DESIGNING NOVEL PEPTIDES FOR INDUCING FIBRONECTIN MATRIX ASSEMBLY

Cross-Reference To Related Applications

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional

Application No. 61/612,606 filed March 19, 2012, the contents of which are incorporated herein by reference in their entirety.

Field of the Invention

[0002] The invention described herein relates to isolated peptides that modulate the assembly, multimerization, aggregation and/or polymerization of proteins comprising one or more domains comprising a fibronectin-like type III domain, or a domain comprising a fibronectin type III fold, or beta sheet, e.g. fibronectin and fibrinogen.

Government Support

[0003] This invention was made in part with U.S. Government support grant POl CA45548 from the National Institute of Health and grant BC074986 from the Department of Defense. The U.S. Government has certain rights in this invention.

Sequence Listing

[0004] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 13, 2013, is named 002806-071971-PCT_SL.txt and is 152,592 bytes in size.

Background of the Invention

[0005] The extracellular matrix (ECM) provides chemical and mechanical cues that support cell survival, adhesion, and growth. The fibronectin component of the ECM is a provisional scaffold that is required for the assembly of other matrix components found in the more mature ECM, like collagen (Sottile and Hocking DC (2002) Molecular biology of the cell 13: 3546-3559). The ability to induce fibronectin polymerization can greatly enhance the rate at which scaffolds integrate into the target site as well as augment their mechanical properties through the highly stretchable character of fibronectin (Gildner et al. (2004) American journal of physiology Heart and circulatory physiology 287: H46-53; Hocking et al. (2000) The Journal of biological chemistry 275: 10673-10682; Ohashi et al. (1999) PNAS 96: 2153-2158). Fibronectin can also modulate availability of growth factors, such as TGF-β (Kawelke et al. PLoS One. 2011 ; 6:e28181. Epub 2011 Nov 28.), that are critical for wound healing, connective tissue integrity and fibrosis. Hence, modulation of fibronection multimerization can influence wound healing, fibrosis, proliferative diseases, tumorigenesis, angiogenesis, and the strength and integrity of connective tissues.

[0006] Anastellin, (US Patent Nos. 5,629,291 and 5,453,489) a 75 amino acid C-terminal protein fragment of the first repeat of FN type III, induces fibronection multimerization. This polypeptide has been shown to be both anti-angiogenic, (i.e. it can halt the cell cycle of endothelial cells and reduce blood vessel density in tumors (Ambesi et al. 2005 Cancer research 65: 148-156; Yi and Ruoslahti 2001 PNAS 98: 620-624; Neskey et al. 2008 Journal of experimental & clinical cancer research:CR 27: 61) and anti-tumorigenic, (i.e., it can suppress tumor growth and metastases).

Anastellin can produce a fibronectin multimer with enhanced capacity to facilitate cell adhesion to surfaces. However, the size of anastellin makes it poorly suited for administration to subjects.

Additionally, the length of anastellin forestalls industrial synthesis of the peptide and necessitates production via recombinant expression, a less desirable alternative. Anginex is a 33 amino acid long β-sheet forming peptide that is also capable of inducing fibronectin polymerization with an activity that is half the strength of anastellin (Akerman et al. (2005) PNAS 102: 2040-2045). Anginex, however, is not derived from fibronectin but instead is designed from combinatorial work on β-sheet containing anti-angiogenic proteins such as interleukin-8, platelet factor-4, and

bactericidal/permeability-increasing protein (Mayo et al. (1996) Protein science 5: 1301-1315).

Because Anginex is not a fibronectin-based molecule, the downstream fibronectin aggregates are obviously non-physiological.

[0007] CLT1 (a cyclic lOmer) and BBK32 (a 22-mer) each initiate fibronectin assembly and have been implicated in accumulation and/or homing to tumors. Notably, as CTL1 is a cyclic decapeptide, it requires additional cyclization steps not required by the, e.g. linear peptide embodiments described herein. Additionally, while CLT1 binds fibrin-FN complexes, it is unable to bind fibrin or immobilized fibronectin (Pilch et al. (2006) PNAS 103:2800-4).

Summary

[0008] Described herein are isolated peptides which can modulate the assembly, multimerization, aggregation and/or polymerization of proteins comprising a fibronectin-like type III domain or beta sheet or a domain comprising a fibronectin type III fold or beta sheet as well as compositions and methods relating to those peptides. These peptides and fragments, derivatives, and variants thereof, are amenable to in vitro synthesis and can be used in vitro or in vivo to modulate production and maintenance of the extracellular matrix, tumor growth and development, angiogenesis, fibrosis, and the mechanical integrity of connective tissues such as bone, muscle, and tendon, and the assembly of clotting proteins. Modulation of assembly, multimerization, aggregation and/or polymerization can encompass both inhibition of extracellular matrix assembly or enhancement and/or induction of extracellular matrix assembly. The nature of the modulation can be dependent upon, for example, the dosage, microenvironment, and the isolated peptide's amino acid sequence. Accordingly, the compositions described herein permit, e.g. modulation of fibronetic multimerization and/or drug loading of super fibronectin as a biologically-derived targeting nanoparticle. Further, embodiments of the compositions described herein can permit the incorporation of bioorthogonal moieties during chemical synthesis.

[0009] In some embodiments, it is advantageous to enhance or induce extracellular matrix assembly. Accordingly, provided herein are isolated peptides which induce and/or enhance the assembly, multimerization, aggregation and/or polymerization of proteins with one or more domains comprising a fibronectin-like type III domain, or a domain comprising a fibronectin type III fold or beta sheet as well as compositions and methods relating to those peptides. Aspects of the invention described herein are based on the inventors' discovery of a number of short fibronectin-derived peptides which can induce polymerization of fibronectin and/or fibrinogen. These peptides and fragments, derivatives, and variants thereof, are amenable to in vitro synthesis and can be used in vitro or in vivo to modulate production and maintenance of the extracellular matrix, tumor growth and development, angiogenesis, fibrosis, the mechanical integrity of connective tissues such as bone, muscle, and tendon, cell adhesion to ECM proteins, and the assembly of clotting proteins.

[0010] In some aspects, the technology described herein relates to an isolated peptide comprising the amino acid sequence of

Vali-Ser2-Asp3-Val4-X5-X6-X ₇ -X8- 9- io- ii- i2- i3- hri4-Xi5-Xi6- Seri ₇ -Xi ₈ - Xi ₉ -X ₂ o-Ser2i-X22-X23 (SEQ ID NO: 71) _; wherein X ₅ is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val; wherein X ₆ is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, and Asn; wherein X ₇ is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His; wherein X ₈ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser; wherein X ₉ is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro; wherein X ₁₀ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid; wherein X _n is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, Asn, Glu, and any beta-branched amino acid; wherein X ₁₂ is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X ₁₃ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin; wherein X _i5 is selected from the group consisting of Pro, Gly, Ala, Ser, Thr, Asp, Glu, Val, He and Leu; wherein X _i6 is selected from the group consisting of Thr, Gly, Ala, Ser, Gin, Glu, Asp, Asn, Leu, and Val; wherein X _i8 is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; wherein X _i9 is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, or any beta-branched amino acid; wherein X ₂ o is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein X ₂₂ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; wherein X ₂₃ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some aspects, the technology described herein relates to an isolated peptide comprising the amino acid sequence of

Vali-Ser ₂ -Asp ₃ -Val ₄ -X5-X6-X7-X ₈ -X ₉ -Xio-Xii-Xi ₂ - i3-Thri ₄ -Xi5-Xi6- Xi ₉ -X ₂₀ -Ser ₂ i-X ₂₂ -X ₂₃ (SEQ ID NO: 39) _; wherein X ₅ is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val; wherein X ₆ is selected from the group consisting of Arg, Ser, Thr; wherein X ₇ is selected from the group consisting of Asp, Lys, Asn, and His; wherein X ₈ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, and Ser;wherein X ₉ is selected from the group consisting of Glu, Gin, and Pro; wherein Xi ₀ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, and Thr; wherein Xn is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, and any beta-branched amino acid; wherein X _i2 is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X _i3 is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser; wherein X _i5 is selected from the group consisting of Pro, Gly, Ala, Val, He and Leu; wherein X _i6 is selected from the group consisting of Thr, Gly, Ala, Ser, and Val; wherein X _i8 is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, Thr; wherein X ₁₉ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, or any beta-branched amino acid; wherein X ₂₀ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp; wherein X ₂₂ is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr; wherein X ₂₃ is selected from the group consisting of Asp, Gin, Thr; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Val ₁ -Ser ₂ -Asp ₃ -Val ₄ -Pro ₅ -Arg ₆ -Asp ₇ -Leu ₈ - Glu ₉ -Val ₁₀ - Valn-Ala ₁₂ - Ala ₁₃ - Thr _i4 - Proi ₅ - Thr _i6 - Seri ₇ -Leui ₈ -Leui ₉ -Ile ₂₀ -Ser ₂ i-Trp ₂₂ -Asp ₂₃ (SEQ ID NO: 2).

[0011] In some embodiments, the peptide comprises the amino acid sequence of

Cys ₁ -Val ₂ -Ser ₃ -Asp ₄ -Val ₅ -X ₆ - X ₇ -X ₈ -X ₉ -X ₁₀ -Xn-Xi ₂ -Xi ₃ - X ₁₄ -Thr ₁₅ -X ₁₆ -X ₁₇ - Ser ₁₈ -X ₁₉ - X ₂₀ -X ₂ i-Ser ₂₂ -X ₂₃ -X ₂₄ (SEQ ID NO: 72) _; wherein X ₆ is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val; wherein X ₇ is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, Asn; wherein X ₈ is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His; wherein X ₉ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser;wherein Xi ₀ is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro; wherein Xn is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid; wherein X _i2 is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, Asn, Glu, and any beta-branched amino acid; wherein X _i3 is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X ₁₄ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin; wherein X ₁₆ is selected from the group consisting of Pro, Gly, Ala, Ser, Thr, Asp, Glu, Val, He and Leu; wherein X ₁₇ is selected from the group consisting of Thr, Gly, Ala, Ser, Gin, Glu, Asp, Asn, Leu, and Val; wherein X ₁₉ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; wherein X ₂₀ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, or any beta-branched amino acid; wherein X ₂ i is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein X ₂₃ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; and wherein X ₂ 4 is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe. In some embodiments, the peptide comprises the amino acid sequence of

Cys ₁ -Val ₂ -Ser ₃ -Asp4-Val5-X6- X ₇ -X ₈ -X ₉ -Xio-Xn-Xi2-Xi3- Xi ₄ -Thr ₁₅ -Xi ₆ -Xi7- Ser ₁₈ -X ₁₉ - X ₂₀ -X ₂ i-Ser ₂₂ -X ₂₃ -X ₂₄ (SEQ ID NO: 40) _; wherein X ₆ is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val; wherein X ₇ is selected from the group consisting of Arg, Ser, Thr; wherein X ₈ is selected from the group consisting of Asp, Lys, Asn, and His; wherein X ₉ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, and Ser;wherein Xi ₀ is selected from the group consisting of Glu, Gin, and Pro; wherein Xn is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, and Thr; wherein X _i2 is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, and any beta-branched amino acid; wherein X ₁₃ is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X ₁₄ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser; wherein X ₁₆ is selected from the group consisting of Pro, Gly, Ala, Val, He and Leu; wherein X ₁₇ is selected from the group consisting of Thr, Gly, Ala, Ser, and Val; wherein X ₁₉ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, Thr; wherein X ₂₀ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, or any beta-branched amino acid; wherein X ₂₁ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp; wherein X ₂₃ is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr; and wherein X ₂₄ is selected from the group consisting of Asp, Gin, and Thr. In some embodiments, the peptide comprises the amino acid sequence of Cysi-Val ₂ -Ser ₃ -Asp ₄ -Val ₅ - Pro ₆ - Arg ₇ - Asp ₈ -Leu ₉ - Gluio-Valn-Vali ₂ -Alai ₃ - Alai ₄ -Thri ₅ - Proi ₆ -Thri ₇ -Seri ₈ -Leui ₉ -Leu ₂₀ - Ile ₂ i-Ser ₂₂ -Trp ₂₃ -Asp ₂₄ (SEQ ID NO: 3).

[0012] In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Seri-X ₂ -X ₃ -X ₄ -Ser ₅ -X ₆ -X ₇ (SEQ ID NO: 73) _; wherein X ₂ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; wherein X ₃ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, or any beta-branched amino acid; wherein X ₄ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein X ₆ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; wherein X ₇ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Cys ₁ -Ser ₂ -X3-X4-X5-Ser ₆ -X ₇ -X ₈ (SEQ ID NO: 74); wherein X ₃ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; X ₄ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, and any beta-branched amino acid; X ₅ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; X ₇ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; and X ₈ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe. In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Seri-X ₂ -X3-X4-Ser ₅ -X ₆ -X7 (SEQ ID NO: 12) _; wherein X ₂ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, Thr; wherein X ₃ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, or any beta-branched amino acid; wherein X ₄ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp; wherein X ₆ is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr; wherein X ₇ is selected from the group consisting of Asp, Gin, Thr; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Cys ₁ -Ser ₂ -X3-X4-X5-Ser ₆ -X ₇ -X ₈ (SEQ ID NO: 41); wherein X ₃ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; X ₄ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, and any beta-branched amino acid; X ₅ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; X ₇ is selected from the group consisting ofTrp, Val, He, Leu, Phe, and Tyr; and X ₈ is selected from the group consisting of Asp, Gin, and Thr. In some embodiments, the peptide comprises the amino acid sequence of Ser ₁ -Leu ₂ -Leu ₃ -Ile ₄ -Ser ₅ -Trp ₆ -Asp ₇ (SEQ ID NO: 5) _. In some embodiments, the peptide comprises the amino acid sequence of Cys ₁ -Ser ₂ -Leu ₃ -Leu ₄ -Ile ₅ -Ser ₆ -Trp ₇ -Asp ₈ (SEQ ID NO: 14)

[0013] In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Χ ₁ -Χ ₂ -Χ ₃ -Χ ₄ -Χ ₅ -Χ ₆ -Χ ₇ -Χ ₈ -Τ1ΐΓ ₉ (SEQ ID NO: 75);wherein is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, and Asn; wherein X ₂ is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His; wherein X ₃ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser; wherein X ₄ is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid; wherein X ₆ is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, Asn, Glu, and any beta-branched amino acid; wherein X ₇ is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X ₈ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Tyr ₁ -X ₂ -X3-X ₄ -X5-X6-X7-X8-X9-Trir ₁ o (SEQ ID NO: 76);wherein X ₂ is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, and Asn; wherein X ₃ is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His; wherein X ₄ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser; wherein X ₅ is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro; wherein X ₆ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid; wherein X ₇ is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, Asn, Glu, and any beta-branched amino acid; wherein X ₈ is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; and wherein X ₉ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin.

[0014] In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Xi-X ₂ -X3-X4-X5-X6-X7-X8-Thr ₉ (SEQ ID NO: 15);wherein Xi is selected from the group consisting of Arg, Ser, Thr; wherein X ₂ is selected from the group consisting of Asp, Lys, Asn, and His; wherein X ₃ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, and Ser; wherein X ₄ is selected from the group consisting of Glu,Gln, and Pro; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, and Thr; wherein X ₆ is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, and any beta-branched amino acid; wherein X ₇ is selected from the group consisting of Ala,Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X ₈ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Arg ₁ -Asp ₂ -Leu ₃ -Glu ₄ -Val ₅ -Val ₆ -Ala ₇ -Ala ₈ -Thr ₉ (SEQ ID NO: 7). In some embodiments, the peptide comprises the amino acid sequence of

Tyri-X ₂ -X ₃ -X ₄ -X5-X6-X7-X8-X9-Thrio (SEQ ID NO: 26);wherein X ₂ is selected from the group consisting of Arg, Ser, Thr; wherein X ₃ is selected from the group consisting of Asp, Lys, Asn, and His; wherein X ₄ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, and Ser; wherein X ₅ is selected from the group consisting of Glu,Gln, and Pro; wherein X ₆ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, and Thr; wherein X ₇ is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, and any beta-branched amino acid; wherein X ₈ is selected from the group consisting of Ala,Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; and wherein X ₉ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, and Ser. In some embodiments, the peptide comprises the amino acid sequence of Tyr ₁ -Arg ₂ -Asp ₃ -Leu ₄ -Glu ₅ -Val ₆ -Val ₇ -Ala ₈ -Ala ₉ -Thr ₁ o (SEQ ID NO: 8).

[0015] In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Thr ₁ -Ala ₂ -Thr ₃ -Ile ₄ -Ser ₅ (SEQ ID NO: 9); or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Tyr ₁ -Thr ₂ -Ala ₃ -Thr ₄ -Ile ₅ -Ser ₆ (SEQ ID NO: 10).

[0016] In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Tyr ₁ -X ₂ -Arg ₃ -X4-Thr ₅ -X ₆ -X ₇ -Glu ₈ (SEQ ID NO: 77); wherein X ₂ is selected from a group consisting of Tyr, Ser, Ala, Val, He, Leu, Phe, and Trp; wherein X ₄ is selected from a group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein X ₆ is selected from a group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, His, Thr, and Cys; wherein X ₇ is selected from the group consisting of Gly, Arg, Glu, Ser, He, Thr, Val, His, Trp, and Cys; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of

Tyri-X ₂ -Arg ₃ -X ₄ -Thr ₅ -X6-X7-Glu ₈ (SEQ ID NO: 16); wherein X ₂ is selected from a group consisting of Tyr, Ala, Val, He, Leu, Phe, and Trp; wherein X ₄ is selected from a group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein Xe is selected from a group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, His, Thr, and Cys; wherein X ₇ is selected from the group consisting of Gly, Arg, Glu, Trp, and Cys; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of

Tyr ₁ -Tyr ₂ -Arg ₃ -Ile ₄ -Thr ₅ - Tyr ₆ -Gly ₇ -Glu ₈ (SEQ ID NO: 11) _.

[0017] In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Gln ₁ -Glu ₂ -X ₃ -Thr ₄ -X ₅ -Pro ₆ (SEQ ID NO: 78); wherein X ₃ is selected from the group consisiting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, Thr, Pro, and Glu; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, Pro, Glu, Lys, Ser, and any beta-branched amino acid; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Tyri-Gln ₂ -Glu ₃ -X ₄ -Thr ₅ -X ₆ -Pro ₇ (SEQ ID NO: 79); wherein X ₄ is selected from the group consisiting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, Thr, Pro, and Glu; and wherein X ₆ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, Pro, Glu, Lys, Ser, and any beta-branched amino acid. In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Glni-Glu ₂ -X ₃ -Thr ₄ -X ₅ -Pro ₆ (SEQ ID NO: 17); wherein X ₃ is selected from the group consisiting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, Thr; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, Pro, Glu, Lys, Ser; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Tyr ₁ -Gln ₂ -Glu ₃ -X ₄ -Thr ₅ -X ₆ -Pro ₇ (SEQ ID NO: 27); wherein X ₄ is selected from the group consisiting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, Thr; and wherein X ₆ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, Pro, Glu, Lys, and Ser. In some embodiments, the peptide comprises the amino acid sequence of Gln ₁ -Glu ₂ -Phe ₃ -Thr ₄ -Val ₅ -Pro ₆ (SEQ ID NO: 18) _. In some embodiments, the peptide comprises the amino acid sequence of Tyri-Gln ₂ -Glu ₃ -Phe ₄ -Thr ₅ -Val ₆ -Pro ₇ (SEQ ID NO: 19) _.

[0018] In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Xi-Thr ₂ -X3-Thr ₄ -X5-Tyr ₆ -X7-Val ₈ (SEQ ID NO: 20); wherein Xi is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, and Trp; wherein X ₃ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, and Gly; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, and Trp; wherein X ₇ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Ser, Thr, and Gin; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Xi-Thr ₂ -X3-Thr ₄ -X5-Tyr ₆ -X7-Val ₈ (SEQ ID NO: 80); wherein is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp; wherein X ₃ is selected from the group consisting of He , Ala, Val, Leu, Phe, Tyr, Trp, Gly; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp; wherein X ₇ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Ser, Thr, Gin; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Tyr ₁ -Thr ₂ -Ile ₃ -Thr ₄ -Val ₅ - Tyr ₆ -Ala ₇ -Val ₈ (SEQ ID NO: 21) _.

