Protein substrate to bind growth factor

Abstract

To provide protein substrates comprising integrin binding motif and growth factor (GF) binding peptide motif that enable binding to or activation of both a growth factor receptor and an integrin, simultaneously or selectively.SOLUTION: Substrate proteins may be in the form of a mussel adhesive protein, where an extracellular matrix (ECM) protein derived integrin binding peptide motifs and GF binding peptide motifs are recombinantly incorporated into the mussel adhesive protein. Also there is provided use of the protein substrate for regulating a cellular behavior including cell adhesion, migration, or proliferation which are essential processes in bioprocess, wound healing, tissue engineering, and therapeutic application. In other embodiments, the present invention provides a method and composition of stabilizing and protecting a growth factor from protease digestion or internalization into cells (or cellular uptake) in order to maintain the persistent and durable function of the growth factors.SELECTED DRAWING: Figure 1a

Description

[Technical field]

[0001] The present invention relates to ECM mimetics and growth factor complexes comprising protein substrates having both integrin-binding peptide motifs and growth factor / cytokine-binding peptide motifs. In certain embodiments, the present invention relates to protein substrates comprising one of FGF, TGFβ, PDGF, and VEGF-binding peptide motifs directly, and one of integrin αv, α2, α4, α5, and α9-binding motifs derived from fibronectin, collagen, laminin, vitronectin, and tenascin. [Background technology]

[0002] Cell adhesion receptors, integrins, and growth factor receptors are important molecular determinants in providing specificity for signal transduction during developmental and / or pathological processes. Although integrins and growth factor receptors can independently transmit intracellular signals, the synergy of signals provided by the extracellular matrix (ECM) and growth factors (GFs) appears to regulate complex processes including vascular development during embryogenesis, wound healing, as well as tumor growth / metastasis.

[0003] GFs are involved in the regulation of various cellular processes and typically act as signaling molecules between cells. GFs promote cell proliferation, differentiation and maturation and are diverse in growth factors. Most growth factors act in a diffusible manner and are generally unstable in tissue environments. Such extended maintenance maintains the activity of growth factors in cells or their environment and is considered advantageous in bioprocess and regenerative medicine applications.

[0004] Therefore, many attempts have been made to improve the performance of growth factors (e.g., their active period and safety). The most common strategy to extend the maintenance of growth factors in the environment is to immobilize growth factors on solid substrates by chemical binding, which can be used for many medical and biological applications including wound healing, tissue engineering, etc. (Mirhamed Hajimiri, et al, Growth factor conjugation: Strategies and applications, J. Biomedical Materials Research, Volume 103, Issue 2. 2015 p819-838). In addition, it is very important to add biofunctionality, such as regulating cell function, to biomaterials used for artificial organs. Modification of growth factors for immobilization or high affinity binding to cells or scaffold biomaterials has been performed by various researchers (see Seiichi Tada, et al, Design and Synthesis of Binding Growth Factors, Int J Mol Sci. 2012; 13(5): 6053-6072). However, most of these have limited efficacy, mainly because growth factor activity is lost upon association with the carrier, the growth factors are inefficiently controlled-released, and are poorly protected from proteolysis and / or degradation.

[0005] The extracellular matrix contains many components, such as adhesion molecules, notch signaling molecules, traction-enabling adhesion molecules, and proteoglycan molecules, that bind growth factors and regulate their activity (Cao L., et al. 2009 Promoting angiogenesis via manipulation of VEGF responsiveness with notch signaling. Biomaterials 30, 4085-4093; Discher DE, et al. 2005 Tissue cells feel and respond to the stiffness of their substrate. Science 310, 1139-1143; Ramirez F. & Rifkin DB, 2003 Cell signaling events: a view from the matrix. Matrix Biol. 22, 101-107).

[0006] Many ECM proteins have binding sites for both growth factors and cell attachment, allowing growth factors to be released locally and bind to their cell surface receptors. Thus, the ECM acts as a cofactor, providing growth factors to cell surface receptors. Localization of growth factors by ECM binding also contributes to the establishment of concentration gradients of soluble chemokines and growth factor morphogens, which play a key role in developmental processes. Growth factors may also be sequestered in the ECM, which may function as a localized reservoir. Then, as the ECM breaks down, it should release solid inactive growth factors that are converted into soluble active ligands (Kim, S.-H., et al. 2011. Extracellular matrix and cell signalling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor. The Journal of endocrinology, 209(2), p139-51; Hynes, et al. 2012. Overview of the matrisome-an inventory of extracellular matrix constituents and functions. Cold Spring Harbor perspectives Hynes, RO, 2009; The extracellular matrix: not just pretty fibrils. Science (New York, NY), 326(5957), pp. 1216-9). Some growth factors are known to act in a non-diffusible manner, such as HB-EGF, TGF, TNF, and CSF, while the majority of growth factors act in a diffusible manner (Seiichi Tada, et al. Int J Mol Sci. 2012; 13(5): 6053-6072. Design and Synthesis of Binding Growth Factors).

[0007] It is an object of the present invention to provide a simpler and more reliable protein substrate for immobilizing growth factors with long-lasting safety and functionality by using ECM-derived GF-binding peptide motifs and integrin-binding motifs. Summary of the Invention [Problem to be solved by the invention]

[0008] It is an object of the present invention to provide a protein substrate comprising recombinant adhesion proteins genetically functionalized with integrin binding motifs and heparin binding motifs capable of binding to or sequestering growth factors.

[0009] Another object of the present invention is to provide an extracellular microenvironment surface for regulating cell plasticity, wherein the microenvironment surface comprises a protein substrate according to any one of claims 1 to 17, capable of inducing combinatorial signal transduction by simultaneously activating integrins and growth factor receptors. [Means for solving the problem]

[0010] To achieve the above objectives, in one aspect, the present invention provides a protein substrate comprising a recombinant adhesion protein genetically functionalized with an integrin binding motif and a heparin binding motif capable of binding to or sequestering a growth factor.

[0011] In one embodiment of the present invention, the heparin-binding motif may be derived from fibronectin domain III of collagen, vitronectin, or bone sialoprotein, laminin globular domain, or heparin-binding domain.

