Matrix metalloproteinase inhibitors and uses thereof

Abstract

The present invention provides specific matrix metalloproteinase (MMP) polypeptide inhibitors and uses thereof in treating MMP-related diseases, in particular cancer, such as breast cancer and glioblastoma.

Description

[0001] MATRIX METALLOPROTEINASE INHIBITORS AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to therapeutic polypeptides, specifically Matrix metalloproteinase (MMP) inhibitors, and their use in treating MMP-9-related diseases, in particular cancer.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under CA258274 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0006] Matrix Metalloproteinases (MMPs) are a family of zinc-dependent, calcium- containing, endopeptidases that degrade extracellular matrix (ECM) proteins as well as regulate the activity of other proteinases, growth factors, cytokines, chemokines, and cell receptors (Cui, N., et al., (2017) Prog. Mol. Biol. Transl. Sci. 147: 1-73). In humans, the MMP family comprises 23 different enzymes. MMPs are commonly classified based on their structure and substrate preferences into 5 subgroups: collagenases, gelatinases, matrilysins, stromelysins, and membrane-type (MT) MMPs.

[0007] Generally, MMPs are multidomain proteins with similar, yet not identical structures. All MMPs are expressed with a pro-domain, which keeps the MMP inactive until it is cleaved by another protease. Additionally, all MMPs share a highly conserved catalytic domain with a zinc ion in the active site. Additional domains are present only in some MMPs, including a transmembrane domain in MT-MMPs, a hemopexin-like domain in some MMPs, and a fibronectin domain in gelatinases; these domains are important for substrate recognition (Laronha, H. and Caldeira, J. (2020) Cells 9, 1076; Zitka, O. et al., (2010) Curr. Med. Chem. 17, 3751-3768).

[0008] MMPs mediate various functions of cell signaling and tissue homeostasis and are important for tissue remodeling, wound healing, embryogenesis, morphogenesis, and other physiological processes. However, as MMPs are capable of degrading extracellular matrix (ECM) proteins, their synthesis and activity are kept under tight control. Dysregulation of MMPs contributes to several pathological conditions including tissue destruction, fibrosis, and cancer progression and metastasis (Nagase, H., et al., (2006) Cardiovasc. Res. 69, 562-573; Cabral-Pacheco, G. A. et al., (2020) Int. J. Mol. Sci. 21, 9739). In cancer, MMP overexpression is well documented in various cancer types and high levels of some MMPs are correlated with tumor aggressiveness and poor prognosis (Gobin, E. et al., (2019) BMC Cancer 19, 581; Winer, A., et al., (2018) Mol. Cancer Ther. 17, 1147-1155; Hadler-Olsen, E., et al., (2013) Tumor Biology, 34, 2041-2051). While many MMPs exhibit cancer-promoting effects, including for example, MMP-9 and MMP- 14, it has become apparent that some MMPs such as MMP-8 possess anti-tumorigenic effect.

[0009] Many MMP inhibitors (MMPIs) have been developed in the last years, yet despite promising preclinical data, most MMPIs failed clinical trials, and there is currently one FDA-approved MMPI, Periostat, for periodontal disease treatment (Fields, G. B. (2019) Cells, 8, 984).

[0010] The failure of the MMPIs in clinical trials is owing to several reasons, one of which is serious side effects, due to unselective inhibition. Initial strategies for MMPI design focused on targeting the catalytic zinc, which is necessary for MMP activity by small molecules with strong Zn+2 chelating properties. The poor selectivity of these broad- spectrum inhibitors resulted in serious side effects such as, for example, musculoskeletal syndrome and gastrointestinal disorders that led to discontinuing and failing clinical trials (Vandenbroucke, R. E. and Libert, C., (2014) Nat. Rev. Drug Discov., 13, 904-927). The risk of non-specific inhibition is particularly pronounced in light of the different effects of each MMP on cancer development.

[0011] The high sequence and structural homology within the MMP catalytic domain make the development of selective inhibitors challenging. Several strategies have recently been implemented in an attempt to overcome this difficulty. One strategy involves the development of specific inhibitory anti-MMP antibodies. The antibody is designed to have an epitope that is specific to a single MMP isoform and thereby, bind and inhibit a particular MMP (Fischer, T. and Riedl, R. (2019) Molecules, 24, 2265; Goldberg, G. I. (2015) Structure, 23, 6-7).

[0012] Another strategy for MMPI design is based on engineering of Tissue Inhibitors of Metalloproteinases (TIMPs). TIMPs are natural endogenous metalloproteinases inhibitors of the MMP family and are tight regulators of ECM remodeling. The TIMP family consists of four members (TIMP1-TIMP4) that inhibit MMPs in 1: 1 stoichiometry by binding to their active site and the neighboring areas utilizing the TIMP N-terminal residues and two adjacent loops. TIMPs are made up of two domains, an N-terminal domain of 126 residues and a smaller C-terminal domain. The N-terminal domain folds if produced independently and is capable of inhibiting MMPs with an inhibitory constant (Ki) in the lower nanomolar range (Raffetto, J. D. and Khalil, R. A. (2008) Biochem. Pharmacol., 75, 346-359; Eckfeld, C., HauBler, et al., (2019) Cancer and Metastasis Reviews, 38, 469-481). All TIMPs bind to MMPs non- specifically, inhibiting most family members with similar inhibitory constants. Recently, TIMP2 was shown to exert an inhibitory effect on tumor growth and metastasis in animal models when administered exogenously (Peeney, D. et al., (2020) Carcinogenesis, 41, 313— 325).

[0013] Yosef, G. et al. (Jour. Bio. Chem., 293, 13310-13326 (2018)), discloses protein inhibitors targeting the collaborative activity of MMP-14 and integrin avP3, which promote cancer progression. A bi-specific heterodimer was produced that binds both targets, intended to inhibit MMP-2 activation, MMP-14-, MMP-2-, and integrin avp3- dependent glioblastoma cell invasion, and endothelial cell invasiveness and capillary tube formation.

[0014] Arkadash, V. et al. (Jour. Bio. Chem., 292, 3481-3495 (2017)), discloses a selective inhibitor for MMP-14 enzyme. A modified N-TIMP2 protein was engineered having five mutations relative to the WT, said mutations based on computational methods combined with yeast surface display technique. This variant exhibited a 900- fold improved affinity for MMP-14 and up to 16,000-fold greater specificity compared to other MMPs.

[0015] Shirian, J. et al. (FEBS Lett., 592, 1122-1134 (2018)), to some of the present inventors, discloses two engineered N-TIMP2 variants, 9D_2 and 9D_6, that exhibited up to 2000-fold enhanced selectivity toward MMP-9 compared to other MMPs. However, the affinity of these engineered N-TIMP2 mutants to MMP-9 was similar to, or even slightly worse, than that of wild-type N-TIMP2.

[0016] There is an unmet need to provide means and methods, which are safe and effective, for treating diseases associated with aberrant MMP activity, such as cancer.

[0017] SUMMARY OF THE INVENTION

[0018] The present invention provides in certain aspects polypeptides that inhibit Matrix metalloproteinases (MMPs) and use thereof in treating MMP-related diseases, particularly cancer. The polypeptides are derived from Tissue Inhibitor of Metalloproteinases 2 (TIMP2) and function as highly specific inhibitors of MMP-9, an enzyme that plays a critical role in cell invasion and metastasis.

[0019] It is now disclosed that the polypeptides described herein may be used in the treatment of MMP-9-related diseases, in particular in anti-tumor therapy. Unexpectedly and advantageously, the polypeptides of the invention exhibit increased affinity for MMP-9 compared to the wild- type N-TIMP2 and further demonstrates significantly higher binding specificity for MMP-9 over five other MMPs, thereby enabling precise targeting of MMP-9 and enhancing therapeutic potential.

[0020] The present invention is based in part on the unexpected finding that the polypeptides described herein showed a significantly greater inhibition of MDA-MB- 231 breast cancer cell invasion and proliferation compared to the wild-type N-TIMP2.

[0021] According to certain aspects, the present invention provides a polypeptide of 58-200 amino acids comprising an amino acid sequence selected from (i) SEQ ID NO: 1; (ii) a fragment comprising residues 14-70 of SEQ ID NO: 1; or (iii) an analog of SEQ ID NO: 1 having 1-10 deletions, additions and / or substitutions of amino acid residues other than residues 14, 69 and 70 of SEQ ID NO: 1, wherein the polypeptide is capable of binding to matrix metalloproteinase 9 (MMP-9).

[0022] According to some embodiments, the polypeptide comprises SEQ ID NO: 20. According to some embodiments, the polypeptide comprises SEQ ID NO: 20 wherein Xi is arginine, X2 is glutamic acid or threonine and X3 is tyrosine or tryptophane. According to some embodiments, the polypeptide comprises SEQ ID NO: 20 wherein Xi is arginine, X2 is glutamic acid and X3 is tyrosine. According to some embodiments, the polypeptide comprises SEQ ID NO: 20 wherein Xi is arginine, X2 is threonine and X3 is tryptophane.

[0023] According to some embodiments, the polypeptide comprises an arginine residue at a position corresponding to position 14 of SEQ ID NO: 1; a residue selected from the group consisting of threonine and glutamate at a position corresponding to position 69 of SEQ ID NO: 1; and a residue selected from the group consisting of tryptophane and tyrosine at a position corresponding position 70 of SEQ ID NO: 1.

[0024] According to some embodiments, the polypeptide comprises up to 190, 180, 170, 160 150, or 140 amino acids. According to some embodiments, the polypeptide comprises 100-150 amino acids. According to some embodiments, the polypeptide comprises 120-135 amino acids. According to some embodiments, the polypeptide comprises 124-130 amino acids. According to some embodiments, the polypeptide comprises about 127 amino acids.

[0025] According to some embodiments, the polypeptide analog comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. Each possibility represents a separate embodiment of the invention.

[0026] According to some embodiments, the polypeptide analog comprises 1-2, 1-3, 1- 4, or 1-5 substitutions compared to SEQ ID NO: 1. According to some embodiments, the analog comprises 1, 2, 3, 4, or 5 substitutions compared to SEQ ID NO: 1. Each possibility represents a separate embodiment of the invention. It is to be understood that the possible substitutions are not at positions corresponding to residues 14, 69 and 70 of SEQ ID NO: 1. According to some embodiments, one or more of the substitutions are conservative substitutions. According to certain embodiments, the substitutions are conservative substitutions.

[0027] According to some embodiments, the analog is a polypeptide comprising up to 10 chemical modifications. According to some embodiments, the analog is a polypeptide comprising 1-3 or 1-5 chemical modifications.

[0028] According to certain exemplary embodiments, the polypeptide comprises amino acid sequence SEQ ID NO: 1.

[0029] According to some embodiments, the polypeptide comprises: arginine at a position corresponding to position 14 of SEQ ID NO: 1; glutamate at a position corresponding to position 69 of SEQ ID NO: 1; and tyrosine at a position corresponding to position 70 of SEQ ID NO: 1. According to some embodiments, the polypeptide comprises SEQ ID NO: 2. According to some embodiments, the polypeptide consists of SEQ ID NO: 2 (denoted herein ‘REY’ mutant or variant).