[0019] In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of X _! -Ser ₂ -X ₃ -Asn ₄ -X ₅ (SEQ ID NO: 81); wherein is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, Lys, Arg, Asp, Gin, Thr, and Pro; wherein X ₃ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, Gly, Gin, Asp, Thr, Ser, Arg, and Asn; wherein X ₅ is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, Lys, Gin, Ser, Thr, and Pro; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In one aspect, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Xi-Ser ₂ -X ₃ -Asn ₄ -X ₅ (SEQ ID NO: 22); wherein Xi is selected from the group consisting of He , Ala, Val, Leu, Phe, Tyr, Trp, Lys, Thr, and Pro; wherein X ₃ is selected from the group consisting of He , Ala, Val, Leu, Phe, Tyr, Trp, Gly, Gin, Asp, Thr, Ser, and Asn; wherein X ₅ is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, Lys, Gin, Ser, and Pro; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of

He ₁ -Ser ₂ -Ile ₃ -Asn ₄ -Tyr ₅ (SEQ ID NO: 23) _.

[0020] In some embodiments, the peptide comprises the corresponding amino acid sequence of a homolgous fibronectin gene.

[0021] In some embodiments, the peptide is comprised by a polypeptide comprising multiple or tandem occurrences of one or more of the peptides or segments of the peptides described herein. In some embodiments, the multiple occurrences of the peptides have a physical arrangement selected from the group consisting of: linear, branched; arrayed; multiplexed; cyclized.

[0022] In some embodiments, the peptide is comprised by a polypeptide comprised of at least two of the peptides linked by peptide bonds,chemical cross linkers, or other chemical bonds. The amino acid segment can comprise a chimera that contains contiguous, sequential, substitutional, intervening, or a combination of identical or non-identical peptide sequences or subsequences. The peptide can be embedded in a larger protein sequence.

[0023] In some embodiments, the peptide comprises a mutation elongating the loop region defined by Proi ₅ -Thri ₆ of SEQ ID NO:2, by Proi ₆ -Thri ₇ of SEQ ID NO:3, or by Xi ₅ -Xi ₆ of SEQ ID NO: 1.

[0024] In some embodiments, the peptide comprises a mutation that locks the peptide into a beta strand conformation or otherwise constrained conformation. In some embodiments, the mutation comprises a mutation selected from the group consisting of double Cys mutations or click chemistry or other crosslinking methodologies.

[0025] In some embodiments, the peptide comprises at least one D-amino acid. In some embodiments, the peptide comprises at least one beta-amino acid. In some embodiments, the peptide comprises at least one synthetic amino acid.

[0026] In some embodiments, the peptide comprises at least one peptide bond replacement. In some embodiments, the peptide comprises at least one peptide bond replacement selected from the non-limiting group consisting of: urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid, para-(aminoalkyl)-phenylacetic acid,

meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronic ester, olefinic group, and derivatives thereof.

[0027] In some embodiments, the peptide comprises at least one amino aid selected from the non-limiting group consisting of: amino acid analogs, chemically modified amino acids, non-natural amino acids, homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, hydroxylysine, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3 -mercapto-D -valine), ornithine, citruline, alpha-methyl-alanine, para-benzoylphenylalanine, para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine), diaminobutyric acid, 7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine,

biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine,

cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1-amino-l-cyclopentanecarboxylic acid, 1-amino-l-cyclohexanecarboxylic acid, amino-benzoic acid, amino-naphthoic acid,

gamma-aminobutyric acid, difluorophenylalanine, nipecotic acid, alpha-amino butyric acid, thienyl-alanine, t-butylglycine, trifluoro valine; hexafluoroleucine; fluorinated analogs; azide-modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and derivatives thereof.

[0028] In some embodiments, the peptide has a modification selected from the non-limiting group consisting of: PEGylation; post-translational derivatizations; glycosylation; hydroxylation;

methylation; HESylation; ELPylation; lipidation; acetylation; amidation; biotinylation; end-capping modifications; cyano group modifications; phosphorylation; cyclization; or other conjugation moieties (e.g. protein, antibody, peptide, nucleotide, virus, phage, matrix, insoluble support, particle, etc.).

Modification can be either covalent or non-covalent incorporating a linker and/or spacer or not.

[0029] In some embodiments, the peptide comprises a conservative substitution, insertion, or deletion of one or more amino acids.

[0030] In some embodiments, the peptide is a fusion peptide. In some embodiments, the peptide is coupled to a targeting molecule or inserted intrasequence to the targeting molecule in a single or multivalent fashion. In some embodiments, the peptide is coupled to or comprises a tag molecule selected from the non-limiting group consisting of: a contrast agent; a dye; a radioactive dye; a fluorescent molecule; 19 F; 2 H; 13 C; 15 N; and an isotope. In some embodiments, the peptide is coupled to a therapeutic molecule. In some embodiments, the therapeutic molecule is a chemotherapeutic molecule. In some embodiments, the therapeutic molecule is a fibrosis treatment molecule. In some embodiments, the fibrosis treatment molecule is selected from the non-limiting group consisting of: a steroid; a corticosteroid; an anti-inflammatory agent; and an immunosuppressant. In some embodiments, the therapeutic molecule is a cancer treatment molecule selected from the non-limiting group consisting of: a cytotoxic agent, a pro-apoptotic agent; and an anti-angiogenic agent. In some embodiments, the therapeutic molecule is a injury or wound treatment molecule selected from the non-limiting group consisting of: an anti-inflammatory agent and an antimicrobrial agent.

[0031] In one aspect, the technology described herein relates to an isolated nucleic acid encoding any of the peptides described herein. In one aspect, the technology described herein relates to an expression vector comprising an isolated nucleic acid encoding any of the peptides described herein.

[0032] In one aspect, the technology described herein relates to a composition consisting essentially of one or more peptides, nucleic acids, or vectors described herein as an active ingredient. In some embodiments, the composition further comprises a second pharmaceutically active agent. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0033] In one aspect, the technology described herein relates to a method of promoting the production or maintenance of the extracellular matrix in a subject in need thereof, the method comprising administering a peptide, nucleic acid, or composition as described herein. In some embodiments, the subject is in need of treatment for fibrosis. In some embodiments, the peptide is administered in combination with a fibrosis-treating agent. In some embodiments, the subject suffers from a condition selected from the non-limiting group consisting of: pulmonary fibrosis; scarring; scarring of the skin; trauma; a wound; corneal defects; corneal ulceration; diabetic ulcer; ulcer; sepsis; arthritis; idiopathic pulmonary fibrosis; cystic fibrosis; cirrhosis; endomyocardial fibrosis; mediastinal fibrosis; myelofibrosis; retroperitoneal fibrosis; progressive massive fibrosis; nephrogenic systemic fibrosis; Crohn's disease; keloid; scleroderma; systemic sclerosis; arthrofibrosis; adhesive capsulitis; lung fibrosis; liver fibrosis; kidney fibrosis; heart fibrosis; vascular fibrosis; skin fibrosis; eye fibrosis; bone marrow fibrosis; asthama; sarcoidosis; COPD; emphysema; nschistomasomiasis; cholangitis; diabetic nephropathy; lupus nephritis; postangioplasty aterial restenosis; atherosclerosis; burn scarring; hypertrophic scarring; nephrogenic fibrosing dermatopathy; postcataract surgery; proliferative vitreoretinopathy; Peyronie's disease; Duputren's contracture; dermatomyositis; and graft versus host disease.

[0034] In some embodiments, the subject is in need of treatment for a proliferative disease. In some embodiments, the peptide is administered in combination with a chemotherapeutic agent. In some embodiments, the proliferative disease is selected from the non-limiting group consisting of: cancer; a tumor; rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre -neoplastic lesions (e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia), carcinoma, oral hairy leukoplakia, and psoriasis.

[0035] In one aspect, the technology described herein relates to a method of inhibiting fibrosis in a subject in need thereof, the method comprising administering a peptide, nucleic acid, or composition described herein. In some embodiments, the peptide is administered in combination with a fibrosis-treating agent. In some embodiments, the fibrosis is a result of a condition selected from the non-limiting group consisting of: pulmonary fibrosis; scarring; scarring of the skin; trauma; a wound; corneal defects; corneal ulceration; diabetic ulcer; ulcer; sepsis; arthritis; idiopathic pulmonary fibrosis; cystic fibrosis; cirrhosis; endomyocardial fibrosis; mediastinal fibrosis; myelofibrosis; retroperitoneal fibrosis; progressive massive fibrosis; nephrogenic systemic fibrosis; Crohn's disease; keloid;

scleroderma; systemic sclerosis; arthrofibrosis; adhesive capsulitis; lung fibrosis; liver fibrosis; kidney fibrosis; heart fibrosis; vascular fibrosis; skin fibrosis; eye fibrosis; bone marrow fibrosis; asthama; sarcoidosis; COPD; emphysema; nschistomasomiasis; cholangitis; diabetic nephropathy; lupus nephritis; postangioplasty aterial restenosis; atherosclerosis; burn scarring; hypertrophic scarring; nephrogenic fibrosing dermatopathy; postcataract surgery; proliferative vitreoretinopathy; Peyronie's disease; Duputren's contracture; dermatomyositis; and graft versus host disease.

[0036] In one aspect, the technology described herein relates to a method of inhibiting cellular growth in a subject in need thereof, the method comprising administering a peptide, nucleic acid, or composition described herein. In some embodiments, the peptide is administered alone or in combination with a chemotherapeutic agent, cytotoxic agent, pro-apoptotic agent or anti-angiogenic agent. In some embodiments, the subject is a subject in need of treatment for a proliferative disease. In some embodiments, the proliferative disease is selected from the group consisting of: cancer; a tumor; rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre -neoplastic lesions (e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia), carcinoma, oral hairy leukoplakia, and psoriasis.

[0037] In one aspect, the technology described herein relates to a method of inhibiting angiogenesis in a subject in need thereof, the method comprising administering a peptide, nucleic acid, or composition described herein. In some embodiments, the peptide is administered in combination with a chemotherapeutic agent. In some embodiments, the subject is a subject in need of treatment for a proliferative disease. In some embodiments, the angiogenesis is associated with a condition selected from the group consisting of: cancer; a tumor; rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions (e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia), carcinoma, oral hairy leukoplakia, psoriasis, obesity, macular degeneration, and blindness.

[0038] In one aspect, the technology described herein relates to a method of increasing the strength of bone, tendon, ligaments, cartilage, or connective tissue wherein the method comprises administering a peptide, nucleic acid, or composition described herein.

[0039] In one aspect, the technology described herein relates to a method of promoting wound healing wherein the method comprises administering a peptide, nucleic acid, or composition described herein.

[0040] In one aspect, the technology described herein relates to a method of targeting, directing, or homing therapeutic agents, multifunctional moieties, or imaging molecules or moieties to sites of extracellular matrix production or accumulation wherein the method comprises administering to the subject a peptide, nucleic acid, or composition described herein, wherein the peptide is coupled to a therapeutic agent, a cytotoxic agent, a pro-apoptotic agent, an anti-angiogenic agent, a polypeptide, a protein, an antibody, a nucleic acid molecule, a small molecule, and/or an imaging molecule or moiety. In some embodiments, the site of extracellular matrix production or accumulation is a tumor. In some embodiments, the site of extracellular matrix production or accumulation is a fibrotic lesion. In some embodiments, the site of extracellular matrix production or accumulation is an injury or wound site or blood clot.

[0041] In one aspect, the technology described herein relates to a method of inducing the polymerization of a polypeptide comprising one or more domains comprising a fibronectin-like type III domain, or a domain comprising a fibronectin type III fold, or beta sheet, wherein the method comprises contacting at least two polypeptide or protein molecules with a peptide, nucleic acid, or composition described herein. In some embodiments, the polypeptide comprising one or more domains comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet is selected from the non-limiting group consisting of: fibronectin and fibrinogen.

[0042] In one aspect, the technology described herein relates to the use of a composition described herein, for preventing, treating and/or ameliorating fibrosis. In one aspect, the technology described herein relates to the use of a composition described herein, for preventing, treating and/or ameliorating a proliferative disease. In one aspect, the technology described herein relates to the use of a composition described herein, for promoting wound healing. In one aspect, the technology described herein relates to the use of a composition described herein, for increasing the strength of bone, tendon, ligaments, cartilage, or connective tissue.

Brief Description of the Drawings

[0043] Figures 1A-1B depict the sequences of the designed peptides that induce fibronectin assembly described herein. Figure 1 A depicts unfolding of 10FNIII by force applied at the RGD loop (blue) to a predicted intermediate structure. The location of the peptides CP1 (backbone highlighted, SEQ ID NO: 2), CPA (SEQ ID NO: 8), CPB (SEQ ID NO: 5), and CPE (SEQ ID NO: 10) within the predicted unfolded 10FNIII structure are highlighted. Figure IB outlines the sequences of the peptides CP1, CPA, CPB, and CPE with N- and C-terminal modifications.

[0044] Figures 2A-2C depict the multimerization of fibronectin induced by CP1 in a concentration dependent manner. In Figure 2A, increasing concentrations of CP1 (0-500 μΜ) are incubated with rhodamine fibronectin ([Rhod-FN] = 0.3 mg/ml) for 16 h at 37 °C. SDS-PAGE analysis (under non-reducing conditions) of the reaction mixtures shows that the addition of CP1, and not buffer alone (50 mM Tris ^» HCl, pH 7.4), results in assembly of FN into high molecular weight species (multimers) in a concentration dependent manner. Mixing Rhod-FN with control fragments derived from 10FNIII (CPA and CPE at comparable concentrations) did not yield significant increase in detectable multimers above baseline (data not shown). Dimeric FN (FN) and each of its two monomeric arms (arrow) are labeled on the gel. Figure 2B shows densitometric analysis of the gel for the FN and multimeric species labeled in Figure 2A. Figure 2C illustrates the multimerization of FN in the presence of increasing concentrations of CP1 (0-150 μΜ) as monitored by optical density at 590 nm (turbidity) for 4.5 h at 25 °C. At time 0 h, FN is added at a final concentration of 0.2 mg/ml to each peptide sample. The solution turbidity increases only following the addition of FN in a CP1 concentration dependent fashion. [0045] Figures 3A-3D demostrate that the C-terminal fragment of CP1 retains multimerization activity. Figure 3A depicts the results of the quantitative analysis by densitometry for multimers analyzed by non-reducing SDS-PAGE. Comparison of the peptides at 750 μΜ shows that CPB retains the multimerization activity of CP1. Error bars represent one standard deviation of the mean Rhod-FN intensity as determined by densitometric analysis of reaction mixtures analyzed by non-reducing SDS-PAGE (n=2 independent gels not shown). In Figure 3B, 750 μΜ CP1 or varying concentrations of CPB (50-750 μΜ) are incubated with FN and analyzed by non-reducing

SDS-PAGE. Figure 3C compares the multimer density as a function of CPB or CP1 concentration illustrating the enhanced activity of CP1. Figure 3D depicts the aggregation of 5 mg/ml fibrinogen, a protein containing domains with beta strand content, in the presence of increasing concentrations of CPB (0-500 μΜ) as monitored by turbidity for 2 h at 37 °C. The turbidity illustrates the action of CPB on other proteins containing domains with beta strand content.

[0046] Figures 4A-4C depict the exposure of hydrophobic sites in peptide and lOFNIII mixtures as assayed by 8-anilino-l-napthalenesulfonic acid (ANS) fluorescence. Figure 4 A denotes the circular dichroism spectrum of lOFNIII (10 μΜ, in 10 mM sodium phosphate, pH 7.4 at 25°C) showing significant beta strand content. Figure 4B represents the circular dichroism spectrum of CP1 (30 μΜ, in 10 mM sodium phosphate, pH 7.4 at 25°C) displaying a typical random coil signature. Figure 4C demonstrates that lOFNIII exposes hydrophobic sites as shown by the increase in ANS fluorescence over background. Addition of CP1 in the presence of lOFNIII further increases the exposure of hydrophobic sites as shown by the additional increase in ANS fluorescence. ANS (50 μΜ) and lOFNIII (50 μΜ) in the presence or absence of CP1 (50 μΜ) are excited at 360 nm in PBS at 25°C. Emission spectra are measured between 380 and 650 nm and are averaged across at least three measurements.

[0047] Figure 5 depicts enhanced adhesion of FN to lung fibroblast cells in the presence of

CPB. Lung fibroblast cells are cultured in the presense of increasing concentrations of CPB (0-200 μΜ) with biotinylated FN (10 ng/μΐ) for 36 h. Cells are washed and lysed in deoxycholate (DOC) buffer. DOC soluble fractions are separated by reducing SDS-PAGE and analyzed by Western blot (actin serves as the loading control). DOC soluble fractions show that increasing concentrations of CPB enhanced binding of biotinylated FN to the cell surface.

[0048] Figures 6A-6D demonstrate that the C-terminus of CP1 reduces the viability and cell growth of mammary adenocarcinoma cell lines. Weakly tumorigenic (M28) or tumorigenic (M6) cells are plated at low confluency in a 96-well plate and allowed to spread overnight. Anastellin (30 μΜ), CPA (150 μΜ), CPB (150 μΜ), or CPA (150 μΜ) + CPB (150 μΜ) (suspended in 25 mM HEPES, pH 7.1) are added in media the next day and refreshed on Day 3. On Day 6, resazurin, an indicator of metabolic capacity, was added. Percent viability (normalized against the control sample) was calculated by the turnover of resazurin during the 4h incubation for both M28 (Figure 6A) and M6 (Figure 6B) cells. Error bars represent standard error of the mean (n > 3 samples). Figure 6C depicts the results of an EdU-based growth assay analyzed by FACS (10000 events). M6 cells are plated at low density in a 24-well plate and allowed to spread overnight before inoculation with 0, 50, or 200 μΜ of peptide and refreshed every other day. On Day 4, the media was replaced with fresh peptide solution containing 10 μΜ Click-iT EdU for a 24 h incubation before labeling with Pacific Blue azide. Cell growth decreases for carcinoma cells treated with CPB, but not for CPA nor CPE (data not shown). Figure 6D quantifies the decrease in cell growth of carcinoma cells after a three day incubation with increasing CPB concentrations. Error bars represent standard error of the mean across two independent experiments (n=2).

[0049] Figure 7 depicts SMD simulations modeling force applied between the RGD loop and the N-terminus predict unfolding of 10FNIII to an intermediate. When cell traction imparts force (FCell) at the RGD loop (central Gly depicted as a bead) of 10FNIII (left) anchored at the N-terminus (bead), the domain is predicted to unfold to a kinetic intermediate (right) with an unraveled N-terminus exposing β-strands A and B (labeled CPl) but not strand E (26).

[0050] Figures 8 demonstrates the polymerization of fibronectin by CPl, as compared to anastellin. Figure 8 depicts an image of non-reducing SDS-PAGE analysis of rhodamine labeled FN (0.3 mg/ml) incubated with buffer '(-)', anastellin, CPl, or CPlscr at 150 μΜ for 16 h at 37 °C shows that both anastellin and CPl induced the formation of a multimeric FN species ('Multimer') with large discrete molecular weights relative to the disulfide-bonded molecular FN ('FN') (left). Preparations of labeled fibronectin contain reduced monomeric fibronectin arms near the indicated 250 kD standard. Figure 8 also depicts a graph of densitometry of rhodamine fluorescence (right) revealing significant multimerization of fibronectin by anastellin and CPl (p < 0.01, two-tailed). CPl shows significantly higher activity than anastellin (*, p < 0.05, one-tailed), and scrambling the CPl sequence reduced its activity (**, p < 0.01, one-tailed) to background levels (p > 0.05, two-tailed).

[0051] Figures 9A-9B demonstrate the effect of CPE and 10FNIII on fibronectin

multimerization. Figure 9A depicts a graph of densitometry of rhodamine fluorescence of labeled fibronectin analyzed by SDS-PAGE showing low-level multimerization of labeled fibronectin induced by CPE (50-400 μΜ). Multimers were induced significantly above background by high concentrations of CPE (150 & 250 μΜ, *, p < 0.05, two-tailed; 400 μΜ: **, p < 0.01, two-tailed). Figure 9B depicts a graph of densitometry of rhodamine fluorescence revealing that recombinant 10FNIII (50-500 μΜ) does not induce significant fibronectin multimerization above background (p > 0.05, two-tailed).