[0012] In a preferred embodiment of the present invention, the heparin-binding motif derived from fibronectin domain III may be a peptide of KYILRWRPKNS (SEQ ID NO: 7), YRVRVTPKEKTGPMKE (SEQ ID NO: 8), SPPRRARVT (SEQ ID NO: 9), ATETTITIS (SEQ ID NO: 10), VSPPRRARVTDATETTITISWRTKTETITGFG (SEQ ID NO: 11), ANGQTPIQRYIK (SEQ ID NO: 12), KPDVRSYTITG (SEQ ID NO: 13), PRARITGYIIKYEKPGSPPREVVPRPRPGV (SEQ ID NO: 14), WQPPRARI (SEQ ID NO: 15), WQPPRARITGYIIKYEKPG (SEQ ID NO: 16), YEKPGSPPREVVPRPRP (SEQ ID NO: 17), or KNNQKSEPLIGRKKT (SEQ ID NO: 18). In another preferred embodiment of the present invention, the heparin binding motif derived from the laminin globular domain is selected from the group consisting of GLIYYVAHQNQM (SEQ ID NO: 19), RKRLQVQLSIRT (SEQ ID NO: 20), GLLFYMARINHA (SEQ ID NO: 21), KNSFMALYLSKG (SEQ ID NO: 22), VVRDITRRGKPG (SEQ ID NO: 23), RAYFNGQSFIAS (SEQ ID NO: 24), GEKSQFSIRLKT (SEQ ID NO: 25), TLFLAHGRLVFMFNVGHKKL (SEQ ID NO: 26), TLFLAHGRLVFM (SEQ ID NO: 27), LVFMFNVGHKKL (SEQ ID NO: 28), GAAWKIKGPIYL (SEQ ID NO: 29), VIRDSNV The peptide may be VQLDV (SEQ ID NO:30), GKNTGDHFVLYM (SEQ ID NO:31), RLVSYSGVLFFLK (SEQ ID NO:32), GPLPSYLQFVGI (SEQ ID NO:33), RNRLHLSMLVRP (SEQ ID NO:34), LVLFLNHGHFVA (SEQ ID NO:35), AGQWHRVSVRWG (SEQ ID NO:36), KMPYVSLELEMR (SEQ ID NO:37), RYVVLPR (SEQ ID NO:38), VRWGMQQIQLVV (SEQ ID NO:39), TVFSVDQDNMLE (SEQ ID NO:40), APMSGRSPSLVLK (SEQ ID NO:41), VLVRVERATVFS (SEQ ID NO:42), or RNIAEIIKDI (SEQ ID NO:43).In another preferred embodiment of the present invention, the heparin-binding motif derived from the heparin-binding domain of collagen may be a peptide of KGHRGF (SEQ ID NO: 44), TAGSCLRKFSTM (SEQ ID NO: 45), or GEFYFDLRLKGDK (SEQ ID NO: 46). In another preferred embodiment of the present invention, the heparin-binding motif derived from the heparin-binding domain of vitronectin may be a peptide of KKQRFRHRNRKGYRSQ (SEQ ID NO: 47). In another preferred embodiment of the present invention, the heparin-binding motif derived from the heparin-binding domain of bone sialoprotein may be a peptide of KRSR (SEQ ID NO: 48), or KRRA (SEQ ID NO: 49).

[0013] In one embodiment of the present invention, the heparin-binding motif can bind to basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), or platelet-derived growth factor (PDGF).

[0014] In other embodiments of the invention, the integrin binding motif may be an αvβ3-, αvβ6-, αvβ8-, α5β1-, or α9β1-binding peptide. In a preferred embodiment of the invention, the integrin binding motif is capable of activating integrin αvβ6, and the heparin binding motif is capable of binding to TGF-β. In other preferred embodiments of the invention, the integrin binding motif is capable of activating integrin α5β1 or α9β1, and the heparin binding motif is capable of binding to bFGF.

[0015] In one embodiment of the invention, the recombinant adhesive protein may be derived from a recombinant mussel adhesive protein. In a preferred embodiment of the invention, the recombinant mussel adhesive protein may comprise, consist essentially of, or consist of the peptide sequences of SEQ ID NOs: 1-6, and 60-74. In another preferred embodiment of the invention, the integrin binding motif and / or heparin binding motif may be attached to the N-terminus and / or C-terminus of the recombinant adhesive protein. In another preferred embodiment of the invention, the integrin binding motif and / or heparin binding motif may be attached to either the N-terminus or the C-terminus of the recombinant adhesive protein.

[0016] In one embodiment of the present invention, the integrin binding motif and the heparin binding motif may be linked via a spacer linker peptide. In a preferred embodiment of the present invention, the spacer linker peptide may be the peptide of SEQ ID NO: 75.

[0017] In another aspect, the invention provides an extracellular microenvironment surface for modulating cellular plasticity.

[0018] In one embodiment of the present invention, the microenvironment surface may include a protein substrate of the present invention capable of inducing combinatorial signal transduction by simultaneously activating integrins and growth factor receptors. In a preferred embodiment of the present invention, the plasticity of the cells may be epithelial-mesenchymal transition.

[0019] The protein substrate is provided in the form of a recombinant adhesion protein that contains a GF-binding peptide motif and an integrin-binding peptide motif, which induces or manipulates a wide range of cellular behaviors, including cell attachment, migration, growth, and survival, by simultaneously or sequentially activating growth factor receptors or integrins.

[0020] In one aspect, the invention provides a protein substrate in the form of a recombinant adhesion protein comprising the following three domains: 1) Mussel adhesive protein domains that adhere to cells, tissues, or any substrate, such as plastic and glass surfaces; 2) a growth factor binding domain capable of immobilizing or sequestering a growth factor to activate or inhibit a cognate growth factor receptor; and 3) an integrin-binding domain that can activate integrins.

[0021] According to the present invention, any recombinant mussel adhesive protein can be used for the purpose of the present invention. In one embodiment of the present invention, the recombinant mussel adhesive protein can comprise, consist essentially of or consist of peptides of SEQ ID NOs: 1-6 and 60-74. In a preferred embodiment of the present invention, the recombinant mussel adhesive protein can be selected from byssus protein 1 decapeptide repeats (SEQ ID NO: 1), byssus protein 3 (SEQ ID NO: 2), byssus protein 5 (SEQ ID NOs: 3-4) or combinations thereof. Preferably, byssus protein 1 decapeptide repeats and byssus protein 3, byssus protein 1 decapeptide repeats and byssus protein 5, or a hybrid of byssus protein 1, byssus protein 3 and byssus protein 5. Preferably, hybrid proteins (SEQ ID NO: 5 and SEQ ID NO: 6) consisting of six repeats of the byssus protein 1 decapeptide at the N- and C-termini of byssus protein 5 of Mytilus edulis (SEQ ID NO: 3) or byssus protein 5 of Mytilus galloprovincialis (SEQ ID NO: 4) are used for the present invention.

