WO2012150869A1 - Antibodies that bind trefoil factors and methods of using same - Google Patents

Abstract

The invention relates generally to monoclonal antibodies that bind to TFF and methods of using such to modulate, treat, prevent or delay the progression of a disease or disorder such as cancer or a proliferative disorder.

Description

ANTIBODIES THAT BIND TREFOIL FACTORS AND METHODS OF USING SAME

FIELD OF THE INVENTION

[0001] The invention relates generally to monoelonal antibodies that bind to TFF and methods of using such antibodies to modulate, treat, prevent or delay the progression of a disease or disorder such as cancer or a proliferative disorder.

BACKGROUND OF THE INVENTION

[0002] The regulation and control of proliferation and/or survival of cells in animals is a complex process involving a number of cellular factors and their interactions with one another. Mutations or alteration in expression in any number of these cellular factors can result in

uncontrolled proliferation or growth of cells and ultimately lead to the development of tumors and cancer.

[0003] Hormones and/or growth factors are involved in the normal regulation and control of cellular growth and development. Alterations in expression levels of hormones can result in aberrant proliferation of cells. Such alterations in expression of a hormone may result in the transformation of a normal cell to a cancer cell.

[0004] There is a need to understand further the effects of hormones and/or growth factors on the development of proliferative disorders, including identifying any cellular factors which promote cell proliferation, cell survival and/or oncogenic transformation. This will aid in the identification of means for the regulation of proliferation and/or survival, and particularly means for the treatment of proliferative disorders such as cancer.

SUMMARY OF THE INVENTION

[0005] The invention provides antibodies that bind to a trefoil factor (TFF) polypeptide, such as, for example, TFFl , TFF3 and/or any derivatives or fragments thereof, and methods of using such antibodies to modulate, e.g., reduce, inhibit, treat or prevent cancer or a proliferative disorder, and/or to modulate, e.g., reduce, inhibit, or delay the progression of a cancer or proliferative disorder.

[0006] The antibodies to TFF and/or antigen-binding fragments or derivatives thereof described herein include antibodies that specifically bind a trefoil factor 1 (TFFl) polypeptide in any form, such as, for example, monomer and/or dimer. The antibodies to TFF and/or antigen-binding fragments or derivatives thereof described herein include antibodies that specifically bind a trefoil factor 3 (TFF3) polypeptide in any form, such as, for example, monomer and/or dimer.

[0007] The antibodies of the invention include antibodies that bind TFF1 and/or any antigen- binding fragments or derivatives thereof, that include (a) a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 6, 12, 18, 42 or 47 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6, 12, 18, 42 or 47; (b) a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 7, 13, 19, 38, 43 or 48 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7, 13, 19, 38, 43 or 48; (c) a VH CDR3 region comprising the amino acid sequence of SEQ ID NO: 8, 14, 20, 39, 44 or 49 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8, 14, 20, 39, 44 or 49; (d) a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 9, 15, 21, 40, 45 or 50 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9, 15, 21, 40, 45 or 50; (e) a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 10, 16, 46 or 51 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, 16, 46 or 51 ; and (f) a V_L CDR3 region comprising the amino acid sequence of SEQ ID NO: 1 1, 17, 22, 41 or 52 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 1 , 17, 22, 41 or 52.

[0008] The antibodies of the invention include antibodies that bind TFF3 and/or any antigen- binding fragments or derivatives thereof, that include (a) a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 23 or 29 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 23 or 29; (b) a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 24 or 30 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 24 or 30; (c) a VH CDR3 region comprising the amino acid sequence of SEQ ID NO: 25 or 31 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 25 or 31 ; (d) a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 26 or 32 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 26 or 32; (e) a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 27 or 33 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 27 or 33; and (f) a V_L CDR3 region comprising the amino acid sequence of SEQ ID NO: 28 or 34 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 28 or 34. [0009] For example, the anti-TFFl antibody includes a V_H CDRl region comprising the amino acid sequence of SEQ ID NO: 6; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR3 region comprising the amino acid sequence of SEQ ID NO: 8; a VL CDRl region comprising the amino acid sequence of SEQ ID NO: 9; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 10; and a VL CDR3 region comprising an amino acid sequence of SEQ ID NO: 1 1.

[00010] For example, the anti-TFFl antibody includes a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 12; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 13, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 14; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 15; a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 16; and a VL CDR3 region comprising an amino acid sequence of SEQ ID NO: 17.

[00011] For example, the anti-TFFl antibody includes a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 18; a VH CDR2 region comprising the amino acid sequence of SEQ ID NO: 19, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 20; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 21 ; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 16; and a VL CDR3 region comprising an amino acid sequence of SEQ ID NO: 22.

[00012] For example, the anti-TFFl antibody includes a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 18; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 38, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 39; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 40; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 16; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO: 41.

[00013] For example, the anti-TFFl antibody includes a V_H CDRl region comprising the amino acid sequence of SEQ ID NO: 42; a VH CDR2 region comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR3 region comprising the amino acid sequence of SEQ ID NO: 44; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 45; a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 46; and a VL CDR3 region comprising an amino acid sequence of SEQ ID NO: 41.

[00014] For example, the anti-TFFl antibody includes a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 47; a VH CDR2 region comprising the amino acid sequence of SEQ ID NO: 48, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: ;49 a V_L CDR1 region comprising the amino acid sequence of SEQ ID NO: 50; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 51 ; and a VL CDR3 region comprising an amino acid sequence of SEQ ID NO: 52.

[00015] For example, the anti-TFF3 antibody includes a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 23; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 24, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 25; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 26; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 27; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO: 28.

[00016] For example, the anti-TFF3 antibody includes a V_H CDRl region comprising the amino acid sequence of SEQ ID NO: 29; a VH CDR2 region comprising the amino acid sequence of SEQ ID NO: 30, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 31 ; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 32; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 33; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO: 34.

[00017] The antibodies to TFF provided herein specifically bind to a trefoil factor polypeptide. For example, in some embodiments, the antibodies to TFF bind to TFF1. In some embodiments, the antibodies to TFF bind to TFF3. The invention also provides multivalent antibodies that recognize both TFF1 and TFF3. These antibodies are also referred to herein as multimeric antibodies. For example, in some embodiments, the TFF specific antibody is a heterodimer. In some embodiments, the TFF specific antibody is a chimeric antibody in which the antigen-binding fragment of the antibody from one species is fused with constant region from another species. For example, the TFF specific antibody is a humanized antibody.

[00018] The term "antibody" is used herein in the broadest sense and is intended to include intact monoclonal antibodies and polyclonal antibodies, as well as antigen-binding or otherwise immunologically active derivatives, variants, fragments and/or any other modification thereof so long as they exhibit the desired biological activity. Antibodies encompass immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. These include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fc, Fab, Fab', and Fab₂ fragments, and a Fab expression library. Antibody molecules relate to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from one another by the nature of heavy chain present in the molecule. These include subclasses as well, such as IgGl, IgG2, and others. The light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all classes, subclasses, and types. Also included are chimeric antibodies, for example, monoclonal antibodies or modifications thereof that are specific to more than one source, e.g., a mouse or human sequence. Further included are camelid antibodies or nanobodies. TFF binding antibodies also include multi- specific, e.g., bispecific, antibodies and functional fragments thereof. The terms "TFF binding antibodies" and "TFF antibodies" are used interchangeably herein. It will be understood that each reference to "antibodies" or any like term, herein includes intact antibodies, as well as any modifications thereof.

[00019] Antibodies to TFF include those that bind to domains or residues that are exposed, e.g., outer loop structure residues in the tertiary structure of the protein in solution, participate in TFF dimerization, aggregation, as well as domains responsible for promoting cellular proliferation, survival, and oncogenicity. For example, the epitope binding specificity of the antibody includes a TFF sequence that contains a domain involved in stimulation of cell proliferation, survival and oncogenicity. The invention encompasses not only an intact monoclonal antibody, but also an immuno logically-active antibody fragment, e.g., a Fab or (Fab)₂ fragment; an engineered single chain Fv molecule; or a chimeric molecule, e.g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and the remaining portions of another antibody, e.g., of human origin.

[00020] TFF binding antibodies are used to modulate, e.g., reduce or otherwise inhibit, completely or partially, the ability of the target TFF molecule (i.e., the TFF antigen to which a given TFF binding antibody binds) to bind or otherwise interact with another TFF molecule. The additional TFF molecule can be the same as the target TFF molecule, as in the case of homo- multimerization, e.g., homodimerization, or the additional TFF molecule can differ from the target TFF molecule.

[00021] TFF binding antibodies are also used to modulate, e.g., reduce or otherwise inhibit, completely or partially, the ability of the target TFF molecule to bind or otherwise interact with a second molecule, such as, for example, a cognate TFF receptor molecule, an extracellular receptor or other cell-surface and/or intracellular signaling molecules.

[00022] Other TFF-binding antibodies are used to directly target TFF-over-expressing cells for destruction. In the latter case, the antibody, or fragment thereof, activates complement in a patient treated with the antibody. In some instances, the antibody mediates antibody-dependent cytotoxicity of tumor cells in the patient treated with the antibody. The antibody, or fragment thereof, is administered alone or conjugated to a cytotoxic agent. Binding of the antibody to a tumor cell expressing TFF results in impairment or death of the cell, thereby reducing tumor load. The antibody is optionally conjugated to a radiochemical, or a chemical tag which sensitizes the cell to which it is bound to radiation or laser-mediated killing.

[00023] Optionally, the antibody compositions contain a pharmaceutically acceptable carrier and/or a second compound. For example, the second compound is a chemotherapeutic or antineoplastic agent. Such agents are administered sequentially, e.g., prior to, or after the administration of the TFF antibody, or simultaneously, e.g. , co-administration or co-therapy.

[00024] The invention provides methods of alleviating a symptom of a cancer, a cell proliferation disorder or a cell survival disorder by administering an anti-TFF antibody described herein to a subject in need thereof in an amount sufficient to alleviate the symptom of the cancer, cell proliferation disorder or cell survival disorder in the subject. For example, the subject is a human. For example, cancer is an epithelial cancer. The epithelial cancer is, for example, selected from lung cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, hepatic carcinoma, gastric carcinoma, endometrial carcinoma, renal carcinoma, thyroid cancer, biliary duct cancer, esophageal cancer, brain cancer, melanoma, multiple myeloma, hematologic tumor, and lymphoid tumor. The cell proliferation disorder or cell survival disorder is, for example, selected from the group consisting of keratinocyte hyperproliferation, inflammatory cell infiltration, endometriosis, cytokine alteration, epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myo fibromatosis, osteoplastic tumors, and other dysplastic masses. These methods can also include the administration of a second compound in conjunction with the anti-TFF antibody. For example, the second compound is a chemotherapeutic or anti-neoplastic agent.

[00025] The antibodies to TFF described herein are used in methods of inhibiting

proUferation and/or survival of a tumor cell by contacting the cell, a biological sample suspected of containing a tumor cell, an extracellular receptor, such as a TFF receptor, or another cell-surface protein on the tumor cell with any of the antibodies to TFF described herein, or with combinations of these antibodies. [00026] The antibodies to TFF described herein are used in methods of preventing cancer or a cell proliferation and/or survival disorder in a subject in need thereof by administering any of the antibodies to TFF described herein, or by administering combinations of these antibodies.

[00027] The antibodies to TFF described herein are used to treat a cancer or tumor. For example, the tumor or cancer is an epithelial tumor such as, e.g., lung cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, hepatic carcinoma, gastric carcinoma, endometrial carcinoma, renal carcinoma, thyroid cancer, biliary duct cancer, esophageal cancer, brain cancer, melanoma, multiple myeloma, hematologic tumor, and lymphoid tumor.

[00028] The antibodies to TFF described herein are used to treat a proliferative disorder. Proliferative disorders include, e.g., keratinocyte hyperproliferation, inflammatory cell infiltration, cytokine alteration, endometriosis, epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myo fibromatosis, osteoplastic tumors, and other dysplastic masses.

[00029] The subject is a mammal, preferably a human suffering from or at risk of developing a tumor or cancer or proliferative disorder. The compositions and methods are also useful for veterinary use, e.g., in treating, cats, dogs, and other pets in addition to livestock, horses, cattle and the like.

[00030] The antibodies to TFF described herein are also useful in a variety of diagnostic applications. The invention features a method for diagnosing cancer or a cell proliferation and/or survival disorder in a mammal by contacting a tissue or bodily fluid from the mammal with an antibody to TFF under conditions sufficient to form an antigen-antibody complex and detecting the antigen-antibody complex. Cancers or tumors detected using the antibodies to TFF described herein include an epithelial tumor such as, e.g., lung cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, hepatic carcinoma, gastric carcinoma, endometrial carcinoma, renal carcinoma, thyroid cancer, biliary duct cancer, esophageal cancer, brain cancer, melanoma, multiple myeloma, hematologic tumor, and lymphoid tumor. Proliferative disorders detected using the antibodies to TFF described herein include, e.g., keratinocyte hyperproliferation, inflammatory cell infiltration, cytokine alteration, endometriosis, epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteoplastic tumors, and other dysplastic masses. [00031] Patient derived tissue samples, e.g., biopsies of solid tumors, as well as bodily fluids such as a CNS-derived bodily fluid, blood, serum, urine, saliva, sputum, lung effusion, and ascites fluid, are contacted with an antibody to TFF.