[0052] Figures 10A-10E demonstrate that CPl interacts with 10FNIII. Figure 10A depicts a graph of densitometry of rhodamine labeled fibronectin multimers separated by SDS-PAGE demonstrating that fibronectin multimer formation initiated by 150 μΜ CPl was reduced in the presence of 75 μΜ 10FNIII (**, p < 0.01, one-tailed). The multimerization by CPl (150 μΜ) in the presence of 10FNIII (75 μΜ) is significantly different from the samples for 10FNIII alone (*, p < 0.05, two-tailed) or background (**, p < 0.01, two-tailed). Figure 10B depicts a graph comparing of ANS (50 μΜ) emission intensities in the presence of buffer, CPl (150 μΜ), lOFNIII (50 μΜ), anastellin (50 μΜ), and a comixture of lOFNIII (50 μΜ) and CPl (150 μΜ). Figure IOC depicts a graph of the CD spectrum of 10 μΜ lOFNIII. Figure 10D depicts a graph of CD spectrum of 30 μΜ CPl. Figure 10E depicts a graph of quantification of ANS-dependent emission spectra maxima above background as a percentage of the maximal ANS emission in the presence of anastellin (50 μΜ) for samples containing lOFNIII (50 μΜ) with or without CPl or CPlscr (150 μΜ). ANS fluorescence due to anastellin and coniixtures of lOFNIII with CPl or CPlscr are significantly higher than ANS background (*, p < 0.05, one -tailed).

[0053] Figures 11 A-l IF demonstrate the effect of CPB on fibronectin and lOFNIII. Figure

11 A depicts a graph of densitometry analysis of rhodamine labeled FN multimers separated by SDS-PAGE showed significant fibronectin multimerization by 500 μΜ CPl, CPB, or equimolar coniixtures of CPA and CPB (p < 0.01, two-tailed). Combination of CPA and CPB minimally enhanced CPB activity (p = 0.05, two-tailed) but is significantly higher than that for equimolar CPl (**, p < 0.01, two-tailed). A W6A point mutation in CPB abolished multimerization activity. Figure 11B depicts a graph of densitometry analysis of CPl or CPB (150-750 μΜ) mixtures with rhodamine labeled fibronectin separated by SDS-PAGE showing significant fibronectin multimerization (p < 0.01, two-tailed). CPl induced more multimers at 150 μΜ than at 500 μΜ (**, p < 0.01, one-tailed) and was significantly different from CPB at 150 μΜ (**, p < 0.01, two-tailed) but similar to 500-750 μΜ CPB (p > 0.05, two-tailed). Figure 11C depicts a graph of a comparison of ANS (50 μΜ) emission intensities in the presence of buffer, CPB (500 μΜ), lOFNIII (50 μΜ), anastellin (50 μΜ), and a comixture of lOFNIII (50 μΜ) and CPB (500 μΜ). Figure 1 ID depicts a graph of quantification of ANS-dependent emission spectra maxima above background as a percentage of the maximal ANS emission in the presence of anastellin (50 μΜ) for samples containing lOFNIII (50 μΜ) with or without 500 μΜ CPB or CPB(W6A). A significant increase in fluorescence was measured for anastellin (*, p < 0.05, one -tailed) and lOFNIII with CPB (**, p < 0.01, one-tailed) or CPB(W6A) (*, p < 0.05, one-tailed). Addition of CPB, but not CPB(W6A), to lOFNIII significantly increased ANS fluorescence above that for lOFNIII (*, p < 0.05, one-tailed). Figure 1 IE depicts a graph of the CD spectrum of 70 μΜ CPB. Figure 1 IF depicts a graph comparison of ThT (20 μΜ) fluorescence at 482 nm after a 60 min incubation at 25 °C for lOFNIII (10 μΜ) in the presence or absence of 500 μΜ CPB or CPB(W6A). Addition of CPB to lOFNIII led to a significant increase in fluorescence above that for CPB (*, p< 0.05, one-tailed) or lOFNIII (**, p < 0.01, one-tailed). Addition of CPB(W6A) to lOFNIII did not lead to a significant change in fluorescence (p > 0.05, two-tailed).

[0054] Figures 12A-12C demonstrates the effect of CPB on FBG polymerization. Figure 12A depicts a graph of the development of turbidity at 590 nm (25 °C) by CPB (500-750 μΜ) as monitored before and after the addition of FBG (5 mg/ml) at t = 0 min (dashed line marked '+FBG'). Figurel2B depicts a graph of time traces of ThT -dependent (20 μΜ) fluorescence at 482 nm (25 °C) for buffer, CPB (500 μΜ), FBG (10 μΜ), and a comixture of FBG (10 μΜ) and CPB (500 μΜ). Figure 12C depicts a graph of ThT-dependent fluorescence for buffer, CPB(W6A) (500 μΜ), FBG (10 μΜ), and a comixture of FBG (10 μΜ) and CPB(W6A) (500 μΜ) over time.

[0055] Figure 13 demonstrates that CPB binds to FN by comparing the fluorescence of rhodamine labeled FN, unlabeled FN, and unlabeled FN incubated with FITC labeled CPB (500 μΜ) separated by non-reducing SDS-PAGE analysis. The FN dimer (labeled FN) and reduced arms near 250 kD are labeled. Addition of FITC labeled CPB to unlabeled FN enables visualization of the protein.

[0056] Figure 14 demonstrates enhanced accumulation of biotin labeled FN into the deoxycholate insoluble pool by lung fibroblast cells in the presence of anastellin and CPB, but not mutant peptide CPB(W6A). Samples loading volumes were normalized based on vimentin band intensity.

[0057] Figure 15 demonstrates the accumulation of the CPB peptide in 4T1 mammary tumors implanted orthotopically in the mammary fat pad of mice following intravenous injection of pre -formed multimers generated by the pre-incubation of FITC labeled CPB with unlabeled FN. Average fluorescence signal represented as percent injected dose per gram ( ID/g) of homogenized tumor tissue (n=2) shows significant fluorescence above untreated samples (*, p < 0.05, two-tailed).

Detailed Description of the Invention

[0058] Aspects of the invention described herein relate to peptides identified by the inventors which have the ability to modulate assembly, multimerization, aggregation and/or polymerization of polypeptides and/or proteins which comprise one or more domains comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet. By virtue of this activity, the isolated peptides described herein can, for example, modulate fibrosis, tumorigenesis, angiogenesis, the formation of extracellular matrix, the strength of connective tissue, the degree of cell adhesion to the ECM, and assembly of clotting proteins. In some embodiments, the isolated peptides described herein can, for example, promote or inhibit the formation of extracellular matrix, inhibit fibrosis, inhibit tumorigenesis and angiogenesis, strengthen connective tissue, enhance cell adhesion to the ECM, and facilitate the assembly of clotting proteins. The isolated peptides discovered by the inventors, as well as methods and compositions relating to the peptides and their applications are described further herein.

[0059] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0061] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0062] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0063] As used in this specification and the appended claims, the singular forms "a," "an," and

"the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0064] Definitions of common terms in cell biology and molecular biology can be found in

"The Merck Manual of Diagnosis and Therapy", 19th Edition, published by Merck Research

Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); The ELISA guidebook (Methods in molecular biology 149) by Crowther J. R. (2000). Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), , Molecular Biology and

Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0065] Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in U. S. Pat. Nos: 4,965,343, and 5,849,954; Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.); which are all incorporated by reference herein in their entireties.

[0066] The terms "decrease," "reduce," "reduced", and "reduction" are all used herein generally to mean a decrease by a statistically significant amount relative to a reference. However, for avoidance of doubt, "reduce," "reduction", or "decrease" typically means a decrease by at least 10% as compared to the absence of a given treatment and can include, for example, a decrease by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , up to and including, for example, the complete absence of the given entity or parameter as compared to the absence of a given treatment, or any decrease between 10-99% as compared to the absence of a given treatment.

[0067] The terms "increased" /'increase", or "enhance" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase", or "enhance" means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0068] As used herein in the context of expression, the terms "treat," "treatment," and the like, as used in the context of the therapeutic methods described herein, refer to a decrease in severity, indicators, symptoms, and/or markers of fibrotic conditions or proliferative diseases as described herein. In the context of the present invention insofar as it relates to any of the conditions recited herein, the terms "treat," "treatment," and the like mean to relieve, alleviate, ameliorate, inhibit, slow down, reverse, or stop the progression, aggravation, deterioration, anticipated progression or severity of at least one symptom or complication associated with a fibrotic condition or proliferative disease. In one embodiment, the symptoms of the fibrotic condition or proliferative disease are alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0069] As used herein, the term "administer" refers to the placement of a composition into a subject by a method or route which results in delivery of at least part of the administered composition to a desired site such that the desired effect is produced. A compound or composition described herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, topical (including buccal and sublingual), intracranial, and intracerebral administration.

[0070] In some embodiments, a "subject" as used herein can be a human or an animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf. Patient or subject includes any subset of the foregoing, e.g., all of the above. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human.

[0071] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of fibrotic conditions or proliferative diseases. In addition, the methods and assays described herein can be used to treat domesticated animals and/or pets. A subject can be one who has been previously diagnosed with or identified as suffering from or having a fibrotic condition or proliferative disease or one or more complications related to a fibrotic condition or proliferative disease, and optionally, but need not have already undergone treatment for a fibrotic condition or proliferative disease or the one or more complications related to a fibrotic condition or proliferative disease. Alternatively, a subject can also be one who has not been previously diagnosed as having a fibrotic condition or proliferative disease or one or more complications related to a fibrotic condition or proliferative disease. For example, a subject can be one who exhibits one or more risk factors for a fibrotic condition or proliferative disease or one or more complications related to a fibrotic condition or proliferative disease or a subject who does not exhibit fibrotic condition or proliferative disease risk factors.

[0072] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one strand nucleic acid of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the template nucleic acid is DNA. In another aspect, the template is RNA. Suitable nucleic acid molecules are DNA, including genomic DNA, ribosomal DNA and cDNA. Other suitable nucleic acid molecules are RNA, including mRNA, rRNA and tRNA. The nucleic acid molecule can be naturally occurring, as in genomic DNA, or it may be synthetic, i.e., prepared based up human action, or may be a combination of the two. The nucleic acid molecule can also have certain modification such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl

(2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA), cholesterol addition, and phosphorothioate backbone as described in US Patent Application

20070213292; and certain ribonucleoside that are is linked between the 2'-oxygen and the 4'-carbon atoms with a methylene unit as described in US Pat No. 6,268,490, wherein both patent and patent application are incorporated hereby reference in their entirety.

[0073] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene.

[0074] The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5' untranslated (5'UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0075] As used herein, the term "heterologous nucleic acid fragments" refers to nucleic acid sequences that are not naturally occurring in that cell.

[0076] The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. As used herein, the term "viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the gene encoding an isolated peptide as described herein in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0077] The term "replication incompetent" when used in reference to a viral vector means the viral vector cannot further replicate and package its genomes. For example, when the cells of a subject are infected with replication incompetent recombinant adeno-associated virus (rAAV) virions, the heterologous (also known as transgene) gene is expressed in the patient's cells, but, the rAAV is replication defective (e.g., lacks accessory genes that encode essential proteins for packaging the virus) and viral particles cannot be formed in the patient's cells. The term "transduction" as used herein refers to the use of viral particles or viruses to introduce exogenous nucleic acids into a cell.

[0078] The term "transfection" as used herein in reference to methods, such as chemical methods, to introduce exogenous nucleic acids, such as the nucleic acid sequences encoding an an isolated peptide as described herein, into a cell. As used herein, the term transfection does not encompass viral-based methods of introducing exogenous nucleic acids into a cell. Methods of transfection include physical treatments (electroporation, nanoparticles, magnetofection, or

microinjections), and chemical-based transfection methods. Chemical-based transfection methods include, but are not limited to those that use cyclodextrin, polymers, liposomes, nanoparticles, cationic lipids or mixtures thereof (e.g., DOPA, Lipofectamine and UptiFectin), and cationic polymers, such as DEAE-dextran or polyethylenimine.

[0079] As used herein, the term "proteins", "peptide", and "polypeptides" are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "proteins", "peptide", and "polypeptides" , which are used interchangeably herein, refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, PEGylated, Updated, etc.) and amino acid analogs, regardless of its size or function. The terms "proteins", "peptide", and "polypeptides" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides, peptides, or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides (e.g.

polypeptides comprising 75 or fewer amino acids), but usage of these terms in the art overlaps.

[0080] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically bind an antigen. The terms also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms besides antibodies; including, for example, Fv, Fab, and F(ab)'2 as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference) or synthetic monobodies and/or adnectins (See, generally, Hood et al., Immunology, Benjamin, N.Y., 2ND ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference).

[0081] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) below and/or above normal, or lower or higher than the concentration of a marker in a reference sample. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.

[0082] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%.

[0083] Other terms are defined herein within the description of the various aspects of the invention.

[0084] Aspects of the invention described herein are directed to peptides, compositions, and methods relating to isolated peptides which induce or modulate the assembly, multimerization, aggregation and/or polymerization of polypeptides comprising one or more domains comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet. As used herein, "fibronectin" refers to the polypeptide of SEQ ID NOs: 32, 33, 34, 35, 36, or 37 (NCBI Gene ID No; 2335), and homologues, variants, and/or fragments thereof having a length of at least 200 amino acids and at least 80% identical to any of SEQ ID NOs: 32-37 or a homologue thereof, e.g. 80% or more identical, 85% or more identical, 90% or more identical, 95% or more identical, 98% or more identical, or 99% or more identical. As used herein, a "fibronectin type III domain" refers to the third type of internal repeat found in fibronectin that folds into an immunoglobin-like beta sandwich structure formed by two anti-parallel beta sheets composed of seven total beta strands that encloses a hydrophobic core. This fibronection type III fold is found in approximately 2% of identified mammalian proteins. A fibronectin type III fold can be identified by the distinctive beta sheet folding pattern described above herein. The fibronectin type III domain is also referred to as "FNIII" and is annotated in the NCBI conserved domain database as cd00063 and is listed as a superfamily (SCOP 49265) in the structural classification of proteins database (available on the world wide web at

http://scop.mrc-lmb.cam.ac.uk/scop/). In one embodiment, a fibronectin type III domain can comprise the amino acid sequence of SEQ ID NO: 38.

[0085] Proteins comprising a fibronectin type III fold can include, but are not limited to, fibronectin, membrane spanning cytokine receptors, growth hormone receptors, tyrosine phosphatase receptors, adhesion molecules, intracellular proteins, extracellular proteins, and bacterial glycosyl hydrolases. Non-limiting examples of proteins comprising a fibronectin-like type III domain or fibronectin-like type III repeat include ABI3BP; ANKFN1 ; ASTN2; AXL; BOC; BZRAP1 ; C20orf75; CDON; CHL1 ; CMYA5; CNTFR; CNTN1 ; CNTN2; CNTN3; CNTN4; CNTN5; CNTN6; COL12A1 ; COL14A1 ; COL20A1 ; COL7A1 ; CRLF1 ;CRLF3; CSF2RB; CSF3R; DCC; DSCAM; DSCAML1; EBI3; EGFLAM; EPHA1 ; EPHA10; EPHA2; EPHA3; EPHA4; EPHA5; EPHA6; EPHA7; EPHA8; EPHB 1 ; EPHB2; EPHB3; EPHB4; EPHB6;EPOR; FANK1 ; FLRT1; FLRT2; FLRT3; FN1 ; FNDC1 ; FNDC3A; FNDC3B; FNDC4; FNDC5; FNDC7; FNDC8; FSD1 ; FSD1L; FSD2; GHR; HCFC1 ; HCFC2; HUGO; IFNGR2; IGF1R; IGSF22;IGSF9; IGSF9B; IL11RA; IL12B; IL12RB1 ; IL12RB2; IL20RB; IL23R; IL27RA; IL31RA; IL6R; IL6ST; IL7R; INSR; INSRR; ITGB4; I16ST; KALI ;

KALRN; L1CAM; LEPR; LIFR; LRFN2; LRFN3; LRFN4;LRFN5; LRIT1 ; LRRN1 ; LRRN3;

MERTK; MIDI ; MID2; MPL; MYBPC1 ; MYBPC2; MYBPC3; MYBPH; MYBPHL; MYLK;

MYOM1 ; MYOM2; MYOM3; NCAM1 ; NCAM2; NEOl ; NFASC; NOPE;NPHSl ; NRCAM; OBSCN; OBSL1 ; OSMR; PHYHIP; PHYHIPL; PRLR; PRODH2; PTPRB; PTPRC; PTPRD; PTPRF; PTPRG; PTPRH; PTPRJ; PTPRK; PTPRM; PTPRO; PTPRS; PTPRT;PTPRU; PTPRZ1 ; PTPsigma; PUNC; RIMBP2; ROBOl ; ROB02; ROB03; ROB04; ROS1 ; SDK1 ; SDK2; SNED1 ; SORL1 ; SPEG; TEK; TIE1 ; TNC; TNN; TNR; TNXB ; TRIM36; TRIM42; TRIM46;TRIM67; TRIM9; TTN; TYR03; UMODL1 ; USH2A; VASN; VWA1 ; dJ34F7.1 ; fmi; proteins comprising beta sheet (e.g. fibrinogen); proteins with a beta sandwich fold (e.g. nectin and the immunoglobin superfamily (cll 1960 in the NCBI CDD)); and proteins containing an immunoglobin-like fold (e.g. tenascin, titin, etc).

[0086] In some aspects, the invention described herein relates to isolated peptides identified by the inventors which induce or modulate the assembly, multimerization, aggregation and/or polymerization of polypeptides comprising a fibronectin type III domain. In some embodiments, the isolated peptides comprise fragments of human fibronectin which have been identified to have the assembly, multimerization, aggregation and/or polymerization activity. In some embodiments, the isolated peptides comprise variants, substitutions, homologues, or derivatives of the fragments of human fibronection which have been identified to have the assembly, multimerization, aggregation, and/or polymerization activity. In some embodiments, an isolated peptide as described herein is not fibronectin. In some embodiments, an isolated peptide as described herein comprises 75 or fewer amino acids.

[0087] In some aspects, the invention described herein relates to isolated peptides identified by the inventors which induce or modulate the assembly, multimerization, aggregation and/or polymerization of polypeptides comprising a fibronectin type III domain. In some embodiments, the isolated peptides comprise fragments of human fibronectin which have been identified to have the assembly, multimerization, aggregation and/or polymerization activity. In some embodiments, the isolated peptides comprise variants, substitutions, homologues, or derivatives of the fragments of human fibronection which have been identified to have the assembly, multimerization, aggregation, and/or polymerization activity. In some embodiments, an isolated peptide as described herein is not fibronectin. In some embodiments, an isolated peptide as described herein comprises 75 or fewer amino acids.

[0088] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Val ₁ -Ser ₂ -Asp ₃ -Val ₄ -X5-X ₆ -X7-X ₈ -X ₉ -X ₁ o-Xii-Xi2-Xi3- hr ₁ 4-X ₁ 5-X ₁₆ - Ser ₁₇ -X ₁₈ - Xi9-X ₂ o-Ser ₂ i-X22-X23 (SEQ ID NO: 71) _; wherein X ₅ is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val; wherein X ₆ is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, and Asn; wherein X ₇ is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His; wherein X ₈ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser; wherein X ₉ is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro; wherein Xi ₀ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid; wherein Xn is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, Asn, Glu, and any beta-branched amino acid; wherein X ₁₂ is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X ₁₃ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin; wherein X ₁₅ is selected from the group consisting of Pro, Gly, Ala, Ser, Thr, Asp, Glu, Val, He and Leu; wherein X ₁₆ is selected from the group consisting of Thr, Gly, Ala, Ser, Gin, Glu, Asp, Asn, Leu, and Val; wherein X _i8 is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; wherein X _i9 is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, or any beta-branched amino acid; wherein X ₂ o is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein X ₂₂ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; wherein X ₂₃ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Val ₁ -Ser ₂ -Asp ₃ -Val ₄ -X5-X ₆ -X7-X ₈ -X ₉ -X ₁ o-Xii-Xi ₂ -Xi3- Thr ₁₄ -X ₁₅ - Xi ₆ -Ser ₁₇ - X ₁₈ - X ₁₉ -X ₂₀ -Ser ₂₁ -X ₂₂ -X ₂₃ (SEQ ID NO: 24). wherein X ₅ is selected from the group consisting of Pro, Gly, Ala, and Leu, Ser, Val; wherein X ₆ is selected from the group consisting of Arg, Ser, Thr; wherein X ₇ is selected from the group consisting of Asp, Lys, Asn, and His; wherein X ₈ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, and Ser;wherein X ₉ is selected from the group consisting of Glu,Gln, and Pro; wherein X ₁₀ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, and Thr; wherein X _n is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, and any beta-branched amino acid; wherein X ₁₂ is selected from the group consisting of Ala,Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X _i3 is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser; wherein X _i5 is selected from the group consisting of Pro, Gly, Ala, Val, He, and Leu; wherein X _i6 is selected from the group consisting of Thr, Gly, Ala, Ser, and Val; wherein X _i8 is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, Thr; wherein X _i9 is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, or any beta-branched amino acid; wherein X ₂₀ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp; wherein X ₂₂ is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr; and wherein X ₂₃ is selected from the group consisting of Asp, Gin, Thr.