[0022] In the present invention, the GF-binding domain may be a heparin-binding or syndecan-binding peptide motif derived from an ECM protein including collagen, fibronectin, laminin, vitronectin, fibrinogen, tenascin, or bone sialoprotein. Growth factors that are bound or sequestered by heparin-binding or syndecan-binding motifs include basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF), and cytokines that are bound or sequestered by heparin or syndecan-binding motifs are transforming growth factor beta (TGF-β), interleukin-2, and interleukin-6.

[0023] In another aspect, the invention provides a protein substrate for generating a synthetic extracellular microenvironment for culturing valuable cells comprising an ECM-derived integrin-binding peptide and one or more GF-binding motifs for immobilizing exogenous growth factors. Exogenous growth factors bound to heparin or syndecan-binding motifs are retained within the protein substrate according to the invention.

[0024] In another aspect, the present invention provides a protein substrate for sustained growth factor delivery for bioprocessing or tissue engineering applications.

[0025] In another aspect, the invention provides a composition for cell therapy, wound healing or tissue engineering comprising a protein substrate of the first aspect and a pharma- ceutically acceptable carrier.

[0026] In another aspect, the invention provides a protein substrate as a synthetic extracellular matrix that retains growth factors or cytokines.

[0027] In another aspect, the invention provides a method of promoting cell migration comprising using a protein substrate that binds to both a growth factor receptor and an integrin.

[0028] The present invention provides a substrate for immobilizing growth factors or cytokines for delivery to cells, tissues, or organs.Embodiments of the present invention as well as features and advantages will be apparent from the further description herein. Effect of the Invention

[0029] The protein substrates of the present invention may be used for surface coatings for cell culture related applications, such as stem cell expansion, delivery of growth factors for tissue engineering, or biological processes for therapeutic applications. [Brief description of the drawings]

[0030] [Figure 1a] Figure 1a shows two basic formats of protein substrates that provide integrin-binding motifs and heparin-binding motifs that can activate or bind various growth factors: Format A is a protein substrate with integrin-binding and GF-binding peptide motifs at the C-terminus and N-terminus, while in Format B, both binding peptide motifs are integrated at the C-terminus of the protein substrate. [Figure 1b] Figure 1b illustrates the mechanism of action of the protein substrate of the present invention: Due to the specificity, the coated surface of the protein substrate forms a topography similar to an island that allows specific interactions between binding motifs and growth factor receptors or integrins, as shown in Figure 1b. [Figure 2a] Figure 2a shows the layout of heparin-binding peptide motif sequences for screening any peptide motif with high affinity to GFs. Figure 2a is a layout of heparin or syndecan-binding peptide motifs derived from fibronectin, collagen, and laminin. [Figure 2b] Figure 2b shows the layout of heparin-binding peptide motif sequences for screening any peptide motif with high affinity to GF. Figure 2b is a layout of heparin or syndecan binding peptides derived from the laminin LG domain. [Diagram 3] FIG. 3 shows calculated GF binding affinities for GF binding motifs. [Figure 4a] FIG. 4a shows the results of screening various ECM-derived GF-binding peptide motifs against bFGF. [Figure 4b] FIG. 4b shows the results of screening various ECM-derived GF-binding peptide motifs against TGF-β. [Figure 4c] FIG. 4c shows the results of screening laminin-derived GF-binding peptide motifs for bFGF, TGF-β, and PDGF, respectively. [Figure 5a] Figure 5a shows the sustained release of TGF-β bound to a protein substrate with high affinity for TGF-β. Figure 5a shows the layout of a TGF-β binding peptide motif with high affinity for TGF-β. [Figure 5b] Figure 5b shows the sustained release of TGF-β bound to a protein substrate with high affinity for TGF-β. Figure 5b shows the absorption profile of a GF binding motif with high affinity for TGF-β showing sustained release of TGF-β over a long period of time. [Figure 6a] FIG. 6a shows the layout of ECM-derived peptide motifs that bind to epithelial-mesenchymal transition (EMT)-inducible integrins αv, α2, and α9. [Figure 6b] Figure 6b shows the results of Western blot of breast epithelial cells, MCF-10A, cultured on surfaces coated with EMT-inducible integrin binding motifs. In Figure 6b, fibronectin expression is shown as a TGF-β-induced EMT marker bound to the protein substrate. Protein substrates with GF binding motifs (YEK & ANG) that have high affinity for TGF-β induced high expression of fibronectin, whereas protein substrates with GF binding motifs (WQ) that have low affinity for TGF-β did not induce fibronectin expression. [Figure 7]Figure 7 shows trypsin-mediated TGF-β cleavage analyzed by Western blot. TGF-β bound to a protein substrate has a low level of trypsin digestion, whereas TGF-β without a protein substrate is largely digested when treated with trypsin. [Figure 8a] Figure 8a shows the effect of GF bound to a protein substrate on the growth and proliferation of human foreskin fibroblasts in low serum conditions (0.5% FBS). Figure 8a shows the effect of PDGF bound to a protein substrate on cell growth and proliferation. [Figure 8b] Figure 8b shows the effect of GF bound to a protein substrate on the growth and proliferation of human foreskin fibroblasts in low serum conditions (0.5% FBS). Figure 8b shows the effect of FGF2 bound to a protein substrate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention relates to a protein substrate that induces signal transduction mediated simultaneously or selectively by integrins and growth factor / cytokine receptors to regulate cell behavior. The protein substrate may be provided in the form of a recombinant adhesion protein comprising the following three domains: 1) a recombinant mussel adhesive protein domain that attaches to cells, tissues, or the surface of any substrate, e.g., plastic or glass; 2) a growth factor binding domain capable of immobilizing or sequestering a growth factor or cytokine to activate or inhibit a cognate growth factor receptor or cytokine receptor; and 3) An integrin-binding domain that can activate the integrin receptor.

[0032] As used herein, the term "substrate" refers to a material to which another material is applied. In biology, the surface on which an organism, e.g., a plant, fungus, or animal, lives can be referred to as a substrate. Any such surface can include biotic or abiotic components.

[0033] As used herein, a recombinant mussel adhesive protein refers to a fusion protein comprising mussel byssus protein FP-5 and mussel byssus protein FP-1 decapeptide. In one embodiment, the protein substrate provided herein comprises a mussel byssus protein selected from the group consisting of SEQ ID NOs: 5-6.