[00032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[00033] Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[00034] Figure 1A is an illustration depicting SDS-PAGE analysis of a first batch of recombinant TFFl and TFF3 antigen polypeptides used to generate the anti-TFF antibodies of the invention, under reducing and non-reducing conditions. The numeric references are: 1. SeeBlue Plus2 Marker; 2. TFF-1 Reduced; 3. TFF-3 Reduced; 4. TFF-1 Non-Reduced and 5. TFF-3 Non- Reduced.

[00035] Figure IB is an illustration depicting SDS-PAGE analysis of a second batch of recombinant TFF3 antigen polypeptides used to generate the anti-TFF antibodies of the invention, under reducing and non-reducing conditions. Numeric references are: 1. TFF3 Batch 1, Reduced; 2. TFF3 Batch 1 , Non-reduced; 3. TFF3 Batch 2, Reduced; and 4. TFF3 Batch 2, Non-reduced. Figure 1C is an illustration depicting the re-analysis of the first batch of TFFl antigen under the same SDS- PAGE conditions as used for Figure 1 A.

[00036] Figures 2 A, 2B and 2C are graphs depicting size-exclusion HPLC analysis of standards (Fig. 2A), TFFl (Fig. 2B) and TFF3 (Fig. 2C) batches.

[00037] Figure 3 is a series of graphs depicting the results of polyclonal ELISA for TFFl and TFF3, and cross-reactivity between the TFFl panned phage and TFF3, and vice versa. Absorbance at 450nm is indicative of the amount of binding present in the library and the phage pools after each round of panning. [00038] Figure 4 is a series of graphs depicting monoclonal ELISA of clones picked from the Round 3 glycerol stock of panning of TFFl . The signal at 450nm indicates binding of individual clones to the immobilized TFFl . Clones chosen for sequencing are shown in the darker color.

[00039] Figure 5 is a series of graphs depicting monoclonal ELISA of clones picked from the Round 3 glycerol stock of panning of TFF3. The signal at 450nm indicates binding of individual clones to the immobilized TFF3. Clones chosen for sequencing are shown in the darker color.

[00040] Figure 6 is a cladogram depicting the sequence identity between anti-TFFl clones. Clones 1-1E3, 1-2C5 and 1-2B10 did not translate to a scFv, explaining their high divergence from the other clones. These clones were disregarded from further analysis. Three unique sequences were identified from the anti-TFFl clones (clone 1-2C7 was found to be identical).

[00041] Figure 7 is a cladogram depicting the sequence identity between anti-TFF3 clones. 2 unique sequences were identified.

[00042] Figure 8 is a graph depicting the results of a cross -reactivity ELISA in which plates were coated with either TFFl or TFF3, and the five unique clones were tested for binding to both antigens.

[00043J Figure 9 is an illustration depicting the plasmid vector map for human IgGl antibody heavy chain.

[00044] Figure 10 is an illustration depicting the plasmid vector map for human IgGl antibody light chain.

[00045] Figure 11 is a graph depicting a typical Protein-A affinity purification chromatogram for all anti-TFFl and anti-TFF3 reformatted antibodies expressed in CHO-S cells.

[00046] Figure 12 is an illustration depicting the results of SDS-PAGE analysis under reduced (left lanes) and non-reduced (right lanes) conditions, of purified anti-TFFl -1 (also referred to herein as anti-TFFl clone 1), anti TFFl -2 (also referred to herein as anti-TFFl clone 2) and anti-TFFl -3 (also referred to herein as anti-TFFl clone 3) human IgG antibodies.

[00047] Figures 13A-13C are a series of graphs depicting single-cycle kinetics analyses of three anti-TFFl IgG for binding to TFFl using Biacore T100. The results for anti-TFFl clone 1 are shown in Figure 13 A, the results for anti-TFFl clone 2 are shown in Figure 13B, and the results for anti-TFFl clone 3 are shown in Figure 13C.

[00048] Figures 14A-14B are a series of graphs depicting single-cycle kinetics analyses of anti-TFF3 clone 1 IgG for binding to TFFl and TFF3 using Biacore TIOO (GE Healthcare Bio- Sciences AB, Sweden). The results for anti-TFF3-l for binding to TFFl are shown in Figure 14A, and the results for anti TFF3-1 for biding to TFF3 are shown in Figure 14B.

[00049] Figure 15 is a series of graphs depicting the results of monoclonal ELISA on Round 2 phage panned on TFFl . Two plates of clones were analyzed (180 clones) and 31 showed binding to TFFl .

[00050] Figure 16 is a series of graphs depicting the results of monoclonal ELISA on Round 2 phage panned on TFF3. Two plates of clones were analyzed (180 clones) and none showed binding to TFF3.

[00051] Figure 17 is a cladogram depicting the sequence identity between anti-TFFl isolated from Round 2 panning. The sequences previously identified from Round 3 panning are included in the cladogram. Most sequences isolated were identical to the three existing sequences, but two additional sequences were identified.

[00052] Figure 18 is a series of graphs depicting the overlay of the results of monoclonal ELISA of anti-TFF3 clones from Round 3 panning, analyzed on the first batch of TFF3 and a new batch of TFF3. Four clones (indicated by the arrows) were sequenced as they were positive on the new batch of TFF3, but were previously negative.

[00053] Figures 19 A, 19B and 19C are a series of graphs depicting typical Protein-A affinity purification chromatograms for all anti-TFFl : clone 4 (Figure 19A), clone 5 (Figure 19B) and clone 6 (Figure 19C) reformatted antibodies expressed in CHO-S cells.

[00054] Figure 20 is an illustration depicting SDS-PAGE analysis under reduced (left lanes) and non-reduced (right lanes) conditions, of purified Anti TFFl -4 (also referred to herein as aTFFl , clone 4), anti-TFFl -5 (also referred to herein as aTFFl, clone 5) and anti-TFFl -6 (also referred to herein as aTFFl , clone 6) human IgG antibodies. Lane numbers denote the following samples:

[00055] .

Lanes Sample Dilution

1 TFFl -4 (Non-reduced) undiluted

2 TFFl^ (Non-reduced) 10X

3 TFFl -5 (Non -reduced) undiluted

4 TFFl -5 (Non-reduced) 10X

5 TFFl -6 (Non-reduced) undiluted

6 TFFl -6 (Non-reduced) 10X

7 TFFl -4 (Reduced) undiluted

8 TFFl -4 (Reduced) 10X

9 TFFl -5 (Reduced) undiluted

10 TFFl -5 (Reduced) 10X

11 TFFl -6 (Reduced) undiluted

12 TFFl -6 (Reduced) 10X

13 Marker

[00056] Figure 21 is a graph depicting single-cycle kinetics analysis of anti-TFFl clone 4 IgG for binding to TFFl using Biacore T200.

[00057] Figure 22 is a graph depicting single-cycle kinetics analysis of anti-TFFl clone 5 IgG for binding to TFFl using Biacore T200.

[00058] Figure 23 is a graph depicting single-cycle kinetics analysis of anti-TFFl clone 6 IgG for binding to TFFl using Biacore T200.

[00059] Figures 24A and 24B are a series of graphs depicting the inhibition of AGS cell proliferation by positive control TFFl antibody (pAb control) (Fig. 24A) and by Paclitaxel (Fig. 24B).

[00060] Figures 25A-25C are a series of graphs depicting the effect of three negative controls on AGS cell proliferation. The three negative controls are control IgG antibody (Fig. 25A), buffer control (Fig. 25B) and hybridoma medium control (Fig. 25C) at 20 and 50 μΐ/well.

[00061] Figures 26A-26F are a series of graphs depicting the inhibition of AGS cell proliferation by the following antibodies: anti-TFFl clone 1 (Fig. 26A), anti-TFFl clone 2 (Fig.

26B), anti-TFFl clone 3 (Fig. 26C), anti-TFFl clone 4 (Fig. 26D), anti-TFFl clone 5 (Fig. 26E) and anti-TFFl clone 5 (Fig. 26F).

[00062] Figure 27 is a graph depicting the inhibition of AGS cell proliferation by anti-TFF3 clone 1 antibody. [00063] Figures 28A-28B are a series of graphs depicting the dose-dependent inhibition of MCF-7 cell proliferation by positive control TFF1 antibody (pAb control) (Fig. 28A) and by Paclitaxel (Fig. 28B).

[00064] Figures 29A-29C are a series of graphs depicting the effect of three negative controls on MCF-7 cell proliferation. The three negative controls are control IgG antibody (Fig. 29A), buffer control (Fig. 29B) and hybridoma medium control (Fig. 29C) at 20 and 50 μΐ/well.

[00065] Figures 30A-30F are a series of graphs depicting the inhibition of MCF-7 cell proliferation by the following antibodies'. anti-TFFl clone 1 (Fig. 30A), anti-TFFl clone 2 (Fig. 30B), anti-TFFl clone 3 (Fig. 30C), anti-TFFl clone 4 (Fig. 30D), anti-TFFl clone 5 (Fig. 30E) and anti-TFFl clone 5 (Fig. 30F).

[00066] Figure 31 is a graph depicting the effect the anti-TFF3 clone 1 antibody on MCF-7 cell proliferation.

[00067] Figures 32A-32C are a series of graphs depicting the effect of a variety of TFF3 antibodies on MCF-7 cells.

DETAILED DESCRIPTION OF THE INVENTION

[00068] The trefoil factor family of proteins is characterized by a 40-amino acid trefoil motif that contains 3 conserved disulfide bonds. The 3 intrachain disulfide bonds form the trefoil motif (TFF domain). The trefoil motif is known in the art, e.g. Taupin and Podolsky, Nat Rev Mol Cell Bio. 4(9):721-32, 2003; Hoffmann et al., Histol Histopathol 16(1):319-34, 2001; and Thim, Cell Mol Life Sci 53(1 1-12):888-903, 1997.

[00069] In humans, three distinct members of the trefoil peptides have been identified. TFF1 or pS2 was first detected in a mammary cancer cell line as an estrogen-inducible gene. In human stomach, it is predominantly located in the foveolar cells of the gastric mucosa. TFF2 (formerly spasmolytic polypeptide or SP) was first purified from porcine pancreas and is expressed in mucous neck cells, deep pyloric glands, and Brunner's glands. TFF3 or intestinal trefoil factor (1TF) was the last to be identified and is predominantly expressed in the goblet cells of the small and large intestine. The trefoil peptides are involved in mucosal healing processes and are expressed at abnormal elevated levels in neoplastic diseases. A wide range of human carcinomas and

gastrointestinal inflammatory malignancies, including peptic ulceration and colitis, Crohn's syndrome, pancreatitis, and biliary disease, aberrantly express trefoil peptides. Orthologues of these human proteins have been identified in other animals; for example, rats, mice and primates. [00070] The trefoil family of peptides possess divergent function in the mammary gland with TFFl functioning as a mitogen and TFF2 stimulating branching morphogenesis and cell survival. TFF3 is widely co-expressed with TFFl in malignancies of the human mammary gland whereas TFF2 is not expressed in the mammary epithelial cells.

[00071] The studies described herein were designed to (i) identify antibody fragment (ScFv) against recombinant human Trefoil Factor-1 (TFFl) and Trefoil Factor-3 (TFF3); (ii) reformat identified VH and VL into full human immunoglobulin- 1 (IgGl); (iii) co-transfect and transiently express reformatted antibodies in suspension mammalian cells (Chinese Hamster Ovary, CHO cells); (iv) purify full IgG-1 antibody from transient expression and provide affinity-purified antibodies; and (v) measure the affinity of reformatted antibodies (both anti TFFl and anti-TFF3) using Surface Plasmon Resonance technology (Biacore, GE Healthcare Bio-Sciences AB, Sweden).

[00072] Briefly, recombinant TFFl and TFF3 antigen polypeptides produced in E. coli and purified were used to generate the antibodies provided herein. SDS-PAGE and size-exclusion HPLC showed the antigens to be pure, and forming dimers and possibly trimers. There was some degradation of the first batch of TFF3 antigen polypeptide, and a second batch of TFF3 antigen polypeptide was produced. (See Figures 1 A-1 B and 2A-2C; Table 1). No degradation of TFFl was observed. (See Figure 1C). The proprietary human naive scFv library known as the Sheets library by University of California, San Francisco (UCSF), was used to perform panning procedures. Fresh phage particles were produced by infecting the supplied phage particles into XL 1 -Blue cells.