[0089] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Vali-Ser ₂ -Asp ₃ -Val ₄ -X5-Arg ₆ -X7-Leu ₈ -X ₉ -Valio-Xii-Xi ₂ -Alai ₃ - Thri ₄ -Xi ₅ - Thri ₆ -Seri ₇ - Leuis- Xi ₉ -Ile ₂₀ -Ser ₂ i-X ₂₂ -Asp ₂₃ (SEQ ID NO: 1) _; wherein X ₅ is selected from the group consisting of Pro, Gly, Ala, and Leu; wherein X ₇ is selected from the group consisting of Asp, Lys, and Asn; wherein X ₉ is selected from the group consisting of Glu and Gin; wherein Xn is selected from the group consisting of Val, Trp, He, Leu, and Phe; wherein X ₁₂ is selected from the group consisting of Ala and Gly; wherein X ₁₅ is selected from the group consisting of Pro, Gly, Ala, and Leu; wherein X ₁₉ is selected from the group consisting of Leu, Trp, Val, He, and Phe; and wherein X ₂₂ is selected from the group consisting of Trp, Val, He, Leu, and Phe. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Val _! -Ser ₂ -Asp ₃ - Val ₄ -Pro ₅ -Arg ₆ -Asp ₇ -Leu ₈ -Glu ₉ -Val ₁₀

-Valii-Alai ₂ -Alai ₃ -Thri ₄ -Proi ₅ -Thri ₆ -Seri ₇ -Leui ₈ -Leui ₉ - Ile ₂ o-Ser ₂ i-Trp ₂₂ -Asp ₂₃ (SEQ ID NO: 2).

[0090] In some embodiments, the peptide comprises the amino acid sequence of

Cys ₁ -Val2-Ser ₃ -Asp4-Val5-X ₆ - X ₇ -X ₈ -X ₉ -X ₁₀ -Xii-Xi ₂ -Xi3- Xi ₄ -Thr ₁₅ -X ₁₆ -X ₁₇ - Ser ₁₈ -X ₁₉ - X ₂ o-X ₂ i-Ser ₂ 2-X ₂₃ -X ₂₄ (SEQ ID NO: 72) _; wherein X ₆ is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val; wherein X ₇ is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, Asn; wherein X ₈ is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His; wherein X ₉ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser;wherein Xi ₀ is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro; wherein Xn is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid; wherein X _i2 is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, Asn, Glu, and any beta-branched amino acid; wherein X _i3 is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X ₁₄ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin; wherein X ₁₆ is selected from the group consisting of Pro, Gly, Ala, Ser, Thr, Asp, Glu, Val, He and Leu; wherein X ₁₇ is selected from the group consisting of Thr, Gly, Ala, Ser, Gin, Glu, Asp, Asn, Leu, and Val; wherein X ₁₉ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; wherein X ₂₀ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, or any beta-branched amino acid; wherein X ₂ i is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein X ₂₃ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; and wherein X ₂₄ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe. In some aspects, the technology described herein relates to an isolated peptide comprising the amino acid sequence of Cysi-Val ₂ -Ser ₃ -Asp ₄ -Val5-X ₆ - Χ ₇ -Χ ₈ -Χ ₉ -Χι ₀ -Χη-Χΐ2-Χΐ3- Xi ₄ -Thri5-Xi ₆ -Xi ₇ - Seri ₈ -Xi ₉ - X ₂ o-X ₂ i-Ser ₂ 2-X ₂₃ -X ₂₄ (SEQ ID NO: 25); wherein X ₆ is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val; wherein X ₇ is selected from the group consisting of Arg, Ser, Thr; wherein X ₈ is selected from the group consisting of Asp, Lys, Asn, and His; wherein X ₉ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, and Ser;wherein Xi ₀ is selected from the group consisting of Glu, Gin, and Pro; wherein Xn is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, and Thr; wherein X _i2 is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, and any beta-branched amino acid; wherein X ₁₃ is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X ₁₄ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser; wherein X ₁₆ is selected from the group consisting of Pro, Gly, Ala, Val, He and Leu; wherein X ₁₇ is selected from the group consisting of Thr, Gly, Ala, Ser, and Val; wherein X ₁₉ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, Thr; wherein X ₂₀ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, or any beta-branched amino acid; wherein X ₂ i is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp; wherein X ₂₃ is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr; wherein X ₂ 4 is selected from the group consisting of Asp, Gin, Thr. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Cysi-Val ₂ -Ser ₃ - Asp ₄ -Val ₅ -Pro ₆ -Arg ₇ -Asp ₈ - Leu ₉ -Glui ₀ - Valn-Vali ₂ -Alai ₃ -Alai ₄ -Thri ₅ -Proi ₆ -Thri ₇ - Serig- Leui ₉ -Leu ₂ o-Ile ₂ i-Ser ₂₂ -Trp ₂ 3-Asp ₂₄ (SEQ ID NO: 3).

[0091] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Seri- X ₂ - X ₃ -X ₄ -Ser ₅ -X ₆ -X7 (SEQ ID NO: 12) wherein wherein X ₂ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, Thr; wherein X ₃ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, or any beta-branched amino acid; wherein X ₄ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp; wherein X ₆ is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr; wherein X ₇ is selected from the group consisting of Asp, Gin, Thr. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Ser ₁ -Leu ₂ -Leu ₃ -Ile ₄ -Ser ₅ -X ₆ -Asp ₇ (SEQ ID NO: 4) _; wherein X ₆ is selected from the group consisting of Trp, Val, He, Leu, and Phe.

[0092] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Ser _! -X ₂ -X ₃ -X ₄ -Ser ₅ -X ₆ -X ₇ (SEQ ID NO: 73) _; wherein X ₂ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; wherein X ₃ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, or any beta-branched amino acid; wherein X ₄ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein X ₆ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; wherein X ₇ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Cysi-Ser ₂ -X ₃ -X ₄ -X ₅ -Ser ₆ -X ₇ -X8 (SEQ ID NO: 74); wherein X ₃ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; X ₄ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, and any beta-branched amino acid; X ₅ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; X ₇ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; and X ₈ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe.

[0093] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Cysi-Ser ₂ -X ₃ -X ₄ -X ₅ -Ser ₆ -X ₇ -X8 (SEQ ID NO: 13); wherein X ₃ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; X ₄ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Ser, He, Tyr, His, Cys, and any beta-branched amino acid; X ₅ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; X ₇ is selected from the group consisting ofTrp, Val, He, Leu, Phe, and Tyr; and X ₈ is selected from the group consisting of Asp, Gin, and Thr. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Cysi-Ser ₂ -X3-X ₄ -X5-Ser ₆ -Trp ₇ -X ₈ (SEQ ID NO: 28); wherein X ₃ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr; X ₄ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Ser, He, Tyr, His, Cys, and any beta-branched amino acid; X ₅ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; and X ₈ is selected from the group consisting of Asp, Gin, and Thr.

[0094] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Seri-Leu ₂ -Leu ₃ -Ile ₄ -Ser ₅ -Trp ₆ -Asp ₇ (SEQ ID NO: 5) _. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of

Cysi-Ser ₂ -Leu ₃ -Leu ₄ -Ile ₅ -Ser ₆ -Trp ₆ -Asp ₈ (SEQ ID NO: 14).

[0095] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Xi-X ₂ -X3-X4-X5-X6-X7-X8-Thr ₉ (SEQ ID NO: 15); wherein Xi is selected from the group consisting of Arg, Ser, Thr; wherein X ₂ is selected from the group consisting of Asp, Lys, Asn, and His; wherein X ₃ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, and Ser; wherein X ₄ is selected from the group consisting of Glu, Gin, and Pro; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, and Thr; wherein X ₆ is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, and any beta-branched amino acid; and wherein X ₇ is selected from the group consisting of Ala Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; and wherein X ₈ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, and Ser. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of

Arg ₁ -X ₂ -X ₃ -X ₄ -Val ₅ -X ₆ -X ₇ -Ala ₈ -Thr ₉ (SEQ ID NO: 6); wherein X ₂ is selected from the group consisting of Asp, Lys, and Asn; wherein X ₃ is selected from the group consisting of Leu, Trp, Val, He, and Phe; wherein X ₄ is selected from the group consisting of Glu and Gin; wherein X ₆ is selected from the group consisting of Val, Trp, He, Leu, and Phe; and wherein X ₇ is selected from the group consisting of Ala and Gly. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Arg Asp ₂ - Leu ₃ -Glu ₄ -Val ₅ -Val ₆ -Ala ₇ -Ala ₈ -Thr ₉ (SEQ ID NO: 7).

[0096] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Tyri-X ₂ -X ₃ -X ₄ -X5-X ₆ -X7-X ₈ -X9-Thri ₀ (SEQ ID NO: 26);wherein X ₂ is selected from the group consisting of Arg, Ser, Thr; wherein X ₃ is selected from the group consisting of Asp, Lys, Asn, and His; wherein X ₄ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, and Ser; wherein X ₅ is selected from the group consisting of Glu,Gln, and Pro; wherein X ₆ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, and Thr; wherein X ₇ is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, and any beta-branched amino acid; wherein X ₈ is selected from the group consisting of Ala,Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; wherein X ₉ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Tyri-Arg ₂ -Asp ₃ -Leu ₄ - Glu ₅ -Val ₆ - Val ₇ -Ala ₈ -Ala ₉ -Thrio (SEQ ID NO: 8).

[0097] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Thri-Alai-Thr ₃ -Ile ₄ -Ser ₅ (SEQ ID NO: 9). In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Tyri-Thr ₂ -Ala ₃ -Thr ₄ -Ile ₅ -Ser ₆ (SEQ ID NO: 10).

[0098] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Tyri-X ₂ -Arg ₃ -X ₄ -Thr ₅ -X6-X7-Glu ₈ (SEQ ID NO: 77); wherein X ₂ is selected from a group consisting of Tyr, Ser, Ala, Val, He, Leu, Phe, and Trp; wherein X ₄ is selected from a group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein X ₆ is selected from a group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, His, Thr, and Cys; wherein X ₇ is selected from the group consisting of Gly, Arg, Glu, Ser, He, Thr, Val, His, Trp, and Cys; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence Tyr ₁ -X ₂ -Arg ₃ -X ₄ -Thr ₅ -X ₆ -X ₇ -Glu ₈ (SEQ ID NO: 16) wherein X ₂ is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, and Trp; wherein X ₄ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein X ₆ is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, His, Thr, and Cys; and wherein X ₇ is selected from the group consisting of Gly, Arg, Glu, Trp, and Cys. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of

Tyr ₁ -Tyr ₂ -Arg ₃ -Ile ₄ -Thr ₅ -Tyr ₆ -Gly ₇ -Glu ₈ (SEQ ID NO: 11) _.

[0099] In one embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Gln ₁ -Glu ₂ -X ₃ -Thr ₄ -X ₅ -Pro ₆ (SEQ ID NO: 78); wherein X ₃ is selected from the group consisiting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, Thr, Pro, and Glu; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, Pro, Glu, Lys, Ser, and any beta-branched amino acid; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, the peptide comprises the amino acid sequence of Tyri-Gln ₂ -Glu ₃ -X ₄ -Thr ₅ -X ₆ -Pro ₇ (SEQ ID NO: 79); wherein X ₄ is selected from the group consisiting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, Thr, Pro, and Glu; and wherein X ₆ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, Pro, Glu, Lys, Ser, and any beta-branched amino acid. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of : Gln ₁ -Glu ₂ -X3-Thr ₄ -X ₅ -Pro ₆ (SEQ ID NO: 17); wherein X ₃ is selected from the group consisting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, and Thr; and wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, Pro, Glu, Lys, and Ser. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of:

Glni-Glu ₂ -Phe ₃ -Thr ₄ -Val5-Pro6 (SEQ ID NO: 18).

[00100] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Tyri-Gln ₂ -Glu ₃ -X ₄ -Thr ₅ -X6-Pro ₇ (SEQ ID NO: 27); wherein X ₄ is selected from the group consisiting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, Thr; wherein X ₆ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, Pro, Glu, Lys, Ser; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of:

Tyri-Gln ₂ -Glu ₃ -Phe ₄ -Thr ₅ -Val ₆ -Pro ₇ (SEQ ID NO: 19).

[00101] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Xi-Thr ₂ -X ₃ -Thr ₄ -X5-Tyr ₆ -X7-Val ₈ (SEQ ID NO: 80); wherein Xi is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, and Trp; wherein X ₃ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, and Gly; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, and Trp; wherein X ₇ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Ser, Thr, and Gin; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of: X ₁ -Thr ₂ -X ₃ -Thr ₄ -X5-Tyr ₆ -X ₇ -Val ₈ (SEQ ID NO: 20); wherein is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, and Trp; wherein X ₃ is selected from the group consisting of He , Ala, Val, Leu, Phe, Tyr, Trp, and Gly; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, and Trp; and wherein X ₇ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Ser, Thr, and Gin. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of:

Tyri-Thr ₂ -Ile ₃ -Thr ₄ -Val ₅ -Tyr ₆ -Ala ₇ -Val ₈ (SEQ ID NO: 21).

[00102] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of Xi-Ser ₂ -X ₃ -Asn ₄ -X ₅ (SEQ ID NO: 81); wherein Xi is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, Lys, Arg, Asp, Gin, Thr, and Pro; wherein X ₃ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, Gly, Gin, Asp, Thr, Ser, Arg, and Asn; wherein X ₅ is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, Lys, Gin, Ser, Thr, and Pro; or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof; wherein the isolated peptide is not fibronectin; and wherein the isolated peptide comprises 75 or fewer amino acids. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of: X ₁ -Ser ₂ -X3-Asn ₄ -X ₅ (SEQ ID NO: 22); wherein is selected from the group consisting of He , Ala, Val, Leu, Phe, Tyr, Trp, Lys, Thr, and Pro; wherein X ₃ is selected from the group consisting of He , Ala, Val, Leu, Phe, Tyr, Trp, Gly, Gin, Asp, Thr, Ser, and Asn; and wherein X ₅ is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, Lys, Gin, Ser, and Pro. In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of: Ilei-Ser ₂ -Ile ₃ -Asn ₄ -Tyr ₅ (SEQ ID NO: 23).

[00103] In some embodiments, an isolated peptide as described herein can be a

pharmaceutically acceptable prodrug. As used herein, a "prodrug" refers to compounds that can be converted via some chemical or physiological process (e.g., enzymatic processes and metabolic hydrolysis) to a therapeutic agent. Thus, the term "prodrug" also refers to a precursor of a biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject, i.e. an ester, but is converted in vivo to an active compound, for example, by hydrolysis to the free carboxylic acid or free hydroxyl. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in an organism. The term "prodrug" is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a subject. Prodrugs of an active compound may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound. Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of an alcohol or acetamide, formamide and benzamide derivatives of an amine functional group in the active compound and the like. See Harper, "Drug Latentiation" in Jucker, ed. Progress in Drug Research 4:221-294 (1962); Morozowich et al, "Application of Physical Organic Principles to Prodrug Design" in E. B. Roche ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs, APHA Acad. Pharm. Sci. 40 (1977); Bioreversible Carriers in Drug in Drug Design, Theory and Application, E. B. Roche, ed., APHA Acad. Pharm. Sci. (1987); Design of Prodrugs, H. Bundgaard, Elsevier (1985); Wang et al. "Prodrug approaches to the improved delivery of peptide drug" in Curr. Pharm. Design. 5(4):265-287 (1999); Pauletti et al. (1997) Improvement in peptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al. (1998) "The Use of Esters as Prodrugs for Oral Delivery of (3-Lactam antibiotics," Pharm. Biotech. ll,:345-365; Gaignault et al. (1996) "Designing Prodrugs and Bioprecursors I. Carrier Prodrugs," Pract. Med. Chem. 671-696; Asgharnejad, "Improving Oral Drug Transport", in Transport Processes in Pharmaceutical Systems, G. L. Amidon, P. I. Lee and E. M. Topp, Eds., Marcell Dekker, p. 185-218 (2000); Balant et al., "Prodrugs for the improvement of drug absorption via different routes of administration", Eur. J. Drug Metab. Pharmacokinet., 15(2): 143-53 (1990); Balimane and Sinko, "Involvement of multiple transporters in the oral absorption of nucleoside analogues", Adv. Drug Delivery Rev., 39(1-3): 183-209 (1999); Browne, "Fosphenytoin (Cerebyx)", Clin. Neuropharmacol. 20(1): 1-12 (1997); Bundgaard, "Bioreversible derivatization of drugs— principle and applicability to improve the therapeutic effects of drugs", Arch. Pharm. Chemi 86(1): 1-39 (1979); Bundgaard H. "Improved drug delivery by the prodrug approach", Controlled Drug Delivery 17: 179-96 (1987); Bundgaard H. "Prodrugs as a means to improve the delivery of peptide drugs",Arfv. Drug Delivery Rev. 8(1): 1-38 (1992); Fleisher et al. "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Arfv. Drug Delivery Rev. 19(2): 115-130 (1996); Fleisher et al. "Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting", Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A): 360-81, (1985); Farquhar D, et al., "Biologically Reversible Phosphate -Protective Groups", Pharm. Sci., 72(3): 324-325 (1983); Freeman S, et al., "Bioreversible Protection for the Phospho Group: Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl) Methylphosphonate with Carboxyesterase," Chem. Soc, Chem. Commun., 875-877 (1991); Friis and Bundgaard, "Prodrugs of phosphates and

phosphonates: Novel lipophilic alphaacyloxyalkyl ester derivatives of phosphate- or phosphonate containing drugs masking the negative charges of these groups", Eur. J. Pharm. Sci. 4: 49-59 (1996); Gangwar et al., "Pro-drug, molecular structure and percutaneous delivery", Des. Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21. (1977); Nathwani and Wood, "Penicillins: a current review of their clinical pharmacology and therapeutic use", Drugs 45(6): 866-94 (1993);

Sinhababu and Thakker, "Prodrugs of anticancer agents", Adv. Drug Delivery Rev. 19(2): 241-273 (1996); Stella et al., "Prodrugs. Do they have advantages in clinical practice?", Drugs 29(5): 455-73 (1985); Tan et al. "Development and optimization of anti-HIV nucleoside analogs and prodrugs: A review of their cellular pharmacology, structure-activity relationships and pharmacokinetics", Adv. Drug Delivery Rev. 39(1-3): 117-151 (1999); Taylor, "Improved passive oral drug delivery via prodrugs", Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino and Borchardt, "Prodrug strategies to enhance the intestinal absorption of peptides", Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus, "Concepts for the design of anti-HIV nucleoside prodrugs for treating cephalic HIV infection", Adv. Drug Delivery Rev.: 39(l-3):63-80 (1999); Waller et al., "Prodrugs", Br. J. Clin.

Pharmac. 28: 497-507 (1989), which are incorporated by reference herein in their entireties.

[00104] In some embodiments, an isolated peptide as described herein can be a

pharmaceutically acceptable solvate. The term "solvate" refers to an isolated peptide as described herein in the solid state, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.

[00105] In some embodiments, an isolated peptide as described herein can be in a non-crystalline, i.e. amorphous solid form.

[00106] In some embodiments, an isolated peptide as described herein can be an analog, derivative, variant, conservative substitution variant, deletion mutant, insertion mutant, or functional fragment of the amino acid sequences described above herein. Variants of the isolated peptides described herein (e.g. SEQ ID NOs: 1-27 and 71-81) can be obtained by mutations of native nucleotide or amino acid sequences, for example SEQ ID NO: 2 or a nucleotide sequence encoding a peptide comprising SEQ ID NO:2. A "variant," as referred to herein, is a polypeptide substantially homologous to an isolated peptide described herein (e.g. SEQ ID NOs: 1-27 and 71-81), but which has an amino acid sequence different from that of an isolated peptide described herein because of one or a plurality of deletions, insertions or substitutions.

[00107] A homolog of an isolated peptide as described herein can also comprise amino acid sequences that are structurally homologous to the regions of 10FNIII from which the isolated peptides described herein were derived. A structural homolog can be from any FNIII repeat, e.g. the first FNIII repeat of a polypeptide, the second FNIII repeat of a polypeptide, or any other FNIII repeat of a polypeptide. For example, a structural homolog of SEQ ID NOs: 1-2 can include the first N-terminal strands of any FNIII repeat that includes the first residue up to the last residue in the second beta strand; a structural homolog of SEQ ID NOs: 5, 12, 41, 14, 73, and/or 74 can be the second beta strand of any FNIII repeat; a structural homolog for SEQ ID NOs: 6, 7, 8, 15, 26, 75, and/or 76 can be the first beta strand of any FNIII repeat; a structural homolog for SEQ ID NO: 9 and/or 10 can be the fifth beta strand of any FNIII repeat; a structural homolog for SEQ ID NO: 11, 16, and/or 77 can be the third beta strand of any FNIII repeat; a structural homolog for SEQ ID NO: 17, 18, 19, 27, 78, and/or 79 can be the fourth beta strand of any FNIII repeat; a structural homolog for SEQ ID NO: 20, 21, 80, and/or 81 can be the sixth beta strand of any FNIII repeat; and a structural homology for SEQ ID NO: 22-23 can be the seventh beta strand of any FNIII repeat.

