cell migration and (C) blocked the survival effect of AGR2, whereas commercial Ab did not. (D) Immunohistochemical analysis using the developed mAb on TMA showed strong labeling of PDAC (indicated by arrows), but normal pancreas was not labeled.

Figure 6A-i. in vivo treatment with AGR2/c4.4a antibody reduced tumor growth and metastasis and improved survival. After injection of a fluorescein substrate (Xenogen, Aramada, Calif.), the use of a fluorescent probe for bioluminescence imaging of living animals was utilizedImaging systemTumor growth and metastasis were measured systematically weekly. The number of mice surviving until the end of the experiment was marked (percent survival). (A, B, G, H) model 1-AspC-1-invasive model. Two weeks after injection of invasive AsPC-1 cells, mice (n ═ 6) were treated with control IgG or mAb (combination AGR2/c4.4a mAb (5 mg each)/kg/body weight/twice a week/i.p) with or without Gem (100mg/kg body weight/once a week/i.p) when tumor weights were less than 0.5g (as surgically confirmed from the parallel untreated group) up to 7 weeks. (A) All mice treated with the combined mabs survived for at least 6 weeks, whereas all control mice died within 6 weeks. (B) Tumor volume was estimated weekly by imaging. The combination IgG showed a decrease in tumor volume with or without Gem compared to the control IgG with or without Gem. At the end of the experiment, tumor weights and metastases to liver and lung were compared ex vivo between control and treatment groups. Mice treated with mabs showed greatly reduced tumor growth (G) and incidence of metastasis (H). Gemcitabine (Gem) alone or in combination with mabs had no significant effect. (C, D) model 2-Capan 2-matrix model. Two weeks after injection of matrix-forming Capan-2 cells, mice (n ═ 7) were treated with 15mg AGR2 or c4.4a mAb/kg/body weight/twice a week/i.p. Treatment was stopped after 15 weeks (13 weeks of treatment) and tumor size in surviving animals was monitored by bioluminescence until 63 weeks. (C) Treatment with either mAb alone showed an improvement in survival at 24 weeks compared to 9-week-dead control mice. After 48 weeks without treatment, mice showed no tumor regeneration. (D) Mean change in tumor volume indicates that mice treated with either mAb showed slower growth. In several mice, disappearance of the tumor was observed. (E, F) model 3-Capan-2-regression study. Four weeks after the injection of Capan-2 cells, when the tumor weight exceeded 1g (as surgically confirmed from the parallel untreated group), mice (n-5) were treated with AGR2 or c4.4a mAb at 15 mg/kg/body weight/twice a week/i.p or with both mAb combinations (7.5 mg each). The treatment was stopped after 12 weeks. Bioluminescence was monitored in surviving animals until 18 weeks. (E) Treatment of mice with greater than 1g tumor with mAb improved their survival for 14 weeks compared to control mice that died within 4 weeks of treatment. (F) Each processing groupAs shown by bioluminescence imaging measurements. (I) Histological examination of tumors developed in model 3 was performed. TUNEL staining of paraffin sections showed increased apoptotic cells in the groups treated with the antibodies alone or in combination compared to the control. Staining for p-ERK and Ki-67 showed increased activity in cancer cells of control mice, whereas antibody treated mice showed no activity in cancer cells. Quantification indicated a significant increase in the number of apoptotic cells per field in sections of tumors from mAb-treated mice (. p)<0.05)。

Fig. 7A-d. extracellular rAGR 2-mediated function. (A) Pancreatic cancer cells (BxPC-3 and MiaPaCa-2) were used for these studies. Extracellular addition of rAGR2 caused a significant dose-dependent increase in proliferation (BxPC-3-3 fold increase; MiaPaCa-2-4 fold increase; p<0.05). Pancreatic cancer cells were plated with and without rAGR2(0-100nM) (2X 10)⁴Individual cells) were used for migration studies (B) on a Boyden chamber and plated on an invasion chamber for invasion studies (C). After 22h, cells were fixed with methanol and stained with hematoxylin, and cells in 10 random fields were photographed and counted at 100-fold magnification. Extracellular addition of rAGR2 caused a significant dose-dependent increase in migration (BxPC-3-7 fold increase; MiaPaCa-2-3 fold increase) and invasion (BxPC-3-10 fold increase; MiaPaCa-2-3 fold increase). (D) For apoptosis assays, BxPC-3 cells were treated with or without Gem (1. mu.M) and AGR2(100 nM). Gem treatment resulted in a significant increase (1-fold) in apoptosis. Extracellular addition of rAGR2 significantly reduced Gem-induced apoptosis (50% reduction), thereby enhancing cancer cell survival. Data shown are mean ± SE (. + -.) of 3 experiments<0.05 control).

Figures 8A-i. candidate receptor binding and silencing. Western blot showing candidate receptor for AGR 2: (A) immunoprecipitation of CD59, (B) uPAR, and (C) C4.4A. IP analysis was performed using a commercially available anti-AGR 2 antibody and IgG was used as a control antibody. Immunoprecipitated samples were loaded for western blot analysis and gels were probed with the respective antibodies. AGR2IP and all three candidate receptors were co-immunoprecipitated. Lane 1: labeling with molecular weight; lane 2: (ii) a single lysate; lane 3: lysate + IgG Ab (mouse); lane 4: lysate + anti-AGR 2 antibody (mouse). The micrographs shown represent three independent experiments. (D) Recombinant AGR2 and c4.4a were combined in solution to test for direct physical interaction, and IP was performed using anti-AGR 2 antibody and c4.4a was probed by western blot. IgG was used as control antibody. As indicated in this pull-down experiment (pull-down experiment), rAGR2 and rc4.4a interacted directly. Lane 1: labeling with molecular weight; lane 2: recombinant protein + IgG Ab (mouse); lane 3: recombinant protein + anti-AGR 2 antibody (mouse). (E) RT-PCR of pancreatic cancer cell lines showed expression of C4.4A in all cell lines tested. The micrographs shown represent three independent experiments. (F) Western blot also showed expression of protein levels of c4.4a in all pancreatic cancer cell line lysates tested. The micrographs shown represent three independent experiments. (G-I) silencing of each candidate receptor was achieved transiently in AsPC-1 cells using siRNA at two final concentrations (5 and 10 nM). Western blots were performed with the respective antibodies, and the same membranes were also blotted with β -actin, which served as a loading control. Lane 1: labeling with molecular weight; lane 2: si control (5 nM); lane 3: si control (10 nM); lane 4: respective siRNA (5 nM); and lane 5: respective siRNA (10 nM). Two concentrations of siRNA showed almost complete silencing of each receptor. The micrographs shown represent three independent experiments.

Figure 9A-d. silencing c4.4a reduces basal and rAGR 2-mediated functions. BxPC-3 pancreatic cancer cells were transiently transfected with Si control, SiC4.4A, SiCD59, or SiuPAR. (A) For proliferation studies, cells were treated without (basal) and with rAGR2(100nM, daily) and cell numbers were estimated by MTS assay after 48 hours. The effect of silencing candidate receptors on migration (B) and invasion (C) was analyzed. C4.4a silencing significantly reduced basal and rAGR 2-stimulated proliferation, migration, and invasion. Silencing of other candidate receptors had no significant effect. (D) For apoptosis studies, cells were treated without or with Gem (0.5. mu.M) and rAGR2(100 nM). In Si control transfected cells, Gem stimulated apoptosis and the effect was improved by the addition of rAGR 2. In these cells, silencing of c4.4a induced apoptosis, however Gem did not further increase apoptosis. After c4.4a silencing, the addition of rAGR2 did not protect the cells from the effects of Gem. Data shown are mean ± SE (./p <0.05) of 3 experiments.

Figure 10A-b. c4.4a and integrin beta1 interact to mediate the effects of AGR 2. As part of the AGR2-C4.4A signaling complex, the effect of various integrins was tested by using a panel of siRNAs. (A) For apoptosis studies, siRNA transfected BxPC-3 cells were treated with none or Gem (0.5. mu.M) and AGR2(100 nM). In Si control transfected cells, addition of rAGR2 reduced Gem-mediated apoptosis. In contrast, when integrin β 1 is silenced, Gem stimulates apoptosis but this effect is not reduced by AGR 2. Silencing of other integrins did not have any significant effect on abrogating the survival effect mediated by AGR 2. Since laminins 1 and 5 are known to interact with c4.4a, these laminins were evaluated for their silencing effect on the survival effect of c4.4a mediated AGR 2. Silencing of laminin 1 and 5 abolished the survival effect mediated by AGR 2. (B) To determine the effect of integrin beta1 silencing on AGR 2-stimulated cell proliferation, cells were treated with different sirnas and allowed to grow for 48h before being analyzed with the MTS assay. Silencing of laminin 1 and 5 and integrin beta1 abrogates AGR 2-mediated proliferation. Data shown are mean ± SE (./p <0.05) of 3 experiments.

The monoclonal antibodies developed in fig. 11A-d bind to AGR2 and c4.4a proteins, respectively, and block their function. A panel of mAbs was used to identify AGR2(18kD) and C4.4A (50kD) from endogenous lysates (SU 86.86/Panc-1). However, commercial Ab showed non-specific bands. Additional bands identified with endogenous proteins were re-confirmed using recombinant proteins. For apoptosis studies, AspC-1 cells were treated without or with Gem (0.5. mu.M) and rAGR2(100nM) and with purified (A) AGR2 and (B) C4.4A Ab (1. mu.M). Commercially available abs were used as controls. Addition of Gem resulted in increased apoptosis, whereas addition of AGR2 resulted in survival benefits. The horizontal line represents the median apoptosis value. Data shown are mean ± SE (./p <0.05) of 3 experiments. (C, D) binding assays were performed using purified (C) AGR2 and (D) C4.4A Ab as selected from apoptosis assays with high blocking efficiency. ELISA assays were performed by coating with antigenic peptides (0.5 μ g) and probing with the respective purified abs. Data shown are mean ± SE (./p <0.05) of 3 experiments. Based on high specificity and binding, clone 28B was selected for AGR2 and 1A for c4.4a. Purified abs were also run on a 4% -20% gradient gel to check for the presence of heavy (50kD) and light (25kD) chains. AGR2 and c4.4a do not show additional bands.

FIG. 12 survival curves in Capan-2 tumors. The figure shows the survival curves of mice with in situ formed Capan-2 tumors and treated with control human igg (huctrl), humanized anti-AGR 2 mAb (HuAGR2) or humanized anti-c4.4a mAb (huc4.4a) antibodies.

Description of illustrative embodiments

The present invention is based, in part, on the discovery that c4.4a (LYPD3) is a functional cell surface receptor for extracellular AGR 2. Here, c4.4a (LYPD3) was identified as a functional cell surface receptor for extracellular AGR 2. To support the notion that the AGR2/c4.4a autocrine loop may be a therapeutic target for cancer, monoclonal blocking antibodies against both AGR2 and c4.4a were developed. In vivo treatment with these antibodies significantly reduced PDAC tumor weight and metastasis and prolonged survival. These results indicate that the AGR2/C4.4A interaction is a target with therapeutic potential for cancer therapy.

AGR2 and C4.4A

AGR2 correlates with poor outcomes in several tumor types (brechthowa et al, 2011) but the mechanism was not previously known. AGR2 has been reported to be involved in protein maturation and folding (Park et al, 2009; Zhao (Zhao) et al, 2010; althull (Altschul) et al, 1997; bijia (Higa) et al, 2011) to modulate cathepsin (dumaitin) et al, 2011) and to modulate MUC-1 levels (Park) et al, 2009; Norris (Norris) et al, 2013). However, the role of these AGR2 does not explain its ability to function as an oncogene (Wang et al, 2008) or AGR 2's ability to increase the aggressiveness of several types of cancer. Thus, it is possible that this protein has multiple intracellular and extracellular functions. Potentially, its physiological and pathological functions differ. In this study, extracellular addition of rAGR2 stimulated proliferation, migration, invasion, and chemoresistance of PDAC cells. These reactions require 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 role in amphibians, where it is a secreted signaling molecule that interacts with specific receptors.