[00073] Three (3x) rounds of antibody phage biopanning were performed on immobilized recombinant TFFl and TFF3 antigen polypeptides. (Figure 3). Phage ELISA on the population of phage after each round showed enrichment of TFFl and TFF3 binders with each round. Cross- reactivity analysis showed that the anti-TFFl pool did not bind TFF3, but the anti-TFF3 pool bound both TFFl and TFF3. Phage ELISA was performed on 180 clones isolated from each Round 3 pool. 94% of clones from the anti-TFFl population were positive for binding to TFFl , and 95% of clones from anti-TFF3 were positive for binding to TFF3 (Figures 4 and 5). 22 positive clones for both TFFl and TFF3 were sequenced. 3 unique sequences were found for anti-TFFl (oTFFl) (Figure 6), with oTFFl -clone 1 being expressed in 18% of clones, <xTFFl -clone2 being expressed in 59% of clones, and aTFFl-clone3 being expressed in 9% of clones. 2 unique sequences were found for anti-TFF3 (o;TFF3) (Figure 7), with TFF3 -clone 1 being expressed in 73% of clones and aTFF3- clone2 being expressed in 27% of clones. All the antibodies are VH3 family, and all have a kappa light chain except oTFF3-clonel which has a lambda light chain (Table 2). All clones are specific for their panned antigen, expect aTFF3- clonel which binds to both TFF1 and TFF3 antigens (Figure 8). The five sequences were reformatted to whole human IgGl (Figures 9 and 10) and were expressed and purified successfully (Figures 1 1 and 12). All 3 oTFFl antibodies showed binding to recombinant TFF1 by Surface Plasmon Resonance (Biacore), with KD ranging from 0.9 to 16.8 nM. None bound to recombinant TFF3 (Table 4 and Figures 13A-13C). One cc-TFF3 antibody (aTFF3- clone 1 ) bound to both recombinant TFF1 and TFF3 (KD = 1 .1 and 5.8 nM respectively), but the other antibody (aTFF3-clone2) did not bind to either antigen (Table 5 and Figures 14A-14B). All 5 unique sequences (3xa-TFFl and 2xo;TFF3) were DNA sequenced, and the CDRs were identified. The sequences of the anti-TFF antibodies are presented below in Example 2.

[00074] Reference herein to "TFF", "TFF protein(s)", or "TFF family of proteins" refers to the group of related proteins including TFF1 , TFF2, and TFF3. TFF proteins share at least approximately 28 to 45% amino acid identity within the same species.

[00075] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F_at_>, F_ab' and F_{(ab )2} fragments, and an F_ab expression library. By "specifically bind" or "immunoreacts with" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e., bind) with other polypeptides or binds at much lower affinity (K_d > 10^"6) with other polypeptides.

[00076] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ea., 2nd ed. Raven Press, N.Y. (1989)). The variable regions of each light/heavy chain pair form the antibody binding site. [00077] The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

[00078] In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, IgG₂, and others.

Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[00079] The term "antigen-binding site" or "binding portion" refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as 'Tiypervariable regions," are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus, the term "FR" refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen- binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs." The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901 -917 (1987), Chothia et al. Nature 342:878-883 (1989).

[00080] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.

Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural

characteristics, as well as specific charge characteristics. An antibody is the to specifically bind an antigen when the dissociation constant is <1 μΜ; preferably <100 nM and most preferably 10 nM.

[00081] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if an antibody has the same specificity as a TFF1 or TFF3 antibody described herein by ascertaining whether the former prevents the latter from binding to a CD3 antigen polypeptide. If the antibody being tested competes with an antibody of the invention, as shown by a decrease in binding by the TFF1 or TFF3 antibody of the invention, then the two antibodies bind to the same, or a closely related, epitope. Another way to determine whether an antibody has the specificity of an antibody of the invention is to pre-incubate the antibody of the invention with the TFF antigen with which it is normally reactive, i.e., TFF1 or TFF3, and then add the antibody being tested to determine if the antibody being tested is inhibited in its ability to bind the TFF antigen. If the antibody being tested is inhibited then, it is likely to have the same, or functionally equivalent, epitopic specificity as the antibody of the invention.

[00082] The data described herein were generated using the following materials and methods. Administration of compositions for cancer therapy

[00083] The TFF-specific antibodies described herein are used to inhibit the growth of a tumor cell, or kill the tumor cell. In addition to cancer therapy, the methods are useful to confer clinical benefit to those suffering from or at risk of developing a precancerous condition or lesion or a non-cancerous hyperproliferative disorder.

[00084] The TFF antibodies and therapeutic formulations thereof are used in methods of treating or alleviating a symptom associated with an immune-related disorder. For example, the compositions of the invention are used to treat or alleviate a symptom of any of the proliferative disorders, neoplastic disorders and/or cancers described herein. The TFF antibody and therapeutic formulations thereof are administered to a subject suffering from proliferation disorder, such as cancer or other neoplastic disorder. A subject suffering from a proliferation disorder is identified by methods known in the art. For example, subjects are identified using any of a variety of clinical and/or laboratory tests such as, physical examination, radiologic examination and blood, urine and stool analysis to evaluate immune status.

[00085] Administration of a TFF antibody to a patient suffering from a proliferation disorder such as cancer or other neoplastic disorder is considered successful if any of a variety of laboratory or clinical results is achieved. For example, administration of a TFF antibody is considered successful one or more of the symptoms associated with the disorder is alleviated, reduced, inhibited or does not progress to a further, i.e., worse, state. Administration of a TFF antibody is considered successful if the disorder, e.g., a cancer or other neoplastic disorder, enters remission or does not progress to a further, i.e., worse, state.

[00086] The agent {e.g., antibodies of the invention) of use in inhibiting a TFF may be used on their own, or in the form of compositions in combination with one or more pharmaceutically acceptable diluents, carriers and/or excipients.

[00087] As used herein, the phrase "pharmaceutically acceptable diluents, carriers and/or excipients" is intended to include substances that are useful in preparing a pharmaceutical composition, may be co-administered with an agent in accordance with the invention while allowing same to perform its intended function, and are generally safe, non-toxic and neither biologically nor otherwise undesirable. Examples of pharmaceutically acceptable diluents, carriers and/or excipients include solutions, solvents, dispersion media, delay agents, emulsions and the like. Diluents, carriers and/or excipients may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability.

[00088] A variety of pharmaceutically acceptable diluents, carriers and/or excipients known in the art may be employed in compositions of the invention. As will be appreciated, the choice of such diluents, carriers and/or excipients will be dictated to some extent by the nature of the agent to be used, the intended dosage form of the composition, and the mode of administration thereof. By way of example, in the case of administration of nucleic acids such as vectors adapted to express antisense or iRNA, suitable carriers include isotonic solutions, water, aqueous saline solution, aqueous dextrose solution, and the like.

[00089] In addition to standard diluents, carriers and/or excipients, a pharmaceutical composition of the invention may be formulated with additional constituents, or in such a manner, so as to enhance the activity of the agent or help protect the integrity of the agent. For example, the composition may further comprise adjuvants or constituents which provide protection against degradation, or decrease antigenicity of an agent, upon administration to a subject. Alternatively, the agent may be modified so as to allow for targeting to specific cells, tissues or tumors.

[00090] Additionally, the antibodies are formulated with other ingredients which may be of benefit to a subject in particular instances. For example, optionally, one or more anti-neoplastic agents are co-administered or incorporated into the formulation. Examples of such agents include: alkylating agents (e.g., chlorambucil (Leukeran™), cyclophosphamide (Endoxan™, Cycloblastin™, Neosar™, Cyclophosphamide™), ifosfamide (Holoxan™, Ifex™, Mesnex™), thiotepa (Thioplex™, Thiotepa™)); antimetabolites/S-phase inhibitors (e.g., methotrexate sodium (Folex™, Abitrexate™, Edertrexate™), 5-fiuorouracil (Efudix™, Efudex™), hydroxyurea (Droxia™, Hydroxyurea, Hydrea™), amsacrine, gemcitabine (Gemzar™), dacarbazine, thioguanine (Lanvis™));

antimetabolites/mitotic poisons (e.g., etoposide (Etopophos™, Etoposide, Toposar™), vinblastine (Velbe™, Velban™), vindestine (Eldesine™), vinorelbine (Navelbine™), paclitaxel (Taxol™)); antibiotic -type agents (e.g., doxorubicin (Rubex™), bleomycin (Blenoxane™), dactinomycin (Cosmegen™), daunorubicin (Cerubidin™), mitomycin (Mutamycin™)); hormonal agents (e.g., amino glutethimide (Cytadren™); anastrozole (Arimidex™), estramustine (Estracyt™, Emcyt™), goserelin (Zoladex™), hexamethylmelanine (Hexamet™), letrozole (Femara™), anastrozole (Arimidex™), tamoxifen (Estroxyn™, Genox™, Novaldex™, Soltamox™, Tamo fen™)); or any combination of any two or more anti-neoplastic agents (e.g., Adriamycin/5- fluorouracil/cyclophosphamide (FAC), cyclophosphamide/methotrexate/5-fluorouracil (CMF)). The antibodies of the invention may also be formulated with compounds and agents, other than those specifically mentioned herein, in accordance with accepted pharmaceutical practice.

[00091] In accordance with the mode of administration to be used, and the suitable pharmaceutical excipients, diluents and/or carriers mentioned herein before, compositions of the invention are converted to customary dosage forms such as solutions, orally administrable liquids, injectable liquids, tablets, coated tablets, capsules, pills, granules, suppositories, trans-dermal patches, suspensions, emulsions, sustained release formulations, gels, aerosols, liposomes, powders and immuno liposomes. The dosage form chosen will reflect the mode of administration desired to be used, the disorder to be treated and the nature of the agent to be used. Particularly preferred dosage forms include orally administrable tablets, gels, pills, capsules, semisolids, powders, sustained release formulation, suspensions, elixirs, aerosols, ointments or solutions for topical administration, and injectable liquids.

[00092] Skilled persons will readily recognize appropriate dosage forms and formulation methods. The compositions can be prepared by contacting or mixing specific agents and ingredients with one another. Then, if necessary, the product is shaped into the desired formulation. By way of example, certain methods of formulating compositions may be found in references such as Gennaro AR: Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins, 2000.

[00093] The amount of an antibody of the invention in a composition can vary widely depending on the type of composition, size of a unit dosage, kind of carriers, diluents and/or excipients, and other factors well known to those of ordinary skill in the art. The final composition can comprise from 0.0001 percent by weight (% w) to 100% w of the actives of this invention, preferably 0.001 % w to 10% w, with the remainder being any other active agents present and/or carrier(s), diluent(s) and/or excipient(s).

[00094] Administration of any of the agents or compositions of the invention can be by any means capable of delivering the desired activity (inhibition of tumor cell proliferation) to a target site within the body of a subject. A "target site" may be any site within the body which may have or be susceptible to a proliferative disorder, and may include one or more cells, tissues or a specific tumor.

[00095] For example, administration may include parenteral administration routes, systemic administration routes, oral and topical administration. For example, administration may be by way of injection, subcutaneous, intraorbital, ophthalmic, intraspinal, intracisternal, topical, infusion (using e.g. slow release devices or minipumps such as osmotic pumps or skin patches), implant, aerosol, inhalation, scarification, intraperitoneal, intracapsular, intramuscular, intratumoral, intranasal, oral, buccal, transdermal, pulmonary, rectal or vaginal. As will be appreciated, the administration route chosen may be dependent on the position of the target site within the body of a subject, as well as the nature of the agent or composition being used.

[00096] The dose of an antibody of the invention or composition administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the nature of the condition to be treated, severity of symptoms of a subject, the size of any tumor to be treated, the target site to be treated, the mode of administration chosen, and the age, sex and/or general health of a subject. Persons of general skill in the art to which the invention relates will readily appreciate or be able to determine appropriate administration regimes having regard to such factors, without any undue experimentation. Administration of an antibody of the invention is in an amount necessary to at least partly attain a desired response. Administration may include a single daily dose or administration of a number of discrete divided doses as may be appropriate. Administration regimes can combine different modes or routes of administration. For example, intratumoral injection and systemic administration can be combined.

[00097] The method may further comprise further steps such as the administration of additional agents or compositions which may be beneficial to a subject having regard to the condition to be treated. For example, other agents of use in treating proliferative disorders (such as the anti-neoplastic agents mentioned above) could be administered. It should be appreciated that such additional agents and compositions may be administered concurrently with the agents and compositions of the invention, or in a sequential manner (for example the additional agents or compositions could be administered before or after administration of the agents or compositions of the invention. It should be appreciated in relation to sequential delivery of agents or compositions, that sequential administration of one agent or composition after the other need not occur

immediately, although this may be preferable. There may be a time delay between delivery of the agents or compositions. The period of the delay will depend on factors such as the condition to be treated and the nature of the compositions or agents to be delivered. However, by way of example, the delay period can be between several hours to several days or months.

Diagnosis of Cancers and/or Proliferation Disorders

[00098] The antibodies to TFF described herein are also useful in a variety of diagnostic applications. The invention features a method for diagnosing cancer or a cell proliferation and/or survival disorder in a mammal by contacting a tissue or bodily fluid from the mammal with an antibody to TFF under conditions sufficient to form an antigen-antibody complex and detecting the antigen-antibody complex. Cancers or tumors detected using the antibodies to TFF described herein include an epithelial tumor such as, e.g., lung cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, hepatic carcinoma, gastric carcinoma, endometrial carcinoma, renal carcinoma, thyroid cancer, biliary duct cancer, esophageal cancer, brain cancer, melanoma, multiple myeloma, hematologic tumor, and lymphoid tumor. Proliferative disorders detected using the antibodies to TFF described herein include, e.g., keratinocyte hyperproliferation, inflammatory cell infiltration, cytokine alteration, endometriosis, epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myo fibromatosis, osteoplastic tumors, and other dysplastic masses.