[0034] As used herein, the term "GF-binding peptide motif" refers to a short peptide derived from the heparin-binding or syndecan-binding domains of extracellular matrix proteins, including, but not limited to, fibronectin domain III, laminin LG domain, collagen heparin-binding domain, vitronectin heparin-binding domain, or fibrinogen. In one embodiment, the GF-binding peptide motif comprises 5-40 amino acids or a combination thereof. In various embodiments, the GF-binding peptide motif may be KYILRWRPKNS (SEQ ID NO: 7), YRVRVTPKEKTGPMKE (SEQ ID NO: 8), SPPRRARVT (SEQ ID NO: 9), ATETTITIS (SEQ ID NO: 10), VSPPRRARVTDATETTITISWRTKTETITGFG (SEQ ID NO: 11), ANGQTPIQRYIK (SEQ ID NO: 12), KPDVRSYTITG (SEQ ID NO: 13), PRARITGYIIKYEKPGSPPREVVPRPRPGV (SEQ ID NO: 14), WQPPRARI (SEQ ID NO: 15), WQPPRARITGYIIKYEKPG (SEQ ID NO: 16), YEKPGSPPREVVPRPRP (SEQ ID NO: 17), KNNQKSEPLIGRKKT (SEQ ID NO: 18) derived from fibronectin, or GLIYYVAHQNQM (SEQ ID NO: 19), RKRLQVQLSIRT (SEQ ID NO: 20), GLLFYMARIN (SEQ ID NO: 21), or GLLFYMARIN (SEQ ID NO: 22) derived from laminin. HA (SEQ ID NO: 21), KNSFMALYLSKG (SEQ ID NO: 22), VVRDITRRGKPG (SEQ ID NO: 23), RAYFNGQSFIAS (SEQ ID NO: 24), GEKSQFSIRLKT (SEQ ID NO: 25), TLFLAHGRLVFMFNVGHKKL (SEQ ID NO: 26), TLFLAHGRLVFM (SEQ ID NO: 27), LVFMFNVGHKKL (SEQ ID NO: 28), GAAWKIKGPIYL (SEQ ID NO: 29), VIRDSNVVQ LDV (SEQ ID NO: 30), GKNTGDHFVLYM (SEQ ID NO: 31), RLVSYSGVLFFLK (SEQ ID NO: 32), GPLPSYLQFVGI (SEQ ID NO: 33), RNRLHLSMLVRP (SEQ ID NO: 34), LVLFLNHGHFVA (SEQ ID NO: 35), AGQWHRVSVRWG (SEQ ID NO: 36), KMPYVSLELEMR (SEQ ID NO: 37), RYVVLPR (SEQ ID NO: 38), VRWGMQQIQLVV (SEQ ID NO: 39),The amino acid sequence is selected from the group of heparin-binding or syndecan-binding peptides consisting of TVFSVDQDNMLE (SEQ ID NO: 40), APMSGRSPSLVLK (SEQ ID NO: 41), VLVRVERATVFS (SEQ ID NO: 42), RNIAEIIKDI (SEQ ID NO: 43), PGRWHKVSVRWE (SEQ ID NO: 76), or KGHRGF (SEQ ID NO: 44), TAGSCLRKFSTM (SEQ ID NO: 45), GEFYFDLRLKGDK (SEQ ID NO: 46), or KKQRFRHRNRKGYRSQ (SEQ ID NO: 47), or KRSR (SEQ ID NO: 48), KRRA (SEQ ID NO: 49), derived from collagen, or bone sialoprotein.

[0035] As used herein, the term "integrin binding motif" refers to a short peptide derived from an extracellular matrix protein, including, but not limited to, fibronectin, laminin, collagen, vitronectin, or tenascin. The integrin binding motif can bind to and activate integrin αv, α5, α8, or α9 to support cell attachment and induce morphogenesis together with a growth factor bound to the heparin binding motif. In various embodiments, the heparin binding peptide comprises an amino acid sequence selected from the group consisting of RGD (SEQ ID NO: 50), RGDV (SEQ ID NO: 51), PQVTRGDVFTMP (SEQ ID NO: 52) for integrin αv binding, or GRGDSP (SEQ ID NO: 53), PHSRNSGGSGSGSGRGDSP (SEQ ID NO: 54) for integrin α5 binding, or EDGIHEL (SEQ ID NO: 55), VAEIDGIEL (SEQ ID NO: 56) for integrin α9 binding, or VFDNFVLK (SEQ ID NO: 57) for integrin α8 binding.

[0036] In one embodiment, the present invention discloses a protein substrate that provides a GF-binding peptide motif that simultaneously or selectively sequesters or binds growth factors. Any suitable GF-binding peptide motif may be selected from the group consisting of heparin-binding or syndecan-binding motifs derived from fibronectin domain III, laminin LG domain, collagen heparin domain, vitronectin heparin domain, or bone sialoprotein, as detailed in the definition of the term heparin-binding motif above.

[0037] In one embodiment, a protein substrate for sequestering or binding basic fibroblast growth factor (bFGF) is disclosed. In general, the GF-binding peptide motif may be selected from heparin / syndecan binding domains of fibronectin, laminin, and collagen for bFGF binding. Preferably, fibronectin-derived peptides PRARITGYIIKYEKPGSPPREVVPRPRPGV (SEQ ID NO: 14), WQPPRARI (SEQ ID NO: 15), laminin-derived peptides RYVVLPR (SEQ ID NO: 38), VRWGMQQIQLVV (SEQ ID NO: 39), VLVRVERATVFS (SEQ ID NO: 42), or collagen-derived peptides KGHRGF (SEQ ID NO: 44) may be selected to sequester or bind bFGF.

