Anti-matrix metalloproteinase-7 (MMP-7) inhibitor antibodies and uses thereof

Abstract

An antibody comprising an antigen recognition region that binds to the catalytic site of MMP-7, having the amino acid sequences of complementarity-determining regions (CDRs) shown in SEQ ID NOs: 3 (CDR1), 4 (CDR2) and 5 (CDR3) arranged in order from N to C on the antibody light chain, and SEQ ID NOs: 6 (CDR1), 7 (CDR2) and 8 (CDR3) arranged in order from N to C on the antibody heavy chain.

Description

Related Applications

[0001] This application claims the benefit of priority from Israel Patent Application No. 264768, filed February 10, 2019, the entire contents of which are incorporated herein by reference.

[0002] An ASCII file entitled 4,599 Sequence Listings.txt, created on February 6, 2020, containing 81,393 bytes, was submitted contemporaneously with the filing of this application and is incorporated herein by reference. [Technical field]

[0003] In some embodiments, the present invention relates to an antibody that binds to the catalytic site of MMP-7. The antibody can be used for the treatment of cancer, more particularly, but not limited to, pancreatic cancer. [Background technology]

[0004] Matrix metalloproteinase 7 (MMP-7, matrilysin) has emerged as an important target for the development of next-generation cancer therapeutics due to its association with the clinical behavior of multiple tumor types. It is an independent prognostic factor for overall survival in patients with advanced colorectal cancer. Ablation of MMP-7 significantly reduced tumor formation in a multiple intestinal neoplasia mouse model. Similarly, overexpression of MMP-7 was found to promote tumor development in an MMTV-neu mouse breast cancer model. The mechanism by which MMP-7 contributes to early tumor growth remains poorly defined. MMP-7 null mice are prone to loss of dysplastic lesions after pancreatic duct ligation, and inhibition of MMP-7 with broad-spectrum MMP inhibitors has been shown to reduce the number of intestinal polyps.

[0005] MMP-7 is unique among MMPs because it lacks a hemopexin-like domain and its main structure consists of a signal peptide, a propeptide and a zinc-containing catalytic domain. 3 MMP-7 is a secreted MMP and, like other MMPs, is activated after cleavage of the propeptide. The proteases responsible for its activation have not yet been fully defined. MMP-7 is involved in the degradation of macromolecules such as type IV collagen, gelatin, laminin, entactin / nidogen and tenascin-C. In addition to these classical enzymatic roles, MMP-7 has been implicated in the modification of signaling pathways and in the regulation of cytokine activity. The best-characterized substrates for MMP-7's non-canonical signaling-related activities include Fas-L, Fas-R / CD-95, TNF-α, VEGF, plasminogen, E-cadherin and integrin β-4. MMP-7 is involved in regulating ErbB4 activity, inducing IL-17-mediated epithelial to mesenchymal transition, and acts as a pro-invasive effector molecule via the Wnt / β-catenin pathway. These effects make MMP-7 an interesting target acting on tumor cells as well as stromal cells with multiple downstream and upstream implications in both the protease web and signaling cascades. In pancreatic cancer, serum MMP-7 was recently shown to be a preoperative prognostic marker, as its increased expression correlated with unresectable disease. Previous evidence has shown that MMP-7 in serum, plasma, and pancreatic juice is useful as a diagnostic marker, along with associated disease indicators.

[0006] Pancreatic cancer is a lethal and aggressive malignancy that is estimated to be the second leading cause of cancer-related deaths in the United States by 2030. Although its incidence is increasing dramatically, treatment options for pancreatic cancer remain scarce and novel treatment strategies are urgently needed.

[0007] The background art includes WO 2012 / 056455, which discloses antibodies directed against MMP-7. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2012 / 056455 Summary of the Invention

[0009] According to some embodiments of the present invention, there is provided an antibody comprising an antigen recognition region that binds to the catalytic site of MMP-7, the antigen recognition region having the amino acid sequences of complementarity determining regions (CDRs) shown in SEQ ID NOs: 3 (CDR1), 4 (CDR2) and 5 (CDR3), arranged in N to C order on the light chain of the antibody, and SEQ ID NOs: 6 (CDR1), 7 (CDR2) and 8 (CDR3), arranged in N to C order on the heavy chain of the antibody.

[0010] According to some embodiments of the present invention, there is provided a method for treating a disease associated with imbalanced or abnormal activity of MMP-7 in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an antibody described in claim 1, thereby treating the disease associated with imbalanced or abnormal activity of MMP-7 in the subject.

[0011] According to an aspect of some embodiments of the present invention, there is provided a method for diagnosing a disease associated with imbalanced or abnormal activity of MMP-7 in a subject, the method comprising contacting a sample from the subject with an antibody described herein to analyze expression of MMP-7, wherein upregulation of MMP-7 expression is indicative of a disease associated with imbalanced or abnormal activity of MMP-7.

[0012] According to an aspect of some embodiments of the present invention, there is provided a method of treating cancer in a subject in need thereof, comprising: (a) analyzing a sample from a subject for the amount of MMP-7; and (b) upon determining that the amount of MMP-7 is above a predetermined level, administering a therapeutically effective amount of an antibody described herein to the subject, thereby treating the cancer.

[0013] According to an aspect of some embodiments of the present invention, there is provided a method of treating cancer in a subject in need thereof, comprising: (a) analyzing in a subject's sample for the amount of MMP-7 using an antibody described herein; and (b) upon determining that the amount of MMP-7 is above a predetermined level, administering to the subject a therapeutically effective amount of an agent that downregulates the amount of MMP-7, thereby treating the cancer.

[0014] According to an aspect of some embodiments of the present invention, there is provided a method of treating pancreatic cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody comprising an antigen recognition region that binds to the catalytic site of MMP-7, wherein the antibody inhibits the activity of MMP-7, and the Ki of the antibody against MMP-7 is at least 5-fold lower than the Ki of the antibody against MMP2 or MMP9, thereby treating the pancreatic cancer.

[0015] According to an aspect of some embodiments of the invention there is provided a pharmaceutical composition comprising an antibody as described herein.

[0016] According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide encoding at least one CDR amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7 and 8.

[0017] According to an aspect of some embodiments of the invention there is provided an article of manufacture comprising an antibody and a chemotherapeutic agent as described herein.

[0018] According to some embodiments of the invention, the antibody has the VL amino acid sequence shown in SEQ ID NO:1.

[0019] According to some embodiments of the invention, the antibody has the VH amino acid sequence shown in SEQ ID NO:2.

[0020] According to some embodiments of the invention, the antibody is attached to a detectable or therapeutic moiety.

[0021] According to some embodiments of the invention, the disease is cancer.

[0022] According to some embodiments of the invention, the cancer is pancreatic cancer.

[0023] According to some embodiments of the invention, the analyzing is performed using an antibody.

[0024] According to some embodiments of the invention, the antibody is an antibody described herein.

[0025] According to some embodiments of the invention, the polynucleotide encodes the CDR amino acid sequences shown in SEQ ID NOs:3-5.

[0026] According to some embodiments of the invention, the polynucleotide encodes the CDR amino acid sequences shown in SEQ ID NOs:6-8.

[0027] According to some embodiments of the invention, the polynucleotide encodes the CDR amino acid sequences shown in SEQ ID NOs:3-8.

[0028] According to some embodiments of the invention, the method further comprises administering to the subject a chemotherapeutic agent.

[0029] According to some embodiments of the invention, the chemotherapeutic agent is a nucleoside analog.

[0030] According to some embodiments of the invention, the nucleotide analog comprises gemcitabine.

[0031] According to some embodiments of the invention, the chemotherapeutic agent is oxaliplatin.

[0032] According to some embodiments of the invention, the dose of the chemotherapeutic agent is less than the standard dose when used as a single agent.

[0033] According to some embodiments of the invention, the chemotherapeutic agent is a nucleoside analog.

[0034] According to some embodiments of the invention, the nucleotide analog comprises gemcitabine.

[0035] According to some embodiments of the invention, the chemotherapeutic agent is oxaliplatin.

[0036] According to some embodiments of the invention, the antibody and the chemotherapeutic agent are formulated in a single composition.

[0037] According to some embodiments of the invention, the article of manufacture is for use in the treatment of cancer.

[0038] According to some embodiments of the invention, the cancer is pancreatic cancer.

[0039] Unless otherwise defined, all technical and / or 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 embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. A simple explanation of the drawings from multiple perspectives

[0040] Certain embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. Referring now specifically to the drawings in detail, it is emphasized that the particulars shown are by way of example for the purpose of an illustrative discussion of embodiments of the present invention. In this regard, from the description considered in conjunction with the drawings, it will become apparent to those skilled in the art how embodiments of the present invention may be practiced. [Brief description of the drawings]

