Sustained release composition of matrix metalloproteinase inhibitors and uses thereof

Abstract

The present invention provides polypeptide conjugates comprising a sequence of matrix metalloproteinases (MMP) inhibitor and a plurality of heparin-binding peptide, biomaterial particles comprising complexes of said conjugates and alginate sulfate and allowing extended release of the MMP inhibitor and use of said biomaterial particles in treating diseases and conditions in which MMP inhibitors are implicated. Specifically, use of such biomaterial particles comprising MMP-9 inhibitor in preventing cardiac remodeling is provided.

Description

SUSTAINED RELEASE COMPOSITION OF MATRIX METALLOPROTEINASE INHIBITORS AND USES THEREOFFIELD OF THE INVENTION

[0001] The present invention relates to extended-release pharmaceutical compositions comprising complexes of alginate sulfate with conjugates comprising an inhibitor of matrix metalloproteinases, uses thereof as well to conjugates per se.BACKGROUND OF THE INVENTION

[0002] Numerous sustained (continuous) drug delivery (release) strategies are used in order to improve the retention time of a drug near its target molecule and within the diseased local site, resulting in enhanced therapeutic effect and reduced off-target toxic activities, thus diminishing the requirement for repeated administration of the drug, which may worsen the well-being of patients. Such systems are especially preferable if a local, long-term treatment is needed, if the target molecule has a delayed expression in the disease site, and if the access to the disease site (target organ) is restricted, such as drug administration directly to the heart muscle, as in case of a heart failure (HF).

[0003] Heart failure (HF), one of the leading causes of death worldwide, is commonly a result of myocardial infarction (MI), which is followed by extracellular matrix (ECM) degradation and long-term remodeling of the heart. Therefore, a possible therapeutic approach against HF would be reducing ECM degradation that is directly associated with a decline in cardiac function.

[0004] ECM degradation is mainly performed by the proteolytic activity of matrix metalloproteinases (MMPs), which are a family of about 24 zinc- and calcium-dependent endopeptidases that function in the ECM environment. MMPs proteolytic activity is regulated by their endogenous inhibitors, the tissue inhibitors of metalloproteinase (TIMPs). While MMPs are highly expressed by inflammatory cells post MI, the levels of TIMPs, expressed by cardiac fibroblasts, remain unchanged. Of note, studies show that an excessive, MMP-dependent, post MI ECM degradation, which is caused mostly due to an imbalance between the levels of MMPs and TIMPs, determines the severity of post MI remodeling. Specifically, it was found that MMP-9 plasma levels were >200% higher than control values one day post MI (reaching a peak level on day 3 post MI), whereas the levels of TIMP-2, the natural inhibitor of MMP-9 and other MMPs, remains unchanged, resulting in uncontrolled proteolytic activity of MMP-9, tissue remodeling and LV dysfunction (R. Mukherjee et al.,“Myocardial infarct expansion and matrix metalloproteinase inhibition,” Circulation, vol. 107, no. 4, pp. 618-625, 2003, doi: 10.1161 / 01.CIR.0000046449.36178.00).

[0005] Over the last years, many attempts to control MMPs activity post MI were made. Daily oral administration of MMP inhibitors has failed in clinical trial due to low target selectivity of the inhibitor and subsequent adverse side effects (M. P. Hudson et al., J. Am. Coll. Cardiol., vol. 48, no. 1, pp. 15-20, 2006; B. J. Dezube et al., J. Clin. Oncol., vol. 24, no. 9, pp. 1389-1394, 2006). Repeated subcutaneous injections of doxycycline antibiotic showed harmful effect on angiogenesis in vivo. To avoid the need of repeated administration, to prevent systemic effects of a drug, and to provide prolonged and local treatment, drug delivery to the heart using biomaterials is preferable (S. D. Anker et al., “A prospective comparison of alginate -hydrogel with standard medical therapy to determine impact on functional capacity and clinical outcomes in patients with advanced heart failure (AUGMENT-HF trial),” Eur. Heart J., vol. 36, no. 34, pp. 2297-2309, 2015). Nevertheless, while the use of cardiac patches, composed of biomaterials, involves invasive surgical implantation procedure, the use of injectable biomaterials like hydrogels or nanoparticles is favorable. In that respect, direct injection to the heart of drug delivery depot, like hydrogels containing MMPs inhibitors is currently studied (B. P. Purcell et al., “Delivery of a matrix metalloproteinase-responsive hydrogel releasing TIMP-3 after myocardial infarction: Effects on left ventricular remodeling,” Am. J. Physiol. - Hear. Circ. Physiol., vol. 315, no. 4, pp. H814-H825, 2018). The release mechanism in such systems is complex and is usually based on environmental, uncontrolled conditions such as body temperature, pH and enzymatic activity in the injected tissue, therefore the release of the drug is not predictable and less controlled. In addition, the released drug does not have a specific, single target and therefore may lead to unwanted side effect within the tissue itself.

[0006] In the past years, it has become clear that inhibitors with narrow or single MMP specificity hold much greater therapeutic potential. The complexity of the MMPs that exert both detrimental and beneficial roles, the high homology between the MMPs, and the difficulty in identifying specificity in binding and inhibition, impede the development of specific MMP inhibitors.

[0007] Such a high-affinity and high-selectivity, potent inhibitor for the MMP-9 catalytic domain (MMP-9CAT), namely C9, as described in G. Yosef, H. Hayun, and N. Papo, (“Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy,” Biochem. J., vol. 478, no. 5, pp. 1139-1157, 2021). However, administration of a small sized protein, such as C9, results in the proteins' short retentiontime in the tissue (E. Ruvinov, J. Leor, and S. Cohen Smadar, “The effects of controlled HGF delivery from an affinity -binding alginate biomaterial on angiogenesis and blood perfusion in a hindlimb ischemia model,” Biomaterials, vol. 31, no. 16, pp. 4573-4582, 2010, doi: 10.1016 / j.biomaterials.2010.02.026 and therefore its use as a therapeutic drug may be limited.

[0008] Alginate sulfate (AlgS) - a semi- synthetic, biocompatible and with multiple affinity binding sites containing biomaterial - serves as a building block for numerous controlled release systems such as alginate hydrogels or nanoparticles. To create these affinity binding sites, the uronic acid monomers of the alginate natural polysaccharide are sulfated. The rationale behind AlgS design is to mimic the natural interactions between heparin / heparan sulfate and various growth factors, cytokines, chemokines, or cell adhesion molecules, collectively known as heparin-binding proteins.

[0009] W02007043050 provides bioconjugates comprising a sulfated polysaccharide such as alginate sulfate and hyaluronan sulfate and at least one bioactive polypeptide capable of binding a sulfate group of said sulfated polysaccharide.

[0010] WO2017175229 discloses compositions comprising alginate, sulfated alginate, or hyaluronan sulfate for repairing or regenerating damaged tissue and in particular joint tissue.

[0011] Despite the long-term need, there are no efficient therapies allowing preventing development of long-term remodeling of the heart after myocardial infarction.SUMMARY OF THE INVENTION

[0012] According to one aspect, the present invention provides biomaterial particles comprising a complex of alginate sulfate and a conjugate of a matrix metalloproteinase (MMP) inhibitor with from 1 to 10 repetitions of a heparin-binding peptide (HBP). According to some examples, the biomaterial particles comprise a complex of alginate sulfate and a conjugate of a matrix metalloproteinase (MMP) inhibitor with from 3 to 10 repetitions of a heparin-binding peptide (HBP). According to some examples, the HBP consists of from 8 to 25 amino acids. According to some examples, the conjugate comprises from 3 to 8 or from 3 to 5 repetitions of the HBP. According to some examples, the MMP inhibitor and the HBPs are conjugated via an amid bond. According to some examples, the HBPs are conjugated to the C-terminus of the MMP inhibitor. According to some examples, the MMP inhibitor is an inhibitor of MMP9. According to some examples, the MMP inhibitor comprises an amino acid sequence selected from SEQ ID NO: 1 and 2. According to some examples, the heparin-binding peptide comprises an amino acid sequence selectedfrom SEQ ID NO: 3 and 4. According to some examples, the conjugate has an average hydrodynamic diameter of from 5.5 to 15 nm and / or an average radius of gyration of from 2.2 to 5 nm. According to some examples, the biomaterial particles have an average hydrodynamic diameter of from 1 to 15 pm. In some examples, the present invention provides biomaterial particles comprising a complex of alginate sulfate and a conjugate of an inhibitor of matrix metalloproteinase 9 with from 3 to 6 repetitions of a heparin -binding peptide (HBP), wherein the MMP inhibitor comprises the amino acid sequence SEQ ID NO: 1 and the HBPs each comprises the amino acid sequence GGGGSPPRRARVTY set forth as SEQ ID NO: 4. According to some embodiments of the invention, the conjugate comprises an amino acid sequence selected from SEQ ID NO: 7-10. According to some examples, the biomaterial particles are hydrogel microparticle. According to some examples, the biomaterial particles provide an extended-release of the MMP inhibitor. According to some examples, the biomaterial particles provide extended release of the MMP inhibitor for at least 30 hours.

[0013] According to yet another aspect, the present invention provides a pharmaceutically acceptable composition comprising biomaterial particles according as described herein, and pharmaceutically acceptable carriers and / or excipients. According to some examples, the pharmaceutically acceptable composition is formulated for oral or parenteral administration. According to some examples, the pharmaceutically acceptable composition is formulated for intracardiac injection. According to any one of the examples, the pharmaceutically acceptable composition is for use in treatment of a disease or condition associated with an increased activity of a matrix metalloproteinase. According to some examples, the matrix metalloproteinase is MMP9. According to some examples, the disease or condition is selected from cardiac remodeling, myocardial infarction, ischemic diseases, cancer, osteoporosis, wounds and muscular dystrophy. According to some examples, the condition is a cardiac remodeling. According to some examples, the treating comprises inhibiting or preventing cardiac remodeling caused by myocardial infarction (MI). According to some examples, the treating comprises inhibiting or preventing left ventricular remodeling. According to some examples, the present invention provides a pharmaceutical composition comprising a conjugate of the present invention comprising MMP-9 inhibitor conjugated to from 3 to 6 copies of HBP for use in treating and preventing left ventricular remodeling after myocardial infarction. According to some examples, the pharmaceutical composition is administered intracardially in proximity to the site of ischemia. According to someexamples, the pharmaceutically acceptable composition is administered within about 1 week after the MI.

[0014] According to yet another aspect, the present invention provides a method of preventing or inhibiting the development of cardiac remodeling after myocardial infarction in a subject in need thereof, the method comprises intracardially administering the biomaterial particles according as described in the application.

[0015] According to a further aspect, the present invention provides a polypeptide comprising an amino acid sequence of a matrix metalloproteinase (MMP) inhibitor and from 1 to 10 sequential repetitions of an amino acid sequence of a heparin-binding peptide (HBP) comprising an amino acid sequence selected from SEQ ID NO: 3 and 4. According to some examples, wherein the amino sequences of the HBP are located at the C terminus of the MMP inhibitor. According to some examples, the MMP is MMP9. According to some examples, the MMP9 inhibitor comprises an amino acid sequence selected from SEQ ID NO: 1 and 2. According to some examples, the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 5-10.

[0016] According to another aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide as described in any one of the embodiments and examples of the application. According to some examples, the nucleic acid molecule comprises from 1 to 10 sequential repetitions of the nucleic acid sequence selected from SEQ ID NO: 13 and 14, and a nucleic acid sequence selected from SEQ ID NO: 11 and 12 encoding an MMP inhibitor. According to some examples, the nucleic acid comprises a nucleic acid molecule selected from SEQ ID NO: 15-21.

[0017] According to another aspect, the present invention provides a nucleic acid construct comprising the nucleic acid molecule as described in the application, operably linked to a promoter.

[0018] According to a further aspect, the present invention provides a cell comprising the nucleic acid construct as described herein.BRIEF DESCRIPTION OF DRAWINGS

[0019] Figs. 1A-1E show in vitro inhibitory activity assays for N-TIMP2 variants. The reaction kinetics between MMP-9CAT and its Anorogenic substrate in the presence of different inhibitor (N-TIMP2 proteins) concentrations (ranging from 0.3125 nM to 1.5 nM) were measured and the slopes (cleavage velocities) at each inhibitor concentration were calculated. Curves were fitted by the Morrison tight binding equation (Equation 1 inMethods section) to obtain the inhibition coefficient (Ki) of N-TIMP2 and C9 proteins. Tight-binding curves of (Fig. 1A) N-TIMP2WT, (Fig. IB) C9, (Fig. 1C) C9-HBP1, (Fig. ID) C9-HBP2; (Fig. IE) C9-HBP3 are shown. Error bars represent standard deviation (STD); n = 3. The inhibition of the gelatinolytic activity of MMP-9 by N-TIMP2 and C9 proteins was measured by zymography. MMP-9FL was resolved on gelatin SDS-PAGE and treated with 100 nM of the following variants: N-TIMP2WT, C9, C9-HBP1 and C9-HBP3 (Fig. IF). The first band represents untreated MMP-9FL. Fig. 1G shows results of a dose dependent assay of C9-HBP3. Panels Fig. 1H and Fig. II represent the quantification of band intensity (in panels Fig. IF and Fig. 1G, respectively) normalized to the intensity of the control (untreated) gel. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test, comparing each treatment with the untreated control. * P < 0.05, *** P < 0.000 , n = 3.

[0020] Figs. 2A-2H show the affinity between C9 variants and AlgS or heparin. SPR measurements reveal that fusion of HBP repeats with C9 enhances its binding affinity to AlgS. Each curve in panels A-C represents a different protein concentration. (A), (B) and (C) represent binding curves of C9-HBP1, C9-HBP2 and C9-HBP3 to AlgS, respectively. (Figs. 2D, 2E) and 2F) represent equilibrium analysis of binding to AlgS, for C9-HBP1, C9- HBP2 and C9-HBP3, respectively. Fig. 2G and 2H shows binding curve of C9 to AlgS and heparin, respectively at different concentrations.

[0021] Figs. 2I-2N shows affinity curves of C9 variants and heparin. Each curve in panels 2I-2N represents a different protein concentration. (21), (2J) and (2K) represent binding curves of C9-HBP1, C9-HBP2 and C9-HBP3 to heparin, respectively. (2L), (2M) and (N) represent equilibrium analysis of binding to heparin, for C9-HBP1, C9-HBP2 and C9-HBP3, respectively.