[0038] In another embodiment, the present invention discloses a protein substrate for providing a GF-binding peptide motif for sequestering or binding transforming growth factor beta (TGF-β), which may be selected from the heparin / syndecan binding domains of fibronectin, laminin, collagen, vitronectin, or bone sialoprotein for TGF-β binding.Preferably, the fibronectin-derived motifs ANGQTPIQRYIK (SEQ ID NO: 12), KPDVRSYTITG (SEQ ID NO: 13), PRARITGYIIKYEKPGSPPREVVPRPRPGV (SEQ ID NO: 14), WQPPRARI (SEQ ID NO: 15), WQPPRARITGYIIKYEKPG (SEQ ID NO: 16), YEKPGSPPREVVPRPRP (SEQ ID NO: 17), or the laminin-derived peptides GLIYYVAHQNQM (SEQ ID NO: 19), RKRLQVQLSIRT (SEQ ID NO: 20), No. 20), GLLFYMARINHA (SEQ ID NO: 21), KNSFMALYLSKG (SEQ ID NO: 22), VVRDITRRGKPG (SEQ ID NO: 23), RAYFNGQSFIAS (SEQ ID NO: 24), GEKSQFSIRLKT (SEQ ID NO: 25), TLFLAHGRLVFMFNVGHKKL (SEQ ID NO: 26), TLFLAHGRLVFM (SEQ ID NO: 27), LVFMFNVGHKKL (SEQ ID NO: 28), GAAWKIKGPIYL (SEQ ID NO: 29), VIRDSNVVQLDV (SEQ ID NO: 30) ), GKNTGDHFVLYM (SEQ ID NO: 31), RLVSYSGVLFFLK (SEQ ID NO: 32), GPLPSYLQFVGI (SEQ ID NO: 33), RNRLHLSMLVRP (SEQ ID NO: 34), LVLFLNHGHFVA (SEQ ID NO: 35), AGQWHRVSVRWG (SEQ ID NO: 36), KMPYVSLELEMR (SEQ ID NO: 37), RYVVLPR (SEQ ID NO: 38), VRWGMQQIQLVV (SEQ ID NO: 39), TVFSVDQDNMLE (SEQ ID NO: 40), APMSGRSPSLVLK ( SEQ ID NO:41), VLVRVERATVFS (SEQ ID NO:42), RNIAEIIKDI (SEQ ID NO:43), or KGHRGF (SEQ ID NO:44), TAGSCLRKFSTM (SEQ ID NO:45), GEFYFDLRLKGDK (SEQ ID NO:46), or KKQRFRHRNRKGYRSQ (SEQ ID NO:47) from vitronectin, or KRSR (SEQ ID NO:48), KRRA (SEQ ID NO:49) from bone sialoprotein may be selected to bind to or sequester TGF-β.More preferably, the heparin-binding motif may be selected from ANGQTPIQRYIK (SEQ ID NO: 12), KPDVRSYTITG (SEQ ID NO: 13), YEKPGSPPREVVPRPRP (SEQ ID NO: 17) from fibronectin, or KNSFMALYLSKG (SEQ ID NO: 22), RYVVLPR (SEQ ID NO: 38), GKNTGDHFVLYM (SEQ ID NO: 31), RLVSYSGVLFFLK (SEQ ID NO: 32), VLVRVERATVFS (SEQ ID NO: 42) from laminin, or KKQRFRHRNRKGYRSQ (SEQ ID NO: 47) from vitronectin, or KRSR (SEQ ID NO: 48), KRRA (SEQ ID NO: 49) from bone sialoprotein.

[0039] In another embodiment, the present invention discloses a protein substrate for providing a GF-binding peptide motif for sequestering or binding platelet-derived growth factor (PDGF). The PDGF binding motif is a laminin-derived motif GLIYYVAHQNQM (SEQ ID NO: 19), RKRLQVQLSIRT (SEQ ID NO: 20), GLLFYMARINHA (SEQ ID NO: 21), KNSFMALYLSKG (SEQ ID NO: 22), VVRDITRRGKPG (SEQ ID NO: 23), RAYFNGQSFIAS (SEQ ID NO: 24), GEKSQFSIRLKT (SEQ ID NO: 25), TLFLAHGRLVFMFNVGHKKL (SEQ ID NO: 26), TLFLAHGRLVFM (SEQ ID NO: 27), LVFMFNVGHKKL (SEQ ID NO: 28), GAAWKIKGPIYL (SEQ ID NO: 29), VIRDSNVVQLDV (SEQ ID NO: 30), GKNTGDHFVLYM (SEQ ID NO: 31), RLVSYSGVLFFLK (SEQ ID NO: 32), GP It may be selected from LPSYLQFVGI (SEQ ID NO: 33), RNRLHLSMLVRP (SEQ ID NO: 34), LVLFLNHGHFVA (SEQ ID NO: 35), AGQWHRVSVRWG (SEQ ID NO: 36), KMPYVSLELEMR (SEQ ID NO: 37), RYVVLPR (SEQ ID NO: 38), VRWGMQQIQLVV (SEQ ID NO: 39), TVFSVDQDNMLE (SEQ ID NO: 40), APMSGRSPSLVLK (SEQ ID NO: 41), VLVRVERATVFS (SEQ ID NO: 42), RNIAEIIKDI (SEQ ID NO: 43), PGRWHKVSVRWE (SEQ ID NO: 76), or KKQRFRHRNRKGYRSQ (SEQ ID NO: 47) from vitronectin, or KRSR (SEQ ID NO: 48), KRRA (SEQ ID NO: 49) from bone sialoprotein. More preferably, the PDGF binding motif may be selected from RKRLQVQLSIRT (SEQ ID NO: 20), KNSFMALYLSKG (SEQ ID NO: 22), RYVVLPR (SEQ ID NO: 38), GKNTGDHFVLYM (SEQ ID NO: 31), VLVRVERATVFS (SEQ ID NO: 42), or KKQRFRHRNRKGYRSQ (SEQ ID NO: 47) from vitronectin, or KRSR (SEQ ID NO: 48) from bone sialoprotein.

[0040] The present invention further discloses a protein substrate for the sustained release of growth factors under physiological conditions.

[0041] It is well known that interaction with heparin sulfate present in the extracellular matrix not only regulates the diffusion of growth factors such as FGF (Duchesne L,et al,transport of fibroblast growth factor 2 in the pericellular matrix is ​​controlled by the spatial distribution of its binding sites in heparan sulfate.PLoS Biol.2012;10(7):e1001361.,Dowd CJ,et al,Heparan sulfate mediatesbFGF transport through basement membrane by diffusion with rapid reversible binding.J Biol Chem.1999 19;274(8):5236-44), but also directly regulates the storage and release of FGF (Bashkin P,et al.,asic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules.Biochemistry.1989 21;28(4):1737-43).

[0042] In one embodiment, the present invention provides a protein substrate as a sustained release system for TGF-β that does not lose function over a period of several days under physiological conditions, the protein substrate providing a TGF-β binding motif selected from KNSFMALYLSKG (SEQ ID NO: 22), RYVVLPR (SEQ ID NO: 38), GKNTGDHFVLYM (SEQ ID NO: 31), RLVSYSGVLFFLK (SEQ ID NO: 32), and VLVRVERATVFS (SEQ ID NO: 42) for sustained release of TGF-β under physiological conditions.

[0043] The present invention also discloses a protein substrate for simultaneously providing an integrin binding peptide motif and a GF binding peptide motif, wherein the integrin binding motif may be integrated into the N-terminus of a recombinant adhesion protein and a growth factor or cytokine binding motif may be integrated into the C-terminus of said adhesion protein or vice versa.