[00099] Methods for diagnosis include detecting a tumor cell in vivo or ex vivo in bodily fluids or in tissue. For example, a biopsied tissue is contacted with an antibody and antibody binding measured. In addition to biopsied tissue samples, whole blood, serum, plasma, stool, urine, cerebrospinal fluid, bronchoalveolar lavage, sputum, exhaled breath condensate, semen, saliva, joint fluid or ulcer secrete is tested. Whole body diagnostic imaging may be carried out to detect microtumors undetectable using conventional diagnostic methods. Accordingly, a method for diagnosing a tumor in a mammal is carried out by contacting a tissue, e.g., a lymph node, of a mammal with a detectably-labeled antibody which binds to a TFF e itope. An increase in the level of antibody binding at a tissue site compared to the level of binding to a normal nonneoplastic tissue indicates the presence of a neoplasm at the tissue site. For detection purposes, the antibody is labeled with a detectable marker, e.g., non-radioactive tag, a radioactive compound, or a

colorimetric agent. For example, the antibody or antibody fragment is tagged with ¹²⁵I, ⁹⁹Tc, Gd^, or Fe^. Green fluorescent protein is used as a colorimetric tag.

[000100] A method for diagnosis or prognosis is carried out by contacting a bodily fluid or tissue sample from the mammal with an antibody under conditions sufficient to form an antigen- antibody complex and detecting the antigen-antibody complex; quantitating the amount of complex to determine the level of TFF, and comparing the level with a normal control level of TFF. For prognostic purposes, an increasing level of TFF over time indicates a progressive worsening of the disease, and therefore, an adverse prognosis.

[000101] Patient derived tissue samples, e.g., biopsies of solid tumors, as well as bodily fluids such as a CNS-derived bodily fluid, blood, serum, urine, saliva, sputum, lung effusion, and ascites fluid, are contacted with an antibody to TFF.

[000102] Detecting an increase in TFF or TFF gene products in a patient-derived tissue sample (e.g., solid tissue or bodily fluid) is carried out using standard methods, e.g., by Western blot assays or a quantitative assay such as ELISA. For example, a standard competitive ELISA format using an antibody to TFF is used to quantify patient TFF levels. Alternatively, a sandwich ELISA using a first antibody as the capture antibody and a second antibody as a detection antibody is used.

[000103] Methods of detecting TFF include contacting a component of a bodily fluid with an antibody bound to solid matrix, e.g., micro titer plate, bead, dipstick. For example, the solid matrix is dipped into a patient-derived sample of a bodily fluid, washed, and the solid matrix is contacted with a reagent to detect the presence of immune complexes present on the solid matrix.

[000104] Proteins in a test sample are immobilized on (e.g., bound to) a solid matrix. Methods and means for covalently or noncovalently binding proteins to solid matrices are known in the art. The nature of the solid surface may vary depending upon the assay format. For assays carried out in microtiter wells, the solid surface is the wall of the micro titer well or cup. For assays using beads, the solid surface is the surface of the bead. In assays using a dipstick (i.e., a solid body made from a porous or fibrous material such as fabric or paper) the surface is the surface of the material from which the dipstick is made. Examples of useful solid supports include nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidine fluoride (known as

IMMULON™), diazotized paper, nylon membranes, activated beads, and Protein A beads. The solid support containing the antibody is typically washed after contacting it with the test sample, and prior to detection of bound immune complexes. Incubation of the antibody with the test sample is followed by detection of immune complexes by a detectable label. For example, the label is enzymatic, fluorescent, chemiluminescent, radioactive, or a dye. Assays which amplify the signals from the immune complex are also known in the art, e.g., assays which utilize biotin and avidin.

[000105] A TFF-detection reagent, e.g., an antibody, is packaged in the form of a kit, which contains one or more antibodies, control formulations (positive and/or negative), and/or a detectable label. The assay may be in the form of a standard two-antibody sandwich assay format known in the art.

EXAMPLES

[000106] The anti-TFF antibodies described herein were generated and evaluated using the following methods.

Example 1 : Methods of Generating anti-TFF 1 and anti-TFF3 antibodies

[000107] Antigen Analysis: Recombinant Trefoil Factor 1 (TFF1) and Trefoil Factor 3 (TFF3) polypeptides were used to generate the antibodies provided herein. The recombinant antigen polypeptides had the following the characteristics: produced in E. coli cells; solubilized from inclusion bodies and refolded; included a purification tag that was cleaved after purification; and a molecular weight range of 7-9 kDa. The TFF1 polypeptide was 60 amino acids long, and the TFF3 polypeptide was 59 amino acids long. The sequence of each recombinant TFF antigen polypeptide is shown below, along with a comparison of the homology of the native TFF1 and TFF3 sequences:

TFF1 Antigen Polypeptide Sequence:

EAQTETCTVAPRERQNCGFPGVTPSQCANKGCCFDDTVRGVP CFYPNTIDVPPEEECEF (SEQ ID NO: 53)

TFF3 Antigen Polypeptide Sequence:

EEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNRGCCFDSRI PGVPWCFKPLQEAECTF (SEQ ID NO: 54)

TFF1 and TFF3 Sequence Homology

43.3% identity in 60 residues overlap; Score: 161.0; Gap frequency: 0.0%

TFF3 6 LALLSSSSAEEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNRGCCFDSRIPGVPWCFKP

TFF1 12 LVLVSMLALGTLAEAQTETCTVAPRERQNCGFPGVTPSQCANKGCCFDDTVRGVPWCFYP [000108] As described in PCT Publication No. WO 06/069253 and PCT Publication No. WO 08/042435, the contents of each of which are hereby incorporated by reference in their entirety, polyclonal sera raised against these recombinant TFFl and TFF3 antigens, was active in a bioassay for neutralizing the proliferative effect of native TFF1/TFF3. See also, Amiry et. al., "Trefoil factor- 1 (TFFl) enhances oncogenicity of mammary carcinoma cells," Endocrinology, vol. 150: 4473- 4483 (2009), the contents of which are hereby incorporated by reference in their entirety.

[000109] Recombinant human TFFl and TFF3 were analyzed by SDS-PAGE under reducing and non-reducing conditions. 13μΙ, of TFFl or TFF3 were added to 5μL· NuPAGE 4X Loading Buffer (Invitrogen) and either 2 μΙ_, of NuPAGE 1 OX Reducing Agent (Invitrogen) for reduced samples or 2μΙ, of water for non-reduced samples. These 20μΙ_, mixtures were incubated at 70°C for lOmin, and then 15μΙ_, loaded onto a 12% Bis-Tris NuPAGE gel (Invitrogen). SeeBlue Plus2 Marker (5μί) (Invitrogen) was used as protein molecular weight standards. The gel was run in MES buffer at 200V for 45mins. The gel was washed in water and then stained with Simply Blue SafeStain (Invitrogen) for lhr and then destained in water. The results of this SDS-PAGE analysis are shown in Figure 1A.

[000110] The sizes of reduced TFFl and TFF3 are consistent with the expected molecular weight range (~7 to 9kDa). Under non-reduced conditions, the antigens are larger and approximate dimer and trimer for each. This indicated the presence of inter-molecular disulfide bonds. The TFF3 antigen preparation also contains an additional, slightly smaller band, which may be a degradation cleavage product. This product also formed dimer and trimer under non-reducing conditions (Fig. lA).

[000111] A second batch of recombinant TFF3 antigen polypeptide was produced and compared with the first batch described above in connection with Figure 1 A, using the same SDS- PAGE analysis, under reducing and non-reducing conditions. As shown in Figure IB, under reducing conditions, the first batch of TFF3 showed a lower molecular weight band than the second batch (See Fig. IB). This appeared to be the same minor band that was present when the sample was analyzed in Figure 1 A (see Fig. 1 A, Lane 3). However, the major band observed in Figure 1 A was no longer present in the second analysis. Additionally, there were no longer minor bands observed in the non-reduced sample. The TFF3 sample from the first batch used to generate the data shown in Figure 1 A was likely partially degraded to a smaller molecular weight in the first analysis and was likely completely degraded by the second analysis shown in Figure IB. The sample from the first batch still formed dimer and trimer moieties, the same as the non-degraded product. The second batch of TFF3 did not show any degradation.

[000112] To confirm that there was no degradation observed in the first batch of recombinant TFF1 antigen polypeptide used to generate the data shown in Figure 1 A, the sample of recombinant TFF1 antigen polypeptide was re-evaluated approximately 11 months after initial SDS-PAGE analysis shown in Figure 1 A. The sample was prepared as described above. The results of this - SDS-PAGE re-analysis are shown in Figure 1C. The sample of recombinant TFF1 antigen polypeptide did not exhibit any degradation to a lower molecular weight, and this sample was detected in a majority as dimer and trimer moieties as was previously seen in Figure 1 A. (Figure 1C). Some higher molecular weight bands were present in the re-analysis, which may indicate some shuffling of disulfide bonds to higher aggregates (See Fig. 1 C).

[000113] Analytical Size-exclusion HPLC: The recombinant TFF 1 and TFF3 antigen polypeptides were also analyzed by size-exclusion HPLC using a BioSep-SEC-S3000

(Phenomenex) on an Agilent 1200 series LC. Calibration was done using gel filtration standards (Bio-Rad). 20μΕ of the antigens was loaded onto the HPLC column. The results of this HPLC analysis are shown in Figures 2A, 2B and 2C, and the retention times of molecular weight standards and sample peaks on size-exclusion HPLC are shown below in Table 1. Molecular weights of sample peaks were calculated from the retention times of the molecular weight standards.

Table 1.

[000114] As shown in Figures 2A-2C and in Table 1 , the TFF1 sample appeared to be mostly an equal mixture of monomer and dimer, with some higher molecular weight aggregates in the leading edge of the dimer peak. This leading edge could be what was appearing as trimer in the SDS-PAGE analysis described above. For TFF3, the sample was mostly monomer, although the peak was slightly asymmetric, possibly due to the apparent degradation product visible on the SDS- PAGE. The dimer was only a small fraction of the sample on HPLC, but it appeared more dominant on the PAGE analysis.

Example 2: Phage Display scFv Library

[000115] A proprietary single chain Fv phage library from the University of California San Francisco, known as the Sheets library, was used to pan for binders against recombinant human TFF1 and TFF3 derived from E. coli cells. The Sheets library is a human, naive scFv library, containing variable heavy and kappa/lambda chains, joined by a glycine-serine polylinker, cloned into the vector pHENl. The scFv coding regions are cloned in-frame with the g3p gene which encodes the pill protein of the bacteriophage M13K07. This enables the scFv to be displayed on the surface of the bacteriophage when the M13K07 phage is being assembled within E. coli. The library has a diversity of 6.7x10⁹, and the production of this library is described in Sheets et al, "Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens," PNAS, vol. 95(11):6157-62 (1998).

[000116] Amplification of Antibody Phage Library: An aliquot of purified phage was provided to AIBN by UCSF, and the titre determined to be 3.9x10¹² cfu/mL. The helper phage M13K07, was purchased from New England Biolabs at lxlO^{1 1} pfii/mL. XLl -Blue E. coli cells were purchased from Stratagene. The M13K07 titre was amplified by infecting XLl-Blue cells with the commercial phage stock, and purifying the phage by precipitation from 500mL of overnight culture. The final helper phage titre was l xl0¹³ pfu/mL.

[000117] To prepare fresh library phage particles for panning, the supplied Sheets library stock was first infected into XLl-Blue cells and new phage particles were produced after super- infection with M13K07 helper phage. This was achieved by infecting 50mL of XLl - Blue culture in 2YT-Tet media, at OD₆oo⁼l -2, with 10¹¹ cfu of the supplied Sheets library stock. After 30min infection at 37°C, the culture was used to inoculate 700mL of 2YT-AmpGlu which was subsequently incubated for lhr at 37°C. 3x10' pfii of M13K07 helper phage was added to the culture and incubated for 2h at 37°C to initiate phage production. After 2h, the culture was centrifuged and then resuspended in 750mL 2YT-AmpKana. The culture was incubated overnight at 30°C for phage production.

[000118] Phage was purified from the culture supernatant by precipitating the phage with 1/5 volume of PEG-NaCl (20% PEG-6000, 2.5M NaCl) and incubating on ice for lhr. The precipitate was resuspended with 120mL PBS and then precipitated again with 1/5 vol PEG-NaCl. The final precipitate was resuspended in lOmL PBS-20% glycerol and stored at -80°C. The phage titre was determined by infecting l OOuL of OD6oo=0.63 XLl-Blue cells with serial dilutions of the phage stock. The infected cells were plated on 2YT-AmpGlu agar and incubated overnight at 37°C. The colonies on the plates were counted to determine the phage titre. The titre of the amplified Sheets library was ~4xl0¹³ cfu/mL.