[0022] Figs. 3A-3I and 3L show C9, C9-HBP1, C9-HBP3 proteins structure and bioconjugates of these proteins with AlgS. (A) DLS profiles (number psd) of the proteins. (B) SAXS profiles of C9, C9-HBP1 and C9-HBP3 (1 mg / ml [58.24 pM, 53.90 pM and 46.78 pM, respectively], Tris buffer, pH 7.5) (C) Kratky plots of C9, C9-HBP1 and C9-HBP3 (1 mg / ml [58.24 pM, 53.90 pM and 46.78 pM, respectively], Tris buffer, pH 7.5). Cryo-TEM images of (D) C9-HBP3, (E) AlgS, (F) a mixture of C9 and AlgS, (G) bioconjugates of C9- HBPl-AlgS, (H) bioconjugates of C9-HBP2-AlgS and (I) bioconjugates of C9-HBP3-AlgS. Fig. 3L shows enlarged scale of Fig. 3A

[0023] Figs. 3J and 3K shows the structure and thermal stability of C9 proteins. (A) SAXS profiles in Guinier plot of C9, C9-HBP1 and C9-HBP3. (B) SAXS profiles (raw data,without background subtraction) of C9-HBP3 at RT and 37 °C showing there is no change in the scattering profile.

[0024] Figs. 4 and 4B demonstrate AlgS particles sustained C9-HBP3 release in vivo. The fluorescence signal after injection to the hip muscle was detected in living mice over time by IVIS. Fig. 4A shows quantification of fluorescence signal over time (47 h) in living mice injected with C9-HBP3 protein or C9-HBP3-AlgS particles. Inset represents quantification of fluorescence signal in living mice during the first 5 h (enlarge view on 0-5 hours is shown in G). Fig. 4B shows a quantification of fluorescence signal in living mice over time, injected with a mixture of C9 and AlgS, C9-HBPl-AlgS particles or C9-HBP3-AlgS particles (enlarge view on 20-45 hours is shown in H). Fig. 4C shows images of Legs extracted from mice injected with a mixture of C9 and AlgS, C9-HBPl-AlgS particles and C9-HBP3-AlgS particles, 45 h after injection are shown. Upper left leg in each panel belongs to a negative control mouse. Fig. 4D shows quantification of fluorescence signal in mice legs. (Vilber, Lourmat, France). Fig. 4E shows hearts following injection of particles composed from AlgS and fluorescence labeled C9-HBP1 or C9-HBP3 in different time points (t=0, t=2 days and t=6 days) after injection. Fig. 4F shows a Quantification of the fluorescence intensity of C9- HBPl-AlgS and C9-HBP3-AlgS injected into mice hearts at different time points. The fluorescence signal after injection of C9-HBPl-AlgS and C9-HBP3-AlgS particles into the left ventricle myocardium was detected by NEWTON 7.0. Fig. 4G is a zoom in view of Fig. 4A (smaller time scale) and Fig 4H shows a zoom in view of Fig. 4B The fluorescence signal of the negative control organ [leg (A,B,D) or heart (F)] was subtracted from the signal of the injected mice. One-way ANOVA with Tukey's multiple comparison test was utilized for statistical analysis, bars represent mean ± SEM; ***p< 0.0001 ; n = 3 or 4.

[0025] Figs. 5A-5F shows that AlgS particles sustains C9-HBP3 release in vivo. The mice were divided into two groups (A and B). Each group contained two mice injected with either C9-HBP3 protein or C9-HBP3-AlgS particles. The fluorescence signal in mice was detected by IVIS (live imaging) over time. Figs. 5A and 5B represent measurements post injection to the hip muscle (time 0) of C9-HBP3 or C9-HBP3-AlgS, of groups A and B, respectively. Figs. 5C and 5D represent measurements 5 h post injection of groups A and B, respectively. Fig. 5E and 5F represent measurements 26 h and 47 h post injection of C9-HBP3-AlgS particles, respectively. The left mouse in each panel represents a negative control (injected with lOmM HEPES).

[0026] Figs. 6A-6F show fluorescence signal of C9-HBP3 protein injected into in a leg of living mice over time. The fluorescence signal was detected by IVIS® over time.Measurement of the fluorescently labeled C9-HBP3 protein right after injection (time 0) Fig. 6A, 30 min - Fig. 6B), 1 h - Fig. 6C, 1.5 h - Fig. 6D, 2.5 h - Fig. 6E and 21 h post injection (Fig. 6F). The left mouse in each panel represents a negative control.

[0027] Figs. 7A-7D shows organs extracted from mice after injection of C9-HBP3-AlgS particles into mice's legs. Fig. 7A shows organs extracted from a mouse 47 h post injection of C9-HBP3-AlgS particles. (Fig. 7B shows legs extracted from five different mice 47 h after injection with C9-HBP3-AlgS particles. The left leg belongs to the negative control mouse. Fig. 7C shows organs extracted from mouse 21 h post injection of C9-HBP3 protein. Fig. 7D shows legs extracted from a control mouse (left) and 21 h after injected with C9- HBP3 protein (right).

[0028] Fig. 8 shows In vitro sustained release of C9-HBP1 and C9-HBP3 from particles. The particles were centrifuged at t=0, t=2 days, t=4 days and t=6 days. The supernatant (unbounded protein) was removed and the precipitate (C9-HBPl-AlgS and C9-HBP3-AlgS particles) were collected. (A) WB analysis of the proteins (C9-HBP1 or C9-HBP3) that were bound to the particles at each time point. (B) Quantification of the bounded C9-HBP1 or C9- HBP3 at each time point, normalized to t=0 for each protein. A Two-way ANOVA with Sidak’s multiple comparison test was utilized for statistical analysis, bars represent mean ± SEM; * P < 0.05 ***P< 0.0001; n = 3.

[0029] Figs. 9A-9I show HBP repetitions prolong the release from AlgS particles in vivo. The fluorescence signal after injection to the hip muscle was detected in living mice over time by IVIS. Images of mice injected with (Fig. 9A) a mixture of C9 and AlgS, (Fig. 9B) C9-HBPl-AlgS particle and (Fig. 9C) C9-HBP3-AlgS particles. Images of mice 20 h after injection with (Fig. 9D) a mixture of C9 and AlgS, (Fig. 9E) C9-HBPl-AlgS particles and (Fig. 9F) C9-HBP3-AlgS particles. Images of mice 45 h after injection with (Fig. 9G) a mixture of C9 and AlgS, (Fig. 9H) C9-HBP-AlgS particle and (Fig. 91) C9-HBP3-AlgS particles. The left mouse in each panel represents a negative control, injected with 10 mM HEPES.

[0030] Figs. 10A-10C show organs extracted from mice 45 h post injection. Organs extracted from injected mice. The left leg in each panel is the non-injected leg, and the right leg is the injected leg. Fig. 10A shows organs of a mouse injected into a leg with a mixture of C9 and AlgS. Fig. 10B shows organs of a mouse injected into a leg with C9-HBPl-AlgS particles. Fig. 10C shows organs of a mouse injected into a leg with C9-HBP3-AlgS particles.

[0031] Figs. 11A-11J show the results of in vivo injection of C9-HBPl-AlgS and C9-HBP3- AlgS particles into the left ventricle myocardium. An experiment tested the protein clearance from the injected area. Hearts following injection of particles composed from AlgS and fluorescence labeled C9-HBP1 or C9-HBP3 at different time points (t=0, 5 h, 24 h, 2 days and 5 days) after injection. Pictures were taken using NEWTON 7.0 (Vilber, Lourmat, France). Figs. 11A, 11B, 11C, 11D and HE show the fluorescence from the labeled C9- HBP3-AlgS at t=0, 5h, 25h, 2days and 5 days, respectively. Figs. HF, 11G, 11H, HI and 11J show the fluorescence from the labeled C9-HBPl-AlgS at t=0, 5h, 25h, 2 days and 5 days, respectively.

[0032] Figs. 12A and 12B shows C9-HBP3 long term activity and stability. Fig. 12A shows tight-binding titration curves of C9-HBP3 at t=0 and after 4 days of incubation at 37 °C. The fluorescence signal of the substrate [Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2-TFA] cleavage by MMP9CAT was measured in different inhibitor concentrations (ranging from 0.3125 nM to 1.5 nM). The slopes (cleavage velocities) at each inhibitor concentration were measured, and the curves were fitted by the Morrison tight binding equation (Equation 1 in Methods section) to obtain the Ki values. Error bars represent STD; n = 3. Fig. 12B show SDS-PAGE analysis on 15% polyacrylamide gel under non-reducing conditions of C9- HBP3 at time t=0 and after 4 days incubation at 37 °C.

[0033] Figs. 13A-13F show the evaluation of therapeutic efficacy in mice MI model. Echocardiogram measurements of the average %EF on day 4 and day 28 after MI for each of the treated groups (n = 13 or 14 in each group) is shown in Fig. 13A. The change in %EF from day 4 to day 28 for each group(n = 13 or 14 in each group) is shown in Fig. 13B. Fig. 13C shows the change in %EF from day 4 to day 28, distinguishing between mice with initial %EF <30 and >30 (n = 5-9 for each group). Fig. 13D shows representative pictures of the histological analysis using Sirius red staining, for each of the treatments. Fig. 13E shows the percentage of fibrosis in the area remoted from the formed scar. Outliers were excluded using ROUT method, n = 12-14 in each group. Fig. 13F shows relative scar thickness [thickness of the scar / thickness of the septum]. Outliers were excluded using ROUT method, n = 8-12 in each group. Fig. 13G shows expansion index of the hearts. Outliers were excluded using ROUT method, n = 8-11 in each group. Statistical analyses were performed using: a two- way ANOVA with Sidak’s multiple comparison test in Fig. 13A, one-way ANOVA with Tukey's multiple comparison test in Figs. 13B, 13E, 13F and 13G, and a one-way ANOVA with Dunn’s multiple comparison test in Fig. 13C. Data represent mean ± SEM, *P< 0.05, **P< 0.01.DETAILED DESCRIPTION OF THE INVENTION

[0034] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In case of conflict, the patent specification, including definitions, will control.

[0035] In nature, an inhibitor of matrix metalloproteinases (MMP), and specifically an inhibitor of MMP9 does not comprise a heparin-binding peptide (HBP). In the present invention, HBP was appended to the MMP inhibitor in order to allow its complexation with alginate sulfate and subsequently to provide its extended-release. Unexpectedly, it was observed that only when an inhibitor matrix metalloproteinase (MMP) 9 was conjugated with more than 2 repetitions of a heparin-binding peptide (HBP) it could complex with alginate sulfate (AlgS) to form stable microparticles and provided a much longer extended release of the MMP9 inhibitor compared to a similar complex in which MMP9 inhibitor was conjugated with one HBP.

[0036] Since AlgS is not immunogenic and is not recognized by enzymes in the human body, the release of the entrapped proteins in those particles is slow, under control and depends solely on the equilibrium binding constant between AlgS and the protein. Administration of heparin-binding peptide (HBP)-containing MMPs entrapped within such particles can thus potentially reduce the dosage required to achieve a superior therapeutic efficacy and an improvement in clinical outcome due to the protein local as well as long-term effect. In addition, considering that HBP may interact with heparin sulfate is an important part of extracellular matrix, HBP further extends the retaining term the MMP conjugated to it in proximity to the tissue into which it is injected. Therefore, fusion of a multiple HBP repeating units to MMPs, a concept that so far has not been reported in the literature, can generate, by modulating the interaction between the protein and AlgS, a tunable release rates of the MMPs from the protein-AlgS particles, a release that is controlled by the equilibrium binding constant.

[0037] According to one aspect, the present invention provides a a particle comprising a complex of an alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor and from 1 to 10 repetitions of a heparin-binding peptide (HBP). According to some embodiments, the particle is a biomaterial particle. According to some embodiments, the present invention provides a biomaterial comprising a complex of an alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor and from 2 to 10 repetitions of a heparin-binding peptide (HBP). According to someembodiments, the present invention provides a biomaterial comprising a complex of an alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor with from 3 to 10 repetitions of a heparin-binding peptide (HBP). According to some embodiments, the biomaterial is in a form of particles. Thus, according to one aspect, the present invention provides a biomaterial particle comprising a complex of alginate sulfate and a conjugate of a matrix metalloproteinase (MMP) inhibitor with from 3 to 10 repetitions of a heparin-binding peptide (HBP). According to some embodiments, the present invention provides a biomaterial particle comprising a complex of alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor with from 2 to 10 repetitions of a heparin-binding peptide (HBP). According to some embodiments, the conjugate comprises a matrix metalloproteinase (MMP) inhibitor and from 3 to 10 repetitions of a heparin -binding peptide (HBP) conjugated thereto. Therefore, according to some embodiments, the present invention provides a biomaterial particle comprising a complex of an alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor and from 3 to 10 repetitions of a heparin-binding peptide (HBP). According to some embodiments, the present invention provides a plurality of particles (e.g. biomaterial particles) each of them as described above. Therefore, according to some embodiments, the present invention provides biomaterial particles each comprising a complex of alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor and from 2 to 10 repetitions or from 3 to 10 repetitions of a heparin-binding peptide (HBP). According to some embodiments, the present invention provides a plurality of biomaterial particles each comprising a complex of alginate sulfate and a conjugate of a matrix metalloproteinase (MMP) inhibitor with from 2 to 10 repetitions or from 3 to 10 repetitions of a heparin-binding peptide (HBP).

[0038] The term "conjugate" as used herein refers to the association MMP with one or more repeats of heparin-binding peptide wherein the MMP and HBP(s) are covalently bound.

[0039] The terms "biocompatible", "biomaterial" and "biocompatible material" are used herein interchangeably and refer to a composition, compound, material or device being compatible with living cells, tissue or body. While biocompatibility can be measured via any number of parameters, compositions that do not elicit an immune response (or only a minimal response) are consistent with being biocompatible. Similarly, compositions that are not toxic to an organism are also consistent with being biocompatible.