[0044] Crosstalk between integrins and growth factor receptors is well known. For example, Intestine et al. reported that FGF2-FNIII9-10 fusion protein significantly increased cell adhesion and proliferation of MG63 cells compared to FNIII9-10 alone (Jun-Hyeog Jang & Chong-Pyoung Chung, Engineering and expression of a recombinant fusion protein possessing fibroblast growth factor-2 and fibronectin fragment. Biotechnology Letters volume 26, p1837-1840 (2004)), and FNIII9-10-mediated adhesion promoted the effect of FGF1 on neurite outgrowth of PC12 cells (Choung PH, et al., Synergistic activity of fibronectin and fibroblast growth factor receptors on neuronal adhesion and neurite extension through extracellular signal-regulated kinase pathway. Biochem Biophys Res Commun, 2002, 295:898-902).

[0045] In one embodiment, the present invention discloses a protein substrate that mimics fibronectin domain III having the sequence RGDSGSGSGSGSGANGQTPIQRYIK (SEQ ID NO:58).

[0046] In another embodiment, the present invention discloses a protein substrate mimicking fibronectin domain III (sequence number 59), in which an integrin binding motif RGD (sequence number 50) is integrated at the C-terminus of the adhesion protein and a growth factor or cytokine, e.g., TGF-β binding motif ANGQTPIQRYIK (sequence number 12) is integrated at the N-terminus of the adhesion protein.

[0047] The present invention also provides microenvironment surfaces that simultaneously activate integrins and growth factor receptors to control phenotypic plasticity directly linked to wound healing, angiogenesis or pathogenesis, such as fibrosis or tumor metastasis.

[0048] In one embodiment, the present invention provides a synthetic tumor microenvironment surface comprising the protein substrate to induce epithelial-mesenchymal transition (EMT) in epithelial cells.

[0049] EMT is a process in which epithelial cells lose epithelial proteins, including E-cadherin, and acquire mesenchymal markers such as N-cadherin, vimentin, and fibronectin. EMT is linked to many processes including embryonic or cancer development and / or progression (Kalluri R, et al. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119:1420-8. Jordan NV, et al. Tracking the intermediate stages of epithelial-mesenchymal transition in epithelial stem cells and cancer. Cell Cycle. 201;10:2865-73), wound healing and tissue repair, and cell migration (Hosaka K, et al. Pericyte-fibroblast transition promotes tumor growth and metastasis. Proc Natl Acad Sci US A. 2016;113:E5618-27. Yang X, et al. Silencing Snail suppresses tumor cell proliferation and invasion by reversing epithelial-to-mesenchymal transition and arresting G2 / M phase in non-small cell lung cancer. Int J Oncol. 2017;50:1251-60). Although the EMT event is essential for development and wound repair, it has also been recognized as a contributing factor to fibrotic diseases and cancer. Many soluble and insoluble factors, including TGF-β and ECM proteins, determine the extent and duration of the EMT event. Specifically, cytokines such as TGF-β, TNFα, and IL6 and hypoxia can induce EMT in a variety of tumors.

[0050] Several extracellular matrix (ECM) proteins, including collagen-I, fibronectin, and hyaluronan, and ECM remodeling by extracellular lysyl oxidase also influence the regulation of EMT (Hae-Yun Jung, et al. Clin Cancer Res; 21(5) March 1, 2015. Molecular Pathways: Linking Tumor Microenvironment to Epithelial-Mesenchymal Transition in Metastasis). Several integrins are known to mediate EMT. For example, EMT is regulated by integrin αvβ6 through activation of the TGF-β1-Smad2 / 3 signaling pathway (Wang J, et al. (2015) Interleukin-1beta promotes epithelial-derived alveolar elastogenesis via αvβ6 integrin-dependent TGF-beta activation. Cell Physiol Biochem 36:2198-2216). The expression of several integrin complexes, including α5β1, which binds to fibronectin, and integrins α1β1 and α2β1, which interact with collagen I, are also upregulated during EMT and have been shown to mediate the disruption of the E-cadherin complex. Cell interactions with the ECM have been shown to be regulated by ECM-associated proteins, such as SPARC, a glycoprotein that promotes the interaction of collagen with α2β1.

[0051] In one embodiment, the present invention provides a protein substrate comprising an integrin binding motif selected from integrin α5β1, α9β1, or αvβ3 binding motifs, and a cytokine binding motif selected from ANGQTPIQRYIK (SEQ ID NO: 12), KPDVRSYTITG (SEQ ID NO: 13), or YEKPGSPPREVVPRPRP (SEQ ID NO: 17) for inducing EMT in a breast cancer cell line, MCF-10A.

[0052] The present invention also provides methods for stabilizing a growth factor or cytokine against loss of biological activity over extended periods of time in cell culture conditions by mixing a protein substrate containing a growth factor or cytokine binding motif with the growth factor or cytokine in cell culture medium or a buffer solution, e.g., PBS buffer.

[0053] In one embodiment, a composition comprising a protein substrate providing a cytokine binding motif ANGQTPIQRYIK (SEQ ID NO: 12) and a growth factor binding motif YEKPGSPPREVVPRPRP (SEQ ID NO: 17) is dissolved in a cell culture medium such as DMEM or a buffer solution such as PBS. The composition can be stored for at least one week without loss of its biological activity, as demonstrated by an EMT promotion test in breast epithelial cell line MCF-10A.

[0054] The present invention will now be described in detail with reference to Preparation Examples, Examples, and Experimental Examples thereof.

[0055] However, it should be understood that the following Preparations, Examples, and Experimental Examples are provided for the purpose of illustrating the present invention only, and are not intended to limit the scope of the present invention.

[0056] Working Example Example 1. Preparation of a protein substrate providing an integrin-binding motif and a heparin-binding motif E. coli-based protein expression systems have been commercialized for the efficient production of various mussel adhesive proteins, including fusion proteins of mussel byssus protein 1 and byssus protein 5 (see US2020 / 0062809A1 and WO2011 / 115420A2), which are commercially available under the brand MAPTrix® marketed by Kollodis Biosciences, Inc. Methods for producing mussel adhesive proteins are fully described in US20200 / 062809A1 and WO2011 / 115420A2, which are incorporated by reference for all purposes as if fully disclosed herein.

[0057] The basic scheme of the protein substrate is illustrated in Figure 1a. As shown in Figure 1a, two peptide motifs can be integrated into the N-terminus and C-terminus of the mussel adhesive protein, respectively (Scheme A). Alternatively, as shown in Figure 1b, both motifs can be integrated into the C-terminus or N-terminus (Scheme B), where a spacer linker peptide such as SGSGSGSGSG effectively separates the two peptides for a synergistic effect.