[000119] Round 1 Panning of recombinant human TFF1 and TFF3 : The TFFl antigens and first batch of TFF3 antigens were panned against the secondary stock of Sheets human naive antibody phage library. The biopanning procedure was identical for TFF1 and TFF3, and the following procedure was performed on each antigen. Nunc Maxisorb Immunotubes were coated with lmL of antigen at 100 μg/mL, diluted in PBS. The tubes were capped and incubated rotating at room temperature overnight. The antigen solutions were discarded and the tubes were washed by filling the tubes three times with PBS and discarding the wash solutions. The immunotubes were filled with 2% skim milk powder in PBS and incubated for lhr at room temperature to block remaining sites on the immunotubes. For each antigen, 10¹³ pfu of the secondary Sheets phage library stock was incubated with 2mL 2% skim milk powder for lhr, rotating, at RT to block any milk binders present in the library. After blocking, the milk solution was discarded from the immunotubes, and the blocked library was added to the immunotubes and incubated for lhr, rotating, at room temperature. After lhr, the library solution was discarded to phage waste, and the tubes were washed three times by filling the tubes with 0.1% Tween-20 in PBS, and then three times with PBS. The bound phage was eluted from the immunotube by adding lmL 200mM Glycine pH 2.5 and incubating for 8min at room temperature, rotating. The eluate was divided into two tubes, each containing 500μΙ, 1M Tris, pH 7.4 to neutralize the acid eluate. Glycerol was added at 20% to one tube for back-up storage at -80°C. The remaining neutralized eluate was used to infect lOmL XLl-Blue culture at OD₆oo=0.58, for 30min at 37°C. A 150μΕ sample was taken after infection to determine the output titre, by plating serial dilutions of the sample onto 2YT-AmpGlu plates, and incubating overnight at 37°C. The infected culture was centrifuged and then resuspended in 0.5mL of 2YT media and spread evenly over two 150mm 2YT-AmpGlu agar plates. The plates were incubated overnight at 30°C. 2YT-AmpGlu media with 20% glycerol was added to the 150mm plates and the colonies scraped off the agar into an uniform slurry using a colony spreader. The slurry was divided into lmL aliquots and stored at -80°C. The output titre was determined by counting colonies of the titre plates.

[000120] Phage particles were prepared from the Round 1 glycerol stocks in preparation for Round 2 panning. Glycerol stock was inoculated into 50mL 2YT-AmpGlu to a starting OD between

12

0.05 and 0.1. The culture was grown to OD6oo⁼ 1.0, and then 10 pfu Ml 3K07 helper phage was added. The culture was left for infection at 37°C with no shaking, following by 37°C with shaking. The culture was centrifuged and then resuspended in lOOmL 2YT-AmpKana and incubated overnight at 30°C. Phage particles were purified from the culture supernatant by precipitation with 1/5 volume PEG-NaCl and incubation at 4°C for lhr. The precipitate was resuspended in lOmL PBS, and then precipitated again in 1/5 volume PEG-NaCl. The final precipitate was resuspended in 3mL PBS with 20% glycerol and stored at -80°C. The titre of the purified phage was determined by infecting XLl-Blue cells with serial dilutions of the phage and plating onto 2YT-AmpGlu plates. After overnight incubation at 37°C, colonies were counted to determine the phage titre.

[000121] Round 2 and 3 panning ofTFFl and TFF3: The Round 2 and 3 panning procedures are identical to the Round 1 procedure, except (1) coating concentration of antigen on immunotube was 100μg/mL for Round 2 and 50μg/mL for Round 3 ; (2) the number of washes after incubation of the immunotube with phage and before elution with acid was increased with each round. Initially, Round 2 was performed with 20 washes of Tween-20 in PBS and 20 washes with PBS. However, this resulted in zero output titre, so the wash was determined to be too stringent. Consequently, Round 2 was repeated using 5 washes with Tween-20 in PBS and 5 washes with PBS. For Round 3, the number of washes was increased to l Ox with Tween-20 in PBS and l Ox with PBS.

[000122] Polyclonal Phage ELISA: The following procedure was performed for both TFF1 and TFF3. Cross-reactivity was also examined by using the TFF1 panned phage against plates coated with TFF3, and vice versa. Consequently, four polyclonal ELISA's were performed.

[000123] The wells of columns 2-6 of a 96-well Nunc Maxisorb plate were coated with 200μΙ_^ antigen at 10μg/mL. Column 1 was filled with 200μί of PBS. The plate was incubated overnight at room temperature .

[000124] The antigen solution was discarded and the wells were washed by filling 3 times with PBS and discarding the wash solution. The wells were filled with 2% milk in PBS to block the remaining binding sites on the plastic. 90μΙ_, of purified phage particles from each round and from the secondary Sheets library stock were separately added to 1 .8mL of 2% milk in PBS. The blocked plate and blocked phage solutions were incubated for 2hr at room temperature. After two hours the blocking solution was discarded from the plate, 180μΙ_, of 2% milk in PBS was added to columns 3- 5, and 200μί blocked phage particles were added as follows:

• Library phage: A1 , B 1 , A2, B2

• Round 1 phage: CI , D 1 , C2, D2

· Round 2 phage: El , F l , E2, F2

• Round 3 phage: Gl, HI , G2, H2

[000125] Serial 1/10 dilutions were made (20μί in 200μΕ) across the plate from Column 2 to Column 5. Column 1 acts as a negative control for no antigen and Column 6 is a negative control for no phage added. The plate was incubated for lhr at room temperature.

[000126] The plate was washed three times with PBS-0.1 % Tween-20. HRP-conjugated Anti- Mi 3 phage (GE Healthcare) was diluted 1/5000 in 2% milk in PBS, and 200μί was added to each well. The plate was incubated at room temperature for lhr.

[000127] The plate was washed three times with PBS-0.1 % Tween-20. TMB solution (Sigma) was added (ΙΟΟμΕ) to each well. This produces a blue color which intensifies with time. The plate is incubated for up to l Omins and then the reaction terminated by addition of Ι ΟΟμί 1M Sulphuric acid. The reaction should be terminated earlier than l Omins if it appears that the signal between dilutions is losing discrimination. Absorbance at 450nm was measured using a microplate reader. The results of the ELISA analysis are shown in Figure 3.

[000128] The specific polyclonal ELISAs for TFF1 and TFF3 showed increasing binding from the library phage through to the Round 3 phage, confirming that the phage population is being enriched for binders against the panned antigen with each round of panning (See Fig. 3).

[000129] The cross-reactivity ELISA shows that the phage panned against TFF1 did not display any binding to TFF3. Conversely, the phage pool after panning against TFF3 does show a similar level of binding to TFF1. Clones that specifically bind to only TFF3 will be tested and evaluated using monoclonal phage ELISA.

[000130] Monoclonal Phage ELISA and Clonal Analysis for Round 3: The glycerol stock of pooled phagemid-containing XL 1 -Blue cells prepared during Round 3 panning of TFF1 and TFF3 were spread over 2YT-AmpGlu plates to achieve single colonies. 180 colonies for both TFF1 and TFF3 were inoculated individually into 150μί of 2YT-AmpGlu media in 96-well round-bottom plates, and incubated overnight with shaking at 37°C. This overnight plate was then used to inoculate a new 96-well plate by transferring 5 to corresponding wells containing 150μΙ, of 2 YT- AmpGlu, and also to create a glycerol stock plate of clones by adding 60μΙ_^ of 50% glycerol to each well of the overnight plate. After three hours incubation at 37°C of the newplate, M13K07 helper phage was added at 4x10⁸ pfu per well. The plate was incubated without shaking at 37°C for 30min for phage infection, and then with shaking for 30min. The plate was then centrifuged and the cell pellets resuspended in 200μί of 2YT-AmpKana, prior to overnight incubation at 30°C for phage production. A Nunc Maxisorb 96-well plate was coated with antigen by adding 200μί of either TFF1 or TFF3 at 3μg/mL in PBS, and incubating overnight at room temperature. Negative control wells containing no antigen were included.

[000131] The overnight culture plate was centrifuged and the supernatant, containing phage particles, was added to a equal volume of 2% milk in PBS to block non-specific phage. The ELISA plate was washed by discarding the antigen solution and filling the wells three times with PBS. The wells were filled with 2% milk in PBS to block the remaining binding sites, and incubated lhr at room temperature.

[000132] The ELISAplate was emptied of blocking solution, and then ΙΟΟμί of 2% milk in PBS and ΙΟΟμί of blocked phage particles were added to corresponding wells. The plate was incubated for lhr at room temperature. Unbound phage was discarded and the wells washed by filling the wells three times with 0.1% Tween-20 in PBS. HRP -conjugated Anti-M13 phage (GE Healthcare) was diluted 1/5000 in 2% milk in PBS, and 200μΙ_ was added to each well. The plate was incubated at room temperature for lhr.

[000133] The plate was washed three times with 0.1% Tween-20 in PBS. TMB solution (Sigma) was added (ΙΟΟμί) to each well. This produces a blue color which intensifies with time. The plate is incubated for up to 1 Omins and then the reaction terminated by addition of 1 ΟΟμί 1 M Sulphuric acid. The reaction should be terminated earlier than lOmins if it appears that

discrimination between clones is being lost. Absorbance at 450nm was measured using a microplate reader.

[000134] The monoclonal ELISA for both TFF1 and TFF3 showed that the majority of clones picked showed binding to the immobilized antigen; 94% for TFF1 and 95% for TFF3. (See Fig. 4 and Fig. 5)

[000135] 22 clones each for TFF1 and TFF3 isolated from the Round 3 glycerol stocks and shown to be positive binders by monoclonal ELISA were chosen for sequencing. Clones were chosen of varying ELISA signal, rather than the highest 22 clones, in the hope of increasing the diversity of clones obtained. The corresponding clones in the 96-well glycerol plate created during the monoclonal ELISA were used to inoculate 5mL of 2YT-AmpGlu media. The culture was grown overnight at 37°C, and the phagemid DNA was extracted from the cell pellet using Qiagen QiaPrep Plasmid DNA Extraction kit, as per the manufacturer's instructions. Approximately 1 OOng of purified phagemid DNA was combined with either pHEN For or pHEN Rev sequencing primers. The DNA and primers solutions were sent to the Australian Genome Research Facility where

Sanger sequencing was performed. Vector NTI was used to create a contiguous sequence from the forward and reverse data for each sample. The translated sequences from the bacterial leader sequence through to the myc tag were aligned using ClustalW, and unique sequences were identified.

[000136] Three unique scFv sequences were identified amongst the 22 anti-TFFl clones sequenced (See Fig. 6). The "a" and "anti-" as in " TFF" and "anti-TFF" are used herein interchangeably. The sequence of TFFl-clonel was represented by 4 clones (18%), oTFFl-clone2 was represented by 13 clones (59%) and aTFFl-clone3 was represented by 2 clones (9%). The remaining three clones did not translate to a complete scFv sequence and thus are not functional.

[000137] Two unique scFv sequences were identified amongst the 22 anti-TFF3 clones sequenced (See Fig. 7). The sequence of TFF3 -clone 1 was represented by 16 clones (73%) and oTFF3-clone2 was represented by 6 clones (27%). [000138] The five isolated sequences were examined by IMGT V-Quest analysis to determine the V-gene family usage. (See e.g., Lefranc MP, "IMGT, the international ImMunoGeneTics database," Nucleic Acids Res., vol. 31 :307-310 (2003). All sequences were in the commonly used V_H3 family, and all of the sequences except aTFF3-clonel were kappa light chains. tfTFF3 -clone 1 contained a lambda light chain (See Table 2 below).

Table 2: Variable gene families of the five unique clones isolated, determined by IMGT V-Quest

Analysis

[000139] The translated sequences of each scFv are shown below, with the complementarity determining regions (CDR) highlighted in the boxes in each sequence, the PelB leader sequence being italicized in each sequence, and the (G₄S)₃ linkers underlined.

aTFFl-clonel

KYLLPTAAAGLLLLAAQPAMAQVQLQQSGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL

EWVSGISGSGDNTYYADSVKGRFAISRDNSKNTLYLQMDSLRAEDTAVYFCARDKGVRSMDVWGLG

TLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRAS|ESI SYWV|AWYQQI PGKAPK

LLIYKASTLE SGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSNFPLTFGGGTKVEIKRAAA

EQKLISEEDLNGAA ( SEQ ID NO: i;

VH CDR1 : GFTFSSYSMN (SEQ ID NO:

VH CDR2 : GISGSGDN (SEQ ID NO: 7)

VH CDR3 : KGVRSMDVWGL (SEQ ID NO:

VL CDR1 : ESISYWV (SEQ ID NO: 9) VL CDR2: KASTLE (SEQ ID NO: 10)

VL CDR3: QQYSNF (SEQ ID NO: 11) aTFFl-clone2

MKyiiPrAAAGLiLLAAQPAMAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYA TnliVRQAPGKGL

E VSGI SGSAGSTYYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCARDRSWYFDLWGMGT

LVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSAAIGDRVTITCRASlQGINNYLlAWYQQKPGKAPKL

LIY^ASTLQSGVPSRFSGSGSGTEFTLTISGLQPEDFATYYCQKLSSYPLTFGGGTKVEIKRAAAE

QKLISEEDLNGAA (SEQ ID NO: 2)

VH CDR1 : GFTFSSYAMT (SEQ ID NO: 12!