[0041] [Figure 1A] Immunization with synthetic Zn Tripod or human MMP-7 catalytic enzyme alone did not yield antibody candidates that specifically bound to the activated enzyme. Immune responses were examined in bleeds from mice immunized with Zn Tripod alone, with sera using ELISA against the antigens Zn-Tripod-KLH (Zn Tripod), MMP-7 activated form, MMP-7 zymogen / pro form, and BSA control. The graph shows binding with various antigen dilutions. ELISA did not show the presence of antibodies that bound to MMP-7 activated conformation or zymogen form. Reactivity against Zn Tripod was observed. [Figure 1B] Immunization with synthetic Zn Tripod or human MMP-7 catalytic enzyme alone did not yield antibody candidates that specifically bound to the activated enzyme. Weak responses were also confirmed by ELISA using bleeds from mice immunized with recombinant human MMP-7 active form alone. Weak responses were characterized by binding to both the MMP-7 proform and the MMP-7 active form. Notably, no binding was observed in Zn Tripod-coated wells. [Figure 2A] Alternating immunization yields anti-MMP-7 antibodies with high binding affinity and selectivity for the active form of the enzyme. Female BALB / c mice were immunized at 3-week intervals with synthetic Zn tripodal emulsified with complete Freund's adjuvant or with active recombinant human MMP-7. Progressive immune responses were observed as a function of repeated injections using ELISA. At 18 weeks, B cells from candidates with high binding affinity were selected for hybridoma generation. [Figure 2B] Cross-immunization yields anti-MMP-7 antibodies with high binding affinity and selectivity for the active form of the enzyme. Serum immune responses of selected pre-fusion mice, when tested using ELISA, showed dual specificity for both Zn tripod and MMP-7, and negligible binding to MMP-14 at various dilutions. [Figure 2C]Cross-immunization yields anti-MMP-7 antibodies with high binding affinity and selectivity for the active form of the enzyme. Purified hybridoma monoclonal antibody GSM-192 binding curve with MMP-7 active enzyme (Kd=43.1±1.43 nM). The antibody did not bind effectively to a panel of other MMPs, demonstrating its high specificity for MMP-7. [Figure 2D] Cross-immunization yields anti-MMP-7 antibodies with high binding affinity and selectivity for the active form of the enzyme. The effect of GSM-192 Fab on the enzymatic activity of human MMP-7 was examined in vitro using a short fluorogenic peptide. GSM-192 Fab inhibited MMP-7 activity with Ki=131.98±10.23nM. Related MMPs such as MMP-14, MMP-9 and MMP-12 showed negligible or no effect in the presence of GSM-192, and continued activity. [Figure 2E] Cross-immunization yields anti-MMP-7 antibodies with high binding affinity and selectivity for the active form of the enzyme. We show the structure of a synthetic Zn tripod that mimics the highly conserved MMP active site. In this structure, Zn coordination with the three histidines (highlighted in red) is conserved, as in all MMP active sites. [Figure 2F] Cross-immunization yields anti-MMP-7 antibodies with high binding affinity and selectivity for the active form of the enzyme. Dot blots showed selective binding of anti-MMP-7 Abs to the active form of the enzyme with high affinity at various concentrations. GSM-192 did not bind to the zymogen form. [Figure 3A] GSM-192 structure and docking shows the unique affinity of the antibody for a binding site close to the conserved active site in the activated form of the enzyme. Crystal structure (2.3 Å) of the Fab fragment of GSM-192 represented as a ribbon diagram. The heavy chain (light green) and light chain (dark green) are indicated (PDB: 6FBJ). [Figure 3B]The GSM-192 structure and docking show the intrinsic affinity of the antibody for a binding site close to the conserved active site within the activated form of the enzyme. A docking model of GSM-192 to activated human MMP-7 revealed direct binding to the rim (exosite) of the enzyme active site. The surface of MMP-7 is shown in beige and semi-transparent. The antibody is outlined in dark green showing three side chains: L100H, R101H and Y33L located near the Leu anchoring spot (purple). The latter interacts with MMP-7 in the active site anchoring the exosite. Acetohydroxamic acid (AHA) is shown as a ball-and-stick model with carbon atoms in yellow, oxygens in red and nitrogens in blue. [Figure 3C] The GSM-192 structure and docking show the unique affinity of the antibody for a binding site close to the conserved active site in the activated form of the enzyme. The docked structure of MMP-7 (beige), complexed with GSM-192 (green) shows excellent surface complementarity and a small "tunnel" through which the small inhibitor AHA can insert into the interface and bind the Zn2+ ion. [Figure 3D] The GSM-192 structure and docking shows the unique affinity of the antibody for a binding site close to the conserved active site within the activated form of the enzyme. Ball and stick model showing key contacts near the active site zinc (grey). [Figure 4A] Both gene silencing and inhibition of MMP-7 by the inhibitory antibody GSM-192 result in tumor cell death in vitro. MMP-7 lentiviral silencing resulted in an increase in the sub-G1 peak in FACS-based cell cycle analysis, indicative of a jump in AsPC-1 cell death. Two vectors, lentivirus 1 (LV-1) and lentivirus 2 (LV-2), showed a similar increase in the sub-G1 peak. [Figure 4B]Both gene silencing and inhibition of MMP-7 by the inhibitory antibody GSM-192 result in tumor cell death in vitro. MMP-7 inhibition results in tumor cell death via apoptosis in AsPC-1 cells. FACS analysis showed increased Annexin V-associated apoptosis (Q1+Q2) after treatment with increasing doses of GSM-192 Fab. The FACS data output here shows early apoptotic cells as Q1 and late apoptotic cells as Q2. The increase in the percentage of cells undergoing apoptosis was plotted using the mean ± sem (grey bars). LOXL-2 Fab, generated using the same pipeline, was used as a treatment control. [Figure 4C] Both gene silencing and inhibition of MMP-7 by the inhibitory antibody GSM-192 results in tumor cell death in vitro. Western blot showing FasL expression in cell lysates with and without GSM-192 Fab treatment. An overall increase in FasL levels in anti-MMP-7 Fab-treated cells was observed. Anti-LOXL-2 Fab at 1.5 μM concentration and the pro-apoptotic drug staurosporine (STS) were used as treatment controls. Alpha-tubulin was used as a sample preparation control. Data in the graph represent alpha-tubulin normalized mean ± sem. [Figure 5A] GSM-192 treatment delays or reduces cell migration in scratch and transwell assays, respectively. Treatment with GSM-192 delayed cell migration in a standard scratch assay. Seven hours after treatment, control antibody GST (non-specific pooled IgG)-treated wells close the scratch, whereas GSM-192-treated wells do not. Data represent the mean ± sem and significance was assessed by two-tailed t-test. Scale bar, 20 μm. **P ≤ 0.01. [Figure 5B]GSM-192 treatment delays or reduces cell migration in scratch and transwell assays, respectively. GSM-192 significantly reduces cells migrating across the transwell membrane after 15 h of treatment. Data represent the mean ± sem and significance was assessed by two-tailed t-test. Scale bar, 20 μm. ***P ≤ 0.001. [Figure 6A] MMP-7 expression and cell survival following GSM-192 treatment. Western blot analysis of cell culture media showing MMP-7 expression in various pancreatic cancer cell lines. [Figure 6B] MMP-7 expression and cell viability after GSM-192 treatment. Using the MTT cell viability assay, dose-response curve fitting analysis was generated to obtain IC50 values ​​for various concentrations of GSM-192 treatment on AsPC-1 and CFPAC-1 pancreatic ductal adenocarcinoma cells. These IC50 concentrations were 2.33 μM for AsPC-1 and 4.34 μM for CFPAC-1, indicating the selection of Ab concentrations for further experiments. [Figure 7A] Immunohistochemical staining of human PDAC tissue with GSM-192. Commercially available anti-MMP-7 Ab staining shows increased expression of MMP-7 protein in the core compared to the periphery of human PDAC tumors. [Figure 7B] Immunohistochemical staining of human PDAC tissue with GSM-192. Comparison of GSM-192 and anti-MMP-7 commercial antibody staining shows differential localization. GSM-192 stains primarily the active enzyme on the cell surface, while the commercial antibody stains both the zymogen and catalytic enzyme in the nuclear and cytoplasmic compartments. Scale=20 μm. [Figure 8] Anti-MMP-7 mAb is not captured by circulating MMPs in RIP1-Tag2 mouse serum. Compared to pure catalytic MMP-7, the antibody does not bind to a wide range of total serum proteins from mice (13.5 weeks) with advanced insulinoma. This indicates the lack of catalytic forms of MMP-7 in blood and the high specificity of the anti-MMP-7 antibody for the catalytic enzyme. Thus, the antibody is less susceptible to removal as a result of removal or binding to the circulating enzyme. [Figure 9A]Early stage treatment overall reduces the number of islets undergoing the angiogenic switch in the RIP1-Tag2 insulinoma (PanNET) model: the number of vascularized islets in GSM-192-treated mice was half the total number seen in control mice. [Figure 9B] In vivo, anti-MMP-7 mAb did not significantly change the numbers of Ki-67 and caspase-3 positive cells, but reduced the area covered by CD34+ cells in vascularized islets. [Figure 9C] Late treatment shows a sustained effect on tumor volume in the RIP1-Tag2 mouse model. Total tumor volume is the sum of all tumor loci, as indicated. Total tumor volume is significantly reduced in treated vs. control mice. **P≦0.01, *P≦0.05. [Figure 10A] Tube formation by HUVEC cells was disrupted when treated with 1 μM GSM-192: the total number of closed loops and branch points was significantly reduced in the treated group compared to the GST control antibody treated group. [Figure 10B] Aortic ring sprout assay showed a significant reduction in neovascular sprouts at 1 μM GSM-192. Vascular sprouts were significantly reduced in the GSM-192-treated group after 7 days of ex vivo incubation. These results demonstrate a direct effect on angiogenesis following treatment with GSM-192. [Figure 11A] MTT cell death assay absorbance data plotted as a function of the log of gemcitabine (GEM) concentration and fitted to a variable slope sigmoidal regression model show a significant decrease in IC50 for the GEM+GSM-192 group compared to the GEM+PBS group. [Figure 11B] The mean IC50 of three such independent experimental curve fitting analyses were subsequently plotted to indicate significant differences between groups. Data represent the mean ± sem and significance was assessed by two-tailed t-test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] In some embodiments, the present invention relates to an antibody that binds to the catalytic site of MMP-7. The antibody can be used for the treatment of cancer, more particularly, but not limited to, pancreatic cancer.

[0043] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0044] Matrix metalloproteinases are involved in many biological processes ranging from cell proliferation, differentiation and remodeling of the extracellular matrix (ECM) to angiogenesis and cell migration. These processes require a delicate balance between the functions of matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs). Loss of this balance is a hallmark of many pathological conditions, including metastatic tumors, neurodegenerative diseases and osteoarthritis.

[0045] A number of MMP inhibitors are known in the art, including small peptide inhibitors such as hydroxomates, non-microbial tetracyclines and monoclonal antibodies.

[0046] The present inventors have previously discovered that antibodies that recognize both electronic and structural determinants of the catalytic site of metalloenzymes can be used as potent inhibitors thereof. The use of haptens that mimic the metal-binding catalytic site of metalloenzymes as immunogens has allowed the generation of highly efficient therapeutic antibodies that have been shown to be capable of treating clinical conditions characterized by elevated metalloprotein activity (see WO 2004 / 087042 and WO 2008 / 102359).

[0047] The inventors now understand that an additional screening step is essential to discover a highly specific MMP-7 antibody. Only antibodies that can bind to the active form of MMP-7 and cannot bind to the zymogen must be selected for further development (i.e., selected for fusion and developed to generate monoclonal antibodies). The inventors carried out this step by performing a dot blot analysis using nitrocellulose membranes coated with various concentrations of active MMP-7 and its zymogen, respectively. By performing this additional assay, the inventors discovered a highly specific monoclonal antibody against MMP-7, referred to herein as GSM-192, that binds to the catalytic domain of the enzyme but not to the zymogen form of the enzyme.

[0048] Docking of the Fv domain of GSM-192 into the available MMP-7 structures suggested that GSM-192 is effective at binding the catalytic groove region (Figures 3A-D), providing steric hindrance that prevents the entry of substrate molecules.

[0049] While analyzing the specificity of this antibody, we found that GSM-192 binds to MMP-7 with impressive selectivity and affinity when compared to related MMPs (Figures 2A-F). Inhibition of MMP-7 with GSM-192 Fab induced cell death in pancreatic cancer cells in a concentration-dependent manner. Treatment of PDAC human cells with GSM-192 resulted in a concentration-dependent stabilization of the major extrinsic death pathway ligand-Fas ligand. Thus, inhibition of active MMP-7 caused cell death in vitro, accompanied by an increase in cell surface Fas ligand in the treated group (Figures 4A-C). Without being bound by theory, it can be concluded that inhibition of MMP-7 by GSM-192 may be due to interference with one of the downstream functions of MMP-7, namely protecting cancer cells from FasL-induced apoptosis. Furthermore, GSM-192 treatment of PDAC cells (CFPAC-1) impaired their ability to migrate through transwell pores and close artificial wounds in a scratch assay (Figure 5A-B). In conclusion, specific inhibition of MMP-7 using a high affinity novel antibody further supports its role as a key target in pancreatic cancer progression.

[0050] The inventors propose that antibodies such as GSM-192 may have significant and direct impact on therapeutic, diagnostic and research applications. Specifically, for diagnostic applications, antibodies that recognize the active conformation of MMPs would be valuable since diseased tissues show marked differential expression of the active forms across their zymogens.

[0051] Thus, according to a first aspect of the present invention, there is provided an antibody comprising an antigen recognition region that binds to the catalytic site of MMP-7, and having the amino acid sequences of complementarity determining regions (CDRs) shown in SEQ ID NOs: 3 (CDR1), 4 (CDR2) and 5 (CDR3), arranged in N to C order on the antibody light chain, and SEQ ID NOs: 7 (CDR1), 8 (CDR2) and 9 (CDR3), arranged in N to C order on the antibody heavy chain.

[0052] The antibody of this aspect of the invention may comprise the VH amino acid sequence shown in SEQ ID NO: 2 and the VL amino acid sequence shown in SEQ ID NO: 1. The CDR sequences of the antibody are provided in SEQ ID NOs: 3-8.

[0053] According to particular embodiments, the antibody comprises an amino acid sequence that is at least 90% homologous, at least 91% homologous, at least 92% homologous, at least 93% homologous, at least 94% homologous, at least 95% homologous, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous or even 100% homologous to the sequences shown in SEQ ID NOs: 1 and 2 (the CDR sequences of the antibodies are always 100% homologous to those provided herein above).

[0054] As used herein, the term "antibody" refers to an intact antibody molecule, and the phrase "antibody fragment" refers to functional fragments thereof, such as Fab, F(ab')2, and Fv, capable of binding to macrophages. These functional antibody fragments are defined as follows: (i) Fab, which is the fragment containing a monovalent antigen-binding fragment of an antibody molecule that can be produced by digestion of whole antibodies with the enzyme papain to produce an intact light chain and a portion of one heavy chain; (ii) Fab', which is the fragment of an antibody molecule that can be obtained by treating whole antibodies with the enzyme pepsin, followed by reduction, to produce an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (iii) Fab', which is the fragment of an antibody molecule that can be obtained by treating whole antibodies with the enzyme pepsin without subsequent reduction to produce an intact light chain and a portion of the heavy chain; (iv) Fv, defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains; (v) single-chain antibody ("SCA"), which is a genetically engineered molecule containing the variable region of a light chain and the variable region of a heavy chain linked by a suitable polypeptide linker as a genetically fused single-chain molecule; and (vi) a peptide encoding a single complementarity determining region (CDR).

[0055] As used herein, the terms "complementarity determining region" or "CDR" are used interchangeably to refer to the antigen-binding regions found in the variable regions of heavy and light chain polypeptides. Generally, an antibody comprises three CDRs in each of the VH (CDRHI or HI; CDRH2 or H2; and CDRH3 or H3) and three CDRs in each of the VL (CDRLI or LI; CDRL2 or L2; and CDRL3 or L3).

[0056] The identity of amino acid residues in a particular antibody that make up a variable region or CDR can be determined using methods well known in the art, including sequence variability as defined by Kabat et al. (see, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC), the location of structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989), a compromise of Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and the world wide web site www(dot)bioinf-org(dot)uk / abs), contact definitions (MacCallum et al. al., J. Mol. Biol. 262:732-745, 1996) and "stereostructure definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008).

[0057] As used herein, "CDR" and "variable region" may refer to variable regions and CDRs defined by any approach known in the art, including a combination of approaches.

[0058] Methods for generating antibodies (i.e., monoclonal and polyclonal) are well known in the art. Antibodies can be produced by any one of several methods known in the art, including inducing in vivo production of antibody molecules, screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed [Orlandi DR et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837, Winter G. et al. (1991) Nature 349: 293-299], or using continuous cell lines in culture to produce monoclonal antibody molecules. These include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and Epstein-Barr virus (EBV)-hybridoma technology [Kohler G., et al. (1975) Nature 256:495-497, Kozbor D., et al. (1985) J. Immunol. Methods 81:31-42, Cote RJ et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030, Cole SP et al. (1984) Mol. Cell. Biol. 62:109-120].

[0059] If the compounds of the present invention are too small to elicit a strong immunogenic response, such antigens (haptens) can be coupled to antigenically neutral carriers such as keyhole limpet hemocyanin (KLH) or serum albumin (e.g., bovine serum albumin (BSA)) carriers (see U.S. Pat. Nos. 5,189,178 and 5,239,078). Coupling to the carrier can be accomplished using methods well known in the art, for example, direct coupling to amino groups can be achieved, optionally followed by reduction of the imino bond formed. Alternatively, the carrier can be attached using condensing agents such as dicyclohexylcarbodiimide or other carbodiimide dehydrating agents. Coupling can also be achieved using linker compounds, both homobifunctional and heterobifunctional linkers available from Pierce Chemical Company, Rockford, Ill. The resulting immunogenic complex can then be injected into a suitable mammalian subject, such as a mouse, rabbit, etc. A suitable protocol involves repeated injections of the immunogen in the presence of an adjuvant according to a schedule that enhances production of antibodies in the serum. The titer of immune serum can be readily measured using immunoassay procedures well known in the art.

[0060] The antisera obtained can be used directly or monoclonal antibodies can be obtained as described above.

[0061] Antibody fragments can be obtained using methods well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference). For example, antibody fragments according to the invention can be prepared by proteolytic hydrolysis of the antibody or by expression of DNA encoding the fragment in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems).