[0040] The term "repetitions" and "copies" may used herein interchangeably. The term "matrix metalloproteinases (MMPs)" also known as matrix metallopeptidases and matrixins refer to metalloproteinases that are calcium-dependent zinc-containing endopeptidases. TheMMPs are numbered from 1 to 28. Therefore, the present invention provides complexes of alginate sulfate and a conjugate comprising an inhibitor of a matrix metalloproteinase selected from MMP1-MMP28 and any combination thereof and from 3 to 10 repetitions of heparin-binding peptides. According to some embodiments, the inhibitor is an inhibitor of a collagenase MMP. According to some embodiments, the collagenase MMP is selected from MMP1, MMP2, MMP8, MMP9, and MMP13. According to some embodiments, the inhibitor is an inhibitor of a gelatinase MMP. According to some embodiments, the gelatinase MMP is selected from MMP9 and MMP2. According to some embodiments, the inhibitor is an inhibitor of MMP2. According to some embodiments, the inhibitor is an inhibitor of MMP9. According to some embodiments, the inhibitor is a polypeptide. According to some embodiments, the inhibitor is a protein. The terms "peptide" and “polypeptide” are used herein interchangeably and refer to a chain of amino acid residues linked by peptide bonds, i.e., a covalent bond formed between the carboxyl group of one amino acid and an amino group of an adjacent amino acid. The term “peptide” refers to short sequences having up to 50 amino acids. A chain of amino acids monomers longer than 50 amino acids is referred as a “polypeptide”. Such polypeptides, when having more than 50 amino acid residues, can also be classified as proteins, more particularly, proteins of low or medium molecular weight. The terms "peptide", "polypeptide" and "protein" encompass also the analogs of these peptides, polypeptide and protein. The term "analog”, “analog” and “sequence analog” are used herein interchangeably and refer to an analog of a peptide, polypeptide or protein having at least 85% sequence identity with the original peptide, wherein the analog retains the activity of the original peptide. Thus, the terms “analog” and “active analog” may be used interchangeably. The term “analog” refers to a peptide, polypeptide or protein which contains substitutions, rearrangements, deletions, additions and / or chemical modifications in the amino acid sequence of the parent peptide. The analog of the present invention is a functional analog. The terms “analog” and "functional analog" are used herein interchangeably refer to polypeptide, peptide or protein that maintains the properties and the functionality of the parent polypeptide, peptide or protein. According to some embodiments, the peptide analog has at least 89%, at least 90% or at least 95% sequence identity to the original peptide. According to one embodiment, the analog has about 85% to about 95%, about 90% to about 95% or about 85% to about 99% sequence identity to the original peptide. According to some embodiments, the term a peptide / polypeptide / conjugate comprising the amino acid sequence SEQ ID N0:X encompass also peptide / polypeptide / conjugate comprising an amino acid sequence havingat least 85% sequence identity to SEQ ID NO:X, and having the properties such as function and structure as the peptide / polypeptide / conjugate comprising the amino acid sequence SEQ ID NO: X. According to any one of the aspects and embodiments of the invention, the terms “peptide comprising the amino acid sequence set forth in SEQ ID NO: X”, “peptide comprising SEQ ID NO: X” and “peptide having SEQ ID NO: X” are used herein interchangeably. The terms “peptide consisting of the amino acid sequence set forth in SEQ ID NO: X”, “peptide consisting of SEQ ID NO: X” and “peptide of SEQ ID NO: X” are used herein interchangeably.

[0041] The same rule holds for nucleic acid sequence. Thus, the terms “nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: X”, “nucleic acid comprising SEQ ID NO: X” and “nucleic acid having SEQ ID NO: X” are used herein interchangeably. The terms “nucleic acid consisting of the nucleic acid sequence set forth in SEQ ID NO: X”, “nucleic acid consisting of SEQ ID NO: X” and “nucleic acid of SEQ ID NO: X” are used herein interchangeably.

[0042] According to some embodiments, the MMP inhibitor is a protein-based inhibitor. According to some embodiments, the MMP inhibitor is a polypeptide. According to some embodiments, the MMP inhibitor comprises a polypeptide consisting of from 10 to 250 amino acids. According to some embodiments, the MMP inhibitor has a molecular weight of from 10 kDa to 30 kDa.

[0043] According to some embodiments, the inhibitor is an inhibitor of MMP9. According to some embodiments, the inhibitor is an inhibitor of MMP-9 catalytic domain. According to some embodiments, the inhibitor of MMP-9 has the amino acid sequence SEQ ID NO: 1 denoted herein as C9. According to some embodiments, the inhibitor of MMP-9 consists of the amino acid sequence SEQ ID NO: 1. According to some embodiments, the inhibitor of the MMP9 comprises the amino acid sequence SEQ ID NO: 2 (herein denoted N-TIMP2WT). According to some embodiments, the inhibitor of MMP-9 consists of the amino acid sequence SEQ ID NO: 2. According to some embodiments, the inhibitor of MMP-9 is REGA-3G12 antibody or an MMP-9 binding region thereof or the scFv thereof as described in Paemen et al., Eur. J. Biochem. 1995, 234, 759-765. According to some embodiments, the inhibitor of MMP-9 is AB0041 or AB0046 monoclonal antibody or an MMP-9 binding region thereof or the scFv thereof as described in Marshall et al., PLoS ONE 2015, 10 or the humanized version thereof e.g. andecaliximab.

[0044] According to some embodiments, the conjugate comprises from 2 to 10 repetitions of the heparin-binding peptide (HBP). According to some embodiments, the conjugatecomprises from 3 to 10 repetitions of the heparin-binding peptide (HBP). According to some embodiments, the conjugate comprises from 2 to 8, from 2 to 6, from 2 to 5, from 3 to 6, from 3 to 5, or from 4 to 5 repetitions of the HBP. According to some embodiments, the conjugate comprises from 3 to 8 or from 3 to 7 repetitions of the HBP. According to some embodiments, the conjugate comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 repetitions of the HBP. According to some embodiments, the conjugate 3 repetitions of the HBP. According to some embodiments, the conjugate 4 repetitions of the HBP. According to some embodiments, the conjugate 5 repetitions of the HBP. According to some embodiments, the conjugate 6 repetitions of the HBP.

[0045] According to the teaching of the present invention, the repetitions of HBPs sequences are sequential, i.e., one sequence follows another. However, the HBP sequences may be separated by a spacer. According to some embodiments, the spaced is a peptide spacer, i.e. a stretch of amino acids. Typically, such spacer has not activity and is meant merely to reduce spatial hindrance or to provide some distance between sequences. Thus, according to some embodiments, the HBPs or some of them are separated from each other by a spacer. According to some embodiments, the HBP as placed in a line and separated from the MMP inhibitor by a spacer. According to some embodiments, the HBPs may be different or the same. According to some embodiments, all HBPs are identical. According to some embodiments, the plurality of the HBPs are branched, e.g. are not linearly connected.

[0046] In some alternative embodiments, the present invention provides biomaterial particle(s) comprising a complex of alginate sulfate and a conjugate comprising a matrix metalloproteinase MMP 1 inhibitor) and from 3 to 10 repetitions of a heparin-binding peptide (HBP), optionally wherein the HBP is separated from the MMP1 inhibitor by a spacer.

[0047] In some alternative embodiments, the present invention provides biomaterial particle(s) comprising a complex of alginate sulfate and a conjugate of an inhibitor of matrix metalloproteinase 2 (MMP2 inhibitor) and from 3 to 10 repetitions of a heparin -binding peptide (HBP), optionally wherein the HBP is separated from the MMP2 inhibitor by a spacer.

[0048] According to some embodiments, the HBP consists of from 8 to 25 amino acids. According to some embodiments, the HBP consists of from 10 to 20, from or from 12 to 16 amino acids.

[0049] According to some embodiments, the HBP comprises the amino acid sequence SPPRRARVTY set forth here as SEQ ID NO: 3. According to some embodiments, the HBPfurther comprises a linker / spacer. According to some embodiments, the linker comprises from 2 to 10 non-negative and non-bulky amino acids. According to some embodiments, the linker comprises from 2 to 10 amino acid selected from the group consisting of Gly, Ala, Vai, Leu, and Cys. According to some embodiments, the linker comprises from 2 to 10 or from 3 to 6 Gly. According to some embodiments, the HBP comprises the amino acid sequence GGGGSPPRRARVTY set forth here as SEQ ID NO: 4. According to some embodiments, the HBP consists of the amino acid sequence SEQ ID NO: 3. According to some embodiments, the HBP consists of the amino acid sequence SEQ ID NO: 4.

[0050] According to some embodiments, the MMP inhibitor and the HBPs are conjugated via an amide bond. According to some embodiments, the MMP inhibitor and the HBPs are conjugated via a spacer. According to some embodiments, the MMP inhibitor and the HBPs are conjugated via a peptide spacer. According to some embodiments, the MMP inhibitor and the HBPs constitute a single polypeptide. According to some embodiments, the conjugate of an MMP inhibitor and the HBPs is a fusion protein. The term “fusion protein” refers to a chimeric protein / polypeptide in which two or more separate protein sequences or a sequence of a protein and a polypeptide that are recombinantly expressed as a single moiety.

[0051] According to some embodiments, the plurality of HBPs is conjugated to the C- terminus of the MMP inhibitor. According to some embodiments, the plurality of HBPs is conjugated to the N-terminus of the MMP inhibitor. According to some embodiments, the plurality of HBPs is conjugated to the MMP inhibitor via a peptide spacer.

[0052] The term "alginate sulfate", synonymously used as "sulfated alginate" in the context of the present specification, refers to a polysaccharide consisting of P-D-mannuronic acid (M) and a-L-guluronic acid (G) organized in homo- or heteropolymeric block structures. Each monosaccharide unit contains 2 hydroxyl (-OH) groups available for sulfation. The alginate polymer can have a degree of substitution of sulfation (DSs) ranging from 0 to 2 per monomer (monosaccharide unit, either P-D-mannuronic acid or a-L-guluronic acid) or 0 to 4 per disaccharide unit (either P-D-mannuronic acid-a-L-guluronic acid, P-D-mannuronic acid- P-D-mannuronic acid or a-L-guluronic acid- a-L-guluronic acid), meaning between none and all of the available -OH groups are sulfated. The degree of sulfation dictates the physical and biological properties of the hydrogel that is composed of such a sulfated alginate polymer.

[0053] According to some embodiments, alginate sulphate has an average molecular weight of from 1 to 20 kDa or from 5 to 15 kD or from 9 to 12 kD or about 10 kDa. According tosome embodiments, the alginate sulphate comprises from 5 to 15 wt% of sulfur. According to another embodiments, the alginate sulphate comprises from of 7 to 12 or from 8 to 13 wt% of sulfur. According to some embodiments, alginate sulphate is prepared as described in Freeman et al., Biomaterials 29 (2008) 3260-3268. According to some embodiments, the alginate sulfate is biodegradable. According to some embodiments, the alginate sulfate is bioerodible.

[0054] As used herein the term "complex" is understood to mean a non-covalent interaction between two or more chemical entities. Therefore, according to some embodiments, the complex is not formed by covalent bonds of a conjugate and alginate sulfate. The non- covalent bonds comprise electrostatic interactions, hydrogen bonds Van der Waals forces, and hydrophobic interactions. Subsequently, according to any one of the embodiments of the application, the present invention provides a biomaterial particle comprising a non- covalent complex of an alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor and from 2 to 10 repetitions of a heparin -binding peptide (HBP). According to some embodiments, the complex is formed by electrostatic interactions of HBPs and alginate sulphate.

[0055] According to some embodiments, the complexes of the alginate sulfate and the conjugate of MMP inhibitor and HBPs form particles. According to some embodiments, the particles are semi- solid particles. According to some embodiments, the microparticles are hydrated.

[0056] According to some embodiments, the present invention provides a biomaterial particle comprising a complex of an alginate sulfate and a conjugate comprising a matrix metalloproteinase 9 inhibitor and from 2 to 10 repetitions of a heparin-binding peptide (HBP). According to some embodiments, the present invention provides biomaterial particles comprising a complex of an alginate sulfate and a conjugate comprising a matrix metalloproteinase 9 inhibitor and from 3 to 10 repetitions of a heparin-binding peptide (HBP). According to some embodiments the MMP9 inhibitor comprises the amino acid sequence SEQ ID NO: 1 and denoted as C9. According to some embodiments the MMP9 inhibitor comprises the amino acid sequence SEQ ID NO: 2. According to some embodiments, the heparin-binding peptide comprises the amino acid sequence SEQ ID NO: 3. According to some embodiments, the heparin-binding peptide comprises the amino acid sequence GGGGSPPRRARVTY set forth here as SEQ ID NO: 4. According to some embodiments, the conjugate comprises from 2 to 4 repetitions of such HPBs. According to some embodiments, the conjugate comprises from 3 to 6 repetitions of such HPBs.According to some embodiments, the conjugate comprises from 3 to 5 repetitions of such HPBs. According to some embodiments, the conjugate comprises 3 repetitions of the HPBs. According to some embodiments, the conjugate comprises 3 repetitions of the HPB each comprising or consisting of the amino acid sequence selected from SEQ ID NO: 4 and 3. According to some embodiments, the conjugate comprises 4 repetitions of the HPBs. According to some embodiments, the conjugate comprises 4 repetitions of the HPB each comprising or consisting of the amino acid sequence selected from SEQ ID NO: 4 and 3. According to some embodiments, the conjugate comprises 5 repetitions of the HPBs. According to some embodiments, the conjugate comprises 5 repetitions of the HPB each comprising or consisting of the amino acid sequence selected from SEQ ID NO: 4 and 3. According to some embodiments, the HBPs are conjugated to the C-terminus of the MMP9 inhibitor. According to some embodiments, the conjugate is a single polypeptide. According to some embodiments, the HBPs are spaced from the MMP inhibitor by a spacer. According to some embodiments, the conjugate comprises the amino acid sequence SEQ ID NO: 5. According to other embodiments, the conjugate comprises the amino acid sequence SEQ ID NO: 6. According to yet another embodiment, the conjugate comprises the amino acid sequence SEQ ID NO: 7. According to a further embodiment, the conjugate comprises the amino acid sequence SEQ ID NO: 8. According to one embodiment, the conjugate comprises the amino acid sequence SEQ ID NO: 9. According to another embodiment, the conjugate comprises the amino acid sequence SEQ ID NO: 10. According to some embodiments, the complex is a non-covalent complex.

[0057] As discussed above, the alginate sulfate and the conjugate of MMP inhibitor and HBPs form a complex. According to some embodiments, the complex is formed via electrostatic interactions of the conjugate and alginate sulfate. According to some embodiments, the complex is formed via electrostatic interactions of the HBPs and alginate sulfate. As discussed above, the present invention relates both to a single biomaterial particle as well as to a population of the biomaterial particles, i.e. to a plurality of the biomaterial particles according to any one of the above aspects and embodiments. According to some embodiments, in the population of the biomaterial particles, from about 5% to about 95% of the conjugates of the MMP inhibitor and HBPs are complexed with alginate sulfate. According to some embodiments, from about 10% to about 90%, from 15% to 85%, from 20% to 80%, from 25% to 70%, from 30% to 60% of the conjugates of the MMP inhibitor and HBPs are complexed with alginate sulfate. According to some embodiments, from about5% to about 30%, from 30% to 60%, from 60% to 90%, from 10% to 40%, or from 20% to 50% of the conjugates of the MMP inhibitor and HBPs are complexed with alginate sulfate.

[0058] The term "linker" is used here to define an entity such as a peptide or polypeptide that links or binds to other entities, e.g., alginate sulfate and MMP inhibitor. The term "spacer" refers to an entity such as a peptide or polypeptide that is placed between two entities and provides a spatial separation, e.g., to prevent spatial hindrance. According to some embodiments, the spacer comprises from 2 to 50 non-bulky amino acids such as Gly, Ala, Vai, and Leu. According to some embodiments, the spacer comprises from 2 to 6 Gly amino acids.