In amphibians, AGR2 promotes limb growth by interacting with Prod1, a GPI-linked receptor associated with the human Ly6 family of receptors (Galat, 2008; charterjee and Mayor, 2001) (kuma (Kumar) et al, 2007; darwa (da Silva) et al, 2002). The Lys6 family includes uPAR, C4.4A and CD59 (Galat, 2008; DaShelv (da Silva), et al, 2002). The present study indicates that the Lys family receptors ((uPAR, c4.4a, and CD59) are co-immunoprecipitated with AGR2, possibly because of structural homology between these receptors (gallant, 2008.) however, this co-immunoprecipitation study cannot confirm this interaction only by blocking the interaction of AGR2 and c4.4a by silencing or blocking antibodies although dystrophin-1 binding to AGR2 was reported in the yeast two-hybrid system this observation surprisingly observed that silencing of the other two receptors CD59 and uPAR slightly increases the migration of PDAC cells this observation was unexpected as previous reports indicate that silencing uPAR inhibits PDAC cell migration (Xue et al, 2009) something unclear caused this difference, but this may be due to the study being performed in different cell lines, the data presented here support a model that AGR2 and c4.4a participate in autocrine loops that activate survival mechanisms.

In the case of a previous gene profiling study,c4.4a was found to be highly expressed in pancreatic cancer, but not in normal or chronic pancreatitis tissues (Logsdon et al, 2003). c4.4A is a regulatory factor previously described as cancer cell metastasis (Ross)Et al, 1998; orphan receptors of Jacobsen (Jacobsen) and pruu (Ploug), 2008). c4.4A in melanoma (Ross)Et al, 1998) and non-small cell lung cancer (Hansen) et al, 2007), and in breast cancer (Hansen) et al, 2007) and colorectal cancer (Paret) et al, 2007; in xiaoshi (Konishi) et al, 2010), c4.4a protein levels were associated with poor prognosis. C4.4a is defined herein as a functional cell surface receptor for AGR 2. Silencing or antibody-mediated blocking of c4.4a abrogated the effect of extracellular AGR2, thus supporting that AGR2 is a ligand for c4.4a. However, the mechanism of action of c4.4a has not been explored before. Thus, the signaling complex molecules interacting with c4.4a were examined to identify specific molecules.

Like other Glycosylphosphatidylinositol (GPI) -linked cell membrane receptors, c4.4a lacks an intracellular domain to mediate downstream signaling mechanisms. Based on the homology of c4.4a and uPAR (another member of the Ly6 family), these interactions may include extracellular matrix proteins and specific integrin receptors. C4.4a is known to promote migration by associating with α 6 β 4 (engora et al, 2012). C4.4a was previously reported to bind to laminin 1 and 5, although no functional studies were performed (Paret et al, 2005). Laminin 1 and 5 are believed to interact primarily with integrin α 3 β 1 (Smith) et al, 2001; bijia (Higa) et al, 2011). Integrin α 3 β 1 is expressed by pancreatic duct cells (ginger (Jiang et al, 2002). Silencing of laminin 1, laminin 5 or integrin beta1 abolished the effect of AGR2 treatment, thus suggesting their involvement in the AGR2 mediated c4.4a receptor complex.

To examine the potential therapeutic benefit of blocking the AGR2/c4.4a autocrine loop, blocking mabs were developed against both ligand (AGR2) and receptor (c4.4a). These two abs block both basal and AGR2 mediated functions. Preclinical studies using blocking mabs in three different types of preclinical models resulted in significant reductions in tumor weight and metastasis and improved survival. Treatment with mAb gave better benefit than treatment with gemcitabine (Gem) (clinical standard of care for PDAC). Partial or complete tumor regression was observed in several mice after treatment with either mAb alone or a combination of both mabs. Even several weeks after treatment, no tumor recurrence was observed.

Thus, AGR2 has an extracellular function that increases cancer cell invasiveness and c4.4a is a functional receptor for AGR 2. The signaling complex of c4.4a may include laminin 1, laminin 5, and β 1 integrin. Blocking mabs against AGR2 and/or c4.4a significantly reduced tumor growth and metastasis and caused tumor regression, resulting in significantly improved survival.

Therapeutic antibodies

In certain embodiments, antibodies or fragments thereof that bind to at least a portion of AGR2 or c4.4a protein and inhibit AGR2/c4.4a binding are contemplated, as well as related uses thereof in the treatment of disease. As used herein, the term "antibody" is intended to broadly refer to any immunobinder, such as IgG, IgM, IgA, IgD, and IgE, as well as polypeptides comprising CDR domains of antibodies that maintain 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 natural or synthetic ligand. Preferably, the anti-AGR 2 or anti-c4.4a antibody is a monoclonal or humanized antibody. By known means and as described herein, polyclonal or monoclonal antibodies, antibody fragments, and binding domains and CDRs (including engineered versions of any of the foregoing), specific for the AGR2 protein or the c4.4a protein, one or more of its respective epitopes, or conjugates of any of the foregoing, can be created, whether such antigens or epitopes are isolated from natural sources or synthetic derivatives or variants of natural compounds.

Examples of antibody fragments suitable for embodiments of the invention include, without limitation: (i) from V_L、V_H、C_LAnd C_H1A Fab fragment consisting of the domain; (ii) from V_HAnd C_H1A "Fd" fragment consisting of a domain; (iii) v from a single antibody_LAnd V_H(iii) an "Fv" fragment consisting of the domain; (iv) from V_HA "dAb" fragment consisting of a domain; (v) an isolated CDR region; (vi) a F (ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules ("scFv"), wherein V_HDomains and V_LThe domains are connected by a peptide linker that allows association of the two domains to form a binding domain; (viii) bispecific single chain Fv dimers (see U.S. Pat. No. 5,091,513); and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (U.S. patent application publication 20050214860). Fv, scFv, or diabodies may be joined by a linker V_HAnd V_LThe incorporation of disulfide bonds of the domains is stabilized. Minibodies comprising a scFv attached to the CH3 domain can also be made (Hu et al, 1996).

Antibody-like binding peptidomimetics are also contemplated in the examples. Liu (Liu) et al (2003) describe "antibody-like binding peptidomimetics" (ABiP), which are peptides that act as reduced antibodies and have certain advantages of long serum half-life and less cumbersome synthetic methods.

As is well known to those skilled in the art, AGR2 and C4.4A mRNA sequences (and SEQ ID NOs: 1 and 2, respectively) can be used to produce recombinant proteins and peptides. For example, such mRNA sequences may be engineered into a suitable expression system, such as yeast, insect cells, or mammalian cells, for the production of AGR2 or c4.4a proteins or peptides.

Animals can be vaccinated with an antigen such as soluble AGR2 or c4.4a protein to produce antibodies specific for AGR2 or c4.4a protein. Typically 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 that binds to an antigen for use in eliciting an immune response in an animal. Antibodies produced in animals in response to antigen vaccination include a variety of different molecules (polyclonal antibodies) made by a variety of B lymphocytes producing individual antibodies. Polyclonal antibodies are a mixed population of antibody species, each of which recognizes a different epitope on the same antigen. Given the correct conditions for polyclonal antibody production in an animal, most antibodies in the serum of the animal will recognize a common epitope on the antigenic compound of the immunized animal. The specificity is further enhanced by affinity purification so that only those antibodies recognizing the antigen or epitope of interest are selected.

Monoclonal antibodies are single species antibodies in which each antibody molecule recognizes the same epitope, since all antibody-producing cells are derived from a single B lymphocyte cell line. The process for producing monoclonal antibodies (mabs) generally begins along the same route as the preparation of polyclonal antibodies. In some embodiments, rodents such as mice and rats are used to produce monoclonal antibodies. In some embodiments, the monoclonal antibody is produced using rabbit, sheep, or frog cells. The use of rats is well known and may provide certain advantages. Mice (e.g., BALB/c mice) are routinely used and typically give a high percentage of stable fusions.

Hybridoma technology involves the fusion of a single B lymphocyte from a mouse previously immunized with AGR2 or the c4.4a antigen with immortal myeloma cells (usually mouse myeloma). This technique provides a method of propagating a single antibody-producing cell for unlimited number of productions, allowing the production of unlimited number of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity.

In one embodiment, the antibody is a chimeric antibody, e.g., an antibody comprising an antigen binding sequence from a non-human donor grafted onto a heterologous non-human, or humanized sequence (e.g., a framework and/or constant domain sequence). Methods have been developed to replace the light and heavy chain constant domains of monoclonal antibodies with similar human-derived domains, 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 the variable domains of monoclonal antibodies to more human forms by recombinantly constructing antibody variable domains having both rodent, e.g., mouse, and human amino acid sequences. In "humanized" monoclonal antibodies, only the hypervariable CDRs are derived from a mouse monoclonal antibody 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 believed that the replacement of an amino acid sequence characteristic of rodents in an antibody with an amino acid sequence found at a corresponding position in a human antibody will reduce the likelihood of an adverse immune response during therapeutic use. Antibody-producing hybridoma cells or other cells may also be genetically mutated or otherwise altered, which may or may not alter the binding specificity of the antibodies produced by the hybridoma cells.

Methods for producing polyclonal antibodies in various animal species, as well as for producing various types of monoclonal antibodies, including humanized, chimeric, and fully human antibodies, are well known in the art and are highly predictable. For example, the following U.S. patents and patent applications provide an enabling description of such methods: U.S. Pat. nos. 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797, respectively; 4,472,509; 4,606,855, respectively; 4,703,003, respectively; 4,742,159, respectively; 4,767,720, respectively; 4,816,567; 4,867,973, respectively; 4,938,948, respectively; 4,946,778; 5,021,236, respectively; 5,164,296, respectively; 5,196,066, respectively; 5,223,409; 5,403,484; 5,420,253, respectively; 5,565,332; 5,571,698; 5,627,052; 5,656,434, respectively; 5,770,376, respectively; 5,789,208; 5,821,337; 5,844,091, respectively; 5,858,657, respectively; 5,861,155, respectively; 5,871,907, respectively; 5,969,108, respectively; 6,054,297; 6,165,464, respectively; 6,365,157, respectively; 6,406,867, respectively; 6,709,659, respectively; 6,709,873, respectively; 6,753,407, respectively; 6,814,965, respectively; 6,849,259, respectively; 6,861,572, respectively; 6,875,434, respectively; 6,891,024, respectively; 7,407,659, respectively; and 8,178,098. All patents, patent application publications, and other publications cited herein or in this application are hereby incorporated by reference.

Antibodies can be produced from any animal source, including birds and mammals. Preferably, the antibody is ovine, murine (e.g., mouse or rat), rabbit, goat, guinea pig, camel, horse, or chicken. Additionally, newer technologies allow the development and screening of human antibodies from human combinatorial antibody libraries. For example, phage antibody expression technology allows for the production of specific antibodies in the absence of animal immunization, as described in U.S. patent No. 6,946,546, which is incorporated herein by reference. These techniques are further described in max (Marks) et al (1992); schtermer (Stemmer) (1994); gram (Gram) et al (1992); mabas (Barbas) et al (1994); and Schier et al (1996).

It is fully expected that antibodies against AGR2 and/or c4.4a will have the ability to block AGR2/c4.4a binding regardless of the animal species, monoclonal antibody cell line, or other source of antibody. Certain animal species are less preferred for the production of therapeutic antibodies because they are more likely to cause allergic reactions due to activation of the complement system by the "Fc" portion of the antibody. However, intact antibodies can be enzymatically digested into "Fc" (complement-binding) fragments and antibody fragments with binding domains or CDRs. Removal of the Fc portion reduces the likelihood that the antigen antibody fragment will elicit an undesirable immune response, and thus, antibodies without Fc may be preferred for prophylactic or therapeutic treatment. As described above, antibodies can also be constructed so as to be chimeric or partially or fully human to reduce or eliminate adverse immune consequences caused by administering to an animal antibodies that have been raised in or have sequences from other species.

Substitution variants typically comprise the replacement of one amino acid with another at one or more positions within a protein, and may be designed to modulate one or more properties of a polypeptide, with or without loss of other function or property. Substitutions may be conservative, i.e., one amino acid is replaced by an amino acid of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following exchanges: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine to serine; glutamine to asparagine; glutamic to aspartic acids; 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, the substitutions may be non-conservative such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve the substitution of a residue with a chemically dissimilar residue, such as changing a polar or charged amino acid to a non-polar or uncharged amino acid, and vice versa.

The protein may be recombinant or synthesized in vitro. Alternatively, the non-recombinant protein or recombinant protein may be isolated from bacteria. It is also contemplated that bacteria comprising such variants can be implemented in compositions and methods. Therefore, protein isolation is not required.

It is contemplated to have between about 0.001mg and about 10mg total polypeptide, peptide, and/or protein/ml in the composition. Thus, the concentration of protein in the 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, 10.0mg/ml or more (or any range derived 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%, 1%, or more, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% can be antibodies that bind to AGR2 or c4.4a.

