CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Serial No 61/667,630 filed July 3, 2012, which is hereby incorporated by reference herein. TECHNICAL FIELD

The technical field, in general, relates to peptides that bind to extracellular matrices via specific binding interactions.

BACKGROUND

The extracellular matrix (ECM) provides structural support for tissue and signaling capabilities for cells. The ECM plays an important role in development and tissue repair.

SUMMARY OF THE INVENTION

As reported herein, it has been discovered that placenta growth factor (PIGF) exhibits specific binding activity towards ECM. PIGF is an angiogenic cytokine that exists in multiple splice variants. PIGF was originally identified in the placenta, where it has been proposed to control trophoblast growth and differentiation. PIGF is expressed during early embryonic development. PIGF has been shown to be expressed in the villous trophoblast, while vascular endothelial growth factor (VEGF) is expressed in cells of mesenchymal origin within the chorionic plate. PIGF is expressed in several other organs including the heart, lung, thyroid, skeletal muscle, and adipose tissue. PIGF acts as a potent stimulator of VEGF secretion by monocytes and significantly increases mRNA levels of the proinflammatory chemokines interleukin-1 beta, interleukin-8, monocyte chemoattractant protein- 1, and VEGF in peripheral blood mononuclear cells of healthy subjects. PIGF induces tumor angiogenesis by recruiting circulating hematopoietic progenitor cells and macrophages to the site of the growing tumors (Ribatti D, 2008).

An embodiment is an isolated polypeptide comprising a sequence chosen from the group consisting of SEQ ID NO:4 having from 0 to 5 conservative substitutions, SEQ ID NO:5 having from 0 to 5 conservative substitutions, and subsequences thereof. Said subsequences may be chosen as exhibiting specific binding to one or more of fibrinogen, fibronectin, vitronectin, tenascin C, osteopontin, and fibrin. A dissociation constant may be specified, for example, wherein the specific binding of the polypeptide to fibrinogen has a dissociation constant (KD) of less than about 100 nM, or less than about 40 nM, or less than about 25 nM.

An embodiment is a biologic delivery vehicle comprising a molecular fusion of a biological agent and a peptide comprising a sequence or subsequence of at least 6 residues of a sequence chosen from the group consisting of SEQ ID NO: 4 having from 0 to about 15% conservative substitutions and SEQ ID NO: 5 having from 0 to about 15% conservative substitutions. As explained in more detail herein, the peptide exhibits specific binding to one or more of, or all, of the extracellular matrix molecules selected from the group consisting of fibrinogen, fibronectin, vitronectin, tenascin C, osteopontin, fibrin, collagen, Collagen I, and heparin sulfate. In fact, the tested peptides exhibited specific binding to all of said extracellular matrix molecules. Examples of biologic agents are those chosen from the group consisting of a protein, a protein drug, a marker, an immunoagent, a chemokine, a cytokine, and a cell adhesion peptide. The term cytokine, as used herein, includes growth factors and morphogens.

An embodiment is a biomaterial comprising a matrix, with the matrix comprising a polypeptide comprising a sequence chosen from the group consisting of SEQ ID NO:4 having from 0 to 5 conservative substitutions, SEQ ID NO: 5 having from 0 to 5 conservative substitutions, and all subsequences thereof, said peptide exhibiting specific binding to an extracellular matrix molecule. The matrix may be natural or synthetic and covalently crosslinked, crosslinked without covalent binds, or free of crosslinks.

An embodiment is a medicament comprising a peptide, vehicle, or biomaterial comprising a P1GF2, e.g., a domain of P1GF2. The medicament may be used, e.g., in a medical treatment, to make a medical composition, e.g., as a vaccine, for drug delivery, wound healing, and tissue healing, e.g., healing of a bone, fistula, or an ulcer.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 : A domain within P1GF2 (PlGF2i ₂ 3-i44) strongly and promiscuously binds ECM proteins, (a) GF binding to ECM proteins, measured by ELISA. A signal over 0.1 (gray box) was considered as representative of a specific binding. P1GF2 strongly binds all ECM proteins tested (gray bars), (b) Alignment of the protein sequences of the splice variants P1GF2 and PlGF-1 (which does not bind). P1GF2 contains an additional 21 amino-acid insert (PlGF2i ₂ 3_i44, in gray) located near the C-terminus. (c) Binding of PlGF2i ₂ 3_i44 to ECM proteins when fused to a non-binding model protein, GST (GST-PIGF2123-144). A scrambled version of PlGF2i ₂ 3-i44 (GST-PlGF2 _scr ) does not bind ECM proteins. In (a) and (c), n > 3, mean ± SEM. The alignment shows sequences of PlGF-1 (PlGF-1 LPAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLVDVVSEYPSEVEHMFS PSCVSLLRCTGCCGDENLHCVPVETANVTMQLLKIRSGDRPSYVELTFSQHVRCECR PLREKMKPERCGDAVPRR (SEQ ID NO:58) as compared to P1GF2 (LPAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLVDVVSEYPSEVEHMFS PSCVSLLRCTGCCGDENLHCVPVETANVTMQLLKIRSGDRPSYVELTFSQHVRCECR PLREKMKPERRRPKRGKRRREKQRPTDCHLCGDAVPRR, SEQ ID NO:59).

FIGURE 2: Binding of various GST-PlGF2i ₂ 3_i44 fragments to fibronectin, collagen I, heparan sulfate, and neuropilin-1. (a) Design of GST-PlGF2i ₂ 3_i44 fragments, (b) Binding of GST-P1GF2123-144 fragments to fibronectin, collagen I, heparan sulfate, and neuropilin-1. The depicted alignments include fragments of GST-P1GF2:

RRRPKGRGKRRREKQRPTDCHLCGDAVPRR (SEQ ID NO: 60), RRRPKGRGKRRREKQRPTDCHL (SEQ ID NO:61), RRPKGRGKRRREKQRPTD (SEQ ID NO:62), RRRPKGRGKRRREKQ (SEQ ID NO: l), GKRRREKQ (SEQ ID NO:2), and RRRPKGRG (SEQ ID NO:3).

FIGURE 3: The heparin-binding domain of VEGF-A165 is substituted with PlGF2i ₂ 3_ 144 (black box) to generate VEGF-A121-PlGF2i ₂₃ _i4 ₄ (SEQ ID NO: 7). PlGF2i ₂₃ _i4 ₄ is fused to the C-terminus of PDGF-BB to generate PDGF-BB-PlGF2i ₂₃ _i ₄ 4 (SEQ ID NO: 9) PlGF2i ₂₃ _ 144* (gray box) containing a point mutation (Cysi ₄₂ to Ser) is inserted at the C-terminus of BMP-2 to generate BMP-2-PlGF2i ₂₃ _i ₄ 4* (SEQ ID NO: 13).

FIGURE 4: has 2 panels, (a) Cytokines-PlGF2i ₂ 3_i44(*) binding to ECM proteins (fibronectin, vitronectin, tenascin C, osteopontin, collagen I, fibrinogen) and heparan sulfate measured by ELISA. ELISA plates were coated with cytokines and further incubated with ECM proteins at increasing concentration (0.02 to 320 nM). Bound ECM proteins were detected using antibodies. The binding curve was fitted by non- linear regression to obtain the dissociation constant (K _D ) using A4 ₅ o _n m ⁼ Bmax*[concentration]/(K _D + [concentration]), n = 3, mean ± SEM. (b) Cytokines-PlGF2i ₂ 3_i44(*) are retained in fibrin matrix. Fibrin matrices were made in the presence of wildtype cytokines (PlGF-1, P1GF2, VEGF-A121, VEGF-A165, PDGF-BB, and BMP-2) or modified cytokines (VEGF-A121-PlGF2i ₂₃ _i ₄ 4, PDGF-BB- P1GF2 i ₂ 3_i44, or BMP-2-PlGF2i ₂ 3_i44(*) and further incubated in 8 volumes of physiological buffer for 7 days. The buffer was changed every day, and cumulative released of cytokines were quantified for each day. Wildtype PlGF-1, VEGF-A121, VEGF-A165, PDGF-BB, and BMP-2 were quickly released, while VEGF-A121-PlGF2i ₂₃ -i44, PDGF-BB-PlGF2i ₂₃ _i ₄₄ , and BMP-2-PlGF2i ₂₃ _i ₄₄ * were sequestered in the matrix.

FIGURE 5: In vitro, PlGF2i ₂₃ _i ₄₄ -fused GFs shows similar bioactivity compared to wild-type GFs. (a) Human ECs were stimulated with VEGF-A121, VEGF-A165, or VEGF- A-PlGF2i ₂₃ _i ₄₄ , and (b) human mesenchymal stem cells were stimulated with PDGF-BB or PDGF-BB-PlGF2i ₂₃ _i ₄₄ . Phosphorylated GF receptors (VEGFR-2 and PDGFR-β) were quantified by ELISA (n = 3, mean ± SEM). The insertion of the PlGF2i ₂₃ _i ₄₄ into VEGF-A and PDGF-BB do not alter their signaling. Moreover, the insertion of PlGF2i ₂₃ _i ₄₄ into VEGF-A121 increases its activity to the level of VEGF-A165. As it is the case for VEGF- A 165, this increased activity on receptor phosphorylation is most likely due the binding of PlGF2i ₂₃ _i ₄₄ to neuropilin-1, which increases VEGF-A potency in stimulating VEGFR-2 phosphorylation(Migdal M, et al, 1998; Pan Q, et al, 2007; Whitaker GB, et al, 2001). The Student t-test was used for statistical comparisons; */?<0.05, **/?<0.01. (c) BMP-2-PlGF2i ₂₃ _ i ₄₄ * was evaluated by its ability to promote ALP activity in human mesenchymal stem cells (induction of osteoblastic differentiation). Cellular ALP was quantified after 14 days of culture in presence of BMP-2 or BMP-2-PlGF2i ₂₃ _i ₄₄ *. No differences in cell number and ALP activity were observed between cells treated with BMP-2 or BMP-2-PlGF2i ₂₃ _i ₄₄ *. Results are expressed as ng of ALP/10k cells (n = 4, mean ± SEM).

FIGURE 6: PlGF2i ₂₃ _i ₄₄ - fused GFs display enhanced affinity for ECM components. (a) Affinity (shown is K _D ) of wild-type versus PlGF2i ₂₃ _i ₄₄ -fused GFs for ECM proteins and heparan sulfate, n = 3, mean ± SEM. (b-f) PlGF2i ₂₃ _i ₄₄ -fused GFs are retained at the site of delivery for an extended period relative to wild-type GFs. (b) VEGF-A165 and VEGF-A- PlGF2i ₂₃ _i ₄₄ retention when injected subcutaneously in the back skin of mice, n = 6 per time point, mean ± SEM. (c-f) Wildtype and PlGF2i ₂₃ _i ₄₄ - fused GF retention when placed in 5 mm diameter defects in the mouse back skin (c,d) or mouse calvarium (e,f) filled with a fibrin matrix. Retention after 3 and 6 days in the fibrin matrix (gray bars) and the tissue surrounding the defect (black bars, 2 mm farther), n > 4 per time point, mean ± SEM. For all panels, Student's t-test; **/?<0.01, ***/?<0.001.

FIGURE 7: VEGF-A-PlGF2i ₂₃ _i ₄₄ and PDGF-BB-PlGF2i ₂₃ _i ₄₄ induce greater skin wound healing and angiogenesis than wildtype VEGF-A and PDGF-BB. (a-j) Delivering low doses (200 ng of each, combined) of VEGF-A-PlGF2i ₂₃ _i ₄₄ and PDGF-BB-PlGF2i ₂₃ _i ₄₄ promoted skin-wound healing in diabetic mice, while the same doses of wild-type VEGF- A165 and PDGF-BB did not. Full -thickness back- skin wounds (6 mm diameter) were treated with GFs delivered topically (at day 0, 3, and 6 for wounds analyzed at day 10; at day 0, 3, 6, and 9 for wounds analyzed at day 15) or delivered once in a fibrin matrix. Six different groups were tested: topically, PBS vehicle only, VEGF-A 165 + PDGF-BB, and VEGF-A- PlGF2i ₂ 3_i44 + PDGF-BB-PlGF2i ₂ 3_i4 ₄ ; in fibrin, fibrin only, fibrin containing VEGF-A 165 + PDGF-BB, and fibrin containing VEGF-A-PlGF2i ₂₃ _i ₄ 4 + PDGF-BB-PlGF2i ₂₃ _i ₄₄ . After 10 and 15 days (topical groups; a-b), or 7 and 10 days (fibrin groups; f-g), wound closure and granulation tissue formation were evaluated by histology. All points are mean ± SEM (n = 8- 10 wounds per group per time point. Student's t-test; *p<0.05, **/?<0.01, ***/?<0.001. (c,h) Representative histology at 10 days for the fibrin groups and at 15 days for the topical groups (hematoxylin and eosin staining). Black arrows indicate wound edges; red arrows indicate tips of healing epithelium tongue. The granulation tissue, stained in pink- violet. Muscle under the wounds is stained in pink-red. Scale bar = 1 mm. (d,e,i,j) Quantification of the angiogenesis within the granulation tissue. After 10 and 15 days (topical groups; d,e), or 7 and 10 days (fibrin groups; I, J), wound tissues were stained for ECs (CD3 T cells) and SMCs (desmin ⁺ cells); dual staining indicates stable vascular morphology (n > 4 per time point, mean ± SEM). Wild-type GFs were compared to PlGF2i ₂₃ _i ₄₄ -fused GFs using the Student's t-test; *p<0.05, **/?<0.01, ***/?<0.001.

FIGURE 8: VEGF-A-PlGF2i ₂ 3_i ₄₄ induces much less vascular permeability than the same dose of wild-type VEGF-A165 (10 μg). (a) The graphs show measurement of vascular permeability in the mouse ear skin, n > 4, mean ± SEM. For statistical comparisons, VEGF- A165 was compared to VEGF-A-PlGF2i ₂ 3_i ₄₄ using non-parametric Mann-Whitney U test; */?<0.05. (b,c) Representative images of the mouse ear skin vasculature 20 min after VEGF- A application. Permeability induced by VEGF-A is visualized by the red-labeled dextran leaking from the vessels. Scale bar = 0.2 mm.

FIGURE 9: Delivering PDGF-BB-PlGF2i ₂₃ _i ₄₄ and BMP-2-PlGF2i ₂₃ _i ₄₄ * induce greater bone regeneration in the rat than wild-type PDGF-BB and BMP-2. Critical-size calvarial defects (6 mm diameter) were treated with GFs delivered topically or in a fibrin matrix. Six different groups were tested: topically, saline vehicle only, BMP-2 + PDGF-BB, and BMP -2 -P1GF2123.144* + PDGF-BB-PlGF2i ₂₃ _i4 ₄ ; and in fibrin, fibrin only, fibrin containing BMP-2 + PDGF-BB, and fibrin containing BMP-2-PlGF2i ₂₃ -i44* + PDGF-BB- PlGF2i23-i44- The doses were 1 μg of each GF, combined, for the groups treated topically to the dura and 200 ng of each GF, combined, for the groups with fibrin, (a-d) Four weeks after treatment, bone repair was measured by μCT as bone volume and coverage of the defect (a,b show groups topical groups; c,d show fibrin groups), (e-j) Representative calvarial reconstructions, e, saline vehicle; f, BMP-2 + PDGF-BB; g, BMP-2-PlGF2i ₂₃ -i44* + PDGF- BB-P1GF2123-144; h, fibrin only, i, fibrin with BMP-2 + PDGF-BB; j, fibrin with BMP-2- PlGF2i23-i44* + PDGF-BB-PlGF2i23-i44). The defect area is shaded. Data are means ± SEM (n = 6 per condition). For statistical comparisons, wild-type GFs were compared to PlGF2i ₂ 3_i44- fused GFs using the Student's t-test; **/?<0.01 , ***/?<0.001.

