WO2018087757A1 - Matrix metalloproteinase inhibitors and methods of use thereof - Google Patents

Matrix metalloproteinase inhibitors and methods of use thereof Download PDF

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Publication number
WO2018087757A1
WO2018087757A1 PCT/IL2017/051218 IL2017051218W WO2018087757A1 WO 2018087757 A1 WO2018087757 A1 WO 2018087757A1 IL 2017051218 W IL2017051218 W IL 2017051218W WO 2018087757 A1 WO2018087757 A1 WO 2018087757A1
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mmp
timp2
polypeptide
binding
seq
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PCT/IL2017/051218
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French (fr)
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Niv Papo
Julia Shifman
Jason SHIRIAN
Valeria ARKADASH
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The National Institute for Biotechnology in the Negev Ltd.
Yissum Research Development Company Of The Hebrew University
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Publication of WO2018087757A1 publication Critical patent/WO2018087757A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • MMP matrix metalloproteinase
  • MMPs matrix metalloproteinases
  • MMPs play a crucial role in the promotion of metastasis and other aspects of tumor growth through cleavage and activation of a variety of different proteins (Egeblad et al., Coussens et al.).
  • MMP- 14 also known as MT1-MMP
  • Elevated MMP- 14 expression is also associated with early death of patients with various cancers (Zhang et al.) and is correlated with cancer progression, invasion, lymph node metastases, a poor clinical stage, larger tumor size, and tumor stage progression (Tetu, et al.). In the laboratory, MMP- 14 has been shown to play a direct role in the
  • MMP inhibitors Because of the importance of MMPs in cancer, many MMP inhibitors have been designed over the past 30 years. Yet, due to their high toxicity, all failed in clinical trials (Overall, C. M. et al., Lopez-Otin, C. et al). The major drawback of MMP inhibitors developed to date was that they targeted the conserved active site of MMPs and thus reacted with other Zn 2+ -containing proteins in the body, resulting in high toxicity (Vandenbroucke et al.).
  • MMP-8 and MMP- 12 exhibit anti-tumorigenic function (Folgueras et al., Decock et al.).
  • MMP-8 and MMP- 12 exhibit anti-tumorigenic function (Folgueras et al., Decock et al.).
  • MMP inhibitors that can inhibit one or a small subset of MMPs with high specificity and high efficacy.
  • polypeptide MMP inhibitors including an isolated polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 1, or a fragment, derivative or analog thereof.
  • the polypeptide MMP inhibitor is a polypeptide at least 70% identical to an amino acid sequence set forth as SEQ ID NOs 2, 4, 6, 8, 24-27 or 47.
  • nucleic acid sequences including a nucleic acid sequence encoding the described isolated polypeptides, such as the nucleic acid sequence set forth as SEQ ID NO: 3, 5, 7, or 9, and expression vectors that contain the provided nucleic acid sequences.
  • the described synthetic polypeptides and polynucleotides can be used in compositions for treatment of a disease or condition characterized by aberrant overexpression of matrix metalloproteinase (MMP) -14, MMP-9, or MMP-2.
  • MMP matrix metalloproteinase
  • MMP matrix metalloproteinase
  • Figure 1 shows library design for identification of the described peptides.
  • Structure of N-TIMP2 shown in cyan
  • MMP-14CAT shown in gray [adapted from PDB ID: 1BUV] [adapted from PDB ID: 1BUV], with positions that were chosen for full randomization shown as red spheres.
  • Purple sphere represents the Zn 2+ atom found in the active site of MMP-14CAT.
  • the plots below the structure summarize AAGbind values obtained from computational saturation mutagenesis performed at the N-TIMP2 positions that are mutated in the YSD library (see Example 1 for details).
  • the vertical axis of each subplot represents the MMP type, and the horizontal axis represents the amino acid identity of the mutation.
  • Blue dots represent AAGbind ⁇ -0.5 kcal/mol
  • green dots represent -0.5 ⁇ AAGbind ⁇ 0.5 kcal/mol
  • yellow dots represent 0.5 ⁇ AAGbind ⁇ 1.5 kcal/mol
  • red dots represent AAGbind ⁇ 1.5 kcal/mol
  • gray dots represent cases where AGA or AGB (AG of single chain) was greater than 2 kcal/mol and AAGbind was negative, indicating single-chain destabilizing mutations leading to an artificial improvement in AAGbind.
  • Figures 2A-2E show random mutagenesis library screening of N-TIMP2.
  • Figure 2A Schematic representation of N-TIMP2WT expressed using the pCTCON construct, with MMP- 14-cAT as a soluble target (left panel), and flow cytometry analysis of yeast expressing N- TEV1P2WT in the pCTCON construct labeled with both 1 ⁇ of MMP-14CAT conjugated to DyLight-488 (em. 525 nm) and with mouse anti-c-Myc antibody followed by sheep anti-mouse secondary antibody conjugated to PE (em. 575 nm) for detection of expression (right panel).
  • FIG. 2B Schematic representation of N-TIMP2WT expressed using the pCHA construct, with MMP- 14CAT as its soluble target (left panel), and flow cytometry analysis of the yeast expressing N-TIMP2WT in the pCHA construct under the same conditions as the N-TIMP2WT in the pCTCON construct.
  • Figure 2C FACS results showing sorting of focused N-TIMP2 libraries.
  • Sort round 1 represents a sort for protein expression in which the library is labeled only for detection of expression, and a population with a high expression level is selected from the representative gate.
  • sort round 2 In sort round 2 (S2), 1 ⁇ of MMP-14CAT conjugated to DyLight-488 was used to select 1.5% (gated pool) of the high-affinity population.
  • Sort 4 and Sort 6 (S6) 50 nM and 5 nM, respectively, of MMP- 14CAT conjugated to DyLight-488 were used as targets to select the high-affinity pool of clones.
  • the x-axis shows c-Myc expression, and the y-axis shows receptor binding.
  • Polygons indicate sort gates used to select the desired yeast cell population.
  • Figure 2D Titration curves of the yeast surface displayed N- TEVIP2WT and four selected N-TIMP2 clones (N-TIMP2 A -D). The yeast cells expressing four different clones were labeled with MMP- 14CAT conjugated to DyLight-488 at a concentration of 1-1000 nM.
  • the binding signal (DyLight-488, em. 525 nm) was normalized to the expression of each clone (PE, em. 575 nm).
  • Figure 2E Sequences of the N-TIMP2 variants identified after the sixth sort (S6).
  • Figure 3 shows logo summaries of all N-TIMP2 clones sequenced after each selective round of sorting.
  • the height of each letter is proportional to its frequency at that position.
  • the total height of the stack represents conservation at that position.
  • Green, purple, blue, red and black letters, respectively, represent polar, neutral, basic, acidic and hydrophobic amino acids.
  • the identity and position number of N-TIMP2WT are denoted at the bottom of the figure.
  • numbers S l-6 represent the sort number.
  • To the right, the average AAGbind value in kcal/mol is shown (see Experimental Procedures for calculation details).
  • the logos were generated by the WebLogo sever (weblogo.berkeley.edu/logo.cgi).
  • Figures 4A-4E show purification, characterization, and MMP-14 inhibitory activity of
  • N-TIMP2 variants Figure 4A: Size-exclusion chromatography (SEC) for clone N-TIMP2B.
  • Figure 4B Mass spectrometry analysis for clone N-TIMP2B after SEC.
  • Figure 4C SDS- PAGE analysis on 15% polyacrylamide gel under reducing conditions for the purified clones N-TIMP2WT, N-TIMP2A, N-TIMP2 b , N-TIMP2 c and N-TIMP2 D .
  • Figure 4D MMP- 14 inhibition by N-TIMP2 W T;
  • Figure 4E MMP- 14 inhibition by N-TIMP2 variants.
  • MMP- 14CAT was incubated with N-TIMP2WT and N-TIMP2 variants at various concentrations.
  • the substrate Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH 2 (SEQ ID NO: 23) was added, and the fluorescent signal upon substrate cleavage by MMP-14CAT was measured.
  • the slopes (cleavage velocities) at each inhibitor concentration were measured, and the curves were fitted by the Morrison equation (eq.1) to obtain the Ki values.
  • Figures 5A-5E show SPR measurements of MMP-14CAT and N-TIMP2 variants interactions.
  • Figure 5A N-TIMP2 W T;
  • Figure 5B , N-TIMP2 A ;
  • Figure 5C N-TIMP2 B ;
  • Figure 5D N-TIMP2c;
  • Figure 5E N-TIMP2D.
  • the binding response (y-axis) was measured for MMP-14CAT at different concentrations, 20 nM (blue), 10 nM (green), 5 nM (purple), 2.5 nM (yellow) and 0 nM (red).
  • Figures 6A-6C show inhibition of gelatin and collagen degradation by N-TIMP2 variants.
  • Figure 6A Representative gelatin zymography of 1 nM MMP-2 and MMP-9 full-length proteins resolved on SDS-PAGE and treated with 100 nM of ⁇ - ⁇ 2 inhibitors. Left lane of each gel represents pro-MMP2 (upper) and active MMP-2 (lower) while the right lane represent active MMP-9.
  • Figure 6B Quantification of band intensity normalized to the intensity of the control (untreated) gel.
  • N-TIMP2 W T 51 %
  • N-TIMP2 A 57%
  • N-TIMP2 B 51 %
  • N-TIMP2 C 41 %
  • N-TIMP2 D 55%
  • N-TIMP2 D 55%
  • N-TIMP2 WT 61 %
  • N-TIMP2 A 68%
  • N-TIMP2 B 68%
  • N-TIMP2 C 79%
  • N-TIMP2 D 74%.
  • Error bars represent SEM;
  • Figure 6C Representative degradation products of bovine type I collagen.
  • Collagen (1.5 ⁇ ) was incubated with (lane 2) or without (lane 1) 2.2 ⁇ of MMP-14CAT, or with 2.2 ⁇ of MMP-14CAT along with 2.5 ⁇ of inhibitors (N-TIMP2 WT , N-TIMP2 A , N-TIMP2 B , N-TIMP2 C , N-TIMP2 D , lanes 3-7, respectively).
  • the labels ' ⁇ and ' ⁇ 2' indicate the al (I) and ⁇ 2 (I) chains of type I collagen, while ' ⁇ chains' indicates the cross-link between two al chains or between al chain and a2 chain.
  • the 3/4 cleavage product of type I collagen by MMP-14 CAT IS indicated as TC A (3/4) fragment.
  • Figures 7A-7C show binding of N-TIMP2 variants to MDA-MB-231 cells and inhibition of cancer cell invasion.
  • Figure 7A Expression of MMP-14 on MDA-MB-231 cells (left panel). Solid line, cells only; dotted line, MMP-14 expression; Right panel, binding of the N-TIMP2 inhibitors, conjugated to DyLight-488 fluorescent dye, to the cells.
  • Figure 7B Representative micrographs of invading MDA-MB-231 cells treated with 7.5 nM of N-TIMP2 inhibitors; the cells were stained with Dipp Kwik Differential Stain and visualized as dark forms on light background by light microscopy using x40 magnification lenses.
  • Figure 7C Calculated fold of invasion. The experiment was repeated 3 times; means and standard error are given. *** P ⁇ 0.001 for t-test comparisons of the indicated condition versus N-TIMP2WT.
  • Figure 8A-8C show structural analysis of MMP-14 affinity enhancing mutations.
  • N-TIMP2 is shown in cyan and MMP-14 is shown in green.
  • Figure 8A Interactions at position 4 of N-TIMP2;
  • Figure 8B Interactions at positions 68 and 71 of N-TIMP2;
  • Figure 8C Interactions at positions 97 and 99 of N-TIMP2.
  • Residues involved in an interaction are shown in stick representation. Mutant residue identities are labeled in red, WT identities of residues in all panels are labeled in black. Polar interactions are shown as dotted red lines.
  • Figure 9 a variation of the model shown in Figure 1, shows N-TIMP2 (red) in complex with MMP-14cat (gold) (adapted from PDB ID: 1BUV). Positions included in the small focused library are highlighted as green spheres.
  • Figure 10A shows schematic representation of N-TIMP2 displayed on the yeast surface (adapted from Chao et al. [13]). The N-terminus of N-TIMP2 is free and facing away from the yeast surface.
  • Figure 10B shows expression of the construct can be monitored by FACS by fluorescently labeled (red burst in Figure 10A) antibodies that bind the c-Myc tag in the construct.
  • Figure IOC shows binding of N-TIMP2 to catalytic domains of MMP proteins in solution can be monitored simultaneously by FACS by directly fluorescent labeled MMP proteins (yellow and blue bursts in Figure 10A). Red polygons represent sample cell collection gates. The figure here was produced in the initial sorting round on the focused library.
  • Figures 11A and 11B show logos based on sequencing of evolved libraries after final selection experiments representing frequency of amino acid residues at mutated positions in each evolved library. The WT identity of each position is shown below each logo.
  • Figure 11 A represents sequencing results of the MMP-14 binding evolved library.
  • Figure 11B represents sequencing results of the MMP-9 binding evolved library.
  • Figure 12 shows a logo of sequencing results from 24 colonies isolated from the third selective sort on a library of randomly mutated N-TIMP2 genes, with selection pressure applied to obtain MMP-14 specific mutants. Mutation is most prominent at position 71, 98 and 116. Positions located in the N-TIMP2:MMP interface are marked with an asterisk.
  • Figures 13A and 13B show binding specificity of select N-TIMP2 mutant yeast colonies assessed by FACS. Binding is determined by the average signal of fluorescence of yeast cells caused by fluorescently labeled MMP. Binding specificity is determined in the following manner in the case of MMP-14 ( Figure 13A): (MMP-14 mut /MMP-14 w t)/(MMP- 9mut/MMP-9wt), and vice-versa for MMP-9 ( Figure 13B). Tables at right indicate identities of mutants as compared to WT, the order of letters corresponds to the following positions on N- TEV1P2: 4, 6, 35, 38, 68, 71, 97, 99. Mutants designated with a '+' contain mutations which were not planned.
  • Figures 14A and 14B show enzyme activity assays with inhibition by N-TIMP-WT or mutants expressed in P. pastoris. Data for WT N-TIMP2 expressed in E. coli is not shown for simplicity. Points ⁇ S.D. represent an average of at least three trials. Curves were fit to equation 1.
  • Figure 14A Enzyme activity experiments performed with MMP-14 ca t.
  • Figure 14B Enzyme activity experiments performed with MMP-9 ca t.
  • Figures 15A-15M Interactions observed in computationally modeled structures of specificity enhancing mutations of N-TIMP2.
  • N-TIMP2 is depicted in cyan and MMP-9 ca t or MMP-14 C at is depicted in green, polar contacts are showed as red dotted lines. MMP residues shown in sticks are not further than 4A away from the highlighted N-TIMP2 residue.
  • Figures 15A-15D depict the interactions at position 4, Figures 15E-15G depict interactions at position 38, Figures 15H-15K depict interactions at position 71, Figures 15L-15M depict interactions at position 97.
  • the WT structure at each position is depicted in Figures 15 A, 15C, 15E, 15H, 15 J, and 15L. All other figures depict the interactions caused by mutation. The bottom right-hand corner of each figure describes the mutation on N-TIMP2 and the MMP type it is interacting with.
  • nucleic and/or amino acid sequences provided herewith are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO 1 is a summary sequence of the N-TIMP2 variants described herein.
  • SEQ ID NO 2 is the amino acid sequence of the N-TIMP2A polypeptide.
  • SEQ ID NO 3 is a nucleic acid encoding the N-TIMP2A polypeptide.
  • SEQ ID NO 4 is the amino acid sequence of the N-TIMP2B polypeptide.
  • SEQ ID NO: 5 is a nucleic acid encoding the N-TIMP2B polypeptide.
  • SEQ ID NO: 6 is the amino acid sequence of the N-TIMP2c polypeptide.
  • SEQ ID NO: 7 is a nucleic acid encoding the N-TIMP2c polypeptide.
  • SEQ ID NO: 8 is the amino acid sequence of the N-TIMP2D polypeptide.
  • SEQ ID NO: 9 is a nucleic acid encoding the N-TIMP2 D polypeptide.
  • SEQ ID NO: 10 is the amino acid sequence of wildtype N-TIMP2.
  • SEQ ID NOs 11 and 12 are forward and reverse primers for cloning N-TIMP2WT into the pCTCON vector.
  • SEQ ID NOs 13 and 14 are forward and reverse primers for cloning N-TIMP2WT into the pCHA-VRCOl-scFv vector.
  • SEQ ID Nos 15 and 16 are forward and reverse primers for use in production of the focused N-TIMP2W T library.
  • SEQ ID NO's 17-21 are forward primers for cloning N-TIMP2WT and variants A-D into the pPICZaA vector.
  • SEQ ID NO: 22 is the reverse primer for cloning N-TIMP2WT and variants A-D into the pPICZaA vector.
  • SEQ ID NO: 23 is the fluorogenic substrate for use in MMP-14 binding assays.
  • SEQ ID NO: 24 is the amino acid sequence of the N-TIMP2 14-4 mutant polypeptide.
  • SEQ ID NO: 25 is the amino acid sequence of the N-TIMP2 14-6 mutant polypeptide.
  • SEQ ID NO: 26 is the amino acid sequence of the N-TIMP2 9-2 mutant polypeptide.
  • SEQ ID NO: 27 is the amino acid sequence of the N-TIMP2 9-6 mutant polypeptide.
  • SEQ ID NOs: 28 and 29 are forward degenerate codon primers used to make mutant N- ⁇ 2 sequences.
  • SEQ ID Nos: 30 and 31 are reverse degenerate codon primers used to make mutant N- TEVIP2 sequences.
  • SEQ ID NO: 32 is a forward N-TIMP2 amplification primer.
  • SEQ ID NOs: 33 and 34 are forward and reverse N-TIMP2 amplification primers.
  • SEQ ID NO: 35 is a forward amplification primer for mutant 14-4.
  • SEQ ID NO: 36 is a forward amplification primer for mutant 14-6.
  • SEQ ID NO: 37 is a forward amplification primer for mutants 9-2 and 9-6.
  • SEQ ID NO: 38 is a forward amplification primer for mutant ATE.
  • SEQ ID NO: 39 is a reverse amplification primer for N-TIMP2 mutants.
  • SEQ ID NO: 40 is a forward sequencing primer for N-TIMP2 mutants.
  • SEQ ID Nos: 41-45 are forward PCR primers for transferring N-TIMP2 mutant sequences into the pet21 expression plasmid.
  • SEQ ID NO: 46 is a reverse PCR primer for transferring N-TIMP2 mutant sequences into the pet21 expression plasmid.
  • SEQ ID NO: 47 is the amino acid sequence of the N-TIMP2 ATE mutant polypeptide.
  • Administration The introduction of a composition into a subject by a chosen route.
  • Administration of an active compound or composition can be by any route known to one of skill in the art.
  • Administration can be local or systemic.
  • local administration examples include, but are not limited to, topical administration, subcutaneous administration, intramuscular administration, intrathecal administration, intrapericardial administration, intra-ocular administration, topical ophthalmic administration, or administration to the nasal mucosa or lungs by inhalational administration.
  • local administration includes routes of administration typically used for systemic administration, for example by directing intravascular administration to the arterial supply for a particular organ.
  • Local administration also includes the incorporation of active compounds and agents into implantable devices or constructs, such as vascular stents or other reservoirs, which release the active agents and compounds over extended time intervals for sustained treatment effects.
  • Systemic administration includes any route of administration designed to distribute an active compound or composition widely throughout the body via the circulatory system.
  • systemic administration includes, but is not limited to intra- arterial and intravenous
  • Systemic administration also includes, but is not limited to, oral, topical, subcutaneous, intramuscular routes, or administration by inhalation, when such administration is directed at absorption and distribution throughout the body by the circulatory system.
  • Analog or Derivative An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28).
  • QSAR quantitative structure activity relationships
  • a derivative is a biologically active molecule derived from the base structure.
  • Autoimmune disease A disease resulting from an aberrant immune response, such as the production of antibodies or cytotoxic T cells specific for a self-antigen or a subject's own cells or tissues.
  • Autoimmune diseases include, but are not limited to, inflammatory bowel disease, diabetes mellitus type 1, systemic lupus erythematosis, Churg-Strauss Syndrome, multiple sclerosis, Graves' disease, idiopathic thrombocytopenic purpura and rheumatoid arthritis.
  • Binding affinity A term that refers to the strength of binding of one molecule to another at a site on the molecule. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association constant and dissociation constant for a pair of molecules, but it is not critical to the methods herein that these constants be measured or determined. The concepts of binding affinity, association constant, and dissociation constant are well known.
  • sample Any sample that may be obtained directly or indirectly from an organism, such as a subject, including whole blood, plasma, serum, tears, mucus, saliva, urine, pleural fluid, spinal fluid, gastric fluid, sweat, semen, vaginal secretion, sputum, fluid from ulcers and/or other surface eruptions, blisters, abscesses, tissues, cells (such as, fibroblasts, peripheral blood mononuclear cells, or muscle cells), organelles (such as mitochondria), organs, and/or extracts of tissues, cells (such as, fibroblasts, peripheral blood mononuclear cells, or muscle cells), organelles (such as mitochondria) or organs.
  • the sample is collected or obtained using methods well known to those skilled in the art.
  • Neoplasia is a neoplasm (a tumor or cancer), which is an abnormal growth of tissue that results from excessive cell division.
  • a tumor that does not metastasize is referred to as "benign.”
  • a tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant.”
  • Neoplasia is one example of a proliferative disorder.
  • a “cancer cell” is a cell that is neoplastic, for example a cell or cell line isolated from a tumor.
  • hematological tumors include leukemias.
  • solid tumors such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma,
  • chondrosarcoma chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers (such as small cell lung carcinoma and non-small cell lung carcinoma), ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma,
  • Cardiac disease Also, cardiovascular disease.
  • Chemotherapeutic agent An agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth or hyperplasia. Such diseases include cancer, autoimmune disease as well as diseases characterized by hyperplastic growth such as psoriasis.
  • a chemotherapeutic agent is a radioactive compound.
  • chemotherapeutic agent for instance, see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et ah, Chemotherapy, Ch.
  • Chemotherapeutic agents include small molecule agents and biologic agents.
  • Contacting Placement in direct physical association. Includes both in solid and liquid form. Contacting can occur in vitro with isolated cells or in vivo by administering to a subject.
  • Detect To determine if an agent (such as a signal or particular probe, such as a MMP inhibitory polypeptide described herein) is present or absent. In some examples, this can further include quantification.
  • Diagnosis The process of identifying a disease or a predisposition to developing a disease, by its signs, symptoms, and results of various tests and methods, for example the methods disclosed herein. The conclusion reached through that process is also called "a diagnosis.”
  • the term "predisposition" refers to an effect of a factor or factors that render a subject susceptible to a condition, disease, or disorder, such as cancer. In some examples, of the disclosed methods, testing is able to identify a subject predisposed to developing a condition, disease, or disorder, such as a malignant carcinoma.
  • Effective amount of a compound A quantity of compound sufficient to achieve a desired effect in a subject being treated; also referred to as a "therapeutically effective amount.”
  • An effective amount of a compound can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of the compound will be dependent on the compound applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the compound.
  • a polynucleotide is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.
  • Expression Control Sequences Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked, for example the expression of a nucleic acid encoding a MMP inhibitory polypeptide described herein.
  • Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included.
  • promoters derived from the genome of mammalian cells such as metallothionein promoter
  • mammalian viruses such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • Functional fragments and variants of a polypeptide include those fragments and variants that maintain one or more functions of the MMP inhibitory polypeptides described herein. It is recognized that the gene or cDNA encoding a polypeptide can be considerably mutated without materially altering one or more the polypeptide's functions. First, the genetic code is well-known to be degenerate, and thus different codons encode the same amino acids. Second, even where an amino acid substitution is introduced, the mutation can be conservative and have no material impact on the essential functions of a protein. See Stryer, Biochemistry 3rd Ed., (c) 1988. Third, part of a polypeptide chain can be deleted without impairing or eliminating all of its functions.
  • insertions or additions can be made in the polypeptide chain for example, adding epitope tags, without impairing or eliminating its functions (Ausubel et al. Short Protocols in Molecular Biology, 4 th ed., John Wiley & Sons, Inc., 1999).
  • Other modifications that can be made without materially impairing one or more functions of a polypeptide include, for example, in vivo or in vitro chemical and biochemical modifications or the incorporation of unusual amino acids.
  • modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquination, labeling, e.g., with radionucleides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art.
  • radioactive isotopes such as 32 P
  • ligands which bind to or are bound by labeled specific binding partners ⁇ e.g., antibodies
  • fluorophores e.g., fluorophores
  • chemiluminescent agents e.g., fluorophores, fluorophores, and chemiluminescent agents.
  • Inhibiting protein activity To decrease, limit, or block an action, function or expression of a protein.
  • the phrase inhibit protein activity is not intended to be an absolute term. Instead, the phrase is intended to convey a wide-range of inhibitory effects that various agents may have on the normal (for example, uninhibited or control) protein activity.
  • Inhibition of protein activity may, but need not, result in an increase in the level or activity of an indicator of the protein's activity. By way of example, this can happen when the protein of interest is acting as an inhibitor or suppressor of a downstream indicator.
  • protein activity may be inhibited when the level or activity of any direct or indirect indicator of the protein's activity is changed (for example, increased or decreased) by at least 10%, at least 20%, at least 30%, at least 50%, at least 80%, at least 100% or at least 250% or more as compared to control measurements of the same indicator.
  • IBD Inflammatory Bowel Disease
  • ulcerative colitis ulcerative colitis and Crohn's disease.
  • a pharmaceutically acceptable fluid composition comprising at least one active ingredient, for example, a polypeptide.
  • the active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, pH buffering agents and the like.
  • non-toxic auxiliary substances such as emulsifying agents, preservatives, pH buffering agents and the like.
  • nucleic acid and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and polypeptides.
  • Label A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule.
  • Specific, non-limiting examples of labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.
  • MMP Matrix metalloproteinase(s)
  • family of enzymes consisting of more than 20 homologous proteins that play a significant role in the degradation of the extracellular matrix, a crucial step in many cellular processes.
  • MMPs are multi-domain proteins that differ in domain architecture and substrate preferences (1), but they all share a catalytic domain with a nearly identical active site containing a Zn 2+ ion (2).
  • MMPs are synthesized in an inactive form and are subsequently activated by the cleavage of a pro-domain, often by other MMPs or by different proteases (3,4), thus producing complex protein-protein interaction (PPI) networks (5).
  • PPI complex protein-protein interaction
  • MMP-2 is activated physiologically by MMP- 14; MMP-9 is activated by active MMP-2 and active MMP-7; and MMP-1 is also activated by active MMP-7 (4).
  • Some MMPs, such as MMP-3, are 'master regulators,' being able to activate themselves as well as other MMPs (6). Imbalance in MMP activity leads to various diseases, including cancers (7), cardiovascular diseases (8) and arthritis (9).
  • Operably linked A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • parenteral Administered outside of the intestine, for example, not via the alimentary tract.
  • parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.
  • compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions for example, powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polypeptide A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred.
  • polypeptide or protein as used herein encompasses any amino acid sequence and includes modified sequences such as glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
  • polypeptide fragment refers to a portion of a polypeptide which exhibits at least one useful epitope.
  • functional fragments of a polypeptide refers to all fragments of a polypeptide that retain an activity, or a measurable portion of an activity, of the polypeptide from which the fragment is derived. Fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
  • An epitope is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen. Thus, smaller peptides containing the biological activity of insulin, or conservative variants of the insulin, are thus included as being of use.
  • substantially purified polypeptide refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • Variations in the cD A sequence that result in amino acid changes, whether conservative or not, are usually minimized in order to preserve the functional and immunologic identity of the encoded protein.
  • the immunologic identity of the protein may be assessed by determining whether it is recognized by an antibody; a variant that is recognized by such an antibody is immunologically conserved. Any cDNA sequence variant will preferably introduce no more than twenty, and preferably fewer than ten amino acid substitutions into the encoded polypeptide.
  • Variant amino acid sequences may, for example, be 80%, 90% or even 95% or 98% identical to the native amino acid sequence. Programs and algorithms for determining percentage identity can be found at the NCBI website.
  • Preventing or treating a disease refers to inhibiting the full development of a disease, for example inhibiting the development of myocardial infarction in a person who has coronary artery disease or inhibiting the progression or metastasis of a tumor in a subject with a neoplasm.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • Sequence identity The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well known in the art.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for
  • Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
  • nucleic acid sequences that do not show a high degree of identity can nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
  • Solid support (or substrate): Numerous and varied solid supports are known to those in the art and include, without limitation, nitrocellulose, the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, membranes, microparticles (such as latex particles), and sheep (or other animal) red blood cells. Any suitable porous material with sufficient porosity to allow access by detector reagents and a suitable surface affinity to immobilize capture reagents ⁇ e.g., monoclonal antibodies) is contemplated by this term.
  • the porous structure of nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents, for instance, capture reagents.
  • Nylon possesses similar characteristics and is also suitable. Microporous structures are useful, as are materials with gel structure in the hydrated state.
  • useful solid supports include: natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfaci
  • the surface of a solid support may be activated by chemical processes that cause covalent linkage of an agent (e.g., a capture reagent) to the support.
  • an agent e.g., a capture reagent
  • any other suitable method may be used for immobilizing an agent (e.g. , a capture reagent) to a solid support including, without limitation, ionic interactions, hydrophobic interactions, covalent interactions and the like.
  • Subject Living multi-cellular organisms, including vertebrate organisms, a category that includes both human and non-human mammals.
  • Sustained release of a pharmaceutical compound: A sustained release compound that can be biologically/chemically available and active upon administration to a subject and contact with a target site, and over an extended period of time. Numerous sustained release additives and formulations are known to the art and can be combined with the MMP inhibitors described herein.
  • Tissue inhibitors of metalloproteinases Broad inhibitors of the MMP family. In nature, there are four such inhibitors (TIMPs 1-4) that show 40-50% sequence identity and exhibit slightly different preferences for various MMPs (31). All TIMPs bind to MMPs using the same interaction mode, coordinating the active site but also interacting with a number of additional sites on MMPs (32-38). As natural effectors, TIMPs are non- immunogenic and non-toxic to humans.
  • Vector A nucleic acid molecule as introduced into a host cell, thereby producing a transfected host cell.
  • Recombinant DNA vectors are vectors having recombinant DNA.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can also include one or more selectable marker genes and other genetic elements known in the art.
  • Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses. II. Overview of Several Embodiments
  • polypeptide MMP inhibitors including an isolated polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 1, or a fragment, derivative or analog thereof.
  • the polypeptide MMP inhibitor is a polypeptide at least 70% identical to an amino acid sequence set forth as SEQ ID NOs 2, 4, 6, 8, 24-27, or 47.