The antibody or preferably the immunological part of the antibody may be chemically conjugated to or expressed as a fusion protein with other proteins. For the purposes of the present specification and appended claims, all such fusion proteins are included within the definition of antibody or antibody immune portion.

The embodiments provide antibodies and antibody-like molecules against AGR2 and c4.4a, polypeptides and peptides linked to at least one agent to form an antibody conjugate or payload. In order to increase the efficiency of an antibody molecule as a diagnostic or therapeutic agent, 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 include molecules having a desired activity, such as cytotoxicity. Non-limiting examples of effector molecules that have been attached to antibodies include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, and the like. In contrast, a reporter is defined as any moiety that can be detected by an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent tags, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.

Several methods are known in the art for attaching or conjugating an antibody to its conjugate moiety. Some attachment methods involve the use of metal chelates, for example, the use of organic chelators attached to antibodies, such as diethylenetriaminepentaacetic acid anhydride (DTPA); ethylene triamine tetraacetic acid; n-chloro-p-toluenesulfonamide; and/or tetrachloro-3-6-biphenylglycoluril. Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein labels are prepared in the presence of these coupling agents or by reaction with isothiocyanates.

Treatment of diseases

Certain aspects of the embodiments of the invention may be used for the prevention or treatment of diseases or disorders associated with the AGR2/c4.4a mediated autocrine survival loop. The functionality of the AGR2/C4.4A autocrine loop may be reduced by any suitable drug to prevent the AGR2/C4.4A interaction. Preferably, such a substance may be an anti-AGR 2 or anti-c4.4a antibody.

"Treatment" or "treating" refers to the administration or application of a therapeutic agent to a subject or the performance of a procedure or modality on a subject with the purpose of obtaining a therapeutic benefit for a disease or health-related disorder. For example, treatment may comprise administering a pharmaceutically effective amount of an antibody that inhibits the AGR 2/c4.4a-mediated autocrine survival loop.

"Subject" and "patient" refer to humans or non-humans, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.

As used throughout this application, the terms "therapeutic benefit" or "therapeutically effective" refer to anything that promotes or enhances the health of a subject with respect to the medical treatment of such a condition. This includes, but is not limited to, a reduction in the frequency or severity of disease signs or symptoms. For example, treatment of cancer may involve, for example, reduction in tumor size, reduction in tumor invasiveness, reduction in cancer growth rate, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject having cancer.

Antibodies that bind to AGR2 or c4.4a can be administered to treat cancer. The cancer may be a solid tumor, metastatic cancer or non-metastatic cancer. In certain embodiments, the cancer may originate from the bladder, blood, bone, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.

The cancer may be specifically the following histological types, but it is not limited to these: neoplasms, malignant; epithelial cancer; undifferentiated epithelial carcinoma; giant fusiform cell carcinoma; small cell carcinoma; small cell lung cancer; non-small cell lung cancer; papillary carcinoma; squamous cell carcinoma; lymphatic epithelial cancer; basal cell carcinoma; hair matrix cancer; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinomas; bile duct cancer; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; familial polyposis coli adenocarcinoma; a solid cancer; malignant carcinoid tumors; bronchioloalveolar carcinoma; papillary adenocarcinoma; a cancer of the chromophobe; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granulosa cell adenocarcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-encapsulated sclerosing cancer; adrenocortical carcinoma; endometrioid carcinoma; skin appendage cancer; hyperhidrosis carcinoma; sebaceous gland cancer; cerumen adenocarcinoma; mucoepidermoid carcinoma; cystic carcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory cancer; paget's disease of the breast; acinar cell carcinoma; squamous carcinoma of gland; adenocarcinoma w/squamous metaplasia; malignant thymoma; malignant ovarian stromal tumors; malignant blastocyst cell tumors; malignant granulosa cell tumors; malignant male blastoma; a supporting cell carcinoma; malignant stromal cell tumors; malignant lipocytoma; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; hemangiospherical sarcoma; malignant melanoma; melanoma-free melanoma; superficial diffusible melanoma; giant pigmented nevus malignant melanoma; epithelial-like cell melanoma; malignant blue nevi; a sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; interstitial sarcoma; malignant mixed tumor; a mullerian mixed tumor; nephroblastoma; hepatoblastoma; a carcinosarcoma; malignant stromal tumors; malignant brenner's tumor; malignant phyllo-tumor; synovial sarcoma; malignant mesothelioma; clonal cell tumors; embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant vascular endothelioma; kaposi's sarcoma; malignant vascular endothelial cell tumors; lymphangioleiomyosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; malignant odontogenic tumors; amelogenic cell dental sarcoma; malignant ameloblastic tumors; amelogenic cell fibrosarcoma; malignant pineal tumor; chordoma; malignant glioma; ependymoma; astrocytoma; primary plasma astrocytoma; fibroid astrocytoma; astrocytomas; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectoderm; cerebellar sarcoma; ganglionic neuroblastoma; neuroblastoma; retinoblastoma; olfactive neurogenic tumors; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granulosa cell tumors; malignant lymphoma; hodgkin's disease; hodgkin; granuloma paratuberis; small lymphocytic malignant lymphoma; large cell diffuse malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other designated non-hodgkin lymphomas; malignant tissue cell proliferation; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphoma cell leukemia; leukemia of the myeloid lineage; basophilic granulocytic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia.

A. Pharmaceutical preparation

In the case where clinical use of therapeutic compositions comprising inhibitory antibodies is initiated, it is often beneficial to prepare pharmaceutical or therapeutic compositions suitable for the intended use. This will typically require the preparation of a pharmaceutical composition that is substantially free of pyrogens and any other impurities that may be harmful to humans or animals. Suitable buffers may also be used to stabilize the complex and allow uptake by the target cells. In certain embodiments, the pharmaceutical composition may include, for example, at least about 0.1% active compound. In other embodiments, the active compound may comprise between about 2% to about 75%, or for example between about 25% to about 60%, and any range derivable therein, by weight of the unit.

The therapeutic compositions of the embodiments of the invention are advantageously administered in the form of an injectable composition as a liquid solution or suspension; solid forms suitable for dissolution or suspension in a liquid prior to injection may also be prepared. These formulations may also be emulsified.

The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, such as a human. The preparation of pharmaceutical compositions comprising antibodies or additional active ingredients will be known to those skilled in the art in view of this disclosure. In addition, for animal (e.g., human) administration, it is understood that the formulation should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA office of biological standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol/water solutions, saline solutions, parenteral carriers such as sodium chloride, ringer's dextrose, and the like), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids, and nutritional supplements, like materials, and combinations thereof, as known to those of ordinary skill in the art. The pH and precise concentration of the various components of the pharmaceutical composition are adjusted according to well-known parameters.

The term "unit dose" or "dose" refers to physically discrete units suitable for use in a subject in connection with administration (i.e., an appropriate route and treatment regimen), each unit containing a predetermined amount of a therapeutic composition calculated to produce a desired response as discussed above. The amount administered, both in terms of number of treatments and unit dose, depends on the desired effect.

The actual dosage of the compositions of the embodiments of the invention administered to a patient or subject may be determined by physical and physiological factors such as the weight, age, health and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic intervention, the patient's primary disease, the route of administration, and the potency, stability and toxicity of the particular therapeutic agent. For example, the dosage per administration may include about 1 μ g/kg/body weight to about 1000 mg/kg/body weight (such ranges include intermediate doses) or more, and any range derivable therein. In non-limiting examples of ranges derived from the numbers recited herein, about 5 μ g/kg/body weight to about 100 mg/kg/body weight ranges, about 5 μ g/kg/body weight to about 500 mg/kg/body weight ranges, and the like can be administered. In any event, the practitioner responsible for administration will determine the concentration of one or more active ingredients in the composition and the appropriate dose or doses for the individual subject.

The active compounds may be formulated for parenteral administration, for example for injection by the intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions may be prepared as liquid solutions or suspensions; solid forms suitable for preparing solutions or suspensions upon addition of liquid prior to injection may also be prepared; and the formulation may also be emulsified.

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 easy injection is possible. It should also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The proteinaceous composition can be formulated as a neutral form or as a salt. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such 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, hydroxides of sodium, potassium, ammonium, calcium, or iron), and organic bases (such as isopropylamine, trimethylamine, histidine, procaine, and the like).

The pharmaceutical composition may comprise a solvent or dispersion medium comprising, for example: water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and vegetable oils. 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 action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, 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 of compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

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

The therapeutic compositions of the present invention are advantageously administered in the form of injectable compositions as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid prior to injection may also be prepared. These formulations may also be emulsified. Typical compositions for such purposes include a pharmaceutically acceptable carrier. For example, the composition may comprise 10mg, 25mg, 50mg, or up to about 100mg of human serum albumin per ml of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers, and the like.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol/aqueous solutions, saline solutions, parenteral carriers such as sodium chloride, ringer's dextrose, and the like. Intravenous vehicles include fluids and nutritional supplements. Preservatives include antimicrobials, antioxidants, chelating agents, and inert gases. The pH and precise concentration of the various components of the pharmaceutical composition are adjusted according to well-known parameters.

In particular embodiments, the compositions of the present invention are suitable for application to a mammalian eye. For example, the formulation may be a solution, suspension or gel. In some embodiments, the composition is administered via a biodegradable implant, such as an intravitreal implant or an intraocular insert, e.g., an intraocular insert designed for placement against the conjunctival surface. In some embodiments, the therapeutic agent coats the medical device or implantable device. The formulations of the present invention may be applied to the eye in drops in an aqueous solution. The drops may be delivered from a single dose ampoule which is preferably sterile and thus makes the bacteriostatic component of the formulation unnecessary. Alternatively, drops may be delivered from a multi-dose bottle, which preferably includes a means to extract the preservative from the formulation upon release of the formulation, such means being known in the art. In other aspects, the components of the invention can be delivered to the eye as a concentrated gel or similar vehicle that forms a soluble insert that is placed under the eyelid.

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

The therapeutic compositions of the present invention may include classical pharmaceutical agents. Administration of the therapeutic compositions according to the invention will be by any general route, as long as the target tissue is available by that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Topical administration is particularly advantageous for the treatment of skin cancer to prevent chemotherapy-induced alopecia or other hyperproliferative disorders of the dermis. Alternatively, administration can be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions are typically administered as pharmaceutically acceptable compositions that include a physiologically acceptable carrier, buffer or other excipient. For treatment of pulmonary or respiratory conditions, aerosol delivery may be used, the volume of the aerosol being between about 0.01mL and 0.5 mL.

The effective amount of the therapeutic composition depends on the intended target. For example, one skilled in the art would readily recognize the appropriate amount of the compound by considering factors such as the size and weight of the subject; the degree of neovascularization or disease penetration; the age, health, and sex of the subject; the route of administration; and whether administration will be local or systemic, the effective amount of an antibody of the invention administered to a given subject can be readily determined. The term "unit dose" or "dose" refers to physically discrete units suitable for use in a subject in connection with administration (i.e., an appropriate route and treatment regimen), each unit containing a predetermined amount of a therapeutic composition calculated to produce a desired response as discussed above. The amount administered, both in terms of number of treatments and unit dose, depends on the desired protection or effect.

The exact amount of therapeutic composition will also depend on the judgment of the practitioner and will be specific to each individual. Factors that affect dosage include the physical and clinical state of the patient, the route of administration, the intended target of treatment (e.g., symptom relief versus cure), and the efficacy, stability, and toxicity of the particular therapeutic substance.

B. Combination therapy

In certain embodiments, the compositions and methods of the present embodiments relate to antibodies or antibody fragments directed to AGR2 or c4.4a in combination with a second or additional therapy to inhibit AGR2/c4.4a interactions. This therapy can be applied to the treatment of any disease associated with the AGR2/C4.4A mediated autocrine survival loop. For example, the disease may be cancer.

Methods and compositions comprising combination therapy enhance therapeutic or protective effects and/or increase the efficacy of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve a desired effect (e.g., killing cancer cells and/or inhibiting cell hyperproliferation). This process may involve contacting the cell with both the antibody or antibody fragment and the second therapy. The tissue, tumor, or cell may be contacted with one or more compositions or pharmacological formulations comprising one or more drugs (i.e., antibodies or antibody fragments or anti-cancer agents), or by contacting the tissue, tumor, and/or cell with two or more different 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 anti-cancer agent. It is further contemplated that such combination therapy may be used in combination with chemotherapy, radiation therapy, surgical therapy, or immunotherapy.