[0062] Alternatively, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds, to produce a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, in Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which are incorporated herein by reference in their entireties. See also Porter, RR, Biochem. J., 73:119-126, 1959. Other methods of cleaving antibodies can also be used, such as separation of heavy chains to form monovalent light and heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical or genetic techniques, so long as the fragments bind to the antigen recognized by the intact antibody.

[0063] The Fv fragment is V H Chain and V L The association may be non-covalent, as described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variable chains may be linked by intermolecular disulfide bonds or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragment comprises V variable chains connected by peptide linkers. H and V L These single-chain antigen-binding proteins (sFv) contain V H and V LThe sFv are prepared by constructing a structural gene containing a DNA sequence encoding the V domain. The structural gene is inserted into an expression vector and subsequently introduced into a host cell, such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFv are described, for example, by Whitlow and Filpula, Methods, 2:97-105, 1991; Bird et al., Science 242:423-426, 1988; Pack et al., Bio / Technology 11:1271-77, 1993; and Ladner et al., U.S. Pat. No. 4,946,778.

[0064] CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDRs of the antibody of interest. Such genes are prepared, for example, by synthesizing the variable regions from the RNA of antibody-producing cells using the polymerase chain reaction. See, for example, Larrick and Fry, Methods, 2:106-10, 1991.

[0065] It will be understood that for human therapy or diagnosis, humanized antibodies are preferably used. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include a human immunoglobulin (recipient antibody) in which residues that form the complementarity determining regions (CDRs) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by the corresponding non-human residues. Humanized antibodies may also include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions, which are of the human immunoglobulin consensus sequence. The humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

[0066] Thus, according to another aspect of the present invention, there is provided an isolated polynucleotide encoding at least one CDR amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7 and 8. The polynucleotide may encode the light chain CDRs (e.g. SEQ ID NOs: 3-5) and / or the heavy chain CDRs (e.g. SEQ ID NOs: 6-8). Optionally, the polynucleotide may encode each of the antibody CDRs. The polynucleotide may further encode a sequence encoding the antibody scaffold (e.g. IgG1, 2, 3 or 4). The scaffold may comprise a human sequence.

[0067] The polynucleotides of some embodiments of the invention may be used, and preferably cloned, in the nucleic acid constructs of some embodiments of the invention to genetically direct the production of antibodies or antibody chains in transformed host cells of some embodiments of the invention.

[0068] Polynucleotides of some embodiments of the present invention can be introduced into cells by any one of a variety of methods known in the art. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, MI (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor MI (1995); Vectors, A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988), and Gilboa et al., Biotechniques 4(6):504-512 (1986), and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors.

[0069] Cells that express the polynucleotides described herein are also contemplated by the present invention.

[0070] Methods for humanizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often called import residues and are usually taken from an import variable domain. Humanization can be carried out essentially according to the method of Winter et al. [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)] by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. Thus, such humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially less than an intact human variable domain is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0071] Human antibodies can also be produced using a variety of techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Mol. Biol., 222:581 (1991)). al., J. Immunol., 147(1):86-95 (1991)]. Similarly, humans can be generated by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon antigenic challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio / Technology 10,779-783 (1992); Lonberg et al., Nature 368:101-103 (1993); 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

[0072] Once the antibodies are obtained, they can be tested for metalloenzyme inhibitory activity and affinity. Suitable assay conditions for metalloprotein inhibitory activity are described in Knight et al., FEBS Letters 296(3):263-266(1992), Cawston et al., Anal.Biochem, 99:340-345(1979), Cawston et al., Methods in Enzymology 80:771 et seq.(1981); Cawston et al., Biochem.J., 195:159-165(1981), Weingarten et al., Biochem.Biophys.Res.Comm., 139:1184-1187(1984), and U.S. Patent Nos. 4,743,587 and 5,240,958.

[0073] The inhibitory antibodies of this aspect of the invention typically have very high binding affinity and specificity for MMP-7, d is 40-50 nM, more typically about 40-45 nM. The antibodies of this aspect of the present invention typically exhibit a tight binding inhibition pattern against MMP-7 (e.g., Ki=1-500 nM), and more preferably have a Ki of 100-150 nM, which is at least 2-fold, at least 5-fold, or even at least 10-fold lower than the Ki of antibodies against other MMPs, such as MMP2, MMP9, or MMP14.

[0074] The antibody may bind to the metal coordinating ion of the catalytic site and not to the metal ion itself, or the antibody may bind to both the metal ion and its coordinating ion present in the active site pocket. It will be appreciated that the antibody may bind to a site on the surface of MMP-7 in addition to binding to the catalytic site.

[0075] The present invention also provides any (poly)peptide sequence comprising at least one, two, three, four, five or six of the CDR sequences of these antibodies and their homologs and fragments, so long as their metalloenzyme inhibitory activity (specific inhibition of the catalytic activity of metalloproteins) is retained.

[0076] According to some embodiments of the invention, antibodies may be conjugated to a functional moiety, such as a detectable or therapeutic moiety (also called an "immunoconjugate"). Immunoconjugates may be isolated molecules, such as soluble and / or synthetic molecules.

[0077] Various types of detectable or reporter moieties can be conjugated to the antibodies of the invention. These include, but are not limited to, radioisotopes (

[0125] Examples of suitable ligands include ligands that are capable of binding to a ligand of interest, such as iodine, phosphorescent chemicals, chemiluminescent chemicals, fluorescent chemicals (fluorophores), enzymes, fluorescent polypeptides, affinity tags, and molecules detectable by positron emission tomography (PET) or magnetic resonance imaging (MRI) (imaging agents).

[0078] Examples of suitable fluorophores include, but are not limited to, phycoerythrin (PE), fluorescein isothiocyanate (FITC), Cy-chrome, rhodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas Red, PE-Cy5, and the like. For further guidance regarding the selection of fluorophores, methods for linking fluorophores to various types of molecules can be found in Richard P. Haugland, "Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992-1994", 5th ed., Molecular Probes, Inc. (1994); U.S. Pat. No. 6,037,137 to Oncoimmunin Inc.; Hermanson, "Bioconjugate Techniques", Academic Press New York, NY (1995); Kay M. et al., 1995. Biochemistry 34:293; Stubbs et al., 1996. Biochemistry 35:937; Gakamsky D. et al., "Evaluating Receptor Stoichiometry by Fluorescence Resonance Energy Transfer,'' in ''Receptors: A Practical Approach,'' 2nd ed., Stanford C. and Horton R. (eds.), Oxford University Press, UK. (2001); U.S. Patent No. 6,350,466 to Targesome, Inc. Fluorescence detection methods that can be used to detect antibodies when conjugated to a fluorescently detectable moiety include, for example, fluorescence activated flow cytometry (FACS), immunofluorescence confocal microscopy, fluorescence in situ hybridization (FISH), and fluorescence resonance energy transfer (FRET).

[0079] Many types of enzymes can be attached to the antibodies of the invention [e.g., horseradish peroxidase (HPR), β-galactosidase, and alkaline phosphatase (AP)] and detection of the enzyme-linked antibody can be carried out using ELISA (e.g., in solution), enzyme-linked immunohistochemical assay (e.g., in fixed tissues), enzyme-linked chemiluminescence assay (e.g., in electrophoretically separated protein mixtures) or other methods known in the art [e.g., Khatkhatay M I. and Desai M, 1999. J Immunoassay 20:151-83; Wisdom G B., 1994. Methods Mol Biol. 32:433-40; Ishikawa E. et al., 1983. J Immunoassay 4:209-327; Oellerich M, 1980. J Clin Chem Clin Biochem. 18:197-208; Schurs A H. and van Weemen (See BK, 1980. J Immunoassay 1:229-49).

[0080] The affinity tag (or member of a binding pair) can be an antigen recognizable by a corresponding antibody (e.g., digoxigenin (DIG) recognized by an anti-DIG antibody) or a molecule with high affinity for the tag (e.g., streptavidin and biotin). The antibody or molecule that binds to the affinity tag can be fluorescently labeled as described above or conjugated to an enzyme.

[0081] Streptavidin or biotin molecules can be attached to the antibodies of the present invention using a variety of methods widely practiced in the art. For example, biotin molecules can be attached to the antibodies of the present invention via a recognition sequence for a biotin protein ligase (e.g., BirA), as described in the Examples section below and in Denkberg, G. et al., 2000. Eur. J. Immunol. 30:3522-3532. Alternatively, streptavidin molecules may be attached to antibody fragments such as single chain Fv, essentially as described in Cloutier SM.et al., 2000.Molecular Immunology 37:1067-1077; Dubel S.et al., 1995.J Immunol Methods 178:201; Huston JS.et al., 1991.Methods in Enzymology 203:46; Kipriyanov SM.et al., 1995.Hum Antibodies Hybridomas 6:93; Kipriyanov SM.et al., 1996.Protein Engineering 9:203; Pearce LA.et al., 1997.Biochem Molec Biol Intl 42:1179-1188).

[0082] Functional moieties such as fluorophores conjugated to streptavidin are commercially available from essentially all major suppliers of immunofluorescence flow cytometry reagents (eg, Pharmingen or Becton-Dickinson).

[0083] According to some embodiments of the invention, biotin-conjugated antibodies are bound to streptavidin molecules to form multivalent compositions (eg, dimeric or tetrameric forms of the antibodies).

[0084] Table 1 provides non-limiting examples of identifiable moieties that can be conjugated to the antibodies of the invention.

[0085] [Table 1]

[0086] As already mentioned, the antibody may be conjugated to a therapeutic moiety, which may be, for example, a cytotoxic moiety, a toxic moiety, a cytokine moiety, and a second antibody moiety containing a different specificity to the antibody of the invention.

[0087] Non-limiting examples of therapeutic moieties that can be conjugated to the antibodies of the invention are provided in Table 2 below.

[0088] [Table 2]

[0089] Functional moieties (detectable or therapeutic moieties of the invention) can be attached or conjugated to antibodies of the invention in a variety of ways, depending on the context, use and purpose.

[0090] When the functional moiety is a polypeptide, the immunoconjugate may be produced by recombinant means. For example, a nucleic acid sequence encoding a toxin (e.g., PE38KDEL) or a fluorescent protein (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP) or yellow fluorescent protein (YFP)) may be linked in-frame to a nucleic acid sequence encoding an antibody of the invention and expressed in a host cell to produce a recombinant conjugated antibody. Alternatively, the functional moiety may be chemically synthesized by the stepwise addition of one or more amino acid residues in a defined order, such as by solid-phase peptide synthesis techniques.

[0091] The functional moiety may also be attached to the antibody of the invention using any suitable chemical bond, directly or indirectly, for example, via a peptide bond (if the functional moiety is a polypeptide) or via covalent attachment to an intervening linker element, such as a linker peptide or other chemical moiety, such as an organic polymer, using standard chemical synthesis techniques widely practiced in the art [see, for example, hypertexttransferprotocol: / / worldwideweb(dot)chemistry(dot)org / portal / Chemistry]. Chimeric peptides may be linked via a bond at the carboxy (C) or amino (N) terminus of the peptide, or via a bond to an internal chemical group, such as a linear, branched or cyclic side chain, an internal carbon or nitrogen atom. A description of fluorescent labeling of antibodies is provided in detail in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110.

[0092] Exemplary methods for conjugating peptide moieties (therapeutic or detectable moieties) to the antibodies of the invention are described herein below.

[0093] SPDP conjugation-Non-limiting examples of methods for SPDP conjugation are described in Cumber et al. (1985, Methods of Enzymology 112:207-224). Briefly, a peptide such as a detectable or therapeutic moiety (e.g., 1.7 mg / ml) is mixed with a 10-fold excess of SPDP (50 mM in ethanol), and an antibody is mixed with a 25-fold excess of SPDP in 20 mM sodium phosphate, 0.10 M NaCl pH 7.2, and each reaction is incubated at room temperature for about 3 hours. The reaction is then dialyzed against PBS. The peptide is reduced, for example, with 50 mM DTT for 1 hour at room temperature. The reduced peptide is desalted by equilibration on a G-25 column (maximum 5% sample / column volume) with 50 mM KH2PO4 pH 6.5. The reduced peptide is combined with the SPDP antibody at a 1:10 antibody:peptide molar ratio and incubated overnight at 4° C. to form the peptide-antibody conjugate.

[0094] Glutaraldehyde conjugation - A non-limiting example of a method for glutaraldehyde conjugation is described in GT Hermanson (1996, 'Antibody Modification and Conjugation, in Bioconjugate Techniques, Academic Press, San Diego). Briefly, antibody and peptide (1.1 mg / ml) are mixed in 10-fold excess with 0.05% glutaraldehyde in 0.1 M phosphate, 0.15 M NaCl pH 6.8 and allowed to react for 2 hours at room temperature. Excess sites can be blocked by adding 0.01 M lysine. After reaction, excess glutaraldehyde is removed using a G-25 column equilibrated with PBS (10% v / v sample / column volume).

[0095] Carbodiimide conjugation-Conjugation of peptides and antibodies can be accomplished using dehydrating agents such as carbodiimides, for example in the presence of 4-dimethylaminopyridine. Carbodiimide conjugation can be used to form covalent bonds between carboxyl groups of peptides and hydroxyl groups of antibodies (resulting in the formation of ester bonds), or amino groups of antibodies (resulting in the formation of amide bonds), or sulfhydryl groups of antibodies (resulting in the formation of thioester bonds). Similarly, carbodiimide coupling can be used to form similar covalent bonds between carbon groups of antibodies and hydroxyl, amino, or sulfhydryl groups of peptides [see J. March, Advanced Organic Chemistry: Reaction's, Mechanism, and Structure, pp. 349-50 & 372-74 (3d ed.), 1985]. For example, peptides can be covalently conjugated to antibodies using carbodiimides, such as dicyclohexylcarbodiimide [B. Neises et al. (1978), Angew Chem., Int. Ed. Engl. 17:522; A. Hassner et al. (1978, Tetrahedron Lett. 4475); EP Boden et al. (1986, J. Org. Chem. 50:2394) and LJ Mathias (1979, Synthesis 561)].