[00108] The variant amino acid or DNA sequence preferably is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to the sequence from which it is derived (referred to herein as an "original" sequence). The degree of homology (percent identity) between an original and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web.The variant amino acid or DNA sequence preferably is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an "original" sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp (available on the world wide web at http://blast.ncbi.nlm.nih.gov).

[00109] Alterations of the original amino acid sequence can be accomplished by any of a number of known techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting

reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide -directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. In some embodiments, an isolated peptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

[00110] Variants can comprise conservatively substituted sequences, meaning that one or more amino acid residues of an original peptide are replaced by different residues, and that the conservatively substituted peptide retains a desired biological activity, i.e., the ability to polymerize polypeptides comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet, that is essentially equivalent to that of the original peptide. Examples of conservative substitutions include substitution of amino acids that do not alter the secondary and/or tertiary structure of SEQ ID NOs: 1-27 and/or 71-81 substitutions that do not change the overall or local hydrophobic character, substitutions that do not change the overall or local charge, substitutions by residues of equivalent sidechain size, or substitutions by sidechains with similar reactive groups.

[00111] Other examples involve substitution of amino acids that have not been evolutionarily conserved in the parent sequence across species. Conserved amino acids, such as the proline residues at the FNIII domain boundaries found at residues 5, 25, 64, and 87 of 10FNIII (SEQ ID NO: 38) as well as conserved residues in the loop between beta strands E and F (Gly61, Leu62, Pro64, and Gly65 of SEQ ID NO: 38 and the equivalents thereof) or residues that make up the hydrophoboic core at positions Leu8, VallO, Alal3, Leul8, Ile20, Trp22, Tyr32, Ile34, Tyr36, Phe48, Val50, Ala57, Ile59, Tyr68, Ile70, Val72, Ala74, Ile88, Ile90, Tyr92 of SEQ ID NO: 38 and the equivalents thereof) and the equivalent residues in other fibronectin type III domain sequences, have been identified. Other conserved features of a fibronectin type III domain include structural features which are known to be conserved, e.g. the backbone hydrogen bond connecting the 6 ^th and 23 ^rd residues of SEQ ID NO: 38 (and the equivalents thereof) is conserved, as is the fact that the turn between the A and B beta strands of SEQ ID NO: 38 has a length of 2 residues. Advantageously, in some embodiments, these conserved amino acids and structures are not altered when generating conservatively substituted sequences. In some embodiments, if altered, amino acids found at equivalent positions in other fibronectin polypeptides are substituted. Among the amino acids in the protein of SEQ ID NOs: 1-2 that are conserved are those at positions Pro5, Leu8, VallO, Alal3, Leul8, Ile20, and Trp22 (and the equivalents thereof).

[00112] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics or substitutions of residues with similar sidechain volume are well known. Isolated peptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. inducing polymerization of polypeptides comprising one or more fibronectin-like type III domains or one or more domains comprising a fibronectin type III fold or beta sheet; or anti-tumorigenic properties, is retained, as determined by the assays described elsewhere herein.

[00113] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).

[00114] Alternatively, naturally occurring residues can be divided into groups based on common side -chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He, Phe, Trp; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin, Ala, Tyr, His, Pro, Gly; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe, Pro, His, or hydroxyproline. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[00115] Particularly preferred conservative substitutions for use in the variants described herein are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu or into Asn; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr or into Phe; Tyr into Phe or into Trp; and/or Phe into Val, into Tyr, into He or into Leu. In general, conservative substitutions encompass residue exchanges with those of similar physicochemical properties (i.e. substitution of a hydrophobic residue for another hydrophobic amino acid).

[00116] Any cysteine residue not involved in maintaining the proper conformation of the isolated peptide as described herein can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the isolated peptide as described herein to improve its stability or facilitate multimerization. [00117] The inventors' have further identified particular domains and/or structures of the isolated peptides described herein and their relation to the activity of the peptides, e.g. the ability to induce polymerization of fibronectin. By way of non-limiting example, the loop comprised by amino acid 15 or 16 of SEQ ID NOs: 1-2 (and the equivalents thereof) can be mutated for altered

conformational flexibility. In some embodiments, a variant of an isolated peptide as described herein can comprise a mutation affecting the beta turn. By way of non-limiting example, peptides comprising the amino acid sequence Val ₁ -Ser ₂ -Asp ₃ -Val4-Pro ₅ -Arg ₆ -Asp ₇ -Leu ₈ -Glu ₉ -Val ₁₀ - Valn-Ala ₁₂ -Ala ₁₃ -Thri ₄ - Xi ₅ -Xi ₆ -Seri ₇ -Leui ₈ -Leui ₉ -Ile ₂ o-Ser ₂ i-Trp ₂₂ -Asp ₂₃ (SEQ ID NO: 42); wherein X _i5 is selected from the group consisting of Pro, Gly, Ala, Val, He, and Leu; wherein X _i6 is selected from the group consisting of Thr, Gly, Ala, Ser, and Val are variants comprising mutations affecting the beta turn.

[00118] The conserved core residue of SEQ ID NOs: 1-2, comprising the 22nd amino acid of those sequences, can be mutated such that the hydrophobic nature of the residue is maintained. In some embodiments, a variant of an isolated peptide as described herein can comprise a mutation wherein hydrophobic substitutions are made in the conserved core residue. By way of non-limiting example, peptides comprising the amino acid sequence Vali-Ser ₂ -Asp ₃ -Val ₄ -Pro ₅ -Arg ₆ -Asp ₇ -Leu ₈ -Glu ₉ -Valio- Valn-Alai ₂ -Alai ₃ -Thri ₄ -Proi ₅ -Thri ₆ -Seri ₇ - Leui ₈ -Leui ₉ -Ile ₂ o-Ser ₂ i-X22-Asp ₂₃ (SEQ ID NO: 43) _; wherein X ₂ 2 is selected from the group consisting of Trp, Val, He, Leu,and Phe are variants comprising mutations wherein hydrophobic substitutions are made in the conserved core residue. Further non-limiting examples include variants of the amino acid sequence non-limting, peptides comprising the amino acid sequence Val ₁ -Ser ₂ -Asp ₃ -Val ₄ -X5-Arg ₆ -Asp ₇ -X ₈ - Glu ₉ -X ₁₀ - Valn-Ala ₁₂ -Xi ₃ -Thr ₁₄ - Pro ₁₅

-Thr ₁₆ -Ser ₁₇ -X ₁₈ -Leu ₁ 9-X2o-Ser ₂ i-X22-Asp ₂₃ (SEQ ID NO: 44); wherein X ₅ is selected from the group consisting of Pro, Ala, Ser, and Val; wherein X ₈ is selected from the group consisting of Leu, He, Met, Ala, Pro, Glu, Phe, and Ser; wherein X ₁₀ is selected from the group consisting of Val, Phe, Leu, Tyr, and Thr; wherein X ₁₃ is selected from the group consisting of Ala, Thr, Val, He, Pro, Glu, and Ser; wherein X ₁₈ is selected from the group consisting of Leujle, Phe, Met, Val, His, and Thr; and wherein X ₂₀ is selected from the group consisting of He, Val, and Ala; and wherein X ₂₂ is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr. Further non-limting examples include variants of the amino acid sequence Vali-Ser ₂ -Asp ₃ -Val ₄ -X5-Arg ₆ -Asp ₇ -X ₈ - Glu ₉ -Xi ₀ - Valn-Ala^-X^ -Thri ₄ - Proi ₅ -Thri ₆ -Seri ₇ -Xi ₈ -Leui ₉ -X ₂ o-Ser ₂ i-X22-Asp ₂₃ (SEQ ID NO: 44); wherein X ₅ is selected from the group consisting of Pro, Ala, Ser, and Val; wherein X ₈ is selected from the group consisting of Leu, He, Met, Ala, Pro, Glu, Phe, and Ser; wherein Xi ₀ is selected from the group consisting of Val, Phe, Leu, Tyr, and Thr; wherein X _i3 is selected from the group consisting of Ala, Thr, Val, He, Pro, Glu, and Ser; wherein Xis is selected from the group consisting of Leujle, Phe, Met, Val, His, and Thr; wherein X ₂ o is selected from the group consisting of He, Val, and Ala; and wherein X ₂ 2 is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr. Further non-limiting examples include variants of the amino acid sequence of Cysi-Val ₂ -Ser ₃ - Asp ₄ -Val ₅ -X ₆ -Arg ₇ -Asp ₈ - X ₉ -Glui ₀ - Xn-Vali ₂ -Alai ₃ -Xi ₄ -Thri ₅ - Pro ₁₆ -Thr ₁₇ - Ser ₁₈ - X ₁₉ -Leu ₂ o-X2i-Ser ₂ 2-X23-Asp ₂ (SEQ ID NO: 45) _; wherein X ₆ is selected from the group consisting of Pro, Ala, Ser, and Val; wherein X ₉ is selected from the group consisting of Leu, He, Met, Ala, Pro, Glu, Phe, and Ser; wherein Xn is selected from the group consisting of Val, Phe, Leu, Tyr, and Thr; wherein X ₁₄ is selected from the group consisting of Ala, Thr, Val, He, Pro, Glu, and Ser; wherein X ₁₉ is selected from the group consisting of Leujle, Phe, Met, Val, His, and Thr; wherein X ₂ i is selected from the group consisting of He, Val, and Ala; and wherein X ₂₃ is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr. Further non-limiting examples can include

Seri-X ₂ -Leu ₃ -X ₄ -Ser ₅ -X6-Asp ₇ (SEQ ID NO: 46) _; wherein X ₂ is selected from the group consisting of Leujle, Phe, Met, Val, His, and Thr; wherein X ₄ is selected from the group consisting of He, Val, and Ala; and wherein X ₆ is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr. Further non-limiting examples can include Argi-Asp ₂ - X ₃ -Glu ₄ -X5-Val ₆ -Ala ₇ -X ₈ -Thr ₉ (SEQ ID NO: 47) wherein X ₃ is selected from the group consisting of Leu, He, Met, Ala, Pro, Glu, Phe, and Ser; wherein X ₅ is selected from the group consisting of Val, Phe, Leu, Tyr, and Thr; and wherein X ₈ is selected from the group consisting of Ala, Thr, Val, He, Pro, Glu, and Ser Further non-limiting examples can include Tyr Arg ₂ -Asp ₃ - X ₄ -Glu ₅ -X ₆ -Val ₇ -Ala ₈ -X ₉ -Thri ₀ (SEQ ID NO: 48) wherein X ₄ is selected from the group consisting of Leu, He, Met, Ala, Pro, Glu, Phe, and Ser; wherein X ₆ is selected from the group consisting of Val, Phe, Leu, Tyr, and Thr; and wherein X ₉ is selected from the group consisting of Ala, Thr, Val, He, Pro, Glu, and Ser. Further non-limiting examples can include

Tyr ₁ -X ₂ -Arg ₃ -X ₄ -Thr ₅ -X ₆ -Gly ₇ -Glu ₈ (SEQ ID NO: 49) wherein X ₂ is selected from the group consisting of Tyr and Phe; wherein X ₄ is selected from the group consisting of He, Val, and Leu; wherein X ₆ is selected from the group consisting of Tyr, His, Val , Thr, Ala, Trp, and Cys. Further non-limiting examples can include Gln ₁ -Glu ₂ -X ₃ -Thr ₄ -X ₅ -Pro ₆ (SEQ ID NO: 50) wherein X ₃ is selected from the group consisting of Phe, Val, Leu, Asp, He, Lys, Arg, Thr, Tyr, and Ala; wherein X ₅ is selected from the group consisting of Val, Pro, He, Leu, Ala, Glu, Pro, Lys, and Ser. Further non-limiting examples can include Tyri-Gln ₂ -Glu ₃ -X ₄ -Thr ₅ -X ₆ -Pro ₇ (SEQ ID NO: 51) wherein X ₄ is selected from the group consisting of Phe, Val, Leu, Asp, He, Lys, Arg, Thr, Tyr, and Ala; wherein X ₆ is selected from the group consisting of Val, Pro, He, Leu, Ala, Glu, Pro, Lys, and Ser. Further non-limiting examples can include Tyri-Thr ₂ -X ₃ -Thr ₄ -X ₅ -Tyr ₆ -X7-Val ₈ (SEQ ID NO: 52) wherein X ₃ is selected from the group consisting of He, Val, Phe, Tyr, Gly; wherein X ₅ is selected from the group consisting of Val, He, and Leu; wheein X ₇ is selected from the group consisting of Ala, Ser, Thr, Gin, and Val. Further non-limiting examples can include Xi-Ser ₂ -X ₃ -Asn ₄ -X ₅ (SEQ ID NO: 53) wherein Xi is selected from the group consisting of He, Leu, Ala,Val, Lys, and Thr; wherein X ₃ is selected from the group consisting of He, Gly, Gin, Asp, Ala, Thr, Ser, Asn, and Phe; wherein X ₅ is selected from the group consisting of Tyr, Lys, Gin, Val, Ala, He, Ser, and Phe.

[00119] In some embodiments, a variant of an isolated peptide as described herein can comprise a mutation modulating the stability of the 10FNIII precursor. By way of non-limiting example, peptides comprising the amino acid sequence Val ₁ -Ser ₂ -Asp ₃ -Val ₄ -Pro ₅ -X ₆ -X ₇ -Leu ₈ -X ₉ - Val ₁₀ -Val _n -Ala ₁₂ -Ala ₁₃ -Thr ₁₄ - Pro ₁₅ -Thr ₁₆ -Ser ₁₇ -Leu ₁₈ -Leu ₁₉ -Ile ₂₀ -Ser ₂₁ -X ₂₂ -X ₂₃ (SEQ ID NO: 54). wherein X ₆ is selected from the group consisting of Arg, Ser, Thr (change to hydrophyllic residue to break conserved hydrogen bond between Arg6 & Asp23 or prevent thrombin cleavage; Craig et al. (2004) Structure 12: 21-30; Koide et al. (1998) J Mol Biol 284: 1141-1151); wherein X ₇ is selected from the group consisting of Asp, Lys, Asn, and His (surface mutations to enhance solubility by removing negative charge (charged amino acids between Arg6 and Asp23 of SEQ ID NO: 38 decrease lOFNIII mechanical stability); Koide et al. (2001) Biochemistry 40: 10326-10333 & Dutta et al. (2005) Protein Sci 14: 2838-2848); wherein X ₉ is selected from the group consisting of Glu and Gin (surface mutations to enhance solubility; Dutta et al. (2005) Protein Sci 14: 2838-2848); wherein X ₂₂ is selected from the group Trp, Phe (destabilizes the hydrophobic core, Dutta et al. (2008) J Mol Biol 382: 721-733), Val (stabilizes an N-terminal fragment of lOFNIII; Dutta et al. (2005) Prot Sci 14: 2838-2848);or Ala; and wherein X ₂ 3 is selected from the group consisting of Asp, Gin, Thr (change to hydrophyllic residue to break conserved hydrogen bond between Arg6 & Asp23; Craig et al. (2004) Structure 12: 21-30) are variants comprising mutations which affectthe stability of the lOFNIII precursor. Specifically, the listed mutations of X ₆ and X ₂ 3 decrease stability while the listed mutations of X ₇ and X ₉ increase stability. By way of non-limiting examples, the equivalent mutations in the peptide of SEQ ID NO: 3 can have the same effects. By way of non-limiting example, peptides comprising the amino acid sequence

Xi-X ₂ -Leu ₃ -X ₄ -Val ₅ -Val ₆ - Ala ₇ -Ala ₈ -Thr ₉ (SEQ ID NO: 55) _; wherein Xi is selected from the group consisting of Arg, Ser, and Thr (change to hydrophyllic residue to break conserved hydrogen bond between Arg6 & Asp23; Craig et al. (2004) Structure 12: 21-30; Koide et al. (1998) J Mol Biol 284: 1141-115Γ) to destabilize the lOFNIII precursor; wherein X ₂ is selected from the group consisting of Asp, Lys, Asn, and His (surface mutations to enhance solubility; Koide et al. (2001) Biochemistry 40: 10326-10333 & Dutta et al. (2005) Protein Sci 14: 2838-2848); and wherein X ₄ is selected from the group consisting of Glu and Gin (surface mutations to enhance solubility; Dutta et al. (2005) Protein Sci 14: 2838-2848); are variants comprising mutations stabilizing the lOFNIII precursor. By way of non-limiting example, peptides comprising the amino acid sequence Seri-Leu ₂ -Leu ₃ -Ile ₄ -Ser ₅ -X6-X7 (SEQ ID NO: 56) _; wherein Xe is selected from the group Trp, Phe (destabilizes the hydrophobic core, Dutta et al. (2008) J Mol Biol 382: 721-733), Val (stabilizes an N-terminal fragment of lOFNIII; Dutta et al. (2005) Prot Sci 14: 2838-2848) or Ala;and wherein X ₇ is selected from the group consisting of Asp, Gin, Thr (change to hydrophyllic residue to break conserved hydrogen bond between Arg6 & Asp23; Craig et al. (2004) Structure 12: 21-30) are variants comprising mutations stabilizing the lOFNIII precursor. By way of non-limiting example, peptides comprising the amino acid sequence

Tyri-Tyr ₂ -Arg ₃ -Ile ₄ -Thr ₅ -X ₆ -X7-Glu ₈ (SEQ ID NO: 57) wherein X ₆ is selected form the group consisting of Tyr, Phe, Cys; and wherein X ₇ is selected from the group consisting of Gly, Arg, Glu, or Trp are variants comprising mutations stabilizing or destabiling the lOFNIII precursor (Dutta et al. (2005) Protein Sci 14: 2838-2848). By way of non-limiting example, peptides comprising the amino acid sequence Gln ₁ -Glu ₂ -Phe ₃ -Thr ₄ -X ₅ -Pro ₆ (SEQ ID NO: 58) wherein X ₅ is selected from the group consisting of Val or Ala are variants comprising mutations that destabilize the lOFNIII precursor and show decreased fragment compelementation by yeast two-hybrid experiments (Dutta et al. (2005) Protein Sci 14: 2838-2848). By way of non-limiting example, peptides comprising the amino acid sequence X ₁ -Ser ₂ -Ile ₃ -Asn ₄ -Tyr ₅ (SEQ ID NO: 59) wherein is selected from the group consisting of He, Val, or Ala are variants comprising mutations that destabilize the lOFNIII precursor and show decreased fragment complementation by yeast two-hydrid experiments (Dutta et al. (2005) Protein Sci 14: 2838-2848). All of the references in the foregoing paragraph are incorporated by reference herein in their entireties.