DETAILED DESCRIPTION

As reported herein, it has been discovered that placenta growth factor (PIGF) exhibits specific binding activity towards ECM. Aspects of the invention include PIGF polypeptides, molecular fusions of PIGF for delivery of biologies, biomaterials incorporating PlGFs, and drug delivery. The PIGF polypeptides may include or be limited to, e.g., one or more domains or fragments of PIGF.

Fibronectin

Fibronectin (FN) is widely expressed by multiple cell types and is critically important in many ECM-dependent (Krammer A, et ah, 2002) processes in the vertebrate, by playing important roles in cell adhesion, migration, growth and differentiation (Mao Y and Schwarzbauer JE, 2005; Pankov R and Yamada KM, 2002). FN is a dimeric glycoprotein composed of two nearly identical 230-270 kDa subunits linked covalently near their C- termini by a pair of disulfide bonds. Each subunit consists of three types of repeating modules, type I, II and III. These modules comprise functional domains that mediate interactions with other ECM components, with cell surface receptors and with FN itself. FN contains 12 type I repeats, 2 type II repeats and 15-18 type III repeats. FN can be subdivided into two forms, soluble plasma FN (abundant soluble constituent of plasma [300 μg/mL]) and less-soluble cellular FN. Plasma FN is secreted by hepatocytes and enriched in blood whereas cellular FN is secreted by fibroblasts and many other cell types and is incorporated into a fibrillar matrix at the cell surface. Cellular FN consists of a much larger and more heterogeneous group of FN isoforms that result from cell-type specific splicing patterns producing FNs with different cell-adhesive, ligand-binding, and solubility properties that provide a mechanism for cells to precisely alter the composition of the ECM in a developmental and tissue-specific manner.

FN is a ligand for several members of the integrin receptor family. The most well studied recognition sequence, the tripeptide RGD, is located in the 10 ^th type III repeat (FN III 10). The recognition of this simple tripeptide sequence is complex and depends on flanking residues, its three dimensional presentation and individual features of the integrin-binding pockets. For example, a second site in the 9 type III repeat (FN ΙΠ9), the "synergy site" comprising the pentapeptide PHSRN (SEQ ID NO:50) {Mardon HJ and Grant KE, 1994), promotes specific α5β1 integrin binding to FN and in FN 1119-10, via interactions with the a5 subunit {Mould AP, et al, 1997) whereas ανβ3 integrin binding to RGD is independent of the synergy site {Damn EH, et al., 1995). Integrin α5β1 is the initial receptor mediating assembly of FN in fibrillar matrix formation {Mao Y and Schwarzbauer JE, 2005; Pankov R and Yamada KM, 2002).

In addition to integrin binding, FN also binds cytokines. The second heparin binding domain of FN (FN III 12- 14) binds most growth factors (cytokines capable of stimulating cellular growth) from the platelet-derived growth factor and fibroblast growth factor families, and some growth factors from the transforming growth factor beta and neurotrophin families {Martino MM and Hubbell J A, 2010).

Although FN molecules are the product of a single gene, the resulting protein can exist in multiple forms that arise from alternative splicing of a single pre-mRNA that can generate as many as 20 variants in human FN. A major type of splicing occurs within the central set of type III repeats (FN III7 to FN III 15). Exon usage or skipping leads to inclusion or exclusion of either of two type III repeats - EDB (also termed EIIIB or EDII and located between FN repeats III7 and III8) and EDA (also called EIIIA or EDI and located between FN repeats III 11 and III 12). The alternatively spliced EDA and EDB domains are almost always absent from plasma FN. Binding of a ₄ ias well as α,9βι to an EDGIHEL sequence (SEQ ID NO: 51) located within the alternatively spliced EDA segment has been reported, suggesting a possible adhesive function for the increased EDA-containing FN species. FN EDA has been explored as a platform for subunit vaccines. Based on the observation that FN EDA ligates and activates Toll-like receptor 4 (TLR4), one research group has explored using FN EDA as an adjuvant DAMP in subunit vaccines, generating the fusion protein FN III EDA-antigen (Lasarte JJ, et al., 2007). A fusion protein containing EDA and the MHC I epitope SIINFEKL derived from ovalbumin at the C-terminus as well as a fusion protein containing EDA and the full ovalbumin improved ovalbumin presentation by DCs and induced cytotoxic response in vivo. These EDA recombinant proteins were shown to protect mice from a challenge with tumor cells expressing ovalbumin. In spite of a useful effect of FN EDA in recombinant subunit vaccines, the adjuvancy of FN EDA has not been adequate to confer protection in viral challenge models in the mouse (Mansilla C, et al., 2009). Indeed, a combination with another adjuvant, poly(LC), and anti-CD40 was needed to downregulate intrahepatic expression of hepatitis virus R A. As such, FN EDA has been found to be insufficiently potent for the arts of vaccinology.

Tenascin C

Tenascin C (TNC) is a large multifunctional extracellular matrix glycoprotein that is present during development and re-expressed in adult life in the case of tissue remodeling, such as wound healing (Trebaul A, et al., 2007), cancer (Orend G, 2005), and inflammation (Udalova IA, et al., 2011). During development, tenascin C plays a highly restricted and dynamic role in the patterning of the neural and vascular networks and the skeleton. It has shown to affect cell adhesion, proliferation, and migration via direct interaction with cells or indirectly through binding to other extracellular matrix molecules, such as fibronectin (Jones FS and Jones PL, 2000).

In a healthy adult organism, tenascin C is produced in a tightly controlled, rapid, and transient manner and contained to specific locations where tissue repair, such as wound healing and nerve regeneration (Joester A and Faissner A, 2001), is necessary and infection needs to be resolved (Udalova IA, et al, 2011). However, in the case of uncontrolled tenascin C production, this molecule becomes pathological resulting in abnormal tissue growth, such as cancer, restenosis after percutaneous coronary angioplasty (Imanaka-Yoshida K, et al., 2001) and stent implantation, fibrotic diseases, chronic wounds, cardiovascular diseases (Golledge J et al., 2011), and autoimmune diseases (Udalova IA, et al., 2011). Recently, tenascin C has been linked to cardiac and arterial injury, tumor angiogenesis and metastasis (O'Connell JT, et al., 2011; Oskarsson T, et al., 2011), as well as in modulating stem cell behavior (Midwood KS, et al., 2011). In the case of cancer metastasis, it has been shown that cancer cells, responsible for metastasis, produce tenascin C, with inhibition of this tenascin C production resulting in reduced metastasis (Oskarsson T, et al., 2011). Therefore, tenascin could be an important target in the development of diagnostic and therapeutic treatments, especially when particular functions in this large molecule can be defined and localized to a narrowed, specific region.

Human tenascin C is a disulfide-bonded hexabranchion containing 4 major domains: First, an assembly domain at the N-terminal forms a coiled coil structure and interchain disulfide bonds that mediates the hexamer formation. Second, a series of 14.5 epidermal growth factor-like repeats, which are between 30 and 50 amino acids long and each contain six cysteines, have shown to obtain anti-adhesive properties. Third, a series of 15 fibronectin type III repeats, which are approximately 90 amino acids long and form two sheets of antiparallel beta-strands, contain several integrin binding regions {Jones FS and Jones PL, 2000). Fourth, a fibrinogen like globular domain is located at the C terminal {Midwood KS, et al, 2011; Udalova IA, et al, 2011). This fibrinogen-like globular domain has been shown to agonize TLR4 {Midwood K et al, 2009). As such, this domain is a signal of danger to the body and initiates immunological reactions.

The fibronectin type III domain region of tenascin has shown a large variability due to alternative splicing depending on the TNC source {Jones FS and Jones PL, 2000). The numbers (x-y) of fibronectin type III domains of TNC will be defined in this report as TNC IIIx-y. Domain TNC III3 {Peng Q, et al., 2009) contains an RGD peptide and multiple integrin binding domains (for example: α _ν β ₃ , ο¾>βι, α ₃ β ₆ , , α ₈ βι {Yokosaki Υ, et al., 1998), α _χ βι, α ₈ βι) ( for a large variety of cell types (for example: smooth muscle cells, endothelial cells, neurons, astrocytes, glioma) {Jones FS and Jones PL, 2000). Domain TNC III5 has demonstrated to bind heparin {Weber P, et al, 1995). As reported herein, the domain TNC III5, and longer domains comprising the TNC III5 domain such as TNC III 1-5 and TNC III3- 5, have been shown to bind chemokines.

Fibrinogen and Fibrin

Fibrinogen is a soluble plasma glycoprotein that is synthesized by the liver and the precursor protein during blood coagulation. The proteolytic enzyme thrombin, coagulation factor II, will polymerize fibrinogen into fibrin during coagulation by cleaving fibrinopeptides from its central domain, preventing physicochemical self-assembly or polymerization of the molecule {Weisel JW, 2007). Fibrin is sequentially chemically cross- linked by factor XHIa forming the primary structural protein of a viscoelastic blood clot {Mosesson MW, 2005), and functioning as a specialized provisional protein network that is formed principally in spontaneous tissue repair. The stability of fibrin depends on its interplay with molecular/cellular components of the hemostatic system {Hantgan RR, et al, 1994). In addition to cross-linking fibrin to itself, factor XHIa cross-links other adhesive proteins into the blood clot. Fibrin can bind several cell-adhesion receptors such as integrins and notably promotes the adhesion of platelet and leukocytes such as monocytes and neutrophils {Flick MJ, et al, 2004; Ugarova TP and Yakubenko VP, 2001).

Fibrin matrices were one of the first biomaterials used to prevent bleeding and promote wound healing {Janmey PA, et al, 2009). Fibrin is available from autologous sources and from cryoprecipitated pooled human blood plasma. Today, fibrin is one of the most used hydrogel in the clinic. The complex fibril structure and cross-linked character of fibrin matrix can be controlled by the details of its formation (Lorand L and Graham RM, 2003; Standeven KF, et al, 2007; Weisel JW, 2004). Importantly, in contrast to fibrillar collagen matrices where cell migration occurs both through mechanisms that are dependent and independent of proteolytic degradation, cell migration in fibrin is almost exclusively dependent upon cell-associated proteolytic activity (essentially from plasmin and matrix metalloproteinases {Mosesson MW, 2005)). One of the main advantages of fibrin is that several proteins are naturally incorporated into fibrin matrix during the coagulation such as fibronectin and alpha-2 -plasmin inhibitor, by covalent cross-linking via the transglutaminase factor XHIa {Mosesson MW, 2005). Therefore, this natural reaction can be easily exploited to functionalize fibrin with multiple cell-signaling molecules {Patterson J et al, 2010; Schense JC and Hubbell JA, 1999). In addition, fibrinogen is known to possess specific interactions with fibroblast growth factor (FGF)-2, VEGF-A165 and insulin-like growth factor binding protein (IGFBP)-3 {Peng H, et al, 2004; Sahni A, et al, 1998; Sahni A, et al, 2006; Werner S and Grose R, 2003).

Fibrin is a useful base matrix, and heparin binding peptides and molecular fusions described herein may be used with the same. Other materials may also be engineered to include TG or moieties that interact with transglutaminases to receive a TG molecular fusion. US 7241730, 6,331,422, US 6,607,740, US 6,723,344, US Pub 2007/0202178, US Pub 2007/0264227 are hereby incorporated herein by reference for all purposes; in case of conflict, the specification is controlling.

Fibrin matrices are subject to degradation by proteases in vivo, and protease inhibitors are frequently formulated in fibrinogen / fibrin matrixes to prolong their lifetime in vivo. This renders the fibrin matrices more useful in applications of tissue adhesives and sealants, and in applications of tissue engineering. One such protease inhibitor is aprotinin. A fibrin-binding form of aprotinin has been engineered by including a factor XHIa substrate within a fusion protein comprising aprotinin {Lorentz KM, et al, 2011).

Matrices are useful for purposes of sustained release of drugs. Drugs may be entrapped in the matrix and slowly diffuse from the matrix. Affinity may be engineered between a drug and components of the matrix. For example, affinity for heparin has been used to prolong the release of heparin-binding cytokines from fibrin-based matrices, incorporating binding sites for heparin into the fibrin matrix and employing heparin as an intermediate in that binding interaction {Sakiyama SE, et al, 1999). Tissue Repair and Regeneration

After damage, tissue repair or regeneration is the result of a spatio-temporal coordination of cell fate processes that are controlled by a multitude of cell-signaling events coming from the extracellular microenvironment and recruited cells at the site of injury {Gurtner GC, et al., 2008). Within a biomechanical context provided by this elastic milieu {Discher DE, et al., 2009), cells adhere by receptor-mediated interactions with extracellular matrix components such as fibronectin and laminin (among many others), mediated by specialized adhesion receptors such as integrins and others (Berrier AL and Yamada KM, 2007). These receptors transmit stress from the extracellular matrix, through the membrane, to the cytoskeleton within the cell in a dynamic and concerted manner {Hinz B, 2009). The adhesion receptors do much more than transmit stress, however; in particular within clusters of adhesion receptors in the membrane, biochemical signal transduction takes place through kinase activation and other mechanisms {Berrier AL and Yamada KM, 2007; Hinz B, 2009). In addition to adhesion proteins, the extracellular matrix also sequesters and presents a number of morphoregulatory molecules including, morphogens, cytokines, and growth factors, which control processes of cell division, and/or migration, and/or differentiation, and/or multicellular morphogenesis {Discher DE, et al, 2009; Schultz GS and Wysocki A, 2009). Morphogens, cytokines, and growth factors are powerful soluble signaling molecules, because they can change cell fate and induce tissue morphogenesis directly. The term morphogen is principally used in developmental biology to describes a particular type of signaling molecule that can induce a cellular response in a concentration-dependent manner {Affolter M and Basler K, 2007), while cytokines and chemokines (small cytokine inducing chemotaxis) are regulatory proteins essential for the development and functioning of both innate and adaptive immune response {Rossi D and Zlotnik A, 2000; Vilcek J and Feldmann M, 2004). By definition growth factors are capable of inducing cell growth, in addition to other cellular response such as migration and differentiation {Cross M and Dexter TM, 1991). A growth factor can be either a morphogen or a cytokine.

For example, key cytokines involved in tissue morphogenesis include vascular endothelial growth factors (VEGFs), platelet derived growth factors (PDGFs), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), bone morphogenetic proteins (BMPs), transforming growth factors beta (TGF-Ps), and neurotrophins (β-NGF, NT-3, BDNF). Many cytokines bind extracellular matrix components such as heparan sulfate proteoglycans {Lindahl U and Li JP, 2009), and reside there until released by enzymatic processes or dissociation. These factors, when released and sometimes also when matrix- bound (Makarenkova HP, et ah, 2009), bind to cell-surface receptors and trigger signaling, principally through kinase activation. Thus, the extracellular matrix serves as a reservoir of signaling molecules, both adhesion molecules and cytokines, that instruct cell decision processes. Angiogenesis, multicellular morphogenesis, and stem cell differentiation are cellular processes that are tightly controlled by the extracellular matrix and cytokines, and especially by their cooperative signaling. Because tissue repair is driven by these processes, the function of the extracellular matrix guides the design of biomaterials in tissue engineering and regenerative medicine, with the overall goal of mimicking the following key features: the presentation of adhesion molecules and the release of cytokines.