[0096] As mentioned hereinabove, one particular use of antibodies against MMP-7 is the prevention or treatment of diseases associated with imbalanced or abnormal activity of MMP-7.

[0097] Examples of such diseases include, but are not limited to, arthritic diseases such as osteoarthritis (OA), rheumatoid arthritis (RA), septic arthritis, soft tissue rheumatism, polychondritis and tendonitis, metastatic tumors, periodontal disease; corneal ulcers induced by alkali or other burns, radiation, vitamin E or retinoid deficiency, etc.; glomerular diseases such as proteinuria, dytrophic epidermolysis bullosa, etc.; bone resorption diseases such as osteoporosis, Paget's disease, hyperparathyroidism and cholesteatoma; birth control by preventing ovulation or implantation; angiogenesis associated with tumor growth or associated with diabetic retinopathy and macular degeneration; coronary thrombosis associated with atherosclerotic plaque rupture; emphysema, wound healing and HIV infection.

[0098] According to one embodiment, the disease is cancer. Exemplary cancers include pancreatic cancer, ovarian cancer, renal cell carcinoma, colon cancer, breast cancer, gastric cancer, rectal cancer and prostate cancer.

[0099] The cancer being treated may be at an early or late stage, hi one embodiment, the cancer has metastasized.

[0100] Additional contemplated cancers include, but are not limited to, hereditary adrenocortical carcinoma, bladder cancer; breast cancer; breast cancer, ductal; breast cancer, invasive intraductal; breast cancer, sporadic; breast cancer, susceptible; breast cancer, type 4; breast cancer, type 4; breast cancer-1; breast cancer-3; breast ovarian cancer; Burkitt's lymphoma; cervical cancer; colorectal adenoma; colorectal cancer, hereditary; colorectal cancer, hereditary nonpolyposis, type 1; colorectal cancer, hereditary nonpolyposis, type 2; colorectal cancer, hereditary nonpolyposis, type 3; colorectal cancer, hereditary nonpolyposis Polyposis, type 6;Colorectal cancer, hereditary nonpolyposis, type 7;Dermatofibrosarcoma protuberans;Endometrial cancer;Esophageal cancer;Gastric cancer, fibrosarcoma;Glioblastoma multiforme;Glomus tumors, multiple;Hepatoblastoma;Hepatocellular carcinoma;Hepatocellular carcinoma;Leukemia, acute lymphoblastic;Leukemia, acute myeloid;Leukemia, acute myeloid, with eosinophilia;Leukemia, acute nonlymphocytic;Leukemia, chronic myeloid;Li-Fraumeni syndrome;Liposarcoma;Lung cancer;Lung cancer, small cell;Lymphoma, non-Hodgkin;Lynch syndrome II (lynch cancer family syndrome II);male germ cell tumors;mast cell leukemia;medullary thyroid;medulloblastoma;melanoma, meningioma;multiple endocrine neoplasms;myeloid malignancies, predisposing;myxosarcoma, neuroblastoma;osteosarcoma;ovarian cancer;ovarian cancer, serous;ovarian cancer;ovarian sex cord tumors;pancreatic cancer;pancreatic endocrine tumors;paraganglioma, familial nonchromaffin;pilomatricoma;pituitary tumors, invasive;prostatic adenocarcinoma;prostatic carcinoma;renal cell carcinoma, papillary, familial and sporadic;retinoblastoma;rhabdoid predisposing syndrome, familial;rhabdoid tumor;rhabdomyosarcoma;small cell carcinoma of the lung;soft tissue sarcoma, squamous cell carcinoma, head and neck;T-cell acute lymphoblastic leukemia;Turcot's syndrome with glioblastoma;calcium with esophageal cancer;cervical cancer, Wilms' tumor, type 2; and Wilms' tumor, type 1.

[0101] According to certain embodiments, the cancer is pancreatic cancer.

[0102] According to certain embodiments, the pancreatic cancer is a pancreatic adenocarcinoma, a neuroendocrine tumor, or acinar carcinoma of the pancreas.

[0103] Thus, the present specification provides a method of treating pancreatic cancer in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an antibody comprising an antigen recognition region that binds to the catalytic site of MMP-7, wherein the antibody inhibits the activity of MMP-7, and the Ki of the antibody against MMP-7 is at least 5-fold lower than the Ki of the antibody against MMP2 or MMP9, thereby treating pancreatic cancer.

[0104] Examples of such antibodies are provided herein, as well as in WO 2012 / 056455 and WO 2010 / 012167, the contents of which are incorporated herein by reference.

[0105] The specificity of the antibodies can be confirmed using assays known in the art. Suitable assay conditions for metalloprotein inhibitor activity are described in Knight et al., FEBS Letters 296(3):263-266 (1992), Cawston et al., Anal. Biochem, 99:340-345 (1979), Cawston et al., Methods in Enzymology 80:771 et seq. (1981); Cawston et al., Biochem. J., 195:159-165 (1981), Weingarten et al., Biochem. Biophys. Res. Comm., 139:1184-1187 (1984), and U.S. Pat. Nos. 4,743,587 and 5,240,958.

[0106] According to another embodiment, the disease is inflammatory bowel disease (IBD), which is a severe gastrointestinal disorder characterized by intestinal inflammation and tissue remodeling, which increases in frequency and may prove disabling to the patient. The main forms of IBD, ulcerative colitis (UC) and Crohn's disease, are chronic, relapsing conditions clinically characterized by abdominal pain, diarrhea, rectal bleeding and fever.

[0107] Subjects which may be treated include mammalian subjects, such as humans.

[0108] Another use of antibodies against MMP-7 is in the diagnosis of diseases associated with upregulated expression of MMP-7.

[0109] Thus, according to another aspect of the present invention, there is provided a method for diagnosing a disease associated with imbalanced or abnormal activity of MMP-7 in a subject, comprising contacting a sample from said subject with an antibody described herein (e.g., GSM-192) to analyze expression of MMP-7, wherein upregulation of MMP-7 expression is indicative of a disease associated with imbalanced or abnormal activity of MMP-7.

[0110] Methods for analyzing the expression of MMP-7 using the disclosed antibodies include, but are not limited to, Western analysis, immunoprecipitation, and immunohistochemistry.

[0111] The sample may be a liquid, such as urine, saliva, cerebrospinal fluid, blood, serum, etc.; a solid or semi-solid, such as tissue, faeces; or a solid tissue, such as those commonly used for histological diagnosis.

[0112] Typically, the amount of MMP-7 is compared to a control (a corresponding sample from a healthy subject) or to a known amount of MMP-7 corresponding to a healthy subject.

[0113] After diagnosis, the subject can be informed of the result. Further diagnostic tests may be performed based on the results of the tests using the MMP-7 antibodies disclosed herein.

[0114] It will be appreciated that as well as diagnosis being carried out in vitro (ie on a sample from a subject), diagnosis can also be carried out in vivo.

[0115] Diseases that may be diagnosed include those listed above for diseases that may be treated.

[0116] If the diagnostic result is negative, i.e., there is no significant increase in MMP-7 expression and it is established that the patient has cancer, a chemotherapeutic agent (not an MMP-7 antibody) may be prescribed depending on the type of cancer.

[0117] If the result of the diagnosis is positive, i.e., increased expression of MMP-7, we contemplate treating the disease with an agent that downregulates the amount / activity of MMP-7. In one embodiment, the agent used to treat the disease is an inhibitory MMP-7 antibody, for example, as disclosed herein and in WO2012 / 056455 and WO2010 / 012167, the contents of which are incorporated herein by reference.

[0118] Methods for generating MMP-7 antibodies are known in the art. In one embodiment, the method comprises subjecting a subject (e.g., an animal subject) to: (i) a synthetic zincomimetic compound that has similar structural and electronic properties to the catalytic domain of MMP-7; and (ii) MMP-7, including immunization with

[0119] Synthetic zincomimetic compounds that have similar structural and electronic properties to the catalytic domains of metalloenzymes are typically compounds that contain a chelated metal ion, typically zinc or its analogous ions, cobalt or cadmium.

[0120] According to one embodiment, the chelating agent is a porphyrin.

[0121] Zinc mimetic compounds can be selected based on the structural and electronic properties of the actual catalytic domain in the target polypeptide. Typically, the target polypeptide contains three amino acids that provide the three contact points required for transition metal coordination. Representative coordination complex geometries can be tetrahedral, square planar or trigonal depending on the transition metal ion. In general, the mimetic compositions of the present invention are selected based on the amino acid side chain structure and coordination geometry. Typically, the amino acids that can regulate transition metal binding are histidine, arginine, glutamic acid, cysteine, methionine, tryptophan, serine, threonine and tyrosine, with the first two being preferred.

[0122] Exemplary synthetic zincomimetic compounds are disclosed in WO 2004 / 087042 and WO 2008 / 102359, which are incorporated herein by reference.

[0123] According to one embodiment, the synthetic zincomimetic compound has the general formula (I): JPEG0007631207000003.jpg13287In formula, m and n each independently represent an integer of 1 to 6; X1 to X3 and Y1 to Y3 are each independently O or S; R1-R3 are each independently selected from the group consisting of hydrogen, alkyl, and cycloalkyl; R is O-(CH2)xC(=O)NR'-(CH2)y-NR'R'', on the other hand: x and y are each independently an integer of 1 to 6; R' and R'' are each independently selected from the group consisting of hydrogen, alkyl, and cycloalkyl.

[0124] According to a particular embodiment of this aspect of the invention, the compound has the general formula (II): The compound is [2-(2-aminoethylcarbomoyl)-ethoxymethyl]-tris-[2-(N-(3-imidazol-1-yl-propyl))-ethoxymethyl]methane, designated Imisdp, having the formula JPEG0007631207000004.jpg12785, where R is O-CH2-CH2-CONH-CH2-CH2-NH2.

[0125] As already mentioned, the method of the present invention is carried out by immunizing with both the zincomimetic compounds described above and the MMP-7 metalloenzyme itself.

[0126] The present invention contemplates immunization with full-length MMP-7, or a portion thereof. Typically, the portion should contain an antigenic determinant (i.e., an epitope) specific to MMP-7.

[0127] According to one embodiment, the antigenic determinant is on the surface of MMP-7.

[0128] The MMP-7 used for immunization may be purified from its in vivo environment or may be produced by recombinant means.

[0129] According to one embodiment, the immunization includes an initial immunization with a zincomimetic compound and a subsequent immunization (e.g., 3-12 weeks later) with MMP-7. The exact time of immunization can be determined by examining whether an immune response exists in the immunized animal (e.g., mouse). For example, the titer of antibodies in the serum can be analyzed.

[0130] According to another embodiment, the immunization protocol involves an initial immunization with MMP-7, followed (eg, 3-12 weeks later) by an immunization with the zincomimetic compound.

[0131] According to yet another embodiment, the immunization procedure involves co-immunization (ie, simultaneously) with both MMP-7 and the zincomimetic compound.

[0132] The antibodies of the invention can be administered to the subject itself or as part of a pharmaceutical composition.

[0133] As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0134] As used herein, the term "active ingredient" refers to an antibody capable of accounting for a biological effect.

[0135] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the administered compound. These phrases include adjuvants.

[0136] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0137] Techniques for drug formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.

[0138] Suitable routes of administration may include, for example, oral, rectal, transmucosal, particularly nasal, intestinal, or parenteral delivery, including intramuscular, subcutaneous and intramedullary injection, as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, common coronary, intravenous, inrtaperitoneal, intranasal, or intraocular injection.

[0139] Conventional approaches for drug delivery to the CNS include: neurosurgical strategies (e.g., intracerebral injection or intraventricular infusion); molecular manipulation of the drug in an attempt to exploit one of the endogenous transport pathways of the BBB (e.g., production of chimeric fusion proteins containing a transport peptide with affinity for an endothelial cell surface molecule in combination with a drug that cannot itself cross the BBB); pharmacological strategies designed to increase the lipid solubility of the drug (e.g., conjugation of a water-soluble drug to a lipid or cholesterol carrier); and temporary disruption of the integrity of the BBB by hyperosmotic disruption (resulting from injection of a mannitol solution into the carotid artery or the use of a biologically active drug such as angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with invasive surgery, size limitations imposed by the limitations inherent in endogenous transport systems, potentially undesirable biological side effects associated with systemic administration of chimeric molecules composed of carrier motifs that may be active outside the central nervous system, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, making them suboptimal delivery methods.

[0140] Alternatively, the pharmaceutical composition can be administered in a local rather than systemic manner, for example, by injecting the pharmaceutical composition directly into a tissue area of ​​the patient.

[0141] The term "tissue" refers to a part of an organism that consists of an aggregate of cells having a similar structure and / or a common function. Examples include, but are not limited to, brain tissue, retina, skin tissue, liver tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, heart tissue, brain tissue, vascular tissue, kidney tissue, lung tissue, gonadal tissue, and hematopoietic tissue.

[0142] The pharmaceutical compositions of the present invention may be manufactured by processes that are well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0143] Thus, pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers that contain excipients and auxiliaries that facilitate processing of the active ingredient into a pharma- ceutically usable preparation. Appropriate formulations will vary depending on the route of administration selected.