[0058] Two types of protein substrates with sequence number 58 (hereinafter referred to as "MAP-RGD-GF") and sequence number 59 (hereinafter referred to as "GF-MAP-RGD") were recombinantly designed as disclosed in US2020 / 0062809A1 and WO2011 / 115420A2, and expressed and purified in an E. coli expression system. Multiple protein substrates with GF binding motifs were produced by the same procedure. All protein substrates were lyophilized and stored in a refrigerator for further experiments.

[0059] Example 2. Growth factor binding assay Multiple arrays for growth factor or cytokine binding assays consist of multiple heparin-binding peptide motifs arrayed in a 96-well format as shown in Figures 2a and 2b, where Figure 2a is an array layout of GF-binding motifs derived from fibronectin, collagen, and laminin, and Figure 2b is an array layout of GF-binding motifs derived from the laminin globular domain.

[0060] For specific binding of peptide motifs to growth factors or cytokines, low cell attachment or superhydrophobic surfaces are coated with substrate proteins, where the substrate proteins are formed as particles. Thus, the surfaces look like particle islands, as shown in FIG. 1b. The coating method is state of the art, and the detailed procedure is described in US patent application US16 / 546,966, developed by the present inventors and fully incorporated herein by reference. The surfaces allow biomolecules such as GFs or target cells to specifically bind to the peptide motifs provided on the surface. Non-target biomolecules or cells are forced to suspend and are eventually washed away.

[0061] Recombinant basic FGF, TGF-β, and PDGF-BB were purchased from R&D Systems (Camarillo, CA), Ultralow cell attachment 96-well plates were purchased from Corning, NY, fatty acid-free bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louise, MO), and fetal bovine serum was purchased from Thermo Fisher Scientific (Waltham, MA).

[0062] To eliminate or minimize any non-specific binding of recombinant growth factors, the 96-well plate surface coated with substrate proteins was blocked with 2 wt% BSA in PBST buffer (4 mM phosphate and 155 mM sodium chloride, 0.05 wt% Tween-20, pH 7.4) by agitating the plate for 1 hour at room temperature. Then, individual recombinant growth factors (50 nM) dissolved in PBST were added to the blocked well plate containing heparin-binding motifs, and the plate was agitated overnight at 4°C. The binding of growth factors to the protein substrate coated on the well plate was confirmed by immunoaffinity assay. Briefly, the well plate was treated sequentially with a primary antibody against the growth factor and a secondary antibody labeled with horseradish peroxidase (HRP). Finally, the TMB (3,3',5,5'-tetramethylbenzidine) substrate was chemically exchanged with HRP and the absorbance values ​​at 450 nm were used to analyze the binding of growth factors to each binding motif. Figure 3 shows the TMB absorbance values ​​corresponding to growth factor binding, from which the absorbance values ​​of blank samples lacking growth factors were subtracted. Absorbance readings exceeding 0.1 were considered as significant interactions.

[0063] As shown in Figures 4a and 4b, the present inventors identified GF-binding motifs derived from fibronectin that specifically bind to bFGF, TGF-β, and PDGF-BB. The GF-binding motifs with high affinity for each growth factor are summarized in Table 1 below.

[0064] [Table 1]

[0065] As shown in FIG. 4c, most laminin-derived GF-binding peptide motifs showed higher affinity for TGF-β and PDGF, but relatively low affinity for bFGF, similar to the case of the fibronectin GF-binding motif.

[0066] Example 3. Sustained release of growth factors bound to protein substrates Heparin-binding domains in ECM proteins have been shown to stabilize growth under physiological conditions. To determine whether the GF-binding peptide motif confers any protective effect against growth factors, TGF-β was conditioned with PBS buffer or cell culture medium DMEM in the presence or absence of the GF-binding peptide motif identified in Example 2. bFGF (0.5 μg) was added to the protein substrate-coated surface and incubated at 37° C. for 2, 4, and 7 days. The inventors evaluated the safety of TGF-β as a function of time at 37° C. using enzyme-linked immunosorbent assay (ELISA) and found that it was gradually released from the protein substrate with a half-life of 4 days. As shown in FIG. 5b, the GF-binding motif (ANG-MAP-RGD-IDAP) with high affinity for TGF-β exhibited long-term sustained release of TGF-β under cell culture conditions.

[0067] Example 4. Synthetic microenvironments for inducing transdifferentiation of epithelial cells into mesenchymal cells The TGF-β binding motifs identified in Examples 2 and 3 were arrayed on a 6-well plate. After treatment with TGF-β (5 ng) as disclosed in Example 2, the 6-well plate was stored in a CO2 incubator at 37°C for a given period of time (e.g., 2, 4, and 7 days, respectively). Breast epithelial cells MCF-10A were seeded and cultured on the well plate coated with the TGF-β binding peptide motif. As shown in Figure 6b, the two motifs with high affinity for TGF-β were able to induce MCF-10A to undergo EMT, but the low TGF-β binding affinity motif WQPPRARI (SEQ ID NO: 15) was unable to induce EMT as evidenced by the lack of fibronectin upregulation.

[0068] Example 5. GF protection from trypsin activity The TGF-β binding peptide motifs identified in Examples 2 and 3 were incubated with TGF-β (2 μg) and trypsin (0.3 μg) for 15 minutes at 30° C. After trypsinization, the proteins of the samples were separated by 15% SDS-PAGE in a size-dependent manner. The SDS-PAGE was then stained with Coomassie blue solution for 2 hours. After destaining, the intensity of the bands stained with Coomassie blue solution was analyzed. As shown in FIG. 7, the ANGQTPIQRYIK (SEQ ID NO: 12), ANG-M-RGD (SEQ ID NO: 59), KNSFMALYLSKG (SEQ ID NO: 22), KRSR (SEQ ID NO: 48), and RKRLQVQLSIRT (SEQ ID NO: 20) motifs inhibited the trypsinization of TGF-β compared to the absence of the MAP-fusion motif.

[0069] Example 6. Growth factor binding protein substrate for cell growth A total of 96-well plates (non-tissue culture treated, SPL Life Science) were coated with protein substrates bearing GF-binding peptide motifs at 50 μg / ml in sodium acetate buffer for 1 h at room temperature. 20 ng / ml FGF2 and PDGF-BB (BioVision, Milpitas, CA, USA) in DMEM with 0.5% FBS were added to individual wells and incubated at 37°C in a CO2 incubator for 1 h. Cell growth assays were performed using human foreskin fibroblasts (Hs68, ATCC) in DMEM medium (Invitrogen) supplemented with 0.5% fetal bovine serum (FBS). Cells were seeded at 1,500 cells / well on plates pre-coated with GF-binding substrates and incubated at 37°C in a CO2 incubator for 48 h. CCK-8 assays were then performed to determine cell growth. Hs68 cells were checked for mycoplasma contamination and then used for passages 5 to 10. Protein substrates lacking the GF-binding peptide motif and heparin were used as negative and positive controls, respectively.