When the terms "contacting" and "exposing" are applied to a cell, this is used herein to describe a process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct proximity to a target cell. For example, to achieve cell killing, the two agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

The inhibitory antibodies can be administered before, during, after, or in various combinations relative to the anti-cancer treatment. The time interval for administration may range from simultaneous to several minutes to several days to several weeks. In embodiments where the antibody or antibody fragment is provided to the patient separately from the anti-cancer agent, it is generally ensured that a significantly long time does not expire between each delivery, such that the two compounds are still able to exert a favorable combined effect on the patient. In such cases, it is contemplated that one may provide both antibody therapy and anti-cancer therapy to the patient within about 12 to 24 or 72 hours of each other, particularly within about 6-12 hours of each other. When several days (2, 3,4, 5,6, or 7) or weeks (1, 2, 3,4, 5,6, 7, or 8) elapse between administrations, it may be desirable in some cases to significantly extend the period of time for treatment.

In certain embodiments, a course of treatment will last 1-90 days or longer (this range includes the middle days). It is contemplated that one agent is administered on any one day or any combination thereof from day 1 to day 90 (this range includes intermediate days) and another agent is administered on any one day or any combination thereof from day 1 to day 90 (this range includes intermediate days). One or more agents may be administered to a patient one or more times during a day (over 24 hours). In addition, after a course of treatment, a period of time during which no anti-cancer therapy is administered is considered. This period may last from 1 to 7 days, and/or from 1 to 5 weeks, and/or from 1 to 12 months or longer (this range includes intermediate days), depending on the patient's condition, such as their prognosis, strength, health, etc. It is expected that the treatment cycle will be repeated as necessary.

Different combinations may be used. For example, below, the antibody therapy is "a" and the anti-cancer therapy is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

administration of any of the compounds or therapies of the embodiments of the present invention to a patient will follow the general protocol for administering such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring toxicity due to the combination therapy.

1. Chemotherapy

A variety of chemotherapeutic agents may be used in accordance with embodiments of the present invention. The term "chemotherapy" refers to the treatment of cancer with drugs. "chemotherapeutic agent" is used to refer to a compound or composition that is administered in the treatment of cancer. These agents or drugs are classified by the way they are active in the cell, e.g., whether or at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly cross-link DNA, insert into DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, metodopa, and urodopa; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethamine; acetogenins (acetogenins) (especially bullatacin (bullatacin) and bullatacin (bullatacinone)); camptothecin (including the synthetic analogue topotecan); bryostatins; callystatin; CC-1065 (including its Aldocosan, Kazelesin and Bizelesin synthetic analogs); nostoc (especially nostoc 1 and nostoc 8); dolastatin; duocarmycin (duocarmycin) (including synthetic analogs, KW-2189 and CB1-TM 1); eislobin (eleutherobin); coprinus atrata base (pancratistatin); sarcandra glabra alcohol (sarcodictyin); 1 of spongin; nitrogen mustards (nitrogen mustards), such as meconin, chlorambucil, chlorophosphamide, estramustine, ifosfamide, mechlorethamine (mechlorethamine), mechlorethamine hydrochloride, melphalan, neoentin, benzene mustard cholesterol, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorourethrin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin γ 1 and calicheamicin ω I1); dynemicin, including dynemicin a; bisphosphonates, such as clodronate; an esperamicin; and neocarzinostatin chromophores and related tryptophane diyne antibiotic chromophores, aclacinomycin, actinomycin, anthranilic acid, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, carcinomycin, tryptophysin, chromomycin, dactinomycin, daunomycin, mitorubicin, 6-diazo-5-oxo-L-leucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mariomycin, mitomycins (such as mitomycin C, mycophenolic acid, noramycin, olivomycin, pelomomycin, pofiomycin, puromycin, ferrirubicin, roxobicin, streptonigrin, streptozotocin, tubercidin, ubenicillin, staphylol, diclodine, and the like, And zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as carpoterone, drotandrosterone propionate, epitioandrostanol, meperiane, testolactone; anti-adrenal agents such as mitotane and troostine; folic acid replenisher such as folinic acid; acetic acid glucurolactone; (ii) an aldophosphamide glycoside (aminolevulinic acid); eniluracil; amsacrine; betabucin (betastaucil); a bisantrene group; edatrexae; desphosphamide (defofamine); dimecorsine; diazaquinone; eflornithine (elformithine); ammonium etiolate; an epothilone; etoglut; gallium nitrate; a hydroxyurea; lentinan; lonidamine (lonidainine); maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanol; diamine nitracridine; pentostatin; methionine; pirarubicin; losoxanthraquinone; podophyllinic acid; 2-ethyl hydrazide; procarbazine; PSK polysaccharide complex; lezoxan; rhizomycin; a texaphyrin; helical germanium; alternarionic acid; a tri-imine quinone; 2,2' -trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucosin A, bacillocin A and snakesin); uratan; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulcitol; pipobroman; capping neomycin (glycine); cytarabine ("Ara-C"); cyclophosphamide; taxanes, 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; nuantro (novantrone); (ii) teniposide; edatrexae; daunomycin; aminopterin; (xiloda); ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoid acids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicamycin (plicomycin), gemcitabine, navelbine, farnesyl protein transferase inhibitors, antiplatin, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

2. Radiotherapy

Other factors that cause DNA damage and have been widely used include the so-called targeted delivery of gamma rays, X-rays, and/or radioisotopes to tumor cells. Other forms of DNA damage factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Pat. nos. 5,760,395 and 4,870,287), and UV-irradiation. Most likely, all of these factors cause a wide range of damage to DNA, to precursors of DNA, to DNA replication and repair, and to assembly and maintenance of chromosomes. The dose of X-rays ranges from a daily dose of 50 to 200 roentgens for a prolonged period of time (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dosage of radioisotopes varies widely, and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by neoplastic cells.

3. Immunotherapy

The skilled person will appreciate that additional immunotherapies may be combined or used in conjunction with the methods of the examples. Generally, in the context of cancer treatment, immunotherapeutics rely on the use of immune effector cells and molecules to target and destroy cancer cells.(rituximab) is one such example.(Yipriomama; Bezishi Meishibao) is an example of an approved anti-CTLA-4 antibody.(pembrolizumab; merck) and(nivolumab; Beshizubao corporation, Behcet.) is an example of an approved anti-PD-1 antibody. The immune effector may be, for example, an antibody specific for a marker on the surface of a tumor cell. The antibody may act alone as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody may also be conjugated to one drug or toxin (chemotherapeutic, radionuclide, ricin a chain, cholera toxin, pertussis toxin, etc.) and serve as only one targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

In one aspect of immunotherapy, tumor cells necessarily carry some sort of marker suitable for targeting, i.e., the marker is not present on most other cells. There are many tumor markers and any of these may be suitable for targeting in the context of embodiments of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (P97), gp68, TAG-72, human milk fat globules, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and P155. An alternative aspect of immunotherapy is the combination of anti-cancer and immunostimulatory effects. Immunostimulatory molecules also exist, including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-interferon, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

Examples of immunotherapies currently being explored or used are immunological adjuvants such as mycobacterium bovis, plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. nos. 5,801,005 and 5,739,169); hui (Hui) and Qianban (Hashimoto), 1998; kreiss duride (Christodoulides) et al, 1998); cytokine therapy: such as interferons alpha, beta, and gamma, IL-1, GM-CSF and TNF (Bukowski et al, 1998; Davison et al, 1998; Hersterland et al, 1998); gene therapy, such as TNF, IL-1, IL-2, and p53 (Qin et al, 1998; Hausdin-Wood (Austin-Ward) and Villaseca (Villaseca), 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD 20, anti-ganglioside GM2, and anti-p 186 (Hollander, 2012; Hanibuchi et al, 1998; U.S. Pat. No. 3, 5,824,311). It is contemplated that one or more anti-cancer therapies may be used with the antibody therapies described herein.

4. Surgical operation

About 60% of the population with cancer will undergo some type of surgery, including prophylactic, diagnostic or staged, curative, and palliative surgery. Curative surgery includes resection (in which all or part of the cancerous tissue is physically removed, resected, and/or destroyed), and may be used in conjunction with other therapies, such as the treatments, chemotherapies, radiation therapies, hormonal therapies, gene therapies, immunotherapies, and/or alternative therapies of embodiments of the present invention. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (morse's surgery).

When a part or all of cancer cells, tissues or tumors are excised, a cavity is formed in the body. Treatment may be accomplished by regional perfusion, direct injection, or topical application with additional anti-cancer therapies. 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 also have varying doses.

5. Other agents

It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the efficacy of the treatment. These additional agents include agents that affect upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions increases the anti-hyperproliferative effect on the adjacent hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the embodiments of the invention to improve the anti-hyperproliferative efficacy of the treatments. Cell adhesion inhibitors are contemplated to improve the efficacy of embodiments of the invention. Examples of cell adhesion inhibitors are Focal Adhesion Kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the present embodiments to improve therapeutic efficacy.

Kit and diagnosis

In various aspects of the embodiments, kits comprising therapeutic and/or other therapeutic agents and delivery agents are contemplated. In some embodiments, kits for preparing and/or administering the therapies of the embodiments are contemplated. The kit may include one or more sealed vials containing any of the pharmaceutical compositions of the embodiments of the present invention. The kit may include, for example, at least one AGR2 or c4.4a antibody and reagents for making, formulating, and/or administering the components of the examples or performing one or more steps of the methods of the invention. In some embodiments, the kit further comprises a suitable container, which is a container that is non-reactive with the kit components, such as an Eppendorf tube, assay plate, syringe, vial, or tube. These containers may be made of sterilizable materials such as plastic or glass.

The kit may further comprise a leaflet summarizing the procedural steps of the methods described herein and will essentially follow the same procedures as described herein or be known to one of ordinary skill in the art. The instructional information can be in a computer-readable medium comprising machine-readable instructions which, when executed by a computer, cause the display of a real or virtual program for delivering a pharmaceutically effective amount of the therapeutic agent.

Examples III

The following examples are included to illustrate preferred embodiments of the invention. 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 invention, 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 invention.

Materials and methods

A cell line. NIH 3T3, BXPC3, su86.86, MiaPaCa-2, AsPC-1 and Capan-2 cells were obtained from ATCC (Manasas, Va.). Confirmation of cell line identity using DNA fingerprinting (16 systems, plomega). Cells were routinely cultured in DMEM containing 10% FBS and maintained at 5% CO at 37 ℃₂In a humid atmosphere.

Antibodies and recombinant proteins. Purchase antibodies against: AGR2 (mouse polyclonal), DAG-1, CD59(Cat # ab56703, ab105504, ab9182, Abcam, cambriqi, massachusetts), laminin 1, laminin 5, ITG β 1, α 6, β 4(sc-74417, sc-20145, sc-9936, sc-10730, santal cruz biotechnology, dallas, texas), c4.4a, uPAR (Cat # AF5428, MAB807, R & D Systems, minneapolis, minnesota), p-ERK (Cat #9160, cell signaling, denves, massachusetts), β -actin (Cat # a2066, sigma, louis, missouri); and control IgG (Cat # OB 010701; southern Biotechnology, Bermingham, Alabama). Human and mouse AGR2 proteins have 96% homology (brechthowa (Brychtova) et al, 2011) so human recombinants (rAGR2) (ab64013, AbCam, cambridge, massachusetts) were used for all studies. Recombinant c4.4a was also purchased (5428-C4-050, R & D Systems, minneapolis, mn).

The following pre-designed and pre-validated siRNAs were purchased from Qiagen (Qiagen) (los Angeles, Calif.) SI control (Cat #1027281), siAGR2(Cat # SI04274522), siC4.4A (Cat # SI00105700,707,714,721), siUPAR (Cat # SI03033289), siCD59(Cat # SI03052616), laminin (α 1) (Cat # SI02779511), laminin 5(β 3) (Cat # SI02664116), integrin α 6(Cat # SI02654078), integrin β 1(Cat # SI 00500373), integrin β 2(Cat # SI03648848), and integrin β 4(Cat # SI 02664109). cells were used with siRNAs (5 or 10nM)Transfection reagent (Cat # 301705; Qiagen, los Angeles, Calif.) and lysates were prepared after 72 h.

And (4) IP research. Commercial AGR2 antibody (2. mu.g; Abcam) was used to co-immunoprecipitate AGR2 from SU86.86 lysate (100. mu.g) and subjected to Western blotting. The same membrane was then probed individually for each antibody and with mixed antibodies. In addition, rAGR2 and rC4.4A (2. mu.g each) were suspended in lysis buffer and subjected to IP. IgG (mouse) was used as control. Western blot imaging and processingImaging systems (LI-COR bioscience, Lincoln, Nebraska).