[0144] For injection, the active ingredients of the pharmaceutical composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0145] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compounds with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient. Pharmacological preparations for oral use can be made using solid excipients, optionally milling the resulting mixture and processing the mixture of granules to obtain tablets or dragee cores, after adding suitable auxiliaries as necessary. Suitable excipients are, in particular, sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carbomethylcellulose; and / or fillers, such as physiologically acceptable polymers, such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0146] The dragee core is provided with a suitable coating.For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution, and concentrated sugar solution, which may optionally contain suitable organic solvent or solvent mixture, can be used.Dyes or pigments can be added to the tablet or dragee coating for identification or to characterize different combinations of doses of active compound.

[0147] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. All formulations for oral administration should be in a dosage suitable for the chosen route of administration.

[0148] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0149] For administration by nasal inhalation, the active ingredient for use according to the invention is conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide.In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges, for example of gelatin, for use in a dispenser can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0150] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Preparations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, optionally containing preservatives. The compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending, stabilizing and / or dispersing agents.

[0151] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredient in water-soluble form. Furthermore, suspensions of the active ingredient can be prepared as suitable oil-based or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. If necessary, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredient to allow for the preparation of highly concentrated solutions.

[0152] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, a sterile pyrogen-free water-based solution, before use.

[0153] Pharmaceutical compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, eg, conventional suppository bases such as cocoa butter or other glycerides.

[0154] Pharmaceutical compositions suitable for use in the context of the present invention include compositions in which the active ingredient is contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredient (antibody) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer / anthrax infection) or prolong the survival of the subject being treated.

[0155] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0156] For any preparation used in the methods of the present invention, the therapeutically effective amount or dose can be initially estimated from in vitro and cell culture assays. For example, a dose can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0157] Toxicity and therapeutic effects of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or in experimental animals. Data obtained from these in vitro and cell culture assays, as well as animal studies, can be used in formulating a range of dosages for use in humans. Dosages can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition. (See, for example, Fingl, et al., 1975, in ''The Pharmacological Basis of Therapeutics'', Ch.1 p.1).

[0158] Dosage amount and interval can be adjusted individually to provide tissue or blood levels of the active ingredient sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC varies from preparation to preparation but can be estimated from in vitro data. The dosage required to achieve the MEC varies with individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0159] Depending on the severity and responsiveness of the condition to be treated, dosage may be single or multiple administrations, with the course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the disease is achieved.

[0160] The amount of composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0161] The inventors have shown that the antibodies of this aspect of the invention enhance chemotherapeutic agent sensitivity, and therefore, the inventors contemplate administering the antibodies in conjunction with chemotherapeutic agents.

[0162] Examples of chemotherapeutic agents include, but are not limited to, acivicin; aclarubicin; acodazole hydrochloride; acronine; adriamycin; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizeresin; bleomycin sulfate; brequinar sodium ;Bropirimine;Busulfan;Cactinomycin;Calsterone;Caracemide;Carbetimer;Carboplatin;Carmustine;Carubicin hydrochloride;Carzelesin;Cedefingol;Chlorambucil;Ciloremycin;Cisplatin;Cladribine;Crisnatol mesylate;Cyclophosphamide;Cytarabine;Dacarbazine;Dactinomycin;Daunorubicin hydrochloride;Decitabine;Dexormaplatin;Desaguanine;Desaguanine mesylate;Diaziquone;Docetaxel;Doxorubicin;Doxorubicin hydrochloride;Droloxifene;Que Droloxifene phosphate;Drostanolone propionate;Duazomycin;Edatrexate;Eflornithine hydrochloride;Elsamitrucin;Enloplatin;Enpromate;Epipropizine;Epirubicin hydrochloride;Elbrozole;Esorubicin hydrochloride;Estramustine;Estramustine phosphate sodium;Etanidazole;Etoposide;Etoposide phosphate;Etoprine;Fadrozole hydrochloride;Fazarabine;Fenretinide;Floxuridine;Fludarabine phosphate;Fluorouracil;Flurocitabine;Fos Quidone;Fostriecin sodium;Gemcitabine;Gemcitabine hydrochloride;Hydroxyurea;Idarubicin hydrochloride;Ifosfamide;Irmofosine;Interferon alpha-2a;Interferon alpha-2b;Interferon alpha-n1;Interferon alpha-n3;Interferon beta-Ia;Interferon gamma-Ib;Iproplatin;Irinotecan hydrochloride;Lanreotide acetate;Letrozole;Leuprolide acetate;Riazole hydrochloride;Lometrexol sodium;Lomustine;Losoxantrone hydrochloride;Massoprotocol;Maytansine;Mechlorethamine hydrochloride;Megestrol acetate;Melengestrol acetate;Melphalan;Menogaril;Mercaptopurine;Methotrexate;Methotrexate sodium;Metoprine;Meturedepa;Mitindomide;Mitocalcin;Mitochromine;Mitogillin;Mitomalcin;Mitomycin;Mitospar;Mitotane;Mitoxantrone hydrochloride;Mycophenolic acid;Nocodazole;Nogalamycin;Ormaplatin;Oxisuran;Pac Ritaxel;Oxaliplatin;Pegaspargase;Periomycin;Pentamustine;Peplomycin sulfate;Perfosfamide;Pipobroman;Piposulfan;Piroxantrone hydrochloride;Plicamycin;Promestane;Porfimer sodium;Porfiromycin;Prednimustine;Procarbazine hydrochloride;Puromycin;Puromycin hydrochloride;Pirazofurin;Riboprin;Rogletimide;Safingol;Safingol hydrochloride;Semustine;Cyclo Mutrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Surofenur; Tallysomycin; Taxol; Tecogalan sodium; Tegafur; Teroxantrone hydrochloride; Temoporfin; Teniposide; Teroxylon; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofirin; Tirapazamine; Topotecan hydrochloride; Toremifene citrate; Treston acetate;Triciribine phosphate;Trimetrexate;Trimetrexate glucuronate;Triptorelin;Tubrozole hydrochloride;Uracil mustard;Uredep;Vapreotide;Verteporfin;Vinblastine sulfate;Vincristine sulfate;Vindesine;Vindesine sulfate;Binepidine sulfate;Vingrisinate sulfate;Vinleurosine sulfate;Vinorelbine tartrate;Vinrocidine sulfate;Vinzolidine sulfate;Vorozole;Zeniplatin;Zinostatin;Additional antineoplastic agents include those disclosed in Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division), Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner) and its introduction, pp. 1202-1263;

[0163] In certain embodiments, the chemotherapeutic agent is a nucleoside analog, examples of which include, but are not limited to, gemcitabine, methotrexate, 5-fluorouracil, cytosine arabinoside, behenoyl cytosine arabinoside, tegafur, UFT, and the like.

[0164] According to certain embodiments, the nucleotide agent is gemcitabine.

[0165] According to a particular embodiment, the chemotherapeutic agent is oxaliplatin.

[0166] In the context of combination therapy, the chemotherapeutic agent may be administered by the same route of administration as the antibody is administered (e.g., pulmonary, oral, enteral, etc.) Alternatively, the chemotherapeutic agent may be administered by a different route of administration than the antibody.

[0167] The chemotherapeutic agent can be administered immediately before (or immediately after) the antibody, on the same day as the antibody, one day before (or after), one week before (or after), one month before (or after), or two months before (or after) the antibody, etc.

[0168] The chemotherapeutic agent and the antibody can be administered simultaneously, i.e., the administration of each of these agents can be performed at time intervals that partially or completely overlap with each other. The chemotherapeutic agent and the antibody can be administered during non-overlapping time intervals. For example, the chemotherapeutic agent can be administered within a time frame of t=0-1 hours, and the antibody can be administered within a time frame of t=1-2 hours. Also, the chemotherapeutic agent can be administered within a time frame of t=0-1 hours, and the antibody can be administered anywhere within a time frame of t=2-3 hours, t=3-4 hours, t=4-5 hours, t=5-6 hours, t=6-7 hours, t=7-8 hours, t=8-9 hours, t=9-10 hours, etc. Furthermore, the antibody can be administered anywhere within a time frame of t=minus 2-3 hours, t=minus 3-4 hours, t=minus 4-5 hours, t=5-6 minus hours, t=minus 6-7 hours, t=minus 7-8 hours, t=minus 8-9 hours, t=minus 9-10 hours.

[0169] The antibody and chemotherapeutic agent of the present invention are typically provided in a combined amount to achieve therapeutic, preventive and / or pain relieving effectiveness. This amount obviously depends on the specific compound selected for use, the nature and number of other treatment modalities, the symptom(s) to be treated, prevented and / or alleviated, the species, age, sex, weight, health condition and prognosis of the subject, the mode of administration, the targeting effectiveness, residence time, mode of clearance, the type and severity of side effects of the pharmaceutical composition, and many other factors that will be apparent to those skilled in the art. The antibody is used at a level at which therapeutic, preventive and / or pain relieving effectiveness in combination with the chemotherapeutic agent is observed.

[0170] The chemotherapeutic agent may be administered (together with the antibody) in standard dosages as a single agent, in dosages less than the standard dosage as a single agent, or in dosages greater than the standard dosage as a single agent.

[0171] According to a specific embodiment, the chemotherapeutic agent is administered in standard doses as a single agent.

[0172] As used herein, the term "standard dose" refers to the dose recommended by a regulatory agency (e.g., the FDA) for a given tumor at a given stage.

[0173] According to other specific embodiments, the chemotherapeutic agent is administered at a dose that does not exert at least one side effect associated with the standard dose, non-limiting examples of side effects of chemotherapeutic agent treatment include skin rash, diarrhea, stomatitis, paronychia, fatigue, hyperglycemia, hepatotoxicity, renal failure, cardiovascular effects, electrolyte abnormalities, and GI perforation.

[0174] Thus, in one embodiment, the amount of chemotherapeutic agent, when used as a sole therapy (e.g., 10-99%, preferably 25-75% of that minimum dose), is below the minimum dose required for therapeutic, prophylactic and / or pain relieving efficacy. This allows for a reduction in side effects caused by the chemotherapeutic agent, but in combination with the antibody, the therapy is effective because the combination is effective overall.

[0175] In one aspect of the invention, the antibody and the chemotherapeutic agent are synergistic with respect to their dosage, i.e., the effect provided by the antibody of the invention is greater than would be expected from the additive effect of the chemotherapeutic agent and the antibody when used separately. In an alternative embodiment, the chemotherapeutic agent and the antibody of the invention are synergistic with respect to their side effects, i.e., the side effects caused by the antibody in combination with the chemotherapeutic agent are less than would be expected if an equivalent therapeutic effect were provided by either the chemotherapeutic agent or the antibody when used separately.

[0176] The compositions of the present invention may be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by administration instructions. The pack or dispenser may also be adapted with a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the composition or form of administration for human or animal use. Such notice may, for example, be on a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. Compositions containing the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for the treatment of a designated condition, as further detailed above.

[0177] The term "treating" refers to inhibiting, preventing or arresting the onset of a pathology (disease, disorder or condition) and / or causing the alleviation, remission or regression of a pathology. Those skilled in the art will appreciate that a variety of methodologies and assays can be used to assess the progression of a pathology, and similarly, a variety of methodologies and assays can be used to assess the reduction, remission or regression of a pathology.

[0178] As used herein, the term "prevent" refers to keeping a disease, disorder, or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

[0179] As used herein, the term "subject" includes mammals, preferably humans of any age, suffering from a pathological condition. Preferably, the term encompasses individuals who are at risk of developing a pathological condition.

[0180] The terms "comprises," "comprising," "includes," "including," "having," and combinations thereof mean "including but not limited to."

[0181] As used herein, the term "method" refers to manners, means, techniques, and procedures for accomplishing a given task, and includes, but is not limited to, those manners, means, techniques, and procedures known to or readily developed by those skilled in the art of chemistry, pharmacology, biology, biochemistry, and medicine from known manners, means, techniques, and procedures.

[0182] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention that are described in the context of a single embodiment may also be provided separately or in any suitable subcombination or as appropriate with any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0183] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims below find experimental support in the following examples. EXAMPLES

[0184] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0185] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully described in the literature, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); methodologies described in U.S. Patents 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), ``Basic and Clinical Immunology'' (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), ``Selected Methods in Cellular Immunology'', WHFreeman and Co., New York (1980); available immunoassays are described extensively in the patent and scientific literature, e.g., in U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521; Synthesis''Gait,MJ,ed.(1984);''Nucleic Acid Hybridization''Hames,BD,and Higgins SJ,eds.(1985);''Transcription and Translation''Hames,BD,and Higgins SJ,eds.(1984);''Animal Cell Culture''Freshney,RI,ed.(1986);''Immobilized Cells and Enzymes''IRL Press, (1986);''A Practical Guide to Molecular Cloning''Perbal, B., (1984) and''Methods in Enzymology'' Vol.1-317, Academic Press;''PCR Protocols: A Guide To Methods And Applications'', Academic Press, San Diego, CA (1990);Marshak et al.See, "Strategies for Protein Purification and Characterization-A Laboratory Course Manual," CSHL Press (1996), all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0186] Materials and Methods Antibody generation and purification: Female BALB / c mice were immunized with complete Freund's adjuvant (Disco) and 30 μg of catalytic domain of MMP-7 or Zn-tripod-KLH and boosted every 2 weeks with incomplete Freund's adjuvant by subcutaneous injection. Spleens were harvested and B cells were fused with NSO mouse myeloma cells. Hybridomas were screened for immunoreactivity against catalytic domain of MMP-7 and Zn-tripod by ELISA, and selected hybridomas were subcloned and expanded in tissue culture. Hybridoma cells of GSM-192 were grown in DCCM (serum-free medium designed for hybridoma cell growth and monoclonal antibody production, purchased from Biological Industries, Israel). Cells were pelleted by centrifugation at 193×3 g and the supernatant was collected. The supernatant was dialyzed against 20 mM phosphate buffer (pH 8). A 1 ml HiTrap protein A high performance column was equilibrated with 100 mM phosphate buffer (pH 8) and the supernatant was loaded at 1 ml / min. The antibody was eluted with 100 mM citrate buffer (pH 6) and dialyzed against 50 mM Tris (pH 7.5) and 150 mM NaCl.