[0030] According to some embodiments, the polypeptide comprises arginine at a position corresponding to position 14 of SEQ ID NO: 1; threonine at a position corresponding to position 69 of SEQ ID NO: 1; and tryptophane at a position corresponding to position 70 of SEQ ID NO: 1. According to some embodiments, the polypeptide comprises SEQ ID NO: 3. According to some embodiments, the polypeptide consists of SEQ ID NO: 3 (denoted herein ‘RTW’ mutant or variant). The invention encompasses, according to some embodiments, fragments and variants of the disclosed polypeptide, provided that they maintain one or more functional characteristics. According to some embodiments, the fragment comprises at least 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110 or more amino acid. Each possibility represents a separate embodiment of the invention.

[0031] According to some embodiments, the polypeptide is capable of inhibiting MMP-9 activity. According to certain embodiments, the polypeptide is capable of reducing or altering MMP-9 activity. Each possibility represents a separate embodiment of the present invention.

[0032] According to some embodiments, the polypeptide is characterized by higher binding specificity to MMP-9 relative to at least one of MMP-1, MMP-3, MMP-8, MMP-10 and MMP-14. According to certain embodiments, the polypeptide is characterized by higher binding specificity to MMP-9 relative to MMP-1, MMP-3, MMP-8, MMP-10 and MMP-14.

[0033] According to some embodiments, a conjugate or fusion protein comprising the polypeptide described herein is provided.

[0034] According to some embodiments, the conjugate comprises at least one non- proteinaceous moiety. According to some embodiments, the conjugate comprises a detectable moiety. According to certain embodiments, the detectable moiety is selected from the group consisting of a fluorescent label, a radioactive isotope, an enzymatic label, a chromogenic label, and an affinity label. According to certain embodiments, the conjugate comprises one or more stabilizing moieties.

[0035] According to an additional aspect, the present invention provides a polynucleotide comprising a sequence encoding the polypeptide as described herein.

[0036] According some embodiments, a nucleic acid construct comprising the polynucleotide is provided. According to some embodiments the nucleic acid construct is a plasmid.

[0037] The present invention provides, according to another aspect, a pharmaceutical composition comprising the polypeptide described herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0038] According to some embodiments the polypeptide is of 58-200 amino acids comprising an amino acid sequence selected from (i) SEQ ID NO: 1; (ii) a fragment comprising residues 14-70 of SEQ ID NO: 1; or (iii) an analog of SEQ ID NO: 1 having 1-10 deletions, additions and / or substitutions of amino acid residues other than residues 14, 69 and 70 of SEQ ID NO: 1, wherein the polypeptide is capable of binding to matrix metalloproteinase 9 (MMP-9).

[0039] According to some embodiments, the pharmaceutical composition is formulated for injection or infusion. According to some embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intratumoral administration.

[0040] According to some embodiments, the pharmaceutical composition is for use in treating or preventing a disease or pathological condition associated with MMP dysregulation.

[0041] According to some embodiments, the disease or pathological condition is associated with MMP overexpression. According to some embodiments, the disease or pathological condition is associated with increased activity of MMP.

[0042] According to some embodiments, the MMP is MMP-9. According to certain embodiments, the disease is associated with elevated MMP-9 levels.

[0043] According to some embodiments, the disease is selected from the group consisting of cancer, cardiovascular disease, autoimmune disease or disorder, and inflammation. According to some embodiments, the disease is selected from the group consisting of cancer, neurodegeneration, arthritis, cardiovascular and fibrosis. Each possibility represents a separate embodiment of the invention.

[0044] According to some embodiments, the pharmaceutical composition is for use in treating cancer.

[0045] According to some embodiments, the pharmaceutical composition is for use in treating cancer associated with elevated MMP levels. According to some embodiments, the pharmaceutical composition is for use in treating cancer associated with elevated MMP-9 levels.

[0046] According to some embodiments, the cancer comprises a solid tumor.

[0047] According to some embodiments, the cancer is selected from the group consisting of breast cancer, glioblastoma, colorectal cancer, pancreatic cancer, esophageal cancer, prostate cancer, liver cancer, ovarian cancer, endometrial cancer, stomach cancer, thyroid cancer, carcinoid tumor, head and neck cancer, testis cancer, urothelial cancer, cervical cancer, melanoma, lymphoma and lung cancer. Each possibility represents a separate embodiment of the invention. According to some embodiments, the cancer is selected from the group consisting of breast cancer and glioblastoma. According to some embodiments, the breast cancer is triple negative breast cancer (TNBC).

[0048] According to another aspect, the present invention provides a method of treating an MMP-9 related disease or condition comprising administering to a subject in need thereof, a therapeutically effective amount of at least one polypeptide or a pharmaceutical composition comprising same as described herein.

[0049] The disease, polypeptide and pharmaceutical composition are as described hereinabove. In certain embodiments, the disease or condition is characterized by aberrant overexpression of MMP-9.

[0050] According to yet another aspect, the present invention provides a method of treating cancer comprising administering to a subject in need thereof, a therapeutically effective amount of at least one polypeptide or a pharmaceutical composition comprising same as described herein.

[0051] According to some embodiments, the subject is human.

[0052] According to some embodiments, the method of treating cancer comprises administering or performing at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiotherapy, or immunotherapy.

[0053] According to some embodiments, the method of treating cancer comprises administering the polypeptide described herein and an anti-cancer agent. According to some embodiments, the anti-cancer agent is selected from the group consisting of immune-modulator, activated lymphocyte cell, kinase inhibitor and chemotherapeutic agent.

[0054] According to some embodiments, the method of treating cancer involves preventing or reducing formation, growth or spread of metastases in a subject.

[0055] According to another aspect, the present invention provides a method of detecting a cell that is overexpressing MMP-9 by contacting a cell with at least one of the polypeptides described herein.

[0056] According to some embodiments, the method is of detecting cancer cells associated with MMP-9 overexpression.

[0057] According to certain embodiments, the polypeptide is labeled. According to some embodiments, the polypeptide is fluorescently labeled. According to certain embodiments, the method comprises the steps of (i) providing a biological sample comprising cells; (ii) contacting the labeled polypeptide with the biological sample; (iii) washing unbound polypeptide from the biological sample; and (iv) detecting bound polypeptide.

[0058] According to another aspect, the present invention provides a kit for detecting the presence of MMP-9 in a biological sample, the kit comprises a conjugate comprising at least one polypeptide as described herein and a detectable moiety.

[0059] According to some embodiments, the detectable moiety is selected from the group consisting of fluorescent dyes, radioisotopes, epitope tags, biotin, and nanoparticles.

[0060] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0061] BRIEF DESCRIPTION OF THE FIGURES

[0062] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the characteristics and advantages of the features described herein will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the features described herein are utilized, and the accompanying drawings of which:

[0063] Figures 1A-1C show results of a sorting assay performed on a library of N- TIMP2 variants selected according to binding affinity to MMP-9 protein, the N-TIMP2 variants derived from the 9D-2 variant, including randomized mutations at positions 14, 69 and 70, and expressed on yeast cells, where: Figure 1A is an illustration of the Myc-tagged N-TIMP2 variant expressed on the yeast cell and the MMP-9E402Q protein which it potentially binds (marked by Daylight-488 fluorophore); Figure IB is a representation of the FACS sorting process, including the progression through sorting rounds SI, S2, and S3 to eventual protein sequencing, and the respective FACS results (histograms) of each of the sorting rounds; and Figure 1C is a WebLogo visual depiction for the three randomized amino acid positions of the unique sequences found in the S3 library.

[0064] Figure 2 is a graph representation of the results of an enzyme activity assay, measuring the activity of MMP-9 as a function of the concentration of N-TIMP2 variants added to the medium thereof, for each of WT, 9D-2, RTW and REY N-TIMP2 variants.

[0065] Figures 3A-3C are graph representations of the results of enzyme activity assays, measuring the activity of MMP-1, MMP-3, MMP-8, MMP-9, MMP-10, and MMP-14 proteins, as a function of the concentration of different N-TIMP2 variants, where: Figure 3A shows the results for WT N-TIMP2; Figure 3B shows the results for REY mutated N-TIMP2; and Figure 3C shows the results for RTW mutated N-TIMP2.

[0066] Figure 4 is a bar plot representing the cell proliferation of MDA-MB-231 breast cancer cells 3 days after treatment with either 100 nM, 300 nM, or 600 nM of either mutated REY or WT N-TIMP2 variants, the proliferation levels represented as a percent of proliferation of untreated cells which were set as 100% (control) (at least 3 repeats; *** indicate a P-value of <0.001 in the standard T-test).

[0067] Figures 5A-5B show results of treatment of MDA-MB-231 breast cancer with mutated REY or WT N-TIMP2 variants, testing the inhibitory effect thereof on cancer cell invasion using Matrigel transwell assay, where: Figure 5A is an image of the MDA- MB-231 cells 24 hours after treatment with either 100, 300, and 600 nM of either REY or WT N-TIMP2, as indicated in the figure (untreated cells control, scale bar = 100 pm); and Figure 5B is a bar plot representing the percent of cell invasion under each treatment, with the control cells set at 100% cell invasion (ImageJ quantification of the cells; at least 2 repeats; ** and *** indicate a P-value of < 0.01 and <0.001, respectively).

[0068] Figures 6A-6B show results of treatment of U251-MG adult glioma cancer cells with mutated REY or WT N-TIMP2 variants, testing the inhibitory effect thereof on cancer cell invasion using Matrigel transwell assay, where: Figure 6A is an image of the U251-MG cells 24 hours after treatment with either 100, 300, and 600 nM of either REY or WT N-TIMP2, as indicated in the figure (untreated cells control); and Figure 6B is a bar plot representing the percent of inhibition of cell invasion under each treatment, with the control cells set at 0% inhibition (i.e., 100% cell invasion) (ImageJ quantification of the cells; at least 2 repeats; ** and *** indicate a P-value of < 0.01 and <0.001, respectively).

[0069] DETAILED DESCRIPTION OF THE INVENTION

[0070] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0071] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0072] The present invention provides polypeptide inhibitors that are specific to Matrix metalloproteinases 9 (MMP-9) and uses thereof in the treatment of MMP-related diseases or disorders, in particular cancer. The polypeptides of the invention, derived from the TIMP2 protein, were shown to have higher affinity to MMP-9 compared to WT N-TIMP2 (N-terminal domain of TIMP2) and between 6- to 1. Ixl04-fold enhanced binding specificity toward MMP-9 relative to five alternative MMPs, demonstrating superior results compared to previously engineered mutants. It is now disclosed that cell-based assays performed with triple-negative breast cancer MDA-MB-231 cells demonstrated that treatment with nanomolar concentrations of the polypeptides described herein resulted in greater than 90% inhibition of cell invasion and proliferation, without inducing cytotoxicity. Accordingly, in some aspects, the polypeptides described herein provide novel compounds for the treatment or prevention of MMP-9-associated diseases.

[0073] Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are crucial for breaking down components of the extracellular matrix (ECM). They are involved in various physiological and pathological processes, including tissue remodeling, wound healing, angiogenesis, and cancer metastasis. MMPs are secreted as inactive zymogens and are activated by the removal of a pro- peptide domain. Their activity is tightly regulated by tissue inhibitors of metalloproteinases (TIMPs) and other cellular mechanisms.

[0074] MMP-9, also known as gelatinase B, is a key member of the MMP family. It's a zinc-dependent enzyme primarily known for its ability to degrade denatured collagen (gelatin), as well as native type IV and V collagen, which are major components of the basement membrane. MMP-9 plays a significant role in both normal physiological processes, like bone development and angiogenesis, and in pathological conditions, such as inflammation and cancer invasion. Its expression is often upregulated in various cancers, where it facilitates tumor growth and metastasis by degrading the basement membrane and promoting the release of growth factors. An example of MMP-9 protein sequence is GeneBank Accession: CAC07541.1.