Vaccinology

As mentioned above, cytokines play a fundamental role in tissue morphogenesis.

Cytokines also play a fundamental role in immunology, by regulating proliferation, maturation and migration of different immune cell types, thus driving the appropriate immune response to different types of antigens. The cytokine TGF-β is a particularly important cytokine in immunology.

Chemokines are small proteins that also play fundamental roles in immunology.

Among the chemokines, interferon-γ (IFN-γ) is a critical immunomodulatory chemokine for innate and adaptive immunity against viral and bacterial antigens and for tumor control. IFN- γ is mainly expressed by natural killer (NK) and natural killer T-cells (NKT) as part of the innate immune response, and by CD4 and CD8 T cells during the adaptive immune response.

IFN-γ is the most important chemokine in regulating the balance between Thl and Th2 cells:

Thl cells express IFN-γ, which in turn causes Thl differentiation and Th2 differentiation suppression. The different cellular response to IFN-γ are activated by its binding to an heterodimeric receptor (IFNGR1 and IFNGR2) that activates JAK/STAT1 signaling pathway.

The activation of this intracellular signaling triggers the expression of multiple downstream genes, among them the chemokine interferon gamma-induced protein 10 (CXCL10) and chemokine (C-X-X motif) ligand 11 (CXCL11). These two chemokines elicit their effect by binding CXCR3 receptor on the cell surface and are considered potent chemoattractants for monocyte/macrophages, dendritic cells, NK and T-cells, respectively.

In vaccinology, antigens are peptide or protein domains or whole proteins of pathogen or self-origin (Hubbell JA, et al, 2009). Vaccine antigens in infectious diseases are based on proteins found in the pathogens of interest, such as influenza antigens or tuberculosis antigens. The number of antigens targeted in infectious disease, both in prophylactic and therapeutic vaccines, are myriad. Vaccine antigens in cancer are based on proteins found in the tumor cell type, such as the antigen survivin to be highly expressed in many tumor types or the antigen TRP-2 expressed in melanocytes and a target for cancer vaccination in melanoma. The number of antigens targeted in cancer are myriad.

A vaccine may be made that comprises a P1GF2 domain and an antigen, for instance a vehicle or a matrix as described herein. The P1FG2 provides attachment to native tissue or ECM in the matrix. A vaccine composition may comprise adjuvants, danger signals, and/or chemokines, which may be part of a matrix, a molecular fusion that comprises a P1GF2 domain, or may be added in addition to the P1FG2.

PIGF

Peptides that mimic a domain from P1GF2 are described herein. The cytokine PIGF exists in multiple isoforms. P1GF2 is an elongated isoform of PIGF- 1, containing an insert of sequence R RPKGRGKR REKQRPTDCHL (SEQ ID NO:4) in the human, RRKTKGKRKRSRNSQTEEPHP (SEQ ID NO:5) in the mouse, and related sequences in other mammalian species. Herein the unexpected surprising discovery is reported that this peptide binds very strongly to fibrinogen and fibrin, as well as the extracellular matrix proteins fibronectin, vitronectin, osteopontin, tenascin C, and to lesser extent collagen I. This domain is referred to as the PlGF2i ₂ 3-i44. The term P1GF2 domain is used to refer to this domain and to subdomains that demonstrate specific binding for extracellular matrix. The strong binding between the PlGF2i ₂₃ _i ₄₄ and fibrinogen/fibrin can be used to bind proteins comprising PlGF2i ₂₃ _i ₄₄ , including protein drugs and antigens, in fibrin matrices. The strong binding between PlGF2i ₂ 3_i ₄₄ and fibrinogen/fibrin and/or extracellular matrix proteins can be used to prolong the presence of proteins comprising PlGF2i ₂ 3_i ₄₄ that have been administered in fibrin matrices, that have been administered upon or within the site of an injury, or that have been administered upon or within a tissue site. The strong binding between the P1GF2 domain and extracellular matrix proteins can be used to prolong the retention of proteins comprising the P1GF2 domain in tissues by virtue of binding to extracellular matrix endogenously present in the tissue or tissue lesion site. The discovered affinity between PlGF2i ₂ 3_i ₄₄ and fibrinogen/fibrin and the affinity that exists between PlGF2i ₂ 3_i ₄₄ and extracellular matrix molecules leads to a number of preferred embodiments. The term P1GF2 or P1GF2 domain includes the peptides of SEQ ID NO:4 and 5, and subsequences thereof, as well as the variations of those sequences. SEQ ID NO: 4 and 5 are embodiments of a P1GF2 domain. Further embodiments of a P1GF2 domain include conservative substitutions of the sequences and also truncated forms, with N-terminal and/or C-terminal residues being truncated. Identifying truncations can be readily accomplished by the artisan reading the instant disclosure. The number of consecutive residues that provide specific binding is between about 4 and about 15 residues, with longer sequences also showing specific binding. Accordingly, embodiments of P1GF2 include an isolated polypeptide comprising a sequence chosen from the group consisting of SEQ ID NO:4 having from 0 to 5 conservative substitutions, SEQ ID NO: 5 having from 0 to 5 conservative substitutions, and subsequences thereof, said subsequences exhibiting specific binding to one or more of: fibrinogen, fibronectin, vitronectin, tenascin C, osteopontin, and fibrin. The subsequences include all subsequences of 4 to 15 residues in length, e.g., all 4, 5, 6, and 7— residue subsequences, and all 7-12 and all 5-15 residue subsequences. The value of the dissociation constant for the sequences is low, e.g., wherein the specific binding of the polypeptide to fibrinogen has a dissociation constant (KD) of less than about 40 nM. Moreover, the substitution of L-amino acids in the discovered sequence with D-amino acids can be frequently accomplished, as in Giordano.

Referring to Figure 2, panel a, data for the testing subsequences of the PlGF2i ₂ 3_i52 showed that fragments of 7 residues retained specific binding for extracellular matrix (ECM). The larger fragments, however, showed higher affinity. This data indicates that even shorter sequences can reasonably be expected to show specific binding to appropriate ECM, including all subsequences of four or more residues. Further, many sequences in the biological arts are known to be effective when they are part of even very large molecules, e.g., the RGD cell adhesion motif. Even though some molecules will fold in a way that confounds the specific binding of such relatively small sequences, artisans are very familiar with techniques for creating even very large molecules that employ such sequences in an effective manner. On the other hand, there are a certain number of natural biomolecules that may have one or more such sequences occurring as a result of random chance, considering that there are many natural biomolecules and only about 20 natural amino acids. Such sequences should not be assumed to be active for specific binding because such biomolecules have been evolutionarily tuned to accomplish specific functions. Binding to ECM is a very important naturally-occurring, specific function that should not be attributed to particular biomolecules without suitable biological evidence in such instances. Most adhesion binding motifs can undergo some conservative substitutions and retain functionality. Although not all such substitutions will be effective, such changes are often effective. There are a variety of conservative changes that can generally be made to an amino acid sequence without altering activity. These changes are termed conservative substitutions or mutations; that is, an amino acid belonging to a grouping of amino acids having a particular size or characteristic can be substituted for another amino acid. Substitutes for an amino acid sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations are not expected to substantially affect apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point. Conservative substitutions also include substituting optical isomers of the sequences for other optical isomers, specifically D amino acids for L amino acids for one or more residues of a sequence. Moreover, all of the amino acids in a sequence may undergo a D to L isomer substitution. Exemplary conservative substitutions include, but are not limited to, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free— OH is maintained; and Gin for Asn to maintain a free NH ₂ . Moreover, point mutations, deletions, and insertions of the polypeptide sequences or corresponding nucleic acid sequences may in some cases be made without a loss of function of the polypeptide or nucleic acid fragment. Substitutions may include, e.g., 1 , 2, 3, or more residues. The amino acid residues described herein employ either the single letter amino acid designator or the three- letter abbreviation. Abbreviations used herein are in keeping with the standard polypeptide nomenclature, J. Biol. Chem., (1969), 243, 3552-3559. All amino acid residue sequences are represented herein by formulae with left and right orientation in the conventional direction of amino-terminus to carboxy-terminus. Accordingly, conservative substitutions of the peptides set forth herein are contemplated and may be described in terms of quantity, e.g., 1 to 5, or percent, e.g., 0% to 33%. Artisans will immediately appreciate that all values and ranges within the expressly stated limits are contemplated, e.g., about 5%, 7 about %, or about 15%. In the case of 1 substitution in 7 residues, the substitution is 14.2%, which is about 15%>. In the case of 2 substitutions in 22, the percentage is 9.1 , which is about 10%>. Certain embodiments provide various polypeptide sequences and/or purified or isolated polypeptides. A polypeptide is a term that refers to a chain of amino acid residues, regardless of post-translational modification (e.g., phosphorylation or glycosylation) and/or complexation with additional polypeptides, synthesis into multisubunit complexes, with nucleic acids and/or carbohydrates, or other molecules. Proteoglycans therefore also are referred to herein as polypeptides. As used herein, a "functional polypeptide" is a polypeptide that is capable of promoting the indicated function. Polypeptides can be produced by a number of methods, many of which are well known in the art. For example, polypeptides can be obtained by extraction (e.g., from isolated cells), by expression of a recombinant nucleic acid encoding the polypeptide, or by chemical synthesis. Polypeptides can be produced by, for example, recombinant technology, and expression vectors encoding the polypeptide introduced into host cells (e.g., by transformation or transfection) for expression of the encoded polypeptide.

In some cases a determination of the percent identity of a peptide to a sequence set forth herein may be required. In such cases, the percent identity is measured in terms of the number of residues of the peptide, or a portion of the peptide. A polypeptide of, e.g., 90% identity, may also be a portion of a larger peptide

The term purified as used herein with reference to a polypeptide refers to a polypeptide that has been chemically synthesized and is thus substantially uncontaminated by other polypeptides, or has been separated or purified from other most cellular components by which it is naturally accompanied (e.g., other cellular proteins, polynucleotides, or cellular components). An example of a purified polypeptide is one that is at least 70%>, by dry weight, free from the proteins and naturally occurring organic molecules with which it naturally associates. A preparation of a purified polypeptide therefore can be, for example, at least 80%), at least 90%>, or at least 99%, by dry weight, the polypeptide. Polypeptides also can be engineered to contain a tag sequence (e.g., a polyhistidine tag, a myc tag, or a FLAG ^® tag) that facilitates the polypeptide to be purified or marked (e.g., captured onto an affinity matrix, visualized under a microscope). Thus a purified composition that comprises a polypeptide refers to a purified polypeptide unless otherwise indicated. The term isolated indicates that the polypeptides or nucleic acids of the invention are not in their natural environment. Isolated products of the invention may thus be contained in a culture supernatant, partially enriched, produced from heterologous sources, cloned in a vector or formulated with a vehicle, etc. Polypeptides may include a chemical modification; a term that, in this context, refers to a change in the naturally-occurring chemical structure of amino acids. Such modifications may be made to a side chain or a terminus, e.g., changing the amino-terminus or carboxyl terminus. In some embodiments, the modifications are useful for creating chemical groups that may conveniently be used to link the polypeptides to other materials, or to attach a therapeutic agent.

Specific binding, as that term is commonly used in the biological arts, refers to a molecule that binds to a target with a relatively high affinity compared to non-target tissues, and generally involves a plurality of non-covalent interactions, such as electrostatic interactions, van der Waals interactions, hydrogen bonding, and the like. Specific binding interactions characterize antibody-antigen binding, enzyme-substrate binding, and specifically binding protein-receptor interactions; while such molecules may bind tissues besides their targets from time to time, such binding is said to lack specificity and is not specific binding.

Discussion

Example 1 (see Fig. 1) describes results establishing that the domain PlGF2i ₂ 3-i44 was discovered within P1GF2 that strongly and promiscuously binds ECM proteins. This domain is only a part of P1GF2 and, as such, does not exist in nature. P1GF2 strongly bound all ECM proteins tested (Fig. 1, gray bars). Alignment of the protein sequences of the splice variants P1GF2 and PlGF-1 (which does not bind) illustrates how P1GF2 contains an additional 21 amino-acid insert (PlGF2i ₂ 3_i4 ₄ , in gray) located near the C-terminus. Binding was also shown to be effective when the P1GF2 domain was fused to a protein, GST (GST-PlGF2i ₂₃ _i ₄₄ ). From Example 1, it was concluded that PlGF2i ₂₃ _i ₄₄ comprises a ECM protein binding domain. The binding of various PLGF2 fragments to various ECM proteins, heparan sulfate, and neuropilin-1 was tested, with the results depicted in Figure 2. Example 2 details the experiments as well as describing examples of making truncations and/or substitutions into the sequence.

A variety of cytokines were made as fusion proteins with the P1GF2 domain (Example 3; Figure 3). Fig. 4 (see Example 4) sets forth results for the binding of such fusion proteins with ECM. The dissociation contstants for the specific binding were measured and it was determined that the affinity of P1GF2 for a wide variety of ECM proteins was conferred upon the fusion molecules. These included Vascular endothelial growth factor (VEGF), Platelet- derived growth factor (PDGF), and Bone morphogenetic protein (BMP). Example 5 details the further manufacture of cytokine-PlGF2 domain molecular fustions, including fusions with Insulin Growth Factor-I (IGF-I), Transforming Growth Factor beta 1 (TGF-β Ι), TGF-beta 2 (TGF- 2), Brain-derived neurotrophic factor (BDNF), and a neurotrophin (NT), NT -3. These biological factors were observed (Example 6, Fig. 5) to maintain their biological activity in when fused to the P1GF2 domain. In fact the VEGF fusion molecule had increased activity.

There is a major problem that has arisen in translating VEGF-A to clinical use. Indeed, while VEGF-A activation of VEGF -receptor 2 (VEGF-R2) is potentially a powerful approach to promote angiogenesis, actual administration of VEGF-A has been shown to rapidly induce vascular permeability, which leads to systemic hypotension and edema; this phenomenon has been the dose-limiting toxic response in peripheral and cardio-vascular applications {Simons M and Ware JA, 2003) and presents serious issues in regenerative medicine. It was theorized that combining VEGF and a P1GF2 domain would not affect the potency of the VEGF but would cause it to be released more slowly so that vascular permeability would be lessened and the combination would be more effective than the VEGF by itself. Similarly, the fusion of various cytokines to the P1GF2 domain is similarly theorized to be effective. These theories were supported in a series of experiments. Example 7 (Fig. 6) details how various ECM super-affinity cytokine variants were created that bind to, and are retained by, ECM molecules in vivo. Example 8 (Fig. 7) used clinically important models to test the healing power of molecular fusions of PDGF-BB and VEGF-A with a P1GF2 domain. Wounds treated with the engineered molecular fusions of PDGF-BB and VEGF-A led to significantly faster wound closure, and improved healing was corroborated by observing better granulation tissue and biomarkers (CD31 and desmin) that showed improved angiogenesis.