  • the described polypeptides include a detectable label. In other embodiments, the described polypeptide is conjugated to a solid substrate.
  • the isolated polypeptide is conjugated to a chemotherapeutic agent.
  • nucleic acid sequences including a nucleic acid sequence encoding the described isolated polypeptides, such as the nucleic acid sequence set forth as SEQ ID NO: 3, 5, 7, or 9, and expression vectors that contain the provided nucleic acid sequences.
  • MMP matrix metalloproteinase
  • the disease or condition characterized by aberrant MMP expression is selected from the group consisting of a cancer, such as a carcinoma, an autoimmune disease, such as inflammatory bowel disease, and cardiac disease.
  • the isolated polypeptide is administered following cardiac arrest in the subject.
  • the isolated polypeptide is formulated for immediate or sustained release. In other embodiments, the isolated polypeptide is formulated for local or systemic administration.
  • MMP matrix metalloproteinase
  • MMP matrix metalloproteinase
  • the cell is in a sample isolated from a subject. In other embodiments of the described detection methods, the cell is within a subject.
  • N-TIMP2 TEVIP2 (N-TIMP2) which have been optimized for enhanced MMP-14 or MMP-9 binding affinity and specificity, and which include at least 3 mutations from the wildtype N-TIMP2 sequence.
  • the described N-TIMP2 variants include polypeptides, variants, and fragments thereof, of a polypeptide including the following sequence of N-TIMP2 with variable positions Xi-Xio:
  • Xi is R, A, M, E, P, or S
  • X 2 is V, R, or G
  • X 3 is L, M, I, V, or P
  • X 4 is N, D, Q, or P
  • X 5 is D, G, S, or N
  • X 6 is V, I, A, G, or N
  • X 7 is H, R or L
  • Xs is I or T
  • X 9 is S, N, or T
  • X 10 is K or E (SEQ ID NO: 1).
  • polypeptide includes the following sequence described herein as N-TIMP2 A :
  • polypeptide includes the following sequence described herein as N-TIMP2 B :
  • polypeptide includes the following sequence described herein as N-TIMP2 C :
  • polypeptide includes the following sequence described herein as N-TIMP2 D :
  • polypeptide includes the following sequence described herein as N-TIMP2 mutant 14-4:
  • polypeptide includes the following sequence described herein as N-TIMP2 mutant 14-6:
  • polypeptide includes the following sequence described herein as N-TIMP2 mutant 9-2:
  • polypeptide includes the following sequence described herein as N-TIMP2 mutant 9-6:
  • polypeptide includes the following sequence described herein as N-TIMP2 mutant ATE:
  • polypeptides include polypeptides that share about 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity with the described N-TIMP2 variants.
  • sequence variance from WT is particularly found in those amino acids apart from those described herein as involved in MMP binding (e.g. Xi-X 8 , above).
  • the variation from WT sequence can be conservative substitutions that one of skill will not expect to significantly alter the shape or charge of the polypeptide.
  • the described polypeptides also include those polypeptides that share 100% sequence identity to those indicated, but which differ in post-translational modifications from the native or natively- produced sequence.
  • the described N-TIMP2 variants are provided as a discrete biomolecules.
  • the described polypeptides are a domain of a larger polypeptide, such as an independently-folded structural domain, or an environment-accessible functional domain.
  • the N-TIMP2 polypeptides described herein inhibit MMP activity, but also bind with increased affinity and specificity to MMP- 14 and/or MMP-9, and which allow for strong and specific targeting of a MMP- 14 and/or MMP-9-expressing cell.
  • the described polypeptides are conjugated at the N- and/or C-terminus to at least one of a variety of small molecules, chemotherapeutic agents, labels, and solid substrates, which are thereby targeted to a MMP-14 and/or MMP-9 expressing cell.
  • polypeptides are conjugated to any one of the described polypeptides.
  • the described polypeptides are conjugated to any one of the described polypeptides.
  • chemotherapeutic agent as described herein and as known to the art.
  • agents can include small molecule agents, antibodies, and the like which are designed, for example, to inhibit cell proliferation and/or metastatic cell migration and/or promote cell death.
  • agents include cytotoxic radioisotopes, chemotherapeutic agents and toxins.
  • a detectable label such as a detectable chemical, fluorescent, or radioactive label can be conjugated to the described polypeptides.
  • a detectable label such as a detectable chemical, fluorescent, or radioactive label
  • Antibodies that can be conjugated to N-TIMP2 variant polypeptides, and which themselves are or can be labeled, are standard in the art and are included in the current disclosure.
  • the described N-TIMP2 variants can be conjugated to any suitable affinity tag for specific isolation of MMP-14 and/or MMP-9 expressing cells through standard affinity chromatographic methods.
  • suitable affinity tag for specific isolation of MMP-14 and/or MMP-9 expressing cells through standard affinity chromatographic methods.
  • suitable affinity tag include myc, glutathione- s-transferase (GST), maltose binding protein (MBP), hemagglutinin (HA), and polyhistadine.
  • N-TIMP2 polypeptides described herein can be associated with or conjugated to a solid substrate, alone or in combination with any of the already described modifications.
  • nucleic acids encoding the described N-TIMP2 variant polypeptides such as the nucleic acids set forth herein as SEQ ID NO: 3 (encoding N- TIMP2 A ); SEQ ID NO: 5 (encoding N-TIMP2 B ); SEQ ID NO: 7 (encoding N-TIMP2 C ); and SEQ ID NO: 9 (encoding N-TIMP2 D ).
  • sequences of the described nucleic acids can vary significantly from the sequences set forth herein as SEQ ID NOs 3, 5, 7, and 9; and without any change in the encoded polypeptide.
  • Other and/or additional mutations in the described polypeptides, such as conservative amino acid mutations, can also be included without an appreciable difference.
  • the described nucleic acids share between 60%-100% sequence identity with SEQ ID NOs 3, 5, 7, and 9, such as 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity.
  • nucleic acid sequences are contained within a
  • DNA cloning and/or expression plasmid as are standard in the art. It will be appreciated that any standard expression plasmid can be used to express one or more of the described N-TIMP2 variant-encoding nucleic acids. Such plasmids will minimally contain an origin of replication, selection sequence (such as, but not limited to an antibiotic resistance gene), and expression control sequences operably linked to the N-TIMP2 variant polypeptide.
  • the expression plasmids include post-translational sequences (e.g. signal sequences to direct polypeptide processing and export) that are encoded in-frame with the N- TEV1P2 variant nucleic acids.
  • bacterial expression plasmids include IPTG- inducible plasmids, arabinose-inducible plasmids and the like.
  • Other non-limiting examples of expression induction include light induction, temperature induction, nutrient-induction, and autoinduction, and mammalian- specific DNA expression plasmids.
  • Custom-made expression plasmids are commercially available from suppliers such as New England Biolabs (Ipswich, MA) and DNA 2.0 (Menlo Park, CA).
  • a N-TIMP2 variant- expressing plasmid can be designed for specific localized induction in response to a local cellular micro environment.
  • the peptides and nucleic acids described herein can be supplied in any pharmaceutically acceptable composition.
  • one or more N-TIMP2 variant polypeptide or polypeptide-expressing nucleic are provided in a
  • composition having a therapeutically effective dose of each therapeutic agent, as described herein, and including standard pharmaceutically acceptable salts, excipients, fillers and the like.
  • Various delivery systems are known and can be used to administer polypeptides and nucleic acids as therapeutic agents.
  • Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic molecule(s), construction of a therapeutic nucleic acid as part of a retroviral or other vector, and the like.
  • Methods of introduction include, but are not limited to, intrathecal, intradermal, intramuscular, intraperitoneal (ip), intravenous (iv), subcutaneous, intranasal, epidural, and oral routes.
  • the therapeutics may formulated for administration by any convenient route, including, for example, infusion or bolus injection, topical, absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa, and the like) ophthalmic, nasal, and transdermal, and may be administered together with other biologically active agents.
  • Pulmonary administration can also be employed (e.g., by an inhaler or nebulizer), for instance using a formulation containing an aerosolizing agent.
  • therapeutic agents are delivered in a vesicle, in particular liposomes.
  • the described polypeptides and nucleic acids can be formulated for immediate release, whereby they are immediately accessible to the surrounding environment, thereby providing an effective amount of the active agent(s), upon administration to a subject, and until the administered dose is metabolized by the subject.
  • the described polypeptides and nucleic acids can be formulated in a sustained release formulation or system.
  • the therapeutic agents are provided for an extended duration of time, such as 1, 2, 3, 4 or more days, including 1-72 hours, 24-48 hours,. 16-36 hours, 12-24 hours, and any length of time in between.
  • sustained release formulations are immediately available upon administration, and provide an effective dosage of the therapeutic composition, and remain available at an effective dosage over an extended period of time.
  • the sustained release formulation is not immediately available within the subject and only becomes available, providing a therapeutically effective amount of the active compound(s), after the formulation is metabolized or degraded so as to release the active compound(s) into the surrounding environment.
  • sustained released formulations include polymeric materials commonly used in the art, such as in implants, gels, capsules, and the like.
  • polymers such as bis(p- carboxyphenoxy)propane-sebacic-acid or lecithin suspensions may be used to provide sustained localized release.
  • Other sustained release formulations and systems for use with the described polypeptides and nucleic acids include those such as discussed in the review by Langer (Science 249, 1527 1990).
  • the described polypeptides and nucleic acids are formulated in pharmaceutically acceptable compositions of the compounds, using methods well known to those with skill in the art.
  • the compounds are formulated with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia world-wide for use in animals, and, more particularly, in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • Saline solutions, blood plasma medium, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • the medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like.
  • compositions examples include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the described compositions can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the described compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, all in immediate and sustained-release formulations as understood in the art.
  • the therapeutic can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • Therapeutic preparations will contain a therapeutically effective amount of at least one active ingredient, preferably in purified form, together with a suitable amount of carrier so as to provide proper administration to the patient.
  • the formulation should suit the mode of administration.
  • kits comprising the described N-TIMP2 variant polypeptides or encoding nucleic acids are accordingly also contemplated herein. IV. Methods of treatment of MMP associated diseases
  • MMPs Aberrant overexpression of MMPs, such as MMP- 14, MMP-2, and MMP-9 has been implicated in a variety of conditions and diseases, including various cancers, autoimmune diseases, and cardiovascular diseases.
  • the polypeptides described herein provide a MMP- 14 inhibitory agent with increased MMP- 14 binding affinity and specificity.
  • the described N- TIMP2 variants have also shown increased efficacy against MMP-2 and MMP-9. Accordingly, the described polypeptides, and nucleic acids expressing the described polypeptides, can be used in pharmaceutical compositions in methods for treating diseases and conditions associated with aberrant overexpression of MMP- 14, MMP-2, and MMP-9.
  • the described methods include administering a
  • N-TIMP2 variant polypeptide described herein to a subject in need thereof, thereby treating the disease or condition associated with aberrant expression of MMP- 14, MMP-2, and/or MMP-9.
  • compositions described herein which include the described N-TIMP2 variant polypeptides or polypeptide-encoding nucleic acids, can be used in the described methods of treatment.
  • the disease related to aberrant MMP- 14, MMP-2, and/or MMP-9 expression is a cancer, such as a fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers (such as small cell lung carcinoma and non-small cell lung carcinoma), ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma
  • the described N-TIMP2 variants inhibit cell proliferation. In other embodiments, the described variants inhibit cell migration, thereby inhibiting cancer metastasis. In still other embodiments, the described variants promote cell death, such as by promotion of apoptosis.
  • the disease related to aberrant MMP-14, MMP-2, and/or MMP-9 expression is an autoimmune disease or associated with autoimmunity.
  • diseases include inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease.
  • the disease related to aberrant MMP-14, MMP-2, and/or MMP-9 expression is a cardiovascular disease, such as myocardial infarction, and particularly heart remodeling post myocardial infarction.
  • the described N-TIMP2 variant polypeptides or encoding nucleic acids can be administered to the subject prior to (e.g. to a subject at risk of heart attack) or following a myocardial infarction, in order to decrease the continuing deleterious effects of the cardiac event.
  • diseases related to aberrant MMP-14, MMP-2, and/or MMP-9 expression that can be treated with the described polypeptides include cardiovascular diseases, autoimmune diseases, periodontal disease, diseases of the central nervous system, such as multiple sclerosis and spinal cord injury; and bone degradation.
  • the N-TIMP2 variant is administered to the subject as a polypeptide.
  • the N-TIMP2 variant is administered to the subject by way of an expression vector containing a N-TIMP2 variant-encoded nucleic acid.
  • expression of the polypeptide can be constitutive or induced, as is well-known in the art.
  • inducible expression systems can allow for specific targeting of an area, which contains the inducing signal.
  • the N-TIMP2 variant is administered to the subject in combination with other pharmaceutical agents for treatment of the disease or condition under treatment.
  • the N-TIMP2 variant can be combined with trastuzumab (Herceptin) therapy.
  • administration of the N-TIMP2 variant can be combined with surgery and/or radiation therapy.
  • the one or more therapies in combination with the N-TIMP2 variant can be administered to the subject in sequence (prior to or following) or concurrent.
  • combinations of active ingredients can be administered to a subject in a single or multiple formulations, and by single or multiple routes of administration.
  • each therapeutic agent for use in the described methods will depend on the nature of the disorder or condition to be treated, as well as the stage of the disorder or condition. Therapeutically effective amounts can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each patient's circumstances. The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of
  • the therapeutic compounds and compositions of the present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (e.g., in which the loading dose is about two to five times the maintenance dose).
  • the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art.
  • long-term treatment with the drug is contemplated.
  • the enhanced binding affinity and specificity of the N-TIMP2 variants provided herein allows for methods of detecting a cell that is expressing, such as aberrantly overexpressing MMP-14, MMP-9, or MMP-2.
  • the methods include contacting a cell that is suspected of overexpressing MMP-14, MMP-9, or MMP-2 with an isolated N-TIMP2 variant polypeptide as described herein, washing unbound polypeptide from the cell; and detecting bound
  • the N-TIMP2 variant can be used as any ligand in a variety of in vitro and in vivo labeling and detection assays by standard methods known to the art.
  • the cell or cell-containing tissue is located in a sample that has been taken from a subject by standard methods as described herein. In other embodiments, the cell or cell-containing tissue is located in or on the subject.
  • the cells prior to contacting the cell with the N-TIMP2A variant, are permeablized as standard in the art of cell staining.
  • wash step of the described methods can be done with any suitable variant polypeptide-free buffer or solution. Such washing 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.
  • N-TIMP2 variant polypeptide that has been conjugated to a solid substrate and provides the bait for binding to and detection of a suitable MMP-expressing cell, such as in an ELISA assay or modification thereof.
  • the methods of detection contemplated herein are standard, including but not limited to standard fluorescent, chemiluminescent, and immunocytochemical labeling methods.
  • Kits designed for use in the described detection methods are also described herein.
  • Such kits can contain a N-TIMP2 variant or nucleic acid expressing the polypeptide, and suitable buffers, labels, and as necessary detection reagents.
  • N-TIMP2/MMP-14 (PDB ID: 1BUV)
  • N-TIMP2/MMP-13 (PDB ID: 2E2D)
  • N-TIMP2/MMP-10 (PDB ID: 4ILW).
  • MMP-1 (PBD ID: 3SHI)
  • MMP-2 (PDB ID: 1RTG
  • MMP-3 (PDB ID: 1B3D)
  • MMP-7 (PDB ID: 1MMQ)
  • MMP-8 (PDB ID: 20 Y4)
  • MMP-9 (PDB ID: 1L6J).
  • Unbound MMP structures were first aligned to the MMP- 14 chain of the N-TIMP2/MMP-14 structure, and the original MMP- 14 atoms were removed from the structure by using PyMol.
  • the structure was subsequently refined by using the docking protocol of the ROSIE server (Lyskov et al.), and the structure with the best combination of energy score and small RMSD in comparison to the input structure was chosen as the final complex structure.
  • the energy function optimized by Sharabi et al. was used for calculating the free energy of binding for mutations in the interface. The calculation started with the following stages: a mutation was introduced into N-TIMP2, a shell of neighboring residues around the mutated residue were allowed to repack, and the free energy of the N- TIMP2/MMP complex was calculated for the wild-type complex and for the mutated complex. The two chains were then separated, and the free energies of each single chain in the wild-type complex and the mutant complex were calculated.
  • the change in free energy due to mutation (AG) of the complex and the single chains was calculated by subtracting the free energy of the wild-type structures from the free energy of the mutant structures.
  • the value of AAGbind was determined by subtracting the AG of the individual chains from the AG of the complex.
  • the energy functions are made up of terms that describe van der Waals attractive and repulsive interactions, hydrogen bond interactions, electrostatic interactions, and surface-area-based solvation [see Sharabi et al. for details] .
  • the rotamer libraries used were based on the backbone-dependent library of Dunbrack and Karplus, with additional rotamers expanded by one standard deviation around their mean ⁇ and ⁇ 2 values. The lowest-energy rotameric conformation was found using the dead-end elimination theorem.
  • N-TIMP2 mutants The combinatorial library of N-TIMP2 mutants was designed on the basis of the in silico saturation mutagenesis results, with focus on MMP- 14 as a desired target and MMP-1, MMP-2, MMP-8, MMP-9, MMP- 10, and MMP- 13 as alternative targets. N-TIMP2 positions that contained many affinity-enhancing mutations, particularly towards MMP- 14 were selected.
  • TIMP-4 Structure Modeling A homology model of human TIMP4 was built using the I-
  • TASSER server the input sequence was that deposited in the UNIPROT database (accession #: Q99727) and was modified by removing the signal peptide and truncating the sequence to residue 184.
  • the server produced one output structure.
  • N- TIMP2wT The gene encoding human N- TIMP2wT (residues 1- 127) (Sharabi et al.) was expressed in a Yeast Surface Display (YSD) system in two constructs.
  • YSD Yeast Surface Display
  • N-TIMP2WT was amplified by PGR reaction with Phusion DMA polymerase ( New
  • VRCOl-scFv vector was used (obtained from Dane Wittrup, MIT) (Mata-Fink et al.).
  • the C-terminus of the gene of interest is fused to the N-terminus of Aga-2 (Angelini et al), leaving the N-terminus of the displayed protein exposed to the solvent.
  • the gene was amplified with a forward primer (5'- CTTGTTGGCTATCTTCGCTGCTTTGCCATTGGCCTTAGCTTGCAGCTGCTCCCCGGT G-3' ; SEQ ID NO: 13) containing the KR sequence at the N-terminus of N-TIMP2, which results in a free N-terminus after post-modification cleavage by Kex-2 protease.
  • the reverse primer (5'--
  • the pCT and pCHA vectors were linearized with BamHI-HF and Nhe-HF enzymes (New England Biolabs, USA) and, together with the respective N-TIMP2WT insert, were transformed by homologous recombination into a competent EBYIOO S. cerevisiae yeast strain using a MicroPulser electroporator (Bio-Rad, CA, USA), as previously described.
  • the transformed yeast was first grown on selective SDCAA plates (0.54% Na 2 HP0 4 , 0.856% Na 2 HP0 4 H 2 0, 18.2% sorbitol, 1.5% agar, 2% dextrose, 0.67% yeast nitrogen base, 0.5% Bactocasamino acids) at 30°C. A single colony was then grown in selective SDCAA culture medium (2% dextrose, 0.67% yeast nitrogen base, 0.5% Bactocasamino acids, 1.47% sodium citrate, 0.429% citric acid monohydrate, pH 4.5) at 30°C, and the correct sequence was confirmed by DNA sequencing [sequencing lab of the National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev (BGU), Israel] .
  • the YSD-focused N-TIMP2 library was designed on the basis of positions that were previously shown to be tolerant to mutation by Sharabi and colleagues, and purchased from GenScript (NJ, USA). Briefly, the focused library was prepared using NNS (where N represents A, C, T or G nucleotides, and S represents C and G) degenerate codons at positions 4,35,38,68,71,97 and 99 of the N-TIMP2WT gene.
  • the focused library was amplified with forward and reverse primers (forward-5'-CCGGTTATTTCTACTACCGTCGGTTC-3 ⁇ SEQ ID NO: 15; reverse-5'-GTCAGTTCCTGCAAGTCTTCTTCG-3'; SEQ ID NO: 16) and transformed into competent EBYIOO S. cerevisiae along with the linear pCHA vector.
  • the transformed yeast was grown in selective SDCAA culture medium (2% dextrose, 0.67% yeast nitrogen base, 0.5% Bactocasamino acids, 1.47% sodium citrate, 0.429% cit
  • a library size of 8* 10 6 clones was confirmed by performing serial dilutions of the transformed yeast on SDCAA plates and counting after 48 h of growth.
  • THVIP2W T was grown in the selective and protein-expression-inducing SGCAA medium (2% galactose, 0.67% yeast nitrogen base, 0.5% Bactocasamino acids, 1.47% sodium citrate, 0.429% citric acid monohydrate) overnight at 30 °C to a density of 10 s cells/ml
  • the yeast cells (10 6 cells) were then collected and washed with a buffer containing 50 mM Tris, pH7.5, 100 mM NaCl, 5 mM CaCl 2 and 1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the yeast was incubated with mouse anti-c-Myc antibody (Abeam, Cambridge, UK) at a ratio of 1:50 at room temperature for 1 h. Thereafter, the cells were washed and incubated with sheep anti-mouse secondary antibody conjugated to phycoerythrin (PE) (Sigma- Aldrich, St. Louis, MO, USA) at a 1:50 ratio for 30 min on ice.
  • PE phycoerythrin
  • the catalytic domain of MMP-14 (MMP-14CAT) was purified (see details below) and labeled for 1 h at room temperature with DyLight-488 Amine Reactive Dye, which contains a
  • the cells were incubated with 1 ⁇ of MMP-14CAT labeled with DyLight-488 for 30 min on ice. The yeast was then washed and analyzed using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). In the flow cytometry plots, the horizontal axis provides an indication of the expression of the displayed protein (PE signal), and the vertical axis shows the binding of the displayed protein to the soluble target MMP- 14CAT (DyLight-488 signal).
  • a similar labeling process was performed for yeast expressing the N-TIMP2 focused library for enrichment of the high-affinity cell population.
  • S I the first sort
  • 10 s cells 10 times more than the library diversity size
  • 2-20x 10 6 cells were labeled, depending on the library size.
  • S I sort the high- expressing cells were selected.
  • S2-S6 the cells were labeled for both detection of expression and binding to MMP-14CAT conjugated to DyLight-488, with decreasing concentrations of labeled MMP-14C AT for each round of sorting, beginning with 1 ⁇ of labeled MMP- 14CAT for S2 and ending with 5 nM in the final sort (S6).
  • the affinity maturation screens were performed on an iCyt Synergy FACS apparatus (Sony Biotechnology, San Jose, CA, USA). For each sort, about 1.5% of the binding population normalized to expression was selected using a polygonal gate.
  • N-TIMP2W T and the selected N-TIMP2 variants were measured by incubating the yeast cells with different concentrations (1 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 500 nM, 750 nM and 1000 nM) of labeled MMP-14 C AT for 30 min at room temperature.
  • the plasmid was then transformed into electro-competent E. coli bacteria and grown on ampicillin (100 /g/ml) LB-agar plates. Thereafter, about 20 colonies were transferred to liquid ampicillin-LB culture medium and grown overnight at 37 °C.
  • the plasmid was purified from the bacteria according to the manufacturer's protocol with the HiYield plasmid mini kit (RBC Bioscience, Taiwan).
  • the plasmids were sequenced using the Sanger sequencing method [Genetics Unit, NIBN, BGU, Israel], and the sequences were analyzed using Geneious R7 software (Biomatters, New Zealand).
  • AAGbind was calculated for each variant for which DNA sequencing was performed after each sorting round. Multiple mutations were computationally inserted into the structure, and a AAGbind value was determined as described herein for single mutations (saturation
  • MMP-14 catalytic domain gene (MMP-14CAT, residues 112-292) in a pET3a vector with a C-terminal 6 x His tag was expressed in B121 (DE3) E. coli bacteria and purified as previously described.
  • the human MMP-9 catalytic domain (MMP-9CAT) lacking the fibronectin-like domain (residues 107-215, 391—
  • MMP-2 catalytic domain (MMP2CAT) without the fibronectin-like domain
  • the protein was then loaded on a MonoQ ion-exchange column (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and washed with low- salt buffer (50 mM Tris, pH7.5, 50 mM NaCl, 10% glycerol, 5 ⁇ beta- mercaptoethanol and 0.1% EDTA). The protein was eluted using a gradient up to 1 M NaCl. The protein was then washed with 20 mM Tris, pH 7.5, and 150 mM NaCl buffer and purified on a gel filtration Superdex 200 followed by Superdex 75 column (GE Healthcare Life Sciences, USA) with 20 mM Tris, pH 7.5, and 150 mM NaCl buffer.
  • low- salt buffer 50 mM Tris, pH7.5, 50 mM NaCl, 10% glycerol, 5 ⁇ beta- mercaptoethanol and 0.1% EDTA.
  • the protein was eluted using a gradient up to 1 M NaCl.
  • the protein was then was
  • MMP-1 and MMP-10 The catalytic domains of MMP-1 and MMP-10 (MMP- ICAT and MMP-10CAT, respectively) were purified as previously described.
  • Pro-MMP-2, Pro-MMP-9 and Pro-MMP-8 (residues 21-467) were purchased from R&D Systems (Minneapolis, MN, USA) and were activated before use according to the manufacturer's instructions.
  • MMP-14CAT, MMP-2 C AT, MMP-9CAT, MMP- ICAT, MMP- IOCAT and MMP-8 were determined by UV-Vis absorbance at 280 nm, with extinction coefficients (828o) of 34500, 33920, 18934, 17928, and 79885 M “1 cm “1 , respectively.
  • N-TIMP2WT and its variants were expressed and produced in the methylotrophic X33 P. pastoris yeast strain.
  • the genes were first amplified with forward primers for N-TIMP2WT (5'- GGTATCTCTCGAGAAAAGATGC AGCTGCTCCCCG-3 ' ; SEQ ID NO: 17) and the N-
  • TIMP2A-D variants (5'-GGTATCTCTCGAGAAAAGATGCAGCTGCGCGCCG-3' (SEQ ID NO: 18); 5'-GGTATCTCTCGAGAAAAGATGCAGCTGCAGGCCG-3'(SEQ ID NO: 19); 5 ' -GGTATCTCTCTCGAGAAAAGATGCAGCTGC ATGCCG-3 ' (SEQ ID NO: 20); 5'- GGTATCTCTCGAGAAAAGATGC AGCTGCTCGCCG-3 ' (SEQ ID NO: 21); respectively), and the reverse primer (5 ' -GCTGGCGGCCGCCTCGC AGCCCATCTGGTA-3 ' (SEQ ID NO: 22)).
  • the pPICZaA vector encoding for the Zeocin resistance gene, containing the AOX1 promoter at its N-terminus and a 6 x His tag at the construct' s C-terminus, and the amplified N- TIMP2 insert clones were digested with Xhol and Notl restriction enzymes (New England Biolabs, MA, USA). Thereafter, the inserts and the vector were ligated and transformed into E. coli electro-competent cells. The transformed bacteria were plated on LB agar plates containing 50 ⁇ g/ml Zeocin (Invitrogen, Grand Island, NY, USA).
  • the plasmid was extracted from several colonies, and the correct sequence was verified (Genetics Unit, NIBN, BGU, Israel). Thereafter, 100 ⁇ of plasmids with the correct sequence were linearized by treatment with the Sacl restriction enzyme (New England Biolabs, MA, USA).
  • the N-TIMP2 variants containing the plasmids were transformed into electro-competent X33 P. pastoris according to the pPICZa protocol (Invitrogen, CA, USA).
  • the transformed yeast was grown on YPDS plates (2% peptone, 1% yeast extract, 2% d-glucose, 1 M sorbitol, 2% agar) for 72 h at 30°C.
  • Overexpression of the secreted proteins was determined by western blot, using a 1:3000 dilution of mouse anti- 6xHis antibody (Abeam, Cambridge, UK) primary antibody, followed by a 1 :5000 dilution of anti-mouse secondary antibody conjugated to alkaline phosphatase (Jackson ImmunoResearch, West Grove, PA, USA), and detection by incubation in 2 ml of 5-bromo-4-chloro-3-indolyl phosphate reagent (Sigma- Aldrich, USA).
  • the supernatant was incubated for 1 h at 4°C and then loaded on nickel -nitrilotriacetic acid-Sepharose beads (Invitrogen, USA), washed with 50 mM Tris, pH 7.5, 100 mM NaCl, and 10 mM imidazole, eluted with 20 ml of 50 mM Tris, pH7.5, 100 mM NaCl, 300 mM imidazole, and 5 mM CaCl 2 , and concentrated using a Vivaspin centrifugal concentrator with a 3-kDa cutoff (GE Healthcare Life Sciences, USA). The proteins were further purified using a Superdex 75 column with elution buffer (50 mM Tris, pH 7.5, 100 mM NaCl and 5 mM CaCl 2 ) in an AKTA pure instrument (GE
  • N-TIMP2wT and its variants were tested for inhibitory activity against 0.0075 nM MMP-14 C AT, 0.0075 nM MMP-9CAT, 6.25 nM MMP-2 C AT, 0.2 nM MMP- ICAT, 0.2 nM MMP- 10cAT and 0.075 nM full-length MMP-8 enzymes.
  • MMP-14 C AT was incubated with 0.4-25 nM N-TIMP2WT and with 0.015-0.125 nM of the N-TIMP2 variants in TCNB buffer (50 mM Tris, pH7.5, 100 mM NaCl, 5 mM CaCl 2 , and 0.05% Brij) for 1 h at 37 °C.
  • MMP-9CAT was incubated with 0.03-1.25 nM of N-TIMP2 inhibitors for 1 h at 37 °C in TCNB buffer.
  • MMP-2 C AT was incubated with 50-500 nM of the inhibitors in 50 mM Tris, pH 7.5, and lOmM CaCl 2 for 1 h at 25°C.
  • MMP- ICAT, MMP-10CAT and MMP-8 were incubated in TCNB buffer with inhibitor concentrations of 0.2-12.5 nM for 1 h at 37°C.
  • the fluorogenic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH 2 TFA (SEQ ID NO: 23) [where Mca is (7-methoxycoumarin-4-yl)acetyl, Dpa is N-3-(2,4-dinitrophenyl)-L- 2,3-diaminopropionyl and TFA is trifluoroacetic acid] (Merck Millipore, CA, USA), at a final concentration of 7.5 ⁇ for MMP- 14 or 12.5 ⁇ for the other MMPs, was added to the reaction, and the fluorescence was monitored (with 340/30 excitation and 400/30 emission filters) using a Synergy 2 plate reader (BioTek, Winooski, VT, USA) at 37°C.