[0075] TIMP2 (Tissue Inhibitor of Metalloproteinase-2) is a protein that inhibits matrix metalloproteinases to regulate the extracellular matrix (ECM). TIMP2 also has an MMP-independent function of suppressing endothelial cell proliferation, which is crucial for tissue homeostasis and preventing excessive growth of blood vessels (angiogenesis). TIMP2 is a non-glycosylated protein with a molecular weight of about 21-22 kDa and plays a role in preventing tumorigenesis and maintaining tissue structure. N-TIMP2 (N-terminal domain of TIMP2) refers specifically to the N-terminal portion of the TIMP2 protein. This domain is primarily responsible for the inhibitory activity of TIMP2 against MMPs.

[0076] According to certain aspects, the present invention provides a polypeptide of 58-200 amino acids comprising an amino acid sequence selected from (i) SEQ ID NO: 1; (ii) a fragment comprising residues 14-70 of SEQ ID NO: 1; or (iii) an analog of SEQ ID NO: 1 having 1-10 deletions, additions and / or substitutions of amino acid residues other than residues 14, 69 and 70 of SEQ ID NO: 1, wherein the polypeptide is capable of binding to matrix metalloproteinase 9 (MMP-9).

[0077] According to some embodiments, the polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the amino acid residue corresponding to position 14 of SEQ ID NO: 1 is selected from the group consisting of arginine and lysine; the amino acid residue corresponding to position 69 of SEQ ID NO: 1 is selected from the group consisting of threonine and glutamate; and the amino acid residue corresponding to position 70 of SEQ ID NO: 1 is selected from the group consisting of tryptophane, tyrosine, and phenylalanine; or a fragment, analog or derivative thereof.

[0078] According to some embodiments, the polypeptide comprises arginine at a position corresponding to position 14 of SEQ ID NO: 1; glutamate at a position corresponding to position 69 of SEQ ID NO: 1; and tyrosine at a position corresponding to position 70 of SEQ ID NO: 1. According to some embodiments, the polypeptide comprises SEQ ID NO: 2 (denoted herein ‘REY’ mutant or variant).

[0079] According to some embodiments, the polypeptide comprises arginine at a position corresponding to position 14 of SEQ ID NO: 1; threonine at a position corresponding to position 69 of SEQ ID NO: 1; and tryptophane at a position corresponding to position 70 of SEQ ID NO: 1. According to some embodiments, the polypeptide comprises SEQ ID NO: 3 (denoted herein ‘RTW’ mutant or variant).

[0080] The ‘REY’ and ‘RTW’ variants have 3 mutants compared with ‘9D-2 mutant’, a variant of the wild-type N-TIMP2 that was disclosed in WO2018087757 (as SEQ number 26). The ‘9D-2 mutant’ has five amino acid substitutions compared with the wild-type N-TIMP2. The ‘REY’ and ‘RTW’ variants, in addition to the mutants in positions 14, 69, and 70 of SEQ ID NO: 1 as described herein, have 5 additional substitutions in comparison to wild-type N-TIMP2: S4P, I35V, S68N, V71I, H97R. The wild-type N-TIMP2 sequence is set forth in SEQ ID NO: 18, and the 9D-2 mutant sequence is set forth in SEQ ID NO: 19.

[0081] According to some embodiments, the polypeptide analog of fragment comprises at least one of (i) proline at a position corresponding to position 4 of SEQ ID NO: 1; (ii) valine at a position corresponding to position 35 of SEQ ID NO: 1; (iii) asparagine at a position corresponding to position 68 of SEQ ID NO: 1; (iv) isoleucine at a position corresponding to position 71 of SEQ ID NO: 1; and (v) arginine at a position corresponding to position 97 of SEQ ID NO: 1.

[0082] The terms "polypeptide" and “protein” are used interchangeably herein to refer to a polymer of amino acid residues linked by peptide bonds. The terms apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are an artificial chemical analog of a corresponding naturally occurring amino acid.

[0083] The term “amino acid” is as known in the art and refers to compounds, which have an amino group and a carboxylic acid group, for example in a 1,2- 1,3-, or 1,4- substitution pattern on a carbon backbone. The amino acids include the 20 natural amino acids (which are L-amino acids except for glycine) which are found in proteins as well as selenocysteine and pyrrolysine, the corresponding D-amino acids, the corresponding N-methyl amino acids, side chain modified amino acids, the biosynthetically available amino acids which are not found in proteins (e.g., 4-hydroxy- proline, 5-hydroxy-lysine, citrulline, ornithine, canavanine, djenkolic acid, 0- cy anoalanine), and synthetically derived a-amino acids, such as amino-isobutyric acid, norleucine, norvaline, homocysteine and homoserine. 0-Alanine and y-amino butyric acid are examples of 1,3 and 1,4-amino acids, respectively.

[0084] Within a polypeptide chain, each amino acid is a fundamental building block. Once incorporated into the chain, these amino acids are referred to as residues. The specific location of each residue along the polypeptide chain is its position, which is sequentially numbered from the N-terminus to the C-terminus. This precise numbering provides a consistent and unambiguous way to identify and refer to each specific amino acid residue within the disclosed sequence.

[0085] The polypeptides described herein may be chemically modified. The term "Chemically modified" refers to an amino acid that is modified either by natural processes, or by chemical modification techniques which are well known in the art. Among the numerous known modifications, typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, glycosaminoglycanation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.

[0086] Analogs and derivatives of the polypeptides are also within the scope of the present application.

[0087] The term "Analogs" of the polypeptides of the invention as used herein cover compounds which have the amino acid sequence according to the invention except for one or more amino acid changes, typically, conservative amino acid substitutions. The term also includes deletions or insertions of amino acids. The term “analog" as used herein also covers analogs which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the polypeptide and do not confer toxic properties on compositions containing it and have comparable or improved activity as a wild-type or unmodified polypeptide.

[0088] “Derivatives" of the polypeptides of the invention as used herein cover derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the polypeptide, and do not confer toxic properties on compositions containing it.

[0089] These derivatives may include, for example, aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues, e.g., N-acetyl, formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups), or O-acyl derivatives of free hydroxyl group (e.g., that of seryl or threonyl residues) formed by reaction with acyl moieties.

[0090] The term “derivative” may be used interchangeably with “analog”.

[0091] The term "substitution" or "amino acid substitution" refers to the exchange of an amino acid by another amino acid or by amino acid having a modification at a specific position in the sequence of a parent polypeptide.

[0092] The term “deletion” or “amino acid deletion” refers to the removal of an amino acid at a specific position in a parent polypeptide sequence.

[0093] The term "insertion" or "amino acid insertion" refers to the addition of amino acids into a parent polypeptide sequence.

[0094] According to some embodiments, the analog according to the present invention comprises substitutions, deletions or additions of 1 to 3 amino acids. In some embodiments, the analog has at least about 80% identity to the sequence of the polypeptide of the invention, for example at least about 85%, at least about 90%, at least about 93%, at least about 94%, at least about 95%, 96%, 97%, 98%, or 99% identity to the sequence of the polypeptide of the invention. Each possibility represents a separate embodiment of the invention.

[0095] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0096] Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged. One of skill will recognize that individual substitutions, deletions or additions to polypeptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0097] The following six groups each contain amino acids that are conservative substitutions for one another:

[0098] 1) Alanine (A), Serine (S), Threonine (T);

[0099] 2) Aspartic acid (D), Glutamic acid (E);

[0100] 3) Asparagine (N), Glutamine (Q);

[0101] 4) Arginine (R), Lysine (K), Histidine (H);

[0102] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0103] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

[0104] Analogs according to the present invention may comprise also peptidomimetics. “Peptidomimetic” means that a polypeptide according to the invention is modified in such a way that it includes at least one non-coded residue or non-peptidic bond. Such modifications include, e.g., alkylation and more specific methylation of one or more residues, insertion of or replacement of natural amino acid by non-natural amino acids, replacement of an amide bond with another covalent bond. A peptidomimetic according to the present invention may optionally comprise at least one bond which is an amide replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. The design of appropriate analogs may be computer assisted. Analogs are included in the invention as long as they remain pharmaceutically acceptable and their activity is not damaged.

[0105] "Salts" of the polypeptides of the invention are also included in its scope and are organic and inorganic salts. As used herein the term “salts” refers to both salts of carboxyl groups and to acid addition salts of amino or guanidino groups of the polypeptide molecule. Salts of carboxyl groups may be formed by means known in the art and include inorganic salts, for example sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as salts formed for example with amines such as triethanolamine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, acetic acid or oxalic acid.

[0106] According to some embodiments, the polypeptide analog comprises at least one modification relative to SEQ ID NO: 1, said at least one modification is selected from the group consisting of: deletion, substitution, methylation or addition of amino acids, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0107] According to some embodiments, the analog is a polypeptide comprises up to 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid modifications selected from substitutions, additions, or deletions of amino acids, modification of at least one of the termini of the polypeptide, and combinations thereof.

[0108] According to some embodiments, the analog is a polypeptide comprises up to 10, 9, 8, 7, 6, 5, 4, 3, or 2 chemical modifications. According to some embodiments, the analog is a polypeptide comprises 1-3, 1-4, 2-4, or 1-5 chemical modifications.

[0109] For the purposes of the present invention, a reference to an amino acid at a particular position in a polypeptide, such as position or residue 14 of SEQ ID NO: 1 is intended to encompass any residue that corresponds to that position when the polypeptide is aligned with the reference sequence as is known in the art. As such, additions or deletions of one or more amino acids at the N-terminus, C-terminus, or within the polypeptide sequence do not affect the correspondence of the position. Residues in variants, fragments, or analogs of the polypeptide that align with the designated position in the reference sequence are considered to occupy the corresponding position. Numbering of residues in all polypeptides described herein may thus be determined by sequence alignment or other standard methods that identify equivalent positions relative to the reference sequence.

[0110] Conjugates and fusion proteins comprising the polypeptide described herein are also within the scope of the invention.

[0111] The term "fusion protein" as used herein refers to a chimeric protein containing two polypeptides, proteins or protein fragments fused to each other (i.e., expressed as one polypeptide) often separated by an amino acid linker.

[0112] As used herein, the term "conjugate" relates to a molecule comprising at least, or consisting essentially of, a first polypeptide that is chemically attached to another substance, such as a second polypeptide or non-proteinaceous moiety.

[0113] The polypeptides described herein may be used for treating diseases and conditions associated with aberrant MMP levels, in particular MMP-9.

[0114] According to some embodiments, the pharmaceutical composition is for use in treating or preventing a disease or pathological condition associated with MMP dysregulation.

[0115] According to some embodiments, the disease or pathological condition is associated with MMP overexpression. According to some embodiments, the disease or pathological condition is associated with MMP increased activity.

[0116] According to some embodiments, the MMP is MMP-9. According to certain embodiments, the disease is associated with elevated MMP-9 levels.

[0117] According to some embodiments, the disease is selected from the group consisting of cancer, cardiovascular disease, autoimmune disease or disorder, and inflammation. Each possibility represents a separate embodiment of the invention.

[0118] As used herein, the term “cardiovascular disease” refers to any disease involving the heart and / or the vascular system (all blood vessels incl. arteries, capillaries and veins). Non-limiting examples include atherosclerosis, myocardial infarction (heart attack), and heart failure.