Cell growth, migration, invasion and apoptosis assays. Wild type and siRNA transfected PDAC cells were grown with rAGR2(0-500nM) in the presence or absence of antibody (polyclonal commercial or newly developed mAb) (1. mu.M). The medium was refreshed daily. Cell number was estimated after 48 hours by MTS assay as previously described (Ramachandran et al, 2007). To avoid differences in the basal values of each cell line, the data are presented as a percentage of viable cells compared to the appropriate control. Migration and invasion assays were performed at 24h as previously described (Ramachandran et al, 2007). Apoptosis assays were performed 72h after addition of gemcitabine (Gem) to Gem sensitive BxPC-3 cells (1 μ M) and Gem resistant AsPC-1 cells (5 μ M) (Arumugam et al, 2009) as previously described (Ramachandran et al, 2008). Since siRNA transfection itself induced apoptosis in the substrate, cells transfected with siRNA were treated with lower concentrations of Gem (BxPC 3: 0.5. mu.M; AsPC-1: 1. mu.M).

Immunohistochemical (IHC) staining. IHC was performed with mAb (1:1000) on Tissue Microarray (TMA) slides and using mAb (1:1000) as previously described (Ramachandran et al, 2008)General kit (Vector Laboratories, Burlingham, Calif.) development. The results were blindly evaluated by a pathologist and the expression levels were classified as positive or negative (respectively>10% or<10% cytoplasmic staining of tumor cells) and the staining intensity was classified as strong, medium, or no staining. Apoptotic cells were detected in paraffin sections by fluorescent-labeled TUNEL staining (promega, Cat # G3250). Activation of the MapK pathway was assessed by level analysis of p-ERK (cell signaling, Cat # 9160; 1:1000) and the proliferation index of cancer cells was measured by Ki-67 staining (Thermo Scientific, Cat # RM-9106-S0; 1: 200).

And (3) developing a blocking monoclonal antibody. In the Monoclonal antibody core of UT MDACC (Monoclonal antibody core of UT MDACC), mouse Monoclonal antibodies (mabs) against AGR2 and c4.4a were developed using unconjugated antigenic peptides. Several hybridoma colonies (2400 per antibody) were screened for target recognition by ELISA using KLH conjugated peptide. The selected hybridoma colonies were then cloned and screened again by ELISA to select those with the highest affinity. Selected clones were subcloned and purified using a protein a column. Antibody specificity, blocking ability and purity were confirmed by western blotting, functional screening (apoptosis assay), binding assay (ELISA assay), and purity analysis (SDS-PAGE) of the selected abs against recombinant and cytolytic proteins (as shown in fig. 11A-D). In vitro validation experiments with selected antibodies include inhibition of cancer cell migration and invasion and the ability to increase Gem-induced apoptosis. The first few candidate antibodies with high affinity and functional blocking ability (each against AGR2 and c4.4a) were purified and further used in vivo experiments. The final selected clones were 28B for AGR2 and 1A for c4.4a. Antibodies were typed as IgG1 against AGR2 and IgG2b against c4.4a. Purified antibodies for in vivo experiments were produced in the core of monoclonal antibodies.

In vivo studies. Athymic nude mice were used for in vivo experiments according to the regulatory standards of UT MDACC and approved by the IUCAC Committee (B6.Cg-Foxn 1)^nuFemale-9 weeks old)/J-female (NCI, Besserda, Maryland). Using luciferasesLabeled cells (0.25 × 10)⁶) Establishing in situ tumor. IgG (Cat # OB 010701; southern Biotechnology, Burmingham, Alabama) was used as control Ab.

Model 1 (AsPC-1-invasive cell model) -two weeks after invasive AsPC-1 cell injection, when the tumor weight was less than 0.5g (as surgically confirmed from the parallel untreated group), mice (n ═ 6) were treated with control or mAb (AGR2/c4.4amab 5mg each in combination/kg/body weight/twice a week/i.p) and with or without gems (100mg/kg body weight/once a week/i.p) until all control mice had died (seven weeks). At the end of the experiment, tumor weights and metastases to liver and lung were compared ex vivo between control and treatment groups.

Model 2 (Capan-2-matrix model) -two weeks after injection of matrix-forming Capan-2 cells, mice (n ═ 7) were treated with 15mg AGR2 or c4.4a antibody/kg/body weight/twice a week/i.p. Treatment was stopped after 15 weeks (13 weeks of treatment) and tumor size in surviving animals was monitored by bioluminescence until 63 weeks.

Model 3(Capan 2-regression study) -four weeks after injection of Capan-2 cells when tumor weight exceeded 1g (as surgically confirmed from the parallel untreated group), mice (n ═ 5) were treated with AGR2 or c4.4a mAb at 15 mg/kg/body weight/twice a week/i.p or with both mAb combinations (7.5 mg each). The treatment was stopped after 12 weeks. Bioluminescence was monitored in surviving animals until 18 weeks. After injection of a fluorescein substrate (Xenogen, Aramada, Calif.), utilization was madeLive animal bioluminescence imaging systems measure tumor growth and metastasis weekly. The number of surviving mice was recorded weekly and shown as a percentage of the original group size.

And (5) carrying out statistical analysis. All in vitro experiments were performed in triplicate and on three or more separate occasions. Data are presented as mean ± SEM of three or more independent experiments. In vivo experiments were performed with groups of 7-10 mice. Statistically significant differences were determined by ANOVA analysis (Newman-Keuls multiple comparison test) and were defined as p-values < 0.05.

Example 1 extracellular AGR2 stimulation of PDAC invasiveness and chemoresistance in vitro

AGR2 was previously shown to be highly expressed and secreted by PDAC cells and to contribute to chemoresistance (Ramachandran et al, 2008). Here, it was evaluated whether extracellular AGR2(rAGR2) mimics AGR2 expression. Since the PDAC cell line is heterogeneous, a multicellular model-BxPC-3 (epithelial phenotype, sensitive to Gem), AsPC-1 and MiaPaCa-2 cells (mesenchymal phenotype, high resistance to Gem) were used (Arumugam et al, 2009).

In AspC-1 cells, treatment with rAGR2 increased proliferation (3-fold), migration (10-fold), and invasion (3-fold) in a concentration-dependent manner (FIGS. 1A-C). Similar effects were observed with BxPC-3 and MiaPaCa-2 cell lines (FIGS. 7A-C). To determine the effect of rAGR2 on cancer cell resistance to therapeutic agents, PDAC cells were treated with Gem in the presence or absence of rAGR 2. Although AsPC-1 cells were highly resistant, a significant 3-fold increase in apoptosis was induced at 5 μ M Gem concentration (fig. 1D). Simultaneous treatment with rAGR2 reduced the effect of Gem to near control levels (> 50% reduction), confirming a strong survival effect. Treatment with AGR2 of BxPC3 cells, which are more sensitive to Gem, had an even greater effect (fig. 7D). Thus, extracellular recombinant AGR2 recapitulated the effect on PDAC cells previously observed with AGR2 expression (Ramachandran et al, 2008).

Example 2-C4.4A is a functional receptor for AGR2

Candidate receptors for AGR2 were selected from the literature and examined for importance in AGR2 function. LY6 receptor family members uPAR, C4.4A and CD59 were co-immunoprecipitated with AGR2 (FIG. 2A), whereas DAG-1 was not co-immunoprecipitated (FIGS. 8A-C). To determine the functional importance of each receptor, these receptors were silenced using siRNA and significant silencing was demonstrated (fig. 2C and 8G-I). Only silencing C4.4A significantly reduced basal cell proliferation, migration, and invasion, and almost completely abolished rAGR 2-stimulated cell proliferation, migration, and invasion in AsPC-1 (FIGS. 3A-C) and BxPC-3 cells (FIGS. 9A-C). On the other hand, silencing CD59 and uPAR significantly increased the migration of AsPC-1 cells.

C4.4a silencing also blocked AGR 2-mediated chemoresistance to Gem (fig. 3D and fig. 9D). Silencing of c4.4a alone and in combination with Gem resulted in a significantly increased rate of apoptosis (2-fold), a greater increase than observed with Gem-treated control cells (fig. 3D). Importantly, the ability of AGR2 treatment to protect cells from Gem was abolished after c4.4a silencing. To control off-target effects, four siRNA sequences were examined against c4.4a, each of which showed comparable results (fig. 3E). These data support the notion that the effect of extracellular AGR2 is mediated through interaction with c4.4a.

To determine whether AGR2 interacts with c4.4a directly or by association in the complex, rAGR2 and rc4.4a were combined and co-immunoprecipitated in the absence of other proteins. The direct interaction between rAGR2 and rC4.4A was indicated by the presence of a distinct band in this assay (FIG. 2B). Nine PDAC cell lines were also examined for expression of c4.4a mRNA and protein and observed to be present in all cell lines (fig. 8D-F).

Example 3-C4.4A requires integrin beta1 and laminins 1 and 5 for activity

Surface receptors were studied in view of the previously identified signaling complexes of uPAR (a member of this receptor family) (Smith) and Marshall (Marshall), 2010), including integrins and extracellular matrix components that may be involved in c4.4a signaling. Although functional results are unknown, c4.4a is reported to bind to laminin 1 and 5 (Paret et al, 2005). Thus, candidate integrins and laminins 1 and 5 were silenced and evaluated for AGR 2-mediated Gem resistance effects. Silencing of laminin 1, laminin 5, or integrin beta1 completely abolished the protective effect of AGR2, whereas silencing of integrin beta 2, beta4, or alpha6 had no effect (fig. 4A). Similarly, commercial blocking antibodies to laminin 1, laminin 5, and integrin beta1 also abolished AGR 2-mediated effects of stimulating proliferation and chemoprotection (fig. 4B-C). Similar results for the BxPC-3 cell line are shown in FIGS. 10A-B. In combination with these data, laminin 1 and 5 and integrin beta1 were shown to be involved in the AGR2/C4.4A receptor complex.

Example 4-AGR 2 and C4.4A monoclonal antibodies developed were highly specific and blocked the binding of AGR2 to C4.4A

To further understand the role of AGR2 and c4.4a in cancer, the interaction between them was blocked using antibodies. Commercially available antibodies, while recognizing AGR2(18kD) and c4.4a (50kD) (fig. 5A), did not block AGR 2-induced cell migration (fig. 5B) or Gem resistance (fig. 5C). Thus, AGR2 and C4.4A mAbs (163-28B-1 and 162-1A-1, respectively) were developed that recognized their respective antigens and blocked their interaction (FIG. 5A). The unconjugated antigenic peptide against AGR2 (CIHHLDESPHSQALKKVFAENKEIQKLAEQ; SEQ ID NO:3) and the unconjugated antigenic peptide against C4.4A (CPVRPTSTTKPMPAPTSQTPRQGVEHEASRDEEPRL; SEQ ID NO:4) were injected subcutaneously into Balb/C mice for 6 weeks together with adjuvant. Spleens were collected and fused with myeloma cells (SP2/0-Ag14) and hybridoma cells were screened by ELISA using KLH-conjugated peptides. Candidate hybridoma cells were identified by examining their binding efficiency to antigen by western blot analysis. Both antibodies showed binding to their recombinant and endogenous proteins, respectively, compared to commercially available antibodies (fig. 5A). The new mAb is more specific than the commercial antibody, as indicated by the lack of non-specific bands in western blots of pancreatic cancer cell lysates. Further purification of the hybridoma cells was performed using a protein a column and the purified proteins were tested for functional blocking assays. Although commercially available antibodies had no effect, the novel mAb blocked stimulation of AGR2for cell migration and Gem resistance (fig. 5B-C). Migration assays were performed and the basal and AGR2 mediated migration stimuli were eliminated when two blocking antibodies (163-28B-1 and 162-1A-1) were added compared to the non-specific antibodies, whereas the commercially available AGR2 and c4.4a antibodies did not block the function of AGR 2. Apoptosis assays were performed in AspC-1 cells. AGR2 and the C4.4A antibody (163-28B-1 and 162-1A-1, respectively) abolished the survival effect mediated by AGR2, thereby improving apoptosis, whereas the non-specific antibodies and the commercially available AGR2 and C4.4A antibodies did not.