  • V T _ 1 ([£]+ [/] + K"*" ) - ⁇ ([E]+ [I]+ Kf ) 2 - [E][I]
  • Binding affinity between the N-TIMP2 variants and MMP-14CAT was detected using surface plasmon resonance (SPR) spectroscopy on a ProteOn XPR36 system (Bio-Rad, CA, USA).
  • An HTG chip Bio-Rad, CA, USA
  • 150 ⁇ of 0.033 ⁇ g/ ⁇ l N-TIMP2 variants were immobilized at flow rate of 30 ⁇ /min for 5 min via polyHis-Ni 2+ interactions; an empty channel was used as control.
  • MMP-14CAT was diluted in elution buffer to 2.5 nM, 5 nM, 10 nM and 20 nM and was circulated at a flow rate of 25 ⁇ /min for 200 s, followed by 20 min of dissociation. The response was monitored as a function of time at 25°C.
  • the rate binding constants, k on and k 0 ff, and the equilibrium affinity constant KD between N-TIMP2WT and MMP-14CAT were determined using the Langmuir model.
  • Gelatinase Zymography Assay The gelatinolytic inhibitory activity of the N-TIMP2 variants was tested in a gelatinase zymography assay, as previously described, with the following modifications.
  • Type I Collagen Degradation Assay Inhibition of MMP-14CAT activity on type I collagen was performed as previously described, with the following modifications. Briefly, bovine type I collagen (1.5 mg) (Sigma- Aldrich, St. Louis, MO, USA) was incubated with 2.2 ⁇ of MMP- 14CAT with or without 2.5 ⁇ of each inhibitor in 100 ⁇ of 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM CaCl 2 , 0.05% Brij 35, and 0.02% NaN 3 for 24 h at 27 °C. Each reaction was loaded on 8% SDS-PAGE gel, and the bands were visualized by Instant Blue staining (CBS Scientific, CA, USA). The activity of MMP-14CAT was detected by the characteristic 3/4 fragments (TC A ) of type I collagen, which represent the degradation products of al and a2 collagen type I chains.
  • TC A characteristic 3/4 fragments
  • MDA-MB-231 human breast cancer cell line (kindly provided by Smadar Cohen, BGU, Israel) were maintained in RPMI-1640 (Roswell Park Memorial Institute) medium (Biological Industries Israel Beit-Haemek Ltd., Israel) supplemented with 10% fetal bovine serum (ThermoFisher, MA, USA), 1% 1-glutamine (Biological Industries Israel Beit- Haemek Ltd,. Israel) and 1% penicillin/streptomycin (Biological Industries Beit-Haemek Ltd, Israel).
  • Murine fibroblast NIH-3T3 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in DMEM medium (Biological Industries Israel Beit-Haemek Ltd., Israel) supplemented with 10% fetal bovine serum (Thermo Fisher, MA, USA), 1% 1- glutamine (Biological Industries, Beit-Haemek Ltd., Israel) and 1% penicillin/streptomycin (Biological Industries, Beit-Haemek Ltd., Israel).
  • -TIMP2 proteins were labeled with DyLight-488 (Thermo Fisher, MA, USA) at a ratio of 1:5 (protein:dye) for 1 h at room temperature and washed three times with 50 mM Tris-HCl, pH 7.5, 5 mM CaCl 2 , and 100 mM NaCl using Vivaspin centrifugal concentrators with a 3-kDa cutoff (GE Healthcare Life Sciences, USA).
  • the inhibitory activity of the labeled N-TIMP2 inhibitors was determined as described above. Briefly, MMP-14cAT was incubated with the labeled and non-labeled inhibitors for 1 h at 37 °C.
  • the Boyden chamber chemoinvasion assay was performed as previously described with the following modifications. The experiment was performed with polycarbonate 8- ⁇ filters (Neuoro Probe, Inc, Gaithersburg, MD, USA) coated with Matrigel inside Transwell chambers (Neuoro Probe, Inc, Gaithersburg, MD, USA). The bottom part of each chamber was filled with conditioned medium containing DMEM medium collected from NIH-3T3 fibroblast cells after 24 h of culture and supplemented with 0.1% of BSA. The upper part of the chamber was filled with 200 ⁇ of with MDA-MB-231 (1 ⁇ 10 5 ). Then, 600 ⁇ , of DMEM medium without serum were added to the control chamber or with 7.5 nM of the inhibitors was added to the sample chambers. The cells were incubated for 4 h at 37 °C.
  • the invasive cells were fixed and stained using the Dipp Kwik Differential Stain Kit (American Mastertech Scientific, CA, USA).
  • the cells that had migrated were counted using EVOS FL Cell Imaging System (Thermo Fisher, USA), at 40 x magnification in 2.2 mm 2 fields.
  • the experiment was performed in triplicate, and 10 fields were counted for each sample.
  • This example describes design of the N-TIMP2 library for screening of MMP-14 inhibitors.
  • N-TIMP2 mutants contained mutations at seven positions with the highest probability of mutations for enhancing affinity and specificity of N-TIMP2 towards the catalytic domain of MMP-14 (MMP-14CAT).
  • the library was designed on the basis of previous results of an in silico saturation mutagenesis analysis of N-TIMP2 interacting with eight different MMPs and experimental confirmation for some single N-TIMP2 mutants (44). Based on this analysis, seven N-TIMP2 positions were selected for the current study to be randomized in the N-TIMP2 library, namely, positions 4, 35, 38, 68, 71, 97 and 99 of SEQ ID NO: 1 (and as shown in Fig. 1).
  • the other positions were chosen because they have high potential for improving binding specificity, i.e., for facilitating interactions that are mostly neutral for MMP-14 but destabilize complexes with other MMPs. Rather than focusing the library further by restricting the amino acid choices at each position, we allowed full randomization to twenty amino acids at all seven positions, thus producing a library with a theoretical diversity of 1.3 x 10 9 mutants, which is slightly larger than the limit for YSD.
  • the N-TIMP2 library was then experimentally constructed as detailed in Example 1.
  • This example describes screening of the N-TIMP2 library by yeast surface display (YSD).
  • N-TIMP2WT was cloned into two different YSD plasmids, namely, pCTCON or pCHA, for presentation of the proteins on the
  • N-TIMP2 is placed between the Aga2 protein and the c-Myc epitope tag, whereas the pCHA plasmid allows the N-terminus of N-TIMP2 to be freely exposed.
  • Expression of N-TIMP2W T in both pCHA and pCTCON was verified by using FACS.
  • the designed N-TIMP2 library termed SO
  • SO was subjected to an initial round of expression enrichment, based on c-Myc detection, to yield the S I library (Fig. 2C).
  • Five subsequent rounds of affinity maturation were then performed with decreasing concentrations of MMP-14CAT (ranging from 1 ⁇ in S2 to 5 nM in S6).
  • MMP-14CAT ranging from 1 ⁇ in S2 to 5 nM in S6
  • diagonal sorting gates were used to select cell populations having high target affinity levels relative to protein expression levels, with about 1.5% of the library size being selected each time
  • N-TIMP2 B repeated 12 out of 15 times in S6, and N-TIMP2D repeated twice.
  • N-TIMP2c included one unexpected mutation, Kl 16E, which is located far from the binding interface. It was further confirmed that each of the individual clones identified in S6 bound with higher affinity to MMP-14CAT than to N-TIMP2WT when expressed on the yeast surface (Fig. 2D).
  • Example 3 Characterization of N-TIMP2 Clones with Improved MMP-14CAT Affinity This example describes the characterization of the MMP-14 binding and inhibitory properties of N-TIMP2 A -D.
  • N- TIMP2W T and the four N-TIMP2 variants were expressed and purified (Figs. 4A-C).
  • the expression was performed in Pichia pastoris yeast, which facilitates the correct disulfide bond formation of the N-TIMP2 variants (Fernandez et al.) without the need for refolding, as in the case of expression from Escherichia coli (Williamson et al.).
  • the pPICZaA vector that produces the protein with a free N-terminus and C-terminal His- and c-Myc epitope tags was used.
  • the proteins were using affinity chromatography, followed by size-exclusion chromatography (Fig. 4A). Mass spectrometry analysis confirmed the correct mass for each of the purified variants (Fig. 4B), while SDS-PAGE analysis (Fig. 4C) confirmed their high expression level and purity.
  • MMP-2CAT and MMP-9CAT gelatinases
  • MMP- ICAT and full length MMP-8 collagenases
  • MMP-10CAT stromelysin
  • N-TIMP2W T bound to MMP- 14 with a K of 780 ⁇ 80 pM (Table 1), a finding consistent with previous studies. All the selected N-TIMP2 variants showed picomolar affinities for MMP-14CAT, exhibiting ⁇ 500-fold to ⁇ 900-fold improvement in Ki compared to the K for N- TIMP2W T (Tables 1 and 2). This outstanding enhancement in binding affinity - by almost three orders of magnitude - which was due to only a few mutations, validates the potency of the described combined computational/directed evolution methodology.
  • N-TIMP2WT 100 ⁇ 7 1400 ⁇ 400 1300 ⁇ 50 100 ⁇ 8 2260 ⁇ 150 780 ⁇ 80
  • TIMP2 A Specificity** 48 406 348 286 152 1
  • TIMP2 D Specificity 260 867 340 113 16164 1 * Ki (fold) is calculated as the ratio between the K of N-TIMP2WT and K of N-TIMP2 variant. ** Specificity is calculated as the ratio between the fold of improvement towards MMP- 14 in comparison to other MMPs. [Ki (fold) for MMP-14 C AT/ K (fold) for MMP-X.]
  • KD direct binding constants
  • SPR surface plasmon resonance
  • N-TIMP2WT 1.41 ⁇ 0.02 49.1 ⁇ 0.1 34.9 ⁇ 0.6
  • N-TIMP2A 5.43 ⁇ 0.01 4.65 ⁇ 0.01 0.857 ⁇ 0.002
  • N-TIMP2c 4.72 ⁇ 0.01 5.20 ⁇ 0.01 1.10 ⁇ 0.01
  • Example 4 Inhibition of gelatin and collagen degradation by ⁇ - ⁇ 2 variants
  • This example describes the characterization inhibitory properties of N-TIMP2A-D towards MMP gelatinase and collagenase activities
  • a gelatin zymography assay was performed with pure full-length MMP-2 and MMP-9 proteins.
  • activated MMP-2 and MMP-9 were resolved on gelatin SDS-PAGE incubated with the N-TIMP2 inhibitors, and the gelatinase activity was visualized as clear bands on a dark background (Fig. 6A).
  • N-TIMP2W T and the four tested N-TIMP2 variants showed a significant (40-80%) inhibition of MMP-2 and MMP-9 activity, as expected (Fig. 6B).
  • the inhibitory activity shown by N-TIMP2W T and all the tested N-TIMP2 variants was similar, in agreement with no significant improvement in K values for MMP-9 and MMP- 2 of N-TIMP2WT relative to N-TIMP2 variants as shown by in vitro activity assays (Table 2).
  • inhibition of MMP-9 was stronger than that for MMP-2 for all the proteins, again in agreement with the better K values of the N-TIMP2 variants for this enzyme.
  • N-TIMP2 variants were tested on the collagenolytic function of MMP-14.
  • a type I collagen degradation assay was performed with MMP-14CAT.
  • Bovine type I collagen was incubated with MMP-14CAT and N-TIMP2 variants.
  • Samples were loaded on a SDS-PAGE gel, and the cleavage products were detected.
  • All N-TIMP2 variants, but not N- TIMP2WT showed a significant inhibition of MMP-14CAT (Fig. 6C), with type I collagen cleavage products (TC A fragment) being observed only for the untreated and N-TIMP2WT- treated samples (lanes 2 and 3), but not for the rest of the samples treated with the engineered N-TIMP2 variants (lanes 4-7).
  • This result correlated with the binding affinity (K) values.
  • This example describes the binding and inhibitory properties of N-TIMP2A-D towards MMP-14 expressing cancer cells.
  • N-TIMP2 variant polypeptides could potentially inhibit cell invasion
  • Previously described Matrigel invasion assays showed that MMP-14, MMP-9 and MMP-2 expression and catalytic activity are essential for the invasiveness of different types of cancer cells, such as prostate cancer (PC-3ML), human colon adenocarcinoma, human renal cell carcinoma, the breast cancer cell line MDA-MB-231, fibrosarcoma, and glioma.
  • PC-3ML prostate cancer
  • human colon adenocarcinoma human renal cell carcinoma
  • the breast cancer cell line MDA-MB-231 the breast cancer cell line
  • fibrosarcoma fibrosarcoma
  • glioma glioma.
  • a Boyden chamber chemoinvasion assay was employed. In such assays, the cells were allowed to migrate through a Matrigel-coated membrane and were visualized by light microscopy (Fig. 7B); the number of invading cells was counted (Fig.
  • N-TIMP2WT Addition of the N-TIMP2WT, N- TEVIP2A, N-TIMP2 b and N-TIMP2 C variants resulted in about 40% inhibition of invasion (Fig. 7C). Significantly more effective inhibition of invasion was observed for N-TIMP2D, which inhibits invasion of about 70% of cells, thus making this variant the most promising candidate for drug design. Note that this candidate showed the highest inhibitory activity for both MMP- 14 and MMP-9 (but not for MMP-2) among all the tested variants.
  • N-TIMP2 mutants by far surpass all previous MMP inhibitors based on TEVIP and other scaffolds in terms of their binding affinities and binding specificity shift towards MMP-14CAT.
  • Anti-MMP-14 antibodies previously developed by Udi et al. and by Devy et al. exhibited specific MMP-14 inhibition with nanomolar affinity.
  • N-TIMP2-based MMP inhibitors usually resulted in 10 "10 -10 "9 M K toward the desired MMP compared to 10 "12 K reported here.
  • N-TIMP2 residues mutated in this study do not include the N-terminal residues 1-3 that have been used in most of the previous N-TIMP2 designs, proving that residues that are distant to the catalytic MMP site could be successfully used to modify N-TIMP2's binding specificity.
  • TIMP2 it is also expected to form a new intramolecular H-bond with the backbone of Ala 70 on N-TIMP2 (Fig. 8B), thus contributing to the stability of the N-TIMP2 loop.
  • a Val is substituted by an lie, improving packing against MMP-14 (Fig. 8B).
  • Mutation H97R predominating in all the selected sequences, replaces a small residue that does not contact MMP-14 (Fig. 8C) by a larger one, thereby introducing new hydrophobic interactions and intermolecular hydrogen bonds with Aspl93 and the backbone of Prol91 (Fig. 8C).
  • This mutation has also been previously shown to increase N-TIMP2 affinity for MMP-14 by 14-fold and is also likely to increase its affinity to other MMPs. Finally, at position 99, either a wild- type Thr or a Ser was observed. The mutation to Ser at this position is predicted to lead to a new hydrogen bond with Asp 193 of MMP14 (Fig. 8C); despite this Asp residue being present in most MMPs at this position, this interaction may not be possible in other MMPs due to differences in backbone conformation. In summary, there appear to be two scenarios for improvement of binding to and specificity for MMP- 14, as have been observed in other studies on TIMP/MMP interactions. Some of the acquired mutations are predicted to facilitate favorable intermolecular interactions with MMP-14, while others are expected to stabilize the loops on N-TIMP2 in the MMP-14 bound conformations.
  • TIMP-4 is a tissue specific TIMP, whose expression is normally restricted to the heart, kidney, ovary, pancreas, colon, testes, brain and adipose tissue, and is also evident in breast cancer tumors.
  • TIMP-4 the highest expression levels of TIMP-4 are seen in the heart, and this may be biologically significant as cancer metastasis is rare in heart tissue.
  • Zhao et al. reported a Ki value for TIMP-4 binding to MMP- 14CAT of 340 pM, compared to 70 pM for TIMP2, indicating that TIMP-4 is a strong inhibitor of MMPs upregulated in cancers, such as MMP- 14. Additionally, Ser at position 99 is observed in vertebrate homologues of TIMP- 1.
  • TIMP- 1 exhibits very low affinity for MMP-14 and is not effective in inhibiting its activity (61); therefore, this may indicate that the T99S mutation that we obtained is not effective alone in increasing binding to MMP- 14 and may only be significant when coupled with neighboring mutations, such as at position 97 (namely H97R), as we obtained in this work.
  • Example 7 N-TIMP2/MMP14 and 9 interface-focused and whole-gene randomized mutagenesis
  • This example describes the design and assay of two N-TIMP2 libraries for binding to MMP14 and MMP9, a small focused library combining computationally predicted specificity enhancing mutations and a large library of randomly introduced mutations throughout the N- TEVIP2 gene.
  • YSD Library Preparation Preparation of combinatorial library: A library of N-TIMP2 mutants was created using an assembly PCR method. The following degenerate codon containing primers were used to create mutant N-TIMP2 sequences.
  • the primers contain overlapping regions allowing for assembly of the entire N-TIMP2 gene.
  • the resultant N-TIMP2 sequences were then amplified with the following primers:
  • the polymerase is used to maintain low fidelity and prevent repairs of mismatched nucleotides.
  • the PCR included the following primers containing homology regions to the PCHA vector:
  • GTC AGTTCCTGC AAGTCTTCTTCG-3 ' (SEQ ID NO: 16). These primers were also used to subsequently amplify the PCR products utilizing Phusion high fidelity DNA polymerase, guaranteeing blunt ends, free of adenine overhangs produced by Taq DNA polymerase.
  • the transformation of the libraries to yeast were performed by simultaneously transforming the amplified mutant N-TIMP2 fragments (from PCR) with a cleaved pCHA vector to an EBY100 Saccharomyces cerevisiae yeast strain using a MicroPulser electroporator (Bio-Rad, CA, USA), as previously described (Chao et al.).
  • the primers used for amplification contain pCHA homology regions allowing fragments to be incorporated into the pCHA plasmid by homologous recombination in the yeast cells.
  • the transformed yeast were grown in selective SDCAA medium (0.54% Na 2 HP0 4 , 0.856% Na 2 HP0 4 » H 2 0, 1.5% agar, 2% dextrose, 0.67% yeast nitrogen base, 0.5% Bacto casamino acids).
  • Yeast (EBY100) clones or whole libraries were grown in selective and expression inducing SGCAA medium (2% galactose, 0.67% yeast nitrogen base, 0.5% Bacto casamino acids, 0.54% Na 2 HP0 4 » H 2 0, 0.856% NaH 2 P0 4 ) at 30°C to a density of 10 s cells/ml
  • yeast cells (10 6 cells) were then collected and washed with a buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 5 mM CaCl 2 and 1% bovine serum albumin.
  • yeast cells were incubated with mouse anti-c-Myc antibody (9E10) at a ratio of 1:50 at room temperature for 1 hour. Thereafter the cells were washed and incubated with sheep anti-mouse secondary antibody conjugated to phycoerythrin (PE) at a ratio of 1:50 for 30 minutes on ice.
  • PE phycoerythrin
  • MMP-14 ca t For the detection of binding to MMP-14 ca t, the cells were incubated with MMP-14 ca t labeled with DyLight-488 for 1 hour on ice. The yeast cells were then washed and analyzed using an Accuri C6 flow cytometer (BD Biosciences, CA, USA). An identical process was performed for detection of MMP-9 ca t binding, where MMP-9 ca t is labeled with DyLight-650.
  • yeast cells were simultaneously incubated with varying concentrations (see below) of fluorescently labeled MMP-14 ca t and MMP-9cat.
  • the affinity maturation sorting was performed on an iCyt Synergy FACS apparatus (Sony Biotechnology, CA, USA). For each sort, about 1% of the binding population was selected using a polygonal gate.
  • Reverse primer 5 ' -GCTGCCTTTGCTAGTTGTTGAG-3 ' (SEQ ID NO: 34)
  • Pichia pastoris Expression WT N-TIMP2WT and its mutants were expressed and purified in the X33 Pichia pastoris yeast strain, as described in Example 1 with the following differences.
  • the N-TIMP2 mutant genes were transferred from the pCHA plasmid obtained from yeast lysates (boiling in 0.02 M NaOH) to the pPICZaA P. pastoris expression vector by transfer PCR (Erijman et al) using the following forward primers:
  • Mutants 9-2 and 9-6 5'- GCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGATGC AGCTGCCCGCCG-3 ' (SEQ ID NO: 37);
  • X33 cells were transformed with the WT N-TIMP2 gene cloned into the pPICZaA vector. PCR products were subsequently transformed into chemical competent DH5a E. coli. The transformed bacteria were plated on LB agar plates containing 25 Zeocin. After an overnight incubation at 37°C, three colonies were chosen for growth in liquid LB medium at 37°C for -16 hours, the plasmid was purified by miniprep, and the correct sequence was verified by Sanger sequencing using the following forward primer: 5'- GACTGGTTCC AATTGAC A-3 ' (SEQ ID NO: 40).
  • Bacterial cultures possessing plasmids encoding for the correct sequence were grown for -16 hours at 37°C for large scale plasmid purification by maxiprep.
  • the purified plasmid was then linearized by treatment with the Sacl restriction enzyme.
  • the plasmids were transformed into electro-competent methylotrophic X33 P. pas tons cells according to the Invitrogen pPICZa protocol.
  • the transformed yeast were grown on YPDS plates (1 % yeast extract, 2% peptone, 2% D- glucose, 1 M sorbitol, 2% agar) for 72 hours at 30°C. Thereafter, several colonies from each N-TIMP2 mutant transformation were tested for the presence of the N-TIMP2 gene in the yeast genome by PCR using the following primers:
  • the culture was centrifuged at 3800g for 10 min, the supernatant was collected, imidazole was added to a concentration of 10 mM and the pH raised to 8.0.
  • the subsequent solution was incubated for 1 hour at 4 °C and then purified by nickel bead affinity purification and size exclusion chromatography as described below (see N-TIMP2 Purification). Protein concentrations were determined by UV-Vis absorbance at 280 nm, with an extinction coefficient ( ⁇ 28 ⁇ ) of 13,325 M ⁇ cm "1 for WT N- TEV1P2 and all its mutants.
  • the production yielded an average of about 0.3-1 mg of protein per liter of culture for the WT N-TIMP2 and its mutants.
  • E. coli Expression Yeast colonies were grown and pCHA plasmid DNA extracted and purified by glass bead alkaline lysis. Subsequent purified plasmids were subjected to restriction free cloning as described previously by van den Ent and Lowe in order to transfer N-TIMP2 mutant genes from the pCHA plasmid to the pet21 E. coli expression vector which encodes a C- terminal 6xHis-tag. The following primers were used for the transfer:
  • Mutant 9-2 5'- CCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATTATGTGCAGC TGCCCGCCG-3'(SEQ ID NO: 43);
  • pet21 plasmids were obtained containing the appropriate N-TIMP2 mutant genes, the plasmids were transformed to BL21(DE3)pLysS E. coli cells. Transformed cells were diluted to 5 ml in LB supplemented with ampicillin (100 ⁇ / ⁇ 1), chloramphenicol (25 ⁇ / ⁇ 1) and grown for -16 hours at 37°C. 500 ⁇ of the resulting culture was transferred to 25 ml of LB supplemented with ampicillin (100 ⁇ / ⁇ 1), chloramphenicol (25 ⁇ / ⁇ 1) and 500 ⁇ of dextrose (10% solution) and grown for -16 hours at 37°C.
  • the resulting cell lysate is centrifuged at 18,700xg for 20 min at 4°C.
  • the resulting supernatant is discarded and this process repeated another three times on the resulting pellet containing the unfolded protein sequestered to inclusion bodies.
  • the final pellet is frozen at - 20°C.
  • the resulting solution is centrifuged at 20,800xg for 20 min at 4°C.
  • the eluate is fractionated by FPLC size exclusion chromatography using a 120 ml GE Superdex 75 preparatory gel filtration column equilibrated with the following buffer: 50 mM Tris-HCl, 100 mM NaCl, 50 mM CaCl 2 , pH 7.5.
  • the protein is isolated and concentrated from fractions corresponding to the last major peak corresponding to the monomeric form, and confirmed by SDS-PAGE.
  • Varying concentrations of N-TIMP2 are incubated with 0.4 nM MMP-9 or 0.8 nM MMP-14 for 1 hour at 37°C in 50 mM Tris-HCl, 0.15 M NaCl, 10 mM CaCl 2 , and 0.02% Brij 35 pH 7.5.
  • MCA-Pro-Leu-Gly-Leu- Dpa-Ala-Arg-NH2 TFA [where MCA is (7-methoxycoumarin-4-yl)acetyl; Dpa is N-3-(2,4- dinitrophenyl)-L-2,3 diaminopropionyl; and TFA is trifluoroacetic acid]
  • Fluorescence at 395 nm was measured, at least every ten seconds for at least 6 minutes, immediately after addition of fluoro genie substrate with irradiation at 325 nm on a Biotek Synergy HI plate reader (BioTek, VT, USA) preincubated to 37°C. Controls were also performed to determine uninhibited enzyme activity as well as basal fluorescence levels (without N-TIMP2 or MMP). At least three assays were performed for each N-TIMP2 protein. Ki app Determination
  • Ki app values are an average of the Ki app values determined from single inhibition assay experiments for each N-TIMP2 protein.
  • N-TIMP2 mutants which we had not explored in the past, and to include extra mutants that might be beneficial for MMP-9 binding.
  • Table 4 summarizes the locations at which mutation was allowed, the degenerate codons utilized to generate the library, and the mutations allowed at these positions.
  • This library included 71,680 N-TIMP2 sequences, much less than the theoretical limit of 10 s sequences that could be explored by YSD.
  • the library was constructed using assembly PCR (see methods).
  • A adenosine
  • C cytidine
  • G guanosine
  • T thymidine
  • B C or G or T
  • K G or T
  • M A or C
  • N A or C or G or T
  • R A or G
  • S C or G
  • W A or T
  • Y C or T
  • N-TIMP2 libraries were expressed on the surface of yeast cells with the use of the pCHA expression vector as described above.
  • This vector encodes the Aga2p mating protein on the C-terminus, fused to a c-Myc tag which is fused to the N-TIMP2 protein located on the N- terminus of the construct. It is crucial for the N-terminus of N-TIMP2 to be free as this region of the protein mediates binding to the MMP active site and thereby allows inhibition of MMPs.
  • the Aga2p protein binds by disulfide bonds to the Agalp protein which is encoded for by the yeast cells, thereby allowing the construct to be displayed on the yeast surface ( Figure 10A).
  • N-TIMP2 libraries were incubated simultaneously with two targets, MMP-14 ca t and MMP-9 ca t, labeled respectively with Dylight- 488 and Dylight-650 (Figure 10A). All sorts were performed using FACS (fluorescence activated cell sorting) ( Figure 10B, C). To eliminate N-TIMP2 mutants that are not expressed (either due to deletions or due to frameshifts in the N-TIMP2 gene) we performed an initial pre-sorting round. The pre-sorting was done by targeting the c-Myc tag with an anti-c-Myc antibody and a fluorescently labeled secondary antibody against the anti-c-Myc (see Methods).
  • the c-Myc tag is fused to the C-terminus of N-TIMP2 ( Figure 10A) and therefore single codon frameshift mutations in N-TIMP2 will alter the amino acid sequence of the c-Myc tag peptide and abolish anti-c-Myc antibody binding and fluorescent labeling.
  • yeast cells carrying N-TIMP2 mutants with high binding specificity to MMP-14 cat In order to isolate yeast cells carrying N-TIMP2 mutants with high binding specificity to MMP-14 cat , those cells which bound MMP-14 cat with the highest affinity, 1% of all cells exhibiting binding to MMP-14 ca t alone (gate A in Figure IOC), were isolated for further selection with gradually increasing concentration of MMP-9 ca t and decreasing concentration of MMP-14 ca t from one round of selection to the next.
  • the second selective sorting was therefore performed with 500 nM MMP-14 ca t and 50 nM MMP-9 ca t, whereas the third selective sort was performed with 200 nM MMP-14 cat and 70 nM of MMP-9 cat . In each sort, the 1% of cells exhibiting the highest binding signal to MMP- 14 was collected.
  • N-TIMP2 mutants from fifteen colonies show a substantial consensus at position 4 for proline as opposed to the WT serine as well as a mutation from valine to isoleucine at position 71. Seven out of fifteen of these mutants contain unplanned mutations outside of the N-TIMP2:MMP binding interface.
  • Selective sorting on the larger random library was performed in a similar manner to the smaller interface focused library, as described above.
  • Initial sorting was performed with ⁇ MMP-14 cat and 25 nM MMP-9 cat .
  • the MMP-14 binding population was isolated and subjected to further selective pressure.
  • the second selective sort was performed with 250 nM MMP-14 ca t and 50 nM MMP-9 ca t, and the third selective sort was performed with 200 nM MMP-14 ca t and 70 nM MMP-9cat.
  • N-TIMP2 mutant sequence profiles resulting from the two selection experiments on the focused library show different patterns of mutation at most positions ( Figure 11A and 1 IB). This difference is indicative of the success of the method to produce libraries matured to be specific for one MMP type.
  • mutant 14-4 was determined to be the most specific mutant as determined computationally by analyzing AAGbind of N-TEV1P2 in complex with MMP-14 ca t and MMP-9 ca t (Table 5). Our computational predictions show that this mutant exhibits the greatest difference between negative AAGbind for MMP-14 ca t binding and positive AAGbind for MMP-9 ca t binding. The next best mutant is mutant 14-6, however, it is predicted to improve binding to both MMPs but more so for MMP-14 ca t. We were not able to computationally analyze the MMP-9 specific mutants, since all MMP-9 specific mutants obtained from the final round of sorting contain a proline at position 4.
  • mutants 14-2, -4, -6 were chosen for purification since it exhibited the best specificity in FACS analysis experiments as well as computational predictions.
  • mutant 14-6 was chosen for purification since it repeated at a frequency of 13% in the library resulting from the second selective sort, whereas other mutants did not repeat. This mutant only appears once out of the six sequences obtained from the third selective sort.
  • the mutants chosen for MMP-9 binding specificity are mutants 9-2 and 9-6. Mutant 9-2 performed the best in the FACS analysis ( Figure 13B), whereas mutant 9-6 is repeated 3 out of 15 times among the N-TIMP2 clones selected for sequencing. Table 6 lists the mutations present in the N-TIMP2 mutants chosen for purification and characterization from the focused library. Note that mutant 14-6 contains an additional mutation outside of the binding interface that was not planned and was introduced spontaneously during the YSD selection process.
  • N-TIMP2 variants chosen to be assayed in vitro as purified proteins from the small interface focused N-TIMP2 library. "— " indicates that the WT identity at this particular position was maintained. Mutant 14-6 contains a mutation, V18M, which was not intended to be contained in the library. E. Mutant Characterization
  • the N-TIMP2 mutants chosen for further characterization were purified from Pichia pastoris yeast cells which secrete the properly folded N-TIMP2 proteins into the growth medium and have previously been shown to be effective in in vivo studies (Masuda et al. and Fernandez et al). All mutants were expressed in this system except for mutant 9-2 for which we could not see any expression in P. pastoris. Mutant 9-2 was therefore expressed in Escherichia coli and refolded in vitro from inclusion bodies using the procedure previously established in our lab (Sharabi, 2014). The WT N-TIMP2 was therefore expressed both in P. pastoris as well as in E. coli.