[00120] In some embodiments, it is advantageous to manipulate the stabilizing hydrogen bonds within the peptides of SEQ ID NOs: 1-2, 4-7, 11-12, 16, and 23. By way of non-limiting example, peptides comprising the amino acid sequence Vali-Ser ₂ -Asp ₃ -Val ₄ -Pro ₅ - Arg ₆ -Asp ₇ -Leu ₈ - X ₉ -Vali ₀ - Valn-Alai ₂ -Alai ₃ -Thri ₄ - Pro _i5 -Thri ₆ -Seri ₇ -Leui ₈ -Xi ₉ -Ile ₂ o-Ser ₂ i-Trp ₂₂ -Asp ₂₃ (SEQ ID NO: 60); wherein X ₉ is selected from the group consisting of Glu or Pro; wherein X _i9 is selected from the group consisting of Leu or Pro are variants comprising mutations affecting the backbone hydrogen bonding pattern of lOFNIII within the A and B beta strands to unfold lOFNIII to an intermediate structure (Li et al. (2005) J Mol Biol 345: 817-826). By way of non-limiting example, peptides comprising the amino acid sequence Argi-Asp ₂ -Leu ₃ -X ₄ -Val ₅ -Val ₆ -Ala ₇ - Ala ₈ -Thr ₉ (SEQ ID NO: 61) wherein X ₄ is selected from the group consisting of Glu or Pro comprise mutations affecting the backbone hydrogen bonding pattern of lOFNIII within the A and B beta strands to unfold lOFNIII to an intermediate structure (Li et al. (2005) J Mol Biol 345: 817-826). By way of non-limiting example, peptides comprising the amino acid sequence Ser ₁ -Leu ₂ -X3-Ile ₄ -Ser ₅ -Trp ₆ -Asp ₇ (SEQ ID NO: 62) wherein X ₃ is selected from the group consisting of Leu or Pro are variants comprising mutations affecting the backbone hydrogen bonding pattern of lOFNIII within the A and B beta strands to unfold lOFNIII to an intermediate structure (Li et al. (2005) J Mol Biol 345: 817-826). By way of non-limiting example, peptides cromprising the amino acid sequence Tyr ₁ -Tyr ₂ -Arg ₃ -Ile ₄ -Thr ₅ -Tyr ₆ -X ₇ -Glu ₈ (SEQ ID NO: 63) wherein X ₇ is selected from the group consisting of Gly or Cys are variants comprising mutants that affect the beta strand pattern in lOFNIII given that alteration in amino acid character that is preferred for a beta strand conformation (Dutta et al (2005) Protein Sci 14: 2838 - 2848). By way of non-limiting examples, peptides comprising Xi-Ser ₂ -Ile ₃ -Asn ₄ -X ₅ (SEQ ID NO: 64), Xi is selected from the group consisting of He or Pro; wherein X ₅ is selected from the group consisting of Tyr or Pro are variants comprising mutations that affect the backbone hydrogen bonding pattern between the F and G beta strands in lOFNIII and may or may not destabilize its C-terminal structure (Li et al. (2005) J Mol Biol 345: 817-826). All of the references in the foregoing paragraph are incorporated by reference herein in their entireties.

[00121] In some embodiments, it is advantageous to modulate the beta bulge sequence within the peptides of SEQ ID NOs: l-2 and 4, 5. The beta bulge comprises amino acids Vall l, and Alal2, & Leul9 of SEQ ID NOs: 1-2, Val6, and Ala7 in SEQ ID NOs: 6-7, and Leu3 in SEQ ID NOs: 4, 5 (and the equivalents thereof). In some embodiments, a variant of an isolated peptide as described herein can comprise a mutation which preserves the beta bulge. By way of non-limiting example, peptides comprising the amino acid sequence Val ₁ -Ser ₂ -Asp ₃ -Val ₄ -Pro ₅ -Arg ₆ -Asp ₇ - Leu ₈ - Glu ₉ - Val ₁₀ -Xn- Xi2-Alai ₃ - Thri ₄ -Proi ₅ - Thr _i6 - Seri ₇ -Leui ₈ -Xi9-Ile ₂ o-Ser ₂ i-Trp ₂₂ -Asp ₂ 3 (SEQ ID NO: 65) wherein X _n is selected from the group consisting of Val, He, Leu, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys and any beta-branched amino acid; and wherein Xi ₂ is selected from the group consisting of Ala, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; and wherein X ₁₉ is selected from the group consisting of Leu, Val, Thrjle, Phe, Lys, Met, Gin, Arg, Ser, Tyr, His, Cys, or any beta-branched amino acid comprise mutations which maintain, enhance, or reduce the stability of 10FNIII by mutations in the beta bulge (Dutta et al. (2008) J Mol Biol 382: 721-733). By way of non-limiting example, peptides comprising the amino acid sequence Argi-Asp ₂ -Leu ₃ -Glu ₄ -Val ₅ -X ₆ -X7- Ala ₈ -Thr ₉ (SEQ ID NO: 66) wherein X ₆ is selected from the group consisting of Val, He, Leu, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys and any beta-branched amino acid; and wherein X ₇ is selected from the group consisting of Ala, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, or Asn comprise mutations which maintain, enhance, or reduce the stability of 10FNIII by mutations in the beta bulge (Dutta et al. (2008) J Mol Biol 382: 721-733). By way of non-limiting example, peptides comprising amino acid sequence Ser ₁ -Leu ₂ -X3-Ile ₄ -Ser ₅ -Trp ₆ -Asp ₇ (SEQ ID NO: 67) wherein X ₃ is selected from the group consisting of Leu, Val, Thrjle, Phe, Lys, Met, Gin, Arg, Ser, Tyr, His, Cys, or any beta-branched amino acid comprise mutations which maintain, enhance, or reduce the stability of 10FNIII by mutations in the beta bulge (Dutta et al. (2008) J Mol Biol 382: 721-733). All of the references in the foregoing paragraph are incorporated by reference herein in their entireties.

[00122] In some embodiments, it is advantageous to modulate the tyrosine corner motif within the peptides of SEQ ID NOs:20. By way of non-limiting example, peptides comprising the amino acid sequence X ₁ -Thr ₂ -Ile ₃ -Thr ₄ -Val ₅ -Tyr ₆ -Ala ₇ -Val ₈ (SEQ ID NO: 68) wherein Xi is selected from the group consisting of Tyr or Phe comprises mutations that maintain or destabilize 10FNIII (Batori et al. (2002 Protein Eng 15: 1015-1020; which is incorporated by reference herein in its entirety).

[00123] The loop region of the peptides of SEQ ID NOs: 1-3 can be elongated while maintaining the activity of the isolated peptides. In some embodiments, an isolated peptide as described herein can comprise a mutation elongating the loop region defined by Pro ₁₅ -Thr ₁₆ of SEQ ID NO:2 (and the equivalents thereof).

[00124] In some embodiments, an isolated peptide as described herein can comprise the amino acid sequence of a homologous fibronectin gene corresponding to the amino acids of SEQ ID NOs: 1-27 and 71-81. One of ordinary skill in the art is familiar with how to align the amino acid sequences of SEQ ID NOs: 1-27 and 71-81 with known homologous fibronectin genes or with non-human polypeptide sequences directly or indirectly (e.g. deduced from nucleotide sequences) determined. By way of non-limiting example, the isolated peptides described herein can be aligned with homologous peptides using amino acid alignment programs freely available for that purpose on the world wide wide, e.g. BLAST. These homologous peptides may comprise naturally-occurring variants of the isolated peptides described herein (e.g. SEQ ID NOs. 1-27 and 71-81), that is, the homologous peptides may comprise substitutions, insertions or deletions relative to peptides comprising the amino acid sequences of SEQ ID NOs: 1-27 and 71-81. Homologous peptides can be of any biological origin. By way of non-limiting example, fibronectin polypeptide sequences are known for human (NCBI Gene ID No: 2335), mouse (NCBI Gene ID No: 14268); chimpanzee (NCBI Gene ID No: 459926); rat (NCBI Gene ID No: 25661); and Xenopus laevis (NCBI Gene ID No: 397744).

[00125] As used herein, a "functional fragment" is a fragment or segment of a peptide comprising at least 6 amino acids and which can induce multimerization or assembly of a protein comprising a fibronectin type III domain; or a domain comprising a fibronectin type III fold or beta sheet; or reduce tumor cell viability or growth according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[00126] In some embodiments, an isolated peptide as described herein can be comprised by a polypeptide comprising multiple isolated peptides as described herein, e.g. SEQ ID NOs: 1-27 and 71-81 or variants, substitutions, functional fragments, or derivatives thereof. In some embodiments, a polypeptide can comprise multiple occurrences of one or more peptides as described herein, e.g. SEQ ID NOs: 1-27 and 71-81 or variants, substitutions, functional fragments, or derivatives thereof. In some embodiments, a polypeptide can comprise tandem occurrences of one or more peptides as described herein, e.g. SEQ ID NOs: 1-27 and 71-81 or variants, substitutions, functional fragments, or derivatives thereof. By way of non-limiting example, an isolated peptide as described herein can be comprised by a polypeptide comprising multiple occurrences of SEQ ID NO: 2, that is, at least two occurrences of SEQ ID NO: 2, e.g. 2 or more occurrences of SEQ ID NO: 2, 3 or more occurrences of SEQ ID NO: 2, 4 or more occurrences of SEQ ID NO: 2, 5 or more occurrences of SEQ ID NO: 2, 6 or more occurrences of SEQ ID NO: 2, or 7 or more occurrences of SEQ ID NO: 2. In some embodiments, an isolated polypeptide comprising multiple and/or tandem occurrences of one or more peptides as described herein can be branched. In some embodiments, an isolated polypeptide comprising multiple and/or tandem occurrences of one or more peptides as described herein can be arrayed. In some embodiments, an isolated polypeptide comprising multiple and/or tandem occurrences of one or more peptides as described herein can be multiplexed. Methods of making poplypeptides having any of these various geometries are known in the art and are explained for example in Mammen et al. Angewandte Chemie 1998 37:2755-2797; Cochran et al. Immunity 2000 12:241-250; Terskikh et al. PNAS 1997 94: 1663-8; Carlson et al. ACS Chemical Biology 2007 2: 119-127; Ivanenkov et al. Biochemica et Biophysica Acta-Molecular Cell Research 1999 1448:463-472; Handl et al. Expert Opinion on Therapeutic Targets 2004 8:565-586; Liu et al. Journal of Medicinal Chemistry 2006 49:3436-3439; David et al. Advanced Drug Delivery Reviews 2009 61 :931-9; all of which are incorporated by reference herein in their entireties.

[00127] In some embodiments, the one or more isolated peptides comprised by a polypeptide are linked by peptide bonds, by chemical cross-linkers, linkers, spacers, or by other chemical bonds. Examples of chemical cross-linkers include, but are not limited to gluteraldehyde, formaldehyde, 1 , 1-bi.s (diazoacetyl)-2-phenyIeihane, N-h droxysuccinimide esters (e.g., esters with 4-azidosaiicyIic acid, hoinobifunctional iinidoesters including disucciriimidyl esters such as 3,3'-dithiobis

(suceirdirddylpropionate), and Afunctional nialeiniides such as bis-N-maleiniido-l,S-octane).

Derivatizing agents such as methyl3-[(p-azido-phenyl)dithio] propioimidate yield photoactivatable in ermedia es which are capable of forming cross-links in the presence of light. Alternatively, for example, a lysine residue in a first peptide may be coupled to a C-terminal or other cysteine residue a second peptide, respectively, by treatment with N-y-inaleiniidobutyryloxy-succininiide (Kitagawa and Aikawa (1976) /. Biocherri. 79, 233236). Alternatively, a lysine residue in a first peptide may be conjugated to a glutamic or aspartic acid residue in a second peptide, respectively, using

isobutyichioroformate (Thoreil and De Larson (1978) Radioimmunoassay And Related Techniques: Methodology And Clubucal Applications, p. 288). Other coupling reactions and reagents have been described in the literature and are known to one of ordinary skill in the art.

[00128] In some embodiments, an isolated peptide as described herein can comprise a mutation that locks the peptide into a beta strand conformation or otherwise constrained conformation. In some embodiments, the mutation is a double Cys mutation. In some embodiments the mutation allows assembly of beta strands via click chemistry, e.g. fragments or subsections of a peptide can be expressed and/or synthesized together or separately and joined together by any suitable click chemistry method to form beta strands or click chemistry moieties at at least two locations in the peptide can lock the peptide in a beta strand conformation or otherwise constrained conformationally. Mutations that lock or constrain the peptide can be located at any point along the amino acid sequence of the peptide, e.g. at both ends of the peptide or at any position not at the ends of the peptide. Click chemistry methods and compositions are well known to one of ordinary skill in the art and are described in, for example, U.S. Patent Nos 7,375,234 and U.S. Patent Publications 2005/0032081 ; 2011/0224383; 2010/0136034; and 2010/0081137; and Dieterich et al. PNAS 2006 103:9482-7; Best, M.D. Biochemistry 2009

48:6571-6584; Gunasekaran et al. Protein Engineering 1997 10: 1131-1141 ; and Rajagopal et al.

European Biophysics Journal 2006 35: 162-9; each of which is incorporated by reference herein in its entirety. By way of non-limiting example, for SEQ ID Nos: 1-2 or 24, a Cys can be introduced at the N-terminus of the peptide at position 1 paired with a Cys at the C-terminus following position 23 or a pair of Cys residues can be introduced into the center of the A/B loop defined by Proi ₅ -Thri ₆ .

[00129] In some embodiments, an isolated peptide as described herein can comprise at least one peptide bond replacement. A single peptide bond or multiple peptide bonds, e.g. 2 bonds, 3 bonds, 4 bonds, 5 bonds, or 6 or more bonds, or all the peptide bonds can be replaced. An isolated peptide as described herein can comprise one type of peptide bond replacement or multiple types of peptide bond replacements, e.g. 2 types, 3 types, 4 types, 5 types, or more types of peptide bond replacements. Non-limiting examples of peptide bond replacements include urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid, para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronic ester, olefinic group, and derivatives thereof.

[00130] In some embodiments, an isolated peptide as described herein can comprise naturally occurring amino acids commonly found in polypeptides and/or proteins produced by living organisms, e.g. Ala (A), Val (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His (H). In some embodiments, an isolated peptide as described herein can comprise alternative amino acids, amino acid analogs, chemically modified amino acids, or non-natural amino acids. Non-limiting examples of alternative amino acids include, D-amino acids; beta-amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid,

octahydroindole-2-carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citruline, alpha-methyl-alanine,

para-benzoylphenylalanine, para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine), diaminobutyric acid,

7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine, biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine,

2,2-diethylglycine, 1-amino-l-cyclopentanecarboxylic acid, 1-amino-l-cyclohexanecarboxylic acid, amino-benzoic acid, amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine, nipecotic acid, alpha-amino butyric acid, thienyl-alanine, t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs; azide -modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and derivatives thereof.

[00131] In some embodiments, an isolated peptide can be modified, e.g. a moiety can be added to one or more of the amino acids comprising the peptide. In some embodiments, an isolated peptide as described herein can comprise one or more moiety molecules, e.g. 1 or more moiety molecules per peptide, 2 or more moiety molecules per peptide, 5 or more moiety molecules per peptide, 10 or more moiety molecules per peptide or more moiety molecules per peptide. In some embodiments, an isolated peptide as described herein can comprise one or more types of modifications and/or moieties, e.g. 1 type of modification, 2 types of modifications, 3 types of modifications or more types of modifications. Non-limiting examples of modifications and/or moieties include PEGylation; post-traslational derivitzations; glycosylation; hydroxylation; methylation; HESylation; ELPylation; lipidation;

acetylation; amidation; biotinylation; end-capping modifications; cyano groups; phosphorylation; cyclization; or other conjugation moieties (e.g. protein, antibody, peptide, nucleotide, virus, phage, matrix, insoluble support, particle, etc.). In some embodiments, an end-capping modification can comprise acetylation at the N-terminus, N-terminal acylation, and N-terminal formylation. In some embodiments, an end-capping modification can comprise amidation at the C-terminus, introduction of C-terminal alcohol, aldehyde, ester, and thioester moieties.

[00132] An isolated peptide as described herein can be coupled and or connected to a second functional molecule, peptide and/or polypeptide or inserted intrasequence to the targeting molecule in a single or multivalent fashion, creating, in some emboidments, a "fusion peptide". In some embodiments, an isolated peptide as described herein is coupled to a targeting molecule. In some embodiments, an isolated peptide as described herein is coupled to a targeting molecule by expressing the peptide and the targeting molecule as a fusion peptide, optionally with a peptide linker sequence interposed between them. As used herein a "targeting molecule" can be any molecule, e.g. a peptide, antibody or fragment thereof, antigen, targeted liposome, or a small molecule that can bind to or be bound by a specific cell or tissue type. By way of non-limiting example, if it is desired to target an isolated peptide as described herein to the lung, an isolated peptide comprising the amino acid sequence of SEQ ID NO: 2 could be coupled to an antibody or fragment thereof which is specific for lung cells or tissue, e.g. an antibody or antibody fragment as described in US Patent Publication 2005/0287066. The addition of an antibody to an isolated peptide as described herein permits the peptide to accumulate additively at the desired target site, e.g. the wound or tumor microenvironment.

[00133] In some embodiments, an isolated peptide as described herein can be a fusion peptide or polypeptide. A fusion polypeptide can comprise a peptide linker domain interposed between the first domain of the peptide comprising an amino acid sequence of SEQ ID NOs: 1-27 and 71-81 or derivativatives, variants, functional fragments, prodrug, or analog thereof as described herein and at least a second domain of the fusion peptide. The first peptide domain can be the N-terminal domain or the C-terminal domain or an internal sequence in the case where the partner domain forms after fragment complementation of constituent parts. Methods of synthesizing or producing a fusion protein are well known to those of ordinary skill in the art. Fusion proteins can, e.g., improve production, functionality, stability and/or pharmacological efficacy and reduce immunogenicity.

[00134] The isolated peptides described herein can be integrated at the site of extracellular matrix formation, accumulation and/or maintenance and can therefore be used to target other molecules to those same sites. In one aspect, the invention described herein relates to a method of targeting therapeutic agents or imagining molecules to sites of extracellular matrix production, maintenance, and/or accumulation wherein the method comprises administering to the subject an isolated peptide as described herein or a composition comprising such a peptide, wherein the peptide is coupled to a therapeutic agent or imaging molecule. In some embodiments, the site of extracellular matrix production or accumulation is a tumor. In some embodiments, the site of extracellular matrix production or accumulation is a fibrotic lesion. In some embodiments, the site of extracellular matrix production or accumulation is a wound site.

[00135] In some embodiments, an isolated peptide as described herein can be coupled to a therapeutic molecule. An isolated peptide comprising an amino acid sequence of SEQ ID NOs: 1-27 and 71-81 or derivatives, variants, functional fragments, prodrug, or analog thereof as described herein can, for example, be integrated into the extracellular matrix at sites of binding, assembly, multimerization, aggregation and/or polymerization of fibronectin, thereby targeting a therapeutic molecule to which it is bound to the site of polymerization of fibronectin in addition to the therapeutic effects of the isolated peptide itself. In some embodiments, the therapeutic molecule can be a fibrosis treatment molecule. Non-limiting examples of fibrous treatment molecules include a steroid; a corticosteroid; an anti-inflammatory agent; and an immunosuppressant. In some embodiments, the therapeutic molecule can be a chemotherapeutic molecule. As used herein, "chernotherapeutic agent" includes chemical reagents that inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable or otherwise treat at least one resulting symptom of such a growth.

Chemotherapeutic agents are well known in the art (see e.g.. Oilman A. G., et ai., The Pharmacological Basis of Therapeutics, 8th Ed., Sec .12:1.202-1263 (1990)), and are typically used to treat neoplastic diseases. Non-limiting examples of chemotherapeutic molecules include, bleomycin, docetaxel (Taxotere), doxorubicin, edatrexate, etoposide, finasteride (Proscar), ilutamide (Eulexin), gemcitabine (Gemzar), goserelin acetate (Zoladex), granisetron (Kytril), irinotecan (Carapto/Camptosar), ondansetron (Zofran), paclitaxel (Taxoi), pegaspargase (Oncaspar), pilocarpine hydrochloride (Saiagen), porfimer sodium (Photofrin), inter !eukm-2 (Proleukin), rituximab (Rituxan), topotecan (Hycamtin), trastuzumab (Herceptin), tretinoin (Retin-A), Triapine, vincristine, vinorelbine tartrate (Navelbine); alkylating drugs such as Nitrogen Mustards (e.g., Mechlorethamine (HN2), Cyclophosphamide, ifosfamide, Melphalan (L-sarcoiysin), Chlorambucil, etc.); ethylenimines, methylmelamines (e.g., Hexamethylmelamine, Thiotepa, etc.); Alkyl Sulfonates (e.g., Busulfan, etc.). Nitrosoureas (e.g., Carmustine (BCNU), Lomustine (CCNU), Semustine (niethyl-CCNU), Streptozocin (streptozotocin), etc.), tri.azenes (e.g. Decarbazine (DTTC; dimethyitriazenoimi-dazolecarboxamide)), Aikylators (e.g., cisdianiminedichloropiatirium II (CDDP)), etc.; antimetabolites such as folic acid analogs (e.g., Methotrexate (amefhopterin)); pyrimidine analogs (e.g., fiuorouracil ('5-fluorouracil; 5-FU); floxuri.dine (fluorodeoxyuridine); FUdr; Cytarabine (cyosine arabinoside), etc.); purine analogs, (e.g.,

Mercaptopurine (6~mercaptopurine; 6 -MP); Thioguanine (6~tbioguanine; TG); and Pentostatin

(2 ^' -deoxycoibrmyein)), etc.; vinca alkaloids (e.g., Vinblastin (VLB) and Vincristine); topoisomerase inhibitors (e.g., Etoposide, Tenyposide, Camptothecin, Topotecan, 9-amino-campotothecin CFT-11, etc.); antibiotics (e.g., Dactinomycin (actinomycin D), adriamycin, daunorubicin, doxorubicin, bleomycin, plicamycin (mithramycin), mitomycin (mitomycin C), Taxol, Taxotere, etc.); enzymes (e.g., L-Asparaginase); and biological response modifiers (e.g., interferon-; interleukin 2, etc.). Other chemotherapeutic agents include cis-diaminedichioroplatinum II (CDDP); Carboplatin; Anthracendione (e.g., Mitoxantrone); Hydroxyurea; Procarbazine (N-metiiylhydrazine); and adrenocortical suppressants (e.g., Mitotane, animoglutethimide, etc.); adrenoccaticosteroids (e.g., Prednisone); progestins (e.g., Hydroxyprogesterone caproate, Medroxyprogesterone acetate, Megestrol acetate, etc.); estrogens (e.g., diethylstilbestroi; ethenyl estradiol, etc.); antiestrogens (e.g. Tamoxifen, etc.); androgens (e.g.. testosterone propionate, Fluoxymesterone, etc.); antiandrogens (e.g., Flutaniide); gonadotropiareleasing hormone analogs (e.g., Leuproli.de); and Gleevec.