VH CDR2: GISGSAGS (SEQ ID NO: 13)

VH CDR3 : RS YFDLWGR (SEQ ID NO: 14!

VL CDR1: QGINNYL (SEQ ID NO: 15)

VL CDR2 : AASTLQ (SEQ ID NO: 16)

VL CDR3: QKLSSY (SEQ ID NO: 17) aTFFl-clone3

M YILPTAA¾GIiLI,AAOPAMAQVQLQESGGGLVQPGGSLRLSCAASGFTFSSYA SPVRQAPGKGL EWVS^ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARDRGWYFDLWGPJGT

LVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASIGDRVTITCRASQSISSYIJAWYQQKPGKAPKL

LIY^ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLGSYPLTFGGGTKVEIKRAAAE

QKLISEEDLNGAA (SEQ ID NO: 3)

VH CDR1 : GFTFSSYAMS (SEQ ID NO: 18)

VH CDR2 : AISGSGGS (SEQ ID NO: 19)

VH CDR3: RGWYFDLWGR (SEQ ID NO: 20)

VL CDR1: QSISSYL (SEQ ID NO: 21)

VL CDR2 : AASTLQ (SEQ ID NO: 16)

VL CDR3 : QQLGSY (SEQ ID NO: 22) aTFF3-clonel

M yiLPrAAAGLLLXAAQPAMAQVQLQESGGGLVEPGGSLRLSCAASGFSFSNYAMGWVRQAPGKGL

EWVSTI SGRDDRTYYADSVKGRFTISRDNSKNTLYLQMNTLRVEDTAVYYCARYTGRSLDYWGQGT

LVTVSSGGGGSGGGGSGGGGSQSALTQPPSVS GLRQTATLSCTGN|RNNVGNQGAA|WLQQHQGHPPK

LLSYRNNNRPPGISERLSASRSGNTASLTITGLQPEDEADYYCSAWDSSLRVV GGGTKLTVLGA

AAEQKLISEEDLNGAA (SEQ ID NO: 4

VH CDR1 GFSFSNYAMG (SEQ ID NO: 23)

VH CDR2 TISGRDDRTYYAD (SEQ ID NO: 24;

VH CDR3 YTGRSLDYWGQ (SEQ ID NO: 25)

VL CDR1 RNNVGNQGAA (SEQ ID NO: 26)

VL CDR2 RNNNRP (SEQ ID NO: 27)

VL CDR3 SA DSSLRVVV (SEQ ID NO: 28) aTFF3-clone2

M YLLPTAAAGLLLLAAQPAMAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHKAIVRQAPGKGL

E VSSISSSSKYIYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCETPSGKYWGPJGTLVT

VSSGVGGSGGGGSGGGGSDVVMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIY

|GTSSLQ|SGVPSRFSGSGSGTDFTLTISSLQPEDFATYFC|LQDYNFPYT|FGQGTKLEIKRAAAEQKL ISEEDLNGAA (SEQ ID NO: 5!

VH CDR1 : GFTFSSYGMH (SEQ ID NO : 29)

VH CDR2 : SISSSSKYIYYAD (SEQ ID NO: 30)

VH CDR3 : ETPSGKYWGR (SEQ ID NO : 31)

VL CDR1 : QGIRNDLG (SEQ ID NO: 32)

VL CDR2: GTSSLQ (SEQ ID NO: 33 )

VL CDR3: LQDYNFPYT (SEQ ID NO: 34)

[000140] Round 3 cross -reactivity analysis: Three unique clones were isolated from the panning against TFF1 and two unique clones were isolated for TFF3. The polyclonal ELISA showed cross-reactivity in the anti-TFF3 pool, so these clones were checked for cross-reactivity.

[000141] Nunc ELISAplates were coated with ^g/mL of TFF1 orTFF3 in PBS buffer overnight at room temperature, followed by blocking with 2% milk. Phage corresponding to the unique clones and produced during the monoclonal ELISA were blocked with 2% milk and added to both immobilized antigens. After lhr incubation at room temperature, the plates were washed and bound phage was detected with a 1/5000 dilution of HRP-conjugated Anti-M13 antibody. After lhr incubation at room temperature, the plates were washed and TMB reagent was added for detection.

[000142] The cross-reactivity ELISA showed that all three of the anti-TFFl clones bound only to TFF1, with no cross-reactivity to TFF3. For the anti-TFF3 clones, one (aTFF3-l) bound to both TFF1 and TFF3, whilst the other bound only to TFF3 (See Fig. 8). This cross-reactivity is consistent with the results obtained in the polyclonal ELISA which showed that the anti-TFF3 phage population also bound TFF 1 , but the anti-TFF 1 population was specific for TFF 1.

Example 3. Reformatting to IgGl

[000143] The three unique anti-TFFl clones and the two unique anti-TFF3 clones were reformatted to a whole human immunoglobulin of class IgGl with kappa or lambda light chain as appropriate, using the method of Jones et. al, "A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies," J. Immunol. Methods, vol. 354:85-90 (2010). For each clone, the variable heavy sequence and the variable light sequence were separately PCR amplified from the phagemid DNA, using primers containing an overhang homologous to the MAbXpress suite of expression vectors (Figure 9 and Figure 10 depict the heavy and light chain, respectively). This overhang allows ligation-free insertion of the variable region sequence upstream of the constant region sequence, without addition of extraneous residues.

[000144] The expression vectors were sequenced to confirm the correct insertion of the variable region sequences and to ensure no sequence errors were introduced during PCR. Once confirmed, the expression vector DNA was prepared for transfection by performing DNA purification from 250mL E. coli culture using Hi-Pure Filter Maxiprep kit (Invitrogen).

Example 4. Transient Expression of Heavy and Light Chains

[000145] In the study described herein, transient expression of the heavy and light chains was accomplished using PEI in CHO suspension (CHO-S) cells. Briefly, heavy and light chain expression vectors were co-transfected into lOOmL CHO cultures using PEI. 24 hrs prior to transfection, lOOmL of CHO-S (Invitrogen) in mid-exponential growth phase was seeded in CD- CHO medium supplemented with 8mM Glutamax (Invitrogen) at 1.8 x 10⁶cell/mL in 500mL shaker flask (Corning) and placed in humidified shaking incubator set to 37°C, 7.5% C0₂ and shaking at 170rpm. On the day of transfection, CHO culture was adjusted to 87.5mL at 3 x 10⁶ cell/mL by addition of CD-CHO medium. 2μg plasmid DNA per mL of CHO culture was added to 6.25mL OptiPro medium (Invitrogen) and allowed to incubate at room temperature for 30-60 sec. 4μΙ, of PEI-Max (Polysciences) per μg of plasmid DNA was also added to 6.25mL to OptiPro medium (Invitrogen), and allowed to incubate at room temperature for 30-60 sec. After incubation, the DNA and PEI solutions were combined by mixing gently with pipette, and incubated for 15 min without disturbing. After incubation, add DNA-PEI complex to CHO culture and mix flask gently by swirling and place the culture in humidified incubator. 4-6 hrs post transfection feed the transfected culture by addition of lOOmL CD-CHO medium supplemented with 0.4% (v/v) anti -clumping agent (Invitrogen), 8mM Glutamax (Invitrogen), and placed back in humidified shaking incubator. 48 hrs post transfection, culture was transferred to mild hypothermia condition by lowering the incubator temperature to 32°C. Culture was harvested 7 days post transfection by centrifugation at 230g for 10 min. The supernatant was then collected and filtered through a 0.2μηι PVDF filter. The filtered supernatant was then transferred to downstream processing and purification.

Example 5. Purification of recombinant antibodies

[000146] The antibodies were purified from the culture supernatant after 7 days culture using Protein A chromatography followed by de-salting step for buffer exchange. For the Protein-A chromatography, the 1 mL MabSelect SuRe Protein A column (GE Healthcare) was first cleaned and sanitized with 3 column volumes of 0.5 M sodium hydroxide prior to equilibration in 1 x DPBS. Supernatant was 0.2 μΜ filtered and then was loaded onto the column at a flow rate of 1 mL/min. Antibody was eluted from the column in the reverse direction with a step gradient to 0.1 M Arginine, pH 3.0. A typical purification chromatogram is shown in Figure 11.

[000147] For buffer exchange and filtration, the product was loaded directly onto a 5 mL Sephadex G-25 fine desalting column (GE Healthcare), which was previously cleaned and sanitized with 0.5 M sodium hydroxide and equilibrated in lx DPBS. The product was eluted from the column with 1 x DPBS, pH 7.4. The desalted product was then concentrated with 10 kDa Amicon Ultra 4 centrifugal spin filter device (Millipore). The final product was filtered using 0.2 μΜ filters. The filtered bulk and final product samples were stored at 2-8°C.

[000148] SDS-PAGE analysis showed the expected size heavy and light chains under reducing conditions (50kDa and 25kDa respectively), and complexation of chains to the expected size of antibodies (150kDa) under non-reducing conditions (See Fig. 12). Table 3 below summarizes the yields obtained for the five antibody transfections and purifications. Table 3. Transient expression and purification of all anti-TFFl and TFF3 reformatted antibodies.

Example 6. Biaco re Analysis

[000149] A Biacore T100 instrument was used for analysis of the binding affinity of the expressed antibodies, with running buffer HBS-EP+. Approximately 9000RU of anti-human Fc antibody was immobilized onto a CM5 chip using the Human Antibody Capture Kit (GE

Healthcare) and amine coupling. Each anti-TFFl or anti-TFF3 antibody was prepared in HBS-EP+ buffer at lC^g/mL, and -500RU was captured for kinetic analysis. Single-cycle kinetics was used with 5 concentrations of TFFl or TFF3, and 3M magnesium chloride was used to regenerate the chip between each cycle.

[000150] The three anti-TFFl IgG were all shown to bind TFFl (See Figure 13) with affinity constants ranging from 0.9nM to 16.8nM (See Table 4 below). None of these anti-TFFl antibodies showed binding to TFF3 (data not shown), consistent with the data obtained from phage ELISA. Table 4. Association rate (k_a), dissociation rate (k_d) and affinity constants (KD) for anti TFFl IgG determined using Biacore T 100

[000151] Anti-TFF3 -clonel IgG was shown by Biacore to bind both TFFl and TFF3 (See Figure 14 and Table 5 below), consistent with the observation of its cross-reactivity by phage ELISA. Anti-TFF3-clone2 IgG however, was not able to bind either TFFl orTFF3 and lost its ability to bind after reformatting for unknown reasons. Table 5. Association rate (k_a), dissociation rate (ka) and affinity constants (K_D) for anti TFF3- clonel IgG for binding to TFF1 and TFF3 determined using Biacore T100

Example 7. Re-screening of Round 2 binder from biopanning product against recombinant TFF1 and TFF3 using Sheets scFv phage display library

[000152] The studies described herein were designed to (i) identify additional antibody fragments (ScFv) against recombinant human Trefoil Factor- 1 (TFF1) and Trefoil Factor-3 (TFF3) using the Round 2 binding population described above in Example 2; (ii) reformat the identified VH and VL into full human immunoglobulin- 1 (IgGl); (iii) co-transfect and transiently express reformatted antibodies in suspension mammalian cells (Chinese Hamster Ovary, CHO cells); (iv) purify full IgG-1 antibody from transient expression and provide affinity-purified antibodies, and (v) measure the affinity of reformatted antibodies using Surface Plasmon Resonance technology (Biacore). This Round 2 re-screening identified three additional anti-TFFl antibody sequences, but no new sequences for TFF3. The new anti-TFFl clones are referred to as anti-TFFl -4 or anti-TFFl, clone 4; anti-TFFl-5 or anti-TFFl, clone 5; and anti-TFFl-6 or anti-TFFl, clone 6. The potential new anti-TFF3 clones were identified after analyzing aTFF3 Round 3 with a new batch of TFF3, but these potential clones were found to have the identical sequence of anti-TFF3-l, also referred to herein as anti-TFF3, clone 1.

[000153] Re-screening of Round 2 binders against TFF1 and TFF3: In the hope of obtaining more clones, a monoclonal ELISA was performed on the phage pool from Round 2 panning. Round

2 was expected to contain more diversity, but also have a lower percentage of positive clones. The procedure for Round 2 monoclonal ELISA was as previously described in the Examples above, except that the glycerol stock prepared during Round 2 was used as the starting material.

[000154] For TFF1 panned phage, 31 clones of the 180 screened showed binding to TFF1 (See Figure 15). As expected, this was a lower percentage of positive binders compared with the Round

3 monoclonal ELISA as the pool was less enriched in Round 2. For TFF3 panned phage, there were no positive binders isolated from the 180 clones screened (See Figure 16). Based on these results, it was suspected that the TFF3 may have degraded therefore a new batch of TFF3 antigen was used. Analysis of the old batch and the new batch by SDS-PAGE showed that the old batch has degraded to a smaller molecular weight in the 6 months since the panning described above in the Examples. Consequently, the monoclonal ELISA on the same Round 2 clones was repeated using the new batch of TFF3. However, there were still no positive binders isolated in the 180 clones.