  • N-TIMP-1 is known to contain an acetylated N-terminus and monomeric fractions can contain up to 60% improperly folded protein (Van Doren et al), this is likely to apply to N-TIMP2 as well.
  • the Ki app of the inhibition of MMP-14cat and MMP-9 ca t by N-TIMP2 mutants was determined using an MMP enzyme activity assay in the absence and the presence of the N-TIMP2 inhibitor. In such an assay, the degradation of the fluorogenic MMP substrate is monitored and the initial velocity of the reaction is measured at varying concentrations of N-TIMP2 WT and mutants (see Methods). The reaction was fitted to equation 1 to determine Ki app ( Figures 14A and B).
  • Table 7 summarizes the Ki app values obtained for each N-TIMP2 mutant, for inhibition of MMP-14 ca t as well as MMP-9 ca t. All tested mutants showed a substantial increase in binding specificity towards the desired target MMP. The best mutant from the MMP-14 selection 14-6, exhibited a specificity shift of 59-fold. Interestingly, a very similar 57-fold specificity shift is exhibited by the mutant (ATE) obtained from the random library. The improvements in Ki app of the mutants towards the desired MMP are modest, with the highest improvement of 8.8 fold observed by the ATE mutant. These relatively modest improvements are likely due to our selection protocol that incorporates two MMP targets at the same time.
  • Mutant 14-6 exhibits the largest decrease in binding to MMP-9 ca t with a nearly 20-fold decrease in binding.
  • the mutants selected for specific binding of MMP-9 ca t also show similar results of approximately a 45-fold increase in specificity of binding to MMP-9 ca t over MMP-14 ca t.
  • the specificity increase here is achieved mostly through a great reduction in affinity for MMP-14 despite a slightly worse affinity for MMP-9 compared to WT N-TIMP2.
  • the 9-6 mutant exhibits almost 900-fold better binding affinity towards MMP-9 ca t vs. MMP-14 ca t, which should exhibit a significant difference in cellular experiments.
  • Ki app values were obtained from at least three such assay experiments for each protein and averaged, each value is reported as Ki app ⁇ S.D.
  • AAGbind and specificity shifts were calculated from Ki app values.
  • mutants which specifically bound one type of MMP were not selected in this example, and in essence this feature is an element of negative design in the setup for this example.
  • mutants which bind MMP-14 ca t with high specificity because they have been affinity matured for MMP-14 ca t binding but may also exhibit higher affinity for MMP-9 ca t or other MMPs due to the high sequence and structural homology of the MMPs.
  • N-TIMP2 mutants from both libraries with comparable ⁇ 60-fold increases in binding specificity. This specificity was achieved by only three mutations in the random library mutant ATE, two of them being in the binding interface, and by eight mutations (seven mutations being in the binding interface) in the mutant (14-6) from the focused library.
  • the random library performed worse than the focused library and the selection failed to converge after the three rounds of selection. This could be due to the fact that for error prone PCR, used for generation of our random library, there is a substantial likelihood that mutations will be synonymous or that some amino acid mutations will be very rare since they may require two or three nucleotide mutations in the same codon.
  • success of obtaining a promising sequence is subject to chance to a greater extent, while focused libraries increase our chances of successful design since they are rationally designed and incorporate mutations or positions which are known to be beneficial a priori.
  • the methods used here have produced more superior results than single mutations have achieved in terms of specificity shifts, as well as previous experiments implementing display technology such as phage display to design N-TIMP2.
  • mutations were restricted to residues 1-6, 34-40 and 67-73 of N-TIMP2, similar to the areas mutated here, with the goal of obtaining MMP-l ca t specific mutants, using MMP-3 C at as an anti-target in selection experiments.
  • the affinities of WT N-TEVIP2 are in the same range as those for MMP- 14 and MMP-9 (Bahudhanapati et al).
  • mutants obtained from these experiments ultimately contained mutations only between positions 1-6 and exhibited specificity shifts of only 8.6-fold for MMP-l ca t binding relative to competitor MMPs.
  • the best mutant was further mutated with mutations up to position 9.
  • the resulting mutants exhibited a maximal specificity shift of 77-fold for MMP-l ca t binding relative to competitor MMPs.
  • these mutants exhibited up to 60-fold reduced binding of MMP-l ca t, whereas in our work we only witnessed no greater than a 6.7-fold reduction of affinity in the case of mutant 9-6.
  • Analysis of the libraries after sorting was limited by the number of sequences which could be obtained from single colonies.
  • position 71 of N-TIMP2 is highly enriched for a mutation from valine to asparagine in the library evolved to bind MMP-14 with high specificity ( Figure 11A).
  • the library evolved for high binding specificity to MMP-9 is more enriched for a mutation to isoleucine at this position.
  • This trend may indicate that position 71 of N-TIMP2 can act as a specificity switch, especially since the different selection experiments indicate that each MMP type prefers an amino acid that differs chemically at position 71, MMP-14 preferring a hydrophilic residue at this position and MMP-9 preferring a hydrophobic residue at this position.
  • Another specificity switch was observed at position 4, where proline is strongly preferred for the MMP-9 selection ( Figure 1 IB).
  • Figures 15A-M Structural analysis from computationally generated models ( Figures 15A-M) shows that mutations at position 4 of N-TIMP2 should preferably be polar and large for designing MMP-14 specificity, whereas for MMP-9 specificity the residues should be hydrophobic and small. This is due to the local environments opposite position 4 in MMP-14 and MMP-9.
  • Figure 15A-15D depicts the environment around position 4 and the effect of mutation. On MMP-14 there is a gap that could be filled (characteristic of a cold spot), increasing affinity. Hence, large polar residues are predicted to form favorable contacts with asparagine at position 231 on MMP-14.
  • Mutants 14-4 and 14-6 both contain glutamine at position 38 ( Figure 15E-G). Position 38 is also crucial to binding specificity. For five out of six MMP-14 specific mutants, mutation to a glutamine is observed whereas MMP-9 favoring mutants mostly maintain the WT asparagine at this position. Glutamine and asparagine are chemically similar, however, glutamine is effectively longer than asparagine, and the local environment opposite this position on MMP-9 is hydrophobic. An asparagine at position 208 on MMP-14 is engaged in a hydrogen bond with both the WT asparagine as well as the mutant glutamine; this asparagine at position 208 is occupied by a glycine residue in MMP-9 at the corresponding position 197 ( Figure 15F-15G). Since glutamate is longer than asparagine it is less tolerated by this hydrophobic environment and cannot form any favorable contact with MMP-9.
  • the immediate local environment on MMP-14 opposite position 71 is slightly more hydrophilic than the corresponding immediate local environment on MMP-9 ( Figures 15H, J); a new hydrogen bond is formed between the asparagine residue at position 71 in mutants 14-4 and 14-6 and a neighboring tyrosine at position 203 on MMP-14 ( Figure 151), the
  • this arginine residue may be more preferable for MMP-14 binding since it forms a hydrogen bond with the MMP-14 backbone at position 191 and a salt bridge with an aspartate at position 193 (figure 8M); this arginine only forms one hydrogen bond with the MMP-9 backbone at a nearby position different from that seen with binding to MMP-14 even though the amino-acid sequence in this region is the same. This may be due to slight differences in the backbone. Position 98 is occupied by an isoleucine residue in WT N-TJJV1P2, this residue faces the core of the protein. A mutation to threonine is seen here in the ATE mutant.
  • the ATE mutant of the random library contains a mutation at position 116 from lysine to glutamate, a switch from a positive to a negatively charged residue. In N-TIMP2 this position is surrounded by positive charges. This change in charge may cause new local electrostatic interactions that may have greater long range electrostatic effects on the interface.

Abstract

Described herein are specific matrix metalloproteinase (MMP) polypeptide inhibitors. Uses of the described inhibitors, such as in methods of treatment of MMP-related diseases, and in detection of specific MMP expression is also described.

Description

MATRIX METALLOPROTEINASE INHIBITORS AND METHODS OF USE
THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
Benefit is claimed to US Provisional Patent Application No. 62/419,497, filed
November 9, 2017; the contents of which are incorporated by reference in their entirety.
FIELD
This disclosure relates to specific matrix metalloproteinase (MMP) polypeptide inhibitors. Use of the described inhibitors, such as in methods of treatment of MMP-related diseases, and in detection of specific MMP expression is also described.
BACKGROUND
Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as autoimmune diseases, cardiovascular diseases, and cancer.
In cancer-related processes, some MMPs play a crucial role in the promotion of metastasis and other aspects of tumor growth through cleavage and activation of a variety of different proteins (Egeblad et al., Coussens et al.). In particular, there is elevated expression of a membrane-type MMP, MMP- 14 (also known as MT1-MMP) in various human carcinomas, including uterine, cervical, stomach, lung, breast, colon, melanoma, head and neck, and brain tumors (Bartolome et al). Elevated MMP- 14 expression is also associated with early death of patients with various cancers (Zhang et al.) and is correlated with cancer progression, invasion, lymph node metastases, a poor clinical stage, larger tumor size, and tumor stage progression (Tetu, et al.). In the laboratory, MMP- 14 has been shown to play a direct role in the
multiplication of tumor cells inside a three-dimensional collagen matrix (Hotary, K. B. et al.).
Because of the importance of MMPs in cancer, many MMP inhibitors have been designed over the past 30 years. Yet, due to their high toxicity, all failed in clinical trials (Overall, C. M. et al., Lopez-Otin, C. et al). The major drawback of MMP inhibitors developed to date was that they targeted the conserved active site of MMPs and thus reacted with other Zn2+-containing proteins in the body, resulting in high toxicity (Vandenbroucke et al.).
Moreover, broad inhibition of all MMP family members is not necessarily desirable, since some MMPs, for example, MMP-8 and MMP- 12, exhibit anti-tumorigenic function (Folgueras et al., Decock et al.). In addition, it has been shown in animal cancer models that lack of activity of certain MMPs is correlated with more aggressive cancers (Lopes-Otin et al.).
Thus, a continuing need exists for MMP inhibitors that can inhibit one or a small subset of MMPs with high specificity and high efficacy.
SUMMARY
Provided herein are polypeptide MMP inhibitors including an isolated polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 1, or a fragment, derivative or analog thereof. In particular embodiments, the polypeptide MMP inhibitor is a polypeptide at least 70% identical to an amino acid sequence set forth as SEQ ID NOs 2, 4, 6, 8, 24-27 or 47.
Also described herein are nucleic acid sequences including a nucleic acid sequence encoding the described isolated polypeptides, such as the nucleic acid sequence set forth as SEQ ID NO: 3, 5, 7, or 9, and expression vectors that contain the provided nucleic acid sequences.
The described synthetic polypeptides and polynucleotides can be used in compositions for treatment of a disease or condition characterized by aberrant overexpression of matrix metalloproteinase (MMP) -14, MMP-9, or MMP-2.
Further provided herein are methods for treatment of a disease or condition
characterized by aberrant overexpression of matrix metalloproteinase (MMP) -14, MMP-9, or MMP-2, which include administering to a subject in need thereof a therapeutically effective amount of the described polypeptides, thereby treating the disease or condition.
Additionally described herein are methods of detecting a cell that is overexpressing MMP- 14, MMP-9, or MMP-2 by contacting a cell that is suspected of overexpressing MMP- 14, MMP-9, or MMP-2 with at least one of the described isolated polypeptides; washing unbound polypeptide from the cell; and detecting bound polypeptide.
The foregoing and other objects, features, and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows library design for identification of the described peptides. Structure of N-TIMP2 (shown in cyan) in complex with MMP-14CAT (shown in gray) [adapted from PDB ID: 1BUV], with positions that were chosen for full randomization shown as red spheres. Purple sphere represents the Zn2+ atom found in the active site of MMP-14CAT. The plots below the structure summarize AAGbind values obtained from computational saturation mutagenesis performed at the N-TIMP2 positions that are mutated in the YSD library (see Example 1 for details). The vertical axis of each subplot represents the MMP type, and the horizontal axis represents the amino acid identity of the mutation. Blue dots represent AAGbind<-0.5 kcal/mol, green dots represent -0.5<AAGbind<0.5 kcal/mol, yellow dots represent 0.5<AAGbind<1.5 kcal/mol, red dots represent AAGbind≥1.5 kcal/mol, gray dots represent cases where AGA or AGB (AG of single chain) was greater than 2 kcal/mol and AAGbind was negative, indicating single-chain destabilizing mutations leading to an artificial improvement in AAGbind.
Figures 2A-2E show random mutagenesis library screening of N-TIMP2. Figure 2A: Schematic representation of N-TIMP2WT expressed using the pCTCON construct, with MMP- 14-cAT as a soluble target (left panel), and flow cytometry analysis of yeast expressing N- TEV1P2WT in the pCTCON construct labeled with both 1 μΜ of MMP-14CAT conjugated to DyLight-488 (em. 525 nm) and with mouse anti-c-Myc antibody followed by sheep anti-mouse secondary antibody conjugated to PE (em. 575 nm) for detection of expression (right panel). Figure 2B: Schematic representation of N-TIMP2WT expressed using the pCHA construct, with MMP- 14CAT as its soluble target (left panel), and flow cytometry analysis of the yeast expressing N-TIMP2WT in the pCHA construct under the same conditions as the N-TIMP2WT in the pCTCON construct. Figure 2C: FACS results showing sorting of focused N-TIMP2 libraries. Sort round 1 (S 1) represents a sort for protein expression in which the library is labeled only for detection of expression, and a population with a high expression level is selected from the representative gate. In sort round 2 (S2), 1 μΜ of MMP-14CAT conjugated to DyLight-488 was used to select 1.5% (gated pool) of the high-affinity population. In Sort 4 (S4) and Sort 6 (S6), 50 nM and 5 nM, respectively, of MMP- 14CAT conjugated to DyLight-488 were used as targets to select the high-affinity pool of clones. The x-axis shows c-Myc expression, and the y-axis shows receptor binding. Polygons indicate sort gates used to select the desired yeast cell population. Figure 2D: Titration curves of the yeast surface displayed N- TEVIP2WT and four selected N-TIMP2 clones (N-TIMP2A-D). The yeast cells expressing four different clones were labeled with MMP- 14CAT conjugated to DyLight-488 at a concentration of 1-1000 nM. The binding signal (DyLight-488, em. 525 nm) was normalized to the expression of each clone (PE, em. 575 nm). Figure 2E: Sequences of the N-TIMP2 variants identified after the sixth sort (S6).
Figure 3 shows logo summaries of all N-TIMP2 clones sequenced after each selective round of sorting. The height of each letter is proportional to its frequency at that position. The total height of the stack represents conservation at that position. Green, purple, blue, red and black letters, respectively, represent polar, neutral, basic, acidic and hydrophobic amino acids. The identity and position number of N-TIMP2WT are denoted at the bottom of the figure. To the top left of each logo, numbers S l-6 represent the sort number. To the right, the average AAGbind value in kcal/mol is shown (see Experimental Procedures for calculation details). The logos were generated by the WebLogo sever (weblogo.berkeley.edu/logo.cgi).
Figures 4A-4E show purification, characterization, and MMP-14 inhibitory activity of
N-TIMP2 variants. Figure 4A: Size-exclusion chromatography (SEC) for clone N-TIMP2B. Figure 4B: Mass spectrometry analysis for clone N-TIMP2B after SEC. Figure 4C: SDS- PAGE analysis on 15% polyacrylamide gel under reducing conditions for the purified clones N-TIMP2WT, N-TIMP2A, N-TIMP2b, N-TIMP2c and N-TIMP2D. Figure 4D: MMP- 14 inhibition by N-TIMP2WT; Figure 4E: MMP- 14 inhibition by N-TIMP2 variants. For Figures 4D and 4E, MMP- 14CAT was incubated with N-TIMP2WT and N-TIMP2 variants at various concentrations. The substrate Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (SEQ ID NO: 23) was added, and the fluorescent signal upon substrate cleavage by MMP-14CAT was measured. The slopes (cleavage velocities) at each inhibitor concentration were measured, and the curves were fitted by the Morrison equation (eq.1) to obtain the Ki values.
Figures 5A-5E show SPR measurements of MMP-14CAT and N-TIMP2 variants interactions. Figure 5A: N-TIMP2WT; Figure 5B:, N-TIMP2A; Figure 5C: N-TIMP2B; Figure 5D: N-TIMP2c; Figure 5E: N-TIMP2D. The binding response (y-axis) was measured for MMP-14CAT at different concentrations, 20 nM (blue), 10 nM (green), 5 nM (purple), 2.5 nM (yellow) and 0 nM (red).
Figures 6A-6C show inhibition of gelatin and collagen degradation by N-TIMP2 variants. Figure 6A: Representative gelatin zymography of 1 nM MMP-2 and MMP-9 full-length proteins resolved on SDS-PAGE and treated with 100 nM of Ν-ΤΙΜΡ2 inhibitors. Left lane of each gel represents pro-MMP2 (upper) and active MMP-2 (lower) while the right lane represent active MMP-9. Figure 6B: Quantification of band intensity normalized to the intensity of the control (untreated) gel. The average values for the percentage of inhibition for MMP2 are N-TIMP2WT: 51 %, N-TIMP2A: 57%, N-TIMP2B: 51 %, N-TIMP2C: 41 %, N-TIMP2D: 55% and for MMP9 are N-TIMP2WT: 61 %, N-TIMP2A: 68%, N-TIMP2B: 68%, N-TIMP2C: 79%, N-TIMP2D: 74%. Error bars represent SEM; Statistical analysis was performed by Student's t test compared to untreated control *P< 0.05, ** P< 0.01 n=3. Figure 6C: Representative degradation products of bovine type I collagen. Collagen (1.5 μ ) was incubated with (lane 2) or without (lane 1) 2.2 μ of MMP-14CAT, or with 2.2 μ of MMP-14CAT along with 2.5 μΜ of inhibitors (N-TIMP2WT, N-TIMP2A, N-TIMP2B, N-TIMP2C, N-TIMP2D, lanes 3-7, respectively). The labels 'αΓ and 'α2' indicate the al (I) and α2 (I) chains of type I collagen, while 'β chains' indicates the cross-link between two al chains or between al chain and a2 chain. The 3/4 cleavage product of type I collagen by MMP-14CAT IS indicated as TCA (3/4) fragment. Note the reduction in collagen degradation in the presence of the N-TIMP2 variants. Figures 7A-7C show binding of N-TIMP2 variants to MDA-MB-231 cells and inhibition of cancer cell invasion. Figure 7A: Expression of MMP-14 on MDA-MB-231 cells (left panel). Solid line, cells only; dotted line, MMP-14 expression; Right panel, binding of the N-TIMP2 inhibitors, conjugated to DyLight-488 fluorescent dye, to the cells. Figure 7B: Representative micrographs of invading MDA-MB-231 cells treated with 7.5 nM of N-TIMP2 inhibitors; the cells were stained with Dipp Kwik Differential Stain and visualized as dark forms on light background by light microscopy using x40 magnification lenses. Figure 7C: Calculated fold of invasion. The experiment was repeated 3 times; means and standard error are given. ***P<0.001 for t-test comparisons of the indicated condition versus N-TIMP2WT.
Figure 8A-8C show structural analysis of MMP-14 affinity enhancing mutations.
Computationally modeled interactions of mutations that are common after the 6th round of selection for enhancement of affinity of binding of N-TIMP2 for MMP-14. N-TIMP2 is shown in cyan and MMP-14 is shown in green. Figure 8A: Interactions at position 4 of N-TIMP2; Figure 8B: Interactions at positions 68 and 71 of N-TIMP2; Figure 8C: Interactions at positions 97 and 99 of N-TIMP2. Residues involved in an interaction are shown in stick representation. Mutant residue identities are labeled in red, WT identities of residues in all panels are labeled in black. Polar interactions are shown as dotted red lines.
Figure 9, a variation of the model shown in Figure 1, shows N-TIMP2 (red) in complex with MMP-14cat (gold) (adapted from PDB ID: 1BUV). Positions included in the small focused library are highlighted as green spheres.
Figure 10A shows schematic representation of N-TIMP2 displayed on the yeast surface (adapted from Chao et al. [13]). The N-terminus of N-TIMP2 is free and facing away from the yeast surface. Figure 10B shows expression of the construct can be monitored by FACS by fluorescently labeled (red burst in Figure 10A) antibodies that bind the c-Myc tag in the construct. Figure IOC shows binding of N-TIMP2 to catalytic domains of MMP proteins in solution can be monitored simultaneously by FACS by directly fluorescent labeled MMP proteins (yellow and blue bursts in Figure 10A). Red polygons represent sample cell collection gates. The figure here was produced in the initial sorting round on the focused library.
Figures 11A and 11B show logos based on sequencing of evolved libraries after final selection experiments representing frequency of amino acid residues at mutated positions in each evolved library. The WT identity of each position is shown below each logo. Figure 11 A represents sequencing results of the MMP-14 binding evolved library. Figure 11B represents sequencing results of the MMP-9 binding evolved library.
Figure 12 shows a logo of sequencing results from 24 colonies isolated from the third selective sort on a library of randomly mutated N-TIMP2 genes, with selection pressure applied to obtain MMP-14 specific mutants. Mutation is most prominent at position 71, 98 and 116. Positions located in the N-TIMP2:MMP interface are marked with an asterisk.
Figures 13A and 13B show binding specificity of select N-TIMP2 mutant yeast colonies assessed by FACS. Binding is determined by the average signal of fluorescence of yeast cells caused by fluorescently labeled MMP. Binding specificity is determined in the following manner in the case of MMP-14 (Figure 13A): (MMP-14mut/MMP-14wt)/(MMP- 9mut/MMP-9wt), and vice-versa for MMP-9 (Figure 13B). Tables at right indicate identities of mutants as compared to WT, the order of letters corresponds to the following positions on N- TEV1P2: 4, 6, 35, 38, 68, 71, 97, 99. Mutants designated with a '+' contain mutations which were not planned.
Figures 14A and 14B show enzyme activity assays with inhibition by N-TIMP-WT or mutants expressed in P. pastoris. Data for WT N-TIMP2 expressed in E. coli is not shown for simplicity. Points ± S.D. represent an average of at least three trials. Curves were fit to equation 1. Figure 14A: Enzyme activity experiments performed with MMP-14cat. Figure 14B: Enzyme activity experiments performed with MMP-9cat.
Figures 15A-15M: Interactions observed in computationally modeled structures of specificity enhancing mutations of N-TIMP2. N-TIMP2 is depicted in cyan and MMP-9cat or MMP-14Cat is depicted in green, polar contacts are showed as red dotted lines. MMP residues shown in sticks are not further than 4A away from the highlighted N-TIMP2 residue. Figures 15A-15D depict the interactions at position 4, Figures 15E-15G depict interactions at position 38, Figures 15H-15K depict interactions at position 71, Figures 15L-15M depict interactions at position 97. The WT structure at each position is depicted in Figures 15 A, 15C, 15E, 15H, 15 J, and 15L. All other figures depict the interactions caused by mutation. The bottom right-hand corner of each figure describes the mutation on N-TIMP2 and the MMP type it is interacting with.
BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES
The nucleic and/or amino acid sequences provided herewith are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the attached Sequence Listing:
SEQ ID NO 1 is a summary sequence of the N-TIMP2 variants described herein.
SEQ ID NO 2 is the amino acid sequence of the N-TIMP2A polypeptide.
SEQ ID NO 3 is a nucleic acid encoding the N-TIMP2A polypeptide.
SEQ ID NO 4 is the amino acid sequence of the N-TIMP2B polypeptide. SEQ ID NO: 5 is a nucleic acid encoding the N-TIMP2B polypeptide.
SEQ ID NO: 6 is the amino acid sequence of the N-TIMP2c polypeptide.
SEQ ID NO: 7 is a nucleic acid encoding the N-TIMP2c polypeptide.
SEQ ID NO: 8 is the amino acid sequence of the N-TIMP2D polypeptide.
SEQ ID NO: 9 is a nucleic acid encoding the N-TIMP2D polypeptide.
SEQ ID NO: 10 is the amino acid sequence of wildtype N-TIMP2.
SEQ ID NOs 11 and 12 are forward and reverse primers for cloning N-TIMP2WT into the pCTCON vector.
SEQ ID NOs 13 and 14 are forward and reverse primers for cloning N-TIMP2WT into the pCHA-VRCOl-scFv vector.
SEQ ID NOs 15 and 16 are forward and reverse primers for use in production of the focused N-TIMP2WT library.
SEQ ID NO's 17-21 are forward primers for cloning N-TIMP2WT and variants A-D into the pPICZaA vector.
SEQ ID NO: 22 is the reverse primer for cloning N-TIMP2WT and variants A-D into the pPICZaA vector.
SEQ ID NO: 23 is the fluorogenic substrate for use in MMP-14 binding assays.
SEQ ID NO: 24 is the amino acid sequence of the N-TIMP2 14-4 mutant polypeptide. SEQ ID NO: 25 is the amino acid sequence of the N-TIMP2 14-6 mutant polypeptide. SEQ ID NO: 26 is the amino acid sequence of the N-TIMP2 9-2 mutant polypeptide.
SEQ ID NO: 27 is the amino acid sequence of the N-TIMP2 9-6 mutant polypeptide. SEQ ID NOs: 28 and 29 are forward degenerate codon primers used to make mutant N- ΤΊΜΡ2 sequences.
SEQ ID NOs: 30 and 31 are reverse degenerate codon primers used to make mutant N- TEVIP2 sequences.
SEQ ID NO: 32 is a forward N-TIMP2 amplification primer.
SEQ ID NOs: 33 and 34 are forward and reverse N-TIMP2 amplification primers.
SEQ ID NO: 35 is a forward amplification primer for mutant 14-4.
SEQ ID NO: 36 is a forward amplification primer for mutant 14-6.
SEQ ID NO: 37 is a forward amplification primer for mutants 9-2 and 9-6.
SEQ ID NO: 38 is a forward amplification primer for mutant ATE.
SEQ ID NO: 39 is a reverse amplification primer for N-TIMP2 mutants.
SEQ ID NO: 40 is a forward sequencing primer for N-TIMP2 mutants.
SEQ ID NOs: 41-45 are forward PCR primers for transferring N-TIMP2 mutant sequences into the pet21 expression plasmid. SEQ ID NO: 46 is a reverse PCR primer for transferring N-TIMP2 mutant sequences into the pet21 expression plasmid.
SEQ ID NO: 47 is the amino acid sequence of the N-TIMP2 ATE mutant polypeptide. DETAILED DESCRIPTION
I. Terms
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."
In case of conflict, the present specification, including explanations of terms, will control. In addition, all the materials, methods, and examples are illustrative and not intended to be limiting.
Administration: The introduction of a composition into a subject by a chosen route. Administration of an active compound or composition can be by any route known to one of skill in the art. Administration can be local or systemic.
Examples of local administration include, but are not limited to, topical administration, subcutaneous administration, intramuscular administration, intrathecal administration, intrapericardial administration, intra-ocular administration, topical ophthalmic administration, or administration to the nasal mucosa or lungs by inhalational administration. In addition, local administration includes routes of administration typically used for systemic administration, for example by directing intravascular administration to the arterial supply for a particular organ. Local administration also includes the incorporation of active compounds and agents into implantable devices or constructs, such as vascular stents or other reservoirs, which release the active agents and compounds over extended time intervals for sustained treatment effects.
Systemic administration includes any route of administration designed to distribute an active compound or composition widely throughout the body via the circulatory system. Thus, systemic administration includes, but is not limited to intra- arterial and intravenous
administration. Systemic administration also includes, but is not limited to, oral, topical, subcutaneous, intramuscular routes, or administration by inhalation, when such administration is directed at absorption and distribution throughout the body by the circulatory system.
Analog or Derivative: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base structure.
Autoimmune disease: A disease resulting from an aberrant immune response, such as the production of antibodies or cytotoxic T cells specific for a self-antigen or a subject's own cells or tissues. Autoimmune diseases include, but are not limited to, inflammatory bowel disease, diabetes mellitus type 1, systemic lupus erythematosis, Churg-Strauss Syndrome, multiple sclerosis, Graves' disease, idiopathic thrombocytopenic purpura and rheumatoid arthritis.
Binding affinity: A term that refers to the strength of binding of one molecule to another at a site on the molecule. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association constant and dissociation constant for a pair of molecules, but it is not critical to the methods herein that these constants be measured or determined. The concepts of binding affinity, association constant, and dissociation constant are well known.
Biological Sample: Also, "sample." Any sample that may be obtained directly or indirectly from an organism, such as a subject, including whole blood, plasma, serum, tears, mucus, saliva, urine, pleural fluid, spinal fluid, gastric fluid, sweat, semen, vaginal secretion, sputum, fluid from ulcers and/or other surface eruptions, blisters, abscesses, tissues, cells (such as, fibroblasts, peripheral blood mononuclear cells, or muscle cells), organelles (such as mitochondria), organs, and/or extracts of tissues, cells (such as, fibroblasts, peripheral blood mononuclear cells, or muscle cells), organelles (such as mitochondria) or organs. The sample is collected or obtained using methods well known to those skilled in the art.
Cancer: The product of neoplasia is a neoplasm (a tumor or cancer), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." Neoplasia is one example of a proliferative disorder. A "cancer cell" is a cell that is neoplastic, for example a cell or cell line isolated from a tumor.
Examples of hematological tumors include leukemias. Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers (such as small cell lung carcinoma and non-small cell lung carcinoma), ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma and retinoblastoma).
Cardiac disease: Also, cardiovascular disease. General term to describe diseases and conditions of the heart and vascular system. Particular non-limiting examples include atherosclerosis, myocardial infarction (heart attack), and heart failure.
Chemotherapeutic agent: An agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth or hyperplasia. Such diseases include cancer, autoimmune disease as well as diseases characterized by hyperplastic growth such as psoriasis. In one embodiment, a chemotherapeutic agent is a radioactive compound. One of skill in the art can readily identify a chemotherapeutic agent (for instance, see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et ah, Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., © 2000 Churchill Livingstone, Inc.; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer DS, Knobf MF, Durivage HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).
Chemotherapeutic agents include small molecule agents and biologic agents.
Contacting: Placement in direct physical association. Includes both in solid and liquid form. Contacting can occur in vitro with isolated cells or in vivo by administering to a subject.
Detect: To determine if an agent (such as a signal or particular probe, such as a MMP inhibitory polypeptide described herein) is present or absent. In some examples, this can further include quantification. Diagnosis: The process of identifying a disease or a predisposition to developing a disease, by its signs, symptoms, and results of various tests and methods, for example the methods disclosed herein. The conclusion reached through that process is also called "a diagnosis." The term "predisposition" refers to an effect of a factor or factors that render a subject susceptible to a condition, disease, or disorder, such as cancer. In some examples, of the disclosed methods, testing is able to identify a subject predisposed to developing a condition, disease, or disorder, such as a malignant carcinoma.