[0187] Dot blots were performed by immobilizing 1 μg, 1.5 μg and 2 μg of zymogen and activated MMP-7 recombinant enzyme on nitrocellulose membranes pre-wetted with TBST using a vacuum-based manifold 96-well dot blot system (GE 10447900 Minifold Dot Blot). Blocking was performed with 3% BSA in TBST for 1 hour at room temperature. The membranes were then probed for binding by the antibody eluted in the previous step by incubating overnight. The anti-MMP-7 mAb used was at a concentration of 2ug / ml in TBST. After washing 3 times with TBST, the membranes were incubated with goat anti-mouse HRP antibody and developed using ECL to determine the selectivity of binding for only the catalytic form.

[0188] Antibody digestion with papain for Fab generation. Papain was activated in 0.5 M Tris-HCl (pH 8), 10 mM EDTA and 5 mM dithiothreitol for 15 min at 37°C. Activated papain was added to a solution of intact GSM-192 at a ratio of 1:1,000, and the digestion process was carried out at 37°C for 3 h. The digestion reaction was terminated by adding 20 mM iodoacetamide for 30 min at room temperature in the dark. Fab fragments were isolated from Fc by protein A column, and Fab fragments were collected from the flow-through and dialyzed against 50 mM Tris (pH 7.5) and 150 mM NaCl. The purity of the Fab fragments was estimated by 12% SDS-PAGE gel.

[0189] MMP-7 Elisa binding assay: 96-well plates (Nunc) were coated with 5 mg / ml MMP. After coating, plates were incubated with GSM-192 for 1 h at 25° C. Bound Fab fragments / full length were detected using goat anti-Fab / anti-mouse antibodies followed by peroxidase-conjugated bovine anti-goat antibodies according to standard procedures. Half-maximal effective concentrations were calculated using a four-parametric sigmoidal curve fitting analysis.

[0190] MMP-7 enzyme kinetic assay: The enzymatic activity of MMP-7 in the presence of GSM-192 Fab was measured at 37°C by monitoring the hydrolysis of the fluorescent peptide Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (SEQ ID NO: 10) at λex = 340 nm and GSM-192 = 390 nm as previously described. 30 Various concentrations of GSM-192 Fab (0-200 nM) were incubated with 10 nM MMP-7 in 50 mM Tris-HCl buffer (pH 7.5 at 37 °C), 100 mM NaCl, 5 mM CaCl2 and 0.05% Brij-35 for 2 h at 37 °C. The enzymatic reaction was initiated by adding the fluorogenic peptide to a final concentration of 10 μM. Fluorescence was immediately and continuously recorded for 30 min.

[0191] The inhibition constants were evaluated by measuring the initial reaction rates and fitting the data to an equation where Vi is the initial rate in the presence of inhibitor, V0 is the initial rate in the absence of inhibitor, and I is the inhibitor concentration. To determine the type of inhibition, the initial rates of MMP-7 were measured as a function of substrate concentration (0-30 mM) at several fixed concentrations of Fab GSM-192 (0-500 nM). Apparent KM and Vmax values ​​were derived by linearization. JPEG0007631207000005.jpg3888

[0192] Antibody sequencing: Immunoglobulin V-region genes were cloned and sequenced after amplification by PCR. Total RNA was purified by the phenol-guanidine isothiocyanate method (peqGOLD TriFast from peqlab biotechnologie) at 5 × 10 6The cDNA was prepared from 1000 hybridoma cells. CDNA was obtained and amplified in one step using the ReverseiTTM One-Step RT-PCR Kit (ABgene). The V-region genes were amplified by using degenerate sense primers homologous to mouse heavy and light chain leader sequences and antisense constant primers (Amersham Biosciences). The amplified products were ligated into pGEM-T Easy Vector (Promega) using standard protocols, and both strands of the insert were sequenced with an automated sequencer at the DNA Sequencing Unit (Biological Services, Weizmann Institute of Science).

[0193] GSM-192 light chain sequence (SEQ ID NO:1) DI VT QSPASLAVSLGQRATISCRAS ESFDSYGNTFVHWYQQKPGQP PKLLIYLVSNLESGVPAGFRG RGSRTDFTLTIDPVEADDAAT YYCQQNNEDPYTFGGGTKLEI KRA

[0194] GSM-192 heavy chain sequence (SEQ ID NO:2) EVQLQQSGPELVKPGASVKIP CKASGYTFTDYNMDWMKQSHG KSLEWIGHINPNNGGTFYNQK FKDKATFIVDKSSNTAYMELR SLTSEDTA VYFCARGGGLRRG PFAYWGQGTLVTVS

[0195] Protein crystallography of GSM-192 Fab: Crystals of free GSM-192 Fab obtained using hanging drop vapor diffusion diffracted up to 2.3 Å resolution. Crystals were grown from 0.2 M CaCl2, 0.1 M HEPES pH=7, 20% PEG 6000 and 0.01 M betaine hydrochloride. Crystals formed in the trigonal space group P3121 with cell constants a, b=88.44, c=119.34 Å, g=120° and single copies of light and heavy chains. A full X-ray data set at 2.3 Å resolution was collected at 100 K using a RU-H3R rotating anode with a RAXIS IV++ detector. HKL2000 program 31 The diffraction patterns were indexed and integrated using the SCALEPACK program. 31、39 The processing of X-ray diffraction data collected on vibrational modes and integral reflections was scaled using the CCP4 program suite. 40 Structure factor amplitudes were calculated using TRUNCATE from . Data collection details are also listed in Table 3. The refined structure of anti-Aβ monoclonal antibody mAb (PFA1) (Protein Data Bank (PDB) accession code 2IPU) was used as a model to calculate the structure factor amplitudes. 41 The structure was solved by molecular replacement with the program PHENIX. All steps of atomic refinement were carried out using the program PHENIX. 42 The model was run using the COOT program. 43 The 2mFobs-DFcalc and mFobs-DFcalc maps were fitted using the 2Fobs-DFcalc and mFobs-DFcalc programs. The refinement weights were optimized. The GSM-192 Fab construct consists of 224 amino acid residues in the heavy chain and 215 amino acids in the light chain (439 residues). The final model contains residues 1-221 in the heavy chain and residues 2-185 in the light chain. The Rfree value is 27.85% (for the 5% of reflections not used in refinement) and the Rwork value is 23.22% for 2.3 Å for all data. The GSM-192 Fab model was fitted using the PROCHECK program. 44The refinement statistics of the GSM-192 Fab structure are detailed in Table 3. The coordinates and structure factors of the GSM-192 Fab have been deposited in the PDB under the ID code 6FBJ.

[0196] [Table 3]

[0197] Computational modeling and docking: The Fv domain of the antibody GSM-192 was computationally docked to MMP-7. Comparison of several structures of MMP-7 available in the PDB showed structural variations that affect the width of the active site cleft. Normal mode analysis applied to the experimental structure 45 showed similar mobility of the loops. Therefore, the experimental structure of MMP-7 and several normal mode conformers were used for docking. The FFT-based geometric electrostatic hydrophobic (GEH) version of MolFit 46~48 The method docks molecules using and performs an exhaustive stepwise scan of the relative rotation and translation of the docked molecule, providing a GEH score for all test positions. The resulting poses are filtered using a post-scan propensity and solvation (P&S) filter. 49 was used to filter.

[0198] The filtered models were further screened to include only those whose interactions involved exposed residues in the antibody CDRs. This screen counted the number of atom-atom contacts (distances of 5 Å or less) between exposed CDR residues and the target molecule. The MolFit GEH score is sensitive to small changes in the relative orientation of the molecules. 50,Local rigid-body refinement was previously found to be very effective for identifying truly high-scoring docking models. Therefore, models from several scans were refined by allowing small local rotations in 2° increments. The refinement highlighted one model in the docking results. This model was ranked first in the docking scan employing a normal mode conformer closely resembling structure 2y6a, and its refined score was 3.1σ above the next model and 9σ above the mean score (the mean score and σ were determined by fitting an extreme value distribution function to the distribution of GEH scores). 50 ) Of note, scans with and without AHA as part of the MMP-7 structure yielded identical models; in the latter case, the AHA position was empty and accessible.

[0199] Anchor spots were used to identify preferred binding locations of single amino acid side chains on the surface of the protein. The mapping was performed using ANCHORSmap. 32 This was performed using UCSF-Chimera 51 was used for structural analysis and comparison, and to generate FIG.

[0200] Cell Culture: All cell lines were maintained in a humidified incubator at 37°C with 5% CO2 and cultured in complete medium consisting of DMEM, RPMI medium 1640 or IMDM supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, 1% L-glutamine, 1% sodium pyruvate.

[0201] MMP-7 lentiviral silencing and FACS analysis: Stable knockdown of MMP-7 in AsPC-1 pancreatic cancer cells was achieved by HIV-based stable transduction with shRNA against MMP-7 using the ViraPower Lentiviral Expression System (Thermo Fisher Scientific) in combination with commercially available shRNA oligos (Sigma Aldrich). Recombinant retroviruses were transfected according to standard protocols.52 AsPC-1 cells were transduced at an MOI of 10 in the presence of 8 μg / ml polybrene. 48 hours after transduction, AsPC-1 cells were passaged and allowed to adhere for 6 hours, after which 40 μg / ml puromycin was added to positively select for transduced cells. Antibiotic pressure was maintained for up to 24 hours, after which 100% of non-transduced control cells maintained in the same conditions had died. Knockdown efficacy was determined by qRT-PCR. RNA was isolated from cells using TRIzol (Life Technologies) according to the manufacturer's protocol. Reverse transcription was performed using a High Capacity RT Kit (Life technologies) according to the manufacturer's instructions. Cell cycle analysis using propidium iodide in a FACS was performed on stable knockdown cells maintained at 80% cell confluency.

[0202] MTT cell proliferation assay: MTT solution (M5655 SIGMA) was added at 10% of the total culture volume (0.5 mg / ml MTT in RPMI / IMDM without phenol red) and after 3 h of incubation, plates were washed with PBS and solubilized using MTT solvent (0.1 N HCl in anhydrous isopropanol). Readings were obtained at wavelengths of 630-690 nm in a spectrophotometer plate reader. Relative percentages of cell proliferation were determined compared to control wells. Curve fitting analysis of IC50 was performed using Origin software v8.5.

[0203] FACS Annexin V Apoptosis Detection Assay: 70% confluent cells in 6 cm plates were treated with sterile GSM-192 Fab fragments / control anti-LOXL-2 Fab fragments for 24 h and incubated for 1 h at 4 °C for 1 h as previously described. 53, and prepared for FACS using Annexin V FITC Apoptosis Detection Kit (E Biosciences 88-8005-72). FACS acquisition was performed using a FACS LSR-II machine with Cell Quant software (Becton Dickinson) and further analyzed with FlowJo software (Treestar, Inc., San Carlos, CA).

[0204] Western Blot: Treated cells were lysed with RIPA buffer containing protease inhibitors and kept on ice for 30 min with intermittent shaking. Debris was removed by centrifugation at 14,000×g and the supernatant was used for BCA protein determination assay. Alternatively, culture supernatants were concentrated at least 10-fold using 0.2 μm centricons. Once normalized to equal protein content, sample buffer was added and heated at 95°C for 3 min. Denatured samples were centrifuged at 14,000×g for 5 min, after which 25 μL was loaded onto an SDS page gel and standard blotting procedures were followed (General Protocol for Western Blotting, BioRad bulletin 6376). Primary Abs used were MMP-7 (abcam, ab5706), Fas-L (abcam, ab15285), α-tubulin Ab (sc31779).

[0205] Wound healing assay (scratch assay): The migration behavior of cells was assayed by a wound healing assay using culture inserts (ibidi GmbH, Am Klopferspiel 19, Martinsried, Germany) according to the manufacturer's instructions. After 24 h, the culture inserts were removed, leaving a cell-free gap of approximately 500 μm (defined as the "wound"). The migration speed into this "wounded area" was recorded and measured using a Carl Zeiss microscope (DeltaVision Microscopy Imaging Systems-GE Healthcare Life Sciences, USA). The migration speed was determined by the percentage of the area covered by cells. Each analysis was performed in triplicate and repeated twice.

[0206] Transwell migration assay: Cells were starved overnight in assay medium (IMDM medium containing 1% serum only). Cells (1 × 10 5 ) was added to the top chamber of a 24-well transwell plate (BD Biosciences; pore size 8 μm), and assay medium with or without GSM-192 was added to both chambers. After overnight incubation, cells that had not migrated to the top well were removed, and cells that had migrated to the bottom were fixed with 4% PFA and stained with crystal violet (0.05%). Cell numbers in five fields were counted at 20x magnification, and the average for each chamber was determined.

[0207] Immunohistochemistry: Frozen human PDAC sections in OCT (10 μm thick) were cut on a cryostat. Sections were dried on the benchtop for 30 min at room temperature, rehydrated in PBS for 10 min, preincubated with PNB blocking buffer (PerkinElmer Life Sciences) for 1 h at room temperature, and then incubated with the primary antibody of interest, GSM-192 mAb or MMP-7 (abcam, ab5706), at 4°C overnight. Corresponding secondary HRP antibodies were used at the manufacturer's recommended dilution. Slides were mounted in mounting medium. Images of mounted sections were obtained using a Carl Zeiss light microscope.