Further, the molecular fusion of VEGF and a P1GF2 domain was observed to cause much less vascular permeability despite causing these improved results. Example 9 (Fig. 8) details the results. In brief, the fusion molecule appeared to decouple angiogenesis from hyper-permeability.

In light of these various results showing that the P1GF2 domains could create a desired specific binding in a fusion molecule without disrupting cytokine functions, further tests were conducted to demonstrate the general applicability of such combinations. Example 10 (Fig. 9) details the treatment of bone defects with molecular fusions of cytokines with a P1GF2 domain. In these experiments, a matrix was used to retain and controllably deliver the molecular fusions. In brief, the fusion molecules were much more effective than the cytokines by themselves, and much lower doses were effective (nanograms of the fusion molecule compared to micrograms of the unaltered cytokines). These results demonstrate the effectiveness of a matrix that specifically binds the molecular fusions as well as their effectiveness in a bone healing treatment.

A variety of detailed Examples are further provided that describe how to design and make various molecular fusions. Example 11 details how cell adhesion motifs may be fused to a P1GF2 domain. A fibronectin domain is used as an example. Matrices for delivery drugs and/or promoting cell invasion or healing can be exposed to such molecular fusions to and be modified to carry a drug or other bioactive agent such as a cell adhesion motif. Various matrices are known, including synthetic matrices, fibrin matrices, and natural or synthetic matrices, including those that are covalently crosslinked and those that are not covalently crosslinked. Example 12 details a molecular fusion of a drug for release from a matrix, with Parathyroid Hormone Fragment 1-34 used as an example. Example 13 details a molecular fusion of a P1GF2 domain and a protease inhibitor. The context is a fibrin matrix with aprotinin as an example. Example 14 details a molecular fusion of the chemokines CXCL10, CXCL11, IFN-γ, and CCL21 with P1GF2.

Vaccines may also be made using a P1GF2 domain. Example 15 details the molecular fusion of an immunogenic antigen with a P1GF2 domain. This molecule may be administered in the context of a pharmaceutically acceptable compound and in combination with other features for vaccines, e.g., as detailed elsewhere herein. For instance, Example 16 provides details for engineering the Toll-like receptor agonist fused with a P1GF2 domain.

Drug-delivery and controlled release is generally exemplified by the details of Example 17, which describes a molecular fusion of a bioactive agent with a P1GF2 domain. For instance, an extracellular matrix -binding FGF18 is provided by a fusion protein between FGF18 and a P1GF2 domain. Various alternatives for this fusion are presented.

Molecular Fusion

A preferred embodiment is a molecular fusion between a P1GF2 domain and a therapeutic agent Embodiments include a P1GF2 domain in a molecular fusion with, e.g., a therapeutic agent, marker, cell adhesion molecule, antigen, protein, protein drug, or cytokine. A molecular fusion may be formed between a first P1GF2 peptide and a second peptide. Instead of second peptide a chemical moiety may be used, e.g., a marker, fluorescent marker. The fusion comprises the peptides conjugated directly or indirectly to each other. The peptides may be directly conjugated to each other or indirectly through a linker. The linker may be a peptide, a polymer, an aptamer, a nucleic acid, or a particle. The particle may be, e.g., a microparticle, a nanoparticle, a polymersome, a liposome, or a micelle. The polymer may be, e.g., natural, synthetic, linear, or branched. A fusion protein that comprises the first peptide and the second peptide is an example of a molecular fusion of the peptides, with the fusion protein comprising the peptides directly joined to each other or with intervening linker sequences and/or further sequences at one or both ends. The conjugation to the linker may be through covalent bonds. Methods include preparing a molecular fusion or a composition comprising the molecular fusion, including such a composition in a pharmaceutically acceptable form.

Embodiments include a molecular fusion of a polypeptide that comprises a P1GF2 domain and a transglutaminase substrate (TG). An embodiment of a TG substrate is a peptide that comprises residues 1-8 of alpha 2-plasmin inhibitor (NQEQVSPL) (SEQ ID NO:50). Embodiments include such a polypeptide being a recombinant fusion polypeptide. The molecular fusion may be further comprising a cell adhesion moiety having a specific binding affinity for a cell adhesion molecule. Various cell adhesion moieties are known, for instance, wherein the cell adhesion moiety comprises a ligand for a glycoprotein or a cell surface receptor. Or the cell adhesion moiety may comprise a ligand with specific binding to the cell adhesion molecule and the cell adhesion molecule is a cell surface receptor chosen from the group consisting of an integrin, and a cadherin.

The term molecular fusion, or the term conjugated, refers to direct or indirect association by chemical bonds, including covalent, electrostatic ionic, or charge-charge. The conjugation creates a unit that is sustained by chemical bonding. Direct conjugation refers to chemical bonding to the agent, with or without intermediate linkers or chemical groups. Indirect conjugation refers to chemical linkage to a carrier. The carrier may largely encapsulate the agent, e.g., a polymersome, a liposome or micelle or some types of nanoparticles, or have the agent on its surface, e.g., a metallic nanoparticle or bead, or both, e.g., a particle that includes some of the agent in its interior as well as on its exterior. The carrier may also encapsulate an antigen for immunotolerance. For instance a polymersome, liposome, or a particle may be made that encapsulates the antigen. The term encapsulate means to cover entirely, effectively without any portion being exposed, for instance, a polymersome may be made that encapsulates an antigen or an agent.

Conjugation may be accomplished by covalent bonding of the peptide to another molecule, with or without use of a linker. The formation of such conjugates is within the skill of artisans and various techniques are known for accomplishing the conjugation, with the choice of the particular technique being guided by the materials to be conjugated. The addition of amino acids to the polypeptide (C- or N-terminal) which contain ionizable side chains, i.e. aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, or tyrosine, and are not contained in the active portion of the polypeptide sequence, serve in their unprotonated state as a potent nucleophile to engage in various bioconjugation reactions with reactive groups attached to polymers, i.e. homo- or hetero-bi-functional PEG (e.g., Lutolf and Hubbell, Biomacromolecules 2003;4:713-22, Hermanson, Bioconjugate Techniques, London. Academic Press Ltd; 1996). In some embodiments, a soluble polymer linker is used, and may be adminsited to a patient in a pharmaceutically acceptable form. Or a drug may be encapsulated in polymerosomes or vesicles or covalently attached to the peptide ligand.

The molecular fusion may comprise a particle. The P1GF2 domain may be attached to the particle. An antigen, agent, or other substance may be in or on the particle. Examples of nanoparticles, micelles, and other particles are found at, e.g., US 2008/0031899, US 2010/0055189, US 2010/0003338, which applications are hereby incorporated by reference herein for all purposes, including combining the same with a ligand as set forth herein; in the case of conflict, however, the instant specification controls.

Nanoparticles may be prepared as collections of particles having an average diameter of between about 10 nm and about 200 nm, including all ranges and values between the explicitly articulated bounds, e.g., from about 20 to about 200, and from about 20 to about 40, to about 70, or to about 100 nm, depending on the polydispersity which is yielded by the preparative method. Various nanoparticle systems can be utilized, such as those formed from copolymers of poly(ethylene glycol) and poly(lactic acid), those formed from copolymers of poly(ethylene oxide) and poly(beta-amino ester), and those formed from proteins such as serum albumin. Other nanoparticle systems are known to those skilled in these arts. See also Devalapally et al., Cancer Chemother Pharmacol., 07-25-06; Langer et al., International Journal of Pharmaceutics, 257:169-180 (2003); and Tobio et al., Pharmaceutical Research, 15(2):270-275 (1998).

Larger particles of more than about 200 nm average diameter incorporating the heparin binding ligands may also be prepared, with these particles being termed microparticles herein since they begin to approach the micron scale and fall approximately within the limit of optical resolution. For instance, certain techniques for making microparticles are set forth in U.S. Pat Nos. 5,227,165, 6,022,564, 6,090,925, and 6,224,794. Functionalization of nanoparticles to employ targeting capability requires association of the targeting polypeptide with the particle, e.g., by covalent binding using a bioconjugation technique, with choice of a particular technique being guided by the particle or nanoparticle, or other construct, that the polypeptide is to be joined to. In general, many bioconjugation techniques for attaching peptides to other materials are well known and the most suitable technique may be chosen for a particular material. For instance, additional amino acids may be attached to the polypeptide sequences, such as a cysteine in the case of attaching the polypeptide to thiol-reactive molecules.

The molecular fusion may comprise a polymer. The polymer may be branched or linear. The molecular fusion may comprise a dendrimer. In general, soluble hydrophilic biocompatbile polymers may be used so that the conjugate is soluble and is bioavailable after introduction into the patient. Examples of soluble polymers are polyvinyl alcohols, polyethylyene imines, and polyethylene glycols (a term including polyethylene oxides) having a molecular weight of at least 100, 400, or between 100 and 400,000 (with all ranges and values between these explicit values being contemplated). Solubility in this context refers to a solubility in water or physiological saline of at least 1 gram per liter. Domains of biodegradable polymers may also be used, e.g., polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polycaprolactones, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, and polycyanoacylates.

Embodiments include a polymer comprising a polypeptide comprising a synthetic

P1GF2 peptide. For example embodiments include the polymers listed above as well as a polysaccharide, polyethylene glycol, polyalkylene oxide, collagen, or gelatin. The polymer may further comprises a transglutaminase substrate (TG), a cytokine, and the like.

In some embodiments, a polypeptide-polymer association, e.g., a molecular fusion, is prepared and introduced into the body as a purified composition in a pharmaceutically acceptable condition, or with a pharmaceutical excipient. The site of introduction may be, e.g., systemic, or at a tissue or a transplantation site.

Embodiments include a molecular fusion between a P1GF2 domain and a protein drug, such as a recombinant fusion protein comprising a P1GF2 domain and the protein drug, a chemical conjugate comprising a P1GF2 domain and the protein drug, or an indirect chemical conjugate comprising the P1GF2 domain and the protein drug mediated through joint fusion to a polymer or a polymeric micelle or nanoparticle. Molecular fusions between the P1GF2 domain and the protein drug may serve to anchor the protein drug to tissues when administered in tissue sites, by affinity with fibrinogen/fibrin in injured tissue sites or by affinity to ECM proteins in tissue sites. As such, a preferred embodiment is a molecular fusion of a P1GF2 domain and a protein drug in a pharmaceutically acceptable carrier. Alternatively, molecular fusions between the P1GF2 domain and a protein drug may serve to anchor the protein drug within a fibrin matrix. Fibrin is a commonly used biomaterial matrix, used in sealing and adhering tissues, in regenerative medicine applications, and in drug delivery applications. Anchoring protein drugs within fibrin matrices may provide pharmacological benefits in these and other applications. Peptide and protein antigens may also be linked anchored within fibrin matrices by forming a molecular fusion between the antigen and a P1GF2 domain As such, a preferred embodiment is a molecular fusion of a P1GF2 domain and a protein drug or antigen in a pharmaceutically acceptable formulation of fibrinogen/fibrin. Fibrinogen/fibrin may also be prepared from autologous sources, and as such a preferred embodiment is a molecular fusion of a P1GF2 domain and a protein drug or antigen in a pharmaceutically acceptable carrier for application in autologous fibrin. Vehicle

In many cases, a therapeutic agent, e.g., protein drugs such as cytokines, hormones, or cell-adhesion proteins might be delivered directly at the body site that needs to be treated without the use of any matrix. However, due to interstitial flow and drainage, cytokines or other soluble agents can be rapidly cleared from the site of injection, depending of their binding affinity for the ECM. Since cytokines modified with a P1GF2 sequence, e.g., P1GF2123-144 sequence, show improved binding to several extracellular matrix proteins including fibronectin, tenascin C, vitronectin, osteopontin, and collagen I, they can be better retained at the site of injection, resulting in an improved treatment.

A P1GF2 peptide may be used as a vehicle for delivery of a therapeutic agent. The vehicle is soluble or a colloid in a physiological solution with all components of the vehicle preferably being less than about 500 μιη in maximum dimension when released in the body. Embodiments of the P1GF2 vehicle include a molecular fusion of a biological agent and a peptide comprising a sequence chosen from the group consisting of SEQ ID NO:4 having from 0 to 5 conservative substitutions, SEQ ID NO:5 having from 0 to 5 conservative substitutions, and subsequences thereof, said nucleic acid exhibiting specific binding to one or more of fibrinogen, fibronectin, vitronectin, tenascin C, osteopontin, and fibrin. The biological agent may be chosen from the group consisting of a protein, a protein drug, a marker, an immunoagent, a chemokine, a cytokine, and a cell adhesion peptide. In use, a P1GF2 peptide, by itself or as part of a molecular fusion, exhibits binding specificity for various ECM molecules, including fibrinogen, fibronectin, vitronectin, tenascin C, osteopontin, and fibrin. In this context, fibrinogen and fibrin may be viewed as temporary ECM. Placement of the P1GF2 vehicle into a tissue results in localized immobilization of the vehicle at or near the site of placement, and is not systemic. The agent(s) carried by the vehicle will be released over time or be consumed where they are immobilized by cells that are interacting with the tissue. The patient's own tissue can thus serve as a biomatrix for delivery of factors. Many uses for biomatrices are known, including extended release of drugs.

Matrices

Embodiments include a biomaterial incorporating a P1GF2 domain in a matrix. The term matrix refers to a synthetic three-dimensional structure, including a block, sheet, or film; it is a term used in contrast to a soluble or fluid material. The term synthetic means not native to the patient, and being exogenous relative to the patient. The matrices, when used internally as scaffolds, have to withstand mechanical loads, contain suitable degradation kinetics, and present bioactive molecules. Scaffolds function as a fusion of cell carrier and drug delivery device for the purpose of tissue engineering. To mimic the natural microenvironment for cells in order to induce tissue repair and regeneration, synthetic materials can be modified with ECM fragments. ECM fragments described in this report may be designed to form a molecular fusion with a transglutaminase (TG) substrate at the N terminus, consisting of residues 1-8 of the protein alpha2 plasmin inhibitor (α2ΡΙ1-8, NQEQVSPL (SEQ ID NO:50)). Factor XHIa can therefore be used as a transglutaminase to catalyze the reaction between the glutamines of this sequence (NQEQVSPL) and the lysines of different biomaterials. The coagulation enzyme, factor XHIa, will covalently bind the free amine group of the lysines (Lys) to the gamma-carboxamid group of glutamine (Gin), resulting in bonds that exhibit high resistance to proteolytic degradation. For example, natural fibrin hydrogels are cross-linked by this mechanism and a TG- P1GF2 domain can therefore be cross-linked inside the gel (Schense and Hubbell, 1999).

With regard to preferred embodiments to anchor biomolecules to a fibrin matrix, the biomolecule may be a recombinant protein drug for local delivery in tissue repair, including cytokines. Thus, a preferred embodiment for tissue repair is a pharmaceutical formulation of a tissue repair matrix comprising fibrinogen or fibrin and a molecular fusion between the P1GF2 ₁₂₃ - ₁₄₄ and a recombinant cytokine, including members of the epidermal growth factor (EGF), VEGF, PDGF, FGF, IGF, BMP, TGF-β and neurotrophin families and superfamiles. The fibrin matrix may also serve as a controlled release matrix for sustained delivery of molecular fusions of protein drugs with a PLGF2 domain or PlGF2i ₂ 3_i44 and protein drugs.