Effective amount of a compound: A quantity of compound sufficient to achieve a desired effect in a subject being treated; also referred to as a "therapeutically effective amount." An effective amount of a compound can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of the compound will be dependent on the compound applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the compound.
Encode: A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.
Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked, for example the expression of a nucleic acid encoding a MMP inhibitory polypeptide described herein.
Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Functional fragments and variants of a polypeptide: Included are those fragments and variants that maintain one or more functions of the MMP inhibitory polypeptides described herein. It is recognized that the gene or cDNA encoding a polypeptide can be considerably mutated without materially altering one or more the polypeptide's functions. First, the genetic code is well-known to be degenerate, and thus different codons encode the same amino acids. Second, even where an amino acid substitution is introduced, the mutation can be conservative and have no material impact on the essential functions of a protein. See Stryer, Biochemistry 3rd Ed., (c) 1988. Third, part of a polypeptide chain can be deleted without impairing or eliminating all of its functions. Fourth, insertions or additions can be made in the polypeptide chain for example, adding epitope tags, without impairing or eliminating its functions (Ausubel et al. Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons, Inc., 1999). Other modifications that can be made without materially impairing one or more functions of a polypeptide include, for example, in vivo or in vitro chemical and biochemical modifications or the incorporation of unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquination, labeling, e.g., with radionucleides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art. A variety of methods for labeling polypeptides and labels useful for such purposes are well known in the art, and include radioactive isotopes such as 32P, ligands which bind to or are bound by labeled specific binding partners {e.g., antibodies), fluorophores, and chemiluminescent agents.
Immediate release (of a pharmaceutical compound): A compound that is
immediately biologically/chemically available and active upon administration to a subject and contact with a target site.
Inhibiting protein activity: To decrease, limit, or block an action, function or expression of a protein. The phrase inhibit protein activity is not intended to be an absolute term. Instead, the phrase is intended to convey a wide-range of inhibitory effects that various agents may have on the normal (for example, uninhibited or control) protein activity.
Inhibition of protein activity may, but need not, result in an increase in the level or activity of an indicator of the protein's activity. By way of example, this can happen when the protein of interest is acting as an inhibitor or suppressor of a downstream indicator. Thus, protein activity may be inhibited when the level or activity of any direct or indirect indicator of the protein's activity is changed (for example, increased or decreased) by at least 10%, at least 20%, at least 30%, at least 50%, at least 80%, at least 100% or at least 250% or more as compared to control measurements of the same indicator. Inflammatory Bowel Disease (IBD): A group of diseases characterized by
inflammation or all or part of the gastrointestinal tract. Particular non-limiting examples include ulcerative colitis and Crohn's disease.
Injectable composition: A pharmaceutically acceptable fluid composition comprising at least one active ingredient, for example, a polypeptide. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, pH buffering agents and the like. Such injectable compositions that are useful for use with the compositions of this disclosure are conventional; appropriate formulations are well known in the art.
Isolated: A biological component (such as a nucleic acid molecule or polypeptide) that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs or in which it has been produced, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and polypeptides.
Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non-limiting examples of labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.
Matrix metalloproteinase(s) (MMP): family of enzymes consisting of more than 20 homologous proteins that play a significant role in the degradation of the extracellular matrix, a crucial step in many cellular processes. MMPs are multi-domain proteins that differ in domain architecture and substrate preferences (1), but they all share a catalytic domain with a nearly identical active site containing a Zn2+ ion (2). MMPs are synthesized in an inactive form and are subsequently activated by the cleavage of a pro-domain, often by other MMPs or by different proteases (3,4), thus producing complex protein-protein interaction (PPI) networks (5). For example, MMP-2 is activated physiologically by MMP- 14; MMP-9 is activated by active MMP-2 and active MMP-7; and MMP-1 is also activated by active MMP-7 (4). Some MMPs, such as MMP-3, are 'master regulators,' being able to activate themselves as well as other MMPs (6). Imbalance in MMP activity leads to various diseases, including cancers (7), cardiovascular diseases (8) and arthritis (9). Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Parenteral: Administered outside of the intestine, for example, not via the alimentary tract. Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The term polypeptide or protein as used herein encompasses any amino acid sequence and includes modified sequences such as glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
The term polypeptide fragment refers to a portion of a polypeptide which exhibits at least one useful epitope. The phrase "functional fragments of a polypeptide" refers to all fragments of a polypeptide that retain an activity, or a measurable portion of an activity, of the polypeptide from which the fragment is derived. Fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell. An epitope is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen. Thus, smaller peptides containing the biological activity of insulin, or conservative variants of the insulin, are thus included as being of use.
The term substantially purified polypeptide as used herein refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In one embodiment, the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Variations in the cD A sequence that result in amino acid changes, whether conservative or not, are usually minimized in order to preserve the functional and immunologic identity of the encoded protein. The immunologic identity of the protein may be assessed by determining whether it is recognized by an antibody; a variant that is recognized by such an antibody is immunologically conserved. Any cDNA sequence variant will preferably introduce no more than twenty, and preferably fewer than ten amino acid substitutions into the encoded polypeptide. Variant amino acid sequences may, for example, be 80%, 90% or even 95% or 98% identical to the native amino acid sequence. Programs and algorithms for determining percentage identity can be found at the NCBI website.
Preventing or treating a disease: Preventing a disease refers to inhibiting the full development of a disease, for example inhibiting the development of myocardial infarction in a person who has coronary artery disease or inhibiting the progression or metastasis of a tumor in a subject with a neoplasm. Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for
Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
Nucleic acid sequences that do not show a high degree of identity can nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
Solid support (or substrate): Numerous and varied solid supports are known to those in the art and include, without limitation, nitrocellulose, the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, membranes, microparticles (such as latex particles), and sheep (or other animal) red blood cells. Any suitable porous material with sufficient porosity to allow access by detector reagents and a suitable surface affinity to immobilize capture reagents {e.g., monoclonal antibodies) is contemplated by this term. For example, the porous structure of nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents, for instance, capture reagents. Nylon possesses similar characteristics and is also suitable. Microporous structures are useful, as are materials with gel structure in the hydrated state.
Further examples of useful solid supports include: natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass (these materials may be used as filters with the above polymeric materials); and mixtures or copolymers of the above classes, such as graft copolymers obtained by initializing polymerization of synthetic polymers on a pre-existing natural polymer.
The surface of a solid support may be activated by chemical processes that cause covalent linkage of an agent (e.g., a capture reagent) to the support. However, any other suitable method may be used for immobilizing an agent (e.g. , a capture reagent) to a solid support including, without limitation, ionic interactions, hydrophobic interactions, covalent interactions and the like.
Subject: Living multi-cellular organisms, including vertebrate organisms, a category that includes both human and non-human mammals.
Sustained release (of a pharmaceutical compound): A sustained release compound that can be biologically/chemically available and active upon administration to a subject and contact with a target site, and over an extended period of time. Numerous sustained release additives and formulations are known to the art and can be combined with the MMP inhibitors described herein.
Tissue inhibitors of metalloproteinases (TIMPs): Broad inhibitors of the MMP family. In nature, there are four such inhibitors (TIMPs 1-4) that show 40-50% sequence identity and exhibit slightly different preferences for various MMPs (31). All TIMPs bind to MMPs using the same interaction mode, coordinating the active site but also interacting with a number of additional sites on MMPs (32-38). As natural effectors, TIMPs are non- immunogenic and non-toxic to humans.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transfected host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses. II. Overview of Several Embodiments
Provided herein are polypeptide MMP inhibitors including an isolated polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 1, or a fragment, derivative or analog thereof. In particular embodiments, the polypeptide MMP inhibitor is a polypeptide at least 70% identical to an amino acid sequence set forth as SEQ ID NOs 2, 4, 6, 8, 24-27, or 47.
In some embodiments, the described polypeptides include a detectable label. In other embodiments, the described polypeptide is conjugated to a solid substrate.
In particular embodiments the isolated polypeptide is conjugated to a chemotherapeutic agent.
Also described herein are nucleic acid sequences including a nucleic acid sequence encoding the described isolated polypeptides, such as the nucleic acid sequence set forth as SEQ ID NO: 3, 5, 7, or 9, and expression vectors that contain the provided nucleic acid sequences.
Further provided herein are methods for treatment of a disease or condition
characterized by aberrant overexpression of matrix metalloproteinase (MMP) -14, MMP-9, or MMP-2, which include administering to a subject in need thereof a therapeutically effective amount of the described polypeptides, thereby treating the disease or condition.
In particular embodiments the disease or condition characterized by aberrant MMP expression is selected from the group consisting of a cancer, such as a carcinoma, an autoimmune disease, such as inflammatory bowel disease, and cardiac disease.
In particular embodiments of the described treatment methods, the isolated polypeptide is administered following cardiac arrest in the subject.
In some embodiments of the described methods, the isolated polypeptide is formulated for immediate or sustained release. In other embodiments, the isolated polypeptide is formulated for local or systemic administration.
Further described herein are methods for treatment of a disease or condition
characterized by aberrant overexpression of matrix metalloproteinase (MMP)- 14, MMP-9, or MMP-2, the including administering to a subject in need thereof a nucleic acid expressing the described MMP inhibitory polypeptides, wherein expression of the nucleic acid provides a therapeutically effective amount of the isolated polypeptide to the subject.
It will be appreciated that the described synthetic polypeptide and synthetic nucleic acid compositions can be used as described and as formulated in the above methods of treatment above for use in the treatment of a disease or condition characterized by aberrant
overexpression of matrix metalloproteinase (MMP) -14, MMP-9, or MMP-2. Additionally described herein are methods of detecting a cell that is overexpressing MMP-14, MMP-9, or MMP-2 by contacting a cell that is suspected of overexpressing MMP- 14, MMP-9, or MMP-2 with at least one of the described isolated polypeptides; washing unbound polypeptide from the cell; and detecting bound polypeptide.
In particular embodiments of the described detection methods, the cell is in a sample isolated from a subject. In other embodiments of the described detection methods, the cell is within a subject.
III. MMP Inhibitory Polypeptides
Described herein are synthetic variants of the MMP-inhibiting N-terminal domain of
TEVIP2 (N-TIMP2) which have been optimized for enhanced MMP-14 or MMP-9 binding affinity and specificity, and which include at least 3 mutations from the wildtype N-TIMP2 sequence. The described N-TIMP2 variants include polypeptides, variants, and fragments thereof, of a polypeptide including the following sequence of N-TIMP2 with variable positions Xi-Xio:
CSCXiPX2HPQQAFCNADVVIRAKAVSEKEVDSGNDX3YGX4PIKRIQYEIKQIK MFKGPEKDIEFIYTAPX5SAX6CGVSLDVGGKKEYLIAGKAEGDGKMX7X8X9LCDFIVP WDTLS TTQKXioS LNHR YQMGCE ; wherein: Xi is R, A, M, E, P, or S; X2 is V, R, or G; X3 is L, M, I, V, or P; X4 is N, D, Q, or P; X5 is D, G, S, or N; X6 is V, I, A, G, or N; X7 is H, R or L; Xs is I or T; X9 is S, N, or T; and X10 is K or E (SEQ ID NO: 1).
In particular embodiments the polypeptide includes the following sequence described herein as N-TIMP2A:
CSCAPVHPQQAFCNADVVIRAKAVSEKEVDSGNDPYGDPIKRIQYEIKQIKMFK GPEKDIEFIYTAPDSAICGVSLDVGGKKEYLIAGKAEGDGKMRISLCDFIVP WDTLS TT QKKSLNHRYQMGCE (SEQ ID NO: 2).
In particular embodiments the polypeptide includes the following sequence described herein as N-TIMP2B:
CSCRPVHPQQAFCNADVVIRAKAVSEKEVDSGNDLYGDPIKRIQYEIKQIKMFK GPEKDIEFIYTAPDSAICGVSLDVGGKKEYLIAGKAEGDGKMRISLCDFIVP WDTLS TT QKKSLNHRYQMGCE (SEQ ID NO: 4).
In particular embodiments the polypeptide includes the following sequence described herein as N-TIMP2C:
CSCMPVHPQQAFCNADVVIRAKAVSEKEVDSGNDMYGDPIKRIQYEIKQIKMF KGPEKDIEFIYTAPNSAGCGVSLDVGGKKEYLIAGKAEGDGKMLITLCDFIVP WDTLS TTQKKS LNHR YQMGCE (SEQ ID NO: 6). In particular embodiments the polypeptide includes the following sequence described herein as N-TIMP2D:
CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDMYGDPIKRIQYEIKQIKMF KGPEKDIEFIYTAPNSAGCGVSLDVGGKKEYLIAGKAEGDGKMRITLCDFIVPWDTLS TTQKKS LNHR YQMGCE (SEQ ID NO: 8).
In particular embodiments the polypeptide includes the following sequence described herein as N-TIMP2 mutant 14-4:
CSCEPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGQPIKRIQYEIKQIKMFK GPEKDIEFIYTAPSSANCGVSLDVGGKKEYLIAGKAEGDGKMRISLCDFIVPWDTLSTT QKKSLNHRYQMGCE (SEQ ID NO: 24).
In particular embodiments the polypeptide includes the following sequence described herein as N-TIMP2 mutant 14-6:
CSCEPRHPQQAFCNADVMIRAKAVSEKEVDSGNDVYGQPIKRIQYEIKQIKMF KGPEKDIEFIYTAPNSANCGVSLDVGGKKEYLIAGKAEGDGKMRISLCDFIVPWDTLST TQKKS LNHRYQMGCE (SEQ ID NO: 25).
In particular embodiments the polypeptide includes the following sequence described herein as N-TIMP2 mutant 9-2:
CSCPPVHPQQAFCNADVVIRAKAVSEKEVDSGNDVYGNPIKRIQYEIKQIKMFK GPEKDIEFIYTAPNSAICGVSLDVGGKKEYLIAGKAEGDGKMRITLCDFIVPWDTLSTT QKKSLNHRYQMGCE (SEQ ID NO: 26).
In particular embodiments the polypeptide includes the following sequence described herein as N-TIMP2 mutant 9-6:
CSCPPGHPQQAFCNADVVIRAKAVSEKEVDSGNDVYGNPIKRIQYEIKQIKMFK GPEKDIEFIYTAPNSAICGVSLDVGGKKEYLIAGKAEGDGKMHINLCDFIVPWDTLSTT QKKSLNHRYQMGCE (SEQ ID NO: 27).
In particular embodiments the polypeptide includes the following sequence described herein as N-TIMP2 mutant ATE:
CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFK GPEKDIEFIYTAPSSAACGVSLDVGGKKEYLIAGKAEGDGKMHTTLCDFIVPWDTLST TQKESLNHRYQMGCE (SEQ ID NO: 47).
Variants, fragments, and analogs of the described polypeptides are included in the current disclosure. Such polypeptides include polypeptides that share about 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity with the described N-TIMP2 variants. In particular embodiments, sequence variance from WT is particularly found in those amino acids apart from those described herein as involved in MMP binding (e.g. Xi-X8, above). In other embodiments the variation from WT sequence can be conservative substitutions that one of skill will not expect to significantly alter the shape or charge of the polypeptide. The described polypeptides also include those polypeptides that share 100% sequence identity to those indicated, but which differ in post-translational modifications from the native or natively- produced sequence.
In particular embodiments, the described N-TIMP2 variants are provided as a discrete biomolecules. In other embodiments, the described polypeptides are a domain of a larger polypeptide, such as an independently-folded structural domain, or an environment-accessible functional domain.
The N-TIMP2 polypeptides described herein inhibit MMP activity, but also bind with increased affinity and specificity to MMP- 14 and/or MMP-9, and which allow for strong and specific targeting of a MMP- 14 and/or MMP-9-expressing cell. In some embodiments the described polypeptides are conjugated at the N- and/or C-terminus to at least one of a variety of small molecules, chemotherapeutic agents, labels, and solid substrates, which are thereby targeted to a MMP-14 and/or MMP-9 expressing cell.
In particular embodiments, the described polypeptides are conjugated to any
chemotherapeutic agent as described herein and as known to the art. Such agents can include small molecule agents, antibodies, and the like which are designed, for example, to inhibit cell proliferation and/or metastatic cell migration and/or promote cell death. Particular non- limiting examples of such agents include cytotoxic radioisotopes, chemotherapeutic agents and toxins.
In other embodiments, a detectable label, such as a detectable chemical, fluorescent, or radioactive label can be conjugated to the described polypeptides. Antibodies that can be conjugated to N-TIMP2 variant polypeptides, and which themselves are or can be labeled, are standard in the art and are included in the current disclosure.
In still other embodiments, the described N-TIMP2 variants can be conjugated to any suitable affinity tag for specific isolation of MMP-14 and/or MMP-9 expressing cells through standard affinity chromatographic methods. Particular non-limiting examples of such tags include myc, glutathione- s-transferase (GST), maltose binding protein (MBP), hemagglutinin (HA), and polyhistadine.
In additional further embodiments, the N-TIMP2 polypeptides described herein can be associated with or conjugated to a solid substrate, alone or in combination with any of the already described modifications.
Also provided herein are nucleic acids encoding the described N-TIMP2 variant polypeptides, such as the nucleic acids set forth herein as SEQ ID NO: 3 (encoding N- TIMP2A); SEQ ID NO: 5 (encoding N-TIMP2B); SEQ ID NO: 7 (encoding N-TIMP2C); and SEQ ID NO: 9 (encoding N-TIMP2D).
It will be appreciated that due to degeneracy of the genetic code, the sequences of the described nucleic acids can vary significantly from the sequences set forth herein as SEQ ID NOs 3, 5, 7, and 9; and without any change in the encoded polypeptide. Other and/or additional mutations in the described polypeptides, such as conservative amino acid mutations, can also be included without an appreciable difference. Accordingly, in some embodiments, the described nucleic acids share between 60%-100% sequence identity with SEQ ID NOs 3, 5, 7, and 9, such as 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity.
In particular embodiments, the described nucleic acid sequences are contained within a
DNA cloning and/or expression plasmid as are standard in the art. It will be appreciated that any standard expression plasmid can be used to express one or more of the described N-TIMP2 variant-encoding nucleic acids. Such plasmids will minimally contain an origin of replication, selection sequence (such as, but not limited to an antibiotic resistance gene), and expression control sequences operably linked to the N-TIMP2 variant polypeptide. In particular embodiments, the expression plasmids include post-translational sequences (e.g. signal sequences to direct polypeptide processing and export) that are encoded in-frame with the N- TEV1P2 variant nucleic acids.
Particular non-limiting examples of bacterial expression plasmids include IPTG- inducible plasmids, arabinose-inducible plasmids and the like. Other non-limiting examples of expression induction include light induction, temperature induction, nutrient-induction, and autoinduction, and mammalian- specific DNA expression plasmids. Custom-made expression plasmids are commercially available from suppliers such as New England Biolabs (Ipswich, MA) and DNA 2.0 (Menlo Park, CA). In a particular embodiment, a N-TIMP2 variant- expressing plasmid can be designed for specific localized induction in response to a local cellular micro environment.
In particular embodiments, the peptides and nucleic acids described herein can be supplied in any pharmaceutically acceptable composition. In such embodiments, one or more N-TIMP2 variant polypeptide or polypeptide-expressing nucleic are provided in a
pharmaceutical formulation having a therapeutically effective dose of each therapeutic agent, as described herein, and including standard pharmaceutically acceptable salts, excipients, fillers and the like.
Various delivery systems are known and can be used to administer polypeptides and nucleic acids as therapeutic agents. Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic molecule(s), construction of a therapeutic nucleic acid as part of a retroviral or other vector, and the like. Methods of introduction include, but are not limited to, intrathecal, intradermal, intramuscular, intraperitoneal (ip), intravenous (iv), subcutaneous, intranasal, epidural, and oral routes. The therapeutics may formulated for administration by any convenient route, including, for example, infusion or bolus injection, topical, absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa, and the like) ophthalmic, nasal, and transdermal, and may be administered together with other biologically active agents. Pulmonary administration can also be employed (e.g., by an inhaler or nebulizer), for instance using a formulation containing an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the described
pharmaceutical treatments by injection, catheter, suppository, or implant (e.g. , implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like. In another embodiment, therapeutic agents are delivered in a vesicle, in particular liposomes.
In particular embodiments, the described polypeptides and nucleic acids can be formulated for immediate release, whereby they are immediately accessible to the surrounding environment, thereby providing an effective amount of the active agent(s), upon administration to a subject, and until the administered dose is metabolized by the subject.
In yet another embodiment, the described polypeptides and nucleic acids can be formulated in a sustained release formulation or system. In such formulations, the therapeutic agents are provided for an extended duration of time, such as 1, 2, 3, 4 or more days, including 1-72 hours, 24-48 hours,. 16-36 hours, 12-24 hours, and any length of time in between. In particular embodiments, sustained release formulations are immediately available upon administration, and provide an effective dosage of the therapeutic composition, and remain available at an effective dosage over an extended period of time. In other embodiments, the sustained release formulation is not immediately available within the subject and only becomes available, providing a therapeutically effective amount of the active compound(s), after the formulation is metabolized or degraded so as to release the active compound(s) into the surrounding environment.
In one embodiment, a pump may be used. In another embodiment, the sustained released formulations include polymeric materials commonly used in the art, such as in implants, gels, capsules, and the like. By way of example, polymers such as bis(p- carboxyphenoxy)propane-sebacic-acid or lecithin suspensions may be used to provide sustained localized release. Other sustained release formulations and systems for use with the described polypeptides and nucleic acids include those such as discussed in the review by Langer (Science 249, 1527 1990).
In particular embodiments, the described polypeptides and nucleic acids are formulated in pharmaceutically acceptable compositions of the compounds, using methods well known to those with skill in the art. For instance, in some embodiments, the compounds are formulated with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia world-wide for use in animals, and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions, blood plasma medium, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like.
Examples of pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The described compositions can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The described compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, all in immediate and sustained-release formulations as understood in the art. The therapeutic can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
Therapeutic preparations will contain a therapeutically effective amount of at least one active ingredient, preferably in purified form, together with a suitable amount of carrier so as to provide proper administration to the patient. The formulation should suit the mode of administration.
The ingredients of the described formulations can be supplied either separately or mixed together in unit dosage form, for example, in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, or suspensions, or as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Kits comprising the described N-TIMP2 variant polypeptides or encoding nucleic acids are accordingly also contemplated herein. IV. Methods of treatment of MMP associated diseases
Aberrant overexpression of MMPs, such as MMP- 14, MMP-2, and MMP-9 has been implicated in a variety of conditions and diseases, including various cancers, autoimmune diseases, and cardiovascular diseases. The polypeptides described herein provide a MMP- 14 inhibitory agent with increased MMP- 14 binding affinity and specificity. The described N- TIMP2 variants have also shown increased efficacy against MMP-2 and MMP-9. Accordingly, the described polypeptides, and nucleic acids expressing the described polypeptides, can be used in pharmaceutical compositions in methods for treating diseases and conditions associated with aberrant overexpression of MMP- 14, MMP-2, and MMP-9.
In particular embodiments, the described methods include administering a
therapeutically effective amount of a N-TIMP2 variant polypeptide described herein to a subject in need thereof, thereby treating the disease or condition associated with aberrant expression of MMP- 14, MMP-2, and/or MMP-9.
Any of the compositions described herein which include the described N-TIMP2 variant polypeptides or polypeptide-encoding nucleic acids, can be used in the described methods of treatment.
In particular embodiments, the disease related to aberrant MMP- 14, MMP-2, and/or MMP-9 expression is a cancer, such as a fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers (such as small cell lung carcinoma and non-small cell lung carcinoma), ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma and retinoblastoma). In some embodiments, the described N-TIMP2 variants inhibit cell proliferation. In other embodiments, the described variants inhibit cell migration, thereby inhibiting cancer metastasis. In still other embodiments, the described variants promote cell death, such as by promotion of apoptosis.
In other particular embodiments, the disease related to aberrant MMP-14, MMP-2, and/or MMP-9 expression is an autoimmune disease or associated with autoimmunity. Non- limiting examples of such diseases include inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease.
In additional embodiments, the disease related to aberrant MMP-14, MMP-2, and/or MMP-9 expression is a cardiovascular disease, such as myocardial infarction, and particularly heart remodeling post myocardial infarction. In such embodiments, the described N-TIMP2 variant polypeptides or encoding nucleic acids can be administered to the subject prior to (e.g. to a subject at risk of heart attack) or following a myocardial infarction, in order to decrease the continuing deleterious effects of the cardiac event.
Other diseases related to aberrant MMP-14, MMP-2, and/or MMP-9 expression that can be treated with the described polypeptides include cardiovascular diseases, autoimmune diseases, periodontal disease, diseases of the central nervous system, such as multiple sclerosis and spinal cord injury; and bone degradation.
In particular embodiments, the N-TIMP2 variant is administered to the subject as a polypeptide. In other embodiments, the N-TIMP2 variant is administered to the subject by way of an expression vector containing a N-TIMP2 variant-encoded nucleic acid. It will be appreciated that in such embodiments, expression of the polypeptide can be constitutive or induced, as is well-known in the art. In some embodiments, inducible expression systems can allow for specific targeting of an area, which contains the inducing signal.
In some embodiments, the N-TIMP2 variant is administered to the subject in combination with other pharmaceutical agents for treatment of the disease or condition under treatment. For example, in methods for treating breast cancer the N-TIMP2 variant can be combined with trastuzumab (Herceptin) therapy. In other examples of cancer treatment, administration of the N-TIMP2 variant can be combined with surgery and/or radiation therapy. The one or more therapies in combination with the N-TIMP2 variant can be administered to the subject in sequence (prior to or following) or concurrent. Where applicable, in particular embodiments, combinations of active ingredients can be administered to a subject in a single or multiple formulations, and by single or multiple routes of administration.
The amount of each therapeutic agent for use in the described methods, and that will be effective, will depend on the nature of the disorder or condition to be treated, as well as the stage of the disorder or condition. Therapeutically effective amounts can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each patient's circumstances. The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy.
The therapeutic compounds and compositions of the present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (e.g., in which the loading dose is about two to five times the maintenance dose). In some embodiments, the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art. In some embodiments long-term treatment with the drug is contemplated.
V. Detection Methods
The enhanced binding affinity and specificity of the N-TIMP2 variants provided herein allows for methods of detecting a cell that is expressing, such as aberrantly overexpressing MMP-14, MMP-9, or MMP-2.
In particular embodiments, the methods include contacting a cell that is suspected of overexpressing MMP-14, MMP-9, or MMP-2 with an isolated N-TIMP2 variant polypeptide as described herein, washing unbound polypeptide from the cell; and detecting bound
polypeptide. In such methods, the N-TIMP2 variant can be used as any ligand in a variety of in vitro and in vivo labeling and detection assays by standard methods known to the art.
In particular embodiments, the cell or cell-containing tissue is located in a sample that has been taken from a subject by standard methods as described herein. In other embodiments, the cell or cell-containing tissue is located in or on the subject.
In particular embodiments, prior to contacting the cell with the N-TIMP2A variant, the cells are permeablized as standard in the art of cell staining.
It will be appreciated the "wash" step of the described methods can be done with any suitable variant polypeptide-free buffer or solution. Such washing 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.
Other embodiments include use of an N-TIMP2 variant polypeptide that has been conjugated to a solid substrate and provides the bait for binding to and detection of a suitable MMP-expressing cell, such as in an ELISA assay or modification thereof.
The methods of detection contemplated herein are standard, including but not limited to standard fluorescent, chemiluminescent, and immunocytochemical labeling methods.
Kits designed for use in the described detection methods are also described herein. Such kits can contain a N-TIMP2 variant or nucleic acid expressing the polypeptide, and suitable buffers, labels, and as necessary detection reagents.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
EXAMPLES
Example 1: Methods
Methods in in the following examples are as described herein unless otherwise noted. Computational Saturation Mutagenesis of N-TIMP2/MMP Complexes: Calculations were performed using the following crystal structures: N-TIMP2/MMP-14 (PDB ID: 1BUV), N-TIMP2/MMP-13 (PDB ID: 2E2D), and N-TIMP2/MMP-10 (PDB ID: 4ILW). Since crystal structures for the remaining N-TIMP2/MMP complexes do not exist, we constructed models for such structures using the unbound MMP structures: MMP-1 (PBD ID: 3SHI), MMP-2 (PDB ID: 1RTG, MMP-3 (PDB ID: 1B3D), MMP-7 (PDB ID: 1MMQ), MMP-8 (PDB ID: 20 Y4) and MMP-9 (PDB ID: 1L6J). Unbound MMP structures were first aligned to the MMP- 14 chain of the N-TIMP2/MMP-14 structure, and the original MMP- 14 atoms were removed from the structure by using PyMol. The structure was subsequently refined by using the docking protocol of the ROSIE server (Lyskov et al.), and the structure with the best combination of energy score and small RMSD in comparison to the input structure was chosen as the final complex structure. For calculating the free energy of binding for mutations in the interface, the energy function optimized by Sharabi et al. was used. The calculation started with the following stages: a mutation was introduced into N-TIMP2, a shell of neighboring residues around the mutated residue were allowed to repack, and the free energy of the N- TIMP2/MMP complex was calculated for the wild-type complex and for the mutated complex. The two chains were then separated, and the free energies of each single chain in the wild-type complex and the mutant complex were calculated. The change in free energy due to mutation (AG) of the complex and the single chains was calculated by subtracting the free energy of the wild-type structures from the free energy of the mutant structures. The value of AAGbind was determined by subtracting the AG of the individual chains from the AG of the complex. The energy functions are made up of terms that describe van der Waals attractive and repulsive interactions, hydrogen bond interactions, electrostatic interactions, and surface-area-based solvation [see Sharabi et al. for details] . The rotamer libraries used were based on the backbone-dependent library of Dunbrack and Karplus, with additional rotamers expanded by one standard deviation around their mean χΐ and χ2 values. The lowest-energy rotameric conformation was found using the dead-end elimination theorem. The combinatorial library of N-TIMP2 mutants was designed on the basis of the in silico saturation mutagenesis results, with focus on MMP- 14 as a desired target and MMP-1, MMP-2, MMP-8, MMP-9, MMP- 10, and MMP- 13 as alternative targets. N-TIMP2 positions that contained many affinity-enhancing mutations, particularly towards MMP- 14 were selected.
TIMP-4 Structure Modeling: A homology model of human TIMP4 was built using the I-
TASSER server the input sequence was that deposited in the UNIPROT database (accession #: Q99727) and was modified by removing the signal peptide and truncating the sequence to residue 184. The server produced one output structure.