[0070] As shown in Figures 8a and 8b, the GF-bound protein substrate supported cell growth and proliferation. GF bound to the protein substrate strongly supported the growth and proliferation of human foreskin fibroblast cells in low serum conditions (0.5% FBS) when compared to heparin-bound GF.

[0071] It will be apparent from the foregoing teachings that modifications and variations can be made to the disclosed subject matter to be adapted to various uses and conditions, such embodiments also falling within the scope of the following claims.

[0072] The recitation of a list of elements in any definition of a variable disclosed herein includes a definition of that variable as any single element or combination (or subcombination) of the elements listed. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0073] All patents and publications cited in this specification are herein incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

Claims

1. A protein substrate comprising a recombinant adhesion protein genetically functionalized with an integrin binding motif and a heparin binding motif capable of binding to or sequestering a growth factor.

2. 2. The protein substrate of claim 1, wherein the heparin-binding motif is derived from the group consisting of fibronectin domain III, laminin globular domain, and heparin-binding domain of collagen, vitronectin, and bone sialoprotein.

3. The heparin binding motifs derived from fibronectin domain III are KYILRWRPKNS (SEQ ID NO: 7), YRVRVTPKEKTGPMKE (SEQ ID NO: 8), SPPRRARVT (SEQ ID NO: 9), ATETTITIS (SEQ ID NO: 10), VSPPRRARVTDAETTTITISWRTKTETITGFG (SEQ ID NO: 11), ANGQTPIQRYIK (SEQ ID NO: 12), KPDV The protein substrate of claim 2, selected from the group consisting of RSYTITG (SEQ ID NO: 13), PRARITGYIIKYEKPGSPPREVVPRPRPGV (SEQ ID NO: 14), WQPPRARI (SEQ ID NO: 15), WQPPRARITGYIIKYEKPG (SEQ ID NO: 16), YEKPGSPPREVVPRPRP (SEQ ID NO: 17), and KNNQKSEPLIGRKKT (SEQ ID NO: 18).

4. The heparin binding motifs derived from the laminin globular domain include GLIYYVAHQNQM (SEQ ID NO: 19), RKRLQVQLSIRT (SEQ ID NO: 20), GLLFYMARINHA (SEQ ID NO: 21), KNSFMALYLSKG (SEQ ID NO: 22), VVRDITRRGKPG (SEQ ID NO: 23), RAYFNGQSFIAS (SEQ ID NO: 24), GEKSQFSIRLKT (SEQ ID NO: 25), TLFLAHGRLVFMFNVGHKKL (SEQ ID NO: 26), TLFLAHGRLVFM (SEQ ID NO: 27), LVFMFNVGHKKL (SEQ ID NO: 28), GAAWKIKGPIYL (SEQ ID NO: 29), VIRDSNVVQLDV (SEQ ID NO: 30), GKNTGDHFVLYM ( 31), RLVSYSGVLFFLK (SEQ ID NO:32), GPLPSYLQFVGI (SEQ ID NO:33), RNRLHLSMLVRP (SEQ ID NO:34), LVLFLNHGHFVA (SEQ ID NO:35), AGQWHRVSVRWG (SEQ ID NO:36), KMPYVSLELEMR (SEQ ID NO:37), RYVVLPR (SEQ ID NO:38), VRWGMQQIQLVV (SEQ ID NO:39), TVFSVDQDNMLE (SEQ ID NO:40), APMSGRSPSLVLK (SEQ ID NO:41), VLVRVERATVFS (SEQ ID NO:42), PGRWHKVSVRWE (SEQ ID NO:76) and RNIAEIIKDI (SEQ ID NO:43).

5. 3. The protein substrate of claim 2, wherein the heparin binding motif derived from the heparin binding domain of the collagen is selected from the group consisting of KGHRGF (SEQ ID NO: 44), TAGSCLRKFSTM (SEQ ID NO: 45), and GEFYFDLRLKGDK (SEQ ID NO: 46).

6. 3. The protein substrate of claim 2, wherein the heparin-binding motif derived from the heparin-binding domain of vitronectin is KKQRFRHRNRKGYRSQ (SEQ ID NO: 47).

7. 3. The protein substrate of claim 2, wherein the heparin binding motif derived from the heparin binding domain of bone sialoprotein is KRSR (SEQ ID NO: 48) or KRRA (SEQ ID NO: 49).

8. 2. The protein substrate of claim 1, wherein the heparin-binding motif is capable of binding to basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), or platelet-derived growth factor (PDGF).

9. The protein substrate of claim 1 , wherein the integrin binding motif is selected from αvβ3-, αvβ6-, αvβ8-, α5β1-, and α9β1-binding peptides.

10. The protein substrate of claim 1 , wherein the integrin binding motif is capable of activating integrin αvβ6 and the heparin binding motif is capable of binding to TGF-β.

11. The protein substrate of claim 1 , wherein the integrin binding motif is capable of activating integrin α5β1 or α9β1 and the heparin binding motif is capable of binding to bFGF.

12. 2. The protein substrate of claim 1, wherein the recombinant adhesive protein is derived from a recombinant mussel adhesive protein.

13. 2. The protein substrate of claim 1, wherein the recombinant mussel adhesive protein comprises a peptide selected from the group consisting of SEQ ID NOs: 1-6, and 60-74.

14. 2. The protein substrate of claim 1, wherein the integrin binding motif and / or the heparin binding motif are attached to the N-terminus and / or the C-terminus of the recombinant adhesion protein.

15. 2. The protein substrate of claim 1, wherein the integrin binding motif and the heparin binding motif are both attached to the N-terminus or C-terminus of the recombinant adhesion protein.

16. The protein substrate of claim 15 , wherein the integrin binding motif and the heparin binding motif are linked via a spacer linker peptide.

17. The protein substrate of claim 16 , wherein the spacer linker peptide is a peptide of SEQ ID NO:

75.

18. An extracellular microenvironment surface for modulating cellular plasticity, comprising:

18. An extracellular microenvironment surface comprising a protein substrate according to any one of claims 1 to 17, wherein the microenvironment surface is capable of inducing combinatorial signal transduction by simultaneously activating integrins and growth factor receptors.

19. The external microenvironment surface of claim 18, wherein the plasticity of the cells is cells that are at an epithelial-mesenchymal transition.

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