A preferred embodiment is a fusion protein comprising the PLGF2 domainor

P1GF2 ₁₂ 3- ₁₄₄ and the cytokine VEGF -A, the denotation VEGF -A referring to any of the iso forms of VEGF -A.

The P1GF2 ₁₂₃ - ₁₄₄ may be used to engineer fibrin matrixes for local immunomodulation and immunopotentiation, including vaccination. Preferred embodiments are molecular fusions comprising the PlGF2i ₂ 3-i44 and a chemokine, chemokines of interest including INF-β, CXCLIO, CXCLl l, and CCL21 or cytokines including TGF-βΙ, TGF- β2 or TGF-P3. Preferred embodiments are an immunomodulation or immunopotentiation matrix comprising fibrinogen or fibrin and a molecular fusion between the PLGF2 domain or P1GF2123-144 and a recombinant chemokine, chemokines of interest including INF-β, CXCLIO, CXCLl l, and CCL21 or cytokines including TGF-β Ι, TGF-p2 or TGF- 3. PlGF2i ₂₃ -i44 may be used to incorporate immunological danger signal extracellular matrix proteins in fibrin. PlGF2i ₂ 3-i44 may be used to incorporate danger signal extracellular matrix proteins in fibrin, including the fibrinogen-like globular domain of tenascin C, an immunological danger signal. A preferred embodiment is a molecular fusion of a PLGF2 domain and the fibrinogen-like globular domain of tenascin C.

An important application in immunopotentiation is vaccination. A preferred embodiment is a vaccine matrix comprising fibrinogen or fibrin and a molecular fusion of the PLGF2 domain and a peptide or protein antigen. A preferred embodiment is a molecular fusion between a PLGF2 domain and a peptide or protein antigen. A further preferred embodiment is a vaccine matrix comprising fibrinogen or fibrin, a molecular fusion between PLGF2 domain and a chemokine, a molecular fusion of PLGF2 and the fibrinogen-like globular domain of tenascin C, and a molecular fusion between a PLGF2 domain and a peptide or protein antigen.

Fibrin matrices also provide an adhesive environment within which cells migrate, infiltrate and invade. It is useful to be able to modulate this adhesion environment, and this may be done by making molecular fusions of adhesion peptides or adhesion protein domains, such as FN 1119-10, or many corresponding domains found in fibronectin, vitronectin, laminin, and tenascin C, for example. Preferred embodiments are molecular fusions of PlGF2i ₂ 3-i44 and adhesion domains, the adhesion domains including the integrin-binding peptides derived from fibronectin, adhesion domains comprising the amino acid sequences RGD, RGDS, RGDSP (SEQ ID NO: 52), KLDAPT (SEQ ID NO:51), IDGIHEL (SEQ ID NO: 49), ID APS (SEQ ID NO: 48), LDV, and REDV, and the fibronectin adhesion domains FN III 10, FN 1119-10, as well as the 1 ^8ΐ -5 ⁴ FN type III repeats of tenascin, and the 3 ^th FN type III repeat of tenascin C.

In addition to adhesion domains, it is useful to anchor cytokine- and chemokine- binding domains within fibrin matrices. This can be accomplished with molecular fusions of a PLGF2 domain and cytokine- and chemokine -binding domains, for example from fibronectin, tenascin C, vitronectin, laminin and other matrix molecules. Preferred embodiments are a molecular fusion of a PLGF2 domain and FN III 12- 14, a molecular fusion of a PLGF2 domain and TNC III 1-5, a molecular fusion of a PLGF2 domain and TNCIII 3-5, and a molecular fusion of a PLGF2 domain and TNCIII5.

It is also of value to anchor protease inhibitors within fibrin, to delay degradation of fibrin after implantation within or on the surface of the body. This can be accomplished with molecular fusions of a PLGF2 domain and a protease inhibitor, such as aprotinin. A preferred embodiment is a molecular fusion of PLGF2 and aprotinin. A preferred embodiment is a fibrin formulation comprising a molecular fusion of a PLGF2 domain and aprotinin. Administration

Pharmaceutically acceptable carriers or excipients may be used to deliver embodiments as described herein. Excipient refers to an inert substance used as a diluent or vehicle for a therapeutic agent. Pharmaceutically acceptable carriers are used, in general, with a compound so as to make the compound useful for a therapy or as a product. In general, for any substance, a carrier is a material that is combined with the substance for delivery to an animal. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. In some cases the carrier is essential for delivery, e.g., to solubilize an insoluble compound for liquid delivery; a buffer for control of the pH of the substance to preserve its activity; or a diluent to prevent loss of the substance in the storage vessel. In other cases, however, the carrier is for convenience, e.g., a liquid for more convenient administration. Pharmaceutically acceptable salts of the compounds described herein may be synthesized according to methods known to those skilled in the arts. Pharmaceutically acceptable substances or compositions are highly purified to be free of contaminants, are sterile, and are biocompatible. They further may include a carrier, salt, or excipient suited to administration to a patient. In the case of water as the carrier, the water is highly purified and processed to be free of contaminants, e.g., endotoxins.

The compounds described herein may be administered in admixture with suitable pharmaceutical diluents, excipients, extenders, or carriers (termed herein as a pharmaceutically acceptable carrier, or a carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. Thus the deliverable compound may be made in a form suitable for oral, rectal, topical, intravenous injection, intra-articular injection, parenteral administration, intra-nasal, or tracheal administration. Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used. Suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents may be included as carriers, e.g., for pills. For instance, an active component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. The compounds can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. The active compounds can also be administered parentally, in sterile liquid dosage forms. Buffers for achieving a physiological pH or osmolarity may also be used.

EXAMPLES

Example 1 : A short amino-acid sequence within P1GF2 (PIGF2 ₁₂₃ - ₁₄₄ ) strongly binds ECM proteins.

A domain was discovered within P1GF2 (PIGF2123-144) strongly and promiscuously binds ECM proteins. GF binding to ECM proteins was measured by ELISA. A signal over 0.1 (gray box) was considered as representative of a specific binding. P1GF2 strongly bound all ECM proteins tested (gray bars). Alignment of the protein sequences of the splice variants P1GF2 and PlGF-1 (which does not bind) illustrates how P1GF2 contains an additional 21 amino-acid insert (P1GF2 ₁₂₃ - ₁₄₄ , in gray) located near the C-terminus. Binding of PlGF2i ₂₃ _i ₄₄ to ECM proteins when fused to a non-binding model protein, Glutathione S-transferase (GST) (GST-PlGF2i23_i44) was tested. A scrambled version of PlGF2i ₂₃ -i44 (GST-PlGF2 _scr ) does not bind ECM proteins. Fig. 1 sets forth experimental data for the same. Example 2: Optimization of the ECM binding domain of P1GF2.

From Example 1, it was concluded that P1GF2123-144 comprises a ECM protein binding domain. The binding of various GST-PLGF2 fragments to various ECM proteins, heparan sulfate, and neuropilin-1 was tested, with the results depicted in Figure 2.

This domain may be further engineered through removal of sequences that are not critical for binding ECM proteins through experimentation. Such experimentation can be carried out as follows. The ELISA assay described in Example 1 is useful as a read-out in such experimental optimization. Fusion proteins are made from a protein such as GST that comprise the full-length domain PlGF2i ₂ 3-i44 at one terminus, for example the C-terminus, and binding to surface-bound fibrinogen is measured by an ELISA assay using an antibody that detects the protein GST to establish a baseline of binding induced by the full-length P1GF2 ₁₂₃ -144 domain. Further fusion proteins are made, comprising the P1GF2123-144 domain that has been trimmed by one or more amino acid residues from the C-terminal end of the full-length PlGF2i ₂ 3-i44 or from the N-terminal end of the full-length PlGF2i ₂ 3-i44. Thus, two families of fusion proteins are formed, one with shortening at the N-terminal end of PlGF2i ₂₃ _ 144 and one with shortening at the C-terminal end of P1GF2123-144. Measurement of binding to the surface-bound ECM allows determination of the structure-function relationship between PlGF2i ₂ 3-i44 length (from either end) and affinity for ECM proteins. Conservative substitutions of amino acids within this domain may be similarly characterized.

Example 3: Design and production of ECM-binding cytokines containing PlGF2i ₂ 3-i44.

Sequences encoding for molecular fusions, in particular fusion proteins, of human cytokines (VEGF-A165, PDGF-BB and BMP -2) and the PlGF2i ₂ 3-i ₄₄ domain were amplified by the polymerase chain reaction and were assembled into the mammalian expression vector pXLG, in order to obtain cytokine-PlGF2i ₂ 3-i44 (SEQ ID NO:s 7, 9, 11, 12, and 13). In order to avoid a protein-misfolding issue due to the inclusion of P1GF2123-144, the single cysteine

142

within the P1GF2123-144 (Cys ), can be removed or substituted with another amino acid such as a serine (PlGF2i ₂ 3-i44*). The fusion proteins were expressed in HEK cells and purified by immobilized metal affinity chromatography using a binding buffer containing 500 mM NaCl, 20 mM sodium phosphate and 10 mM imidazole, pH 7.4. The protein was further dialyzed against Tris buffer (20 mM Tris, 150 mM NaCl, pH 7.4). Design examples of cytokines containing P1GF2123-144* are shown in Fig. 3. SEQ ID NO: 6: human VEGF-A121

APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRC

GGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQE

CDKPRR

SEQ ID NO: 7: human VEGF-A121-PlGF2i ₂₃ -i44

APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRC

GGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQE

RRRPKGRGKRRREKQRPTDCHLCDKPRR

The denotation VEGF-A-PlGF2i ₂₃ _i44 is used to refer to SEQ ID NO: 7 and to other fusion designs of VEGF-A comprising the PlGF2i ₂ _i ₄₄ domain.

SEQ ID NO: 8: human PDGF-BB

SLGSLTIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPC VEVQRCSGCCNNRNV QCRPTQVQLRPVQVRKIEIVRK PIFKKATVTLEDHLACKCETVAAARPVT

SEQ ID NO: 9: human PDGF-BB-PlGF2i ₂₃ _i ₄₄

SLGSLTIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNV QCRPTQVQLRPVQVRKIEIVRK PIFKKATVTLEDHLACKCETVAAARPVTRRRPKG RGKRRREKQRPTDCHL

SEQ ID NO: 10: human BMP-2

QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHL NSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGC GCR

SEQ ID NO: 11 : human BMP-2-PlGF2i ₂₃ _i ₄₄

QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHL NSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGC GCRRRPKGRGKRRREKQRPTDCHL

SEQ ID NO: 12: human PlGF2i ₂₃ _i ₄₄ - BMP-2

RRRPKGRGKRRREKQRPTDCHLSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHG ECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLK Y QDMVVEGCGCR

SEQ ID NO: 13: human BMP-2-PlGF2i ₂₃ -i44*

QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHL NSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGC GCRRRPKGRGKRRREKQRPTDSHL

Example 4: Cytokines modified with PlGF2i ₂₃ _i4 ₄ , or PlGF2i ₂₃ _i ₄₄ * display enhanced affinity for ECM components.

The binding of various cytokines modified with P1GF2123-144(*) to various ECM proteins and heparan sulfate was tested, with the results depicted in Figure 4 panels a and b. Dissociation constants were determined as shown in Table 1 , which sets forth the cytokines- PlGF2i ₂₃ _i ₄₄ (*) affinity constants to various ECM proteins and heparan sulfate, measured by ELISA. The dissociation constant (K _D ) was obtained by non-linear regression using A450 nm = Bmax*[concentration]/(K _D + [concentration]). The affinity to ECM protein and heparan sulfate of cytokines modified with PlGF2i ₂₃ _i ₄₄₍ *) (VEGF-A121-PlGF2i ₂₃ _i ₄₄ , PDGF-BB- PlGF2i ₂₃ _i ₄₄ , and BMP-2-PlGF2i ₂₃ _i ₄₄ *) was observed to be much higher (lower K _D ) than wild-type cytokines. As such, the affinity of P1GF2 for ECM proteins was conferred upon VEGF-A165, PDGF-BB, and BMP-2 by fusion of the PlGF2i ₂₃ _i ₄₄ to VEGF-A165, PDGF- BB, and BMP-2, respectively.

TABLE 1

Example 5: Design of ECM-binding cytokines fused to PlGF2i ₂ 3_i44(*) or with a cytokine domain substituted with P1GF2123-133(*).

Sequences encoding for molecular fusions, in particular fusion proteins, of cytokines and the PlGF2i ₂₃ _i44(*) domain were amplified by the polymerase chain reaction and were assembled into the mammalian expression vector pXLG, in order to obtain cytokine - PlGF2i23-i44(*) or PlGF2i23-i44(*)-cytokine. A fusion protein between PlGF2i23-i44(*) and the human forms of IGF-I, TGF-βΙ, TGF- 2, BDNF, and NT-3 are designed in SEQ ID NO:s 15, 17, 18, 20, 22, and 24. A shorter sequence from PlGF2i ₂ 3_i44(*) can also be used. SEQ ID NO:s 1-20 were actually made, and SEQ ID NO: Nos 21-24 are shown as examples of further embodiments.

SEQ ID NO: 14: human IGF-I:

GPETLCGAEL VD ALQF VCGDRGF YFTSiKPTGYGS S SRRAPQTGI VDECCFRSCDLRRL EMYCAPLKPAKSA

SEQ ID NO: 15: human IGF-I-PlGF2i ₂₃ -i44:

GPETLCGAEL VD ALQF VCGDRGF YFNKPTGYGSS SRRAPQTGI VDECCFRSCDLRRL EMYCAPLKPAKSARRRPKGRGKRRREKQRPTDCHL SEQ ID NO: 16: human TGF-βΙ :

ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDT QYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

SEQ ID NO: 17: human TGF-pl-PlGF2i ₂₃ -i44:

ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDT QYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCSR RRPKGRGKRRREKQRPTDCHL

SEQ ID NO: 18: human PlGF2i ₂₃ -i44 _* -TGF-pl :

RRRPKGRGKRRREKQRPTDSHLALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIH EPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYY VGRKPKVEQLSNMIVRSCKCS SEQ ID NO: 19: human TGF-p2:

ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSD TQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS SEQ ID NO: 20: human PlGF2i ₂₃ _i _44* -TGF-p2

RRRPKGRGKRRREKQRPTDSHLALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIH EPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYI GKTPKIEQL SNMI VKS CKC S SEQ ID NO : 21 : human BDNF

HSDPARRGELSVCDSISEWVTAADK TAVDMSGGTVTVLEKVPVSKGQLKQYFYET

KCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCT

LTIKRGR SEQ ID NO: 22: human BDNF-PlGF2i ₂₃ _i ₄₄

HSDPARRGELSVCDSISEWVTAADK TAVDMSGGTVTVLEKVPVSKGQLKQYFYET

KCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCT

LTIKRGRRRPKGRGKRRREKQRPTDCHL SEQ ID NO: 23: human NT-3 :

YAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRC KEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALS RKIGRT SEQ ID NO: 24: human NT-3-PlGF2i ₂₃ _i ₄₄

YAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRC KEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALS RKIGRTRRRPKGRGKRRREKQRPTDCHL Example 6: Activity of cytokines fused to PlGF2i ₂ _i .