Preparation ofDNA Constructs and Library in Yeast: The gene encoding human N- TIMP2wT (residues 1- 127) (Sharabi et al.) was expressed in a Yeast Surface Display (YSD) system in two constructs. For expression in the pCTCON vector (Boder, E. T., and Wittrup, K. D.), N-TIMP2WT was amplified by PGR reaction with Phusion DMA polymerase ( New
England Biolabs, MA, USA) with primers containing homology regions to pCTCON and recognition sites for the restriction enzymes he and BamHI (forward-5'- GGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCTGCAGCTGCTC CCCGGTG-3' (SEQ ID NO: 1 1);
reverse-5'-
GCTATTACAAGTCCTCTTCAGAAATAAGCTTTTGTTCAGATGGATCTTGGAAAGCC AATGGTTTATCTGGCAAGGATCCCTCGCAGCCCATCTGGTAC-3' (SEQ ID NO: 12)). For the construction of yeast-displayed N-TIMP2WT with a free N-terminus, the pCHA-
VRCOl-scFv vector was used (obtained from Dane Wittrup, MIT) (Mata-Fink et al.). In this construct, the C-terminus of the gene of interest is fused to the N-terminus of Aga-2 (Angelini et al), leaving the N-terminus of the displayed protein exposed to the solvent. The gene was amplified with a forward primer (5'- CTTGTTGGCTATCTTCGCTGCTTTGCCATTGGCCTTAGCTTGCAGCTGCTCCCCGGT G-3' ; SEQ ID NO: 13) containing the KR sequence at the N-terminus of N-TIMP2, which results in a free N-terminus after post-modification cleavage by Kex-2 protease. The reverse primer (5'-
GTCTTCTTCGGAGATAAGCTTTTGTTCTGCACGCGTGGATCCCTCGCAGCCCATCTG GTACC-3'; SEQ ID NO: 14) contains homologous regions to the vector and the BamHI restriction site at the 3' end of the reverse primer. The pCT and pCHA vectors were linearized with BamHI-HF and Nhe-HF enzymes (New England Biolabs, USA) and, together with the respective N-TIMP2WT insert, were transformed by homologous recombination into a competent EBYIOO S. cerevisiae yeast strain using a MicroPulser electroporator (Bio-Rad, CA, USA), as previously described. The transformed yeast was first grown on selective SDCAA plates (0.54% Na2HP04, 0.856% Na2HP04H20, 18.2% sorbitol, 1.5% agar, 2% dextrose, 0.67% yeast nitrogen base, 0.5% Bactocasamino acids) at 30°C. A single colony was then grown in selective SDCAA culture medium (2% dextrose, 0.67% yeast nitrogen base, 0.5% Bactocasamino acids, 1.47% sodium citrate, 0.429% citric acid monohydrate, pH 4.5) at 30°C, and the correct sequence was confirmed by DNA sequencing [sequencing lab of the National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev (BGU), Israel] .
The YSD-focused N-TIMP2 library was designed on the basis of positions that were previously shown to be tolerant to mutation by Sharabi and colleagues, and purchased from GenScript (NJ, USA). Briefly, the focused library was prepared using NNS (where N represents A, C, T or G nucleotides, and S represents C and G) degenerate codons at positions 4,35,38,68,71,97 and 99 of the N-TIMP2WT gene. The focused library was amplified with forward and reverse primers (forward-5'-CCGGTTATTTCTACTACCGTCGGTTC-3\ SEQ ID NO: 15; reverse-5'-GTCAGTTCCTGCAAGTCTTCTTCG-3'; SEQ ID NO: 16) and transformed into competent EBYIOO S. cerevisiae along with the linear pCHA vector. The transformed yeast was grown in selective SDCAA culture medium (2% dextrose, 0.67% yeast nitrogen base, 0.5% Bactocasamino acids, 1.47% sodium citrate, 0.429% citric acid
monohydrate, pH 4.5). A library size of 8* 106 clones was confirmed by performing serial dilutions of the transformed yeast on SDCAA plates and counting after 48 h of growth.
Flow Cytometry Analysis and Cell Screening— The yeast containing the gene for N-
THVIP2WT was grown in the selective and protein-expression-inducing SGCAA medium (2% galactose, 0.67% yeast nitrogen base, 0.5% Bactocasamino acids, 1.47% sodium citrate, 0.429% citric acid monohydrate) overnight at 30 °C to a density of 10s cells/ml
(OD6oonm=10.0). The yeast cells (106 cells) were then collected and washed with a buffer containing 50 mM Tris, pH7.5, 100 mM NaCl, 5 mM CaCl2 and 1% bovine serum albumin (BSA). For the detection of protein expression, the yeast was incubated with mouse anti-c-Myc antibody (Abeam, Cambridge, UK) at a ratio of 1:50 at room temperature for 1 h. Thereafter, the cells were washed and incubated with sheep anti-mouse secondary antibody conjugated to phycoerythrin (PE) (Sigma- Aldrich, St. Louis, MO, USA) at a 1:50 ratio for 30 min on ice. The catalytic domain of MMP-14 (MMP-14CAT) was purified (see details below) and labeled for 1 h at room temperature with DyLight-488 Amine Reactive Dye, which contains a
hydroxy succinimide (NHS) ester that forms a covalent bond with primary amines
(Thermo Fisher, Waltham, MA, USA), at a ratio of 1:5 (protein:dye). For the detection of binding to MMP-14CAT, the cells were incubated with 1 μΜ of MMP-14CAT labeled with DyLight-488 for 30 min on ice. The yeast was then washed and analyzed using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). In the flow cytometry plots, the horizontal axis provides an indication of the expression of the displayed protein (PE signal), and the vertical axis shows the binding of the displayed protein to the soluble target MMP- 14CAT (DyLight-488 signal).
A similar labeling process was performed for yeast expressing the N-TIMP2 focused library for enrichment of the high-affinity cell population. For the first sort (c-Myc-clear, S I), 10s cells (10 times more than the library diversity size) were labeled, and for the following sorts, 2-20x 106 cells were labeled, depending on the library size. In the S I sort, the high- expressing cells were selected. In the subsequent sorts (S2-S6), the cells were labeled for both detection of expression and binding to MMP-14CAT conjugated to DyLight-488, with decreasing concentrations of labeled MMP-14CAT for each round of sorting, beginning with 1 μΜ of labeled MMP- 14CAT for S2 and ending with 5 nM in the final sort (S6). The affinity maturation screens were performed on an iCyt Synergy FACS apparatus (Sony Biotechnology, San Jose, CA, USA). For each sort, about 1.5% of the binding population normalized to expression was selected using a polygonal gate.
To obtain the YSD-based affinity titration curves, the same labeling process was implemented, and the levels of binding of N-TIMP2WT and the selected N-TIMP2 variants were measured by incubating the yeast cells with different concentrations (1 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 500 nM, 750 nM and 1000 nM) of labeled MMP-14CAT for 30 min at room temperature.
DNA Sequencing: To sequence the pre- and post-sorted N-TIMP2 libraries, the plasmid DNA was purified from the yeast using the Zymoprep™ yeast plasmid miniprep I kit
(ZymoResearch, Irvine, CA, USA). The plasmid was then transformed into electro-competent E. coli bacteria and grown on ampicillin (100 /g/ml) LB-agar plates. Thereafter, about 20 colonies were transferred to liquid ampicillin-LB culture medium and grown overnight at 37 °C. The plasmid was purified from the bacteria according to the manufacturer's protocol with the HiYield plasmid mini kit (RBC Bioscience, Taiwan). The plasmids were sequenced using the Sanger sequencing method [Genetics Unit, NIBN, BGU, Israel], and the sequences were analyzed using Geneious R7 software (Biomatters, New Zealand).
In-silico Thermodynamic and Structural Analysis of Affinity-Enhanced Variants:
AAGbind was calculated for each variant for which DNA sequencing was performed after each sorting round. Multiple mutations were computationally inserted into the structure, and a AAGbind value was determined as described herein for single mutations (saturation
mutagenesis). This calculation provided both AAGbind values and structural models for these variants. The average AAGbind value for each sorting round was determined by multiplying the AAGbind value of each variant present in the sorting round by the number of times it appeared in the sorting round. The sum of these values was then divided by the total number of sequences obtained for each sorting round. The standard deviations of those values were calculated in a likewise manner. Variants containing a proline residue at position 35 were excluded, since they exhibited unusually high energy values, indicative of artifacts resulting from inaccuracies of our energy function or the need to account for backbone flexibility.
Protein Expression and Purification— The human MMP-14 catalytic domain gene (MMP-14CAT, residues 112-292) in a pET3a vector with a C-terminal 6xHis tag was expressed in B121 (DE3) E. coli bacteria and purified as previously described. The human MMP-9 catalytic domain (MMP-9CAT) lacking the fibronectin-like domain (residues 107-215, 391—
443) was purified as previously described (20), with the following modifications: the gene was expressed in B121 (DE3) pLysS bacteria cells in a pet28 vector (with an N-terminal 6xHis tag) and induced with 1 mM isopropyl β-d-l-thiogalactopyranoside (IPTG) overnight at 30 °C.
The MMP-2 catalytic domain (MMP2CAT) without the fibronectin-like domain
(residues 110-226, 395-446) was cloned into the pET3a vector with a C-terminal 6xHis tag and purified from the Rosetta E. coli strain. Induction was performed with 1 mM IPTG for 18 h at 18 °C. The cells were then harvested, and three rounds of sonication and centrifugation at 12000 g were performed. Thereafter, the protein was loaded onto a nickel column and eluted with 20 mM Tris, pH 7.5, 150 mM NaCl and 500 mM imidazole. The protein was then loaded on a MonoQ ion-exchange column (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and washed with low- salt buffer (50 mM Tris, pH7.5, 50 mM NaCl, 10% glycerol, 5 μΜ beta- mercaptoethanol and 0.1% EDTA). The protein was eluted using a gradient up to 1 M NaCl. The protein was then washed with 20 mM Tris, pH 7.5, and 150 mM NaCl buffer and purified on a gel filtration Superdex 200 followed by Superdex 75 column (GE Healthcare Life Sciences, USA) with 20 mM Tris, pH 7.5, and 150 mM NaCl buffer. The catalytic domains of MMP-1 and MMP-10 (MMP- ICAT and MMP-10CAT, respectively) were purified as previously described. Pro-MMP-2, Pro-MMP-9 and Pro-MMP-8 (residues 21-467) were purchased from R&D Systems (Minneapolis, MN, USA) and were activated before use according to the manufacturer's instructions.
The concentrations of MMP-14CAT, MMP-2CAT, MMP-9CAT, MMP- ICAT, MMP- IOCAT and MMP-8 were determined by UV-Vis absorbance at 280 nm, with extinction coefficients (828o) of 34500, 33920, 18934, 17928, and 79885 M"1 cm"1, respectively.
N-TIMP2WT and its variants were expressed and produced in the methylotrophic X33 P. pastoris yeast strain. The genes were first amplified with forward primers for N-TIMP2WT (5'- GGTATCTCTCGAGAAAAGATGC AGCTGCTCCCCG-3 ' ; SEQ ID NO: 17) and the N-
TIMP2A-D variants (5'-GGTATCTCTCGAGAAAAGATGCAGCTGCGCGCCG-3' (SEQ ID NO: 18); 5'-GGTATCTCTCGAGAAAAGATGCAGCTGCAGGCCG-3'(SEQ ID NO: 19); 5 ' -GGTATCTCTCGAGAAAAGATGCAGCTGC ATGCCG-3 ' (SEQ ID NO: 20); 5'- GGTATCTCTCGAGAAAAGATGC AGCTGCTCGCCG-3 ' (SEQ ID NO: 21); respectively), and the reverse primer (5 ' -GCTGGCGGCCGCCTCGC AGCCCATCTGGTA-3 ' (SEQ ID NO: 22)). The pPICZaA vector, encoding for the Zeocin resistance gene, containing the AOX1 promoter at its N-terminus and a 6xHis tag at the construct' s C-terminus, and the amplified N- TIMP2 insert clones were digested with Xhol and Notl restriction enzymes (New England Biolabs, MA, USA). Thereafter, the inserts and the vector were ligated and transformed into E. coli electro-competent cells. The transformed bacteria were plated on LB agar plates containing 50 μg/ml Zeocin (Invitrogen, Grand Island, NY, USA). The plasmid was extracted from several colonies, and the correct sequence was verified (Genetics Unit, NIBN, BGU, Israel). Thereafter, 100 μ of plasmids with the correct sequence were linearized by treatment with the Sacl restriction enzyme (New England Biolabs, MA, USA). The N-TIMP2 variants containing the plasmids were transformed into electro-competent X33 P. pastoris according to the pPICZa protocol (Invitrogen, CA, USA). The transformed yeast was grown on YPDS plates (2% peptone, 1% yeast extract, 2% d-glucose, 1 M sorbitol, 2% agar) for 72 h at 30°C. Thereafter, expression levels of several colonies from each N-TIMP2 variant transformation were determined; for this purpose, four colonies from each variant were taken from the plates and grown in 5 ml of BMGY medium [2% peptone, 1% yeast extract, 0.23% K2H(P04),
1.1812% KH2(P04), 1.34% yeast nitrogen base, 4x l0"5 % biotin, 1% glycerol]. After overnight growth at 30°C, the cultures were grown in inductive BMMY medium [2% peptone, 1% yeast extract, 0.23% K2H(P04), 1.1812% KH2 (P04), 1.34% yeast nitrogen base, 4x l0"5 % biotin, 0.5% methanol] for 72 h at 30°C, with the addition of 1% methanol each day. Overexpression of the secreted proteins was determined by western blot, using a 1:3000 dilution of mouse anti- 6xHis antibody (Abeam, Cambridge, UK) primary antibody, followed by a 1 :5000 dilution of anti-mouse secondary antibody conjugated to alkaline phosphatase (Jackson ImmunoResearch, West Grove, PA, USA), and detection by incubation in 2 ml of 5-bromo-4-chloro-3-indolyl phosphate reagent (Sigma- Aldrich, USA). Large-scale production of the proteins was performed by growth of the N-TIMP2-expressing yeast exhibiting the highest protein overexpression in 50 ml of BMGY medium overnight, followed by 72 h of growth in BMMY medium, with daily additions of 1% methanol. The proteins were purified by centrifugation of the yeast cell suspension at 3800g for 10 min and filtration of the supernatant, followed by addition of 500 mM NaCl and 10 mM imidazole in pH 8.0. The supernatant was incubated for 1 h at 4°C and then loaded on nickel -nitrilotriacetic acid-Sepharose beads (Invitrogen, USA), washed with 50 mM Tris, pH 7.5, 100 mM NaCl, and 10 mM imidazole, eluted with 20 ml of 50 mM Tris, pH7.5, 100 mM NaCl, 300 mM imidazole, and 5 mM CaCl2, and concentrated using a Vivaspin centrifugal concentrator with a 3-kDa cutoff (GE Healthcare Life Sciences, USA). The proteins were further purified using a Superdex 75 column with elution buffer (50 mM Tris, pH 7.5, 100 mM NaCl and 5 mM CaCl2) in an AKTA pure instrument (GE
Healthcare Life Sciences, USA). SDS-PAGE analysis on a 15% polyacrylamide gel under reducing conditions for the purified proteins was then performed. Bands were visualized by staining with Instant Blue (CBS Scientific, CA, USA). Protein samples were concentrated using a Vivaspin centrifugal concentrator with a 3-kDa cutoff (GE Healthcare Life Sciences, USA) and subjected to mass spectrometry analysis (Use Katz Institute for Nanoscale Science and Technology, BGU, Israel). Protein concentrations were determined by UV-Vis absorbance at 280 nm, using a NanoDrop Spectrophotometer (Thermo Scientific, USA), with an extinction coefficient (ε28ο) of 13,500 M^cm"1 for N-TIMP2WT and all its variants. The production yielded an average of about 1 mg protein for all variants.
MMP Inhibition Studies: N-TIMP2wT and its variants were tested for inhibitory activity against 0.0075 nM MMP-14CAT, 0.0075 nM MMP-9CAT, 6.25 nM MMP-2CAT, 0.2 nM MMP- ICAT, 0.2 nM MMP- 10cAT and 0.075 nM full-length MMP-8 enzymes. MMP-14CAT was incubated with 0.4-25 nM N-TIMP2WT and with 0.015-0.125 nM of the N-TIMP2 variants in TCNB buffer (50 mM Tris, pH7.5, 100 mM NaCl, 5 mM CaCl2, and 0.05% Brij) for 1 h at 37 °C. MMP-9CAT was incubated with 0.03-1.25 nM of N-TIMP2 inhibitors for 1 h at 37 °C in TCNB buffer. MMP-2CAT was incubated with 50-500 nM of the inhibitors in 50 mM Tris, pH 7.5, and lOmM CaCl2 for 1 h at 25°C. MMP- ICAT, MMP-10CAT and MMP-8 were incubated in TCNB buffer with inhibitor concentrations of 0.2-12.5 nM for 1 h at 37°C.
Thereafter, the fluorogenic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 TFA (SEQ ID NO: 23) [where Mca is (7-methoxycoumarin-4-yl)acetyl, Dpa is N-3-(2,4-dinitrophenyl)-L- 2,3-diaminopropionyl and TFA is trifluoroacetic acid] (Merck Millipore, CA, USA), at a final concentration of 7.5 μΜ for MMP- 14 or 12.5 μΜ for the other MMPs, was added to the reaction, and the fluorescence was monitored (with 340/30 excitation and 400/30 emission filters) using a Synergy 2 plate reader (BioTek, Winooski, VT, USA) at 37°C. Reactions were followed spectroscopically for 120 min, and initial rates were determined from the linear portion of the increase in fluorescence signal caused by the cleavage of the fluorescent substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 TFA ((SEQ ID NO: 23). Data were globally fitted by multiple regression to Morrison' s tight binding inhibition equation (84) (see Eq. 1) using Prism (GraphPad Software, San Diego, CA, USA). Ki was calculated by plotting the initial velocities against different concentrations of the inhibitors. Reported inhibition constants are average values obtained from three independent experiments. Calculations were performed using Km values of 1.31± 0.2 μΜ for MMP- 14CAT, 2±0.57 μΜ for MMP-9CAT, 14±0.7 μΜ for MMP-2CAT, 19±4.3 μΜ for MMP- ICAT, 13.8±1.9 μΜ MMP-10CAT and 38±7 μΜ for MMP-8, as determined from at least three Michaelis-Menten kinetic experiments in our laboratory.
VT _ 1 - ([£]+ [/] + K"*" ) - ^([E]+ [I]+ Kf ) 2 - [E][I]
t = (1) 2[E]
(Equation 1)
where Vi - enzyme velocity in the presence of inhibitor; Vo - enzyme velocity in the absence of inhibitor; E - enzyme concentration; I - inhibitor concentration; S - substrate concentration, Km - Michaelis-Menten constant; and Κ?νν - apparent inhibition constant, which
= jr. (i + I¾
K
is given by: m .
Binding Measurements between MMP-14CAT and N-TIMP2 Variants using SPR:
Binding affinity between the N-TIMP2 variants and MMP-14CAT was detected using surface plasmon resonance (SPR) spectroscopy on a ProteOn XPR36 system (Bio-Rad, CA, USA). An HTG chip (Bio-Rad, CA, USA) was loaded with 150 μ\ of 10 mM NiS04 (pH 6.3) at a rate of 30 μΐ/min for 5 min. Then, 150 μ\ of 0.033 μg/μl N-TIMP2 variants (tagged with His6) were immobilized at flow rate of 30 ΐ/min for 5 min via polyHis-Ni2+ interactions; an empty channel was used as control. MMP-14CAT was diluted in elution buffer to 2.5 nM, 5 nM, 10 nM and 20 nM and was circulated at a flow rate of 25 ΐ/min for 200 s, followed by 20 min of dissociation. The response was monitored as a function of time at 25°C. The rate binding constants, kon and k0ff, and the equilibrium affinity constant KD between N-TIMP2WT and MMP-14CAT were determined using the Langmuir model. Gelatinase Zymography Assay: The gelatinolytic inhibitory activity of the N-TIMP2 variants was tested in a gelatinase zymography assay, as previously described, with the following modifications. Inhibition of gelatinolytic activity of MMP-2 and MMP-9 full-length proteins was performed on 8% SDS-PAGE with embedded 1% gelatin. The proteins were first activated according to manufacturer's protocol and then resolved on SDS-PAGE at a concentration of 1 nM. Afterwards, the gels were rinsed for 1 h with gentle agitation in 2.5% Triton X-100 at room temperature and then incubated overnight with 100 nM of N-TIMP2 inhibitors in 50 mM Tris-HCl, pH 7.5, 10 mM CaCl2 and 200 mM NaCl at 37°C. After incubation, the gels were stained with Simply-Blue SafeStain (Thermo Fisher, USA), and the gelatinolytic activity was visualized as clear bands. The signal was quantified using ImageJ software.
Type I Collagen Degradation Assay: Inhibition of MMP-14CAT activity on type I collagen was performed as previously described, with the following modifications. Briefly, bovine type I collagen (1.5 mg) (Sigma- Aldrich, St. Louis, MO, USA) was incubated with 2.2 μ of MMP- 14CAT with or without 2.5 μΜ of each inhibitor in 100 μ\ of 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij 35, and 0.02% NaN3 for 24 h at 27 °C. Each reaction was loaded on 8% SDS-PAGE gel, and the bands were visualized by Instant Blue staining (CBS Scientific, CA, USA). The activity of MMP-14CAT was detected by the characteristic 3/4 fragments (TCA) of type I collagen, which represent the degradation products of al and a2 collagen type I chains.
Cell Culture: MDA-MB-231 human breast cancer cell line (kindly provided by Smadar Cohen, BGU, Israel) were maintained in RPMI-1640 (Roswell Park Memorial Institute) medium (Biological Industries Israel Beit-Haemek Ltd., Israel) supplemented with 10% fetal bovine serum (ThermoFisher, MA, USA), 1% 1-glutamine (Biological Industries Israel Beit- Haemek Ltd,. Israel) and 1% penicillin/streptomycin (Biological Industries Beit-Haemek Ltd, Israel). Murine fibroblast NIH-3T3 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in DMEM medium (Biological Industries Israel Beit-Haemek Ltd., Israel) supplemented with 10% fetal bovine serum (Thermo Fisher, MA, USA), 1% 1- glutamine (Biological Industries, Beit-Haemek Ltd., Israel) and 1% penicillin/streptomycin (Biological Industries, Beit-Haemek Ltd., Israel).
Cell Binding Assay— -TIMP2 proteins were labeled with DyLight-488 (Thermo Fisher, MA, USA) at a ratio of 1:5 (protein:dye) for 1 h at room temperature and washed three times with 50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, and 100 mM NaCl using Vivaspin centrifugal concentrators with a 3-kDa cutoff (GE Healthcare Life Sciences, USA). The inhibitory activity of the labeled N-TIMP2 inhibitors was determined as described above. Briefly, MMP-14cAT was incubated with the labeled and non-labeled inhibitors for 1 h at 37 °C. Thereafter, the cleavage of the fluorescent substrate Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala- Arg-NH2 (SEQ ID NO: 23) was measured. For the binding assays, various concentrations of N- TEV1P2 proteins (10 nM-1 μΜ) were added to 1 x 105 cells in volumes that were appropriate to prevent ligand depletion. Cells were incubated at room temperature for 2 h with gentle agitation, centrifuged at 150xg at 4 °C for 5 min, washed twice with 50 mM Tris-HCl pH 7.5, 5 mM CaCl2, 100 mM NaCl and 0.1% BSA, and analyzed by flow cytometry using an Accuri flow-cytometer (BD Biosciences, USA). Mean fluorescence values were measured using the Accuri C6 software (BD Biosciences, USA) and were plotted against the log of the
concentration of the inhibitors. Error is reported herein as the standard deviation of triplicate measurements.
Invasion Assay— The Boyden chamber chemoinvasion assay was performed as previously described with the following modifications. The experiment was performed with polycarbonate 8-μιη filters (Neuoro Probe, Inc, Gaithersburg, MD, USA) coated with Matrigel inside Transwell chambers (Neuoro Probe, Inc, Gaithersburg, MD, USA). The bottom part of each chamber was filled with conditioned medium containing DMEM medium collected from NIH-3T3 fibroblast cells after 24 h of culture and supplemented with 0.1% of BSA. The upper part of the chamber was filled with 200 μ\ of with MDA-MB-231 (1 χ 105). Then, 600 μ\, of DMEM medium without serum were added to the control chamber or with 7.5 nM of the inhibitors was added to the sample chambers. The cells were incubated for 4 h at 37 °C.
Thereafter, the invasive cells were fixed and stained using the Dipp Kwik Differential Stain Kit (American Mastertech Scientific, CA, USA). The cells that had migrated were counted using EVOS FL Cell Imaging System (Thermo Fisher, USA), at 40x magnification in 2.2 mm2 fields. The experiment was performed in triplicate, and 10 fields were counted for each sample.
Example 2: Combinatorial Library Design based on Computational Saturation
Mutagenesis Analysis
This example describes design of the N-TIMP2 library for screening of MMP-14 inhibitors.
To maximize the chances of selecting the most effective MMP-14 inhibitors, a focused library of N-TIMP2 mutants was designed that contained mutations at seven positions with the highest probability of mutations for enhancing affinity and specificity of N-TIMP2 towards the catalytic domain of MMP-14 (MMP-14CAT). The library was designed on the basis of previous results of an in silico saturation mutagenesis analysis of N-TIMP2 interacting with eight different MMPs and experimental confirmation for some single N-TIMP2 mutants (44). Based on this analysis, seven N-TIMP2 positions were selected for the current study to be randomized in the N-TIMP2 library, namely, positions 4, 35, 38, 68, 71, 97 and 99 of SEQ ID NO: 1 (and as shown in Fig. 1). All seven positions lie in the direct binding interface of N-TIMP2/MMP complexes, and six of them are coupled in pairs as a result of close proximity (no greater than 5.7 A) to one another (35 and 38, 68 and 71, 97 and 99 of SEQ ID NO: 1), suggesting that a mutation at one such position is likely to influence the effect of a mutation at another position that is paired with it. Among these chosen positions, positions 4, 35, 38, 68 and 99 of SEQ ID NO: 1 were included because they contained a large number of mutations with predicted improvement in the affinity of N-TIMP2 for MMP-14CAT. The other positions were chosen because they have high potential for improving binding specificity, i.e., for facilitating interactions that are mostly neutral for MMP-14 but destabilize complexes with other MMPs. Rather than focusing the library further by restricting the amino acid choices at each position, we allowed full randomization to twenty amino acids at all seven positions, thus producing a library with a theoretical diversity of 1.3 x 109 mutants, which is slightly larger than the limit for YSD. The N-TIMP2 library was then experimentally constructed as detailed in Example 1.
Example 3: Sorting of the N-TIMP2 Focused Library and Identification of N-TIMP2
Clones with Improved MMP-14CAT Affinity
This example describes screening of the N-TIMP2 library by yeast surface display (YSD).
A YSD platform was used to select N-TIMP2 variants that bind with high affinity to MMP-14CAT. For this purpose, the coding region of N-TIMP2WT was cloned into two different YSD plasmids, namely, pCTCON or pCHA, for presentation of the proteins on the
Saccharomyces cerevisiae yeast surface as a fusion with the Aga2p/Agalp system (see Figs. 2A-C). In the pCTCON plasmid, N-TIMP2 is placed between the Aga2 protein and the c-Myc epitope tag, whereas the pCHA plasmid allows the N-terminus of N-TIMP2 to be freely exposed. Expression of N-TIMP2WT in both pCHA and pCTCON was verified by using FACS. Binding of expressed pCHA/N-TIMP2WT but not of the pCTCON/N-TIMP2WT clone to the fluorescently labeled MMP-14CAT was then detected, demonstrating the critical role of a free N-terminus for N-TIMP2 binding to MMPs. We therefore continued all selection experiments using the pCHA construct with the N-TIMP2WT gene being substituted by the designed N- TEVIP2 library.
The designed N-TIMP2 library, termed SO, was subjected to an initial round of expression enrichment, based on c-Myc detection, to yield the S I library (Fig. 2C). Five subsequent rounds of affinity maturation were then performed with decreasing concentrations of MMP-14CAT (ranging from 1 μΜ in S2 to 5 nM in S6). In sorting rounds 2 to 6, diagonal sorting gates were used to select cell populations having high target affinity levels relative to protein expression levels, with about 1.5% of the library size being selected each time
(Fig. 2C). Such a selection protocol allowed us to dramatically decrease the bias of selecting variants because of their high expression level but possibly low binding level.
After each round of selection, 15-20 different N-TIMP2 clones were sequenced and analyzed with the aim to follow the progression of the selection (Fig. 3). Sequence analysis showed increased amino acid conservation as the selection progressed, indicating convergence upon a small set of high-affinity sequences. Computational modeling of the selected sequences in the context of N-TIMP2/MMP-14CAT structure confirmed that the average change in free energy of binding, AAGbind, did indeed decrease as the selection progressed (Fig 3, right of logos). Sequencing of 15 clones from the final round of selection (S6) led to the identification of four different N-TIMP2 variants, N-TIMP2A-D (Fig. 2E). Clone N-TIMP2B repeated 12 out of 15 times in S6, and N-TIMP2D repeated twice. N-TIMP2c included one unexpected mutation, Kl 16E, which is located far from the binding interface. It was further confirmed that each of the individual clones identified in S6 bound with higher affinity to MMP-14CAT than to N-TIMP2WT when expressed on the yeast surface (Fig. 2D).
Example 3: Characterization of N-TIMP2 Clones with Improved MMP-14CAT Affinity This example describes the characterization of the MMP-14 binding and inhibitory properties of N-TIMP2A-D.
To assess in vitro N-TIMP2 affinity to MMP-14 and other MMPs, soluble forms of N- TIMP2WT and the four N-TIMP2 variants were expressed and purified (Figs. 4A-C). The expression was performed in Pichia pastoris yeast, which facilitates the correct disulfide bond formation of the N-TIMP2 variants (Fernandez et al.) without the need for refolding, as in the case of expression from Escherichia coli (Williamson et al.). For expression, the pPICZaA vector that produces the protein with a free N-terminus and C-terminal His- and c-Myc epitope tags was used. The proteins were using affinity chromatography, followed by size-exclusion chromatography (Fig. 4A). Mass spectrometry analysis confirmed the correct mass for each of the purified variants (Fig. 4B), while SDS-PAGE analysis (Fig. 4C) confirmed their high expression level and purity.
To determine the binding affinity of each of the purified N-TIMP2 variants to MMP-14 and other MMPs, an enzyme activity essay was utilized. In such an assay, MMP-14CAT was incubated with increasing concentrations of N-TIMP2WT or an N-TIMP2 mutant, and the cleavage of the MMP-14 fluorogenic substrate was determined as a function of time. The slope of each reaction was calculated and fitted to Morrison's tight binding equation (Eq. 1) to determine the Ki value (Figs. 4D-E, Table 1). To determine binding specificity of our N-TIMP2 mutants, we also measured K values for five additional MMPs belonging to different MMP subgroups, namely, gelatinases (MMP-2CAT and MMP-9CAT), collagenases (MMP- ICAT and full length MMP-8), and stromelysin (MMP-10CAT).