[0119] The term "autoimmune disease" or "autoimmune disorder" refers to any disease / disorder in which the body produces an immunogenic (i.e., immune system) response against some component of its own tissues. In other words, the immune system loses its ability to recognize any tissue or system in the body as self, and targets and attacks it as if it were foreign. Autoimmune diseases include, but are not limited to, Crohn’s disease, Addison’s disease, autoimmune parotitis, ankylosing spondylitis, alopecia areata, autoimmune hepatitis, type I diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Guillain-Barre syndrome, Hashimoto’s disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren’s syndrome, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0120] According to some embodiments, the pharmaceutical composition is for use in treating cancer.

[0121] The cancer amendable for treatment by the present invention includes, but is not limited to: carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include breast cancer, glioblastoma, squamous cell cancer, lung cancer (including small-cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade / follicular nonHodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and posttransplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, and edema (such as that associated with brain tumors).

[0122] According to some embodiments, the pharmaceutical composition is for use in treating cancer associated with elevated MMP levels.

[0123] The inhibitory polypeptides described herein can be administered to a subject per se, or in a pharmaceutical composition where they are mixed with suitable carriers or excipients. 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. Herein the term "active ingredient" refers to the agent accounting for inhibition of MMP; the active ingredient of the invention is a polypeptide as described herein, inhibiting the metastasis or invasion of cancerous cell and / or reducing the growth of a cancerous tumor.

[0124] The phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0125] The terms "excipient" and “carrier” as used herein refer to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. The terms refer to a non-toxic solid, semisolid or liquid filler, diluent, vehicle, solubilizing agent, encapsulating material or formulation auxiliary of any conventional type, and encompasses all of the components of the composition other than the active pharmaceutical ingredient. The carrier may contain additional agents such as wetting or emulsifying agents, or pH buffering agents. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.

[0126] Furthermore, the term “carrier" or "excipient” includes mixtures of carriers and or excipients.

[0127] According to another aspect, the present invention provides a method of treating an MMP-9 related disease or condition comprising administering to a subject in need thereof, a therapeutically effective amount of at least one polypeptide as described herein.

[0128] The disease and polypeptide are as described hereinabove. In certain embodiments, the disease or condition is characterized by aberrant overexpression of MMP-9. According to certain embodiments, the disease or condition is characterized by overexpression or increased activity of MMP-9. According to yet another aspect, the present invention provides a method of treating cancer comprising administering to a subject in need thereof, a therapeutically effective amount of at least one polypeptide as described herein.

[0129] According to some embodiments, the subject is human.

[0130] As referred to herein, the term "treating a disease" or "treating a condition" is directed to administering a composition, which includes at least one agent, effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring in a subject. Administration may include any administration route. In some embodiments, the disease is a disease that is caused by or related to the dysregulation of MMP in a cell, tissue, organ, body, and the like. According to some embodiments, the disease is associated with overexpression of MMP or increased activity. According to some embodiments, the MMP is MMP-9. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, glioblastoma, colon cancer and lung cancer. Each possibility represents a separate embodiment of the invention. In some embodiments, the subject is a mammal, such as a human. In some embodiments, the subject is a mammal animal.

[0131] The term "Effective amount" means the amount of a polypeptide of the present invention or pharmaceutical composition comprising a polypeptide of the present invention that will elicit the biological or medical response of or desired therapeutic effect on a tissue, system, mammal or human that is being sought by the researcher, medical doctor, or other clinician. An effective amount of the compound may vary according to factors such as the disease state, age, sex, and weight of the individual.

[0132] The term “Administering” means the actual physical introduction of the composition into or onto (as appropriate) the host. Any and all methods of introducing the composition into the host are contemplated according to the invention; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well-known to those skilled in the art, and also are exemplified herein.

[0133] According to some embodiments, the method of treating cancer comprises administering or performing at least one additional anti-cancer therapy. According to certain embodiments, the additional anticancer therapy is surgery, chemotherapy, radiotherapy, or immunotherapy. According to some embodiments, the method of treating cancer comprises administering the polypeptide described herein and an anti-cancer agent. According to some embodiments, the anti-cancer agent is selected from the group consisting of immune-modulator, activated lymphocyte cell, kinase inhibitor and chemotherapeutic agent.

[0134] According to some embodiments, the immune-modulator is an antibody against an immune checkpoint molecule. According to some embodiments, the immune modulator is an antibody against an immune checkpoint molecule selected from the group consisting of human programmed cell death protein 1 (PD-1), PD-L1 and PD- L2, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, 0X40 (also referred to as CD134), killer cell immunoglobulin-like receptors (KIR), TIGIT, PVR, CTLA-4, NKG2A, GITR, and any other checkpoint molecule or a combination thereof. Each possibility represents a separate embodiment of the invention.

[0135] Any administration route may be used in the method of treating or alleviating a disorder, disease, or complication. According to some embodiments, administration routes include parenteral or enteral routes. According to some embodiments, the method of treating or alleviating a disorder, disease or complication comprises administering the polypeptide of the invention, or a pharmaceutical composition comprising said polypeptide, by an administration route selected from intravenous, subcutaneous, intertumoral, and oral. Each possibility represents a separate embodiment of the present invention.

[0136] The compounds of the present invention can be formulated into various pharmaceutical forms for purposes of administration. Pharmaceutical composition of interest may comprise at least one additive selected from a disintegrating agent, binder, flavoring agent, preservative, colorant and a mixture thereof. For example, a compound of the invention, or its salt form or a stereochemically isomeric form, can be combined with a pharmaceutically acceptable carrier. Such a carrier can depend on the route of administration, such as intravenous, subcutaneous, oral, nasal, percutaneous or parenteral injection.

[0137] According to some embodiments, the pharmaceutical composition is formulated in a form suitable for subcutaneous or intravenous, oral, nasal, intramuscular, administration. According to particular embodiments, the pharmaceutical composition is formulated in a form suitable for nasal or oral administration. According to more particular embodiments, the pharmaceutical composition is formulated in a form suitable for intratumoral administration. Each possibility represents a separate embodiment of the present invention.

[0138] It can be advantageous to formulate the compositions of the invention in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” refers to physically discrete units suitable as unitary dosages, each unit containing a pre-determined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the chosen carrier.

[0139] The formulation of the polypeptides described herein for administering requires careful selection of carriers, additives, and excipients to ensure its stability, efficacy, and appropriate delivery for each administration route. The choice depends on the specific route and the challenges associated with delivering a large, complex molecule.

[0140] For intravenous (IV) and subcutaneous (SC) administration, different excipients for stabilizing and preventing aggregation and denaturation may be used as known in the art. Common excipients include buffers, stabilizers, certain surfactants (e.g., Polysorbate 80) and antimicrobial preservatives. For intravenous use, the formulation must be sterile and free of pyrogens. Subcutaneous formulations often contain excipients that enhance absorption from the injection site into the bloodstream, as the polypeptide needs to diffuse through the subcutaneous tissue. For intratumoral administration, different carriers may be used, for example, polymeric nanoparticles or micelles, hydrogels, liposomes.

[0141] For oral and nasal administration different delivery systems may be used, including enteric-coated capsules or tablets that protect the polypeptide from stomach acid, allowing it to reach the intestine where the coating dissolves, absorption enhancers, and nanoparticles. For nasal administration, formulation may include permeation enhancers (e.g., chitosan, certain surfactants), mucoadhesive excipients and carriers like liposomes or nanoparticles.

[0142] According to some embodiments, the carrier is Phosphate buffered saline (PBS). According to some embodiments, the pharmaceutical composition comprises thickening or gelling agent e.g., polyvinyl alcohol, chitosan, Carboxymethyl cellulose (CMC), carbomers and Hydroxypropyl methylcellulose (HPMC). According to some embodiments, the pharmaceutical composition comprises lipophilic transport carrier e.g., Lauric acid, oleic acid, stearic acid, and lecithin. According to some embodiments, the pharmaceutical composition comprises mucoadhesive agent e.g., sodium hyaluronate. According to some embodiments, the pharmaceutical composition comprises vasoconstrictor, e.g., phenylephrine (PHE), naphazolineand, and oxymetazoline.

[0143] Other routes of administration are intra- articular, intravenous, intramuscular, subcutaneous, topical, transdermal, intradermal, or intrathecal.

[0144] Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0145] For administration by inhalation, the polypeptides for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.

[0146] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form. The polypeptide described herein may be formulated in sterile water or in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, physiological saline buffer or glucose buffer or appropriate combinations thereof. Other ingredients may be included to aid in solubility. In addition, solid preparations that are converted to liquid form shortly before use can be made. For percutaneous administration, the carrier can include a penetration enhancing agent or a wetting agent.

[0147] Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable natural or synthetic carriers are well known in the art. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds, to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. The polypeptides, analogs, fragments, derivatives and conjugates of the present invention may be produced by any method known in the art, including recombinant and synthetic methods and combinations thereof.

[0148] The procedures utilized to construct polypeptides of the present invention generally rely on the known principles of polypeptide synthesis. In certain embodiments, the polypeptides are generated by molecular genetic methods as are known in the art. However, it will be appreciated that accommodation of the procedures to the specific sequences of the present invention may be required.

[0149] The polypeptides of the invention may also be produced by solid phase peptide synthesis. The methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis.

[0150] According to an additional aspect, the present invention provides a method of detecting a cell that is overexpressing MMP-9 by contacting a cell with at least one of the polypeptides as described herein.

[0151] According to certain embodiments, the polypeptide is labeled. According to some embodiments, the polypeptide is fluorescently labeled. According to certain embodiments, the method comprises the steps of (i) providing a biological sample comprising cells; (ii) contacting the labeled polypeptide with the biological sample; (iii) washing unbound polypeptide from the biological sample; and (iv) detecting bound polypeptide.

[0152] The washing step of the described methods can be done with any suitable polypeptide-free buffer or solution as known in the art. The swashing step does not require complete removal of all unbound polypeptide, but rather removal of sufficient excess polypeptide such that the signal to noise ratio of bound polypeptide is sufficient for detection.

[0153] In particular embodiments, the biological sample contains cells or cellcontaining tissue that has been taken from a subject by standard methods as known in the art. According to certain embodiments, the cell or cell-containing tissue is located in or on the subject.

[0154] According to some embodiments, the methods include contacting a cell that is suspected of overexpressing MMP-9 with a polypeptide as described herein, washing unbound polypeptide from the cell; and detecting bound polypeptides. According to another aspect, the present invention provides a kit for detecting the presence of MMP-9 in a biological sample, the kit comprises a conjugate comprising the polypeptide described herein and a detectable moiety.

[0155] According to some embodiments, the detectable moiety is selected from the group consisting of fluorescent dyes, radioisotopes, epitope tags, biotin, and nanoparticles.

[0156] According to some embodiments, the kit further comprises additional component selected from the group consisting of buffers, reagents, substrates, stabilizers, preservatives, controls, solid supports, instructional materials and any combinations thereof.

[0157] As used herein, the term "about", when referring to a measurable value is meant to encompass variations of + / -10%, more preferably + / -5%, even more preferably + / - 1%, and still more preferably + / -0.1% from the specified value.