[0059] According to any one of the above embodiments, the fused proteins of the present invention (i.e. the conjugate of an inhibitor of MMP, such as MMP9, and repeats of HBPs) have an average hydrodynamic diameter of from 5.5 to 15 nm. According to some embodiments, the fused proteins have an average hydrodynamic diameter of from 6 to 12, from 6.5 to 11 or from 7 to 10 nm. According to some embodiments, the fused proteins have an average hydrodynamic diameter of from 7 to 12, from 8 to 12 or from 8 to 10 or about 8.6 nm. According to some embodiments, the conjugate comprising a MMP9 inhibitor and from 3 to 10 copies of HBPs have an average hydrodynamic diameter of from 5.5 to 15 nm or as described in the above embodiments.

[0060] According to any one of the above embodiments, the fused proteins of the present invention have an average radius of gyration of from 2.2 to 5 nm. According to some embodiments, the fused proteins have an average radius of gyration of from about 2.5 to about 3.5, from about 2.5 to about 3.2, from about 2.6 to about 3.2, from about 2.6 to about 3.2 or about 2.8. The hydrodynamic diameter and radius of gyration may be measured and calculated as described in Examples, e.g., using DLS and SAXS. According to some embodiments, the conjugate comprising a MMP9 inhibitor and from 3 to 10 copies of HBPs have an average radius of gyration of from 2.2 to 5 nm or as described in the above embodiments.

[0061] According to any one of the above embodiments, the biomaterial particles of the present invention have an average hydrodynamic diameter of from 1 to 15 pm. According to some embodiments, the biomaterial particles of the present invention have an average hydrodynamic diameter of from 1 to 12 pm, from 1 to 10 pm, from 2 to 10 pm, from 2 to 8 pm, from 3 to 6, from 4 to 6 or about 5 pm. According to some embodiments, the conjugate comprising a MMP9 inhibitor and from 3 to 10 copies of HBPs have an average hydrodynamic diameter of from 1 to 15 pm or as described in the above embodiments.

[0062] According to any one of the above embodiments, the biomaterial particles of the present invention provide an extended release of a MMP inhibitor. According to any one of the above embodiments, the biomaterial particles of the present invention provide an extended release of a conjugate of the MMP inhibitor with HBP repeats. The terms "extended release " and “sustained release” are used herein interchangeably and refer to a release of an active pharmaceutical agent over a prolonged or extended period of time, such as for example over a period of 8, 12, 16 or 24 hours. The active agent is consequently absorbed by the body over a (long) period of time. According to some embodiments, the biomaterial particles of the present invention provide extended release of the MMP inhibitor or the conjugate of the MMP inhibitor with HBP repeats for at least 30 hours. According to some embodiments, the biomaterial particles provide extended release of the MMP inhibitor for at least 35 or at least 40 hours. According to some embodiments, the MMP inhibitor or the conjugate of the MMP inhibitor with HBP repeats is as describe herein above in any one of the above embodiments. According to some embodiments, the present invention provides extended-release biomaterial particles each comprising a complex of an alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor and from 2 to 10 repetitions of a heparin-binding peptide (HBP).

[0063] The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)" are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated or listed. The term “consisting essentially of’ means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

[0064] According to some embodiments, the main components of the particles are the complex of an alginate sulfate and the conjugate. According to some embodiments, the complex of an alginate sulfate and the conjugate constitutes from 80% to 100wt%, from 85% to 99%, from 90% to 98%, from 90wt% to 100wt%, from 92wt% to 98wt%, from 95wt% to 100wt% of the particle. According to some embodiments, alginate sulphate is the sole (only) polysaccharide in the complex. According to some embodiments, alginate sulphate is thesole (only) polysaccharide in the particle. According to some embodiments, alginate sulphate is the sole (only) polysaccharide in the composition. According to some embodiments, the conjugate is the sole (only) polypeptide / protein in the complex. According to some embodiments, conjugate is the sole (only) polypeptide / protein in the particle. According to some embodiments, conjugate is the sole (only) polypeptide / protein in the composition. According to some embodiments, the conjugate is the sole (only) polypeptide and alginate sulphate is the only polysaccharide in the particle. According to some embodiments, the conjugate is the sole (only) polypeptide and alginate sulphate is the only polysaccharide in the complex. According to some embodiments, the conjugate is the sole (only) polypeptide and alginate sulphate is the only polysaccharide in the composition. According to any one of the embodiments of the present application, the particle is devoid of any other MMP inhibitors other than the stated one. According to any one of the embodiments of the present application, the complex is devoid of any other MMP inhibitors other than the stated one. According to any one of the embodiments of the present application, the particle is devoid of any other polysaccharides other than the stated one. According to any one of the embodiments of the present application, the complex is devoid of any other polysaccharide other than the stated one. According to any one of the embodiments of the present application, the particle is devoid of any other HBPs other than the stated one. According to any one of the embodiments of the present application, the complex is devoid of any other HBPs other than the stated one. According to some embodiments, the particle and / or the complex may further comprise pharmaceutically acceptable excipients such as salts, stabilizers, diluents, surfactants etc. According to any one of the According to some embodiments, the conjugate is as described in any one of the embodiments of the present application. According to some embodiments, the conjugate comprises an MMP9 inhibitor, e.g. C9 and 3 or 4 copies of HBPs as described above, e.g. comprising the amino acid sequence selected from SEQ ID NO: 3 and 4. According to some embodiments, the conjugate consists of an MMP9 inhibitor, e.g. C9, from 3 to 6 copies of HBPs as described above, e.g. comprising the amino acid sequence selected from SEQ ID NO: 3 and 4 and optionally spacers.

[0065] According to a further aspect, the present invention provides a composition comprising a plurality of biomaterial particles according to any one of the above aspects and embodiments, and a carrier. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

[0066] The term “carrier” as used herein refers to as a class any compound or composition useful in facilitating storage, stability, administration, cell targeting and / or delivery of the topical composition, including, without limitation, suitable vehicles, skin conditioning agents, skin protectants, diluents, emollients, solvents, excipients, pH modifiers, salts, colorants, rheology modifiers, thickeners, lubricants, humectants, antifoaming agents, erodeable polymers, hydrogels, surfactants, emulsifiers, emulsion stabilizers, adjuvants, surfactants, preservatives, chelating agents, fatty acids, mono-di- and tri-glycerides and derivates thereof, waxes, oils and water. According to some embodiments, the carrier is a pharmaceutically acceptable carrier and the composition is a pharmaceutical composition. Thus, according to another aspect, the present invention provides a pharmaceutically acceptable composition comprising the biomaterial particles of the present invention, and a pharmaceutically acceptable carriers and / or excipients. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well. Therefore, according to some embodiments, the present invention provides a pharmaceutically acceptable composition comprising biomaterial particles comprising a complex of an alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor and from 2 to 10 repetitions of a heparin-binding peptide (HBP). According to some embodiments, the present invention provides a pharmaceutically acceptable composition comprising biomaterial particles comprising a complex comprising an alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor and from 3 to 10 repetitions of a heparin-binding peptide (HBP). According to some embodiments, the present invention provides a pharmaceutically acceptable composition comprising biomaterial particles comprising a complex of alginate sulfate and a conjugate comprising a matrix metalloproteinase 9 inhibitor and from 2 to 10 or from 3 to 10 repetitions of a heparin- binding peptide (HBP). According to some embodiments the MMP9 inhibitor comprises the amino acid sequence SEQ ID NO: 1 and denoted as C9. According to some embodiments the MMP9 inhibitor comprises the amino acid sequence SEQ ID NO: 2. According to some embodiments, the heparin-binding peptide comprises the amino acid sequence GGGGSPPRRARVTY set forth here as SEQ ID NO: 4. According to some embodiments, the heparin-binding peptide comprises the amino acid sequence SEQ ID NO: 3. According to some embodiments, the conjugate comprises from 3 to 5 repetitions of the HPB. According to some embodiments, the conjugate comprises from 2 to 4 repetitions of the HPB. According to some embodiments, the conjugate comprises 3 repetitions of the HPB each comprising or consisting of the amino acid sequence selected from SEQ ID NO: 4 and3. According to some embodiments, the conjugate comprises 4 repetitions of the HPB each comprising or consisting of the amino acid sequence selected from SEQ ID NO: 4 and 3. According to some embodiments, the conjugate comprises 5 repetitions of the HPB each comprising or consisting of the amino acid sequence selected from SEQ ID NO: 4 and 3. According to some embodiments, the HBPs are conjugated to the C-terminus of the MMP9 inhibitor. According to some embodiments, the conjugate is a single polypeptide. According to other embodiments, the conjugate comprises the amino acid sequence selected from SEQ ID NO: 5-10. According to some embodiments, the conjugate comprises the amino acid sequence SEQ ID NO: 7. According to another embodiment, the conjugate comprises the amino acid sequence SEQ ID NO: 8. According to one embodiment, the conjugate comprises the amino acid sequence SEQ ID NO: 9. According to other embodiments, the conjugate comprises the amino acid sequence selected from SEQ ID NO: 7-10.

[0067] According to any one of the above embodiments, the conjugate may comprise a tag. According to some embodiments, the tag is a His. According to some embodiments, the tag, such as His tag is at the C-terminus of the conjugate. According to any one of the above embodiments, the conjugate comprises the amino acid sequence SEQ ID NO: 22 C- terminally to the HBP sequences. According to some embodiments, the conjugate comprises both the amino acid sequence SEQ ID NO: 22 and his tag C terminally to the HBP sequences.

[0068] The term “pharmaceutical composition” as used herein refers to a composition comprising at least one active agent as disclosed herein optionally formulated together with one or more pharmaceutically acceptable carriers.

[0069] Formulation of the pharmaceutical composition may be adjusted according to applications. In particular, the pharmaceutical composition may be formulated using a method known in the art so as to provide rapid, continuous or delayed release of the active ingredient after administration to mammals. For example, the formulation may be any one selected from among plasters, granules, lotions, liniments, lemonades, aromatic waters, powders, syrups, ophthalmic ointments, liquids and solutions, aerosols, extracts, elixirs, ointments, fluidextracts, emulsions, suspensions, decoctions, infusions, ophthalmic solutions, tablets, suppositories, injections, spirits, capsules, creams, troches, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0070] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents,enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may contain other active compounds providing supplemental, additional, or enhanced therapeutic functions, solid carriers or excipients such as, for example, lactose, starch or talcum or liquid carriers such as, for example, water, fatty oils or liquid paraffins.

[0071] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application typically include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol (or other synthetic solvents), antibacterial agents (e.g., benzyl alcohol, methyl parabens), antioxidants (e.g., ascorbic acid, sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetates, citrates, phosphates), and agents that adjust tonicity (e.g., sodium chloride, dextrose). The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, for example. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose glass or plastic vials.

[0072] Pharmaceutical compositions adapted for parenteral administration include, but are not limited to, aqueous and non-aqueous sterile injectable solutions or suspensions, which can contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Such compositions can also comprise water, alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets. Such compositions preferably comprise a therapeutically effective amount of a compound of the invention and / or other therapeutic agent(s), together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0073] The terms "pharmaceutically acceptable" and "pharmacologically acceptable" include molecular entities and compositions that do not produce an adverse, allergic, or other untoward reactions when administered to an animal, or human, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by a government drug regulatory agency, e.g., the United States Food and Drug Administration (FDA) Office of Biologies standards.

[0074] The composition of the present invention may be administered by any known method. The term "administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously,arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. According to some embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a day. According to other embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a month. In some embodiments, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and / or who provides a patient with a prescription for a drug is administering the drug to the patient.

[0075] According to some embodiments, the pharmaceutically acceptable composition of the present invention is formulated for oral administration. According to some embodiments, the pharmaceutically acceptable composition of the present invention is formulated for parenteral administration. The term “parenteral” refers to subcutaneous, intracutaneous, intravenous, intracardiac, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intraperitoneal and intracranial injection, as well as various infusion techniques.

[0076] According to some embodiments, the pharmaceutically acceptable composition of the present invention is formulated for intracardiac injection.

[0077] According to any one of the above embodiments, the pharmaceutical composition of the present invention provides an extended release of the MMP inhibitor. Therefore. The pharmaceutical composition of the present invention is an extended-release composition.

[0078] The terms "extended -release composition" and “sustained-release composition” are used herein interchangeably and refer to a property of a composition wherein the contained active medicament becomes bioavailable over an extended period of time after being administered.

[0079] According to some embodiments, the pharmaceutical composition comprising the particles as described in any one of the above embodiments and aspects provides extended release of the MMP inhibitor for at least 30 hours. According to some embodiments, the biomaterial particles provide extended release of the MMP for at least 35 or at least 40 hours.According to some embodiments, the biomaterial particles provide extended release of the MMP for from 1 to 8 days. According to some embodiments, the biomaterial particles provide extended release of the MMP for from 1 to 7 days. According to some embodiments, the biomaterial particles provide extended release of the MMP for from 1 to 6 days. According to some embodiments, the biomaterial particles provide extended release of the MMP for from 1 to 5 days. According to some embodiments, the biomaterial particles provide extended release of the MMP for from 30 to 72 hour. According to some embodiments, the biomaterial particles provide extended release of the MMP for from 36 to 72 hour. According to some embodiments, the biomaterial particles provide extended release of the MMP for from 30 to 60 hour.

[0080] According to any one of the above embodiments, the pharmaceutically acceptable composition of the present invention is for use in the treatment of a disease or condition associated with or mediated by increased activity of a matrix metalloproteinase. The term “treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, ameliorating abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating or alleviating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and / or (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).

[0081] According to some embodiments, the pharmaceutical composition is for use in treating diseases of conditions associated with increased activity of gelatinase MMPs. According to some embodiments, the pharmaceutical composition is for use in treating diseases or conditions associated with increased activity of an MMP selected from MMP2 and MMP9. According to some embodiments, the pharmaceutical composition is for use in treating a disease of condition associated with increased activity of MMP9. According to some embodiments, the disease or condition is selected from cardiac remodeling, myocardial infarction, ischemic diseases, cancer including metastasis generation, osteoporosis, wounds and muscular dystrophy, such as Duchene muscular dystrophy. According to someembodiments, the disease or condition is selected from autoimmune diseases, atherosclerosis, Alzheimer’s disease, COPD and tissue destruction. Non-limiting examples of a chronic inflammatory disease or an autoimmune disease are rheumatoid arthritis, multiple sclerosis, type-1 diabetes, type-2 diabetes, Crohn's disease, ulcerative colitis, and psoriasis. According to some embodiments, the condition is a cardiac remodeling. Therefore, according to some embodiments, the present invention provides the pharmaceutical composition of the present invention for use in treating cardiac remodeling. According to some embodiments, the use comprises inhibiting or preventing cardiac remodeling. According to some embodiments, the cardiac remodeling is caused by myocardial infarction. According to certain embodiments, the cardiac remodeling comprises left ventricular remodeling. According to some embodiments, the use comprises the treating such as inhibiting or preventing left ventricular remodeling after myocardial infarction.