[00136] An isolated peptide as described herein can comprise, be coupled to, and/or connected to a detectable tag molecule. As used herein, a "tag molecule" is a molecule that is readily detectable or which can be imaged, e.g. a fluorescent molecule, a radiolabeled molecule, or a dye. Readily detectable tag molecules, methods of detecting tag molecules, and methods of coupling tag molecules to a peptide or synthesizing a peptide comprising a tag molecule are well known to one of ordinary skill in the art. Tag molecules can be detected in vitro or in vivo. In some embodiments, a tag molecule can be detected or imaged in vivo after it is administered to a subject. In some embodiments tag molecule is not toxic to a subject. Non-limting examples of methods that can be used to detect or image a tag molecule include fluorescence microscopy, MRI, X-rays, NMR, and ultrasound.

[00137] Exemplary tag molecules include a detectable agent, a contrast agent, electron dense material, magnetic resonance imaging agents, isotopes, radioactive molecule, non-radioactive detectable agents, a dye, paramagnetic contrasting agents, compounds that enhances magnetic resonance imaging (MRI), radiopharmaceuticals, ¹⁹ F, and fluorescent molecules. Radionuclides useful for imaging include radioisotopes of copper, gallium, indium, rhenium, and technetium, including isotopes ⁿ C, ¹³ C, ⁶⁷ Cu, ^m In, "mTc, ⁶⁷ Ga, ⁶⁸ Ga, or a combination. Imaging agents disclosed by Low et al. in U.S. Pat. No. 5,688,488, incorporated herein by reference, are useful in the compositions and methods described herein. In order to function as a suitable tag for medical imaging, the tag molecule is useful in a molecular imaging diagnosis procedure, for example but not limited to, magnetic resonance (MR) imaging. Delivery of such imaging agents using the methods and compositions as disclosed herein can be used to image the extent of fibrotic lesions, wounds, or tumors by MRI or PET for example. Contrast enhancement can be provided by gadolinium, for example, gadolinium in the form of

Gd-DTPA-aminohexanoic acid. Other imaging agents are useful in the methods as disclosed herein include, for example other lanthanide ion coordination complexes can allow for even greater enhanced relaxation at higher field strength (Aime, S., et al. , Chem. Soc. Rev. 27: 19-29, 1998; Aime et al. , . Mannet. Reson. Iman. 16:394-406, 2002). Paramagnetic CES T agents are useful as imaging agents in the methods and compositions as disclosed herein, for example as Eu+3, Tb+3, Dy+3, Er+3, Tm+3, or Yb+ 3 alter tissue contrast via chemical exchange saturation transfer of presaturated spins to bulk I water (Elst, L.V., et al. , Mann. Reson. Med. 47: 1121-1130, 2002). In some embodiments, more than one tag molecule can be used simultaneously in the composition and methods of the present invention, with techniques available for attachment of multiple tag molecules, for example Gd-DTPA to proteins to enhance the MR signal known by persons of ordinary skill in the art.

[00138] Non-limiting examples of fluorescent tag molecules include fluorescein, phycoerytllrin.

Cy3TM, CySTM, ailophycocyanine, Texas Red, perklenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-CySTM, green fluorescent protein and other variants (e.g. YFP and die like), rhodaniine, fluorescein isothiocyanate (F1TC) and Oregon GreenTM. rhodamine and derivatives (e.g., Texas red and tetrarhodiimne isothiocynate (TRITC)), biodn, phycoesythrin, AMCA, CyDyesTM, 6-carboxyfhioreseein (commonly known by the abbreviations FAM and F),

6-carboxy-2\4\7\4,7-hexachlorofiuorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'

-dimethoxyfiuoresceiii (JOE or J). N.N,N^ '--tetramethyL-6carboxyrhodaniine (TAMRA or T), 6-carboxy-X-rhodaniine (ROX or R), 5-cai'boxyrhodaniine-6(} (R6G5 or G5), 6-cai¾oxyrhodaniine-6G (R6G6 or G6), and rhodamine J.1.0; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g urnbelS iferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5 _t etc; BODIPY dyes; quinoline dyes; and commercially available dyes such as the Aie Fiuors.

[00139] The isolated peptides described herein induce and/or enhance the assembly, multimerization, aggregation and/or polymerization of a protein comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet. This activity can be directly measured and/or determined in vitro. For example, the aggregation of a protein comprising a fibronectin type III domain or a domain comprising a fibronectin type III fold or beta sheet can be determined by SDS-PAGE analysis. Fibronection labeled with rhodamine (0.3 mg/mL) at any temperature, including 25°C, 30 °C, and 37°C can be contacted with an isolated peptide as described herein and incubated for 16 hour or less. The reactions can then be mixed with SDS sample buffer (non-reducing conditions) and analyzed by gel electrophoresis. The presence of high molecular weight aggregates (above native monomeric (M) and dimeric (D) species) indicates that the isolated peptide has the ability to induce and/or enhance multimerization of a protein comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet. The results of electrophoretic analysis can be quantified by densitometry analysis. Other methods for monitoring multimerization include spectroscopic techniques such as analysis by turbidity.

[00140] Additionally, the activity of the isolated peptides described herein can be measured or deteremined by assays to determine their anti-angiogenic or anti-tumorigenic activity. By way of non-limiting example, the anti-tumorigenic activity of an isolated peptide as described herein can be determined by administering the isolated peptide to a subject having a tumor and monitoring the size and/or progression of the tumor. A decrease in size of the primary tumor or disease state of the tumor or number of metastatic foci, or a failure of the tumor to grow and/or progress at the rate that would occur in the absence of administration of an isolated peptide as described herein is indicative of

anti-tumorigenic/anti-metastatic activity of the isolated peptide. A further non-limiting example of an assay for anti-tumorigenic activity is to contact tumorigenic cells with an isolated peptide as described herein and determine their viability in vitro. Briefly, tumorigenic cells (e.g. M28 or M6 cells derived from a mammary adenocarcinoma) are plated at low confluency in a 96-well plate and allowed to spread overnight. The isolated peptide is added to at least one well (e.g. at a concentration of 150 μΜ). Optionally, additional doses of the isolated peptide can be added to additional samples over the duration of the experiment. Six days after contacting the cell with the isolated peptide, resazurin, an indicator of metabolic capacity, is added to each well. Percent viability (normalized against the untreated control sample) can be calculated by the turnover of resazurin during the 4h incubation. Examples of well described angiogenesis assays that can be used to test or confirm anti-angiogenic activity of the isolated peptides described herein include, but are not limited to in vitro endothelial cell assays, rat aortic ring angiogenesis assays, cornea micro pocket assays (corneal neovascularization assays), retinal angiogenesis assays, and chick embryo chorioallantoic membrane assays (Erwin, A. et al. (2001) Seminars in Oncology 28(6):570-576).

[00141] The isolated peptides described herein can induce and/or enhance the multimerization and/or polymerization of polypeptides comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet. Accordingly, in one aspect, the invention described herein is directed to a method of inducing the multimerization and/or polymerization of a polypeptide comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet, wherein the method comprises contacting at least two polypeptide molecules comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheetwith an isolated peptide as described herein or composition comprising such a peptide. In some embodiments, the polypeptide comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet is selected from the group consisting of fibronectin and fibrinogen.

[00142] As polypeptides comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet, e.g. fibronectin, are a notable component of the extracellular matrix, the isolated peptides described herein promote the formation and/or maintenance of the extracellular matrix. Accordingly, in one aspect the invention described herein is directed to a method of promoting the production and/or maintenance of the extracellular matrix in a subject in need thereof, the method comprising administering an isolated peptide as described herein or a composition comprising such a peptide. As used herein, a subject in need of promotion of the production and/or maintenance of the extracellular matrix can be any subject having, or diagnosed as having, or at risk of having a condition that would benefit from the production or maintenance of the extracellular matrix. Examples of such conditions include, but are not limited to, fibrotic conditions and proliferative diseases, including tumors and cancers.

[00143] Fibrotic conditions benefit from the production and/or maintenance of the extracellular matrix by reducing the accumulation of scar tissue in favor of extracellular matrix. In some embodiments, the invention as described herein relates to a method of inhibiting fibrosis, the method comprising administering an isolated peptide as described herein or a composition comprising such a peptide. As used herein, "fibrosis" refers to the formation of fibrous tissue as a reparative or reactive process, rather than as a normal constituent of an organ or tissue. Fibrosis is characterized by fibroblast accumulation and collagen deposition in excess of normal deposition in any particular tissue. Fibrosis can occur as the result of inflammation, irritation, or healing. A subject in need of treatment for a fibrotic condition is any subject having, or diagnosed as having, or at risk of having a fibrotic condition. Non-limiting examples of fibrotic conditions include, but are not limited to pulmonary fibrosis; scarring; scarring of the skin; trauma; a wound; chronic wounds (e.g. as in diabetes patients), corneal defects; corneal ulceration; corneal wounds; diabetic ulcer; ulcer; sepsis; arthritis; idiopathic pulmonary fibrosis; cystic fibrosis; cirrhosis; endomyocardial fibrosis; mediastinal fibrosis; myelofibrosis; retroperitoneal fibrosis; progressive massive fibrosis; nephrogenic systemic fibrosis; Crohn's disease; keloid;

scleroderma; systemic sclerosis; arthroiibrosis; adhesive capsulitis; lung fibrosis; liver fibrosis; kidney fibrosis; heart fibrosis; vascular fibrosis; skin fibrosis; eye fibrosis; bone marrow fibrosis; asthma; sarcoidosis; COPD; emphysema; nschistomasomiasis; cholangitis; diabetic nephropathy; lupus nephritis; postangioplasty aterial restenosis; atherosclerosis; burn scarring; hypertrophic scarring; nephrogenic fibrosing dermatopathy; postcataract surgery; proliferative vitreoretinopathy; Peyronie's disease; Duputren' s contracture; dermatomyositis; and graft versus host disease. In some embodiments, an isolated peptide as described herein or composition comprising such a peptide can be administered in combination with a fibrosis-treating agent, examples of which are described above herein. In some embodiments, the invention described herein is directed to the use of a composition comprising an isolated peptide or nucleic acid as described herein for preventing, treating and/or ameliorating fibrosis.

[00144] In one aspect, the technology described herein relates to a method of promoting the production or maintenance of the extracellular matrix in a subject in need thereof, the method comprising administering a peptide, nucleic acid, or composition as described herein. In some embodiments, the subject is in need of treatment for fibrosis. In some embodiments, the peptide is administered in combination with a fibrosis-treating agent. In some embodiments, the subject suffers from a condition selected from the group consisting of: pulmonary fibrosis; scarring; scarring of the skin; trauma; a wound; chronic wounds (e.g. as in diabetes patients), corneal defects; corneal ulceration; corneal wounds; diabetic ulcer; ulcer; sepsis; arthritis; idiopathic pulmonary fibrosis; cystic fibrosis; cirrhosis; endomyocardial fibrosis; mediastinal fibrosis; myelofibrosis; retroperitoneal fibrosis;

progressive massive fibrosis; nephrogenic systemic fibrosis; Crohn's disease; keloid; scleroderma; systemic sclerosis; arthroiibrosis; adhesive capsulitis; lung fibrosis; liver fibrosis; kidney fibrosis; heart fibrosis; vascular fibrosis; skin fibrosis; eye fibrosis; bone marrow fibrosis; asthma; sarcoidosis; COPD; emphysema; nschistomasomiasis; cholangitis; diabetic nephropathy; lupus nephritis; postangioplasty aterial restenosis; atherosclerosis; burn scarring; hypertrophic scarring; nephrogenic fibrosing dermatopathy; postcataract surgery; proliferative vitreoretinopathy; Peyronie's disease; Duputren' s contracture; dermatomyositis; and graft versus host disease.

[00145] Proliferative diseases benefit from the production and/or maintenance of the extracellular matrix by consequence of the fact that increased extracellular matrix production inhibits angiogenesis, cell growth, and metastasis. In some embodiments, the invention as described herein relates to a method of inhibiting cellular growth, the method comprising administering an isolated peptide as described herein or a composition comprising such a peptide. As used herein, a "proliferative disease" refers to a condition characterized by a failure of regulation of tissue growth. The "proliferative disease" is typically a cancer. The cancer may be any kind of cancer or neoplasia. A subject in need of treatment for a proliferative disease is a subject having, or diagnosed as having, or at risk of having a proliferative disease. Non-limiting examples of proliferative diseases include, but are not limited to cancer; a tumor; rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre -neoplastic lesions (e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia), carcinoma, oral hairy leukoplakia, and psoriasis. In some embodiments, angiogenesis can be associated with a proliferative disease, e.g. cancer, obesity, macular degeneration, and blindness. In some embodiments, an isolated peptide as described herein or composition comprising such a peptide can be administered in combination with a chemotherapeutic agent, examples of which are described above herein. In some embodiments, the invention described herein is directed to the use of a composition comprising an isolated peptide or nucleic acid as described herein for preventing, treating and/or ameliorating a proliferative disease. In some embodiments a proliferative disease is treated by increasing the number of fibronectin molecules bound to the integrin receptors on the tumor cell surface, which may modulate integrin-mediated death signaling processes. One class of anti-tumor drugs comprises peptides that bind to integrins, e.g. via RGD motifs (for example, cilengitde) is known in the art and such peptides can be administered in combination with the isolated peptides herein as separate peptides and/or as part of a fusion peptide. In some embodiments, these anti-tumor drugs (e.g. cilengitde) can be administered concurrently with the isolated peptides described herein or separately (e.g. before or after the isolated peptides described herein are administered).

[00146] The production and/or maintenance of the extracellular matrix contributes to the strength and integrity of connective tissues, e.g. bone, tendon, ligaments, mesenchyme and cartilage. Increasing the strength of connective tissues can be advantageous, for example, in order to promote wound healing (e.g. healing of a broken bone or damaged, torn, or ruptured tendon or ligament), treat an elastic tissue disorder, or treat emphysema. Increasing the strength of connective tissues can be used to treat any disorder, disease, or trauma in which a connective tissue is damaged, subject to trauma, atrophied, or weakened. Accordingly, in one aspect, the invention described herein is directed to a method of increasing the strength of bone, tendon, ligaments, cartilage, or connective tissue wherein the method comprises administering an isolated peptide as described herein or a composition comprising such a peptide. As used herein, a "connective tissue" refers to those animal tissues that support organs, fill spaces between them, or perform mechanical functions such as connecting muscles to bone (tendons and ligaments) or providing low friction weighing surface as in articular cartilage. Connective tissues are characterized by their relatively avascular matrices and low cell densities. The most abundant connective tissues are the reticular stroma, muscle, adipose tissue, cartilage and bone. Further examples of connective tissue include, but are not limited to, mesenchyme, mucous connective, areolar (loose), elastic, or blood. Included within the definition of "connective tissue" are terminally differentiated cells as well as precursor cells that have the potential to differentiate into connective tissue cells and tissues. In some embodiments, the invention described herein is directed to the use of a composition comprising an isolated peptide or nucleic acid as described herein for increasing the strength of a connective tissue.

[00147] Angiogenesis can contribute to the pathology of a number of conditions. Accordingly, provided herein are methods of inhibiting angiogenesis in a subject in need thereof, the method comprising administering an isolated peptide, nucleotide, or composition as described herein to the subject. In some embodiments, the subject in need of inhibition of angiogenesis is a subject in need of treatment for a proliferative disease. In some embodiments, the isolated peptide, nucleotide, or composition as described herein can be administered in combination with a chemotherapeutic agent. Non-limiting examples of a proliferative disease include cancer; a tumor; rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions (e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia), carcinoma, oral hairy leukoplakia, psoriasis, obesity, macular degeneration, and blindness. In some embodiments, the invention described herein is directed to the use of a composition comprising an isolated peptide or nucleic acid as described herein for inhibiting angiogenesis.

[00148] By virtue of increasing or promoting the maintenance or production of the extracellular matrix and strengthening connective tissues, the methods described herein can be used to promote wound healing. A wound can be an epithelial, endothelial, connective tissue, ocular, or any other kind of wound in which the strength and/or integrity of a tissue has been reduced, e.g. trauma has caused damage to the tissue. Accordingly, provided herein is a method of promoting wound healing comprising administering a peptide, nucleic acid, or composition as described herein to a subject in need thereof. A subject in need of promotion of wound healing can be a subject with a wound. In some embodiments, the invention described herein is directed to the use of a composition comprising an isolated peptide or nucleic acid as described herein for promoting wound healing.

[00149] For therapeutic applications, the appropriate dosage of compositions will depend upon the type of tissue needing production and/or maintenance of the extracellular matrix or other beneficial effects of an isolated peptide as described herein, the associated medical conditions to be treated, the severity and course of the medical conditions, whether the compositions are administered for preventative or therapeutic purposes, previous therapy, the patient's clinical history and response to the compositions, and the discretion of the attending physician. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the route of administration, and the seriousness of the condition being treated and should be decided according to the judgment of the practitioner and each subject's circumstances in view of, e.g., published clinical studies. Suitable effective dosage amounts for topical administration of the isolated peptide compositions described herein range from about 10 micrograms to about 5 grams applied or administered about every 4 hours, although they are typically about 500 mg or less per every 4 hours. In one embodiment the effective dosage for topical administration is about 0.01 mg, 0.5 mg, about 1 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 g, about 1.2 g, about 1.4 g, about 1.6 g, about 1.8 g, about 2.0 g, about 2.2 g, about 2.4 g, about 2.6 g, about 2.8 g, about 3.0 g, about 3.2 g, about 3.4 g, about 3.6 g, about 3.8 g, about 4.0 g, about 4.2 g, about 4.4 g, about 4.6 g, about 4.8 g, or about 5.0 g, every 4 hours. Equivalent dosages may be administered over various time periods including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every two weeks, about every three weeks, about every month, and about every two months. The effective dosage amounts described herein refer to total amounts administered.

[00150] For systemic administration, the dosage ranges are typically from O.OOlmg/kg body weight to 5 g/kg body weight. In some embodiments, the dosage range is from 0.001 mg/kg body weight to lg/kg body weight, from 0.001 mg/kg body weight to 0.5 g/kg body weight, from 0.001 mg/kg body weight to 0.1 g/kg body weight, from 0.001 mg/kg body weight to 50 mg/kg body weight, from 0.001 mg/kg body weight to 25 mg/kg body weight, from 0.001 mg/kg body weight to 10 mg/kg body weight, from 0.001 mg/kg body weight to 5 mg/kg body weight, from 0.001 mg/kg body weight to 1 mg/kg body weight, from 0.001 mg/kg body weight to 0.1 mg/kg body weight, from 0.001 mg/kg body weight to 0.005 mg/kg body weight. Alternatively, in some embodiments the dosage range is from 0.1 g/kg body weight to 5 g/kg body weight, from 0.5 g/kg body weight to 5 g/kg body weight, from 1 g/kg body weight to 5 g/kg body weight, from 1.5 g/kg body weight to 5 g/kg body weight, from 2 g/kg body weight to 5 g/kg body weight, from 2.5 g/kg body weight to 5 g/kg body weight, from 3 g/kg body weight to 5 g/kg body weight, from 3.5 g/kg body weight to 5 g/kg body weight, from 4 g/kg body weight to 5 g/kg body weight, from 4.5 g/kg body weight to 5 g/kg body weight, from 4.8 g/kg body weight to 5 g/kg body weight. In one embodiment, the dose range is from 5μg/kg body weight to 30μg/kg body weight. Alternatively, the dose range will be titrated to maintain serum levels between 5 μg/mL and 30 μg/mL.