[0208] Animal models RIP1-Tag2: The RIP1Tag2 (RT2) mouse PanNET model was developed by fusing the rat insulin II promoter region with the SV40 early region encoding the large T and small t antigens. Oncogene expression begins during embryonic development (E8.5), but pancreatic islets initially have a normal anatomical and histological appearance ("normal" stage). Beginning at 4-5 weeks of age, hyperplastic and dysplastic islets begin to appear, with 50% of all islets regressing by 10 weeks of age. Vascularized islets appear around 6 weeks of age and comprise 10% of all islets at 10.5 weeks of age. Vascularized islets are recognized by their dilated blood vessels and microhemorrhages. Tumors begin at 9-10 weeks and comprise 2-4% of all islets by 14 weeks. Approximately half of the end-stage tumors show focal or extensive infiltration into the surrounding acinar tissue. RIP-Tag2 mice die at approximately 14 weeks of age, primarily due to hyperinsulinemia. Additionally, we used RIP1-Tag2-Vecad-tdTomato mice to demonstrate postmortem vascular collapse (n=1) and RIP1-Tag2-CX3CR1 to visualize macrophages in the tumor capsule (n=1).

[0209] Gemcitabine co-treatment: 10,000 AsPC-1 cells / well were seeded in a 96-well flat-bottom plate and incubated overnight at 37°C, 5% CO2 in a humidified incubator. The medium in the wells was replaced with serum-free RPMI medium containing 2.33 μM GSM-192 or PBS and 0.05, 0.1, 0.5, 1, 5, 10, 40 and 80 μM gemcitabine. The plates were further incubated for 72 hours at 37°C, 5% CO2 in a humidified incubator. MTT solution (M5655 SIGMA) was added at 10% of the total culture volume (0.5 mg / ml MTT in RPMI / IMDM without phenol red), and after 3 hours of incubation, the plates were washed with PBS and solubilized using MTT solvent (0.1 N HCl in anhydrous isopropanol). Readouts were obtained on a spectrophotometer plate reader at a wavelength of 650 nm, with background readings at 570 nm. Cell death data were plotted as a function of the logarithm of gemcitabine concentration and fitted to a variable slope sigmoidal regression model (GraphPad prism 8.3.0). Additionally, IC values ​​from three experimental replicates were calculated. 50 The mean and corresponding standard deviation were calculated.

[0210] Statistics: For all representative results, experiments were repeated at least twice and sem was calculated using replicates. Normally distributed data were tested by Student's t-test with unequal variances to compare continuous variables between two groups. Statistical analysis was performed using GraphPad Prism 7.

[0211] Data availability: The GSM-192 X-ray structure has been deposited in the Protein Data Bank under accession number 6DBJ.

[0212] result Individual immunization with Zn tripod or activated recombinant human MMP-7 did not result in conformational selectivity: The conserved structure of the metalloenzyme MMP-7 contains a catalytic zinc-histidine complex present within the enzyme active site. Immunization of female BALB / c mice with repeated administration of small amounts of synthetic Zn tripod-KLH emulsified in complete Freund's adjuvant alone did not result in anti-MMP-7 antibodies (Figure 1A), nor did similar immunization with MMP-7-activated enzyme alone result in affinity-matured antibodies with inherent specificity for this form of the enzyme (Figure 1B). Anti-Zn tripod, anti-MMP-7 immune responses were examined in mouse sera using direct enzyme-linked immunosorbent assays (ELISAs), respectively. These two immunizations failed to show either cross-reactivity or conformational selectivity. These results highlighted the limitations in generating affinity maturation in vivo using individual immunization with active site mimetic molecules (Zn tripod) or with the traditional approach of using activated enzyme to generate the antibodies of interest.

[0213] Alternating immunization strategies yield high affinity mAbs selectively targeting MMP-7: Alternating immunization of mice with Zn tripod and recombinant human MMP-7 (hMMP-7) active enzyme was used to generate GSM-192. Female BALB / c mice were immunized every 3 weeks with the small Zn tripod and the catalytic domain of hMMP-7 (see Methods). Anti-Zn tripod, anti-MMP-7 immune responses were examined in mouse sera using ELISA. Progressive responses were observed as a function of repeated injections (Figure 2A). The specificity of the immune response in mouse sera was examined using ELISA against the Zn tripod (Figure 2E) and the catalytic domain of MMP-7 (Figure 2B), respectively. This analysis showed the generation of cross-reactive antibodies that recognize both the Zn tripod and MMP-7 (Figure 2B).

[0214] Only antibodies that were able to bind to the active form of MMP-7, but not to the zymogen, were selected for further development (i.e., selected for fusion and developed to generate monoclonal antibodies). This was performed by dot blot analysis using nitrocellulose membranes coated with various concentrations of active MMP-7 and its zymogen, respectively. It was shown that GSM-192 bound to the catalytic domain of the enzyme, but not to the zymogen form of the enzyme (Figure 2F).

[0215] Selective binding of MMP-7 catalytic domain and inhibition of catalytic activity: The binding constant of the selected anti-MMP-7 mAb GSM-192 was measured using ELISA (Figure 2C). The Kd was determined to be 43.1 ± 1.43 nM, and the antibody showed no cross-reactivity with closely related MMPs with conserved sequence homology and functional similarity, such as MMP-9, MMP-14, MMP-12 and MMP-13. The lack of binding to a panel of other MMPs served to determine the affinity of the novel antibody for an epitope unique specifically to MMP-7.

[0216] Remarkably, the antibody is completely selective for MMP-7, considering that the immune booster also contained the common zinc motif. The effect of GSM-192 Fab fragment on the enzymatic activity of human MMP-7 was investigated in vitro using a short fluorogenic peptide according to standard protocols. The enzymatic activity of recombinant active MMP-7 was measured in the presence of increasing concentrations of GSM-192 Fab fragment. GSM-192 Fab was found to inhibit MMP-7 activity with a Ki of 131.98 ± 10.23 nM. Concurrently, the effect of the antibody on the catalytic activity of related MMPs was measured. The in vitro catalytic activities of MMP-9, MMP-14 and MMP-12 were not affected by anti-MMP-7 Fab treatment, or were only negligibly affected in the case of MMP-14 (Figure 2D).

[0217] Fab fragment crystal structure and docking model analysis with active MMP-7: To determine where the antibody is binding and inhibiting the catalytic activity of MMP-7, GSM-192 Fab was characterized using X-ray crystallography. The crystal structure was determined at 2.3 Å resolution (Figure 3A). The GSM-192 Fab construct consists of 224 amino acid residues in the heavy chain and 215 amino acids in the light chain (439 residues in total). The final model includes residues 1-221 in the heavy chain and residues 2-185 in the light chain. The Rfree value is 27.85% (for the 5% of reflections not used for refinement) and the Rwork value is 23.22% for 2.3 Å for the entire data (Table 1). The antigen-binding surface is folded and shows two protruding elements, one consisting of the light chain CDR1 (residues 31-DSYGN-35L) and the other of the heavy chain CDR3 (residues 101-GLRR-105H). This antigen-binding surface is unique and novel. Docking of GSM-192 (Fv domain) to MMP-7 led to a model in which the antibody loop CDR-H3 binds inside the active site of the enzyme (Figure 3B).

[0218] The Protein Data Bank (PDB) contains several structures of the catalytic domain of MMP-7, which, when superimposed, show flexibility of the loops near the active site. Similar flexibility was seen in normal mode analysis, and therefore antibody GSM-192 (Fv domains L2-L112 and Q0-Q116) was docked into several MMP-7 conformers exhibiting different openings of the active site. Docking into an MMP-7 conformer with an open active site, as seen in structure 2y6c31, produced a statistically superior model with a complementarity score of more than 3σ than the next model shown in Figure 3B.

[0219] The docking model implies that binding is stabilized by strong hydrophobic anchoring of L100H in a pocket within the MMP-7 active site bounded by amino acids L181, A216 and Y241. 32Mapping identified this pocket as a strong binder of hydrophobic residues, especially Leu (Figure 3B,D). Another strong hydrophobic interaction is the binding of Y33L in an elongated cavity on the surface of MMP-7 at the edge of the active site pocket. Additional predicted interactions are R105H-N243, T28HY172, Y32H-T180, N33H-P239 / T240, N52H-H229, N55H-S101 / G99, Y33L-P246 / Q247, F37L-Y241, Y54L-G178, and E60L-N179.

[0220] Remarkably, the model shows that acetohydroxamic acid (AHA), a reversible zinc-binding hydroxamate that binds directly to the catalytic groove, can be accommodated in the binding site along with the antibody. Figure 3C presents a diagram of the predicted structure of the MMP-7 / GSM-192 complex, highlighting on the one hand the excellent surface complementarity and on the other hand the small opening through which AHA can insert and bind to MMP-7 in the presence of GSM-192.

[0221] In summary, the proposed binding mode of the antibody consists of hydrophobic anchorage and numerous other contacts, does not involve Zn2+, and allows simultaneous binding of AHA to Zn2+.

[0222] Lentiviral silencing reveals a critical role for MMP-7 in pancreatic cancer cell survival in vitro: Western blot analysis was used to visualize the presence of MMP-7 in pancreatic cancer cell lines AsPC-1, Su86.86, BxPC-3, CFPAC-1. Cell lysates were blotted onto membranes and probed with an anti-MMP-7 commercial antibody (monoclonal mouse IgG2B, R&D systems). Protein levels of active MMP-7, zymogen and intermediate forms were found to vary in the cell lines (Figure 6A). CFPAC-1 and ASPC-1 had high expression of all MMP-7 forms. We tested whether ablation of MMP-7 is, in principle, important for the survival of monocultured pancreatic cancer cells and performed MMP-7 lentiviral silencing in AsPC-1 cells. Silencing resulted in cells that showed an increased sub-G1 peak in propidium iodide (PI) staining and subsequent FACS analysis, indicating increased cell death compared to non-targeted controls (Figure 4A). Thus, pancreatic cancer cells were found to be sensitive to removal of MMP-7.

[0223] Anti-MMP-7 Fab induces pancreatic tumor cell death via apoptosis: To test the effect of antibody-mediated MMP-7 inhibition on pancreatic cancer cell death, 70% confluent cultures were treated with various GSM-192 Ab concentrations for 24 hours. GSM-192 treatment resulted in induction of cell death. Cells were treated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to confirm cell viability and calculate IC50 for standardized treatment in subsequent studies. CFPAC-1 and AsPC-1 PDAC cells were killed with IC50 of 4.34 μM and 2.33 μM, respectively (Figure 6B).

[0224] Anti-LOXL-2 (lysyl oxidase-like 2) mAb33, produced in a similar pipeline, was used as an isotype control. GSM-192 Fab was shown to induce cell death via apoptosis, and the percentage of the cell population undergoing apoptosis increased in response to increasing concentrations of anti-MMP-7 treatment (Figure 4B). To test for molecular markers of apoptosis, Fas Ligand expression was analyzed by Western blot analysis. AsPC-1 cells were treated with GSM-192 Fab at sublethal concentrations for 24 hours. Indeed, these cells showed increased Fas Ligand (FasL) expression compared to untreated controls (Figure 4C).

[0225] GSM-192 Fab reduces cell motility in vitro: MMP-7-mediated E-cadherin cleavage promotes cell migration 19 Therefore, it was hypothesized that an effective anti-MMP-7 antibody against the activating enzyme should be able to affect migration. In a standard scratch assay (Figure 5A), GSM-192 treatment did not completely stop cell migration, but did show a significant reduction in the rate of wound closure (when controlling for antiproliferative effects using mitomycin C on CFPAC-1 cells in previous experiments, data not shown). Indeed, GSM-192 treatment at sublethal concentrations reduced by almost half the number of cells able to migrate across the transwell membrane after 15 hours of treatment (Figure 5B).

[0226] Evaluate the ability of native MMP7 in RIP1-Tag2 mouse serum to bind to anti-MMP-7 antibodies: Anti-MMP-7 mAb was found to effectively bind low concentrations of MMP-7 catalytic enzyme. Serum isolated from RIP1-Tag2 mice with late-stage insulinoma was used to determine whether anti-MMP-7 mAb is bound and cleared by the circulating enzyme. Very high concentrations of serum loaded onto dot blot membranes did not show any binding of anti-MMP-7 mAb (Figure 8). The total serum protein loaded for this experiment was 335-fold the protein concentration used for the pure MMP-7 catalytic control. The selective binding of anti-MMP-7 antibodies to the catalytic form demonstrated hereinabove may be the reason for no observed binding to the circulating enzyme, which exists predominantly in its zymogen form.

[0227] GSM-192 treatment reduces the development of advanced tumor lesions in early-treated RIP1-Tag2 (PanNET) mice: RIP1-Tag2 mice (n=15) at the transition stage from hyperplasia to the neovascularization stage ranging from 6 to 8 weeks of age were treated intraperitoneally (IP) with GSM-192 and GST control antibody at a concentration of 3 mg / kg daily. After 8 weeks of treatment, animals were sacrificed and their pancreases were embedded in paraffin blocks and sectioned. Several sections at different depths in the tissue were made for subsequent histological analysis or immunofluorescence staining. Tumor size and staging were performed according to the RIP1-Tag2 manual. This treatment was found to reduce the progression of islets towards late-stage neovascularization; in other words, the percentage of islets progressing to post-vascularization lesions was reduced by almost half in the treated group (Figure 9A). As previously observed in in vitro cell cultures, the tissues were examined for differences in cell proliferation rates (using the cell division marker Ki-67) and cell apoptosis rates (using the effector apoptosis marker caspase-3). No significant changes in the number of cells undergoing apoptosis or cell proliferation were observed (Figure 9B). A significant change in the area covered by CD34+ cells was observed, indicating that neovascular progenitor cells were less abundant in the treated group compared to the control Ab group.