[0082] According to some embodiments, the use comprises administering the pharmaceutical composition intracardially in proximity to the site of ischemia.

[0083] According to some embodiments, the pharmaceutically acceptable composition of the present invention is administered up to 2 weeks after the occurrence of the ischemia. According to some embodiments, the pharmaceutically acceptable composition of the present invention is administered as close as possible to the occurrence of the ischemia, e.g., within several minutes or several hours after ischemia. According to some embodiments, the pharmaceutically acceptable composition of the present invention is administered from 1 minute to 7 days after the occurrence of ischemia or MI. According to some embodiments, the pharmaceutically acceptable composition of the present invention is administered on day 1, day 2, day 3, day 4, day 5, day 6 or day 7 day after the occurrence of ischemia or MI.

[0084] According to some embodiments, the present invention provides a pharmaceutically acceptable composition comprising biomaterial particles comprising a complex comprising an alginate sulfate and a conjugate comprising a MMP9 inhibitor and from 3 to 10 repetitions of a HBP, for use in treating, inhibiting or preventing cardiac remodeling. According to some embodiments, the cardiac remodeling is caused by myocardial infarction. According to certain embodiments, the cardiac remodeling comprises left ventricular remodeling. According to some embodiments the MMP9 inhibitor comprises the amino acid sequence SEQ ID NO: 1 and denoted as C9. According to some embodiments the MMP9 inhibitor comprises the amino acid sequence SEQ ID NO: 2. According to some embodiments, the heparin-binding peptide comprises the amino acid sequence GGGGSPPRRARVTY set forth here as SEQ ID NO: 4. According to some embodiments, the heparin -binding peptidecomprises the amino acid sequence SEQ ID NO: 3. According to some embodiments, the conjugate comprises from 3 to 5 repetitions of the HPB. According to some embodiments, the conjugate comprises from 2 to 4 repetitions of the HPB. According to some embodiments, the conjugate comprises 3, 4 or 5 repetitions of the HPB each comprising or consisting of the amino acid sequence selected from SEQ ID NO: 4 and 3. According to some embodiments, the HBPs are conjugated to the C-terminus of the MMP9 inhibitor. According to some embodiments, the conjugate is a single polypeptide. According to some embodiments, the present invention provides a pharmaceutically acceptable composition comprising biomaterial particles comprising a complex comprising an alginate sulfate and a conjugate comprising an amino acid sequence selected from SEQ ID NO: 5-10, for use in treating, inhibiting or preventing cardiac remodeling. According to some embodiments, the conjugate comprises the amino acid sequence SEQ ID NO: 7. According to another embodiment, the conjugate comprises the amino acid sequence SEQ ID NO: 8. According to one embodiment, the conjugate comprises the amino acid sequence SEQ ID NO: 9. According to other embodiments, the conjugate comprises the amino acid sequence selected from SEQ ID NO: 7-10. According to some embodiments, the use comprises administering the pharmaceutical composition intracardially in proximity to the site of ischemia within 0- 14 days after the occurrence of ischemia or MI.

[0085] According to yet another aspect, the present invention provides a method of preventing or inhibiting the development of cardiac remodeling after myocardial infarction in a subject in need thereof, the method comprises intracardially administering the biomaterial particles of any one of the above aspects and embodiments or the pharmaceutical composition comprising the biomaterial particles to the subject.

[0086] According to yet another aspect, the present invention provides a polypeptide comprising an amino acid sequence of a matrix metalloproteinase (MMP) inhibitor and a from 1 to 10, from 2 to 10 or from 3 to 10 sequential repetitions of an amino acid sequence of a heparin-binding peptide (HBP) comprising the amino acid sequence SPPRRARVTY set forth here as SEQ ID NO: 3. Therefore, the polypeptide is a conjugate of an MMP inhibitor and repetitions of HBPs. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well. According to some embodiments, the polypeptide comprises from 2 to 10 or from 3 to 10 repetitions of the amino acid sequence GGGGSPPRRARVTY set forth here as SEQ ID NO: 4. According to some embodiments, the polypeptide comprises from 2 to 8, from 2 to 6, from 2 to 5, from 3 to 8, from 3 to 6, from 3 to 5, or from 4 to 5 repetitions of the HBP. According to some embodiments, thepolypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 repetitions of the amino acid sequence GGGGSPPRRARVTY (SEQ ID NO: 4). According to some embodiments, the polypeptide comprises 3 repetitions of the amino acid sequence GGGGSPPRRARVTY (SEQ ID NO: 4). According to some embodiments, the polypeptide comprises 4 repetitions of the amino acid sequence GGGGSPPRRARVTY (SEQ ID NO: 4). According to some embodiments, the polypeptide comprises 5 repetitions of the amino acid sequence GGGGSPPRRARVTY (SEQ ID NO: 4).

[0087] According to some embodiments, the HBPs are located at the C terminus of the MMP inhibitor. According to some embodiments, the HBPs are spaced from the MMP inhibitor by a spacer, e.g. peptide spacer. According to some embodiments, the HBPs are separated by a spacer, e.g. peptide spacer. According to some embodiments, the HBPs are located at the C terminus of the MMP inhibitor. According to some embodiments, the MMP inhibitor is an inhibitor of MMP9. According to some embodiments, the MMP9 inhibitor comprises the amino acid sequence SEQ ID NO: 1 denoted as C9. According to some embodiments, the MMP9 inhibitor comprises the amino acid sequence SEQ ID NO: 2. According to some embodiment, the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NO: 5-10. According to other embodiments, the conjugate comprises an amino acid sequence selected from SEQ ID NO: 7-10. According to other embodiments, the polypeptide comprises the amino acid sequence SEQ ID NO: 7. According to one embodiment, the polypeptide comprises the amino acid sequence SEQ ID NO: 8

[0088] According to another aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of the present invention. Specifically, according to some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence of a matrix metalloproteinase (MMP) inhibitor and a from 1 to 10, from 2 to 10 or from 3 to 10 sequential repetitions of an amino acid sequence of a heparin -binding peptide (HBP) selected from SEQ ID NO: 3 and 4. According to some embodiments, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a MMP inhibitor having an amino acid sequence selected from SEQ ID NO: 1 and 2. According to some embodiments, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NO: 5-10. According to some embodiments, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising or consisting of the amino acid sequence SEQ ID NO: 7.

[0089] According to some embodiments, the nucleic acid molecule comprises from 1 to 10, from 2 to 10 or from 3 to 10 sequential repetitions of the nucleic acid sequence SEQ ID NO:13. According to some embodiments, the nucleic acid molecule comprises from 1 to 10, from 2 to 10 or from 3 to 10 sequential repetitions of the nucleic acid sequence SEQ ID NO:14. According to some embodiments, the nucleic acid molecule comprises the nucleic acid sequence SEQ ID NO: 11. According to some embodiments, the nucleic acid molecule comprises the nucleic acid sequence SEQ ID NO: 12. According to some embodiments, the nucleic acid molecule comprises the nucleic acid sequence SEQ ID NO: 11 and from 2 to 5 repetitions for the nucleic acid sequence SEQ ID NO: 14. According to some embodiments, the nucleic acid molecule comprises the nucleic acid sequence selected from SEQ ID NO: 15-20. According to some embodiments, the nucleic acid molecule comprises the nucleic acid sequence SEQ ID NO: 18.

[0090] According to another aspect, the present invention provides a nucleic acid construct comprising the nucleic acid molecule of the present invention, operably linked to a promoter.

[0091] According to yet another aspect, the present invention provides a vector comprising the nucleic acid construct of the present invention.

[0092] The term “nucleic acid” refers to single stranded or double stranded sequence (polymer) of deoxyribonucleotides or ribonucleotides. In addition, the polynucleotide includes analogues of natural polynucleotides, unless specifically mentioned. According to an embodiment, the nucleic acid may be” selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), and analogues thereof, but is not limited thereto. The term encompasses DNA, RNA, single stranded or double stranded and chemical modifications thereof.

[0093] The terms “operably linked”, “operatively linked”, “operably encodes”, and “operably associated” are used herein interchangeably and refer to the functional linkage between a promoter and nucleic acid sequence, wherein the promoter initiates transcription of RNA corresponding to the DNA sequence. A heterologous DNA sequence is “operatively associated” with the promoter in a cell when RNA polymerase which binds the promoter sequence transcribes the coding sequence into mRNA which then in turn is translated into the protein encoded by the coding sequence.

[0094] The terms “vector” and “expression vector” are used herein interchangeably and refer to any viral or non-viral vector such as plasmid, virus, retrovirus, bacteriophage, cosmid, artificial chromosome (bacterial or yeast), phage, binary vector in double or single stranded linear or circular form, or nucleic acid, sequence which is able to transform host cells andoptionally capable of replicating in a host cell. The vector may be integrated into the cellular genome or may exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). The vector may contain an optional marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resistance. A cloning vector may or may not possess the features necessary for it to operate as an expression vector

[0095] According to yet another aspect, the present invention provides a cell comprising the nucleic acid molecule, construct or vector of the present invention. According to some embodiments, the cell is a procaryotic cell. According to some embodiments, the cell is a eukaryotic cell.

[0096] The terms “a,” “an,” and “the” ” are used herein interchangeably and mean one or more.

[0097] The term “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes, (A and B) and (A or B).

[0098] The term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination if the combination is not mutually exclusive.

[0099] The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)," are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated or listed. The term “consisting essentially of’ means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

[0100] As used herein, the term “about”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / -10%, or + / -5%, + / -!%, or even + / -0.1% from the specified value.

[0101] Sequence Table

[0102] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.EXAMPLES

[0103] Materials and Methods

[0104] Expression and purification of N-TIMP2 variants and MMP-9 catalytic domain (MMP-9CAT)

[0105] C9 (SEQ ID NO: 1) is a high affinity N-TIMP2 variant that was previously developed and purified in our laboratory (G. Yosef, H. Hayun, and N. Papo, Biochem. J., vol. 478, no. 5, pp. 1139-1157, 2021) and includes seven mutations in the N-TIMP2WT gene: S4M, I35M, N38S, S68G, V71I, H97R and T99S (Integrated DNA Technologies). N-TIMP2WT, C9 and three other proteins: C9-HBP1, C9-HBP2 and C9-HBP3, comprising C9 fused to 1, 2 and 3 repeats of heparin-binding peptide (HBP, GGGGSPPRRARVTY (SEQ ID NO: 4), respectively, were expressed and produced in the methylotrophic X33 P. pastoris yeast strain according to previously published protocol with modifications. In order to generate C9-HBP proteins, the C9 gene was first amplified using PCR with the forward primer ((SEQ ID NO: 23), and reverse primers for C9, C9-HBP1, C9-HBP2 and C9-HBP3 clones SEQ ID NO: 24- 27, respectively. The sequences of the resulted C9-HBP1, C9-HBP2 and C9-HBP3 are set forth is SEQ ID NO: 5-7, and may comprise also a his-tag for simplicity of their purification. For N-TIMPWT, primers were used according to Arkadash et al. (V. Arkadash et al., J. Biol. Chem., vol. 292, no. 8, pp. 3481-3495, 2017). The pPICZaA vector (Invitrogen, MA, USA), encoding for the Zeocin resistance gene, containing the A0X1 promoter and 6xHis tag respectively preceding and succeeding the N- and C-terminus of the C9-HBP genes to be inserted, and the amplified C9-HBP clones were digested with Xhol and Notl restriction enzymes (New England Biolabs, MA, USA). Thereafter, the digested C9-HBP clones andthe vector were ligated and transformed into E. coli electro-competent cells. The transformed bacteria were plated on LB agar plates containing 50 pg / ml Zeocin (Invitrogen, MA, USA). The plasmid was extracted from several randomly selected colonies, and the correct sequence was verified (Genetics Unit, NIBN, BGU, Israel). Thereafter, 100 pg of plasmids with the correct sequence were linearized using SacI restriction enzyme (New England Biolabs, MA, USA). The digested plasmids, each containing one of the clones (N-TIMP2WT, C9, C9-HBP1, C9-HBP2 or C9-HBP3) were transformed into electro-competent X33 Pichia pastoris according to the pPICZa protocol (Invitrogen, MA, 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 transformation were determined; for this purpose, four colonies from each protein were taken from the plates and grown in 5 ml of BMGY medium [2% peptone, 1% yeast extract, 0.23% K2H(PO4), 1.1812% KH2(PO4), 1.34% yeast nitrogen base, 4xl0'5% biotin, 1% glycerol]. After overnight growth at 30 °C, the cultures were grown in 5 ml inductive BMMY medium [2% peptone, 1% yeast extract, 0.23% K2H(PO4), 1.1812% KH2(PO4), 1.34% yeast nitrogen base, 4xl0'5% biotin, 0.5% methanol] for 72 h at 30 °C, with the addition of 1% methanol each day. Cells were centrifuged and the supernatant was analyzed by western blot (WB) to determine the expression of the secreted proteins. Protein detection was performed using a 1:3000 dilution of mouse anti-6xHis primary antibody (Abeam, Cambridge, UK) 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, MO, USA). Large-scale production of the proteins was performed by growth of the N-TIMP2WT, C9 and C9-HBP- expressing yeast exhibiting the highest protein expression in 50 ml of BMGY medium overnight, followed by 72 h of growth in inductive BMMY medium, with daily additions of 1% methanol. On the second and third days of induction, ImM of Phenylmethylsulfonyl fluoride (PMSF) (Roche, Basel, Switzerland) and one tablet of cOmplete™ ULTRA Tablets, EDTA-free, Protease Inhibitor Cocktail (Roche, Basel, Switzerland) were added to the growth medium (500 ml). The proteins were purified by centrifugation of the yeast cell suspension at 4000 g for 30 min and filtration of the supernatant, followed by addition of 300 mM NaCl, 10 mM imidazole and 1% glycerol and titration with NaOH to pH 8.0. The supernatant was incubated for 1 h at 4 °C, followed by 10 min centrifugation 4000 g and filtration. The filtered solution was then loaded on HisTrap™ Fast Flow nickel column (Cytiva, Marlborough, US), washed with two column volumes of 50 mM Tris, pH 7.5, 1 MNaCl, followed by a wash with 50 mM Tris, pH 7.5, 300 mM NaCl, 10 mM imidazole and 5% glycerol. The protein was then eluted with 50 mM Tris, pH 7.5, 300 mM NaCl, 250 mM imidazole, and concentrated using a Vivaspin centrifugal concentrator with a 5-kDa cutoff (Cytiva, Marlborough, US). The proteins were further purified using a Superdex 75 column with phosphate buffered saline (PBS) in an AKTA pure instrument (Cytiva, Marlborough, US). SDS-PAGE analysis on a 15% polyacrylamide gel under non-reducing conditions for the purified proteins was then performed. Bands were visualized by staining with InstantBlue® Coomassie (Abeam, Cambridge, UK). If needed, protein samples were concentrated using a Vivaspin centrifugal concentrator with a 5-kDa cutoff (Cytiva, Marlborough, US) to reach final protein concentration of ~1 mg / ml. Protein concentrations were determined by UV-Vis absorbance at 280 nm, using a NanoDrop Spectrophotometer (Thermo Scientific, MA, USA), with an extinction coefficient (s2so) of 13,325 M^cm’1for C9; 14,815 M’1for C9-HBP1; 16,305 M’1for C9-HBP2; 17,795 M’1for C9- HBP3 and 13,325 M^cnT1for N-TIMP2WT. The production yielded quantities of approximately 0.3-2 mg protein per liter for all proteins.