Figure 5 sets forth the results. In vitro, PlGF2i ₂ _i -fused growth factors (GFs) showed similar bioactivity compared to wild-type GFs. Human ECs were stimulated with VEGF-A121, VEGF-A165, or VEGF-A-PlGF2i ₂₃ _i ₄₄ , and human mesenchymal stem cells were stimulated with PDGF-BB or PDGF-BB-PlGF2i ₂ 3-i ₄₄ . Phosphorylated GF receptors (VEGFR-2 and PDGFR-β) were quantified by ELISA (n = 3, mean ± SEM). The insertion of the PlGF2i ₂₃ _i ₄₄ into VEGF-A and PDGF-BB did not alter their signaling. Moreover, the insertion of PlGF2i ₂₃ _i ₄₄ into VEGF-A121 increased its activity to the level of VEGF-A165. BMP-2-PlGF2i ₂ 3-i ₄₄ * was evaluated by its ability to promote ALP activity in human mesenchymal stem cells (induction of osteoblastic differentiation). Cellular ALP was quantified after 14 days of culture in presence of BMP -2 or BMP-2-PlGF2i ₂₃ _i _44* . No differences in cell number and ALP activity were observed between cells treated with BMP-2 or BMP-2-PlGF2i23-i44*.

Example 7: In vivo retention of cytokines fused to PlGF2i ₂₃ _i _44(*) .

Results are shown in Fig. 6. ECM super-affinity cytokine variants were created that bind to and are retained by ECM molecules in vivo. For example, when injected subcutaneously in the back skin of mice, VEGF-A 165 rapidly disappeared from the injection site, with only 10% remaining in the skin tissue after 3 days. In contrast, about 50% of the injected VEGF-A-P1GF2123-144 remained after 3 days, and more than 10%> could be detected after 6 days. Additionally, in the back skin or calvarium of mice filled with a fibrin matrix containing wild-type or PlGF2i ₂₃ -i ₄₄ -fused cytokines, low amounts of wild-type cytokines were detectable within the delivery site after 3 and 6 days, while PlGF2i ₂₃ -i ₄₄ -fused cytokines were significantly retained in the fibrin matrix and within the tissue surrounding the defects.

Example 8: Treatment of skin wounds with fibrin matrix comprising cytokines fused to

Results are shown in Figure 7. Preclinical evaluations of cytokines for chronic skin- wound healing are generally performed in rodents and most commonly in the db/db diabetic mouse (Hanft JR, et al, 2008; Robson MC, et al, 1992; Robson MC, et al, 1992; Robson MC, et al, 2001), despite the fact that the optimal disease model does not yet exist for human chronic wounds. Nevertheless, there is consensus that the genetically modified db/db mouse represents a clinically relevant model for diabetes-impaired skin-wound healing {Davidson JM, 1998; Sullivan SR, et al, 2004). Success in the db/db mouse model directly opens the way for clinical trials {Hanft JR, et al, 2008; Robson MC, et al, 1992). Full-thickness back- skin wounds were treated with a roughly 100-fold lower dose of cytokines (200 ng of each PDGF-BB and VEGF-A, combined) delivered once in a fibrin matrix or simply applied topically three to four times. These low doses of wild-type PDGF-BB and VEGF-A (delivered in fibrin or topically) did not significantly enhance wound healing compared to untreated or fibrin alone -treated wounds as indicated by either extent of wound closure (the latter indicated by re-epithelialization) or amount of granulation tissue. In contrast, wounds treated with the engineered ECM super-affinity PlGF2i ₂ 3-i4 ₄ -fused PDGF-BB and VEGF-A led to significantly faster wound closure and to more granulation tissue, both topically and in fibrin. Because angiogenesis is a crucial step in sustaining newly formed granulation tissue {Gurtner GC, et ah, 2008), we focused on the extent to which angiogenesis differed between the treatments. Immunohisto logical analysis for CD31 (highly expressed by ECs) and desmin (expressed by smooth muscle cells (SMCs) stabilizing blood vessels) revealed that angiogenesis within the granulation tissues was much more pronounced when P1GF2123-144- fused GFs were delivered. . For example, 20 μg/wound of VEGF-A165 or 10 μg/wound of PDGF-BB (REGRANEX®) applied topically for five consecutive days has been reported to be efficient in the db/db mouse {Chan RK, et ah, 2006; Galiano RD, et ah, 2004). Example 9: Vascular permeability induced by VEGF-A fused to PlGF2i ₂ 3_i ₄₄ .

Results are shown in Figure 8. VEGF-A-PlGF2i ₂ 3_i ₄₄ induces much less vascular permeability than the same dose of wild-type VEGF-A165 (10 μg). Vascular permeability was measured in the mouse ear skin. Permeability induced by VEGF-A was visualized by the red-labeled dextran leaking from the vessels. VEGF-A165 was compared to VEGF-A- PlGF2i ₂₃ _i ₄₄ . Images of the mouse ear skin vasculature were analyzed after VEGF-A application. The results indicated that this approach could resolve a major problem that has arisen in translating VEGF-A to clinical use. Indeed, while VEGF-A activation of VEGF- receptor 2 (VEGF-R2) may be a powerful approach to promote angiogenesis, actual administration of VEGF-A has been shown to rapidly induce vascular permeability, which leads to systemic hypotension and edema; this phenomenon has been the dose-limiting toxic response in peripheral and cardio-vascular applications {Simons M and Ware JA, 2003) and presents serious issues in regenerative medicine. Because VEGF-A-PlGF2i ₂ 3_i ₄₄ has an enhanced capacity to bind endogenous ECM, VEGF-A-PlGF2i ₂ 3_i ₄₄ might induce less vascular permeability. In a model of dextran extravasation from vessels in the skin of the mouse ear {Kilarski WW, et ah, 2013), the rate of leakage due to application of 10 μg VEGF- A-PlGF2i ₂₃ _i ₄₄ was only 19 ± 7% of that due to application of wild-type VEGF-A165, even though it showed equivalent activity in phosphorylation of VEGFR-2 as VEGF-A 165. As such, engineering of VEGF-A to form VEGF-A-P1GF2123 144 appears to decouple angiogenesis (as shown in the model of skin wound healing) from hyper-permeability, potentially solving a major problem with VEGF-A's clinical translation.

Example 10: Treatment of bone defects with fibrin matrix comprising cytokines fused to PlGF2 ₁₂₃ _i44.

Results are shown in Figure 9. Cytokines fused to PlGF2i ₂₃ _i44 are useful in engineering a microenvironment for bone healing. Since, the cytokines BMP-2 and PDGF- BB are beneficial for bone repair {Hollinger JO, et al., 2008), fibrin matrices containing a low dose of combined BMP-2 (200 ng) and PDGF-BB (200 ng), were evaluated for bone repair. A relevant model to illustrate human translational potential is the critical-size calvarial defect in a skeletally mature rat, which is a standard and clinically relevant model for nonunion bone healing {Hollinger JO and Kleinschmidt JC, 1990; Muschler GF, et al). Preclinical evaluations of bone repair materials and osteoinductive proteins commonly include critical-size bone defect models, such as the critical-size calvarial defect in the rat {Hollinger JO and Kleinschmidt JC, 1990). A combination of BMP-2-PlGF2i ₂₃ _i44* and PDGF-BB-PlGF2i ₂₃ _i44 (200 ng of each) were delivered in a fibrin matrix, or delivered topically to the dura prior to surgical skin closure at a somewhat higher dose (1 μg of each, combined). After 4 weeks, bone healing— characterized by bone tissue deposition and coverage of the defects— was analyzed using microcomputed tomography (microCT). The delivery of wild-type GFs alone or within fibrin slightly increase bone healing when compared to the defects without treatment or treated with fibrin only. In contrast, treatment with PlGF2i ₂₃ _i44-fused GFs led to a marked increase of bone tissue deposition compared to wild-type GF. For comparison, 1 μg is usually insufficient to treat calvarial defect of 6mm in the rat {Schmoekel HG, et al., 2005), and milligram-quantities of BMP-2 are needed to treat tibial fractures in humans {Gautschi OP, et al., 2007).

Example 11 : Engineering the adhesion domain of ECM proteins fused to the PlGF2i ₂₃ _i44 domain.

To incorporate a cell adhesion-promoting domain within fibrin matrices, molecular fusions of FN III10 and FN 1119-10 and PlGF2i ₂₃ _i ₄₄ are useful. SEQ ID NO: 25 presents a design using FN 1119-10 that may easily be made by the artisan reading this specification.

SEQ ID NO: 25: human FN III9-10-PlGF2i ₂₃ _i ₄₄

GLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLT NLTPGTEYVVSIVALNGREESPPLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVT

VRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASS K

PISINYRTRRRPKGRGKRRREKQRPTDCHL Example 12: Engineering a protein drug for sustained release from fibrin matrices utilizing the PlGF2i23-i44 domain.

PTHl-34 is known to be useful in regulating system bone mass, and local application of fibrin-binding PTHl-34 variants has been shown to stimulate local bone formation {Arrighi I, et al, 2009). A fusion protein of PTHl-34 and PlGF2i ₂ 3-i44 is designed as in SEQ ID NO: 27; this protein may be readily made by the artisan reading this specification.

SEQ ID NO: 26: human PTHl-34

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF SEQ ID NO: 27: human PTH1-34-P1GF2I ₂₃ _I ₄ 4

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFRRRPKGRGKRRREKQRPTDCH L

Example 13: Engineering a protease inhibitor fused to PlGF2i ₂₃ _i44.

Fibrin has been long used clinically for hemostasis and sealing, yet extension of use in other applications has been limited due to its relatively rapid resorption in vivo, even with addition of aprotinin or other protease inhibitors. Retention of the protease inhibitor aprotinin in fibrin matrices can be accomplished by design and use of a fusion of aprotinin with PlGF2i ₂ _i44. This fusion is designed as in SEQ ID NO: 29; this protein may be readily made by the artisan reading this specification.

SEQ ID NO: 28: bovine aprotinin

RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGG A

SEQ ID NO: 29: bovine aprotinin-PlGF2i ₂₃ _i ₄ 4

RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGG ARRRPKGRGKRRREKQRPTDCHL Example 14: Engineering a chemokine fused to P1GF2123-144.

Fibrin-binding chemokines are useful in immunomodulation and immunotherapy, including vaccination. Fusions of the chemokines CXCL10, CXCL11, IFN-γ, and CCL21 with PlGF2i ₂₃ _i44 are designed in SEQ ID NO: 31, 33, 35 and 37, respectively. These proteins may be readily made by the artisan reading this specification.

SEQ ID NO: 30: human CXCL10

VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKK GEKRCLNPESKAI KNLLKAVSKERSKRSP

SEQ ID NO: 31 : human CXCL10-PlGF2i ₂₃ -i44

VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKK GEKRCLNPESKAI KNLLKAVSKERSKRSPRRRPKGRGKRRREKQRPTDCHL SEQ ID NO: 32: human CXCL1 l-PlGF2i ₂₃ -i44

FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKS KQARLIIKKVERKNF

SEQ ID NO: 33: human CXCL1 l-PlGF2i ₂₃ -i44

FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPK S KQARLIIK VERKNFRRRPKGRGKRRREKQRPTDCHL

SEQ ID NO: 34: human IFN-γ

QDPYVKEAENLK YFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLF KNFKDDQSIQKSVETIKEDMNVKFFNSNKK RDDFEKLTNYSVTDLNVQRKAIHELI QVMAELSPAAKTGKRKRSQMLFRG

SEQ ID NO: 35: human IFN-y-PlGF2i ₂₃ _i ₄₄

QDPYVKEAENLK YFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLF KNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELI QVMAELSPAAKTGKRKRSQMLFRGRRRPKGRGKRRREKQRPTDCHL SEQ ID NO: 36: human CCL21

SDGGAQDCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFLPR RSQAELCADPKEL WVQQLMQHLDKTPSPQKPAQGCRKDRGASKTGK GKGSKGCKRTERSQTPKGP SEQ ID NO: 37: human CCL21-PlGF2i ₂₃ -i44

SDGGAQDCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKEL WVQQLMQHLDKTPSPQRRRPKGRGKRRREKQRPTDCHL

Example 15: Engineering a peptide and a protein antigen fused to PlGF2i ₂₃ _i44.

L-dopachrome tautomerase, also called tyrosinase-related protein 2 (TRP-2), has been identified as a human melanoma-associated antigen and it is expressed by most melanomas as well as normal melanocytes in humans and mice. Human TRP-2 protein or peptide -pulsed dendritic cells have shown the induction of specific CD8+T cells, suggesting that self- reactive TRP-2 CD81 T-cell epitope 180-188 (trp2)-specific cells may escape thymic selection (Sierro SR, et ah, 2011). The fibrin-binding affinity of PlGF2i ₂ _i ₄₄ can be used to incorporate antigens into fibrin matrices used as vaccines. An antigen relevant for cancer vaccine in treatment of melanoma is designed as SEQ ID NO: 39, comprising a specific peptide antigen from TRP-2 and as SEQ ID NO: 41, comprising the entire protein TRP-2, in both cases fused to PlGF2i ₂ _i . These examples are designed and presented to show how artisans can readily adapt these methods to use these or other antigens.

SEQ ID NO: 38: human L-dopachrome tautomerase 180-188

SVYDFFVWL SEQ ID NO: 39: human PlGF2i ₂₃ _i ₄₄ /plasmin cleavage site derived from factor X/L- dopachrome tautomerase 180-188

RRRPKGRGKRRREKQRPTDCHLITFRSVYDFFVWL

SEQ ID NO: 40: human L-dopachrome tautomerase

QFPRVCMTVDSLVNKECCPRLGAESANVCGSQQGRGQCTEVRADTRPWSGPYILRN QDDRELWPRKFFHRTCKCTGNFAGYNCGDCKFGWTGPNCERKKPPVIRQNIHSLSP QEREQFLGALDLAKKRVHPDYVITTQHWLGLLGPNGTQPQFANCSVYDFFVWLHY YSVRDTLLGPGRPYRAIDFSHQGPAFVTWHRYHLLCLERDLQRLIGNESFALPYWNF ATGRNECDVCTDQLFGAARPDDPTLISRNSRFSSWETVCDSLDDYNHLVTLCNGTYE GLLRR QMGR SMKLPTLKDIRDCLSLQKFDNPPFFQNSTFSFRNALEGFDKADGTL DSQVMSLHNLVHSFLNGTNALPHSAANDPIFVVLHSFTDAIFDEWMKRFNPPADAW PQELAPIGHNRMYNMVPFFPPVTNEELFLTSDQLGYSYAIDLPVSVEETPGWPTTLLV VMGTL VALVGLF VLLAFLQ YRRLR GYTPLMETHLS SKRYTEE A

SEQ ID NO: 41 : human L-dopachrome tautomerase-PlGF2123-144

QFPRVCMTVDSLVNKECCPRLGAESANVCGSQQGRGQCTEVRADTRPWSGPYILRN

QDDRELWPRKFFHRTCKCTGNFAGYNCGDCKFGWTGPNCERKKPPVIRQNIHSLSP

QEREQFLGALDLAKKRVHPDYVITTQHWLGLLGPNGTQPQFANCSVYDFFVWLHY YS VRDTLLGPGRPYRAIDFSHQGPAFVTWHRYHLLCLERDLQRLIGNESFALPYWNF ATGRNECDVCTDQLFGAARPDDPTLISRNSRFSSWETVCDSLDDYNHLVTLCNGTYE GLLRRNQMGRNSMKLPTLKDIRDCLSLQKFDNPPFFQNSTFSFRNALEGFDKADGTL DSQVMSLHNLVHSFLNGTNALPHSAANDPIFVVLHSFTDAIFDEWMKRFNPPADAW PQELAPIGHNRMYNMVPFFPPVTNEELFLTSDQLGYSYAIDLPVSVEETPGWPTTLLV VMGTL VALVGLFVLLAFLQYRRLRKGYTPLMETHLSSKRYTEEARRRPKGRGKRRR EKQRPTDCHL

Example 16: Engineering the Toll-like receptor agonist fused to P1GF2123-144.