[0158] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0159] EXAMPLES

[0160] Methods and Materials

[0161] MMP-9E402Q Expression and Purification

[0162] The gene for the human MMP-9 catalytic domain, residues 107-215 and 391-443 inside the pET28a vector (under Lac operon and with N-terminal 6xHis-tag) was used as a template for mutagenesis. Forward and reverse primers were designed, where the reverse primer contained the E402Q mutation. The following primers were used: Forward primer:

[0163] CCAGACCTTTGAAGGCGACTTGAAGTGGCATCACCATAATATTACC (SEQ ID NO: 4)

[0164] Reverse primer (E402Q mutation in bold):

[0165] CCAGTGCATGCCCAAACTGATGTGCGGCAACCAAAAAC (SEQ ID NO: 5) The MMP-9 E402Q (MMP-9E402Q) mutant was generated using the TPCR technique (Erijman, A. et al., (2011) J. Struct. Biol. 175, 171) with the above primers and using the WT MMP-9 gene as a template. TPCR products were transformed into E. coli TOPIO cells, plasmids were purified using plasmid purification kit (Presto™ Mini Plasmid Kit, Cat. No. PDH100), sent for sequencing, and MMP-9E402Q sequence was confirmed.

[0166] MMP-9E402Qlabeling

[0167] MMP-9E402Q was dialyzed against buffer (0.05M sodium borate, 150 mM NaCl pH 8.6) using a dialysis cassette with 10k MWCO (slide-A-lyzer dialysis cassette, cat. no. 66380). The MMP-9E402Q was then labeled with DyLight™488 amine reactive dye (Thermo Fisher cat. no. 46402) according to manufacturer’s protocol and stored in aliquots at -80°C.

[0168] MMP expression and purification

[0169] Both the active WT MMP-9 and inactive MMP-9E402Q proteins were expressed using the same protocol. The MMP-9 gene constructs were transformed into E. coli B121(DE3)pLysS for expression. B121(DE3)pLysS transformants were grown in LB medium with kanamycin (50 pg / ml) at 37°C until an optical density at 600 nm of 0.6- 0.8 was reached. Then MMP-9 expression was induced by 1 mM Isopropyl B-D-l- thiogalactopyranoside (IPTG) and cultures were grown overnight post- induction for 18 hours. MMP-9 was over-expressed in inclusion bodies. IL of induced B121(DE3)pLysS bacterial suspension was pelleted and cells were incubated with 30 ml lysis buffer (50 mM Tris, pH 7.5-8.0, 0.5 M NaCl, 0.5% v / v triton, lysozyme (100 pg / ml)), overnight at 37°C. The lysed cells were further disrupted by four rounds of sonication (60% amplitude, 5 seconds on / off for 2 minutes) coupled with pelleting and resuspension of the pellet in 30 ml buffer (50mM Tris, pH 7.5-8.0, 0.5M NaCl, 0.5% v / v triton). In the fourth round, soluble denatured MMP-9 was extracted from the inclusion bodies by sonicating the pellet with denaturing buffer (8M urea, 25 mM Tris pH 8). The denatured MMP-9 was incubated with 5 ml of pre-equilibrated nickel resin (INDIGO Ni- Agarose resin, PureCube, Cat. No. 75105), overnight at room temperature. The protein was purified with affinity chromatography, where the washing step was done with 10 column volumes of buffer (8M urea, 25mM Tris pH 7.5-8.0, 30mM NaCl and 30mM imidazole), and elution step with at least 3 column volumes of buffer (8M urea, 25mM Tris pH 7.5-8.0, 30mM NaCl and 200mM imidazole). The eluted protein was diluted in the buffer (6M urea, 25mM Tris pH 7.5-8.0, 30 mM NaCl) to a final concentration of 50 pg / ml, and incubated with pre-equilibrated nickel resin, overnight at 4°C. Then, the protein-bound resin was collected and transferred into 45 ml of the refolding buffer (50mM Tris, 30mM NaCl, lOmM CaCl2, 20pM ZnCl2pH 8, 0.2% Zwittergent and 0.1% Brij). Protein was incubated in the refolding buffer overnight at 4°C, then collected on a gravity column and eluted with elution buffer (50mM Tris, 30mM NaCl, lOmM CaCl2, 20pM ZnCl2pH 8, 0.2% Zwittergent and 0.1% Brij, 200 mM imidazole). Finally, the refolded protein was purified using Ion-exchange chromatography (AIEX) with HiTrapQ HP AIEX 1ml column on AKTA Pure™ chromatography system, at 4°C, using buffer A (20mM Tris, 50mM NaCl, 5mM CaCl2, pH 8) and buffer B (20mM Tris, IM NaCl, 5mM CaCl2, pH 8). MMP-9E4O2Q monomers were eluted in 35% buffer B (350 mM NaCl).

[0170] The catalytic domain of MMP-14 was expressed and purified as previously described (Arkadash, V. et al. (2017), J. Bio. Chem. 292, 3481-3495; Batra, J., et al., (2013), PLoS One 8, e75836 (2013)). Constructs containing the prodomain and catalytic domain of human MMP-1, MMP-3, and MMP-10 were expressed in E. coli, extracted from inclusion bodies, purified by ion exchange chromatography under denaturing conditions, refolded, and activated as previously described (Gabasa, M. et al., (2021) Cancer Lett 507, 1-12; Batra, J. et al., (2012) J. Bio. Chem. 287, 15935-15946), to generate the active catalytic domains. A construct for expression of the catalytic domain only of human MMP-8 (residues 100-262) was generated by subcloning into the pET3a expression vector. Recombinant protein was expressed in E. coli strain BL21(DE3) grown in LB medium at 37 °C. Cultures were grown to an ODeoo of 0.4-0.5, induced by addition of 0.4 mM IPTG, and grown for 4 more hours before harvesting cell pellets and extracting inclusion bodies. Inclusion bodies were solubilized in 6M Urea, 20 mM Tris pH 7.5, 100 mM 2-mercaptoethanol, and recombinant MMP-8 was purified under denaturing conditions on a Q-sepharose column eluted with a linear gradient to 0.5 M NaCl. The purified protein was then refolded by dialysis vs. 20 mM Tris pH 7.5, 100 mM NaCl, 5 mM CaCl2, 0.5 mM ZnCl2, as previously described (Betz, M. et al., (1997), Eur. J. Biochem. 247, 356-363). Refolded active MMP-8 was subsequently clarified by centrifugation and further purified to homogeneity on a Superdex-75 column. N-TIMP2 library construction

[0171] An N-TIMP2 library was designed based on the N-TIMP2 9D-2 mutant that was previously obtained (Shirian, J. et al., (2018). FEBS Lett 592, 1122-1134). The library was constructed by randomizing three positions (14, 69, 70) in the N-TIMP29D-2 gene. PCR reactions were performed with the following primers to create the library (mutated codons in bold):

[0172] Forward 1:

[0173] CCGTCGGTTCCGCTGCAGAAGGCTCTTTGGACAAGAGATGCAGCTGCCCA

[0174] CCGGTG (SEQ ID NO: 6)

[0175] Forward2:

[0176] CCGGTGCACCCGCAACAGGCGTTTTGCNNKGCAGATGTAGTGATCAGGGC

[0177] CAAAGCGGTC (SEQ ID NO: 7)

[0178] Reverse 1:

[0179] GTCTTCTTCGGAGATAAGCTTTTGTTCTGCACGCGTGGATCCCTCGCAGCCC

[0180] ATCTGGTACC (SEQ ID NO: 8)

[0181] Reverse2:

[0182] CCTCCAACGTCCAGCGAGACCCCACAGATMNNMNNGTTGGGGGCCGTGT AGATAAACTCTATATCC (SEQ ID NO: 9)

[0183] The first PCR reaction was performed with forward 1 and reverse2 primers, while the second PCR reaction was performed with forward2 and reverse 1 primers. The conditions for both PCR reactions were as following: 30 cycles of denaturing at 95°C for 30 seconds, annealing at 60 °C for 1 minute and elongation at 72 °C for 1 minute, and finally, incubation at 72 °C for 7 minutes. A sample from each PCR reaction was run on a 1% agarose gel to indicate the desired products, then 15 pl of both PCR1 and PCR2 products were mixed for a third PCR reaction producing the full N-TIMP2 library. The third PCR reaction was performed with the same conditions as mentioned above, yet for 15 cycles. pCHA plasmids were linearized with BamHI and Nhel restriction enzymes, and the products of the third PCR reaction were transformed with the linearized plasmids to S. cerevisiae EBY100 yeast cells by electroporation. Transformants were randomly sampled by colony PCR to confirm the presence of the N-TIMP2 gene insert in the pCHA plasmid. The obtained clones were sequenced to validate the mutations in positions 14, 69 and 70. Yeast Surface Display (YSD) and FACS Analysis and Sorting

[0184] WT N-TIMP2 gene and N-TIMP2 library were subcloned into the pCHA vector and were expressed in Saccharomyces cerevisiae EBY 100 yeast strain (Mata-Fink, J. et al., (2013), J. Mol. Biol. 425, 444-456).

[0185] EBY100 yeasts containing the gene for N-TIMP2 (WT or library) were grown in SDCAA media (2% dextrose, 0.67% Difco yeast nitrogen base, 0.5% Bacto casamino acids, 0.54% NaiHPCE and 0.86% NaH2PO4*H2O), overnight at 30°C, until an ODeoo of 7-8 was reached. Then, N-TIMP2 library expression was induced with galactose by growing a sample of the SDCAA-suspension in the inductive SGCAA media (2% galactose, 0.67% Difco yeast nitrogen base, 0.5% Bacto casamino acids, 0.54% NaiHPCU and 0.86% NaH2PO4*H2O), overnight at 30°C, until an ODeoo of 3-4 was reached. After induction, cells were incubated with anti c-Myc antibody conjugated to phycoerythrin (PE-anti c-Myc antibody. Santa Cruz cat. sc-40) for 45-60 minutes at 4°C. Thereafter, labelled MMP-9E402Q (MMP-9E402Q-DyLight488) was added and the cells were incubated for another 60 minutes at 4°C. Finally, cells were washed three times with buffer (50mM Tris, lOOmM NaCl, 5mM CaCh, pH 8.0, 1% Bovine Serum Albumin) and then analysed by FACS.

[0186] Four sorting rounds were performed with the library (S0-S3) using the FACS sorter. The initial round (SO) was a pre-sorting round, where high expressing cells were collected. In the following sorting rounds, cells were sorted for binding such that in each round MMP-9E402Q concentration was decreased, and high binding cells were collected and continued to the next sorting round. The first round of sorting for binding (SI) was performed with 500 nM MMP-9E402Q-DyFight488, the second round (S2) was performed with 300 nM MMP-9E402Q-DyFight488 and the third and final round (S3) was performed with 150 nM MMP-9E402Q-DyFight488. The cells collected in the S3 round and named S3 library were then plated for sequencing.

[0187] Library sequencing & Logo construction

[0188] S3 library was plated on SDCAA agar plates (2% dextrose, 0.67% Difco yeast nitrogen base, 0.5% Bacto casamino acids, 0.54% Na2HPO4, 0.86% NaH2PO4*H2O, 18.2% sorbitol and 1.5% agar) and grown for three days at 30°C. Several different colonies were sampled by colony PCR using the following primers: Forward: CCGGTTATTTCTACTACCGTCGGTTC (SEQ ID NO: 10)

[0189] Reverse: GTCAGTTCCTGCAAGTCTTCTTCG (SEQ ID NO: 11)

[0190] Colony PCR products were run on 1% agarose gel and 15 samples that indicated N- TIMP2 gene were sent for sequencing. Sequences were analyzed and amino acid sequence logo was created with WebLogo (https: / / weblogo.berkeley.edu / logo.cgi). N- TIMP2 mutants that were considered as representative of all the mutations obtained were selected for expression.