[0106] The human enzyme MMP-9CAT, residues 107-215, 391-443, was purified as previously described in N. Sela-Passwell et al., Nat. Med., vol. 18, no. 1, pp. 143-147, 2012, with the following modifications. The gene was expressed in B121(DE3) pLysS E. coli cells in a pET28 vector (with an N-terminal 6xHis tag) and induced with 1 mM isopropyl b-D-1- thiogalactopyranoside (IPTG, Sigma-Aldrich, MO USA) overnight at 30 °C. The cells were then harvested, and three rounds of sonication and centrifugation at 12,000 g were performed, to isolate the inclusion bodies. The inclusion bodies were then solubilized in 8 M urea and 25 mM Tris, pH 7.5. Thereafter, the enzyme was loaded onto a nickel column and eluted with 8 M urea, 25 mM Tris, pH 7.5, 30 mM NaCl and 200 mM imidazole. Refolding was carried out by slow dialysis over 3 days at 4 °C with a gradient of decreasing urea concentrations, starting from 8 M to 0 M. Finally, the enzyme was purified by size exclusion on a gel filtration column, Superdex 75 column (Cytiva, Marlborough, US) with 20 mM Tris, pH 7.5, 50 mM NaCl and 5 mM CaCh buffer. MMP-9CAT concentration was determined by UV-Vis absorbance at 280 nm, using a NanoDrop Spectrophotometer (Thermo Scientific, MA, USA), with an extinction coefficient (s2so) of 33,920 M^cnT1. The production yielded about 0.1 mg enzyme per liter. The purity of the MMP-9CAT was determined by SDS-PAGE (sodium dodecyl sulfate-poly acrylamide gel electrophoresis) analysis.

[0107] MMP -9 inhibition studies

[0108] The inhibitory activity of N-TIMP2wt, C9 and C9-HBP proteins was evaluated against 0.325 nM MMP-9CAT. MMP-9CAT was incubated with different inhibitor concentrations (0-1.5 nM) in TCNB buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM CaCh, and 0.05% Brij) at 37 °C for 1 h. The Anorogenic 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 triAuoroacetic acid] (Merck Millipore, CA, USA) was added, at a final concentration of 7.5 pM. The Auorescence was monitored (with 340 / 30 excitation and 400 / 30 emission filters) using a Synergy 2 plate reader (BioTek, VT, USA) for 60 min at 37 °C. The reactions initial rates in different inhibitor concentration were determined from the linear increase in Auorescence intensity caused by the cleavage of the Anorogenic substrate. Data were fitted to Morrison's equation (Equation 1) for tight binding inhibition using Prism (GraphPad Software). Ki was calculated by plotting the initial velocities against different concentrations of the inhibitors. Ki and its standard deviation (STD) values were the average values obtained from three independent experiments. Calculations were performed using a Km value of 6.175 pM for MMP-9CAT, as determined by measuring MMP-9CAT activity against different concentrations (0-50 pM) of theAnorogenic substrate and fitting the data to the Michaelis -Menten equation (Equation 2) using Prism. The calculated value of Km and its standard error of the mean (SEM) are the average values that were obtained from three independent experiments .

[0109] Equation

[0110] 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 Kiapp- apparent inhibition constant, which is given by Equation 3.

[0111] Equation

[0112] where V is enzyme velocity; Vmax is enzyme maximum velocity achieved at maximum substrate concentration; S is substrate concentration, and Km is Michaelis -Menten constant.

[0113] Equation

[0114] where Ki is the inhibition constant.

[0115] Gelatinase zymography assay

[0116] Inhibition of gelatinolytic activity of human recombinant MMP-9FL full-length MMP-9, (Ala20-Asp707; R&D Systems, MN, USA) was performed on 8% SDS-PAGE with 1% gelatin (Sigma- Aldrich, MO, USA) embedded. MMP-9FL was activated by addition of 4- Aminophenylmercuric acetate (APMA), to a final concentration of 1 mM, and incubation at 37 °C for 24 hours. Following activation, MMP-9FL was resolved on SDS-PAGE. Afterwards, the gels were rinsed for 1 h with gentle agitation in 2.5% Triton X-100 (Sigma- Aldrich, MO, USA) at room temperature and then incubated at 37 °C overnight with 100 nM of N-TIMP2WT, C9 and C9-HBP inhibitors (or 2-70 nM for C9-HBP3) in a development buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCh and 0.02% NaNs), or only in a development buffer used as control. After incubation, the gels were stained with SimplyBlue™ SafeStain (Thermo Fisher, MA, USA), and the gelatinolytic activity was visualized as clear bands. The signal was quantified using ImageJ software. The experiment was performed in triplicate, and the average values were determined with standard error of the mean (SEM).

[0117] Surface plasmon resonance binding experiments

[0118] Binding affinity between the proteins (C9, C9-HBP1, C9-HBP2 or C9-HBP3) and alginate- sulfate (AlgS) or heparin (1 mg / ml, dissolved in Deionized water (DIW)) was detected by surface plasmon resonance (SPR) spectroscopy using a ProteOn XPR36 system (Bio-Rad, CA, USA). Biotinylated AlgS, alginate and heparin were generated as described in Polyak et al. Biomacromolecules, vol. 6, no. 6, pp. 3315-3318, 2005), and were immobilized (0.5 pg in 150 pl of PBS and 0.05% tween (PBST) solution), using an NLC chip (Bio-Rad, CA, USA), to three channels coated with a NeutrAvidin at a flow rate of 30 pl / min for 5 min; a forth channel immobilized with PBST was used as control. The proteins were diluted in a mixture of 50 mM Tris, pH 7.5, 300 mM NaCl and 5 mM CaCh, and were circulated at a flow rate of 40 pl / min for 613 s, followed by 10 min of dissociation. C9 was diluted to 50, 100, 200, 400 and 800 nM; C9-HBP1 to 25, 50, 100, 200 and 400 nM; and C9- HBP2 and C9-HBP3 to 6.25, 12.5, 25, 50 and 100 nM. The binding response, reflected as response units (RU), was monitored as a function of time at 25 °C, and the equilibrium affinity constants (KD) between the proteins and AlgS or heparin were determined using equilibrium binding analysis. The %2values used for the analyses were less than 10% in all cases.

[0119] Particle formation

[0120] Particles were generated by mixing AlgS solution (Img / ml, dissolved in DIW) with the C9 and C9-HBP proteins at 1:2 molar ratio, respectively. The mixture was vortexed andincubated at 37 °C for 1 h to achieve equilibrium in binding between the protein and AlgS and to allow particles formation, followed by overnight incubation at 4 °C, to stabilize the particles. Before injection to mice, the particles were washed three times with 10 mM HEPES, using a Vivaspin with a 30-kDa cutoff.

[0121] Dynamic light scattering (PLS) measurements

[0122] DLS measurements were performed using a Zetasizer Nano-ZS (Malvern Panalytical, Malvern, UK). Samples were dissolved in buffer (50 mM Tris, pH 7.5, 300 mM NaCl and 5 mM CaCh) to a final concentration of 0.3 mg / ml. Samples were equilibrated for 5 min at 4 °C, 25 °C or 37 °C prior to data collection. Correlograms were collected at 173° for at least 10 runs of 10 s. The recorded correlograms were analyzed with the CONTIN procedure using the software provided with the instrument. The size of a particle and standard distribution is calculated from the translational diffusion coefficient by using the Stokes-Einstein equation (Equation 4).

[0123] Equation

[0124] Where dhis the hydrodynamic diameter, d is the translational diffusion coefficient, k is Boltzmann's constant, T is the absolute temperature and q is the viscosity.

[0125] Small angle X-ray scattering (SAXS)

[0126] Small angle X-ray scattering patterns of C9, C9-HBP1 and C9-HBP3 protein solutions were performed at the bio-SAXS BM29 beamline at the European synchrotron radiation facility (ESRF) in Grenoble, France. An energy of 12.5 keV was selected. The scattering intensity was recorded using a Pilatus3 2M in-vac detector, in the interval 0.004 < q < 0.5 A-l. Ten frames with 2 s exposure time were recorded for each sample. Measurements were performed at RT and 37 °C in flow mode, where samples were pushed through the capillary at a constant flow rate. The dedicated beamline software was used for data collection and processing. The scattering spectra of the solvent were subtracted from the corresponding solution data using the Irena package for analysis of small-angle scattering data. The concentration of the studied proteins (C9, C9-HBP1 and C9-HBP3) was 1 mg / ml. The concentrations of the mixed samples of protein with AlgS (Mw=10 kDa) was 0.74 mg / ml protein and 0.18 mg / ml AlgS.

[0127] The size of the proteins was evaluated using Guinier approximation (Equation 5).

[0129] Cryo-transmission electron microscopy (TEM)

[0130] TEM at cryogenic temperature (Cryo-TEM) was used for direct imaging of solutions and dispersions. Vitrified specimens were prepared on a copper grid coated with a perforated lacy carbon 300 mesh (Ted Pella Inc., CA, USA). A typically 3 pl drop from the solution is applied to the grid and blotted with a filter paper to form a thin liquid film of solution. The blotted sample were immediately plunged into liquid ethane at its freezing point (-183 °C). The procedure was performed automatically in the EM GP Plunger (Leica microstsystems, Wetzlar, Germany). The vitrified specimens are transferred into liquid nitrogen for storage. The samples were studied using a FEI Talos F200C TEM, at 200 kV maintained at -180 °C, and images were recorded on a slow scan cooled charge -coupled device CCD at low dose conditions, to minimize electron beam radiation damage.

[0131] In vitro sustained release studies

[0132] C9-HBPl-AlgS and C9-HBP3-AlgS particles were prepared as mentioned above (one sample of 120 pl for each time point). At each time point, t=0, t=2 days, t=4 days and t=6 days the samples were centrifuged at 4 °C for 1 h at top speed (21,380 g) and the supernatant (100 pl) was discarded. The pellet (precipitated particles) from one of the tubes was collected (for both C9-HBPl-AlgS and C9-HBP3-AlgS) and stored at -80 °C until the end of the analysis. The pellets from the other tubes were re-suspended in 100 pl of fresh PBS (to simulate body conditions in which the released protein is cleared from the particles surrounding) and incubated at 37 °C until the next time point. After 6 days, the collected pellets were analyzed by WB analysis, using a 1:3000 dilution of mouse anti- 6xHis primary antibody (Abeam, Cambridge, UK) followed by a 1:2000 dilution of anti-mouse secondary antibody conjugated to HRP (Cell Signaling Technology, MA, USA), and detected using chemiluminescence (EZ-ECL, Biological Industries, Beit Haemek, Israel). The signal was quantified using ImageJ software. The experiment was performed in triplicate, and the average values were determined with SEM.

[0133] In vivo sustained release studies

[0134] All the animal experiments were conducted according to protocols approved by the Ben-Gurion University of the Negev Committee of Use and Care of Animals, (permit number IL-29-06-2020(C)) and the animals were housed and handled according to the university’s Unit for Laboratory Animal Medicine guidelines.

[0135] Hip muscle injection

[0136] C9, C9-HBP1, and C9-HBP3 proteins were labeled with Alexa flour 680 (Thermo Fisher, MA, USA) at 1 :2 molar ratios, according to the manufacturer’s instructions. Particlescomposed of each of the proteins and AlgS were generated, as mentioned above. The fluorescence of the injected samples (C9-HBP3 protein, the mixture of C9 and AlgS, C9- HBPl-AlgS particles and C9-HBP3-AlgS particles) was measured using an in-vivo imaging system (IVIS, Xenogen Corporation, CA, USA) and diluted to reach the same fluorescence signal before injection into mice. Female BALB / C mice at 12 weeks of age (Envigo, Jerusalem, Israel) were used in the experiment. The mouse legs were shaved and 50 pl of sample, either C9-HBP3, a mixture of C9 and AlgS, C9-HBPl-AlgS particles, or C9-HBP3- AlgS particles (10 pl) were injected into the hip muscle at five different positions. The fluorescence signal was measured in living mice over time using IVIS. At the end of the experiment, mice were sacrificed, internal organs and legs were removed, and the fluorescence signal of the dissected organs (i.e., legs, heart, liver, lungs, and kidneys) was measured. For each treatment group, 3 or 4 mice were used. A one-way ANOVA with Tukey's multiple comparison was utilized for statistical analysis of the fluorescence signals, n = 3.

[0137] LV myocardium injection

[0138] C9-HBP1 and C9-HBP3 proteins were labeled as described above. Particles composed of each of the labeled proteins and AlgS were generated, as mentioned above. The fluorescence of the C9-HBPl-AlgS and C9-HBP3-AlgS particles was measured using NEWTON 7.0 (Vilber, Lourmat, France). The solution of C9-HBPl-AlgS was diluted, in order to reach the same fluorescence level as C9-HBP3-AlgS, before injection into mice. BALB / C female mice (11 weeks old) were anesthetized, their chest was surgically opened, and the beating heart was exposed. C9-HBP3-AlgS or control (C9-HBPl-AlgS) were injected, 5 doses of 10 pl each (total 50 pl sample) were injected to the left ventricle of each mouse, using Stepper™ pipette (Dymax, CT, USA) for an accurate volume injection. At different time points following injection, the mice were sacrificed, and the hearts were removed. The fluorescence intensity of the labeled proteins for each of the dissected hearts was measured using NEWTON 7.0. The fluorescence intensity was quantified and analyzed using Prism (GraphPad Software). Error is reported as the SEM of triplicate measurements.