Vaccines with incorporated danger signals provide signals to activate immune responses to incorporated antigens. The ECM protein fragment TNC fibrin globular domain (also referred to as the fibrinogen globe domain) is such a danger signal. The danger signal domain can be incorporated into fibrin matrixes by affinity of PlGF2i ₂ 3-i44 for fibrin. A fusion protein of TNC fibrin globe (SEQ ID NO: 42) and PlGF2i ₂₃ -i44 is designed in SEQ ID NO: 43.

SEQ ID NO: 42: human TNC fibrinogen globular domain

GLLYPFPKDCSQAMLNGDTTSGLYTIYLNGDKAQALEVFCDMTSDGGGWIVFLRRK NGRENFYQNWKAYAAGFGDRREEFLHWLGLDNLNKITAQGQYELRVDLRDHGETA FAVYDKFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFSTFDKDTDSAITNCALS YKGAFWYRNCHRVNLMGRYGDNNHSQGVNWFHWKGHEHSIQFAEMKLRPSNFRN LEGRRKRA

SEQ ID NO: 43: human TNC fibrinogen globular domain-PlGF2i ₂ 3-i44

GLLYPFPKDCSQAMLNGDTTSGLYTIYLNGDKAQALEVFCDMTSDGGGWIVFLRRK NGRENFYQNWKAYAAGFGDRREEFLHWLGLDNLNKITAQGQYELRVDLRDHGETA FAVYDKFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFSTFDKDTDSAITNCALS YKGAFWYRNCHRVNLMGRYGD NHSQGVNWFHWKGHEHSIQFAEMKLRPSNFRN LEGRRRPKGRGKRRREKQRPTDCHL

Example 17: Tissue retention of cytokines containing the PlGF2i ₂ 3-i44.

The cytokine FGF18 has been shown to lead to improved cartilage repair when injected in the joints of animals in osteoarthritis models {Moore EE, et al., 2005). Elimination from the site of injection limits the efficacy of this approach. An extracellular matrix -binding FGF18 variant is provided by a fusion protein between FGF18 and PlGF2i ₂ 3_i44, designed in SEQ ID NO: 45. This protein may be readily made by the artisan reading this specification, as well as other vehicles for other agents or cytokines.

SEQ ID NO: 44: human FGF18

EENVDFRIHVENQTRARDD VSRKQLRLYQLYSRTSGKHIQVLGRRIS ARGEDGDKYA QLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTAL MSAKYSGWYVGFTK GRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTK RSRRIRPTHPA SEQ ID NO: 45: human FGF18-PlGF2i ₂₃ _i ₄ 4

EENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYA QLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTAL MSAKYSGWYVGFTK GRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTK RSRRIRPTHPARRRPKGRGKRRREKQRPTDCHL

One can make other FGF-18 variants in which a native domain within FGF-18 is replaced with a PlFG-2 domain. A hypothetical heparin binding domain exists within FGF-18, namely KRYPKGQPELQKPFKYTTVTKRSRRIR (SEQ ID NO:56), the key domain of which is KRSRRIR (SEQ ID NO: 57). Thus, one substitutional implementation is to replace the KRSRRIR domain with a P1GF2 domain, for example SEQ ID NO: 53.

SEQ ID NO:53: human FGF18-PlGF2i ₂₃ _i ₃₈

EENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYA QLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTAL MSAKYSGWYVGFTK GRPR GPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTR RRPKGRGKRRREKQRPTHPA

A second substitutional example is to extend the P1GF2 domain on its N terminal end so as to better match the amino acids within FGF-18, SEQ ID NO:54, using P1GF2119 44, namely MKPERRRPKGRGKRRREKQRPTDCHL (SEQ ID NO:55) Other possible implementations exist as well.

SEQ ID NO:54: human FGF18-PlGF2 _m -i38

EENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRIS ARGEDGDKYA QLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLE NYTAL

MSAKYSGWYVGFTK GRPRKGPKTRENQQDVHFMKPERRRPKGRGKRRREKQRPT HPA

The cytokine TGF-P3 has been extensively explored in limitation of dermal scars, for example post-surgical incisional scars. The cytokine has been injected along such incision lines {Ferguson MW, et al., 2009). Elimination from the site of injection limits the efficacy of this approach. An extracellular matrix -binding TGF-P3 variant is provided by a fusion protein between TGF-P3 and P1GF2123-144*, designed in SEQ ID NO: 47. This protein may be readily made by the artisan reading this specification, as well as other vehicles for other agents or cytokines.

SEQ ID NO: 46: human TGF- β3:

ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADT THSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS

SEQ ID NO: 47: human PlGF2i ₂₃ -i44*-TGF-p3:

RRRPKGRGKRRREKQRPTDSHLALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVH EPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYV GRTPKVEQLSNMVVKSCKCS

Further Disclosure

1. A biologic delivery vehicle comprising a molecular fusion of a biological agent and a peptide comprising a sequence or subsequence of at least 5, or 6, or 7, residues of a sequence chosen from the group consisting of SEQ ID NO: 4 having from 0 to about 15% conservative substitutions and SEQ ID NO: 5 having from 0 to about 15% conservative substitutions. Said peptide exhibits specific binding to fibrinogen. 2. The vehicle of 1 with the peptide exhibiting specific binding to fibrinogen, fibronectin, vitronectin, tenascin C, osteopontin, fibrin, and heparan sulfate. 3. The vehicle of 1 or 2 wherein the peptide has a specific binding to fibrinogen with a dissociation constant (Kd) of less than about 24, about 40, or about 100 iiM. 4. The vehicle of any of 1-3 wherein the biological agent is chosen from the group consisting of a protein, a protein drug, a marker, an immunoagent, a chemokine, a cytokine, and a cell adhesion peptide. 5. The vehicle of any of 1-4 wherein the molecular fusion comprises a recombinant protein comprising the biologic agent and the peptide. 6. The vehicle of any of 1-4 wherein the molecule fusion comprises a linker covalently bonded with the agent and the peptide. 7. The vehicle of 6 wherein the linker comprises a polymer having a first covalent bond to an N-terminus or a C-terminus of the peptide and a second covalent bond to the biological agent. 8. The vehicle of any of 1-4 wherein the molecule fusion comprises a particle that is joined to the biological agent and to the peptide. 9. The vehicle of 8 wherein the particle is chosen from the group consisting of a microparticle, a nanoparticle, a polymersome, a micelle, and a liposome. 10. The vehicle of 8 being soluble or a colloid in a physiological solution with all components of the vehicle being less than about 500 μιη in maximum dimension. 11. The vehicle of 8 wherein the particle comprises a plurality of amines and/or thiols that participate in a covalent bond to the biological and/or the peptide. 12. The vehicle of any of 1-11 wherein the biological agent comprises a cytokine chosen from the group consisting of epidermal growth factors (EGFs), VEGFs, VEGF-A, VEGF-C, PDGFs, PDGF-AB, PDGF-BB, FGFs, FGF-2, FGF-18, IGFs, IGF-1, BMPs, BMP-2, BMP-7, TGF-Ps, TGF-βΙ, TGF- β2, TGF-P3, the neurotrophins, NT-3, and BDNF. 13. The vehicle of any of 1-11 wherein the biological agent comprises a chemokine chosen from the group consisting of interferons, INF-beta, CXCL chemokines, CXCL10, CXCL11, CXCL 12, CCL chemokines, and CCL21. 14. The vehicle of any of 1-11 wherein the biological agent comprises an immunoagent. 15. The vehicle of 14 wherein the immunoagent provides an antigen. 16. The vehicle of 15 wherein the antigen is at least a portion of tyrosine-related protein 2 (TRP-2). 17. The vehicle of 14 wherein the immunoagent comprises a danger signal. 18. The vehicle of 17 wherein the danger signal comprises a globular domain of tenascin or an EDA domain of fibronectin. 19. The vehicle of any of 1-11 wherein the biological agent comprises a cell adhesion peptide. 20. The vehicle of 19 wherein the cell adhesion peptide comprises a ligand for a cell surface receptor chosen from the group consisting of integrin and cadherin. 21. The vehicle of 19 wherein the cell adhesion peptide comprises a cell adhesion motif chosen from the group consisting a fibronectin cell adhesion domain, a vitronectin cell adhesion domain, a laminin cell adhesion domain, a tenascin cell adhesion domain, a fibronectin FN III 10 domain, a fibronectin FN 1119-10 domain, a tenascin domain taken from one or more of a fibronectin type III repeats 1 to 5, a 3 ^rd FN type III repeat of tenascin C, a FN 1119-10 domain of tenascin, RGD, RGDS, RGDSP, KLDAPT, IDGIHEL, IDAPS, LDV, and REDV. 22. The vehicle of any of 1-11 wherein the biologic agent comprises a protease inhibitor.

23. A biomolecule comprising a cytokine derivatized to include a P1GF2 domain. 24.

The biomolecule of 23 wherein an endogenous extracellular-matrix binding domain of the cytokine has been removed or disabled. 25. The biomolecule of 23 or 24 wherein the derivatized cytokine has specific binding to an extracellular matrix molecule selected from the group of fibrinogen, fibronectin, vitronectin, tenascin C, osteopontin and fibrin. 26. The biomolecule of 25 wherein the dissociation constant of binding of the derivatized cytokine with the extracellular matrix molecule is less than 50% of a dissociation constant of binding of the underivatized cytokine to the same extracellular matrix molecule. 27. The biomolecule of any of 23-26 wherein the cytokine is selected from the group consisting of epidermal growth factors (EGFs), VEGFs, VEGF-A, VEGF-C, PDGFs, PDGF-AB, PDGF-BB, the FGFs, FGF-2, FGF-18, IGFs, IGF-1, BMPs, BMP-2, BMP-7, TGF-Ps, TGF-βΙ, TGF- β2, TGF-P3, neurotrophins, NT-3, and BDNF. 28. The biomolecule of any of 23-27 wherein the biomolecule is a fusion protein or a molecular fusion that further comprises a biologic agent.

An isolated polypeptide comprising a sequence or subsequence of at least 6 residues (or at lest 5, or at least 7, or at least 8) of a sequence chosen from the group consisting of SEQ ID NO: 4 having from 0 to about 15% conservative substitutions and SEQ ID NO: 5 having from 0 to about 15 > conservative substitutions, said peptide exhibiting specific binding to fibrinogen. 29. The polypeptide of 28 further exhibiting specific binding to fibronectin, vitronectin, tenascin C, osteopontin, and fibrin. 30. The polypeptide of 28 or 29 wherein the specific binding of the polypeptide to fibrinogen has a dissociation constant (Kd) of less than about 25 nM. 31. The polypeptide of any of 28-30 wherein the sequence is chosen from the group consisting of SEQ ID NO:4 and SEQ ID NO:5. 32. A fusion protein comprising the polypeptide of any of 28-31.

33. A biomaterial comprising a matrix, with the matrix comprising a peptide comprising a sequence or subsequence of at least 6 residues of a sequence chosen from the group consisting of SEQ ID NO: 4 having from 0 to about 15% conservative substitutions and SEQ ID NO:5 having from 0 to about 15% conservative substitutions, said peptide exhibiting specific binding to the matrix. 34. The biomaterial of 33 wherein the specific binding of the peptide to the matrix has a dissociation constant (Kd) of less than about 100 nM. 35. The biomaterial of 33 wherein the specific binding of the peptide to the matrix has a dissociation constant (Kd) of less than about 25 nM. 36. The biomaterial of any of 33-35 wherein the peptide is specifically bound to the matrix and is available for binding to biomolecules. 37. The biomaterial of 33 or 34 wherein the peptide is free of covalent bonds to the matrix. 38. The biomaterial of any of 33-37 comprising an extracellular matrix domain that specifically binds to the peptide. 39. The biomaterial of 38 wherein the extracellular matrix domain is a domain of a biomolecule chosen from the group consisting of fibrinogen, fibronectin, vitronectin, tenascin C, osteopontin, and fibrin. 40. The biomaterial of any of 33-39 comprising hydrophilic polymers, wherein the peptide is attached to the matrix though a transglutaminase substrate, with a bond being formed by a transglutaminase enzyme between the substrate and the polypeptide. 41. The biomaterial of 40 wherein the polymers or the peptide comprise a transglutaminase substrate that comprises NQEQVSPL (SEQ ID NO: 50). 42. The biomaterial of any of 33-41 further comprising a molecular fusion of the peptide and a biologic agent. 43. The biomaterial of 42 wherein the biological agent comprises a cytokine is selected from the group consisting of epidermal growth factors (EGFs), VEGFs, VEGF-A, VEGF-C, PDGFs, PDGF-AB, PDGF-BB, the FGFs, FGF-2, FGF-18, IGFs, IGF-1, BMPs, BMP-2, BMP-7, TGF-Ps, TGF-βΙ, TGF- β2, TGF-P3, neurotrophins, NT-3, and BDNF. 44. The biomaterial of 42 wherein the biological agent comprises a the biological agent comprises a chemokine chosen from the group consisting of interferons, INF-D , CXCL chemokines, CXCL10, CXCL11, CXCL12, CCL chemokines, and CCL21. 45. The biomaterial of 42 wherein the biological agent comprises an immunoagent. 46. The biomaterial of 42 wherein the immunoagent provides an antigen. 47. The biomaterial of 42 wherein the antigen is at least a portion of tyrosine -related protein 2 (TRP-2). 48. The biomaterial of 42 wherein the immunoagent comprises a danger signal. 49. The biomaterial of 48 wherein the danger signal comprises a globular domain of tenascin or an EDA domain of fibronectin. 50. The biomaterial of 42 wherein the biological agent comprises a cell adhesion peptide. 51. The biomaterial of 42 wherein the cell adhesion peptide comprises a ligand for a cell surface receptor chosen from the group consisting of integrin, cadherin. 52. The biomaterial of 42 wherein the cell adhesion peptide comprises a cell adhesion motif chosen from the group consisting a fibronectin cell adhesion domain, a vitronectin cell adhesion domain, a laminin cell adhesion domain, a tenascin cell adhesion domain, a fibronectin FN III 10 domain, a fibronectin FN 1119-10 domain, a tenascin domain taken from one or more of a fibronectin type III repeats 1 to 5, a 3 ^rd FN type III repeat of tenascin C, a FN 1119-10 domain of tenascin, RGD, RGDS, RGDSP, KLDAPT, IDGIHEL, ID APS, LDV, and REDV. 53. The biomaterial of 42 wherein the biologic agent comprises a protease inhibitor. 54. The biomaterial of any of 28-53 wherein the biologic agent comprises a protease inhibitor. 55. The biomaterial of 54 wherein the protease inhibitor comprises aprotinin and the matrix comprises fibrin. 56. The biomaterial of any of 28-55 further comprising a plurality of molecular fusions, with each of the plurality of the fusions having a distinct biologic agent fused with at least one of the peptides. 57. The biomaterial of 56 comprising between 2 and 10 molecular fusions, with the biologic agent for each of the fusions being independently chosen. 58. The biomaterial of 57 wherein the plurality of molecular fusions have a biologic agent independently chosen from the group consisting of epidermal growth factors (EGFs), VEGFs, VEGF-A, VEGF-C, PDGFs, PDGF-AB, PDGF- BB, FGFs, FGF-2, FGF-18, IGFs, IGF-1, BMPs, BMP-2, BMP-7, TGF-Ps, TGF-βΙ, TGF- β2, TGF-P3, the neurotrophins, NT-3, BDNF, interferon- β, interferons, CXCL chemokines, CXCL10, CXCL11, CXCL 12, CCL chemokines, and CCL21, a globular domain, a fibronectin cell adhesion domain, a vitronectin cell adhesion domain, a laminin cell adhesion domain, a tenascin cell adhesion domain, a fibronectin FN III 10 domain, a fibronectin FN 1119-10 domain, a tenascin domain taken from one or more of a fibronectin type III repeats 1 to 5, a 3 ^rd FN type III repeat of tenascin C, a FN 1119-10 domain of tenascin, RGD, RGDS, RGDSP, KLDAPT, IDGIHEL, ID APS, LDV, and REDV.