[000155] This indicates that the frequency of positive binders within the Round 2 population was less than 0.5%, so a greater number of clones would need to be screened to isolate a positive binder.

[000156] Monoclonal Phage ELISA and clonal analysis: The glycerol stock of pooled phagemid-containing XLl-Blue cells prepared during Round 2 panning of TFFl and TFF3 were spread over 2YT-AmpGlu plates to achieve single colonies. 180 colonies for both TFFl and TFF3 were inoculated individually into 150μί of 2YT-AmpGlu media in 96-well round-bottom plates, and incubated overnight with shaking at 37°C. This overnight plate was then used to inoculate a new 96-well plate by transferring 5μΙ_- to corresponding wells containing 150μ1_, of 2YT-AmpGlu, and also to create a glycerol stock plate of clones by adding 60μΙ_, of 50% glycerol to each well of the overnight plate. After three hours incubation at 37°C of the new plate, M13K07 helper phage was added at 4x10⁸ pfu per well. The plate was incubated without shaking at 37°C for 30min for phage infection, and then with shaking for 30min. The plate was then centrifuged and the cell pellets resuspended in 200μ1_, of 2YT-AmpKana, prior to overnight incubation at 30°C for phage production. A Nunc Maxisorb 96-well plate was coated with antigen by adding 200μί of either TFFl or TFF3 at 3μg/mL in PBS, and incubating overnight at room temperature. Negative control wells containing no antigen were included.

[000157] The overnight culture plate was centrifuged and the supernatant, containing phage particles, was added to an equal volume of 2% milk in PBS to block non-specific phage. The

ELISA plate was washed by discarding the antigen solution and filling the wells three times with PBS. The wells were filled with 2% milk in PBS to block the remaining binding sites, and incubated lhr at room temperature.

[000158] The ELISA plate was emptied of blocking solution, and then ΙΟΟμΙ. of 2% milk in PBS and ΙΟΟμί of blocked phage particles were added to corresponding wells. The plate was incubated for lhr at room temperature. Unbound phage was discarded and the wells washed by filling the wells three times with 0.1% Tween-20 in PBS. HRP-conjugated Anti-M13 phage (GE Healthcare) was diluted 1/5000 in 2% milk in PBS, and 200μί was added to each well. The plate was incubated at room temperature for lhr.

[000159] The plate was washed three times with 0.1% Tween-20 in PBS. TMB solution (Sigma) was added (ΙΟΟμΙ,) to each well. Presence of the HRP conjugate causes the conversion of TMB to a blue colored product, which intensifies with time. The plate is incubated for up to lOmins and then the reaction terminated by addition of ΙΟΟμί 1M Sulphuric acid. The reaction should be terminated earlier than lOmins if it appears that discrimination between clones is being lost.

Absorbance at 450nm was measured using a microplate reader.

[000160] All of the positive clones from the anti-TFFl Round 2 Monoclonal ELISA were sequenced (Figure 17). It was found that the majority of clones were identical to the three anti- TFFl sequences previously isolated from the Round 3 panning: 33.3% of the clones were identical to oTFFl-clone 1, 33.3% were identical to aTFFl-clone 2 and 10% were identical to aTFFl-clone 3. However, three additional clones were identified: aTFFl -clone 4 (6%), aTFFl -clone 5 (6%) and aTFFl -clone 6 (9%). One clone (4) returned bad quality sequence data but it is likely to be identical to aTFFl -clone 1. The variable gene families of the unique clones isolated, as determined by IMGT V-Quest Analysis are shown below in Table 6.

Table 6.

[000161] The translated sequences of each scFv are shown below, with the complementarity determining regions (CDR) highlighted in the boxes in each sequence, the PelB leader sequence being italicized in each sequence, and the (G₄S)₃ linkers underlined.

Anti TFFl-clone 4

MKYLLPTAAAGLLLLAAQPAMAQVQ LQSAGGVVQPGRS RLSCAASGFTFSSYAMSMVRQAPGKGL

EWVS|SI SGSGGS|TYYADSVKGRFTISRD SKNTLYLQM SLRAEDTAVYYCAKD|RYDSSGY FDYW| [G^GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPSSVSASVGDRVTITCRAS|QDjjsWl^WYQQKPG KAPKLLIY^ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFClQQASVFlPVTFGGGTKLEIK

RAAAEQKLISEEDLNGAA (SEQ ID NO: 35)

VH CDR1: GFTFSSYAMS (SEQ ID NO: 18)

VH CDR2: SISGSGGS (SEQ ID NO: 38)

VH CDR3: RYDSSGYWFDYWGQ (SEQ ID NO:

VL CDR1: QDIGSWL (SEQ ID NO: 40)

VL CDR2: AASTLQ (SEQ ID NO: 16)

VL CDR3: QQASVF (SEQ ID NO: 41)

Anti-TFFl-clone 5

W yiiPTAAAGLXXIAAQPAMAQVQLQESGGGVVQPGRSLRLSCAASGSTFSTYGMHfciVRQAPGKGL

EWVA|AI SKDGSNK|YYADSVKGRFTISRDNPKNTLYLQMNSLRAEDTAVYYCAR|GPQGAFDIWGQ|GT

TVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRAS|EGIYH L|A YQQKPGRAPKL LIYKASSLAEGAPSRFSGSGSGTDFTLTISSLQSEDSASYFCQQASVFPVTFGGGTKVDIKRAAAE

QKLISEEDLNGAA (SEQ ID NO: 36)

VH CDR1 : GSTFSTYGMH (SEQ ID NO: 42)

VH CDR2 : AISKDGSNK (SEQ ID NO: 43)

VH CDR3: GPQGAFDIWGQ (SEQ ID NO : 44

VL CDR1: EGIYHWL (SEQ ID NO: 45 )

VL CDR2 : KASSLA (SEQ ID NO: 46)

VL CDR3 : QQASVF (SEQ ID NO: 41) Anti-TFFl-clone 6

MKYLIPTAAAGiLILAAOPA AQVNLRESGGGVVQPGRSLRLSCAASGFTVSNPYMTfciVRQAPGKGL

EWVteVIYTGGSTYYAEPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAWDLGSRFDHWGQGTL

VTVSSGGGGSGGGGSGGGGSHVILTQPASVSGSPGQAITISCTGT|SSDIGGYNYV|S YQQHPGKAPK

LMIYDVSYRPSGVSNRFSGSKSANTASLTISGLQAEDEADYYCA^ DDSLNGWK/FGGGTKVTVLGA AAEQKLISEEDLNGAA (SEQ ID NO: 37)

VH CDR1: GFTVSNPYMT (SEQ ID NO: 47] VH CDR2: SVIYTGGST (SEQ ID NO: 48)

VH CDR3: DLGSRFDH GQ (SEQ ID NO: 49)

VL CDR1: SSDIGGYNYV (SEQ ID NO: 50)

VL CDR2: DVSYRP (SEQ ID NO: 51)

VL CDR3: AWDDSLNGW (SEQ ID NO: 52)

[000162] Repeat of Round 3 monoclonal ELISA with new TFF3 antigen: From the Round 3 pool of binder panned against TFF3, the same 180 clones initially analyzed by monoclonal ELISA using the original batch of TFF3 were analyzed again using the new batch of TFF3, as the previous batch showed degradation. The procedure was as previously described above in the Examples, except that the method was started from the 96-well glycerol stock plate prepared during the first monoclonal ELISA. The results show that each clone behaved similarly on the new batch of TFF3 compared with the previous monoclonal ELISA performed on the old batch of TFF3 (See Figure 18). However, four clones (A 12 and B12 on the first plate, and HI and H2 on the second plate) were positive on the new batch but were negative on the old batch. These four clones were sequenced but they were found to be identical to TFF3-l previously isolated, so no new sequences were isolated in this re- screening.

Example 8. Reformatting to IgGl of Binders Identified in Re-screening

[000163] The three new anti-TFFl clones identified in Example 7 were reformatted to whole human immunoglobulin of class IgGl with kappa light chain, using the method of Jones et. al, 2010, supra. For each clone, the variable heavy sequence and the variable light sequence were separately PCR amplified from the phagemid DNA, using primers containing an overhang homologous to the MAbXpress suite of expression vectors (Figures 9 and 10 for the heavy and light chain, respectively). This overhang allows ligation-free insertion of the variable region sequence upstream of the constant region sequence, without addition of extraneous residues.

[000164] The expression vectors were sequenced to confirm the correct insertion of the variable region sequences and to ensure no sequence errors were introduced during PCR. Once confirmed, the expression vector DNA was prepared for transfection by performing DNA purification from 250mL E. coli culture using Hi-Pure Filter Maxiprep kit (Invitrogen).

Example 9. Transient Expression of Heavy and Light Chains for Reformatted Binders Identified in Re-screening

[000165] Heavy and light chain expression vectors were co-transfected into lOOmL CHO cultures using PEL Briefly, 24 hrs prior to transfection, lOOmL of CHO-S (Invitrogen) in mid- exponential growth phase was seeded in CD-CHO medium supplemented with 8mM Glutamax (Invitrogen) at 1.8 x 10⁶cell/mL in 500mL shaker flask (Corning) and placed in humidified shaking incubator set to 37°C, 7.5% C0₂ and shaking at 170rpm. On the day of transfection, CHO culture was adjusted to 87.5mL at 3 x 10⁶ cell/mL by addition of CD-CHO medium. 2μg plasmid DNAper mL of CHO culture was added to 6.25mL OptiPro medium (Invitrogen) and allowed to incubate at room temperature for 30-60 sec. 4μΙ, of PEI-Max (Polysciences) per μg of plasmid DNA was also added to 6.25mL to OptiPro medium (Invitrogen), and allowed to incubate at room temperature for 30-60 sec. After incubation, complex DNA with PEI solution by mixing gently with pipette.

Incubate for 15 min without disturbing. After incubation, add DNA-PEI complex to CHO culture and mix flask gently by swirling and place the culture in humidified incubator. 4-6 hrs post transfection feed the transfected culture by addition of 1 OOmL CD-CHO medium supplemented with 0.4% (v/v) anti-clumping agent (Invitrogen), 8mM Glutamax (Invitrogen), and placed back in humidified shaking incubator. 48 hrs post transfection, culture was transferred to mild hyperthermia condition by lowering the incubator temperature to 32°C. Culture was harvested 7 days post transfection by centrifugation at 23 Og for 10 min. The supernatant was then collected and filtered through a 0.2μηι PVDF filter. The filtered supernatant was then transferred to downstream processing and purification.

Example 10. Purification of Recombinant Antibodies based on Binders Identified in Re-screening

[000166] The antibodies were purified from the culture supernatant after 7 days culture using Protein A chromatography (Figures 19A, 19B and 19C), followed by de-salting step for buffer exchange. Final protein concentrations are listed in Table 7. Experimental procedures are as follows: for Protein-A chromatography, the 1 mL Mab SelectSure Protein A column (GE

Healthcare) was first cleaned and sanitized with 3 CV of 0.5 M sodium hydroxide prior to equilibration in 1 x DPBS. Supernatant was 0.2 μΜ filtered and then was loaded onto the column at a flow rate of 1 mL/min. Antibody was eluted from the column in the reverse direction with a step gradient to 0.1 M Arginine, pH 3.0. For buffer exchange and filtration, the product was loaded directly onto a 5 mL desalting column (GE Healthcare, Sephadex G-25 fine), which had previously been cleaned and sanitized with 0.5 M sodium hydroxide and equilibrated with lx DPBS. The product was eluted from the column with 1 x DPBS, pH 7.4. The desalted product was then concentrated with 10 kDa Amicon Ultra 4 centrifugal spin filter device (Millipore). Final product was filtered using 0.2 μΜ filters. The filtered bulk and final product samples were stored at 2-8°C. Table 7. Amount of each TFFl antibody and concentration of each transient expression experiment

[000167] SDS-PAGE analysis showed the expected size heavy and light chains under reducing conditions (50kDa and 25kDa respectively), and complexation of chains to the expected size of antibodies (150kDa) under non-reducing conditions (See Figure 20).

Example 11. Biacore Analysis of Antibodies based on Binders Identified in Re-screening

[000168] A Biacore T200 instrument was used for analysis of the binding affinity of the expressed antibodies, with running buffer HBS-EP+. Approximately 9000RU of anti-human Fc antibody was immobilized onto a CM5 chip using the Human Antibody Capture Kit (GE

Healthcare) and amine coupling. Each anti-TFFl antibody was diluted to 10μg/mL in HBS-EP+ buffer, and -500RU was captured for kinetic analysis. Single-cycle kinetics was used with 5 concentrations of TFFl or TFF3, and 3M magnesium chloride was used to regenerate the chip between each cycle.

[000169] The three anti-TFFl IgG were all shown to bind TFFl (See Figures 21 , 22, and 23) with affinity constants ranging from 58nM to 89nM (See Table 8). None of these anti-TFFl antibodies showed binding to TFF3.