[0139] MI induction by surgical procedure and C9-HBP3-AlgS injection

[0140] The chest and abdomen of BALB / C female mice (11 weeks old) were shaved. Mice were anesthetized using isoflurane and intubated. Mice were ventilated using a rodent ventilator and placed on isothermal pad. Under sterile conditions, the chest was surgically opened, and the beating heart was exposed. The pericardium was pulled away from the heart and the left anterior descending (LAD) coronary artery was permanently ligated using 8-0polyethylene suture. The treatment (C9-HBP3-AlgS or 10 mM HEPES as control) was injected to the post MI heart (5 injections of 10 pl each). Air was expelled from the chest by gentle compression prior to incision closing using Histoacryl® (B. Braun, Melsungen, Germany). The mice recovered from anesthesia were returned to their cages. Post-surgical analgesia was conducted using buprenorphine injection (0.1 mg / kg subcutaneously) and Dipyrone in the drinking water (1.25 mg / mL) for 3 days after surgery.

[0141] Heart function assessment following MI

[0142] On days 4 and 28 to the MI surgery - each mouse was anesthetized with 3% isoflurane mixed with 0.5 L / min 100% O2 in an induction chamber. The mouse was then removed from the induction chamber and placed in a supine position atop a heating pad in order to maintain body temperature, the mouse snout was placed within a nose cone connected to the anesthesia system to maintain a steady-state sedation level throughout the procedure. The level of anesthesia decreased to 1.5% isoflurane mixed with 0.5 L / min 100% O2, and the chest hair was shaved by clipper. Echocardiograms were performed with a commercially available echocardiography system Vevo® 3100 (VisualSonics, Toronto, Canada). The flow of isoflurane was controlled to maintain the heart rate at >400 bpm. The heart was first imaged in the 2D mode in the parasternal long and short axis views of the left ventricle. M-mode images were obtained at the level of the papillary muscles. Left Ventricular Ejection Fraction (EF), a measurement of the percentage of blood leaving the heart each time it contracts, was calculated as:

[0144] Where LVEDV is LV end diastolic volume and LVESV is LV end systolic volume.

[0145] Care was taken to avoid excessive pressure on the mice during the examination. All measurements were averaged for 3 consecutive cardiac cycles and performed by an experienced technician, blinded to the treatment group. Only mice with initial %EF (performed on day 4 after surgery) that was lower than 40% (the upper limit to define MI) were taken for further analyses.

[0146] Heart extraction

[0147] The mice were injected intraperitoneal (i.p) with heparin (10 pl / g) 28 days following MI to prevent blood clots. After 10 minutes the mice were euthanized by i.p injection of sodium pentobarbital 1 (10 pl / g) . When the mice were unresponsive to toe-pinch, the thoracic cavity was opened, and the still-beating heart was removed and inserted into 1 M KC1 solution (pre-cooled, 4 °C) in order to keep the heart in diastole position. The hearts werethen washed and cleaned with PBS and inserted into 4% Formaldehyde (FA) solution for 48 h at 4 °C for fixation. The hearts were then kept in 1% FA (4% FA diluted with PBS) at 4 or

[0148] Histological and morphometric analyses

[0149] After fixation, the hearts were laterally cut into 3 pieces (base, middle and apex sections) and embedded in paraffin blocks. The hearts were sectioned to 5 pm slices (Patho- lab diagnostics, Nes Ziona, Israel). Paraffine sections were deparaffinized, hydrated and stained for fibrosis using Sirius Red solution (0.1% in saturated picric acid), “Direct Red 80” (Sigma Aldrich, MO, USA) according to the manufacture instruction. Sections were then scanned using Pannoramic MIDI II (3DHISTECH, Budapest, Hungary). The morphological analyses were performed using Caseviewer software (3DHISTECH, Budapest, Hungary).The parameters that were measured included average wall thickness (from 3 measurements of each heart septum thickness [mm]), average scar thickness (from 3 measurements of each heart scar thickness [mm]), LV cavity area [mm2] and whole LV area [mm2]. Relative scar thickness was calculated as average scar thickness divided by average wall thickness.Expansion index was calculated as follows:

[0151] The fibrosis (collagen content, measured as the % collagen in the remote zone) assessments were performed using QuPath software.

[0152] Statistical analyses

[0153] Statistical analysis was performed using GraphPad Prism version 6.01 for Windows (GraphPad Software, CA, USA). All experimental results are reported as means (± STD or SEM) (the number of repetitions for each experiment is given in the figure legends). Oneway ANOVA was utilized to compare parameters between experimental groups. Comparison between the changes in echocardiographic measurement between day 4 and 28 for each group performed using two-way ANOVA (Figure 5, Results section). Outliers of the in vivo functional experiments (Figure 5, Results section) were excluded using ROUT method.

[0154] Example 1. C9 proteins exhibit similar MMP-9CAT inhibition potencies

[0155] In the current study our aim was to use C9 variant (as described in materials and methods section) to inhibit MMP-9, for its role as a key instigator of post-MI left ventricle(LV) remodeling. To control the release of C9 to the injured myocardium, we generated particles composed of mixtures of C9-HBP fusions and AlgS.

[0156] To increase the affinity of C9 to AlgS, and by that form particles nano-particles, 1, 2 or 3 repeats of HBP sequence (GGGGSPPRRARVTY (SEQ ID NO: 4) were fused to the C- terminal of C9. C9 fused in its C-terminal to 1, 2 and 3 repeats of HBP sequence were expressed in P. pastoris strain X33, and were purified using nickel affinity chromatography followed by size exclusion chromatography, to yield approximately 0.3-2 mg protein per liter for each protein.

[0157] To verify that the HBP does not deteriorate the potency of the fused proteins in terms of MMP-9 inhibition, an MMP-9 activity assay was performed. MMP-9 catalytic domain (designated MMP-9CAT) was incubated with increasing concentrations of N-TIMP2WT, C9 or C9-HBP proteins, and the cleavage of the MMP-9 Anorogenic substrate was determined as a function of time. The initial linear slope of each reaction was fitted to Morrison’s tight binding equation (Equations 1-3 in Methods section) to determine the Ki values (Figs. 1A- 1E). Although fusion with HBP resulted in a small reduction in the inhibitory activity of C9, all C9-HBP proteins exhibited improved inhibitory activity (having Ki in the picomolar range) against MMP-9CAT, compared to N-TIMP2WT (Table 1).

[0158] Table 1: Inhibition constants (Ki) of N-TIMP2 variants towards MMP-9CAT.

[0159] Ki values are obtained from fitting the data shown on Figs. 1A-1E to the Morrison tight binding equation (Equation 1 in Methods section). Fold improvement was calculated as the ratio between the Ki of N-TIMPWT and the Ki of each of the C9 proteins.

[0160] Example 2. C9-HBP3 reduces MMP-9 gelatinase activity

[0161] To test the inhibition of the gelatinolytic activity of MMP-9 (also known as gelatinase B) by the C9 proteins (i.e. C9 and C9-HBP(l-3)), a gelatin zymography assay was performed with recombinant, full length MMP-9 (MMP-9FL). In this assay, MMP-9FL was resolved on gelatin SDS-PAGE embedded in N-TIMP2WT or C9 proteins solutions, and the gelatinase activity was visualized as clear bands on a dark background (Figs. IF, 1G). All tested C9 proteins (C9, C9-HBP1 and C9-HBP3) showed a significant (32-96%) inhibition in MMP- 9FL activity (Fig. 1H). The inhibitory activity obtained by all the C9 proteins was greater than that of N-TIMP2WT- C9-HBP3 exhibited the strongest inhibition of MMP-9FL gelatinolytic activity, despite having a higher Ki value for MMP-9CAT (i.e., lower inhibition ability) than that of C9 and C9-HBP1 (Table 1). This disagreement may result from the ability of HBP, which is fused to C9, to bind gelatin. Moreover, C9-HBP3 demonstrated a dose dependent inhibitory effect on the gelatinolytic activity of MMP-9FL (Fig. II).

[0162] Example 3. Fusion of HBP repeats with C9 enhances its binding affinity to alginate-sulfate

[0163] After confirming that fusing HBP repeats to C9 retains MMP-9 inhibitory activity of C9, we examined whether these repeats improve the ability of C9 to bind alginate sulfate (AlgS), an interaction that is required to generate C9-HBP protein-AlgS particles for the controlled release of the C9-HBP proteins. For that, we measured the equilibrium affinity constants (KD) between C9 or C9-HBP proteins and AlgS, using surface plasmon resonance (SPR) (Fig. 2A-2F). The affinity of C9-HBP proteins to AlgS increased with the increase number of HBP repeats, with C9 having the lowest affinity to AlgS (Table 2).

[0164] Table 2: Binding affinity constants (KD) for C9 variants and AlgS, measured by SPR

[0165] Specifically, C9, C9-HBP1, C9-HBP2 and C9-HBP3 had KD values of 1040 + 64, 182 + 3 nM, 31 + 4 nM and 20 + 1 nM for binding to AlgS, respectively. The SPRsensograms showed that such improvement in affinity of the C9-HBP proteins is a result of both faster association and slower dissociation rates than that of C9.

[0166] Beyond the ability to bind AlgS, an addition of the positively charged HBP to a therapeutic protein can provide the protein the ability that was previously attributed to heparin-binding proteins, i.e. to interact with other components such as heparin / heparan sulfate, laminin, cellular syndecans and other glycosaminoglycans. Therefore, upon release from the particles in the tissue, the fusion protein can interact with the local environment of the tissue extracellular matrix (ECM). The ability to interact with the ECM may preserve the protein in the tissue, prevent its rapid clearance, and by that to enhance the treatment efficacy. This idea was demonstrated by SPR measurement that emphasized the increased ability of C9-HBP3 to bind heparin compared to C9 and also to C9-HBP1 and C9-HBP2 (Fig. 2H, Fig 2I-2N, and Table 3).

[0167] Table 3: Binding affinity constants (KD) for C9 variants and heparin, measured by SPR.

[0168] Example 4. The structure and thermal stability of C9 proteins

[0169] In order to examine the effect HBP has on protein nano -structure and thermal stability, dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) complemented with cryogenic transmission electron microscopy (Cryo-TEM) measurements were performed. As can be seen from the DLS profiles (Fig. 3A), a gradual increase in the hydrodynamic diameter (dh) (i.e., protein size) was correlated with increasing the number of the fused HBP repeats (Table 4), with C9 and C9-HBP3 having dh values of 5.2 ± 0.4 nm and 8.6 ± 0.6, respectively (Equation 4 in Methods section).

[0170] Table 4: Hydrodynamic Diameter of the proteins, based on DLS measurement (Fig. 3A and 3L).

[0171] The SAXS profiles and Kratky plots of C9, C9-HBP1 and C9-HBP3 (Fig. 3B,C ) exhibited a power law of 0, indicating that those proteins are roughly globular. Using Guinier approximation (Equation 5 in Methods section) to determine the Radius of gyration (Rg) of C9, C9-HBP1 and C9-HBP3 yielded Rgof ~2.1 nm, 2.5 nm and 2.8 nm, respectively (Fig. 3 J, Table 5).

[0172] Table 5: Radius of Gyration (Rg) of C9, C9-HBP1 and C9-HBP3. Calculated using Guinier approximation (equation 5 in Methods section) based on SAXS measurements (Fig. 3B).

[0173] The SAXS results are correlated with the previous DLS results, since the dh, measured by DLS includes the water molecules surrounding the protein (hydrodynamic shell), resulting in higher diameter compare to the SAXS measurement. Cryo-TEM images of C9-HBP3 confirm the existence of spherical structures with an average diameter of 10 nm (Fig. 3D). Not surprisingly, C9, C9-HBP1 and C9-HBP2 could not be detected by Cryo- TEM because of their small size. SAXS measurements of C9-HBP3 at 25 °C and 37 °C (Fig. 3K ) show that there is no change in the scattering profiles upon heating, indicating no structural changes in the protein within this temperature range, suggesting preservation of therapeutic potential of C9-HBP3 in body temperature.

[0174] Example 5. C9-HBP3-AlgS particles sustains C9-HBP3 release in vivo

[0175] As a next step of the retention time of C9-HBP3-AlgS particles in the tissue was measured. To compare the time C9-HBP3 and C9-HBP3-AlgS accumulates in the tissue after injection, we used a simple model of muscle injection, in which we injected the protein alone (C9-HBP3) or the C9-HBP3 -AlgS particles into the hip muscles of mice. The presence of fluorescently labeled (using Alexa flour 680) C9-HBP3 protein compared to labeled C9- HBP3-AlgS particles in the mice hip muscle was monitored over time (Fig. 4A and 4G). Before injection, C9-HBP3 proteins or C9-HBP3-AlgS particles were diluted such that theywould have the same fluorescence levels (i.e., the same signal from all of the injected protein) (data not shown). As observed in Fig. 4A and 4G, although injected having the same fluorescence level, C9-HBP3 showed lower fluorescence signal already at t=0 (2-3 minutes after injection), probably due to its rapid clearance (within seconds) from the injected tissue. The fluorescence signal of C9-HBP3 in the hip muscle of living mice decayed completely after 1.5 h (Fig. 4A and 4G), indicating that the protein was rapidly cleared from the mice hip muscle, while the C9-HBP3-AlgS particles showed a prolonged retention time of 26 h in the tissue (Fig. 5A-F vs Fig. 6A-6F). When internal organs (heart, liver, lungs and kidneys) and legs (Figs. 7A and7B) were extracted 47 h after the injection from mice injected with C9-HBP3-AlgS particles, all injected legs exhibited a flourescent signal, whereas the internal organs did not show any signal, indicating the protein was not distributed or accumulated in the body. In organs and legs that were extracted from C9-HBP3 injected mice, already at 21 h after injection, a minor signal was observed in the legs and no signal was observed in the organs (Fig. 7C and 7D). The short retention time of C9-HBP3 (without AlgS) in the hip muscle (Fig. 4A) highlights the need of AlgS particles, a sustained release system, that will preserve the levels of the inhibitor in the tissue and prolong its activity.