[0228] Late-stage treatment shows sustained effects on tumor volume in the RIP1-Tag2 (PanNET) mouse model: Late-stage tumors in 10-12 week-old RIP1-Tag2 mice (n=15) after exponential growth and angiogenesis were treated with GSM-192 and GST control antibody at a concentration of 5 mg / kg every other day by intraperitoneal (IP) injection. Total tumor burden, calculated as tumor volume, was significantly reduced in the GSM-192-treated group (Figure 9C). Tumor volume was measured using x, y, and z dimensions from a caliper scale. Tumor volume in the GSM-192-treated group was less than half that of the untreated group (approximately 60 mm 3 ~24mm 3 ) Thus, the role of MMP-7 is important for late- and early-stage tumor growth through overlapping or distinct mechanisms.

[0229] Tube formation by HUVEC cells was disrupted when treated with GSM-192: Human umbilical vein endothelial cells (HUVECs) were cultured in Matrigel for analysis of angiogenic tube formation. TM or similar ECM substrates. The formation of tubes or capillary-like structures by HUVECs is a measure of the promotion or reduction of angiogenic potential. After 8 hours of incubation with anti-MMP-7 mAb GSM-192, both the number of branching points / nodes and the number of closed loops under phase contrast inverted microscope were found to be significantly decreased compared to control Ab-treated HUVECs. The number of closed loops decreased from an average of 99 loops in the GST control Ab-treated group to an average of 78 loops in the GSM-192 wells (Figure 10A). The number of branching points also decreased from an average of 107 to 83 nodes in the treated group.

[0230] Aortic ring sprouting assay showed a significant reduction in neovascular sprouts with GSM-192 treatment: The aortic ring assay is an ex vivo assay to study angiogenic potential without cell dissociation and provides a more complete depiction of the complex events that lead to microvessel formation. Eight-week-old mouse aortas embedded in type 1 collagen were treated with GSM-192 and control GST mAb for 7 days. Fixed samples were stained with crystal violet and imaged under a microscope. The number of vascular sprouts was quantified. The average number of sprouts was significantly higher in the control mAb-treated samples than in the GSM-192-treated samples (Figure 10B). The number of sprouts decreased from an average of approximately 73 sprouts in the GST-treated samples to 43 sprouts in the GSM-192 samples.

[0231] Co-treatment with GSM-192 increased the IC 50 Pancreatic ductal adenocarcinoma is inherently capable of strong resistance to chemotherapeutic agents. Dysregulation of the Wnt / β-catenin pathway, counting MMP-7 as a downstream target gene, is involved in the acquisition of this drug resistance. MMP-7, via the release of Fas receptor, has been shown to be involved in the reduced sensitivity to the chemotherapeutic drug oxaliplatin. We confirmed the efficacy of GSM-192 in rescuing PDAC AsPC-1 cells from MMP-7-mediated drug resistance. Treatment of AsPC-1 cells reduced the IC of the GEM+PBS group to 126.6 ± 40.5 nM. 50 Co-treatment with 2.33 uM GSM-192 and various concentrations of GSM-192 resulted in IC values ​​of GEM up to 14.2 ± 1.52 nM. 50 The results showed a significant decrease in the expression of GEM in cancer cells (Figures 11A-B). GSM-192 increases the sensitivity of cancer cells to GEM in vitro by inactivating MMP-7. REFERENCES 1. White, RJ, Margolis, PS, Trias, J. & Yuan, Z. Targeting metalloenzymes: a strategy that works. Curr Opin Pharmacol 3, 502-7 (2003). 2. Aharoni, A. et al. The 'evolvability' of promiscuous protein functions. Nat Genet 37, 73-6 (2005). 3. Bonnans, C., Chou, J. & Werb, Z. Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 15, 786-801 (2014). 4. Shiomi, T. & Okada, Y. MT1-MMP and MMP-7 in invasion and metastasis of human cancers. Cancer Metastasis Rev 22, 145-52 (2003). 5. Jackson, H.W., Defamie, V., Waterhouse, P. & Khokha, R. TIMPs: versatile extracellular regulators in cancer. Nat Rev Cancer 17, 38-53 (2017). 6. Murphy, G. Tissue inhibitors of metalloproteinases. Genome Biol 12, 233 (2011). 7. Jung, Y.S. et al. TIMP-1 induces an EMT-like phenotypic conversion in MDCK cells independent of its MMP-inhibitory domain. PLoS One 7, e38773 (2012). 8. Drews, J. Drug discovery: a historical perspective. Science 287, 1960-4 (2000). 9. Overall, C.M. & Kleifeld, O. Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat Rev Cancer 6, 227-39 (2006). 10. Maurel, J. et al. Serum matrix metalloproteinase 7 levels identifies poor prognosis advanced colorectal cancer patients. Int J Cancer 121, 1066-71 (2007). 11. Wilson, C.L., Heppner, K.J., Labosky, P.A., Hogan, B.L. & Matrisian, L.M. Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin. Proc Natl Acad Sci U S A 94, 1402-7 (1997). 12. Rudolph-Owen, L.A., Chan, R., Muller, W.J. & Matrisian, L.M. The matrix metalloproteinase matrilysin influences early-stage mammary tumorigenesis. Cancer Res 58, 5500-6 (1998). 13. Crawford, H.C., Scoggins, C.R., Washington, M.K., Matrisian, L.M. & Leach, S.D. Matrix metalloproteinase-7 is expressed by pancreatic cancer precursors and regulates acinar-to-ductal metaplasia in exocrine pancreas. J Clin Invest 109, 1437-44 (2002). 14. Wagenaar-Miller, R.A. et al. Cooperative effects of matrix metalloproteinase and cyclooxygenase-2 inhibition on intestinal adenoma reduction. Br J Cancer 88, 1445-52 (2003). 15. Dufour, A. & Overall, C.M. Missing the target: matrix metalloproteinase antitargets in inflammation and cancer. Trends Pharmacol Sci 34, 233-42 (2013). 16. Strand, S. et al. Cleavage of CD95 by matrix metalloproteinase-7 induces apoptosis resistance in tumour cells. Oncogene 23, 3732-6 (2004). 17. Ito, T.K., Ishii, G., Chiba, H. & Ochiai, A. The VEGF angiogenic switch of fibroblasts is regulated by MMP-7 from cancer cells. Oncogene 26, 7194-203 (2007). 18. Patterson, B.C. & Sang, Q.A. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B / type IV collagenase (MMP-9). J Biol Chem 272, 28823-5 (1997). 19. McGuire, J.K., Li, Q. & Parks, W.C. Matrilysin (matrix metalloproteinase-7) mediates E-cadherin ectodomain shedding in injured lung epithelium. Am J Pathol 162, 1831-43 (2003). 20. McCawley, L.J. & Matrisian, L.M. Matrix metalloproteinases: they're not just for matrix anymore! Curr Opin Cell Biol 13, 534-40 (2001). 21. Lynch, C.C. et al. Matrix metalloproteinase 7 mediates mammary epithelial cell tumorigenesis through the ErbB4 receptor. Cancer Res 67, 6760-7 (2007). 22. Zhang, Q. et al. Interleukin-17 promotes prostate cancer via MMP7-induced epithelial-to-mesenchymal transition. Oncogene (2016). 23. Brabletz, T., Jung, A., Dag, S., Hlubek, F. & Kirchner, T. beta-catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer. Am J Pathol 155, 1033-8 (1999). 24. Wang, S.C. et al. A Pilot Study Evaluating Serum MMP7 as a Preoperative Prognostic Marker for Pancreatic Ductal Adenocarcinoma Patients. J Gastrointest Surg 20, 899-904 (2016). 25. Park, H.D. et al. Serum CA19-9, cathepsin D, and matrix metalloproteinase-7 as a diagnostic panel for pancreatic ductal adenocarcinoma. Proteomics 12, 3590-7 (2012). 26. Kuhlmann, K.F. et al. Evaluation of matrix metalloproteinase 7 in plasma and pancreatic juice as a biomarker for pancreatic cancer. Cancer Epidemiol Biomarkers Prev 16, 886-91 (2007). 27. Tamburrino, D. et al. Selection criteria in resectable pancreatic cancer: a biological and morphological approach. World J Gastroenterol 20, 11210-5 (2014). 28. Chari, S.T. et al. Early detection of sporadic pancreatic cancer: summative review. Pancreas 44, 693-712 (2015). 29. Sela-Passwell, N. et al. Antibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential. Nat Med 18, 143-7 (2011). 30. Knight, C.G., Willenbrock, F. & Murphy, G. A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases. FEBS Lett 296, 263-6 (1992). 31. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307-26 (1997). 32. Ben-Shimon, A. & Eisenstein, M. Computational mapping of anchoring spots on protein surfaces. J Mol Biol 402, 259-77 (2010). 33. Grossman, M. et al. Tumor Cell Invasion Can Be Blocked by Modulators of Collagen Fibril Alignment That Control Assembly of the Extracellular Matrix. Cancer Res 76, 4249-58 (2016). 34. Page-McCaw, A., Ewald, A.J. & Werb, Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 8, 221-33 (2007). 35. Egeblad, M. & Werb, Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2, 161-74 (2002). 36. Fukuda, A. et al. Stat3 and MMP7 contribute to pancreatic ductal adenocarcinoma initiation and progression. Cancer Cell 19, 441-55 (2011). 37. Almendro, V. et al. The role of MMP7 and its cross-talk with the FAS / FASL system during the acquisition of chemoresistance to oxaliplatin. PLoS One 4, e4728 (2009). 38. Ametller, E. et al. Differential regulation of MMP7 in colon cancer cells resistant and sensitive to oxaliplatin-induced cell death. Cancer Biol Ther 11, 4-13 (2011). 39. Terwilliger, T.C. Maximum-likelihood density modification. Acta Crystallogr D Biol Crystallogr 56, 965-72 (2000). 40. French, S. & Wilson, K. On the treatment of negative intensity observations. Acta Crystallographica Section A: Crystal Physics, Diffraction, Theoretical and General Crystallography 34, 517-525 (1978). 41. McCoy, A.J. et al. Phaser crystallographic software. J Appl Crystallogr 40, 658-674 (2007). 42. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr D Biol Crystallogr 55, 849-61 (1999). 43. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-32 (2004). 44. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. Journal of applied crystallography 26, 283-291 (1993). 45. Suhre, K. & Sanejouand, Y.H. ElNemo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement. Nucleic Acids Res 32, W610-4 (2004). 46. Katchalski-Katzir, E. et al. Molecular surface recognition: determination of geometric fit between proteins and their ligands by correlation techniques. Proc Natl Acad Sci U S A 89, 2195-9 (1992). 47. Heifetz, A., Katchalski-Katzir, E. & Eisenstein, M. Electrostatics in protein-protein docking. Protein Sci 11, 571-87 (2002). 48. Berchanski, A., Shapira, B. & Eisenstein, M. Hydrophobic complementarity in protein-protein docking. Proteins 56, 130-42 (2004). 49. Kowalsman, N. & Eisenstein, M. Combining interface core and whole interface descriptors in postscan processing of protein-protein docking models. Proteins 77, 297-318 (2009). 50. Kowalsman, N. & Eisenstein, M. Inherent limitations in protein-protein docking procedures. Bioinformatics 23, 421-6 (2007). 51. Pettersen, EF et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25, 1605-12 (2004). 52. Yee, JK, Friedmann, T. & Burns, JC Generation of high-titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol 43 Pt A, 99-112 (1994). 53. Vermes, I., Haanen, C., Steffens-Nakken, H. & Reutelingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J Immunol Methods 184, 39-51 (1995).

[0232] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0233] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, citation or identification of a reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting.

[0234] Additionally, the priority document(s) of this application are incorporated herein by reference in their entirety.

Claims

1. A pharmaceutical composition for use in treating pancreatic adenocarcinoma, comprising an antibody having an antigen recognition region that binds to the catalytic site of MMP-7, the antibody having the amino acid sequences of complementarity determining regions (CDRs) shown in SEQ ID NOs: 3 (CDR1), 4 (CDR2) and 5 (CDR3) arranged in N to C order on the antibody light chain, and SEQ ID NOs: 6 (CDR1), 7 (CDR2) and 8 (CDR3) arranged in N to C order on the antibody heavy chain.

2. The pharmaceutical composition of claim 1 , wherein the antibody has a VL amino acid sequence as shown in SEQ ID NO:

1.

3. The pharmaceutical composition of claim 1 , wherein the antibody has a VH amino acid sequence as shown in SEQ ID NO:

2.

4. The pharmaceutical composition of any of claims 1 to 3, wherein the antibody is attached to a detectable or therapeutic moiety.

5. The pharmaceutical composition of any one of claims 1 to 4, further comprising a chemotherapeutic agent.

6. A kit for use in treating pancreatic adenocarcinoma, comprising a pharmaceutical composition containing an antibody having an antigen recognition region that binds to the catalytic site of MMP-7, the antibody having the amino acid sequences of complementarity determining regions (CDRs) shown in SEQ ID NOs: 3 (CDR1), 4 (CDR2) and 5 (CDR3) arranged in N to C order on the antibody light chain, and SEQ ID NOs: 6 (CDR1), 7 (CDR2) and 8 (CDR3) arranged in N to C order on the antibody heavy chain, and a chemotherapeutic agent.

7. 7. The kit of claim 6, wherein the chemotherapeutic agent is a nucleoside analog selected from the group consisting of gemcitabine, cytosine arabinoside, and behenoyl cytosine arabinoside.

8. The kit of claim 7, wherein the nucleoside analog is gemcitabine.

9. The kit of claim 6, wherein the chemotherapeutic agent is oxaliplatin.

10. The kit of claim 6 , wherein the antibody and the chemotherapeutic agent are formulated in a single composition.

Images