[0191] N-TIMP2 expression and purification

[0192] Cloning the selected N-TIMP2 mutants into pPICZaA vector

[0193] The TPCR technique was performed to clone the selected N-TIMP2 mutants into the pPICZaA vector. This technique was utilized with its site-directed mutagenesis application, using mutagenic primers and the 9D_2 mutant subcloned into pPICZaA plasmid as a template for the reaction (9D_2 gene subcloning into pPICZaA plasmid is described in Shirian, J. et al., (2018) FEBS Lett 592, 1122-1134). The following primers were used to to introduce the desired mutations - N14R, S69T, S69E, A70W and A70Y (mutated codons in bold):

[0194] Forward TIMP2 N14R:

[0195] CGCAACAGGCGTTTTGCAGAGCAGATGTAGTGATCAGG (SEQ ID NO: 12)

[0196] Forward TIMP2 S69T_A70W:

[0197] CACGGCCCCCAACACTTGGATTTGTGGGGTC (SEQ ID NO: 13)

[0198] Forward TIMP2 S69E_ A70Y:

[0199] CTACACGGCCCCCAACGAATACATTTGTGGGGTCTC (SEQ ID NO: 14) Reverse TIMP2 R97 :

[0200] GTCACAGAGGGTGATTCTCATCTTGCCGTCCCCC (SEQ ID NO: 15)

[0201] TPCR products were treated with Dpnl and transformed into TOP 10 bacteria. Transformants were plated on LB agar plates containing Zeocin (25 pg / ml) and grown at 37°C overnight. Several colonies were sampled and grown in LB medium with Zeocin (25 pg / mL), at 37°C, overnight, then plasmids were purified using a plasmid purification kit (Presto™ Mini Plasmid Kit, Cat. No. PDH100) and sent for sequencing. Protein Expression and purification pPICZaA plasmids containing the N-TIMP2 mutants were amplified and purified from TOPIO bacteria using a DNA extraction kit (Maxi plasmid DNA kit, Geneaid Cat. No. PM010). Plasmids were then linearized with SacI restriction enzyme and transformed into Pichia pastoris X-33 yeast strain by electroporation. P. pastoris transformants were plated on YPDS agar plates (1% yeast extract, 2% peptone, 2% D-glucose, 1 M sorbitol, 2% agar) with Zeocin (100 pg / ml) and grown for 3 days at 30°C. Several colonies of each mutant were sampled by colony PCR using the following primers:

[0202] AOX1 forward primer: GGTATCTCTCGAGAAAAGATGCAGCTGCTCCCCG (SEQ ID NO: 16)

[0203] AOX1 reverse primer: GCTGGCGGCCGCCTCGCAGCCCATCTGGTA (SEQ ID NO: 17)

[0204] Colony PCR products were run on a 1% agarose gel and samples that indicated the N- TIMP2 gene were sent for sequencing. P. pastoris clones containing WT or mutated N- TIMP2 genes were grown in 50 ml BMGY medium (2% peptone, 1% yeast extract, 0.23% K2H(PO4), 1.18% KH2(PO4), 1.34% yeast nitrogen base, 4x10-5 % biotin, 1% glycerol) until an ODeoo of 10 was reached. Then, the cultures were transferred into 500 ml inductive BMMY medium (2% peptone, 1% yeast extract, 0.23% K2H(PO4), 1.18% KH2 (PO4), 1.34% yeast nitrogen base, 4x10-5 % biotin, 0.5% methanol) and induction was continued for 72 hours at 30°C, with the addition of 0.5% methanol every 22-26 hours. After 72 hours of induction, cells were pelleted at 817xg for 5 minutes, supernatant was collected, the pH of the solution was raised to 8, and the solution was incubated at 4°C for 1 hour. Imidazole was added to a final concentration of lOmM, and the solution was filtered with 0.22 pm filter.

[0205] Nickel resin (1 ml; INDIGO Ni-Agarose, PureCube, Cat. No. 75105) was added to the solution, and the solution was incubated for 1-2 hours at room temperature. N-TIMP2 proteins were purified by affinity chromatography where the washing step was done with 10 column volumes of washing buffer (50mM Tris-HCl, 300mM NaCl, pH 7.5, lOmM imidazole) and the elution step was done with 10 column volumes of elution buffer (50mM Tris-HCl, 300mM NaCl, pH 7.5, 250mM imidazole). The N-TIMP2 elution fraction was concentrated to 1ml or less, using a 3 kDa MWCO centrifugal concentrator (Amicon® Ultra- 15 Centrifugal Filter, Cat. No. UFC9003), and then purified with size exclusion chromatography (SEC) using a Superdex75, 120ml column with buffer (50 mM Tris, 100 mM NaCl pH 7.5). N-TIMP2 monomers were collected, and concentration was determined with the Bradford assay (Pierce™ Bradford Protein Assay Kit, Cat. No. 23200), using the manufacturer’s protocol.

[0206] Enzymatic inhibition assay and Kiappdetermination

[0207] The apparent Ki of MMP inhibition by the N-TIMP2 mutants, as well as by the WT N- TIMP2 and 9D_2 mutant, was determined using an MMP enzymatic activity assay with decreasing concentration of the N-TIMP2 inhibitors. N-TIMP2 variants were incubated with each of 0.1 nM MMP-9, 0.1 nM MMP-8, 0.2 nM MMP- 14, 0.5 nM MMP- 10, 4 nM MMP-3 or 0.5 nM MMP-1, respectively, for 1 hour at 37°C in buffer (50 mM Tris- HC1, 0.15 M NaCl, 10 mM CaC12, and 0.02% Brij 35 pH 7.5). After incubation, 50 pl of Anorogenic MMP substrate MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 TFA (Sigma-Aldrich, MMP Substrate, cat. No. SCP0193) was added to the N-TIMP2:MMP solution in a final concentration of 7.5 pM. Fluorescence at 395 nm was immediately measured at 37°C, at least every 30 seconds for at least 30 minutes, with irradiation at 325 nm, on a Biotek Synergy Hl plate reader (BioTek, VT, USA). Uninhibited enzyme activity (MMP without N-TIMP2) and basal Auorescence (buffer without N-TIMP2 or MMP) were performed as controls. At least 3 assays were performed for each N-TIMP2 protein.

[0208] Mean velocities (Vm) of the enzymatic cleavage of the Anorogenic substrate were derived from the Auorescence generated by this cleavage. Fractions of residual MMP activity were determined by dividing Vmby the uninhibited velocity (V0) and were plotted against the corresponding concentration of N-TIMP2. From this plot the apparent Ki (Kiapp) value was calculated based on the following equation (Murphy, D. J. (2004) Anal. Biochem. 327, 61-67) using MATLAB:

[0209] [([MMP] —[TIMP] - Kiapp)+ / [MMP] + [TIMP] +Kiapp2-4 [MMP] [77MP] ] ® ~ 2 [MMP]

[0210] Where: f= fraction of activity, Vm / V0; and

[0211] Kjappvalues are an average of the Kiappvalues determined from a single inhibition assay for each N-TIMP-2 protein.

[0212] Binding specificity was determined by dividing the Kiappof the tested MMP (referred to also as MMP-X) by the Kiappof MMP-9. Colony formation assay

[0213] Triple negative breast cancer MDA-MB-231 cells were treated with different concentrations of either WT N-TIMP2 or REY N-TIMP2 mutant. Untreated cells were set as control. The cells were seeded on 12-well plates at a density of 1 x 103cells and cultured in DMEM medium supplemented with 10% of FBS for 3 days in standard conditions (37 °C and 5% CO2). The growing medium was replaced once after 48 hrs and WT N-TIMP2 or REY mutant were re-added to the medium in the appropriate concentrations. After 3 days of incubation, the resulting colonies were fixed with 100% methanol for 20 min at -20°C, stained with 0.4% crystal violet for 30 min at RT, and finally washed with DDW to remove the excessive stain. Each sample was imaged using a Nikon microscope at a magnification of lOx and the colonies of each sample were counted using ImageJ.

[0214] Cell invasion assay

[0215] Cell invasion was assayed using Corning Matrigel invasion chamber 24-well plate. Matrigel (CLS356234 CORNING(R)MATRIGEL(R)BASEMENT MEMBRANE) was diluted in cold PBS in 1:4 ratio. The upper compartment of the trans-well chamber was coated with 100 pl Matrigel and incubated overnight at 37°C until gelled. 2xl05of confluent TNBC MDA-MB-231 cells were resuspended in serum-free DMEM medium in the absence or presence of N-TIMP2 variants at different concentrations and then transferred into the upper chamber of each trans-well. Lower chambers contained fresh DMEM medium supplemented with 20% of FBS. After culturing for 24 hours, the cells at the upper surface of the membrane were removed using a swab, while the cells that migrated to the lower membrane surface were fixed with 100% cold methanol for 30 min and stained with 0.4% crystal violet for 1 hr. The cells were imaged using a Nikon microscope at a magnification of lOx. Images were processed and cells were counted with ImageJ.

[0216] MTT assay

[0217] The N-TIMP2 variants were tested for vitro cytotoxicity, using MDA-MB-231 (Breast cancer) and Vero (Normal, African green monkey kidney) cells by conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the above cell lines were incubated at a density of 5xl04cells / well in 96-well plates for 24 h in 100 pL of DMEM with 10% FBS, at 37°C in a humidified 5% CO2 incubator. Following seeding, the culture supernatant was removed and serum-free DMEM medium containing various concentrations of N-TIMP2 variants, REY and WT, were added and the cells were incubated for 24 h. Wells without N-TIMP2 variants served as controls. MTT dye (10 pL, 5 mg / mL) was added to each well and the plate was incubated for an additional 4 h. The medium together with MTT were aspirated off the wells, DMSO (100 pL) was added and the plates shaken for 5 min. The absorbance of each well was measured at 540 nm in a micro plate reader and the percentage of cell viability (CV) was calculated using the formula: CV=[Average absorbance of N-TIMP2 variant replicates / Average absorbance of control replicates] x 100%.

[0218] Modeling of N-TIMP2 / MMP Complexes

[0219] The crystal structure of WT N-TIMP2 in complex with MMP-14 (PDB 1BUV) and the crystal structure of MMP-9 catalytic domain (PDB 6ESM) were used to generate N- TIMP2 / MMP-9 complex by the structural alignment of MMP-9 and MMP-14 and subsequent removal of MMP-14. The N-TIMP2 / MMP-9 modeled structure was then minimized and the mutations corresponding to the mutants REY and RTW were introducing using the Rosetta flexbb_design protocol. The complexes of alternative MMPs with N-TIMP2 variants were modeled by the structural alignment and substitution of MMP-9 by alternative MMP in the N-TIMP2 variant / MMP-9 complex using the crystal structures of MMP- 1 catalytic domain (PDB 3SHI), MMP-3 catalytic domain (PDB 4G9L), MMP-8 catalytic domain (PDB 3DPE), MMP- 10 catalytic domain (PDB 1Q3A) and MMP-14 catalytic domain (PDB 1BUV). Procedure

[0220] 1: Selection of mutated N-TIMP2 variants

[0221] In an attempt to procure a N-TIMP2 variant which would effectively bind MMP-9, the previously produced 9D-2 variant of N-TIMP2 was selected, which had shown high- specificity to MMP-9 relative to its binding to MMP-14 (Shirian, J. et al., (2018). FEBS Lett 592, 1122-1134). Positions 14, 69, and 70 of the 9D-2 variant were selected for randomization. Randomization of these three positions to all twenty amino acids produced a combinatorial library of 8xl03N-TIMP2 variants, all containing the five mutations of the 9D-2 variant relative to WT N-TIMP2 (see also Rotenberg et al. J Biol Chem. 2024 Nov;300(ll): 107867, Figure 1). The N-TIMP2 library was constructed and subcloned into the pCHA plasmid compatible with the YSD technology. In this construct, the N-TIMP2 gene was followed by a Myc tag while the N-terminus of the protein was kept free to allow for interaction with MMP-9 (see Figure 1A).