N-TIMP2WT bound to MMP- 14 with a K of 780±80 pM (Table 1), a finding consistent with previous studies. All the selected N-TIMP2 variants showed picomolar affinities for MMP-14CAT, exhibiting ~500-fold to ~900-fold improvement in Ki compared to the K for N- TIMP2WT (Tables 1 and 2). This outstanding enhancement in binding affinity - by almost three orders of magnitude - which was due to only a few mutations, validates the potency of the described combined computational/directed evolution methodology.
TABLE 1
Inhibition constants (Kj) of MMP with N-TIMP2 variants
Figure imgf000041_0001
N-TIMP2WT 100±7 1400±400 1300±50 100±8 2260± 150 780±80
N-TIMP2A 10±2 1 159± 10 928±50 58±10 700±20 1.6±0. 1
N-TIMP2B 50±2 2730±400 200±3 20±5 350±40 1.0±0.1
N-TIMP2c 96±0.8 16550±3 336±30 68±3 32600±2755 1.5±0.4
N-TIMP2D 30±9 1300±140 510±300 13±4 42150±900 0.9±0. 1
* Values (pM) obtained from fitting the data shown on Fig. 4 to the Morrison tight binding equation.
It was observed that while TIMP2A-D showed greatly improved binding affinity toward MMP- 14 vis-a-vis N-TIMP2WT, affinity improvements toward MMP-9CAT, MMP-2CAT, MMP 1 CAT and full-length MMP-8 were relatively small, and for N-TIMP2C and N-TIMP2D, affinity was substantially weakened toward the physiological target MMP-10CAT. Thus, the mutations present in N-TIMP2c and especially N-TIMP2D resulted in enhancement of specificity toward MMP-14CAT over other MMPs by 2 to 4 orders of magnitude (Table 2). These results demonstrate the high potency and selectivity of all the tested variants towards MMP-14CAT, with N-TIMP2D showing the highest promise as a candidate for in-vivo applications targeting MMP- 14. TABLE 2
Inhibition specificity of N-TIMP2 variants
MMP- MMP- MMP- MMP- MMP-
Clone/MMP MMP-8
lcAT 2CAT 9CAT 10c AT 14CAT
Ki (fold)* 10 1.2 1.4 1.7 3.2 487
N-
TIMP2A Specificity** 48 406 348 286 152 1
Ki (fold) 2 0.5 6.5 5 6.5 780
N-
TIMP2B Specificity 390 1560 120 156 120 1
Ki (fold) 1 0.1 4 1.5 0.07 520
N-
TIMP2c Specificity 520 5200 130 346 7428 1
N- K (fold) 3 1 2.5 8 0.05 867
TIMP2D Specificity 260 867 340 113 16164 1 * Ki (fold) is calculated as the ratio between the K of N-TIMP2WT and K of N-TIMP2 variant. ** Specificity is calculated as the ratio between the fold of improvement towards MMP- 14 in comparison to other MMPs. [Ki (fold) for MMP-14CAT/ K (fold) for MMP-X.]
The direct binding constants (KD) for N-TIMP2 variants binding to MMP- 14CAT were measured using surface plasmon resonance (SPR). KD for N-TIMP2WT binding to MMP- 14CAT was determined to be 34.9 nM, while for the selected N-TIMP2 variants it was improved to ~1 nM for N-TIMP2A-c and to 0.6 nM for N-TIMP2D. The SPR sensorgrams (Figs. 5A-E) show that the improvement in KD for the selected N-TIMP2 variants was mostly achieved due to a slower dissociation rate than that for N-TIMP2WT (Table 3). Improvement in the dissociation rate rather than the association rate is expected for variants selected in YSD experiments, and the slow dissociation rate is indeed one of the most important characteristics of a drug
candidate molecule.
TABLE 3
Kinetic rates and binding affinity constants for N-TIMP2 variants measured by SPR
Clone kon t^ M- 1] k0ff [10-5 s-1] KD [nM]
N-TIMP2WT 1.41±0.02 49.1±0.1 34.9±0.6
N-TIMP2A 5.43±0.01 4.65±0.01 0.857±0.002
N-TIMP2B 6.14±0.01 6.65±0.01 1.08±0.002
N-TIMP2c 4.72±0.01 5.20±0.01 1.10±0.01
N-TIMP2D 6.20±0.01 3.74±0.01 0.603±0.001
Example 4: Inhibition of gelatin and collagen degradation by Ν-ΤΙΜΡ2 variants
This example describes the characterization inhibitory properties of N-TIMP2A-D towards MMP gelatinase and collagenase activities To test the inhibitory activity of N-TIMP2 variants on the gelatinolytic function of MMP-2 and MMP-9, a gelatin zymography assay was performed with pure full-length MMP-2 and MMP-9 proteins. In this assay, activated MMP-2 and MMP-9 were resolved on gelatin SDS-PAGE incubated with the N-TIMP2 inhibitors, and the gelatinase activity was visualized as clear bands on a dark background (Fig. 6A). Both N-TIMP2WT and the four tested N-TIMP2 variants showed a significant (40-80%) inhibition of MMP-2 and MMP-9 activity, as expected (Fig. 6B). The inhibitory activity shown by N-TIMP2WT and all the tested N-TIMP2 variants was similar, in agreement with no significant improvement in K values for MMP-9 and MMP- 2 of N-TIMP2WT relative to N-TIMP2 variants as shown by in vitro activity assays (Table 2). In addition, inhibition of MMP-9 was stronger than that for MMP-2 for all the proteins, again in agreement with the better K values of the N-TIMP2 variants for this enzyme.
To test the inhibitory activity of N-TIMP2 variants on the collagenolytic function of MMP-14, a type I collagen degradation assay was performed with MMP-14CAT. Bovine type I collagen was incubated with MMP-14CAT and N-TIMP2 variants. Samples were loaded on a SDS-PAGE gel, and the cleavage products were detected. All N-TIMP2 variants, but not N- TIMP2WT, showed a significant inhibition of MMP-14CAT (Fig. 6C), with type I collagen cleavage products (TCA fragment) being observed only for the untreated and N-TIMP2WT- treated samples (lanes 2 and 3), but not for the rest of the samples treated with the engineered N-TIMP2 variants (lanes 4-7). This result correlated with the binding affinity (K) values.
Example 5: N-TIMP2 variant binding and inhibition of cancer cells
This example describes the binding and inhibitory properties of N-TIMP2A-D towards MMP-14 expressing cancer cells.
To test whether the identified N-TIMP2 variant polypeptides could potentially inhibit cell invasion, binding was measured of N-TIMP2WT and the engineered N-TIMP2 variants to MDA-MB-231 breast cancer cells, which endogenously express MMP-14 (Fig. 7A). First, binding of the fluorescently labeled N-TIMP2 variants to the human breast cancer MDA-MB- 231 cell line was measured (Fig. 7A, right panel). Control experiments showed that MDA-MB- 231 cells endogenously expressed MMP-14 (Fig. 7A, left panel), and fluorescent labeling of N- TIMP2 variants did not alter the anti-MMP-14 activity of the inhibitors. The binding levels were similar for N-TIMP2WT and the engineered variants N-TIMP2A-D.
Previously described Matrigel invasion assays showed that MMP-14, MMP-9 and MMP-2 expression and catalytic activity are essential for the invasiveness of different types of cancer cells, such as prostate cancer (PC-3ML), human colon adenocarcinoma, human renal cell carcinoma, the breast cancer cell line MDA-MB-231, fibrosarcoma, and glioma. To test the effect of N-TIMP2 inhibitors on in-vitro invasion of breast cancer MDA-MB-231 cells, a Boyden chamber chemoinvasion assay was employed. In such assays, the cells were allowed to migrate through a Matrigel-coated membrane and were visualized by light microscopy (Fig. 7B); the number of invading cells was counted (Fig. 7C). Addition of the N-TIMP2WT, N- TEVIP2A, N-TIMP2b and N-TIMP2C variants resulted in about 40% inhibition of invasion (Fig. 7C). Significantly more effective inhibition of invasion was observed for N-TIMP2D, which inhibits invasion of about 70% of cells, thus making this variant the most promising candidate for drug design. Note that this candidate showed the highest inhibitory activity for both MMP- 14 and MMP-9 (but not for MMP-2) among all the tested variants.
Example 6: Discussion of N-TIMP2 variants
As described in Examples 2-5, we engineered four N-TIMP2 mutants with single-digit picomolar K values for binding to MMP-14, improving affinity towards this target by nearly 900-fold and improving binding specificity relative to other MMP family members by 100- to 16,000-fold. These impressive results demonstrate the enormous potential of the combined computational/directed evolution methodology for protein engineering. In the current study, we used computational methods to reduce the library size to ~108 most promising N-TIMP2 variants, a number that is tractable by the YSD technique. Our strategy allowed full randomization of seven N-TIMP2 positions most critical to MMP-14 binding affinity and binding specificity, thus greatly increasing our chances of selecting variants with superior affinity and specificity.
Our evolved N-TIMP2 mutants by far surpass all previous MMP inhibitors based on TEVIP and other scaffolds in terms of their binding affinities and binding specificity shift towards MMP-14CAT. Anti-MMP-14 antibodies previously developed by Udi et al. and by Devy et al. exhibited specific MMP-14 inhibition with nanomolar affinity. Another MMP-14 specific inhibitor, a short peptide (namely peptide G) developed by Suojanen et al. exhibited micromolar affinity towards MMP-14. Previously explored N-TIMP2-based MMP inhibitors usually resulted in 10"10 -10"9 M K toward the desired MMP compared to 10"12 K reported here.
Previously reported N-TIMP2-based designs mostly increased its binding specificity by reducing N-TIMP2's affinity to alternative MMPs while maintaining the affinity to the desired MMP the same or even slightly reduced. We on the contrary, were able to significantly enhance N-TIMP2's binding affinity toward MMP-14CAT while slightly increasing or decreasing its affinity toward other MMPs, thus making molecules that potentially are much more potent in vivo inhibitors of MMP-14 compared to the previously designed candidates. In addition, N-TIMP2 residues mutated in this study do not include the N-terminal residues 1-3 that have been used in most of the previous N-TIMP2 designs, proving that residues that are distant to the catalytic MMP site could be successfully used to modify N-TIMP2's binding specificity.
Our study demonstrates that introduction of a few mutations into the N-TIMP2 sequence is necessary and sufficient to convert this broad MMP inhibitor into a high-affinity and high- specificity inhibitor of MMP-14. While introduction of single mutations in various studies resulted in 10-100 specificity enhancement towards the desired MMP, introduction of 2-3 mutations in other studies enhanced specificity of N-TIMPs by several hundred fold, and introduction of five mutations in this study resulted in up to 16,000-fold specificity enhancement toward MMP-14 relative to other tested MMPs.
Structural modeling of the N-TIMP2 sequences that appeared after the 6th round of YSD selection in complex with MMP-14CAT (Figs. 8A-C) allows us to explain how each of the individual mutations affects binding affinity for MMP-14. At position 4, an Arg predominates in the selected sequences, while an Ala and a Met are also present. Wild-type Ser at this position has sub-optimal interactions with MMP-14 (Fig. 8A), and single mutations to Arg and Ala at this position were previously shown to result in a ~2- and 4-fold increase in N-TIMP2 affinity to MMP14, respectively. Here, the Arg is predicted to improve intermolecular interactions by introducing additional hydrogen bonds to Asn229 and Asn231 on MMP-14 (Fig. 8A). At position 35, various hydrophobic amino acids (Leu, Met, Pro) substitute for hydrophobic He; such mutations might provide better packing against MMP-14 and against Tyr36 on N-TIMP2. At position 38, an Asp predominates in the selection; this Asp is predicted to form an intramolecular salt bridge to Lys41 on N-TIMP2, thus stabilizing the interacting loop in the MMP-14-bound conformation. Another Asp predominates in the selection at position 68. This Asp packs tightly against Phe204 on MMP-14 and against Ile71 on N-
TIMP2; it is also expected to form a new intramolecular H-bond with the backbone of Ala 70 on N-TIMP2 (Fig. 8B), thus contributing to the stability of the N-TIMP2 loop. At position 71, a Val is substituted by an lie, improving packing against MMP-14 (Fig. 8B). Mutation H97R, predominating in all the selected sequences, replaces a small residue that does not contact MMP-14 (Fig. 8C) by a larger one, thereby introducing new hydrophobic interactions and intermolecular hydrogen bonds with Aspl93 and the backbone of Prol91 (Fig. 8C). This mutation has also been previously shown to increase N-TIMP2 affinity for MMP-14 by 14-fold and is also likely to increase its affinity to other MMPs. Finally, at position 99, either a wild- type Thr or a Ser was observed. The mutation to Ser at this position is predicted to lead to a new hydrogen bond with Asp 193 of MMP14 (Fig. 8C); despite this Asp residue being present in most MMPs at this position, this interaction may not be possible in other MMPs due to differences in backbone conformation. In summary, there appear to be two scenarios for improvement of binding to and specificity for MMP- 14, as have been observed in other studies on TIMP/MMP interactions. Some of the acquired mutations are predicted to facilitate favorable intermolecular interactions with MMP-14, while others are expected to stabilize the loops on N-TIMP2 in the MMP-14 bound conformations.
Some of the mutations appearing after affinity-maturation to MMP- 14 recapitulate evolutionary trends in TIMP homologues. For example, mutations to Asp at positions 38 and 68 are present in wild-type human TIMP-4 as well as in other mammalian homologues of TIMP-4, as observed in the HOVERGEN database and from a structural alignment with a model of TIMP-4. TIMP-4 is a tissue specific TIMP, whose expression is normally restricted to the heart, kidney, ovary, pancreas, colon, testes, brain and adipose tissue, and is also evident in breast cancer tumors. According to Greene et al., the highest expression levels of TIMP-4 are seen in the heart, and this may be biologically significant as cancer metastasis is rare in heart tissue. Zhao et al. reported a Ki value for TIMP-4 binding to MMP- 14CAT of 340 pM, compared to 70 pM for TIMP2, indicating that TIMP-4 is a strong inhibitor of MMPs upregulated in cancers, such as MMP- 14. Additionally, Ser at position 99 is observed in vertebrate homologues of TIMP- 1. It is known that TIMP- 1 exhibits very low affinity for MMP-14 and is not effective in inhibiting its activity (61); therefore, this may indicate that the T99S mutation that we obtained is not effective alone in increasing binding to MMP- 14 and may only be significant when coupled with neighboring mutations, such as at position 97 (namely H97R), as we obtained in this work.
Example 7: N-TIMP2/MMP14 and 9 interface-focused and whole-gene randomized mutagenesis
This example describes the design and assay of two N-TIMP2 libraries for binding to MMP14 and MMP9, a small focused library combining computationally predicted specificity enhancing mutations and a large library of randomly introduced mutations throughout the N- TEVIP2 gene.
A. Methods
Methods are as described in Example 1, except as specified herein. YSD Library Preparation Preparation of combinatorial library: A library of N-TIMP2 mutants was created using an assembly PCR method. The following degenerate codon containing primers were used to create mutant N-TIMP2 sequences.
Forward: 5'- TACCGTCGGTTCCGCTGCAGAAGGCTCTTTGGACAAGAGATGCAGCTGCBMRCCG SKNCACCCGCAACAGGCGTTTTGCAATGCAGATGTAGTGATCAGGGCCAAAGCGG TC AGTGAGAAGGAAGTGGACTCTGG-3 ' (SEQ ID NO: 28)
5'- GAGAAGGAAGTGGACTCTGGAAACGACRWATATGGCMANCCTATCAAGAGGATC CAGTATGAGATCAAGCAGATAAAGATGTTCAAAGGGCCTGAGAAGGATATAGAG- 3' (SEQ ID NO: 29)
Reverse: 5'- CTTTCCTGCAATGAGATATTCCTTCTTTCCTCCAACGTCCAGCGAGACCCCACARW YTGCCGASYWGGGGGCCGTGTAGATAAACTCTATATCCTTCTCAGGCCCTT -3 ' (SEQ ID NO: 30)
5'- CTCGCAGCCCATCTGGTACCTGTGGTTCAGGCTCTTCTTCTGGGTGGTGCTCAGGG TGTCCCAGGGCACGATGAAGTCACAGAGRKWGATRYGCATCTTGCCGTCCCCCTC GGCCTTTCCTGCAATGAGATATTCCTTC -3' (SEQ ID NO: 31)
The primers contain overlapping regions allowing for assembly of the entire N-TIMP2 gene. The resultant N-TIMP2 sequences were then amplified with the following primers:
Forward: 5'-TACCGTCGGTTCCGCTGC-3' (SEQ ID NO: 32)
Reverse: 5'-
GTCTTCTTCGGAGATAAGCTTTTGTTCTGCACGCGTGGATCCCTCGCAGCCCATCTG GTACC-3' (SEQ ID No: 14)
Preparation of a randomly mutated library: The N-TIMP2 gene located in the pCHA vector was subjected to error prone PCR as described by Chao et al. Briefly, the PCR reaction contains high concentrations of MgCl2 and non-standard dNTPs, Taq DNA
polymerase is used to maintain low fidelity and prevent repairs of mismatched nucleotides. The PCR included the following primers containing homology regions to the PCHA vector:
Forward: 5 ' -CCGGTTATTTCTACTACCGTCGGTTC-3 ' (SEQ ID NO: 15), Reverse: 5'-
GTC AGTTCCTGC AAGTCTTCTTCG-3 ' (SEQ ID NO: 16). These primers were also used to subsequently amplify the PCR products utilizing Phusion high fidelity DNA polymerase, guaranteeing blunt ends, free of adenine overhangs produced by Taq DNA polymerase.
The transformation of the libraries to yeast were performed by simultaneously transforming the amplified mutant N-TIMP2 fragments (from PCR) with a cleaved pCHA vector to an EBY100 Saccharomyces cerevisiae yeast strain using a MicroPulser electroporator (Bio-Rad, CA, USA), as previously described (Chao et al.). The primers used for amplification contain pCHA homology regions allowing fragments to be incorporated into the pCHA plasmid by homologous recombination in the yeast cells. The transformed yeast were grown in selective SDCAA medium (0.54% Na2HP04, 0.856% Na2HP04 »H20, 1.5% agar, 2% dextrose, 0.67% yeast nitrogen base, 0.5% Bacto casamino acids).
FACS Analysis and Sorting
Yeast (EBY100) clones or whole libraries were grown in selective and expression inducing SGCAA medium (2% galactose, 0.67% yeast nitrogen base, 0.5% Bacto casamino acids, 0.54% Na2HP04 »H20, 0.856% NaH2P04) at 30°C to a density of 10s cells/ml
(OD6oonm=10). The yeast cells (106 cells) were then collected and washed with a buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 5 mM CaCl2 and 1% bovine serum albumin. For detection of protein expression, yeast cells were incubated with mouse anti-c-Myc antibody (9E10) at a ratio of 1:50 at room temperature for 1 hour. Thereafter the cells were washed and incubated with sheep anti-mouse secondary antibody conjugated to phycoerythrin (PE) at a ratio of 1:50 for 30 minutes on ice. For the detection of binding to MMP-14cat, the cells were incubated with MMP-14cat labeled with DyLight-488 for 1 hour on ice. The yeast cells were then washed and analyzed using an Accuri C6 flow cytometer (BD Biosciences, CA, USA). An identical process was performed for detection of MMP-9cat binding, where MMP-9cat is labeled with DyLight-650.
For sorting, the same induction and labeling methods were used as for FACS analysis. At least 10 times the library diversity of cells were labeled. For initial sorting, for assessment of proper expression, only c-Myc labeling was used. As selective sorting progressed and matured library size decreased the minimum quantity of yeast cells used did not decrease to less than 106 cells. For selection of MMP specific clones, yeast cells were simultaneously incubated with varying concentrations (see below) of fluorescently labeled MMP-14cat and MMP-9cat. The affinity maturation sorting was performed on an iCyt Synergy FACS apparatus (Sony Biotechnology, CA, USA). For each sort, about 1% of the binding population was selected using a polygonal gate.
Yeast Library Sequencing
After each round of sorting, cells collected from the flow cytometry sorter are grown in SDCAA medium (0.54% Na2HP04, 0.856% Na2HP04 »H20, 1.5% agar, 2% dextrose, 0.67% yeast nitrogen base, 0.5% Bacto casamino acids) supplemented with chloramphenicol (25 μ§/ηι1) for -36 hrs. 10 μΐ of resulting culture is diluted in SDCAA to a volume final of 2 ml, 20 μΐ of this diluted solution are plated on SDCAA agar plates supplemented with chloramphenicol (25 μg/ml). Single colonies are picked and dissolved in 10 μΐ of 0.02 M NaOH. Cells are lysed by heating at 99°C for 10 min, for final rounds of sorting 1 μΐ of the NaOH dissolved cells is transferred to SDCAA (before boiling) supplemented with
chloramphenicol before lysis for further FACS analysis and cloning in expression hosts. 1 μΐ of the resulting lysed cells is added to a PCR mix solution and subjected to a PCR program of 45 cycles with annealing at 60°C and elongation at 68°C with Taq DNA polymerase using the following primers:
Forward primer = 5 ' - GGTA ATTA ATC AGCG A AGC G ATG- 3 ' (SEQ ID NO: 33)
Reverse primer = 5 ' -GCTGCCTTTGCTAGTTGTTGAG-3 ' (SEQ ID NO: 34)
N-TIMP2 Expression
Pichia pastoris Expression: WT N-TIMP2WT and its mutants were expressed and purified in the X33 Pichia pastoris yeast strain, as described in Example 1 with the following differences. The N-TIMP2 mutant genes were transferred from the pCHA plasmid obtained from yeast lysates (boiling in 0.02 M NaOH) to the pPICZaA P. pastoris expression vector by transfer PCR (Erijman et al) using the following forward primers:
Mutant 14-4: 5'-
GCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGATGCAGCTGCGAGCCGG-3' (SEQ ID NO: 35);
Mutant 14-6: 5'-
GCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGATGCAGCTGCGAGCCGC- 3'(SEQ ID NO: 36);
Mutants 9-2 and 9-6: 5'- GCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGATGC AGCTGCCCGCCG-3 ' (SEQ ID NO: 37);
Mutant ATE 5'-
GCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGATGC AGCTGCTCCCCGG-3 ' ; (SEQ ID NO: 38) and the following reverse primer for all mutants:
5'-GAGTTTTTGTTCTAGAAAGCTGGCGGCCGCCTCGCAGCCCATCTGGTACC-3' (SEQ ID NO: 39).
X33 cells were transformed with the WT N-TIMP2 gene cloned into the pPICZaA vector. PCR products were subsequently transformed into chemical competent DH5a E. coli. The transformed bacteria were plated on LB agar plates containing 25
Figure imgf000049_0001
Zeocin. After an overnight incubation at 37°C, three colonies were chosen for growth in liquid LB medium at 37°C for -16 hours, the plasmid was purified by miniprep, and the correct sequence was verified by Sanger sequencing using the following forward primer: 5'- GACTGGTTCC AATTGAC A-3 ' (SEQ ID NO: 40). Bacterial cultures possessing plasmids encoding for the correct sequence were grown for -16 hours at 37°C for large scale plasmid purification by maxiprep. The purified plasmid was then linearized by treatment with the Sacl restriction enzyme. The plasmids were transformed into electro-competent methylotrophic X33 P. pas tons cells according to the Invitrogen pPICZa protocol. The transformed yeast were grown on YPDS plates (1 % yeast extract, 2% peptone, 2% D- glucose, 1 M sorbitol, 2% agar) for 72 hours at 30°C. Thereafter, several colonies from each N-TIMP2 mutant transformation were tested for the presence of the N-TIMP2 gene in the yeast genome by PCR using the following primers:
Forward: 5 ' -GGTAATTAATC AGCGAAGCGATG-3 ' (SEQ ID NO: 33), Reverse: 5'- GCTGCCTTTGCTAGTTGTTGAG-3 ' (SEQ ID NO: 34). To this end, three to ten colonies were chosen from the plates for each mutant and grown in 200 μΐ of YPD (1 % yeast extract, 2% peptone, 2% D-glucose) containing 100 μg/m\ Zeocin for 48 hours at 30°C. Cell cultures were pelleted, pellets were pricked and subjected to colony PCR as described above (yeast sequencing) but for 30 cycles. Cultures exhibiting the best positive PCR result were subsequently grown in 50 ml BMGY medium (2% peptone, 1% yeast extract, 0.23%
K2H(P04), 1.1812% KH2(P04), 1.34% yeast nitrogen base, 4 l0"5 % biotin, 1% glycerol) until an Οϋόοο of 10 was reached (16-24 hours). After overnight growth, the cultures were grown in 500 ml inductive BMMY medium (2% peptone, 1% yeast extract, 0.23% K2H(P04), 1.1812% KH2 (P04), 1.34% yeast nitrogen base, 4 l0"5 % biotin, 0.5% methanol) for 72 hours at 30°C, with the addition of 1% methanol after 24 and 48 hours. The culture was centrifuged at 3800g for 10 min, the supernatant was collected, imidazole was added to a concentration of 10 mM and the pH raised to 8.0. The subsequent solution was incubated for 1 hour at 4 °C and then purified by nickel bead affinity purification and size exclusion chromatography as described below (see N-TIMP2 Purification). Protein concentrations were determined by UV-Vis absorbance at 280 nm, with an extinction coefficient (ε28ο) of 13,325 M^cm"1 for WT N- TEV1P2 and all its mutants. The production yielded an average of about 0.3-1 mg of protein per liter of culture for the WT N-TIMP2 and its mutants.
E. coli Expression Yeast colonies were grown and pCHA plasmid DNA extracted and purified by glass bead alkaline lysis. Subsequent purified plasmids were subjected to restriction free cloning as described previously by van den Ent and Lowe in order to transfer N-TIMP2 mutant genes from the pCHA plasmid to the pet21 E. coli expression vector which encodes a C- terminal 6xHis-tag. The following primers were used for the transfer:
Mutant 14-4: 5'-
CCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATTATGTGCAGC TGCGAGCCG-3' (SEQ ID NO: 41);
Mutant 14-6: 5'-
CCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATTATGTGCAGC TGCGAGCCGC-3'(SEQ ID NO: 42);
Mutant 9-2: 5'- CCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATTATGTGCAGC TGCCCGCCG-3'(SEQ ID NO: 43);
Mutant 9-6: 5'-
CCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATTATGTGCAGC TGCCCGCCG-3'(SEQ ID NO: 44);
Mutant ATE: 5'-
CCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATTATGTGCAGC
TGCTCCCCGG-3'(SEQ ID NO: 45);
and the following reverse primer for all mutants:
5'- GATCAAGCTTCGAATTCTTATTAATGATGATGATGATGATGATGCTCGCAGCCCAT CTGG-3' (SEQ ID NO: 46).
Once pet21 plasmids were obtained containing the appropriate N-TIMP2 mutant genes, the plasmids were transformed to BL21(DE3)pLysS E. coli cells. Transformed cells were diluted to 5 ml in LB supplemented with ampicillin (100 μ§/ιη1), chloramphenicol (25 μ§/ιη1) and grown for -16 hours at 37°C. 500 μΐ of the resulting culture was transferred to 25 ml of LB supplemented with ampicillin (100 μ§/ιη1), chloramphenicol (25 μ§/ιη1) and 500 μΐ of dextrose (10% solution) and grown for -16 hours at 37°C. 15 ml of this resultant culture was transferred to 750 ml of LB supplemented with ampicillin (100 μ§/ιη1), chloramphenicol (25 μ§/ιη1) and 25 ml of dextrose (10% solution) and allowed to grow at 30°C until an OD6oo of 0.8 was reached, at this point isopropyl-P-D-l-thiogalactopyranoside (IPTG) was added to attain a final concentration of 1 mM. The culture was allowed to overexpress the recombinant N-TIMP2 protein for a period of -16 hours at 30°C (Sharabi et al., 2014).
Cells were then precipitated at 22,000xg for 20 min at 4°C. Recombinant and unfolded N-TIMP2 is sequestered in inclusion bodies in the resulting bacterial cell pellet, the cell pellet is subsequently frozen at -20°C. Since the protein is sequestered to the inclusion it can be separated from remaining cell components. Pellets of 750 ml culture are unified and transferred to 25 ml of lysis buffer [50 mM NaPi buffer pH = 7.8, 0.5% Triton X-100 (octyl phenol ethoxylate)] . Pellets are lysed by sonication at 65% amplitude for 2 minutes with 5 second pulses. The resulting cell lysate is centrifuged at 18,700xg for 20 min at 4°C. The resulting supernatant is discarded and this process repeated another three times on the resulting pellet containing the unfolded protein sequestered to inclusion bodies. The final pellet is frozen at - 20°C. The frozen pellet is solubilized in 4 ml of solubilization buffer (50 mM Tris-HCl pH = 8.75, 6 M guanidine hydrochloride) and sonicated to attain a homogeneous solution. DTT is added to this solution to attain a final DTT concentration of 10 mM and incubated with rotation at room temperature for one hour. The resulting solution is centrifuged at 20,800xg for 20 min at 4°C. The supernatant is dripped at a rate of one drop per four seconds into 300 ml of refolding buffer (50 mM Tris-HCl pH = 8.75, 0.5 M guanidine hydrochloride, 1 mM reduced glutathione, 0.5 mM oxidized glutathione) to obtain no more than 50 μg of protein per ml with constant low rate stirring. Once dripping is complete the solution is allowed to stir for another 2 hours at room temperature and is then left at 4°C for -16 hours without stirring. The resulting solution is centrifuged at 15,000xg for 10 min and supernatant is purified by nickel bead affinity purification and size exclusion chromatography as described below (see N-TIMP2 Purification). The production yielded an average of about 0.3-0.7 mg of protein per liter of bacterial culture for the WT N-TIMP2 and its mutants.
N-TIMP2 Purification
1 ml slurry of GE Ni Sepharose 6 FF beads was mixed with the solution containing the folded N-TIMP2 protein. Beads were washed of non-specific binders using 50 mM Tris- HCl, 300 mM NaCl, pH 7.5, 10 mM imidazole, protein bound to the beads were eluted using 50 mM Tris-HCl, 300 mM NaCl, pH 7.5, 250 mM imidazole. Protein bound by the beads is eluted to a final volume of 3 ml. In order to isolate monomeric forms, the eluate is fractionated by FPLC size exclusion chromatography using a 120 ml GE Superdex 75 preparatory gel filtration column equilibrated with the following buffer: 50 mM Tris-HCl, 100 mM NaCl, 50 mM CaCl2, pH 7.5. The protein is isolated and concentrated from fractions corresponding to the last major peak corresponding to the monomeric form, and confirmed by SDS-PAGE.