Example 5-AGR2/C4.4A is widely expressed in pancreatic cancer

The expression patterns of AGR2 and c4.4a were also evaluated in patient tissues (TMA-tissue microarrays) using the developed mabs (fig. 5D). Both antibodies showed strong PDAC labeling, but normal pancreas did not. For AGR2, 105 out of 140 (75%) were positive, corresponding to staining rates of 46% (high), 29% (moderate), and 25% (no staining). High levels of AGR2 expression correlated with higher frequency of lymph node metastasis in the overall patient population and stage II patients (p < 0.05). There is also a weak correlation between AGR2 expression and differentiation. For c4.4a, 67 of 74 (91%) were positive, corresponding to staining rates of 52% (high), 39% (moderate), and 9% (no staining). These data confirm that both AGR2 and c4.4a are highly expressed in late-stage PDAC. Since the correlation between the expression of AGR2 and c4.4a was significant in PDAC patients (p <0.0001, correlation coefficient 0.74 (Spearman) r)), these two molecules tended to be expressed together.

Example 6-inhibition of AGR2/C4.4A autocrine Loop provides potential therapeutic benefit

To evaluate the potential therapeutic benefit of inhibiting the AGR2/c4.4a autocrine loop, the therapeutic effect with blocking mabs in a preclinical model was tested. In the invasive cell model (model 1) (FIGS. 6A-B), AsPC-1, a highly tumorigenic, metastatic, and Gem-resistant cell line, was used. The combined effect of the two mabs was tested with or without Gem. Mice were injected with luciferase-expressing AsPC-1 cells in situ and tumor formation was allowed to continue for two weeks before treatment began. After four weeks of treatment (six weeks total), all mice in the control Ab group had died and the remaining mice were sacrificed to compare tumor weight and metastasis. At that time, 30% of the mice treated with the combination of control Ab and Gem, 100% of the mice treated with the combination of AGR2 and C4.4A mAbs (163-28B-1 and 162-1A-1, respectively), and 80% of the mice treated with the combination of mAb and Gem remained alive. The experiment was ended at the end of seven weeks. Combined mAb treatment reduced tumor weight by 33% (p < 0.03) and incidence of metastasis by 66% (p <0.05) compared to control Ab (fig. 6G-H). There was no significant advantage in combining GEM and mAb because this combination resulted in a 40% reduction in tumor weight (p < 0.003) and a 50% reduction in the incidence of metastasis (p < 0.05). Since no substantial benefit is obtained in combination with GEM treatment in model 1, GEM treatment is not considered in models 2 and 3. Treatment with mAb did not reduce the body weight of the animals compared to mice treated with control Ab, indicating a lack of systemic toxicity associated with blocking this pathway.

In the matrix model (model 2) (FIGS. 6C-D), Capan-2, a Gem-resistant, dense matrix-forming but metastasis-free cell line, was used. Mice in the control Ab group all died within nine weeks (seven weeks of treatment) (fig. 6C). At that time, 43% of the mice treated with AGR2 mAb (163-28B-1) and 57% of the mice treated with C4.4A mAb (162-1A-1) survived. Treatment was stopped after 15 weeks (13 weeks of treatment) and animals were allowed to survive until they died or were severely ill. Median survival (point where 50% of animals survived) was six weeks for control Ab, nine weeks for AGR2 Ab, and 10 weeks for c4.4a mAb (p < 0.05). Both AGR2 and c4.4a Ab treatments reduced tumor volume by 50% (p <0.05) compared to control Ab (fig. 6D). Some mice (1/7 treated with AGR2 mAb; 3/7 treated with C4.4A mAb) showed complete tumor regression as indicated by bioluminescent imaging and confirmed by surgical examination. After 63 weeks, one mouse survived in each of the AGR2 Ab and c4.4a Ab groups. After sacrifice, the animals were examined and no evidence of tumor was observed.

Five weeks after the start of cancer cell implantation, when the tumor exceeded 1g, a regression study (model 3) was performed on the mice (fig. 6E-F). In this study, all mice in the control Ab group died three weeks after treatment initiation (total of 8 weeks). At that time, 60% of each mAb-treated group survived. Treatment with mAb was stopped after 12 weeks and mice were allowed to survive until they died or were severely ill. Median survival time was eight weeks for control Ab treated animals, 12 weeks for AGR2 or c4.4a mAb treated animals, and 11 weeks for animals treated with the combination of AGR2 and c4.4a mAb (p < 0.05). For this model, the reduction in tumor volume measured weekly by bioluminescence imaging is shown (fig. 6F). One of the five mice treated with AGR2 mAb showed complete tumor regression. Analysis of tumor residues in surviving mice indicated the presence of high levels of apoptotic cells in the mAb-treated group (fig. 6I). Analysis of p-ERK levels indicated that the activity of this pathway was completely abolished in the antibody treated group. Analysis of proliferation index Ki-67 did not show staining of the mAb treated group. Similar results were observed in other PDAC cell models.

Example 7-162-1A-1 and 163-29B-1 sequencing of the VH and VL regions of the monoclonal antibody

And (5) culturing the cells. 162-1A-1 and 163-28B-1 hybridoma cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS; HyClone, Rougen, Utah), 1mM sodium pyruvate (Mediatech, Helenden, Va.) and 1 Xpenicillin-streptomycin mixture (HyClone) at 37 ℃ in 7.5% CO₂And (5) growing in an incubator.

And (5) carrying out same type parting. The isotype of the mouse monoclonal antibody produced by each of the 162-1A-1 and 163-28B-1 hybridoma cells was determined by ELISA as follows. ELISA plates were coated with 1/1000 diluted 100 μ L/well in PBS of one of the following five goat polyclonal antibodies (all from southern biotechnology, birmingham, alabama), overnight at 4 ℃:

1. anti-mouse IgG, gamma chain specificity

2. Anti-mouse IgG, gamma 1 chain specificity

3. Anti-mouse IgG, gamma 2a chain specificity

4. Anti-mouse IgG, gamma 2b chain specificity

5. Anti-mouse IgM, mu chain specificity

After washing the wells with wash buffer (PBS containing 0.05% tween 20), blocking with ELISA buffer at 300 μ L/well (PBS containing 2% skim milk and 0.05% tween 20) for 30 minutes at room temperature, and washing with wash buffer, ELISA buffer and 100 μ L/well of a 1:1 mixture of each hybridoma cell culture supernatant were applied to the ELISA plates in duplicate. After incubation of the ELISA plates for 1h at room temperature and washing with wash buffer, bound antibodies were detected for 30min at room temperature with 100 μ L/well of one of the following two goat polyclonal antibodies (both from southern biotechnology) diluted in ELISA buffer at 1/1000:

HRP-coupled goat anti-mouse kappa chain polyclonal antibody

HRP-coupled goat anti-mouse lambda chain polyclonal antibody

After washing with washing buffer, by adding 100. mu.L/wellSubstrate (AMRESCO, Clontop, Ohio) was developed and stopped by the addition of 100. mu.L/well of 2% oxalic acid. The absorbance was read at 405 nm.

The typing results were 162-1A-1: IgG2B/κ, and 163-28B-1: IgG 1/kappa.

Cloning and sequencing of mouse immunoglobulin variable region genes from about 5 × 10⁶162-1A-1 and 163-28B-1 in the respective cells according to the supplier's protocolReagents (Invitrogen, Calsbad, Calif.) extracted total RNA. Following the supplier's protocol, SMARTER was used^TMRACE cDNA amplification kit (Crottack, mountain scenery, Calif.) oligo dT-primed cDNA for 5' -RACE was synthesized. The heavy and light chain variable region cDNAs were used by Polymerase Chain Reaction (PCR)DNA polymerase (new england bio laboratories corporation,beverley, ma), using 3' primers that specifically anneal to mouse heavy and light chain constant regions, and in smart er^TMThe 5' -RACE primer (Universal primer A mix) provided in the RACE cDNA amplification kit was amplified.

For PCR amplification of the heavy chain variable region (VH), the 3' primer has the sequence shown below:

MCG1:5'-GCCAGTGGATAGACAGATGG-3' (for gamma 1 chain) (SEQ ID NO:5)

MCG2B:5'-GCCAGTGGATAGACTGATGG-3' (for gamma 2b chain) (SEQ ID NO:6)

For PCR amplification of the kappa light chain variable region (VL), the 3' primer has the sequence shown below:

MCK:5'-GATGGATACAGTTGGTGCAGC-3'(SEQ ID NO:7)

the amplified VH and VL cdnas were subcloned into pjet1.2 vector (seemer science, rockford, il) for sequencing. DNA sequencing was performed on Tocore (menlopak, ca) using the following two primers:

JetFwd:5'-CGACTCACTATAGGGAGAGCGGC-3'(SEQ ID NO:8)

JetRev:5'-AAGAACATCGATTTTCCATGGCAG-3'(SEQ ID NO:9)

several heavy and light chain clones were sequenced and unique sequences homologous to typical mouse heavy and light chain variable regions were identified. The common cDNA sequence for each V gene was obtained using at least four independent clones.

Sequences of the VH and VL genes. The amino acid sequences of the VH and VL regions of the 162-1A-1 and 163-28B-1 monoclonal antibodies are shown in Table 1. The sequences of CDR1, 2 and 3 are shown in underlined solid lines according to the definition of Kabat et al (1991).

Table 1 antibody sequences.

Example 8-regression study-survival Curve in Capan-2 tumors

The in situ tumor was developed with luciferase-labeled CaPan-2 cells (150 ten thousand cells) and tumor growth was allowed to grow until it reached a tumor of about 1g size as measured in parallel experiments (5 weeks). Mice were treated with vehicle indicated HuCtrl (n ═ 5) (human IgG isotype control-Cat #0160-01, southern biotechnology, birmingham, alabama) or with AGR2 humanized Ab indicated HuAGR2(n ═ 7) or with c4.4a humanized Ab indicated huc4.4a (n ═ 7) (25 mg/kgb.wt/i.p./twice a week) for up to 40 weeks. Tumors were measured weekly using bioluminescence imaging. Survival curves were determined as shown in figure 12. At that time, while the control group showed 100% mortality, the hugr 2 group showed 71% survival and huc4.4a showed 87% survival. Median survival was 10 weeks for the control, 16 weeks for HuAGR2, and 18 weeks for the huc4.4a group. Comparison of survival curves (Log-Rank Mantel Cox test) shows that HuAGR2(p ═ 0.0118) and huc4.4a (p ═ 0.0032) show significant improvement in survival as compared to the control group. (p <0.05)

Prophetic example 9-binding epitopes were determined for antibodies 163-28B-1 and 162-1A-1.

The binding epitopes of 163-28B-1 and 162-1A-1 can be determined experimentally. Systematic mutations can be introduced in the AGR2 and c4.4a protein sequences and antibody binding of the resulting sequences can be determined to identify epitope-containing amino acids. This technique can be used to map both linear and conformational epitopes. High-throughput mutagenesis mapping is another approach that utilizes a comprehensive library of mutations in which each clone contains a unique amino acid mutation (conservative, non-conservative, or alanine), and the entire library covers every amino acid in the target protein. Hundreds of plasmid clones from the mutation library were individually arrayed in 384-well microplates, expressed in mammalian cells, and tested for antibody binding. The amino acids required for antibody binding can be identified by loss of fluorescence response and mapped to protein structure to visualize the epitope.

***

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 invention 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 invention. More specifically, it will be apparent that certain agents that 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 invention as defined by the appended claims.

Reference to the literature

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Sequence listing

<110> C.D.Loggesen (Logsdon, Craig D.)