Table 8. Association rate (k_a), dissociation rate (k_d) and affinity constants (KD) for anti TFFl IgG determined using Biacore T 100

[000170] A Biacore T100 instrument was used for analysis of the binding affinity of the expressed antibodies, with running buffer HBS-EP+. Approximately 9000RU of anti-human Fc antibody was immobilized onto a CM5 chip using the Human Antibody Capture Kit (GE

Healthcare) and amine coupling. Purified oTFFl-4, aTFFl -5 and aTFFl -6 hlgGl antibodies were diluted to l(^g/mL in Biacore HBS-EP+ buffer. Approximately 500RU were captured onto a CM5 chip with immobilized Anti-human Fc antibody. Antigens TFFl and TFF3 were diluted in HBS- EP+ buffer to InM, 3nM, ΙΟηΜ, 30nM and l OOnM, and analyzed for binding to each antibody using single-cycle kinetics. Magnesium chloride (3M) was used to regenerate the chip between each cycle. A cycle containing buffer in place of antigen was included for each antibody. This cycle was subtracted from the antigen cycle, and a 1 :1 Langmuir binding model was fitted to the data.

Example 12. Inhibition of Cell Survival and/or Proliferation Using anti-TFF Antibodies

[000171] TFFl and TFF3 are overexpressed in cancer cells of various organs and induce invasion, survival and proliferation of neoplastic cells. The studies described herein were designed to evaluate the in vitro effect of using TFFl and/or TFF3 specific antibodies as cytotoxic agents on cancer cells.

[000172] The following antibodies were tested: anti-TFFl , Clone 1 (also referred to herein as anti-TFF 1-1 or oTFFl -1); anti-TFFl , Clone 2 (also referred to herein as anti-TFF 1 -2 or aTFFl -2); anti-TFFl , Clone 3 (also referred to herein as anti-TFFl-3 or oTFFl -3); anti-TFFl , Clone 4 (also referred to herein as anti-TFF 1 -4 or TFFl -4); anti-TFFl , Clone 5 (also referred to herein as anti- TFFl -5 or aTFFl -5); anti-TFFl , Clone 6 (also referred to herein as anti-TFFl -6 or aTFFl -6) and anti-TFF3, Clone 1 (also referred to herein as anti-TFF3-l or QTFF3-1). A polyclonal TFFl antibody (pAB TFFl) was used as a positive control, and an IgGl isotype antibody was used as the negative control. Additional controls used included a buffer control and a hybridoma medium control. Each antibody was tested at the following concentrations in duplicate or triplicate: 0, 200 and 500 μg/mL.

[000173] AGS cell line: These antibodies were tested using the AGS cell line, a human gastric cancer cell line, in the MTT assay for cell proliferation. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl tetrazolium bromide) is used in a rapid colormetric assay that measures only living cells and can be read on a scanning multiwell spectrophotometer (ELISA reader). MTT is converted to purple formazan only when mitochondrial reductase enzymes are active, and thus conversion is directly related to the number of viable cells. The production of formazan in cells treated with an agent is measured relative to the production in control cells, and a dose-response curve can be generated. [000174] Briefly, in this assay, -2500 cells were seeded in wells of 96 well plates. The following day, test antibodies were added to triplicate wells at the concentrations noted above, 0, 200 and 500 μg/mL. The plates are then incubated in a standard tissue culture incubator for 72 hours in the presence of the test antibodies. As a positive control for an anti-pro liferative effect, a cytotoxic agent (e.g., camptothecin or paclitaxel) is added to a set of wells. One set of wells is treated with a non-specific IgG control antibody spike into random hybridoma conditioned medium at concentrations identical to those used for test antibodies. Vehicle (hybridoma conditioned medium) treated wells are also used as negative controls. Following 72 hours incubation with antibodies, MTT is added to a final concentration recommended by the vendor. Cells are incubated for four hours with MTT, after which they are lysed and absorbance (580 nm) measured on a plate reader.

[000175] Proliferation of AGS cells was inhibited in a dose-dependent manner by both positive control TFFl Ab (pAb Control) and Paclitaxel. (Figure 24A-24B) Proliferation of AGS cells was not affected by three negative controls: control IgG (Figure 25 A), Buffer (Figure 25B) and

Hybridoma Medium (Figure 25C) at 20 and 50 μΐ/well.

[000176] Proliferation of AGS cells was inhibited by all TFFl Clones as shown in Figures 26A-26F, and proliferation of AGS cells was inhibited by TFF3 Clone 1 as shown in Figure 27. Thus, the studies presented herein demonstrate that the proliferation of AGS cells was inhibited in a dose-dependent manner by anti-TFFl and anti-TFF3 antibodies.

[000177] MCF-7 cell line: As described above, the following antibodies were tested: anti- TFFl, Clone 1 ; anti-TFFl, Clone 2; anti-TFFl , Clone 3; anti-TFFl , Clone 4; anti-TFFl , Clone 5; anti-TFFl, Clone 6 and anti-TFF3, Clone 1. As described above, the following controls were used: a polyclonal TFFl antibody (pAB TFFl) was used as a positive control; an IgGl isotype antibody was used as the negative control; and buffer control and hybridoma medium controls were used. Each antibody was tested at the following concentrations in duplicate or triplicate: 0, 200 and 500 μg/mL.

[000178] In particular, these antibodies were tested using the MC7F cell line, a human breast cancer cell line, in the MTT assay described above.

[000179] Proliferation of MCF-7 cells was inhibited in a dose-dependent manner by both positive control TFFl Ab (pAb Control) and Paclitaxel. (Figure 28A, 28B) Proliferation of MCF-7 cells was not affected by three negative controls: control IgG, Buffer and Hybridoma Medium at 20 and 50 μΐ/well. (Figure 29A-29C). [000180] Proliferation of MCF-7 cells was inhibited by all TFF1 Clones as shown in Figures 30A-30F, but proliferation of MCF-7 cells was not inhibited by TFF3 Clone 1 as shown in Figure 31. Thus, the studies presented herein demonstrate that the proliferation of MCF-7 cells was inhibited in a dose-dependent manner by anti-TFFl antibodies.

[000181] The TFF3 antibody clones were also tested using the xCELLigence™ System (Roche Applied Science), which monitors cellular events in real time by measuring electrical impedance across intend igitated micro-electrodes integrated on the bottom of tissue culture E-Plates. 3000 cells per well were grown in 0.5% FCS RPMI and treated with the anti-TFF3 antibody after 24h. The results of these studies are shown in Figures 32A-32C.

[000182] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[000183] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Claims

We claim:

1. An isolated monoclonal anti-trefoil factor 1 (TFF1) antibody or antigen-binding fragment or derivative thereof, wherein the antibody comprises:

(a) a V_H CDRl region comprising the amino acid sequence of SEQ ID NO: 6, 12, 18, 42 or 47;

(b) a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 7, 13, 19, 38,

43 or 48;

(c) a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 8, 14, 20, 39,

44 or 49;

(d) a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 9, 15, 21, 40,

45 or 50;

(e) a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 10, 16, 46 or 51 ; and

(f) a V_L CDR3 region comprising the amino acid sequence of SEQ ID NO: 1 1 , 17, 22, 41 or 52,

wherein the antibody binds TFF1.

2. An isolated monoclonal anti-trefoil factor 3 (TFF3) antibody or antigen-binding fragment or derivative thereof, wherein the antibody comprises:

(a) a V_H CDRl region comprising the amino acid sequence of SEQ ID NO: 23 or 29;

(b) a VH CDR2 region comprising the amino acid sequence of SEQ ID NO: 24 or 30;

(c) a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 25 or 31 ;

(d) a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 26 or 32;

(e) a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 27 or 33; and

(f) a V_L CDR3 region comprising the amino acid sequence of SEQ ID NO: 28 or 34, wherein the antibody binds TFF3.

3. The antibody of claim 1 , wherein the antibody comprises a V_H CDRl region comprising the amino acid sequence of SEQ ID NO: 6; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR3 region comprising the amino acid sequence of SEQ ID NO: 8; a VL CDRl region comprising the amino acid sequence of SEQ ID NO: 9; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 10; and a VL CDR3 region comprising an amino acid sequence of SEQ ID NO: 11.

4. The antibody of claim 1 , wherein the antibody comprises a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 12; a V_H CDR2 region comprising the amino acid sequence of

SEQ ID NO: 13, a VH CDR3 region comprising the amino acid sequence of SEQ ID NO: 14; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 15; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 16; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO: 17.

5. The antibody of claim 1 , wherein the antibody comprises a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 18; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 19, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 20; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 21 ; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 16; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO: 22.

6. The antibody of claim 1 , wherein the antibody comprises a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 18; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 38, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 39; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 40; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 16; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO: 41.

7. The antibody of claim 1, wherein the antibody comprises a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 42; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 43, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 44; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 45; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 46; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO: 41.

8. The antibody of claim 1 , wherein the antibody comprises a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 47; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 48, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: ;49 a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 50; a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 51 ; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO: 52.

9. The antibody of claim 2, wherein the antibody comprises a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 23; a VH CDR2 region comprising the amino acid sequence of SEQ ID NO: 24, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 25; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 26; a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 27; and a VL CDR3 region comprising an amino acid sequence of SEQ ID NO: 28.

10. The antibody of claim 2, wherein the antibody comprises a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 29; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 30, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 31 ; a V_L CDRl region comprising the amino acid sequence of SEQ ID NO: 32; a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 33; and a VL CDR3 region comprising an amino acid sequence of SEQ ID NO: 34.

1 1. An isolated monoclonal anti-trefoil factor 1 (TFF1) antibody or antigen-binding fragment or derivative thereof, wherein the antibody comprises:

(a) a VH CDRl region comprising the amino acid sequence of SEQ ID NO: 6, 12, 18, 42 or 47;

(b) a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 7, 13, 19, 38,

43 or 48;

(c) a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 8, 14, 20, 39,

44 or 49;

(d) a VL CDRl region comprising the amino acid sequence of SEQ ID NO: 9, 15, 21, 40,

45 or 50; (e) a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 10, 16, 46 or 51 ; or

(f) a VL CDR3 region comprising the amino acid sequence of SEQ ID NO: 1 1, 17, 22, 41 or 52,

wherein the antibody binds TFF1.

12. An isolated monoclonal anti-trefoil factor 3 (TFF3) antibody or antigen-binding fragment or derivative thereof, wherein the antibody comprises:

(a) a V_H CDR1 region comprising the amino acid sequence of SEQ ID NO: 23 or 29;

(b) a VH CDR2 region comprising the amino acid sequence of SEQ ID NO: 24 or 30;

(c) a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 25 or 31 ;

(d) a VL CDRl region comprising the amino acid sequence of SEQ ID NO: 26 or 32;

(e) a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 27 or 33; or

(f) a VL CDR3 region comprising the amino acid sequence of SEQ ID NO: 28 or 34, wherein the antibody binds TFF3.

13. A pharmaceutical composition comprising an antibody of any one of the preceding claims and a carrier.

14. A method of treating cancer, a cell proliferation disorder or a cell survival disorder in a subject in need thereof, the method comprising administering an antibody according to any one of claims 1 to 12 to the subject in an amount sufficient to alleviate at least one symptom of the cancer, the cell proliferation disorder or the cell survival disorder disorder in the subject.

15. The method of claim 14, wherein the subject is a human.

16. The method of claim 14 or 15, wherein the cancer is an epithelial cancer.

17. The method of claim 16, wherein the epithelial cancer is selected from lung cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, hepatic carcinoma, gastric carcinoma, endometrial carcinoma, renal carcinoma, thyroid cancer, biliary duct cancer, esophageal cancer, brain cancer, melanoma, multiple myeloma, hematologic tumor, and lymphoid tumor.

18. The method of claim 14, wherein the cell proliferation disorder or a cell survival disorder is selected from the group consisting of keratinocyte hyperproliferation, inflammatory cell infiltration, endometriosis, cytokine alteration, epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myo fibromatosis, osteoplastic tumors, and other dysplastic masses.

19. The method of any one of claims 14 to 18, further comprising the administration of an additional compound wherein the additional compound is a chemotherapeutic or anti-neoplastic agent.

20. A method for detecting or diagnosing a cancer, a tumor, a cell proliferation and/or a cell survival disorder in a mammal, the method comprising

a. contacting a tissue or bodily fluid from the mammal with an anti-TFF antibody as defined in any one of claims 1 to 12 under conditions sufficient to form an antigen- antibody complex, and

b. detecting the antigen-antibody complex,

wherein detection of the complex indicates that a cancer, a cell proliferation and/or a cell survival disorder has been detected.

21. A method according to claim 20 wherein the contacting in a) is in vitro in a sample obtained from the mammal.

22. A method according to clam 20 or 21 wherein the cancer or tumor is an epithelial cancer or tumor.

23. A method according to claim 22 wherein the epithelial cancer or tumor is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, hepatic carcinoma, gastric carcinoma, endometrial carcinoma, renal carcinoma, thyroid cancer, biliary duct cancer, esophageal cancer, brain cancer, melanoma, multiple myeloma, hematologic tumor, and lymphoid tumor.

24. A method according to claim 20 or 21 wherein the cell proliferation disorder is selected from the group consisting of keratinocyte hyperproliferation, inflammatory cell infiltration, cytokine alteration, endometriosis, epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteoplastic tumors, and other dysplastic masses.