[0176] Example 6. HBP repeating units prolong the protein release from AlgS particles

[0177] After we found that the particles prolong the release of the protein in the tissue, we aimed to determine how the addition of HBP units affected the protein release from the particles, first in vitro and then in vivo. The effect of a number of HBP repeats bound to C9 was evaluated. In an in vitro experiment, we showed that C9-HBP3 stayed in the particles for up to 6 days, whereas C9-HBP1 is not detected in the particles already after 4 days (Fig. 8A and 8B). To confirm this finding in vivo, we performed injections to mice hip muscles of a mixture of AlgS with fluorescently labeled C9, (in which particles were not formed), fluorescently labeled C9-HBPl-AlgS, or fluorescently labeled C9-HBP3-AlgS particles. Prior to injections, the fluorescence signal of each treatment was diluted to achieve an equal fluorescence signal. Already at the first measurement (t=0), performed minutes after the injection, we observed differences in the fluorescence signal, that emphasized the fast clearance of the labeled C9 (injected as a mixture with AlgS) and C9-HBP1 (injected as a C9-HBPl-AlgS particle) compared to the release of C9-HBP3 from C9-HBP3-AlgS particles (Fig. 4B). Moreover, we found that only in the treatment with C9-HBP3-AlgS particles, the release of C9-HBP3 protein was sustained (for 45 h), whereas in the treatments with C9 and AlgS mixture or with C9-HBPl-AlgS particles, the C9 or C9-HBP1 proteinswere not visible already at 20 h (Fig. 4B, Fig. 4H and Figs. 9A-9I). When organs were extracted from mice 45 h after the injections of the different proposed treatments, a fluorescence signal was visible only in the legs of mice injected with C9-HBP3-AlgS particles, while neither of the other treatments (the mixture of C9 and AlgS or C9-HBP1- AlgS particles) exhibited a fluorescence signal at that time point (Fig. 4C). The extracted organs (i.e., heart, liver, lungs, and kidneys) (Fig. 10A-10C) showed no fluorescence signal after that period of time for all tested treatments, suggesting that the proteins are not accumulated in the body. Quantification of the fluorescence signal in mice legs 45 h post injection (Fig. 4D), indicated on a significantly higher signal in mice injected with C9- HBP3-AlgS, compared to mice injected with a mixture of C9 and AlgS or C9-HBPl-AlgS particles. These results indicate that a mixture of C9 with AlgS or C9-HBPl-AlgS particles does not form an efficient sustained release system for the proteins, whereas C9-HBP3-AlgS particles are able to retain the protein in the muscle tissue for at least 45 h.

[0178] Fluorescently labeled C9-HBP3-AlgS particles were then injected (transepicardial injection) to the left ventricle (LV) myocardium (fluorescently labeled C9-HBPl-AlgS particles served as control) in order to determine the sustained release of the protein in the heart tissue and to confirm the presence of C9-HBP3 in the heart during the period its inhibitory activity would be mostly efficient against MMP-9 in the myocardium (days 2-4 post MI). Before injection, fluorescently labeled C9-HBPl-AlgS and C9-HBP3-AlgS particles, were diluted to obtain the same fluorescence levels. We found that C9-HBP3-AlgS was detected in the tissue even after 6 days, whereas C9-HBPl-AlgS was almost undetected after a similar period of time (Fig. 4E, Fig 11A-11J). Quantification of the fluorescence signal (Fig. 4F), indicated on a clear trend, that while the initial fluorescence signal (measured 2-3 minutes after the injection) was similar for both C9-HBPl-AlgS and C9- HBP3-AlgS, the signal of C9-HBP3-AlgS decayed slower and was present in the tissue for up to 6 days.

[0179] Example 7. Treatment with C9-HBP3-AlgS particles improves cardiac outcome post MI

[0180] As discussed above, we found that while injected as C9-HBP3-AlgS particles, C9- HBP3 stays in the heart during the time its activity is needed in the tissue (i.e., when MMP- 9 expression is at its highest level, 2-4 days post MI). Further it was shown that the inhibitory activity of C9-HBP3 is not impaired during 4 days ( see Fig. 12 and Table 6), and only slight degradation of the protein was seen (Fig. 12B)

[0181] Table 6. Inhibition constants (Ki) of MMP-9CAT with C9-HBP3 at t=0 and after 4 days incubation at 37 °C. Ki values were obtained from fitting the data shown on Fig. 12A the Morrison tight binding equation (Equation 1 in Methods section).

[0182] Further, a functional experiment to test the therapeutic efficacy of C9-HBP3 using MI model in mice was performed. To do that, an MI model was generated in mice by permanent ligation of the left anterior descending (LAD) coronary artery. C9-HBP3-AlgS (10 pg protein / mice or 50 pg protein / mice, designated as C9-HBP3-AlgS or C9-HBP3-AlgS X 5, respectively) was injected to the ischemic area immediately after LAD ligation (i.e., MI induction), while 10 mM HEPES injected as a control. In an additional example, an inactivat C9- HBP3-AlgS (having an Ala reside at the N-terminus of C9) particles are used as a control. On days 4 and 28 following the ligation and injection of the treatment, the MI degree was assessed by echocardiography, and the ejection fraction (%EF), an indicative measurement of the cardiac pump function, was estimated, together with further functional measurements. The results are presented in Figs. 13A-13D. The post MI remodeling, induced by MMP-9, is a long-term process, lasting for several weeks post ML Therefore, in order to exclude the possible effect our treatment might have during the initial three days post MI and to test only its long-term therapeutic effect on the remodeling process, only the mice whose %EF, measured on day 4, was under 40% (which is the criterion to determine that MI was generated), were included in the analysis. While the %EF measured on day 4 was approximately similar for all the tested groups, on day 28 the group treated with C9- HBP3-AlgS X 5 showed significantly higher %EF, compared to control (Fig. 13A). The average change in %EF change from day 4 to 28 post MI is increased in both treated groups, whereas a statistically significant improvement in cardiac recovery is observed in the group treated with C9-HBP3-AlgS X 5 compared to control (Fig. 13B). Next, we evaluated how the treatments effected mice with severe MI, that are subjected to higher risk of myocardial remodeling and have poor prognosis. For that, we divided each experimental group into 2 sub-populations (initial %EF of >30 or <30, indicating mice with moderate MI or severe MI, respectively). Fig. 13C indicates that mice with severe heart dysfunction (i.e., %EF <30), treated with C9-HBP3-AlgS X 5, showed the greatest improvement in cardiac function compared to the other groups (treated with HEPES or with C9-HBP3-AlgS, initial %EF<30), even though after severe MI the mice are more susceptible to deterioration in cardiac function. From histological analyses, performed using Sirius red staining (Fig. 13D), we observed changes in the % of fibrosis after treatment. C9-HBP3-AlgS treated hearts showed minor tendency of increase in fibrosis at the remote area, whereas hearts treated with C9- HBP3-AlgS X 5 showed significant reduction in % of fibrosis, compared to mice treated with the lower dose (Fig. 13E). The balance between collagen degradation and accumulation in the post MI heart is highly complex. As opposed to the irreversible and unpredictable gene deletion, at higher dose our treatment showed improvement in the percentage of fibrosis, i.e. showed lower fibrosis, which may be a result of inhibiting the proteolytic activity of MMP- 9 only when its levels are elevated post MI. This finding may suggest that at a higher dose, our treatment reduces ECM degradation and limits the replacement of a healthy tissue in a fibrotic scar, tough farther adjustment of the dose should be performed.

[0183] Furthermore, the morphometric measurement of the post MI hearts in mice having a scar on the left ventricular wall showed that the treatment prevented the thickening of the wall in the scar area (Fig. 13F) and reduced the expansion of the heart (Fig. 13G). Conclusively, all the results presented in Figs. 13A-H show that the timely inhibition of MMP-9 activity by the treatment we employed, preserves cardiac function post MI, by preventing ECM degradation and consequent myocardial remodeling.

[0184] Example 8.

[0185] REGA-3G12, AB0041, AB0046 and andecaliximab antibodies, each fused to 3, 4 or 5 repeats of heparin-binding peptide (HBP, GGGGSPPRRARVTY (SEQ ID NO: 4), respectively, are expressed and produced in the methylotrophic X33 P. pastoris yeast strain. The fused antibodies are purified as described in materials and methods. The site of fusion is chosen such that the binding of the antibodies to MMP9 and MMP2 is not affected, e.g. to the constant region of the antibody.

[0186] Particles are generated by mixing AlgS solution (Img / ml, dissolved in DIW) with HBP(3-5)-REGA-3G12, HBP(3-5)-AB0041, HBP(3-5)-AB0046 and HBP(3-5)- andecaliximab proteins at 1:2 molar ratio, respectively. The mixture is vortexed and incubated at 37 °C for 1 h to achieve equilibrium in binding between the protein and AlgS and to allow particles formation, which is followed by overnight incubation at 4 °C, to stabilize the particles.

[0187] Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims

Claims

CLAIMS1. A biomaterial particle comprising a complex of an alginate sulfate and a conjugate comprising a matrix metalloproteinase (MMP) inhibitor and from 3 to 10 repetitions of a heparin-binding peptide (HBP).

2. The biomaterial particle according to claim 1, wherein the HBP consists of from 8 to 25 amino acids.

3. The biomaterial particle according to any one of claims 1 to 2, wherein the conjugate comprises from 3 to 8 or from 3 to 5 repetitions of the HBP.

4. The biomaterial particle according to any one of claims 1 to 3, wherein the MMP inhibitor and the HBPs are conjugated via an amide bond.

5. The biomaterial particle according to any one of claims 1 to 4, wherein the HBPs are conjugated to the C-terminus of the MMP inhibitor.

6. The biomaterial particle according to any one of claims 1 to 5, wherein the MMP inhibitor is an inhibitor of MMP9.

7. The biomaterial particle according to claim 6, wherein the MMP9 inhibitor comprises an amino acid sequence selected from SEQ ID NO: 1 and 2.

8. The biomaterial particle according to any one of claims 1 to 7, wherein the heparin- binding peptide comprises an amino acid sequence selected from SEQ ID NO: 3 and 4.

9. The biomaterial particle according to claim 1, wherein the conjugate comprises an MMP9 inhibitor comprising the amino acid sequence SEQ ID NO: 1 and from 3 to 8 repetitions of a HBP comprising the amino acid sequence SEQ ID NO: 3.

10. The biomaterial particle according to any one of claims 1 to 9, wherein the conjugate has an average hydrodynamic diameter of from 5.5 to 15 nm.

11. The biomaterial particle according to any one of claims 1 to 10, wherein the conjugate has an average radius of gyration of from 2.2 to 5 nm.

12. The biomaterial particle according to any one of claims 1 to 11, having an average hydrodynamic diameter of from 1 to 15 pm.

13. A plurality of biomaterial particle each comprising a complex of alginate sulfate and a conjugate of an inhibitor of matrix metalloproteinase 9 with from 3 to 6 repetitions of a heparin-binding peptide (HBP), wherein the MMP9 inhibitor comprises the amino acid sequence SEQ ID NO: 1 and the HBPs each comprises the amino acid sequence GGGGSPPRRARVTY set forth as SEQ ID NO: 4.

14. The biomaterial particles according to claim 13, wherein the from 3 to 6 repetitions of the HBP are conjugated to the C-terminus of the MMP9 inhibitor.

15. The biomaterial particle(s) according to any one of claims 1 to 14, wherein the conjugate comprises an amino acid sequence selected from SEQ ID NO: 7-10.

16. The biomaterial particle(s) according to any one of claims 1 to 15, wherein the biomaterial particles are hydrogel microparticle.

17. The biomaterial particle(s) according to any one of claims 1 to 16, wherein the biomaterial particles provide an extended-release of the MMP inhibitor.

18. The biomaterial particle(s) according to claim 17, wherein the biomaterial particle(s) provide extended release of the MMP inhibitor for at least 30 hours.

19. A pharmaceutically acceptable composition comprising a plurality of biomaterial particles according to any one of claims 1 to 18, and a pharmaceutically acceptable carriers and / or excipients.

20. The pharmaceutically acceptable composition according to claim 19, formulated for oral or parenteral administration.

21. The pharmaceutically acceptable composition according to claim 20, formulated for intracardiac injection.

22. The pharmaceutically acceptable composition according to any one of claims 19 to 21, for use in treatment of a disease or condition associated with an increased activity of a matrix metalloproteinase.

23. The pharmaceutically acceptable composition according to claim 22, wherein the matrix metalloproteinase is MMP9.

24. The pharmaceutically acceptable composition for use according to claim 22 or 23, wherein the disease or condition is selected from cardiac remodeling, myocardial infarction, ischemic diseases, cancer, osteoporosis, wounds and muscular dystrophy.

25. The pharmaceutically acceptable composition for use according to claim 24, wherein the condition is a cardiac remodeling.

26. The pharmaceutically acceptable composition for use according to claim 25, wherein the treating comprises inhibiting or preventing cardiac remodeling caused by myocardial infarction (MI).

27. The pharmaceutically acceptable composition for use according to claim 26, wherein the treating comprises inhibiting or preventing left ventricular remodeling.

28. The pharmaceutically acceptable composition for use according to any one of claims 24 to 27, wherein the pharmaceutical composition is administered intracardially in proximity to the site of the ischemia.

29. The pharmaceutically acceptable composition for use according to any one of claims 26 to 28, wherein the pharmaceutically acceptable composition is administered within about 1 week after the MI.

30. A method of preventing or inhibiting the development of cardiac remodeling after myocardial infarction in a subject in need thereof, the method comprises intracardially administering a plurality of the biomaterial particles according to any one of claims 1 to 18 to the subject.

31. A polypeptide comprising an amino acid sequence of a matrix metalloproteinase (MMP) inhibitor and from 1 to 10 sequential repetitions of an amino acid sequence of a heparin-binding peptide (HBP) comprising an amino acid sequence selected from SEQ ID NO: 3, 4 and a combination thereof.

32. The polypeptide according to claim 30, wherein the amino sequences of the HBP are located at the C terminus of the MMP inhibitor.

33. The polypeptide according to claim 31 or 32, wherein the MMP is MMP9.

34. The polypeptide according to claim 33, wherein the MMP9 inhibitor comprises amino acid sequence selected from SEQ ID NO: 1 and 2.

35. The polypeptide according to any of claims 31 to 34, comprising an amino acid sequence selected from SEQ ID NO: 5-10.

36. The polypeptide according to any of claims 31 to 34, comprising an amino acid sequence selected from SEQ ID NO: 7-10.

37. A nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide according to any one of claims 31 to 36.

38. The nucleic acid molecule according to claim 37, comprising a nucleic acid sequence selected from SEQ ID NO: 11 and 12 encoding an MMP inhibitor and from 1 to 10 sequential repetitions of the nucleic acid sequence selected from SEQ ID NO: 13 and 14.

39. The nucleic acid molecule according to claim 38, wherein the nucleic acid sequences selected from SEQ ID NO: 13 and 14 are separated by a nucleic acid spacer.

40. The nucleic acid molecule according to any one of claims 37 or 39, comprising a nucleic acid molecule selected from SEQ ID NO: 15-21.

41. A nucleic acid construct comprising the nucleic acid molecule according to any one of claims 36 to 40, operably linked to a promoter.

42. A cell comprising the nucleic acid molecule according to any one of claims 36 to 40 or the nucleic acid construct according to claim 41.

Images