V.lama Qianlan (Ramachandran, Vijaya)

T Arumugam (Arumugam, Thivungadam)

<120> blocking monoclonal antibody against AGR2 and its receptor C4.4A

<130>UTFC.P1242WO

<140> unknown

<141>2015-09-08

<150>62/048,037

<151>2014-09-09

<160>29

<170> PatentIn 3.5 edition

<210>1

<211>528

<212>RNA

<213> Intelligent people

<400>1

auggagaaaa uuccaguguc agcauucuug cuccuugugg cccucuccua cacucuggcc 60

agagauacca cagucaaacc uggagccaaa aaggacacaa aggacucucg acccaaacug 120

ccccagaccc ucuccagagg uuggggugac caacucaucu ggacucagac auaugaagaa 180

gcucuauaua aauccaagac aagcaacaaa cccuugauga uuauucauca cuuggaugag 240

ugcccacaca gucaagcuuu aaagaaagug uuugcugaaa auaaagaaau ccagaaauug 300

gcagagcagu uuguccuccu caaucugguu uaugaaacaa cugacaaaca ccuuucuccu 360

gauggccagu auguccccag gauuauguuu guugacccau cucugacagu uagagccgau 420

aucacuggaa gauauucaaa ucgucucuau gcuuacgaac cugcagauac agcucuguug 480

cuugacaaca ugaagaaagc ucucaaguug cugaagacug aauuguaa 528

<210>2

<211>1041

<212>RNA

<213> Intelligent people

<400>2

auggaccccg ccaggaaagc aggugcccag gccaugaucu ggacugcagg cuggcugcug 60

cugcugcugc uucgcggagg agcgcaggcc cuggagugcu acagcugcgu gcagaaagca 120

gaugacggau gcuccccgaa caagaugaag acagugaagu gcgcgccggg cguggacguc 180

ugcaccgagg ccgugggggc gguggagacc auccacggac aauucucgcu ggcagugcgg 240

gguugcgguu cgggacuccc cggcaagaau gaccgcggcc uggaucuuca cgggcuucug 300

gcguucaucc agcugcagca augcgcucag gaucgcugca acgccaagcu caaccucacc 360

ucgcgggcgc ucgacccggc agguaaugag agugcauacc cgcccaacgg cguggagugc 420

uacagcugug ugggccugag ccgggaggcg ugccagggua caucgccgcc ggucgugagc 480

ugcuacaacg ccagcgauca ugucuacaag ggcugcuucg acggcaacgu caccuugacg 540

gcagcuaaug ugacuguguc cuugccuguc cggggcugug uccaggauga auucugcacu 600

cgggauggag uaacaggccc aggguucacg cucaguggcu ccuguugcca ggggucccgc 660

uguaacucug accuccgcaa caagaccuac uucuccccuc gaaucccacc ccuuguccgg 720

cugcccccuc cagagcccac gacuguggcc ucaaccacau cugucaccac uucuaccucg 780

gccccaguga gacccacauc caccaccaaa cccaugccag cgccaaccag ucagacuccg 840

agacagggag uagaacacga ggccucccgg gaugaggagc ccagguugac uggaggcgcc 900

gcuggccacc aggaccgcag caauucaggg caguauccug caaaaggggg gccccagcag 960

ccccauaaua aaggcugugu ggcucccaca gcuggauugg cagcccuucu guuggccgug 1020

gcugcuggug uccuacugug a1041

<210>3

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>3

Cys Ile His His Leu Asp Glu Ser Pro His Ser Gln Ala Leu Lys Lys

1 5 10 15

Val Phe Ala Glu Asn Lys Glu Ile Gln Lys Leu Ala Glu Gln

20 25 30

<210>4

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>4

Cys Pro Val Arg Pro Thr Ser Thr Thr Lys Pro Met Pro Ala Pro Thr

1 5 10 15

Ser Gln Thr Pro Arg Gln Gly Val Glu His Glu Ala Ser Arg Asp Glu

20 25 30

Glu Pro Arg Leu

35

<210>5

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400>5

gccagtggat agacagatgg 20

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400>6

gccagtggat agactgatgg 20

<210>7

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400>7

gatggataca gttggtgcag c 21

<210>8

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400>8

cgactcacta tagggagagc ggc 23

<210>9

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400>9

aagaacatcg attttccatg gcag 24

<210>10

<211>139

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>10

Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Val Leu Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys

20 2530

Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu

50 55 60

Glu Trp Val Ala Ser Ile Ser Ser Gly Gly Gly Asn Thr Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn

85 90 95

Asn Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu

100 105 110

Tyr Tyr Cys Ala Arg Ser Tyr Tyr Tyr Gly Ile Ser Tyr Asp Thr Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

130 135

<210>11

<211>131

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>11

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 15

Ser Asn Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Leu

35 40 45

Val His Ser Asp Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys

100 105 110

Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

<210>12

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>12

Ser Tyr Thr Met Ser

1 5

<210>13

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>13

Ser Ile Ser Ser Gly Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>14

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>14

Ser Tyr Tyr Tyr Gly Ile Ser Tyr Asp Thr Tyr

1 5 10

<210>15

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>15

Arg Ser Ser Gln Asn Leu Val His Ser Asp Gly Asn Thr Tyr Leu His

1 5 10 15

<210>16

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>16

Lys Val Ser Asn Arg Phe Ser

1 5

<210>17

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>17

Ser Gln Ser Thr His Val Pro Tyr Thr

1 5

<210>18

<211>136

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>18

Met Asp Trp Leu Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser

1 5 10 15

Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Asp Thr Gly Lys Pro Thr Tyr Thr

65 70 75 80

Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Ala Thr SerAla Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Arg Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Gly Arg Val Thr Ala Asp Ser Met Asp Tyr Trp Gly Gln

115 120 125

Gly Thr Ser Val Thr Val Ser Ser

130 135

<210>19

<211>131

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>19

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 15

Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

35 40 45

Val His Ser Asn Gly Asn Ile Tyr Leu His Trp Phe Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys

100 105 110

Ser Gln Ser Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

115 120 125

Glu Leu Lys

130

<210>20

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>20

Asn Tyr Gly Met Asn

1 5

<210>21

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>21

Trp Ile Asn Thr Asp Thr Gly Lys Pro Thr Tyr Thr Glu Glu Phe Lys

1 5 10 15

Gly

<210>22

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>22

Val Thr Ala Asp Ser Met Asp Tyr

1 5

<210>23

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>23

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Ile Tyr Leu His

1 5 10 15

<210>24

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>24

Lys Val Ser Asn Arg Phe Ser

1 5

<210>25

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>25

Ser Gln Ser Thr His Val Pro Leu Thr

1 5

<210>26

<211>175

<212>PRT

<213> Intelligent people

<400>26

Met Glu Lys Ile Pro Val Ser Ala Phe Leu Leu Leu Val Ala Leu Ser

1 5 10 15

Tyr Thr Leu Ala Arg Asp Thr Thr Val Lys Pro Gly Ala Lys Lys Asp

20 25 30

Thr Lys Asp Ser Arg Pro Lys Leu Pro Gln Thr Leu Ser Arg Gly Trp

35 40 45

Gly Asp Gln Leu Ile Trp Thr Gln Thr Tyr Glu Glu Ala Leu Tyr Lys

50 55 60

Ser Lys Thr Ser Asn Lys Pro Leu Met Ile Ile His His Leu Asp Glu

65 70 75 80

Cys Pro His Ser Gln Ala Leu Lys Lys Val Phe Ala Glu Asn Lys Glu

85 90 95

Ile Gln Lys Leu Ala Glu Gln Phe Val Leu Leu Asn Leu Val Tyr Glu

100 105 110

Thr Thr Asp Lys His Leu Ser Pro Asp Gly Gln Tyr Val Pro Arg Ile

115 120 125

Met Phe Val Asp Pro Ser Leu Thr Val Arg Ala Asp Ile Thr Gly Arg

130 135 140

Tyr Ser Asn Arg Leu Tyr Ala Tyr Glu Pro Ala Asp Thr Ala Leu Leu

145 150155 160

Leu Asp Asn Met Lys Lys Ala Leu Lys Leu Leu Lys Thr Glu Leu

165 170 175

<210>27

<211>346

<212>PRT

<213> Intelligent people

<400>27

Met Asp Pro Ala Arg Lys Ala Gly Ala Gln Ala Met Ile Trp Thr Ala

1 5 10 15

Gly Trp Leu Leu Leu Leu Leu Leu Arg Gly Gly Ala Gln Ala Leu Glu

20 25 30

Cys Tyr Ser Cys Val Gln Lys Ala Asp Asp Gly Cys Ser Pro Asn Lys

35 40 45

Met Lys Thr Val Lys Cys Ala Pro Gly Val Asp Val Cys Thr Glu Ala

50 55 60

Val Gly Ala Val Glu Thr Ile His Gly Gln Phe Ser Leu Ala Val Arg

65 70 75 80

Gly Cys Gly Ser Gly Leu Pro Gly Lys Asn Asp Arg Gly Leu Asp Leu

85 90 95

His Gly Leu Leu Ala Phe Ile Gln Leu Gln Gln Cys Ala Gln Asp Arg

100 105 110

Cys Asn Ala Lys Leu Asn Leu Thr Ser Arg Ala Leu Asp Pro Ala Gly

115 120 125

Asn Glu Ser Ala Tyr Pro Pro Asn Gly Val Glu Cys Tyr Ser Cys Val

130 135 140

Gly Leu Ser Arg Glu Ala Cys Gln Gly Thr Ser Pro Pro Val Val Ser

145 150 155 160

Cys Tyr Asn Ala Ser Asp His Val Tyr Lys Gly Cys Phe Asp Gly Asn

165 170 175

Val Thr Leu Thr Ala Ala Asn Val Thr Val Ser Leu Pro Val Arg Gly

180 185 190

Cys Val Gln Asp Glu Phe Cys Thr Arg Asp Gly Val Thr Gly Pro Gly

195 200 205

Phe Thr Leu Ser Gly Ser Cys Cys Gln Gly Ser Arg Cys Asn Ser Asp

210 215 220

Leu Arg Asn Lys Thr Tyr Phe Ser Pro Arg Ile Pro Pro Leu Val Arg

225 230235 240

Leu Pro Pro Pro Glu Pro Thr Thr Val Ala Ser Thr Thr Ser Val Thr

245 250 255

Thr Ser Thr Ser Ala Pro Val Arg Pro Thr Ser Thr Thr Lys Pro Met

260 265 270

Pro Ala Pro Thr Ser Gln Thr Pro Arg Gln Gly Val Glu His Glu Ala

275 280 285

Ser Arg Asp Glu Glu Pro Arg Leu Thr Gly Gly Ala Ala Gly His Gln

290 295 300

Asp Arg Ser Asn Ser Gly Gln Tyr Pro Ala Lys Gly Gly Pro Gln Gln

305 310 315 320

Pro His Asn Lys Gly Cys Val Ala Pro Thr Ala Gly Leu Ala Ala Leu

325 330 335

Leu Leu Ala Val Ala Ala Gly Val Leu Leu

340 345

<210>28

<211>29

<212>PRT

<213> Intelligent people

<400>28

Ile His His Leu Asp Glu Cys Pro His Ser Gln Ala Leu Lys Lys Val

1 5 10 15

Phe Ala Glu Asn Lys Glu Ile Gln Lys Leu Ala Glu Gln

20 25

<210>29

<211>35

<212>PRT

<213> Intelligent people

<400>29

Pro Val Arg Pro Thr Ser Thr Thr Lys Pro Met Pro Ala Pro Thr Ser

1 5 10 15

Gln Thr Pro Arg Gln Gly Val Glu His Glu Ala Ser Arg Asp Glu Glu

20 25 30

Pro Arg Leu

35

Claims (81)

1. An isolated monoclonal antibody, wherein the antibody specifically binds to an AGR2 polypeptide and wherein the antibody competes with 163-28B-1 monoclonal antibody for binding to the polypeptide.

2. The antibody of claim 1, wherein the antibody comprises:

(a) a first VH CDR that is at least 90% identical to VH CDR1 of 163-28B-1 (SEQ ID NO: 20);

(b) a second VH CDR that is at least 90% identical to VH CDR2 of 163-28B-1 (SEQ ID NO: 21);

(c) a third VH CDR that is at least 90% identical to VH CDR3 of 163-28B-1 (SEQ ID NO: 22);

(d) a first VL CDR that is at least 90% identical to VL CDR1 of 163-28B-1 (SEQ ID NO: 23);

(e) a second VL CDR that is at least 90% identical to VL CDR2 of 163-28B-1 (SEQ ID NO: 24); and

(f) a third VL CDR that is at least 90% identical to VL CDR3 of 163-28B-1 (SEQ ID NO: 25).

3. The isolated antibody of claim 2, wherein the antibody comprises:

(a) a first VH CDR identical to SEQ ID NO: 20;

(b) a second VH CDR identical to SEQ ID NO: 21;

(c) a third VH CDR identical to SEQ ID NO 22;

(d) a first VL CDR identical to SEQ ID NO 23;

(e) a second VL CDR identical to SEQ ID NO 24; and

(f) a third VL CDR identical to SEQ ID NO: 25.

4. The isolated antibody of claim 1, wherein the antibody comprises:

(a) a first VH CDR at least 90% identical to SEQ ID NO of 20;

(b) a second VH CDR at least 90% identical to SEQ ID NO 21;

(c) a third VH CDR at least 90% identical to SEQ ID NO 22; and

(d) a VL domain which is at least 90% identical to SEQ ID NO 19.

5. The isolated antibody of claim 4, wherein the antibody comprises:

(a) a first VH CDR identical to SEQ ID NO: 20;

(b) a second VH CDR identical to SEQ ID NO 21;

(c) a third VH CDR identical to SEQ ID NO 22; and

(d) VL domain identical to SEQ ID NO 19.