[0222] The N-TIMP2 library was transformed into S. cerevisiae EBY100 yeast cells and sorted for binding to MMP-9 using Fluorescently Activated Cell Sorting (FACS). An inactive MMP-9E402Q variant was used for the FACS to avoid enzyme autocleavage. Protein expression of N-TIMP2 variants was measured using a PE-conjugated Myc tag antibody, while MMP-9E402Q binding to N-TIMP2 variants was measured by monitoring fluorescence of Dylight-488 fluorophore conjugated to MMP-9E402Q (see Figure 1A). An initial pre-sorting round SO of FACS was performed to collect the top 35% N- TIMP2-variant-expressing cells, followed by three additional rounds of sorting S1-S3, with decreasing concentration of MMP-9, as described in the Methods and Materials section above and presented in Figure IB. In the first sorting round (left histogram), the top 3.7% of the highest-affinity N-TIMP2 binders were collected (box labeled “SI gate”), creating the SI library. In the second sorting round (middle histogram), the top 2% of binders were collected (box labeled “S2 gate”), creating the S2 library. In the third sorting round (right histogram), the top 2% of binders were collected (box labeled “S3 gate”) to create the S3 library. The S3 library of N-TIMP2 derivatives was subsequently sequenced, and a WebLogo visual depiction was created from the 10 unique sequences found in the S3 library (https: / / weblogo.berkeley.edu / logo.cgi), for the three randomized amino acid positions, as shown in Figure 1C. The size of the amino acid one-letter code corresponds to its frequency, where N14, S69 and A70 are the wild-type residues at these positions.

[0223] As can be seen in the Figure, at position 14, arginine, lysine, or asparagine occurred, with arginine being the most favored. At position 69, serine, glutamic acid or threonine were observed with the WT serine being the most favored. At position 70, tryptophan, alanine, phenylalanine, and tyrosine were observed, with tryptophan being the most favored. Out of all sequenced N-TIMP2 variants, two mutants, arginine-threonine- tryptophan (RTW) and arginine-glutamic acid-tyrosine (REY), were chosen for further characterization since these variants contained all the mutations that were observed in other selected variants.

[0224] Example 2: N-TIMP2 variants inhibition of and specificity for MMP-9

[0225] WT N-TIMP2, 9D-2, and the two engineered mutants, REY and RTW, were expressed in P. pastoris and purified first by Ni-affinity chromatography and then by Size Exclusion Chromatography (SEC) to collect the monomeric N-TIMP2 variant species, as explained in the Methods and Materials section above. The ability of the three N- TIMP2 variants to inhibit MMP-9 was then tested, using an enzyme activity assay that measures the cleavage of a Anorogenic peptide substrate and results in Auorescence at 395 nm, as explained in the Methods and Materials section above. The fraction of active MMP-9 was measured in the presence of various concentrations of the N-TIMP2 variants and the data were fit to equation (I) set out in the Methods and Materials section above, to determine the apparent MMP-9 inhibitory activity (Kiapp) for each N-TIMP2 variant. The results of the MMP-9 inhibition assay are presented in Figure 2.

[0226] As can be seen, it was surprisingly found that both of the engineered variants (REY and RTW) demonstrated a 2 to 3.5 fold enhancement in Kiapp(0.10 ± 0.01 nM and 0.15 ± 0.02 nM for RTW and REY, respectively), as compared to both of the WT N-TIMP2 and the 9D-2 mutant, which displayed very similar MMP-9 inhibitory activity (Kiappvalues of 0.34 ± 0.05 nM and 0.31 ± 0.03 nM, respectively).

[0227] To further determine the binding specificity of the mutated N-TIMP2 derivatives to MMP-9, the enzymatic inhibition assay was repeated using five alternative MMPs that represent several MMP subgroups: collagenases (MMP-1 and MMP-8), stromelysins (MMP-3 and MMP- 10), and membrane bound MMPs (MMP- 14). The results are set out in Figures 3A-3C.

[0228] As can be clearly seen, as opposed to the WT N-TIMP2, the two engineered N-TIMP2 variants (REY and RTW) surprisingly demonstrated very high discrimination, i.e., very low inhibition particularly as compared to the inhibition of MMP-9, against MMP- 14, MMP- 10, and MMP-3. Moderate (6-7-fold) discrimination was found against MMP-8, and a trend suggesting light specificity to MMP-9 even as compared to MMP-1 was also shown. Importantly, the binding specificity of the two engineered mutants was equal or improved even as compared to the specificity shown by the previously reported 9D-2 mutant. Example 3: Evaluating N-TIMP2 mutant inhibitory effect on cancer cells

[0229] Having established the specificity of the REY and RTW mutated N-TIMP2 variants to MMP-9 and their MMP-9 inhibiting potency, an in light of the known contribution of MMP-9 to progression of cancers (including tumor growth, cell migration, and invasion), the effectivity of the N-TIMP2 variants in inhibiting cancerous processes was tested. In particular, human MDA-MB-231 cells, as an accepted model of human basal- like triple negative breast cancer, were selected, MMP-9 having been shown to be crucial for progression of this type of cancer both ex-vivo and in vivo (Mehner, C. et al., (2014) Oncotarget 5, 2736-2749). As it was expected that inhibition of cellular phenotypes would require high N-TIMP2 concentrations, the REY mutated N-TIMP2 derivative was selected for the cellular assays, having shown good non-aggregation tendency.

[0230] First, the inhibitory effect of REY N-TIMP2 derivative on the ability of cancer cells to survive and proliferate was evaluated, as compared to WT N-TIMP2. Different concentrations of N-TIMP2 variants were added to MDA-MB-231 cells and then proliferation under each N-TIMP2 treatment was measured. The results of this assay are shown in Figures 4A-4B.

[0231] As can be clearly seen, both WT N-TIMP2 and REY had an inhibitory effect on the cancer cell proliferation, with the inhibition effect increasing in correlation to increased inhibitor concentration and reaching almost total inhibition at the highest tested concentration (600 nM). However, strikingly, the REY mutated N-TIMP2 derivative demonstrated a substantially higher inhibitory effect compared to WT N-TIMP2, with the highly significant difference shown at the lowest, 100 nM concentration (with 88% and 47% inhibition for REY and WT N-TIMP2, respectively). These results indicate that treatment even with a low (e.g., 100 nM) concentration of the REY mutant can be sufficient for -90% inhibition of cancer proliferation, while 6-fold higher concentrations of WT N-TIMP2 are required to achieve similar inhibition. Utilizing the same higher concentrations of REY variant achieve almost total inhibition of cell proliferation (-95%).

[0232] As MMP-9 has been shown to promote invasion and migration of cancer cells by degradation of cell-matrix proteins, particularly in triple negative breast cancer cells (Mehner, C. et al., (2014) Oncotarget 5, 2736-2749), the effect of MMP-9 inhibition on cell invasion was next tested. The inhibitory effect of WT N-TIMP2 and REY mutant on MDA-MB-231 cell invasion was evaluated using Matrigel Transwell invasion assays, which measure the ability of cancer cells to cross an artificial basement membrane, as further described in the Methods and Materials section above. The invasion assays were performed at three different N-TIMP2 concentrations. The results are set out in Figures 5A-5B.

[0233] As is clearly shown in the figures, the inhibitory effect of the treatment, although present in both WT and REY N-TIMP2, was significantly higher for the REY derivative at all tested concentrations, including the relatively low 100 nM concentration. At 300 nM REY achieved -90% inhibition (20% more than WT), and at the highest tested 600 nM concentration REY succeeded in preventing cancer cell invasion almost entirely.

[0234] Example 4: Computational analysis of protein N-TIMP2 MMP-9 protein interaction Following the enhanced potency and specificity of the mutated N-TIMP2 derivatives, the contribution of each of the mutations at positions 14, 69 and 70 to the protein interaction with MMP-9 was analyzed. The selected mutations were introduced into N- TIMP2 sequence using the Rosetta suite (Alford, R. F. et al., (2017) J. Chem. Theory Comput. 13, 3031-3048), and each newly introduced interaction was analyzed. The modeled structure predicted that arginine at position 14, present in both mutants, forms four new hydrogen bonds with MMP-9: two with the backbone of Lysl84, one with the side chain of the Asp 182, and one with the backbone of the same position (see also Rotenberg et al. J Biol Chem. 2024 Nov;300(ll): 107867, Figure 7A). At position 70, A70W and A70Y mutations of the RTW and RWY variants, respectively, were predicted to improve packing and hydrophobic interactions with MMP-9. In addition, Trp70 introduces a new potential H-bond with the backbone of His230 on MMP-9, while Tyr70 may form a new hydrogen bond with a side chain of Asp235 (see also Rotenberg et al. J Biol Chem. 2024 Nov;300(ll): 107867, Figure 7B). At position 69, serine ofWT N-TIMP2 was predicted to form a hydrogen bond with the side chain of Asp235 and the same hydrogen bond was predicted for a threonine in the RTW mutant, while Glu69 of the REY mutant was predicted to form a hydrogen bond with the side chain of His236. Although no new interactions with MMP-9 were predicted to be introduced by mutations at the 69 position, these mutations could be beneficial for MMP-9 affinity in conjunction with the mutations at position 70. In an attempt to analyze the improved selectivity of the REY and RTW N-TIMP2 derivatives compared to both WT and 9D-2 N-TIMP2, the interaction of the engineered mutants was modeled with five alternative MMPs by superimposing the crystal structure of each MMP for MMP-9 in the modeled structure of MMP-9 in complex with RTW and REY. Examination of the binding interfaces of the two engineered N-TIMP2 variants and six MMPs suggested that selectivity of the two N-TIMP2 mutants toward MMP-9 is related to the amino acid at position 70. Replacement of the alanine, which does not contact the MMP surface, forming a cold-spot cavity (Gurusinghe, S. N. S., et al., (2022) Protein Sci. 31, e4435), with large aromatics amino acids such as tryptophan and tyrosine (in RTW and REY mutants, respectively), fills the cavity when the N- TIMP2 variant is interacting with MMP-9 (see also Rotenberg et al. J Biol Chem. 2024 Nov;300(ll): 107867, Figure 8). However, the same mutations were predicted to generate steric clashes with MMP-3, MMP- 10, and MMP- 14, which could explain the significant decrease in affinities for these MMPs. It is noted that no such steric hindrance was predicted with MMP-1 or MMP- 8 upon these mutations in the two N- TIMP2 mutants.

[0235] Example 5: Evaluating N-TIMP2 mutant inhibitory effect on additional cancer-types Having established the high effectiveness of REY mutated N-TIMP2 derivative in inhibiting activity of cancer cells, in particular in cells of human breast cancer, further cancer cell types were tested.