Enzymatic Activity Assay of N-TIMP2:MMP Binding
Varying concentrations of N-TIMP2 are incubated with 0.4 nM MMP-9 or 0.8 nM MMP-14 for 1 hour at 37°C in 50 mM Tris-HCl, 0.15 M NaCl, 10 mM CaCl2, and 0.02% Brij 35 pH 7.5. After incubation, 50 μΐ of the fluorogenic MMP substrate (MCA-Pro-Leu-Gly-Leu- Dpa-Ala-Arg-NH2 TFA [where MCA is (7-methoxycoumarin-4-yl)acetyl; Dpa is N-3-(2,4- dinitrophenyl)-L-2,3 diaminopropionyl; and TFA is trifluoroacetic acid]) at 30 μΜ is added to 150 μΐ of the preincubated N-TIMP2:MMP solution. Fluorescence at 395 nm was measured, at least every ten seconds for at least 6 minutes, immediately after addition of fluoro genie substrate with irradiation at 325 nm on a Biotek Synergy HI plate reader (BioTek, VT, USA) preincubated to 37°C. Controls were also performed to determine uninhibited enzyme activity as well as basal fluorescence levels (without N-TIMP2 or MMP). At least three assays were performed for each N-TIMP2 protein. Kiapp Determination
Initial velocities of the enzymatic (MMP) cleavage of the fluorogenic substrate (described above) were derived from the fluorescence generated by this cleavage. Fraction of MMP activity is determined from the enzymatic assay, expressed as the inhibited velocity (Vi) divided by the uninhibited velocity (Vo), and is plotted against the corresponding concentration of N-TIMP2. From this plot the apparent Ki (Kiapp) value is solved for based on the following equation (Murphy, 2004) using MATLAB: ([MMP] + [T1MP] + K"pp)" ~ J([MMP] + [T1MP] + K' ')2 ~ 4 [MMP] [77 MP] (i)
/ =■ 2 [MMP]
f= fraction of activity, Vi/Vo
Kiapp values are an average of the Kiapp values determined from single inhibition assay experiments for each N-TIMP2 protein.
Specificity was determined according to the following formulas:
Figure imgf000053_0001
Figure imgf000053_0002
B. Library Design
Focused Library Design:
With the combination of prior knowledge from computational analysis which identified mutation tolerant positions in the N-TIMP2 interface and the use of the YSD method we were able to explore the relatively large number of possible combinations of mutations at these positions and find mutations which were only favorable when combined with nearby mutations which would have been overlooked in single mutation studies. For the focused library design, we included eight positions in the N-TIMP2 binding interface (Figure 9) where specificity- enhancing mutations towards MMP-14cat were previously observed (Sharabi, 2014). At each of these positions, we allowed either WT or a beneficial mutation. Additional amino acid choices were included in the library when no degenerate codon could be found to code for only the desired amino acids (Table 4). The use of additional amino acid choices allowed us to explore N-TIMP2 mutants, which we had not explored in the past, and to include extra mutants that might be beneficial for MMP-9 binding. Table 4 summarizes the locations at which mutation was allowed, the degenerate codons utilized to generate the library, and the mutations allowed at these positions. This library included 71,680 N-TIMP2 sequences, much less than the theoretical limit of 10s sequences that could be explored by YSD. The library was constructed using assembly PCR (see methods).
Table 4
N-TIMP2 Desired Amino Acid Degenerate Codon* Amino Acid Mutations
Position Mutations
4 A, Q, E, S BM R A, Q, E, P, S, stop
6 R, Y SKN R, G, L, V
35 E, 1, K RWA E, 1, K, V
38 N, Q. MAN N, Q, H, K
68 S, W, Y WRS R, N, C, K, S, W, Y, stop
71 N, y RWY N, D, 1, V
97 R, H CRY R, id
99 L Y WMY N, s, I, Y
Table 4: Design of the small focused N-TIMP2 library based on mutations tested
experimentally to identify high MMP-14 cat specificity N-TIMP2 binders. WT amino acid identities are underlined.
*A = adenosine, C = cytidine, G = guanosine, T = thymidine, B = C or G or T, K = G or T, M = A or C, N = A or C or G or T, R = A or G, S = C or G, W = A or T, Y = C or T
Random Library Design:
In addition to this rationally designed interface-focused library, we explored a purely random N-TIMP2 library, produced by error prone PCR, that incorporated mutations throughout the entire N-TIMP2 gene at a rate of 1-3 mutations per gene. Such a library was not focused on the N-TIMP2 binding interface and could contain mutations far from the binding site, whose effect we cannot predict computationally.
C. Selection by YSD The N-TIMP2 libraries were expressed on the surface of yeast cells with the use of the pCHA expression vector as described above. This vector encodes the Aga2p mating protein on the C-terminus, fused to a c-Myc tag which is fused to the N-TIMP2 protein located on the N- terminus of the construct. It is crucial for the N-terminus of N-TIMP2 to be free as this region of the protein mediates binding to the MMP active site and thereby allows inhibition of MMPs. The Aga2p protein binds by disulfide bonds to the Agalp protein which is encoded for by the yeast cells, thereby allowing the construct to be displayed on the yeast surface (Figure 10A). To allow for both positive and negative selection, the N-TIMP2 libraries were incubated simultaneously with two targets, MMP-14cat and MMP-9cat, labeled respectively with Dylight- 488 and Dylight-650 (Figure 10A). All sorts were performed using FACS (fluorescence activated cell sorting) (Figure 10B, C). To eliminate N-TIMP2 mutants that are not expressed (either due to deletions or due to frameshifts in the N-TIMP2 gene) we performed an initial pre-sorting round. The pre-sorting was done by targeting the c-Myc tag with an anti-c-Myc antibody and a fluorescently labeled secondary antibody against the anti-c-Myc (see Methods). The c-Myc tag is fused to the C-terminus of N-TIMP2 (Figure 10A) and therefore single codon frameshift mutations in N-TIMP2 will alter the amino acid sequence of the c-Myc tag peptide and abolish anti-c-Myc antibody binding and fluorescent labeling.
After this initial sorting, selective sorting was performed by incubating the library with 1 μΜ of MMP-14Cat and 25 nM of MMP-9cat (Figure IOC). A lower concentration of MMP-9cat compared to MMP-14cat was necessary since N-TIMP2 has a higher natural affinity for MMP-9 as opposed to MMP- 14 (see apparent Ki values below). Under these conditions a distributed binding pattern was observed, where cells bound only MMP-14cat, only MMP-9cat or both MMP-14cat and
MMP-9cat (Figure IOC).
In order to isolate yeast cells carrying N-TIMP2 mutants with high binding specificity to MMP-14cat, those cells which bound MMP-14cat with the highest affinity, 1% of all cells exhibiting binding to MMP-14cat alone (gate A in Figure IOC), were isolated for further selection with gradually increasing concentration of MMP-9cat and decreasing concentration of MMP-14cat from one round of selection to the next. The second selective sorting was therefore performed with 500 nM MMP-14cat and 50 nM MMP-9cat, whereas the third selective sort was performed with 200 nM MMP-14cat and 70 nM of MMP-9cat. In each sort, the 1% of cells exhibiting the highest binding signal to MMP- 14 was collected. After the third round of sorting, twenty-two colonies were randomly chosen for sequencing, only six colonies produced a reliable result containing the whole N-TIMP2 gene without frameshifts or nonsense mutations. The profile of the frequency of mutations at each mutated position of these six mutants is depicted in Figure 11 A, showing that at several positions we have arrived to a consensus. The most outstanding result is a 100% consensus to a mutation to asparagine from valine at position 71. Five out of six of the mutants contained mutations which were not planned and are located outside of the N-TIMP2:MMP binding interface.
In order to isolate yeast cells carrying N-TIMP2 mutants with high binding specificity to MMP-9, selections were performed with gradually increasing concentrations of MMP-14cat and decreasing concentration of MMP-9cat on cells isolated from the right gate B in Figure IOC, in a manner similar to the selection for MMP-14 specific mutants described above. The next selective sort was performed with 1 μΜ of MMP-14cat and 10 nM of MMP-9cat. The third and final selective sort was performed with 2 μΜ MMP-14cat and 5 nM MMP-9cat. The sequences of N-TIMP2 mutants from fifteen colonies (Figure 11B) show a substantial consensus at position 4 for proline as opposed to the WT serine as well as a mutation from valine to isoleucine at position 71. Seven out of fifteen of these mutants contain unplanned mutations outside of the N-TIMP2:MMP binding interface.
Selective sorting on the larger random library was performed in a similar manner to the smaller interface focused library, as described above. Initial sorting was performed with ΙμΜ MMP-14cat and 25 nM MMP-9cat. The MMP-14 binding population was isolated and subjected to further selective pressure. The second selective sort was performed with 250 nM MMP-14cat and 50 nM MMP-9cat, and the third selective sort was performed with 200 nM MMP-14cat and 70 nM MMP-9cat. Likewise, after the initial selective sorting the MMP-9 binding population was isolated and subjected to further selective pressure, where the second selective sort was performed at 1 μΜ MMP-14cat and 10 nM MMP-9cat, and the third selective sort was performed with 2 μΜ MMP-14cat and 5 nM MMP-9cat. From the final MMP-14 selective sort 24 colonies were sequenced (Figure 12), 19 of these colonies (79%) contained the same mutant, namely, V71 A I98T Kl 16E (herein 'ATE'). Position 71 and 98 are located in the N- TIMP2:MMP binding interface, whereas position 116 is not located in the interface but rather on the surface of N-TIMP2 antipodal to the interface.
D. Further Analysis of Selected N-TIMP2 Mutants
The resulting N-TIMP2 mutant sequence profiles resulting from the two selection experiments on the focused library show different patterns of mutation at most positions (Figure 11A and 1 IB). This difference is indicative of the success of the method to produce libraries matured to be specific for one MMP type. In order to avoid expressing all mutants sequenced from the resultant high selectivity libraries, we initially performed computational predictions of AAGbind for all sequenced mutants. The results of this analysis are summarized in Table 5 for mutants evolved for specificity in favor of MMP- 14.
Table 5
Mutants AAGbind AAGbind
Figure imgf000057_0001
(kcal/mol) (kcal/mol)
Mutant 14-1 1.02 -0.14
Mutant 14-2 -0.10 0.78
Mutant 14-3 -3.06 -2.80
Mutant 14-4 -1.58 0.37
Mutant 14-5 -2.17 -3.84
Mutant 14-6 -2.88 -1.00
AAGbinding of N-TIMP2 mutants to MMP-14cat or MMP-9cat as calculated by computational analysis. Negative AAGbinding values indicate improvement in binding and vice- versa.
From this analysis, mutant 14-4 was determined to be the most specific mutant as determined computationally by analyzing AAGbind of N-TEV1P2 in complex with MMP-14cat and MMP-9cat (Table 5). Our computational predictions show that this mutant exhibits the greatest difference between negative AAGbind for MMP-14cat binding and positive AAGbind for MMP-9cat binding. The next best mutant is mutant 14-6, however, it is predicted to improve binding to both MMPs but more so for MMP-14cat. We were not able to computationally analyze the MMP-9 specific mutants, since all MMP-9 specific mutants obtained from the final round of sorting contain a proline at position 4. This proline produces a steric clash causing artificially high AAGbind values requiring conformational changes of the backbone on N-TIMP2 or the MMP to relieve the clash. Our attempts to model such conformational changes did not produce reliable results. Further assessment was performed by FACS analysis, particularly to determine the specificity of MMP-9 specific mutants. Six mutants from each of the final libraries were individually assessed by FACS analysis for binding to MMP-14cat and MMP-9cat while displayed on the yeast surface (Figure 13). The specificity of each mutant was calculated by determining the ratio of the binding signal for the desired MMP to that of the undesired MMP and then dividing by the same ratio for WT N-TIMP2. From such an analysis three mutants stood out as best binders to MMP- 14 (mutants 14-2, -4, -6), although all mutants performed similarly (Figure 13A). Mutant 14-4 was chosen for purification since it exhibited the best specificity in FACS analysis experiments as well as computational predictions. Additionally, mutant 14-6 was chosen for purification since it repeated at a frequency of 13% in the library resulting from the second selective sort, whereas other mutants did not repeat. This mutant only appears once out of the six sequences obtained from the third selective sort. The mutants chosen for MMP-9 binding specificity are mutants 9-2 and 9-6. Mutant 9-2 performed the best in the FACS analysis (Figure 13B), whereas mutant 9-6 is repeated 3 out of 15 times among the N-TIMP2 clones selected for sequencing. Table 6 lists the mutations present in the N-TIMP2 mutants chosen for purification and characterization from the focused library. Note that mutant 14-6 contains an additional mutation outside of the binding interface that was not planned and was introduced spontaneously during the YSD selection process.
Table 6
Position: 4 6 35 38 68 71 97 99 Additional mutations
WT S V 1 N S V H T
14-4 E ~ ~ Q ~ N R s
14-6 E R V Q N N R ~ V18M
9-2 P ~ V ~ N 1 R ~
9-6 P G V — N 1 — N
Mutations in N-TIMP2 variants chosen to be assayed in vitro as purified proteins from the small interface focused N-TIMP2 library. "— " indicates that the WT identity at this particular position was maintained. Mutant 14-6 contains a mutation, V18M, which was not intended to be contained in the library. E. Mutant Characterization
The N-TIMP2 mutants chosen for further characterization were purified from Pichia pastoris yeast cells which secrete the properly folded N-TIMP2 proteins into the growth medium and have previously been shown to be effective in in vivo studies (Masuda et al. and Fernandez et al). All mutants were expressed in this system except for mutant 9-2 for which we could not see any expression in P. pastoris. Mutant 9-2 was therefore expressed in Escherichia coli and refolded in vitro from inclusion bodies using the procedure previously established in our lab (Sharabi, 2014). The WT N-TIMP2 was therefore expressed both in P. pastoris as well as in E. coli. It should be noted that bacterially expressed N-TIMP-1 is known to contain an acetylated N-terminus and monomeric fractions can contain up to 60% improperly folded protein (Van Doren et al), this is likely to apply to N-TIMP2 as well. The Kiapp of the inhibition of MMP-14cat and MMP-9cat by N-TIMP2 mutants was determined using an MMP enzyme activity assay in the absence and the presence of the N-TIMP2 inhibitor. In such an assay, the degradation of the fluorogenic MMP substrate is monitored and the initial velocity of the reaction is measured at varying concentrations of N-TIMP2 WT and mutants (see Methods). The reaction was fitted to equation 1 to determine Kiapp (Figures 14A and B). Table 7 summarizes the Kiapp values obtained for each N-TIMP2 mutant, for inhibition of MMP-14cat as well as MMP-9cat. All tested mutants showed a substantial increase in binding specificity towards the desired target MMP. The best mutant from the MMP-14 selection 14-6, exhibited a specificity shift of 59-fold. Interestingly, a very similar 57-fold specificity shift is exhibited by the mutant (ATE) obtained from the random library. The improvements in Kiapp of the mutants towards the desired MMP are modest, with the highest improvement of 8.8 fold observed by the ATE mutant. These relatively modest improvements are likely due to our selection protocol that incorporates two MMP targets at the same time. Mutant 14-6 exhibits the largest decrease in binding to MMP-9cat with a nearly 20-fold decrease in binding. The mutants selected for specific binding of MMP-9cat also show similar results of approximately a 45-fold increase in specificity of binding to MMP-9cat over MMP-14cat. The specificity increase here is achieved mostly through a great reduction in affinity for MMP-14 despite a slightly worse affinity for MMP-9 compared to WT N-TIMP2. In summary, we have obtained three N-TIMP2 mutants harboring 3-8 mutations that exhibit up to a 60-fold increase in binding specificity towards MMP-14cat and two mutants that exhibit greater than 40-fold increase in binding specificity towards MMP-9cat. The 9-6 mutant exhibits almost 900-fold better binding affinity towards MMP-9cat vs. MMP-14cat, which should exhibit a significant difference in cellular experiments.
Table 7
Kiapp of Kiapp of MMP- AAGbind to AAGbind to Specificity Specificity
MMP-14Cat 9Cat binding MMP-14Cat MMP-9Cat Shift to Shift to binding (nM) (nM) (kcal/mol) (kcal/mol) MMP-14Cat MMP-9Cat
WT 2.64±0.23 0.12±0.02 0.00 0.00 1 1
WTB 1.59±0.24 0.61±0.13 0.00 0.00 1 1
14-4 1.02±0.62 1.28±0.18 -0.59 1.45 27 -
14-6 0.89±0.06 2.43±0.90 -0.67 1.84 59 -
ATE 0.30±0.02 0.80±0.33 -1.34 1.15 57 -
9-2B 133.47±7.81 1.10±0.24 2.73 0.36 - 47
9-6 463.50±33.16 0.50±0.12 3.18 0.86 - 43
In vitro assessment of binding of purified proteins. Purified N-TIMP2 mutants were assayed for their ability to inhibit MMP-14cat and MMP-9cat. Kiapp values were obtained from at least three such assay experiments for each protein and averaged, each value is reported as Kiapp ± S.D.
BBacterially expressed protein.
AAGbind and specificity shifts were calculated from Kiapp values. AAGbind = - RTln(Kiappwt/Kiappmut), where T=310.15 Kelvin. Specificity shifts were rounded to the nearest whole number.
F. Discussion and Conclusions
Utilization of the YSD system allowed for the efficient combination of single mutations previously shown to improve specificity of binding of N-TIMP2 in favor of MMP- 14cat over MMP-9cat. These single mutation experiments showed that several of the positions mutated in this library, such as positions 4, 35, 38 and 68 are tolerant to mutations and contain a number of mutations that exhibit affinity enhancement towards MMP-14cat. The use of degenerate codons, particularly at these mutation-tolerant positions, allowed for the incorporation of additional mutations not explored in the past. Furthermore, such positions can be considered binding "cold spots", as we recently proposed, i.e. positions exhibiting suboptimal interactions where mutation is likely to improve binding affinity. These positions fit into this definition since three or more mutations (experimentally or computationally tested) at these positions exhibit AAGbind values of -0.3 kcal/mol or less for binding with MMP-14cat (Sharabi, 2014). Such cold spots are ideal positions to mutate when enhancing binding specificity of
multispecific proteins.
In comparison to single mutant N-TIMP2 variants, which exhibited a maximum 14-fold increase in binding affinity to MMP-14cat, the best mutants chosen for assessment from the present in vitro evolution experiments exhibited a modest and comparable affinity shift of 8.8- fold for MMP- 14cat. This relatively low affinity increase in the present work is a result of our selection method that uses two competing targets in the experiment and does not select for affinity but rather for specificity. Interestingly, none of the mutants found here by YSD exhibit increased affinity for both targets, as opposed to many of the specificity enhanced single mutants we had explored in the past. The mutants obtained in the present work are much more specific for MMP-14cat, compared to computationally designed single mutants. Our best multiple mutant (14-6) exhibits a ~60-fold specificity shift for MMP-14cat binding relative to MMP-9cat while previously explored single mutants produced specificity shifts of up to 8.6- fold in favor of MMP-14cat. In addition, in the present work, we obtained N-TIMP2 mutants with an ~50-fold specificity shift towards MMP-9cat relative to MMP-14cat that are ~900-fold more potent in MMP-9cat inhibition relative to MMP-14cat (Table 7). Such high differences in Kiapp guarantee high specificity of our inhibitors under physiological conditions.
In each round of selection we chose only those mutants which specifically bound one type of MMP, therefore mutants that increased affinity for both MMP types were not selected in this example, and in essence this feature is an element of negative design in the setup for this example. For instance, if selection were to be performed in the absence of MMP-9cat, and mutants which showed increased affinity for MMP-14cat were chosen in each selection round, we may have obtained mutants which bind MMP-14cat with high specificity because they have been affinity matured for MMP-14cat binding but may also exhibit higher affinity for MMP-9cat or other MMPs due to the high sequence and structural homology of the MMPs..
Since our computational method is only able to assess the effects of single mutations we may overlook mutations which require neighboring mutations to be specificity enhancing when using this method. This problem is avoided to some degree when preparing DNA libraries since multiple mutations can be engineered at certain neighboring positions by rational mutagenesis or by means of random mutagenesis throughout the gene and only those combinations of mutations that possess the desired effects are selected for. In this work, we compared the use of two libraries for MMP inhibitor engineering, one was a focused computationally designed library of -71,000 mutants with mutations only at certain binding interface positions on N-TIMP2, the second was a completely random library, containing 1-3 mutations throughout the N-TIMP2 gene. Interestingly, in the MMP- 14 selection we obtained N-TIMP2 mutants from both libraries with comparable ~60-fold increases in binding specificity. This specificity was achieved by only three mutations in the random library mutant ATE, two of them being in the binding interface, and by eight mutations (seven mutations being in the binding interface) in the mutant (14-6) from the focused library. On the other hand, in the MMP-9 selection, the random library performed worse than the focused library and the selection failed to converge after the three rounds of selection. This could be due to the fact that for error prone PCR, used for generation of our random library, there is a substantial likelihood that mutations will be synonymous or that some amino acid mutations will be very rare since they may require two or three nucleotide mutations in the same codon. Thus, when working with random libraries, success of obtaining a promising sequence is subject to chance to a greater extent, while focused libraries increase our chances of successful design since they are rationally designed and incorporate mutations or positions which are known to be beneficial a priori.
The methods used here have produced more superior results than single mutations have achieved in terms of specificity shifts, as well as previous experiments implementing display technology such as phage display to design N-TIMP2. In an example of experiments performed with phage display (Bahudhanapati et al), mutations were restricted to residues 1-6, 34-40 and 67-73 of N-TIMP2, similar to the areas mutated here, with the goal of obtaining MMP-lcat specific mutants, using MMP-3Cat as an anti-target in selection experiments. The affinities of WT N-TEVIP2 are in the same range as those for MMP- 14 and MMP-9 (Bahudhanapati et al). The mutants obtained from these experiments ultimately contained mutations only between positions 1-6 and exhibited specificity shifts of only 8.6-fold for MMP-lcat binding relative to competitor MMPs. The best mutant was further mutated with mutations up to position 9. The resulting mutants exhibited a maximal specificity shift of 77-fold for MMP-lcat binding relative to competitor MMPs. In contrast to our results, these mutants exhibited up to 60-fold reduced binding of MMP-lcat, whereas in our work we only witnessed no greater than a 6.7-fold reduction of affinity in the case of mutant 9-6. Analysis of the libraries after sorting was limited by the number of sequences which could be obtained from single colonies. Despite this, we believe that further clones were not necessary to be sequenced since the library size had decreased significantly by the third round of selection in the case of the interface focused library. Further sequencing may not have resulted in greater mutational diversity. Utilization of deep sequencing could potentially be a very powerful tool in determining mutational trends in future work. Such a method would be more useful for a randomized library such as the one utilized in this work, since mutations are not likely to be near one another and are therefore sterically and combinatorically independent. Due to the size of the N-TIMP2 gene (381 bp) it was not possible to apply deep sequencing methodologies to analyze whole libraries and provide a better statistical picture of mutational trends for the improvement of affinity and specificity as the accuracy of this method is limited to gene sizes of -300 bp.
Analysis of final yeast display libraries shows trends of consensus at certain positions that can encode specificity. For example, position 71 of N-TIMP2 is highly enriched for a mutation from valine to asparagine in the library evolved to bind MMP-14 with high specificity (Figure 11A). Whereas the library evolved for high binding specificity to MMP-9 is more enriched for a mutation to isoleucine at this position. This trend may indicate that position 71 of N-TIMP2 can act as a specificity switch, especially since the different selection experiments indicate that each MMP type prefers an amino acid that differs chemically at position 71, MMP-14 preferring a hydrophilic residue at this position and MMP-9 preferring a hydrophobic residue at this position. Another specificity switch was observed at position 4, where proline is strongly preferred for the MMP-9 selection (Figure 1 IB).
Several positions show distinct patterns that appear to be important for specificity for one MMP type. Structural analysis from computationally generated models (Figures 15A-M) shows that mutations at position 4 of N-TIMP2 should preferably be polar and large for designing MMP-14 specificity, whereas for MMP-9 specificity the residues should be hydrophobic and small. This is due to the local environments opposite position 4 in MMP-14 and MMP-9. Figure 15A-15D depicts the environment around position 4 and the effect of mutation. On MMP-14 there is a gap that could be filled (characteristic of a cold spot), increasing affinity. Hence, large polar residues are predicted to form favorable contacts with asparagine at position 231 on MMP-14. The residue on MMP-9 corresponding to asparagine 231 is a tyrosine; this large hydrophobic residue will form better contacts with small residues at position 4 on N-TIMP2. These trends are observed experimentally after selection (Figures 11A and 1 IB). Mutations after the final round of selection for MMP-14 binding are glutamate, wild- type serine, asparagine or lysine, while after the final round of selection for MMP-9 they are proline or wild-type serine. The interaction of glutamate at position 4 that appears in mutants 14-4 and 14-6 is depicted in figure 15B, and the interaction of proline at position 4 in mutants 9-2 and 9-6 appears in figure 15D.
Mutants 14-4 and 14-6 both contain glutamine at position 38 (Figure 15E-G). Position 38 is also crucial to binding specificity. For five out of six MMP-14 specific mutants, mutation to a glutamine is observed whereas MMP-9 favoring mutants mostly maintain the WT asparagine at this position. Glutamine and asparagine are chemically similar, however, glutamine is effectively longer than asparagine, and the local environment opposite this position on MMP-9 is hydrophobic. An asparagine at position 208 on MMP-14 is engaged in a hydrogen bond with both the WT asparagine as well as the mutant glutamine; this asparagine at position 208 is occupied by a glycine residue in MMP-9 at the corresponding position 197 (Figure 15F-15G). Since glutamate is longer than asparagine it is less tolerated by this hydrophobic environment and cannot form any favorable contact with MMP-9.
The immediate local environment on MMP-14 opposite position 71 is slightly more hydrophilic than the corresponding immediate local environment on MMP-9 (Figures 15H, J); a new hydrogen bond is formed between the asparagine residue at position 71 in mutants 14-4 and 14-6 and a neighboring tyrosine at position 203 on MMP-14 (Figure 151), the
corresponding position is occupied by a phenylalanine residue in MMP-9 (Figure 15J). The local environment on MMP-9 most likely prefers a hydrophobic residue at position 71 of N- TIMP2 (Figures 15J, K) and is less favorable for larger polar residues. Mutants 9-2 and 9-6 possess an isoleucine at position 71 which is effectively longer than the wild-type valine and therefore forms better contacts with MMP-9. The appearance of mutation at this position in the random library, namely V71A, further highlights the importance of this position as a specificity switch favoring MMP-14 binding despite being a mutation to a hydrophobic residue.
Computationally determined AAGbind for this mutant interacting with MMP-14 and MMP-9 is unfavorable, with respective values of 1.2 and 1.6 kcal/mol, exhibiting a slight preference for MMP-14 binding.
The mutation to arginine at position 97 seems to improve affinity to both MMP-14 and MMP-9 but more so for MMP-14, this is known from previous computational and
experimental results, this arginine residue may be more preferable for MMP-14 binding since it forms a hydrogen bond with the MMP-14 backbone at position 191 and a salt bridge with an aspartate at position 193 (figure 8M); this arginine only forms one hydrogen bond with the MMP-9 backbone at a nearby position different from that seen with binding to MMP-14 even though the amino-acid sequence in this region is the same. This may be due to slight differences in the backbone. Position 98 is occupied by an isoleucine residue in WT N-TJJV1P2, this residue faces the core of the protein. A mutation to threonine is seen here in the ATE mutant. Logically one would expect this mutant residue to become more solvent exposed as it is more hydrophilic than isoleucine, and this may indeed occur in reality, and would require backbone alteration to be accurately modeled computationally; our method is limited in this aspect and we could therefore perhaps not fully assess the potential effect of this mutation. This mutation is computationally shown to produce predicted AAGbind values near 0 kcal/mol for binding with both MMP-14Cat and MMP-9Cat.
The ATE mutant of the random library contains a mutation at position 116 from lysine to glutamate, a switch from a positive to a negatively charged residue. In N-TIMP2 this position is surrounded by positive charges. This change in charge may cause new local electrostatic interactions that may have greater long range electrostatic effects on the interface. References
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Zhao, H. et al. (2004) The Journal of biological chemistry 279, 8592-8601 In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. An isolated polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 8, 2, 4, 6, 24-27, or 47, or a fragment, derivative or analog thereof.
2. The isolated polypeptide of claim 1, further comprising a detectable label.
3. The isolated polypeptide of claim 1 or claim 2, wherein the polypeptide is conjugated to a solid substrate.
4. The isolated polypeptide of any one of claims 1-3, wherein the polypeptide is conjugated to a chemo therapeutic agent.
5. An isolated nucleic acid comprising a nucleic acid sequence encoding the isolated polypeptide of claim 1.
6. The isolated nucleic acid of claim 5, wherein the nucleic acid sequence is set forth as SEQ ID NO: 9, 3, 5, or 7.
7. An expression vector comprising the isolated nucleic acid of claim 5 or claim 6.
8. A composition for use in treatment of a disease or condition characterized by aberrant overexpression of matrix metalloproteinase (MMP) -14, MMP-9, or MMP-2, the composition comprising a therapeutically effective amount of the isolated polypeptide of claim 1.
9. The composition of claim 8, wherein the disease or condition is selected from the group consisting of a cancer, autoimmune disease, and cardiac disease.
10. The composition of claim 9, wherein the cancer is a carcinoma.
11. The composition of claim 9, wherein the autoimmune disease is inflammatory bowel disease.
12. The composition of any one of claims 8-11, wherein the isolated polypeptide is formulated for immediate or sustained release.
13. The composition of any one of claims 8-12, wherein the isolated polypeptide is formulated for local or systemic administration.
14. A method of detecting a cell that is overexpressing MMP-14, MMP-9, or MMP-2, comprising:
contacting a cell that is suspected of overexpressing MMP-14, MMP-9, or MMP-2 with the isolated polypeptide of any one of claims 1-3;
washing unbound polypeptide from the cell; and
detecting bound polypeptide.
15. The method of claim 14, wherein the cell is in a sample isolated from a subject.
16. A method for treatment of a disease or condition characterized by aberrant overexpression of matrix metalloproteinase (MMP) -14, MMP-9, or MMP-2, the method comprising administering to a subject in need thereof a therapeutically effective amount of the isolated polypeptide of claim 1, thereby treating the disease or condition.
17. A method for treatment of a disease or condition characterized by aberrant overexpression of matrix metalloproteinase (MMP)- 14, MMP-9, or MMP-2, the method comprising administering to a subject in need thereof a nucleic acid expressing the isolated polypeptide of claim 1, wherein expression of the nucleic acid provides a therapeutically effective amount of the isolated polypeptide to the subject.
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