6. The isolated antibody of claim 1, wherein the antibody comprises:

(a) a VH domain at least 90% identical to SEQ ID NO 18;

(b) a first VL CDR at least 90% identical to SEQ ID NO 23;

(c) a second VL CDR at least 90% identical to SEQ ID NO 24; and

(d) a third VL CDR at least 90% identical to SEQ ID NO 25.

7. The isolated antibody of claim 6, wherein the antibody comprises:

(a) a VH domain identical to SEQ ID NO 18;

(b) a first VL CDR identical to SEQ ID NO. 23;

(c) a second VL CDR identical to SEQ ID NO. 24; and

(d) a third VL CDR identical to SEQ ID NO 25.

8. The isolated antibody of claim 2, wherein the antibody comprises a VH domain at least about 80% identical to the VH domain of 163-28B-1 (SEQ ID NO:18) and a VL domain at least about 80% identical to the VL domain of 163-28B-1 (SEQ ID NO: 19).

9. The isolated antibody of claim 8, wherein the antibody comprises a VH domain identical to the VH domain of 163-28B-1 (SEQ ID NO:18) and a VL domain identical to the VL domain of 163-28B-1 (SEQ ID NO: 19).

10. The isolated antibody of claim 9, wherein the antibody is 163-28B-1 antibody.

11. A monoclonal antibody or antigen-binding fragment thereof having specificity for an AGR2 polypeptide, wherein the antibody specifically binds to AGR2 epitope NH2-IHHLDECPHSQALKKVFAENKEIQKLAEQ-C (SEQ ID NO: 28).

12. The monoclonal antibody or antigen binding fragment thereof of claim 11, wherein the monoclonal antibody or antigen binding fragment thereof inhibits tumor cell pancreatic ductal adenocarcinoma migration and resistance to gemcitabine-induced apoptosis.

13. The isolated antibody of any one of claims 1-12, wherein the antibody is recombinant.

14. The isolated antibody of any one of claims 1-7, wherein the antibody is an IgG, IgM, IgA or an antigen binding fragment thereof.

15. The isolated antibody of any one of claims 1-7, wherein the antibody is a Fab ', a F (ab ')2, a F (ab ')3, a monovalent scFv, a bivalent scFv, or a single domain antibody.

16. The isolated antibody of any one of claims 1-10, wherein the antibody is a human antibody, a humanized antibody, or a deimmunized antibody.

17. The isolated antibody of any one of claims 1-10, wherein the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

18. An isolated monoclonal antibody, wherein the antibody specifically binds to a c4.4a polypeptide and wherein the antibody competes for binding to the polypeptide with the 162-1A-1 monoclonal antibody.

19. The antibody of claim 18, wherein the antibody comprises:

(a) a first VH CDR that is at least 90% identical to VH CDR1 of 162-1A-1 (SEQ ID NO: 12);

(b) a second VH CDR that is at least 90% identical to VH CDR2 of 162-1A-1 (SEQ ID NO: 13);

(c) a third VH CDR that is at least 90% identical to VH CDR3 of 162-1A-1 (SEQ ID NO: 14);

(d) a first VL CDR that is at least 90% identical to VL CDR1 of 162-1A-1 (SEQ ID NO: 15);

(e) a second VL CDR that is at least 90% identical to VL CDR2 of 162-1A-1 (SEQ ID NO: 16); and

(f) a third VL CDR that is at least 90% identical to VL CDR3 of 162-1A-1 (SEQ ID NO: 17).

20. The isolated antibody of claim 19, wherein the antibody comprises:

(a) a first VH CDR identical to SEQ ID NO 12;

(b) a second VH CDR identical to SEQ ID NO 13;

(c) a third VH CDR identical to SEQ ID NO 14;

(d) a first VL CDR identical to SEQ ID NO. 15;

(e) a second VL CDR identical to SEQ ID NO 16; and

(f) a third VL CDR identical to SEQ ID NO 17.

21. The isolated antibody of claim 18, wherein the antibody comprises:

(a) a first VH CDR at least 90% identical to SEQ ID NO 12;

(b) a second VH CDR at least 90% identical to SEQ ID NO 13;

(c) a third VH CDR at least 90% identical to SEQ ID NO 14; and

(d) a VL domain which is at least 90% identical to SEQ ID NO 11.

22. The isolated antibody of claim 21, wherein the antibody comprises:

(a) a first VH CDR identical to SEQ ID NO 12;

(b) a second VH CDR identical to SEQ ID NO 13;

(c) a third VH CDR identical to SEQ ID NO 14; and

(d) VL domain identical to SEQ ID NO 11.

23. The isolated antibody of claim 18, wherein the antibody comprises:

(a) a VH domain at least 90% identical to SEQ ID NO 10;

(b) a first VL CDR at least 90% identical to SEQ ID NO 15;

(c) a second VL CDR at least 90% identical to SEQ ID NO 16; and

(d) a third VL CDR at least 90% identical to SEQ ID NO 17.

24. The isolated antibody of claim 23, wherein the antibody comprises:

(a) a VH domain identical to SEQ ID NO 10;

(b) a first VL CDR identical to SEQ ID NO. 15;

(c) a second VL CDR identical to SEQ ID NO 16; and

(c) a third VL CDR identical to SEQ ID NO 17.

25. The isolated antibody of claim 19, wherein the antibody comprises a VH domain at least about 80% identical to the VH domain of 162-1A-1 (SEQ ID NO:10) and a VL domain at least about 80% identical to the VL domain of 162-1A-1 (SEQ ID NO: 11).

26. The isolated antibody of claim 25, wherein the antibody comprises a VH domain identical to the VH domain of 162-1A-1 (SEQ ID NO:10) and a VL domain identical to the VL domain of 162-1A-1 (SEQ ID NO: 11).

27. The isolated antibody of claim 26, wherein the antibody is 163-28B-1 antibody.

28. A monoclonal antibody or antigen-binding fragment thereof specific for a C4.4A polypeptide, wherein the antibody specifically binds to the C4.4A epitope NH2-PVRPTSTTKPMPAPTSQTPRQGVEHEASRDEEPRL-C (SEQ ID NO: 29).

29. The monoclonal antibody, or antigen binding fragment thereof, of claim 29, wherein the monoclonal antibody, or antigen binding fragment thereof, inhibits tumor cell pancreatic ductal adenocarcinoma migration and resistance to gemcitabine-induced apoptosis.

30. The isolated antibody of any one of claims 18-27, wherein the antibody is recombinant.

31. The isolated antibody of any one of claims 18-24, wherein the antibody is an IgG, IgM, IgA or an antigen-binding fragment thereof.

32. The isolated antibody of any one of claims 18-24, wherein the antibody is a Fab ', F (ab ')2, F (ab ')3, monovalent scFv, bivalent scFv, or single domain antibody.

33. The isolated antibody of any one of claims 18-27, wherein the antibody is a human antibody, a humanized antibody, or a deimmunized antibody.

34. The isolated antibody of any one of claims 18-27, wherein the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

35. A composition comprising the antibody of any one of claims 1-34 in a pharmaceutically acceptable carrier.

36. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding the antibody of any one of claims 1-33.

37. An antibody V_HRecombinant polypeptide of Domain, the antibody V_HThe domain comprises a V of 163-28B-1_HCDRs 1-3 of the domain (SEQ ID NOS: 20, 21, and 22).

38. An antibody V_HRecombinant polypeptide of Domain, the antibody V_HThe domain comprises a V of 162-1A-1_HCDRs 1-3 of the domain (SEQ ID NOS: 12, 13, and 14).

39. An antibody V_LRecombinant polypeptide of Domain, the antibody V_LThe domain comprises a V of 163-28B-1_LCDRs 1-3 of the domain (SEQ ID NOS: 23, 24, and 25).

40. An antibody V_LRecombinant polypeptide of Domain, the antibody V_LThe domain comprises a V of 162-1A-1_LCDRs 1-3 of the domain (SEQ ID NOS: 15, 16, and 17).

41. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding the polypeptide of any one of claims 37-40.

42. A host cell comprising one or more polynucleotide molecules encoding the antibody of any one of claims 1-33 or the recombinant polypeptide of any one of claims 37-40.

43. The host cell of claim 42, wherein the host cell is a mammalian cell, a yeast cell, a bacterial cell, a ciliate cell, or an insect cell.

44. A method of making an antibody comprising:

(a) expressing a V encoding the antibody of any one of claims 1-33 in a cell_LAnd V_HOne or more polynucleotide molecules of the strand; and is

(b) Purifying the antibody from the cell.

45. A method of treating cancer in a patient, comprising administering to the patient an agent that disrupts the AGR2/c4.4a interaction in an amount effective to treat the cancer.

46. A method of treating a subject having cancer, the method comprising administering an effective amount of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to AGR2 epitope NH2-IHHLDECPHSQALKKVFAENKEIQKLAEQ-C (SEQ ID NO: 28).

47. A method of treating a subject having cancer, the method comprising administering an effective amount of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to the C4.4A epitope NH2-PVRPTSTTKPMPAPTSQTPRQGVEHEASRDEEPRL-C (SEQ ID NO: 29).

48. A method of treating a subject having cancer, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1-34.

49. The method of claim 48, wherein the cancer is breast cancer, lung cancer, head and 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.

50. The method of claim 48, wherein the cancer is pancreatic ductal adenocarcinoma.

51. The method of claim 48, wherein the antibody is administered systemically.

52. The method of claim 48, wherein the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

53. The method of claim 48, further comprising administering to the subject at least a second anti-cancer therapy.

54. The method of claim 53, wherein the second anticancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy or cytokine therapy.

55. The method of claim 48, wherein the subject is a human subject.

56. A composition comprising an AGR2 binding antibody for use in treating cancer in a patient.

57. The composition of claim 56, wherein the antibody competes with the 163-28B-1 monoclonal antibody for binding to AGR 2.

58. A composition comprising a c4.4a binding antibody for use in treating cancer in a patient.

59. The composition of claim 58, wherein the antibody competes for binding to c4.4A with the 162-1A-1 monoclonal antibody.

60. A composition comprising an AGR2 binding antibody and a c4.4a binding antibody for use in treating cancer in a patient.

61. The composition of claim 60, wherein the AGR2 binding antibody competes for binding to AGR2 with the 163-28B-1 monoclonal antibody.

62. The composition of claim 60, wherein the c4.4a binding antibody competes for binding to c4.4a with the 162-1A-1 monoclonal antibody.

63. A composition comprising an antibody of any one of claims 1-34 for use in treating cancer in a patient.

64. The composition of any one of claims 56-63, wherein the cancer is breast cancer, lung cancer, head and 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.

65. The composition of any one of claims 56-63, wherein the cancer is pancreatic ductal adenocarcinoma.

66. The composition of any one of claims 56-63, wherein the patient has been previously determined to have pancreatic ductal adenocarcinoma.

67. The composition of any one of claims 56-63, wherein the patient is determined to have pancreatic ductal adenocarcinoma.

68. The composition of any one of claims 56-63, wherein the antibody is 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.

69. The composition of claim 68, wherein the antibody is a monoclonal antibody.

70. The composition of claim 68, wherein the antibody is a humanized antibody.

71. The composition of claim 68, wherein the antibody fragment is Fab, Fab '-SH, F (ab')₂Or scFv.

72. The composition according to any one of claims 58-63, wherein the antibody is attached to an agent to be targeted to C4.4A-expressing cells.

73. The composition of claim 72, wherein the agent is a cytotoxic agent, cytokine, anti-angiogenic agent, chemotherapeutic agent, diagnostic agent, imaging agent, radioisotope, pro-apoptotic agent, enzyme, hormone, growth factor, peptide, protein, antibiotic, antibody, Fab fragment of an antibody, antigen, survival factor, anti-apoptotic agent, hormone antagonist, virus, phage, bacteria, liposome, microparticle, nanoparticle, magnetic bead, microdevice, cell, nucleic acid, or expression vector.

74. The composition of any one of claims 56-63, wherein the antibody is formulated for systemic administration.

75. The composition of any one of claims 56-63, wherein the antibody is formulated for intravenous, intradermal, intratumoral, intramuscular, intraperitoneal, subcutaneous, or topical administration.

76. The composition of any one of claims 56-63, further comprising administering at least one second anticancer agent.

77. The composition of claim 76, wherein the second anticancer agent is a chemotherapeutic agent, a hormone, an immunotherapeutic agent, or a cytokine.

78.AGR2 binding antibodies in the manufacture of a medicament for the treatment of cancer.

Use of 79.c4.4a binding antibody in the manufacture of a medicament for the treatment of cancer.

80. Use of an antibody of any one of claims 1-34 in the manufacture of a medicament for treating cancer.

81. A kit comprising the antibody of claims 1-34.