59. A medicament comprising pharmaceutically acceptable vehicle of any of 1-22, the biomolecule of any of 23-27, the polypeptide of any of 28-31, the fusion protein of 32, or the biomaterial of any of 33-58. 60. The medicament of 59 for treating a condition of disease, for wound healing, for bone healing, or for vaccination. 61. The medicament of 59 comprising a plurality of molecular fusions, with each of the plurality of the fusions having a distinct biologic agent fused with at least one of the polypeptides. 62. The medicament of 61 comprising between 2 and 10 molecular fusions, with the biologic agent for each of the fusions being independently chosen. 63. A method of treating a patient with a medicament comprising administering a pharmaceutically acceptable vehicle of any of 1-22, the biomolecule of any of 23-27, the polypeptide of any of 28-31 , the fusion protein of 32, or the biomaterial of any of 33-58. 64. A method of treating a patient with a medicament comprising administering a pharmaceutically acceptable molecular fusion of a biological agent and a peptide, or a biomaterial matrix comprising a pharmaceutically acceptable molecular fusion of a biological agent and a peptide, with the polypeptide comprising a sequence or subsequence of at least 6 residues of a sequence chosen from the group consisting of SEQ ID NO: 4 having from 0 to about 15% conservative substitutions and SEQ ID NO: 5 having from 0 to about 15% conservative substitutions. 65. The method of 64 wherein the biologic agent provides an antigen, with the patient being vaccinated by administration of the molecule fusion. 66. The method of 64 wherein the agent comprises a danger signal, with an antigen being administered in combination with the agent. 67. The method of 64 wherein the molecular fusion provides for an extended release of the biologic agent from the site of administration. 68. The method of 64 wherein the biologic agent comprises a cytokine, with the site of administration being chosen from the group consisting of a fistula, a wound, and an ulcer. 69. A vaccine comprising any of the embodiments of 1- 68. 70. A matrix or system comprising any of the embodiments of 1-69 for drug delivery, vaccination, wound healing, or bone healing. 71. A nucleic acid comprising a sequence encoding any of the peptides or proteins of 1 -70.

References:

All references, patents, patent applications, journal articles and publications set forth herein are hereby incorporated by reference herein for all purposes; in case of conflict, the instant specification is controlling.

Affolter M, Basler K (2007). The Decapentaplegic morphogen gradient: from pattern formation to growth regulation. Nat Rev Genet 8:663-674.

Arrighi I, et al. (2009). Bone healing induced by local delivery of an engineered parathyroid hormone prodrug. Biomaterials 30:1763-1771.

Berrier AL, Yamada KM (2007). Cell-matrix adhesion. J Cell Physiol 213:565-573.

Chan RK, et al. (2006). Effect of recombinant platelet-derived growth factor (Regranex) on wound closure in genetically diabetic mice. J Burn Care Res 27:202-205.

Cross M, Dexter TM (1991). Growth factors in development, transformation, and tumorigenesis. Cell 64:271-280.

Danen EH, et al. (1995). Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of integrin alpha 5 beta 1. J Biol Chem 270:21612- 21618.

Davidson JM (1998). Animal models for wound repair. Arch Dermatol Res 290 Suppl:Sl-l l . Discher DE, Mooney DJ, Zandstra PW (2009). Growth factors, matrices, and forces combine and control stem cells. Science 324: 1673-1677.

Ferguson MW, et al. (2009). Prophylactic administration of avotermin for improvement of skin scarring: three double-blind, placebo-controlled, phase I/II studies. Lancet 373:1264-1274.

Flick MJ, et al. (2004). Leukocyte engagement of fibrin(ogen) via the integrin receptor alphaMbeta2/Mac-l is critical for host inflammatory response in vivo. J Clin Invest 113: 1596-1606.

Galiano RD, et al. (2004). Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells. Am J Pathol 164:1935-1947.

Gautschi OP, Frey SP, Zellweger R (2007). Bone morphogenetic proteins in clinical applications. ANZJSurg 77:626-631.

Golledge I et al. (2011). The role of tenascin C in cardiovascular disease. Cardiovasc Res 92:19-28.

Gurtner GC, Werner S, Barrandon Y, Longaker MT (2008). Wound repair and regeneration. Nature 453:314-321.

Hanft JR, et al. (2008). Phase I trial on the safety of topical rhVEGF on chronic neuropathic diabetic foot ulcers. J Wound Care 17:30-32, 34-37.

Hantgan RR, Francis CW, Marder VJ (1994) Chapter 14: Fibrinogen structure and physiology (Lippincott Company, Philadelpia) 3rd Ed.

Hinz B (2009). The myofibroblast: paradigm for a mechanially active cell. J 5ib/wecA:doi: 10.1016/j.jbiomech.2009.1009.1020.

Hollinger JO, Kleinschmidt JC (1990). The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg 1 :60-68.

Hollinger JO, et al. (2008). Recombinant human platelet-derived growth factor: biology and clinical applications. J Bone Joint Surg Am 90 Suppl 1 :48-54.

Hubbell JA, Thomas SN, Swartz MA (2009). Materials engineering for immunomodulation. Nature 462:449-460.

Imanaka-Yoshida K, et al. (2001). Serial extracellular matrix changes in neointimal lesions of human coronary artery after percutaneous transluminal coronary angioplasty: clinical significance of early tenascin-C expression. Virchows Arch 439:185-190.

Janmey PA, Winer JP, Weisel JW (2009). Fibrin gels and their clinical and bioengineering applications. J R Soc Interface 6: 1-10. Joester A, Faissner A (2001). The structure and function of tenascins in the nervous system. Matrix Biol 20: 13-22.

Jones FS, Jones PL (2000). The tenascin family of ECM glycoproteins: structure, function, and regulation during embryonic development and tissue remodeling. Dev Dyn 218:235-259.

Krammer A, et al. (2002). A structural model for force regulated integrin binding to fibronectin's RGD-synergy site. Matrix Biol 21 : 139-147.

Lindahl U, Li JP (2009). Interactions between heparan sulfate and proteins - design and functional implications. Int Rev Cell Molec Biol 276:105-159.

Lorand L, Graham RM (2003). Transglutaminases: crosslinking enzymes with pleiotropic functions. Nature reviews. Molecular cell biology 4: 140-156.

Lorentz KM, Kontos S, Frey P, Hubbell JA (2011). Engineered aprotinin for improved stability of fibrin biomaterials. Biomaterials 32:430-438.

Makarenkova HP, et al. (2009). Differential interactions of FGFs with heparan sulfate control gradient formation and branching morphogenesis. Sci Signal 2:ra55.

Mao Y, Schwarzbauer JE (2005). Fibronectin fibrillogenesis, a cell-mediated matrix assembly process. Matrix Biol 24:389-399.

Mardon HJ, Grant KE (1994). The role of the ninth and tenth type III domains of human fibronectin in cell adhesion. FEB S Lett 340:197-201.

Martino MM, Hubbell JA (2010). The 12th- 14th type III repeats of fibronectin function as a highly promiscuous growth factor-binding domain. Faseb J 24:4711-4721.

Midwood K, et al. (2009). Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease. Nat Med 15:774-780.

Midwood KS, Hussenet T, Langlois B, Orend G (2011). Advances in tenascin-C biology. Cell Mol Life Sci 68:3175-3199.

Moore EE, et al. (2005). Fibroblast growth factor- 18 stimulates chondrogenesis and cartilage repair in a rat model of injury-induced osteoarthritis. Osteoarthritis Cartilage 13:623-631.

Mosesson MW (2005). Fibrinogen and fibrin structure and functions. J Thromb

Haemost 3: 1894-1904.

Mould AP, et al. (1997). Defining the topology of integrin alpha5betal -fibronectin interactions using inhibitory anti-alpha5 and anti-betal monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino -terminal repeats of the alpha5 subunit. J Biol Chem 272: 17283-17292.

Muschler GF, et al. The design and use of animal models for translational research in bone tissue engineering and regenerative medicine. Tissue Eng Part B Rev 16:123-145.

O'Connell JT, et al. (2011). VEGF-A and Tenascin-C produced by S100A4+ stromal cells are important for metastatic colonization. Proc Natl Acad Sci U S A 108: 16002-16007.

Orend G (2005). Potential oncogenic action of tenascin-C in tumorigenesis. Int J Biochem Cell Biol 37: 1066-1083.

Oskarsson T, et al. (2011). Breast cancer cells produce tenascin C as a metastatic niche component to colonize the lungs. Nat Med 17:867-874. -Pankov R, Yamada KM (2002). Fibronectin at a glance. J Cell Sci 115:3861-3863.

Patterson J, Martino MM, Hubbell JA (2010). Biomimetic materials in tissue engineering. Mater Today 13: 14-22.

Peng ¾ et al. (2004). Identification of a binding site on human FGF-2 for fibrinogen. Blood 103:2114-2120.

Peng Q, et al. (2009). Mechanical design of the third Fnlll domain of tenascin-C. J Mol Biol 386: 1327-1342.

Ribatti D (2008). The discovery of the placental growth factor and its role in angiogenesis: a historical review. Angiogenesis 11 :215-221.

Robson MC, et al. (1992). The safety and effect of topically applied recombinant basic fibroblast growth factor on the healing of chronic pressure sores. Ann Surg 216:401 - 406; discussion 406-408.

Robson MC, et al. (1992). Platelet-derived growth factor BB for the treatment of chronic pressure ulcers. Lancet 339:23-25.

Robson MC, et al. (2001). Randomized trial of topically applied repifermin

(recombinant human keratinocyte growth factor-2) to accelerate wound healing in venous ulcers. Wound Repair Regen 9:347-352.

Rossi D, Zlotnik A (2000). The biology of chemokines and their receptors. Annu Rev Immunol 18:217-242.

Sahni A, Odrljin T, Francis CW (1998). Binding of basic fibroblast growth factor to fibrinogen and fibrin. Journal of Biological Chemistry 273:7554-7559.

Sahni A, et al. (2006). FGF-2 binding to fibrin(ogen) is required for augmented angiogenesis. Blood 107: 126-131. Sakiyama SE, Schense JC, Hubbell JA (1999). Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering. Faseb J 13:2214-2224.

Schense JC, Hubbell JA (1999). Cross-linking exogenous bifunctional peptides into fibrin gels with factor XHIa. Bioconjug Chem 10:75-81.

Schmoekel HG, et al. (2005). Bone repair with a form of BMP -2 engineered for incorporation into fibrin cell ingrowth matrices. Biotechnol Bioeng 89:253-262.

Schultz GS, Wysocki A (2009). Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen 17:153-162.

Sierro SR, et al. (2011). Combination of lentivector immunization and low-dose chemotherapy or PD-l/PD-Ll blocking primes self-reactive T cells and induces anti-tumor immunity. Eur J Immunol 41 :2217-2228.

Standeven KF, et al. (2007). Functional analysis of fibrin {gamma} -chain cross- linking by activated factor XIII: determination of a cross-linking pattern that maximizes clot stiffness. Blood 110:902-907.

Sullivan SR, et al. (2004). Validation of a model for the study of multiple wounds in the diabetic mouse (db/db). Plast Reconstr Surg 113:953-960.

Trebaul A, Chan EK, Midwood KS (2007). Regulation of fibroblast migration by tenascin-C. Biochem Soc Trans 35:695-697.

Udalova IA, Ruhmann M, Thomson SJ, Midwood KS (2011). Expression and immune function of tenascin-C . Crit Rev Immunol 31 :115-145.

Ugarova TP, Yakubenko VP (2001). Recognition of fibrinogen by leukocyte integrins. Ann N Y Acad Sci 936:368-385.

Vilcek J, Feldmann M (2004). Historical review: Cytokines as therapeutics and targets of therapeutics. Trends Pharmacol Sci 25 :201 -209.

Weber P, Zimmermann DR, Winterhalter KH, Vaughan L (1995). Tenascin-C binds heparin by its fibronectin type III domain five. J Biol Chem 270:4619-4623.

Weisel JW (2004). The mechanical properties of fibrin for basic scientists and clinicians. Biophys Chem 112:267-276.

Weisel JW (2007). Structure of fibrin: impact on clot stability. J Thromb Haemost 5

Suppl 1 : 116-124.

Werner S, Grose R (2003). Regulation of wound healing by growth factors and cytokines. Physiol Rev 83:835-870. Yokosaki Y, et al. (1998). Identification of the ligand binding site for the integrin alpha9 betal in the third fibronectin type III repeat of tenascin-C. The Journal of biological chemistry 273 : 11423 - 11428.

Kilarski WW, et al. (2013). Intravital immunofluorescence for visualizing the microcirculatory and immune microenvironments in the mouse ear dermis. PLoS One 8:e57135.

Lasarte J J, et al. (2007). The extra domain A from fibronectin targets antigens to TLR4-expressing cells and induces cytotoxic T cell responses in vivo. J Immunol 178:748- 756.

Mansilla C, et al. (2009). Immunization against hepatitis C virus with a fusion protein containing the extra domain A from fibronectin and the hepatitis C virus NS3 protein. J Hepatol 51 :520-527.

Migdal M, et al. (1998). Neuropilin-1 is a placenta growth factor-2 receptor. J Biol Chem 273:22272-22278.

Pan Q, et al. (2007). Neuropilin-1 binds to VEGF121 and regulates endothelial cell migration and sprouting. J Biol Chem 282:24049-24056.

Simons M, Ware JA (2003). Therapeutic angiogenesis in cardiovascular disease. Nat Rev Drug Discov 2:863-871.

Whitaker GB, Limberg BJ, Rosenbaum JS (2001). Vascular endothelial growth factor receptor-2 and neuropilin-1 form a receptor complex that is responsible for the differential signaling potency of VEGF(165) and VEGF(121). J Biol Chem 276:25520-25531.