[0236] Adult glioma cancer cell (U251-MG) invasion was evaluated in the presence of the mutated REY and WT N-TIMP2 variants as MMP-9 inhibitors, using Matrigel transwell invasion assay, as in Example 3. The results are shown in Figures 6A-6B, the bar plot of Figure 6B represented as percent of inhibition (calculated as 100% - [%- invasion], the % -invasion normalized against invasion of non-treated cells which is set as 100%). As can be seen in the figures, while at lower concentrations of 100 and 300 nM the REY and WT N-TIMP2 variants achieved similar inhibition levels (-10% and -40%, respectively), at the concentration of 600 nM the REY variant achieved a substantially higher inhibition of GB cancer cell invasion, reaching almost 90% inhibition.

[0237] Next, several different cell viability assays are conducted, including MTT, Trypan blue exclusion and colony formation assays. The N-TIMP2 variants are tested at several different concentrations, as in Example 3 above. In addition, normal astrocytes are used as a control to evaluate possible toxicity of N-TIMP2 variants to healthy brain cells. The expression of the epithelial-to mesenchymal transition (EMT) activating genes ZEB1 and SLUG transcription factors, as well as the mesenchymal marker vimentin, are measured following 72-hour in-vitro treatment of the cell lines with 1 pg / mL N- TIMP-2 variants.

[0238] Example 6: Evaluating N-TIMP2 variants effect on GB migration in vivo

[0239] In an attempt to evaluate inhibition of the cancer cells in vivo, Luciferase-expressing GL261-luc2 tumor cells (IxlO5) are injected with 50% Matrigel into the flank of immunocompetent C57BL / 6 mice. To allow monitoring N-TIMP2 variant accumulation in the tumor, fluorescent N-TIMP2 variants are constructed. To this end, a C-terminal cysteine is incorporated into the protein gene, which, after expression and purification of the protein is labeled with a Cy3 maleimide fluorophore (Chiu, H. Y., et al., (2016) Sci. Rep. 6, 25019, PMC4865863). Cy3 labeling is quantified by IVIS image analysis.

[0240] Doses of 50 / 100 / 200 pg / kg / day of WT N-TIMP2 or mutated N-TIMP2 derivatives (e.g., REY and RTW) in saline are injected into the area of the tumor on day 10 and continued through day 30. Drugless saline samples are injected into control mice. Statistical analysis is performed on the mean difference in tumor volume between the three dose levels of WT and mutated N-TIMP-2, as well as the vehicle control.

[0241] The growth of tumors is recorded by bioluminescence intensity imaging analysis using the Ivis Spectrum imaging system (Caliper Life Sciences). At day 30 post-implantation, mice are sacrificed, and the excised tumor is sectioned and analyzed. Accumulation of N-TIMP2 (WT or derivatives) in tumors and organs is assessed by immuno staining or by ELISA of homogenized tissues. The expression profiles of ZEB1, SLUG and vimentin is measured following resection of the tumors.

[0242] The effects of the different treatments on tumor progression and on angiogenesis is done by histological examination of the tumor with hematoxylin and eosin staining, and fluorescence immunohistochemistry staining of the subcutaneous tumor block sections to evaluate CD31 (a marker for vascular effect). Example 7: Evaluating N-TIMP2 variants effect on intracranial GB

[0243] For intracranial treatment of GB, intranasal administration of N-TIMP2 variant is utilized. The N-TIMP2 derivative is formulated as a medicament including the following ingredients:

[0244] Other options may also be used (e.g., Dhuria, S. V., et al., (2009) J. Pharmacol. Exp. Ther. 328, 312-320).

[0245] IVIS imaging is employed to longitudinally monitor the penetration of the engineered N-TIMP-2 variant within the brain. Duration and intensity of the signal is recorded every 6 hours for 2 days for each application.

[0246] To evaluate the effect of the N-TIMP2 variants on intracranial glioblastoma (GB), a stereotactic intracranial injection of U87 cells was performed to create an orthotropic tumor xenograft in BALB / cOlaHsd-Foxnlnu mice. The tumor size and detection of spreading is recorded using bioluminescence by the in-vivo imaging system (IVIS) device as average Radiance [p / s / cm2 / sr] every 3 days.

[0247] Sequences

[0248] SEQ ID NO: 1 - Amino acids

[0249] CSCPPVHPQQAFCXiADVVIRAKAVSEKEVDSGNDVYGNPIKRIQYEIKQIKMF KGPEKDIEFIYTAPNX2X3ICGVSLDVGGKKEYLIAGKAEGDGKMRITLCDFIVP WDTLSTTQKKSLNHRYQMGCE

[0250] Xi - arginine or lysine ; X2 - threonine and glutamic acid ; X3 - Tryptophane, tyrosine, or phenylalanine

[0251] SEQ ID NO: 2 - REY mutant - Amino acids CSCPPVHPQQAFCRADVVIRAKAVSEKEVDSGNDVYGNPIKRIQYEIKQIKMF KGPEKDIEFIYTAPNEYICGVSLDVGGKKEYLIAGKAEGDGKMRITLCDFIVP WDTLSTTQKKSLNHRYQMGCE

[0252] SEQ ID NO: 3 - RTW mutant - Amino acids

[0253] CSCPPVHPQQAFCRADVVIRAKAVSEKEVDSGNDVYGNPIKRIQYEIKQIKMF KGPEKDIEFIYTAPNTWICGVSLDVGGKKEYLIAGKAEGDGKMRITLCDFIVP WDTLSTTQKKSLNHRYQMGCE

[0254] SEQ ID NO: 18 Wild type N-TIMP2 - Amino acids

[0255] CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFK GPEKDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPW DTLSTTQKKSLNHRYQMGCE

[0256] SEQ ID NO: 19 - 9D-2 mutant - Amino acids

[0257] CSCPPVHPQQAFCNADVVIRAKAVSEKEVDSGNDVYGNPIKRIQYEIKQIKMF KGPEKDIEFIYTAPNSAICGVSLDVGGKKEYLIAGKAEGDGKMRITLCDFIVP WDTLSTTQKKSLNHRYQMGCE

[0258] SEQ ID NO: 20 - Amino acids

[0259] XiADVVIRAKAVSEKEVDSGNDVYGNPIKRIQYEIKQIKMFKGPEKDIEFIYTAP

[0260] NX2X3

[0261] XI - arginine or lysine ; X2- threonine or glutamic acid ; X3- Tryptophane, tyrosine, or phenylalanine

[0262] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

CLAIMS1. A polypeptide of 58-200 amino acids comprising an amino acid sequence selected from (i) SEQ ID NO: 1; (ii) a fragment comprising residues 14-70 of SEQ ID NO: 1; or (iii) an analog of SEQ ID NO: 1 having 1-10 deletions, additions and / or substitutions of amino acid residues other than residues 14, 69 and 70 of SEQ ID NO: 1, wherein the polypeptide is capable of binding to matrix metalloproteinase 9 (MMP-9).

2. The polypeptide of claim 1, comprising SEQ ID NO: 20.

3. The polypeptide of any one of claims 1 or 2, comprising an arginine residue at a position corresponding to position 14 of SEQ ID NO: 1; a residue selected from the group consisting of threonine and glutamate at a position corresponding to position 69 of SEQ ID NO: 1; and a residue selected from the group consisting of tryptophane and tyrosine at a position corresponding position 70 of SEQ ID NO: 1.

4. The polypeptide of any one of claims 1 or 3, wherein said polypeptide comprising 100-150 amino acids.

5. The polypeptide of any one of claims 1 to 4, wherein said polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

6. The polypeptide of any one of the preceding claims, wherein the polypeptide analog comprises 1-5 substitutions compared to SEQ ID NO: 1.

7. The polypeptide of any one of the preceding claims, wherein one or more of the substitutions are conservative substitutions.

8. The polypeptide of any one of the preceding claims, wherein the polypeptide comprises SEQ ID NO: 2.

9. The polypeptide of any one of claims 1 to 7, wherein the polypeptide comprises SEQ ID NO: 3.

10. The polypeptide of any one of the preceding claims, wherein the polypeptide is capable of inhibiting, reducing or altering MMP-9 activity.

11. The polypeptide of any one of the preceding claims, said polypeptide is characterized by higher binding specificity to MMP-9 relative to at least one of MMP-1, MMP-3, MMP-8, MMP-10 and MMP-14.

12. A conjugate or a fusion protein comprises the polypeptide of any one of the preceding claims.

13. The conjugate of claim 12, said conjugate comprising a detectable moiety.

14. A polynucleotide comprising a sequence encoding the polypeptide according to any one of claims 1 to 11.

15. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 11, and a pharmaceutically acceptable excipient, carrier, or diluent.

16. The pharmaceutical composition of claim 15, wherein said pharmaceutical composition is formulated for injection or infusion.

17. The pharmaceutical composition of claim 15, wherein said pharmaceutical composition is formulated for intravenous or intratumoral administration.

18. The pharmaceutical composition of any one of claims 15 to 17, for use in treating or preventing a disease or pathological condition associated with MMP dysregulation.

19. The pharmaceutical composition for use of claim 18, wherein the disease or pathological condition is associated with MMP overexpression or increased activity.

20. The pharmaceutical composition for use of any one of claims 18 or 19, wherein MMP is MMP-9.

21. The pharmaceutical composition for use of any one of claims 18 to 20, wherein the disease is selected from the group consisting of cancer, cardiovascular disease, autoimmune disease or disorder, and inflammation.

22. The pharmaceutical composition for use of claim 21, wherein the disease is cancer.

23. The pharmaceutical composition for use of claim 22, wherein the cancer is associated with elevated MMP-9 levels.

24. The pharmaceutical composition for use of any one of claims 22 and 23, wherein the cancer comprises a solid tumor.

25. The pharmaceutical composition for use of any one of claims 22 and 23, wherein the cancer is selected from the group consisting of breast cancer, glioblastoma, colorectal cancer, pancreatic cancer, esophageal cancer, prostate cancer, liver cancer, ovarian cancer, endometrial cancer, stomach cancer, thyroid cancer, carcinoid tumor, head and neck cancer, testis cancer, urothelial cancer, cervical cancer, melanoma, lymphoma and lung cancer.

26. The pharmaceutical composition for use of claim 25, wherein the cancer is selected from the group consisting of breast cancer and glioblastoma.

27. A method of treating an MMP related disease or condition comprising administering to a subject in need thereof, a therapeutically effective amount of at least one polypeptide according to any one of claims 1-11 or a pharmaceutical composition according to any one of claims 15-17.

28. The method of claim 27, wherein MMP is MMP-9.

29. The method of any one of claims 27 or 28, wherein the subject is human.

30. The method of any one of claims 27 to 29, wherein the disease is cancer.

31. The method of claim 30, wherein said method comprising at least one additional anti-cancer therapy.

32. The method of any one of claims 30 or 31, wherein said method comprising administering an anti-cancer agent selected from the group consisting of immune-modulator, activated lymphocyte cell, kinase inhibitor and chemotherapeutic agent.

33. The method of any one of claims 30 to 32, wherein said method involves preventing or reducing formation, growth or spread of metastases in the subject.

34. A method of detecting a cell that is overexpressing MMP-9 by contacting a cell with at least one polypeptide according to any one of claims 1-11.

35. The method of claim 34, wherein the polypeptide is labeled.

36. The method of claim 34, wherein the polypeptide is fluorescently labeled.

37. A kit for detecting the presence of MMP-9 in a biological sample, the kit comprises a conjugate comprising at least one polypeptide according to any one of claims 1-11 and a detectable moiety.

38. The kit of claim 36, wherein the detectable moiety is selected from the group consisting of fluorescent dyes, radioisotopes, epitope tags, biotin, and nanoparticles.

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