[00151] The compositions comprising an isolated peptide as described herein are suitably administered to the patient at one time or over a series of treatments. For purposes herein, a

"therapeutically effective amount" of a composition comprising an isolated peptide as described herein is an amount that is effective to either prevent, reduce the likelihood, lessen the worsening of, alleviate, or cure one or more symptoms or indicia of the treated condition.

[00152] Administration of the doses recited above can be repeated for a limited period of time.

In some embodiments, the doses are given once a day, or multiple times a day, for example but not limited to three times a day. In a preferred embodiment, the doses recited above are administered daily for several weeks or months. The duration of treatment depends upon the subject's clinical progress and responsiveness to therapy. Continuous, relatively low maintenance doses are contemplated after an initial higher therapeutic dose.

[00153] Therapeutic compositions containing at least one agent can be conventionally administered in a unit dose. The term "unit dose" when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle.

[00154] In one aspect, the invention described herein is directed to a composition comprising one or more of any of the isolated peptides described herein, e.g. peptides comprising the amino acid sequences of SEQ ID NOs: 1-27 and 71-81 or derivatives, variants, functional fragments, prodrugs, or analogs thereof as described herein. In one aspect, the invention described herein consists essentially of one or more of the isolated peptides described herein as an active ingredient.

[00155] In some embodiments, the invention described herein is directed to a composition comprising a nucleic acid encoding an isolated peptide as described herein. In some embodiments, the nucleic acid comprises RNA or DNA. In some embodiments, the nucleic acid comprises an mRNA. In some embodiments, the nucleic acid encoding an isolated peptide as described herein can be comprised by a vector. Such methods allow clinicians to introduce a nucleic acid sequence encoding an isolated peptide as described herein directly into a patient (in vivo gene therapy) or into cells isolated from a patient or a donor (ex vivo gene therapy). The isolated peptides described herein, when produced by transduced cells after gene therapy, can be maintained at a relatively constant level in a subject, as compared to a protein that is administered directly. Such sustained production of an isolated peptide is particularly appropriate in the treatment of chronic diseases, such as cancers. Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al. , Proc. Natl. Acad. Sci. USA (1995) 92: 1292).

[00156] Further, regulatable genetic constructs using small molecule inducers have been developed that can be included in vectors to be used in some embodiments of the present invention described herein. (Rivera et al. (1996) Nat. Med. 2: 1028-32; No et al. (1996) Proc. Natl. Acad. Sci. USA, 93:3346-51 ; Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-51 ; the GeneS witch® system (Valentis, Inc., Burlingame, Calif.)). These systems are based on the use of engineered transcription factors the activity of which is controlled by a small molecule drug, and a transgene, the expression of which is driven by the regulated transcription factor (Rivera et al. (1996) Nat. Med. 2: 1028-32; Pollock et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13221-26; U.S. Pat. Nos. 6,043,082 and 6,649,595; Rivera et al. (1999) Proc. Natl. Acad. Sci. USA 96:8657-62).

[00157] In some of the aspects described herein, a nucleic acid sequence encoding an isolated peptide as described herein is operably linked to a vector. Vectors can include cloning and expression vehicles, as well as viral vectors. By "recombinant vector" is meant a vector that includes a heterologous nucleic acid sequence, or "transgene" that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. Vectors useful for the delivery of a sequence encoding an isolated peptide as described herein can include onr or more regulatory elements {e.g. , promoter, enhancer, etc.) sufficient for expression of the isolated peptide in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

[00158] Examples of vectors useful in delivery of nucleic acids encoding isolated peptides as described herein include plasmid vectors, non-viral plasmid vectors (e.g. see 6,413,942, 6,214,804, 5,580,859, 5,589,466, 5,763,270 and 5,693,622, all of which are incorporated herein by reference in their entireties); retroviruses (e.g. see U.S. Pat. No. 5,219,740; Miller and Rosman (1989)

BioTechniques 7:980-90; Miller, A. D. (1990) Human Gene Therapy 1 :5-14; Scarpa et al (1991) Virology 180:849-52; Miller et al , Meth. Enzymol. 217:581-599 (1993); Burns et al (1993) Proc. Natl. Acad. Sci. USA 90:8033-37; Boris-Lawrie and Temin (1993) Curr. Opin. Genet. Develop. 3: 102-09. Boesen et al , Biotherapy 6:291-302 (1994); Clowes et al. , 3. Clin. Invest. 93:644-651 (1994); Kiem et al , Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3: 110-114 (1993), the contents of each of which are herein incorporated by reference in their entireties); lentiviruses (e.g., see U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, the contents of which are herein incorporated by reference in their entireties; adenovirus-based expression vectors (e.g., see Haj-Ahmad and Graham (1986) J. Virol. 57:267-74; Bett et al. (1993) J. Virol. 67:5911-21 ; Mittereder et al. (1994) Human Gene Therapy 5:717-29; Seth et al. (1994) J. Virol. 68:933-40; Barr el al. (1994) Gene Therapy 1 :51-58; Berkner, K. L. (1988) BioTechniques 6:616-29; and Rich et al. (1993) Human Gene Therapy 4:461-76; Wu et al. (2001) Anesthes. 94: 1119-32; Parks (2000) Clin. Genet. 58: 1-11 ; Tsai et al. (2000) Curr. Opin. Mol. Ther. 2:515-23; and U.S. Pat. No. 6,048,551; 6,306,652and 6,306,652, incorporated herein by reference in their entireties); Adeno-associated viruses (AAV) (e.g. see U.S. Pat. Nos. 5,139,941 ; 5,622,856; 5,139,941; 6,001,650; and 6,004,797, the contents of each of which are incorporated by reference herein in their entireties); and avipox vectors (e.g. see WO 91/12882; WO 89/03429; and WO 92/03545; which are incorporated by reference herein in their entireties).

[00159] Vectors can be packaged and/or delivered using liposomes (e.g., see U.S. Pat. Nos.

5,580,859, 5,549,127, 5,264,618, 5,703,055; 4,663,161 and 4,871,488; an d Hug and Sleight (1991) Biochim. Biophys. Acta. 1097: 1-17; Straubinger et al. (1983) in Methods of Enzymology Vol. 101, pp. 512-27; de Lima et al. (2003) Current Medicinal Chemistry, Volume 10(14): 1221-31 ; Papahadjopoulos et al. (1975) Biochem. Biophys. Acta. 394:483-491 ; all of which are incorporated by reference herein in their entireties) biolistic delivery; DEAE dextran-mediated transfection, calcium phosphate precipitation, polylysine- or polyornithine-mediated transfection, or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like. Other useful methods of transfection include electroporation, sonoporation, protoplast fusion, peptoid delivery, or microinjection. See, e.g., Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York, for a discussion of techniques for transforming cells of interest; and Feigner, P. L. (1990) Advanced Drug Delivery Reviews 5: 163-87, for a review of delivery systems useful for gene transfer. Exemplary methods of delivering DNA using electroporation are described in U.S. Pat. Nos. 6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No. 2002/0146831, and International Publication No. WO/0045823, all of which are incorporated herein by reference in their entireties.

[00160] In some embodiments, the composition described herein is a pharmaceutical composition. The composition can be administered in a therapeutically effective amount in admixture with pharmaceutical carriers. As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an isolated peptide with which it is admixed, unless so desired. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Other desirable ingredients for use in such preparations include preservatives, co-solvents, viscosity building agents, carriers, etc. The carrier itself or a component dissolved in the carrier may have palliative or therapeutic properties of its own, including moisturizing, cleansing, or anti-inflammatory/anti-itching properties. Penetration enhancers may, for example, be surface active agents; certain organic solvents, such as

di-methylsulfoxide and other sulfoxides, dimethyl-acetamide and pyrrolidone; certain amides of heterocyclic amines, glycols (e.g. propylene glycol); propylene carbonate; oleic acid; alkyl amines and derivatives; various cationic, anionic, nonionic, and amphoteric surface active agents; and the like.

[00161] Pharmaceutical compositions comprising an isolated peptide as described herein can comprise a second pharmaceutically active agent. By way of non-limiting example, a second pharmaceutically active agent can be a fibrosis treatment or a chemotherapeutic, examples of which are described above herein.

[00162] Pharmaceutical compositions administered according to the present invention can be applied, for example, topically to a tissue. Such compositions include solutions, powders, suspensions, lotions, gels, creams, ointments, emulsions, skin patches, sprays, or roll-on products etc. All of these dosage forms, along with methods for their preparation, are known in the pharmaceutical and cosmetic art. Harry's Cosmeticology (Chemical Publishing, 7th ed. 1982); Remington's Pharmaceutical Sciences (Mack Publishing Co., 18th ed. 1990). Typically, such topical formulations contain the active ingredient in a concentration range of 0.1 to 100 mg/ml, in admixture with a pharmaceutically acceptable carrier.

[00163] It is contemplated that the pharmaceutical compositions described herein can also be administered systemically in a pharmaceutical formulation. Systemic routes include but are not limited to oral, parenteral, nasal inhalation, intratracheal, intrathecal, intracranial, and intrarectal. The pharmaceutical formulation is preferably a sterile saline or lactated Ringer's solution. For therapeutic applications, the preparations described herein are administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intrasynovial, intrathecal, oral, or inhalation routes. For these uses, additional conventional pharmaceutical preparations such as tablets, granules, powders, capsules, and sprays may be preferentially required. In such formulations further conventional additives such as binding-agents, wetting agents, propellants, preservatives, lubricants, and stabilizers may also be required. In one embodiment, the pharmaceutical compositions described herein can be administered directly by injection, for example to the affected tissue, such as organ, muscle or tissue, or wound (encompassing but not limited to lacerations, abrasions, avulsions, cuts, velocity wounds, penetration wounds, puncture wounds, contusions, hematomas, tearing wounds, and/or crushing injuries to the skin and subcutaneous tissue). The composition described herein can remain in a liquid form after injection or solidfy into a gel by external activation (e.g. light, sound, etc) to maintain locality of the treatment. A preferred formulation is sterile saline or Lactated Ringer's solution. Lactated Ringer's solution is a solution that is isotonic with blood and intended for intravenous administration. In a further embodiment, ophthalmic isolated peptide compositions are used to treat fibrotic disorders of the eye, e.g.corneal ulceration. In some embodiments, an isolated peptide as described herein is administered in an aqueous solution. In some embodiments, an isolated peptide as described herein is administered in an aqueous buffer with a pH of approximately 7.4.

[00164] In one embodiment, the isolated peptide as described herein, or a nucleic acid encoding the peptide, is administered to a subject for an extended period of time to produce optimum enhancement of the extracellular matrix, anti-tumorigenic, or anti-angiogenic effects. Sustained contact with the isolated peptide composition can be achieved by, for example, repeated administration of the isolated peptide composition over a period of time, such as one week, several weeks, one month or longer. More preferably, the pharmaceutically acceptable formulation used to administer the active compound provides sustained delivery, such as "slow release" of the active compound to a subject. For example, the formulation may deliver the isolated peptide composition for at least one, two, three, or four weeks after the pharmaceutically acceptable formulation is administered to the subject. [00165] As used herein, the term "sustained delivery" is intended to include continual delivery of the composition comprising an isolated peptide as described herein, or a nucleic acid encoding the peptide, in vivo over a period of time following administration, preferably at least several days, a week, several weeks, one month or longer. Sustained delivery of the isolated peptide as described herein, or a nucleic acid encoding the peptide, can be demonstrated by, for example, the continued therapeutic effect of the isolated peptide composition over time. Alternatively, sustained delivery of the the isolated peptide as described herein, or a nucleic acid encoding the peptide, may be demonstrated by detecting the presence of the isolated peptide composition in vivo over time. Preferred approaches for sustained delivery include use of a polymeric capsule, a minipump to deliver the formulation, or a biodegradable implant.

[00166] The compositions can be formulated as a sustained release composition. For example, sustained-release means or delivery devices are known in the art and include, but are not limited to, sustained-release matrices such as biodegradable matrices or semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules that comprise isolated peptides as described herein. A sustained-release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. The sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid).

[00167] Nanoparticles, e.g.microspheres formed of polymers or proteins or biocompatible materials are well known to those skilled in the art, and can, for example, be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the isolated peptide as described herein, or a nucleic acid encoding the peptide, can be incorporated with the microspheres or composite of microspheres, and implanted for slow release over a period of time ranging from days to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673 and 3,625,214, and Jein, TIPS 19: 155-157 (1998), the contents of which are hereby incorporated by reference. Preferred micro particles are those prepared from biodegradable polymers, such as polyglycolide, polylactide and copolymers thereof. Those of skill in the art can readily determine an appropriate carrier system depending on various factors, including the desired rate of drug release and the desired dosage.

[00168] In some embodiments, an isolated peptide as described herein, or a nucleic acid encoding the peptide, can be administered in a pharamaceutical composition comprising liposomes. As used herein, "lipid vesicle" or "liposome" refers to vesicles surrounded by a bilayer formed of lipid components usually including lipids optionally in combination with non-lipidic components. The interior of a vesicle is generally aqueous. One major type of liposomal composition not generally found in nature includes phospholipids other than naturally-derived phosphatidylcholine. Neutral lipid vesicle compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic lipid vesicle compositions generally are formed from dimyristoyl phosphatidylglycerol. Another type of liposomal composition is formed from

phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. Lipids for lipid vesicle or liposome formation are known in the art or described herein below.

[00169] Liposomes are formed by the self-assembly of phospholipid molecules in an aqueous environment. The amphipathic phospholipid molecules form a closed bilayer sphere in an attempt to shield their hydrophobic groups from the aqueous environment, while still maintaining contact with the aqueous phase via the hydrophilic head group. The resulting closed sphere can encapsulate aqueous soluble drugs or agents such as the hemoglobin, enzyme and cofactor compositions described herein, within the bilayer membrane.

[00170] Non-limiting examples of liposome compositions include those described U.S. Pat.

Nos. 4,983,397; 6,476,068; 5,834,012; 5,756,069; 6,387,397; 5,534,241 ; 4,789,633; 4,925,661;

6,153,596; 6,057,299; 5,648,478; 6,723,338; 6,627218; U.S. Pat. App. Publication Nos: 2003/0224037; 2004/0022842; 2001/0033860; 2003/0072794; 2003/0082228; 2003/0212031 ; 2003/0203865;

2004/0142025; 2004/0071768; International Patent Applications WO 00/74646; WO 96/13250; WO 98/33481 ; Papahadjopolulos D, Allen T M, Gbizon A, et al. "Sterically stabilized liposomes.

Improvements in pharmacokinetics and antitumor therapeutic efficacy" Proc Natl Acad Sci U.S.A. (1991) 88: 11460-11464; Allen T M, Martin F J. "Advantages of liposomal delivery systems for anthracyclines" Semin Oncol (2004) 31 : 5-15 (suppl 13). Weissig et al. Pharm. Res. (1998) 15:

1552-1556 each of which is incorporated herein by reference in its entirety. In some embodiments the vesicles comprise a mixture of two or more individual lipids. In some embodiments, in a mixture of lipids, the lipids have similar phase transition temperatures. In some embodiments, where lipids with a melting temperature below room temperature are used, hydration and extrusion procedures used to prepared lipid vesicles can be performed at room temperature. In some embodiments, the lipid vesicle surface is PEG (polyethylene glycol)-adsorbed to prevent vesicle coalescing. The vesicles can be from 150 nm in diameter to 500 nm in diameter. However, very small vesicles are rapidly cleared by the kidneys and very large vesicles present problems for circulation. In some embodiments, the vesicles are from 150 nm in diameter to 500 nm in diameter, for example, 150-450 nm inclusive, 150-400 nm inclusive, 150-300 nm inclusive, 150-220 nm inclusive, 170-210 nm inclusive, 180-210 nm inclusive, 190-200 nm inclusive, or about 200 nm in diameter. Lipid vesicles as described herein can be prepared according to methods used in the preparation of conventional lipid vesicles and PEG-lipid vesicles, as disclosed in e.g. EP-0662820. Passive loading of the active ingredients into the lipid vesicles by dissolving the components in the aqueous phase that is then mixed with a lipid preparation can be sufficient to encapsulate the components, but other methods can also be used.

[00171] In some embodiments the isolated peptide as described herein, or a nucleic acid encoding the peptide, can be included in biodegradable polymeric hydrogels, such as those disclosed in U.S. Pat. No. 5,410,016 to Hubbell et al. These polymeric hydrogels can be delivered to a subject and the active compounds released over time as the polymer degrades. If desirable, the polymeric hydrogels can have microparticles or liposomes which include the active compound dispersed therein, providing another mechanism for the controlled release of the isolated peptide as described herein, or a nucleic acid encoding the peptide.

[00172] For enteral administration, a composition can be incorporated into an inert carrier in discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension or solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or a draught. Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature.

[00173] A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active compound in a free-flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients, e.g., binders, lubricants, inert diluents, surface active or dispersing agents. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.

[00174] A syrup or suspension can be made by adding the active compound to a concentrated, aqueous solution of a sugar, e.g., sucrose, to which can also be added any accessory ingredients. Such accessory ingredients may include flavoring, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, e.g., as a polyhydric alcohol, for example, glycerol or sorbitol.

[00175] Formulations for oral administration can be presented with an enhancer.

Orally-acceptable absorption enhancers include surfactants such as sodium lauryl sulfate, palmitoyl carnitine, Laureth-9, phosphatidylcholine, cyclodextrin and derivatives thereof; bile salts such as sodium deoxycholate, sodium taurocholate, sodium glycochlate, and sodium fusidate; chelating agents including EDTA, citric acid and salicylates; and fatty acids (e.g., oleic acid, lauric acid, acylcarnitines, mono- and diglycerides). Other oral absorption enhancers include benzalkonium chloride,

benzethonium chloride, CHAPS (3-(3-cholamidopropyl)-dimethylammonio-l-propanesulfonate), Big-CHAPS (N, N-bis(3-D-gluconamidopropyl)-cholamide), chlorobutanol, octoxynol-9, benzyl alcohol, phenols, cresols, and alkyl alcohols. An especially preferred oral absorption enhancer for the present invention is sodium lauryl sulfate. [00176] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. The route of administration, dosage form, and the effective amount vary according to the potency of the isolated peptide, its physicochemical characteristics, and according to the treatment location. The selection of proper dosage is well within the skill of an ordinarily skilled physician.

[00177] In one embodiment, the pharmaceutical formulation to be used for therapeutic administration is sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).

[00178] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.

[00179] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[00180] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00181] This invention is further illustrated by the following examples which should not be construed as limiting. [00182] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

1. An isolated peptide comprising the amino acid sequence of:

Val ₁ -Ser ₂ -Asp ₃ -Val ₄ -Xs-X ₆ -X7-X8-X9-Xio-Xii-Xi ₂ -Xi ₃ -Thr ₁ 4-Xis-Xi ₆ - Ser ₁₇ -X ₁₈ -

X ₁₉ -X ₂₀ -Ser ₂₁ -X ₂₂ -X ₂₃ (SEQ ID NO: 71).

wherein X ₅ is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val;

wherein X ₆ is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, and Asn;

wherein X ₇ is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His;

wherein X ₈ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser;

wherein X ₉ is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro;

wherein Xi ₀ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid;

wherein Xn is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, Asn, Glu, and any beta-branched amino acid;

wherein X _i2 is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn;

wherein X ₁₃ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin;

wherein X ₁₅ is selected from the group consisting of Pro, Gly, Ala, Ser, Thr, Asp, Glu, Val, He and Leu; wherein X ₁₆ is selected from the group consisting of Thr, Gly, Ala, Ser, Gin, Glu, Asp, Asn, Leu, and Val;

wherein X ₁₈ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr;

wherein X ₁₉ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, or any beta-branched amino acid;

wherein X ₂₀ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp;

wherein X ₂₂ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala;

wherein X ₂₃ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe;

or a pharmaceutically acceptable salt; analog; prodrug; derivative; solvate; or functional fragment thereof;

wherein the isolated peptide is not fibronectin; and

wherein the isolated peptide comprises 75 or fewer amino acids. The isolated peptide of paragraph 1, wherein the peptide comprises the amino acid sequence of Cys ₁ -Val ₂ -Ser ₃ -Asp ₄ -Val5-X ₆ - X ₇ -X ₈ -X ₉ -X ₁₀ -Xii-Xi ₂ -Xi ₃ - Xi ₄ -Thr ₁₅ -X ₁₆ -X ₁₇ - Ser ₁₈ -X ₁₉ - X ₂₀ -X ₂ i-Ser ₂₂ -X ₂₃ -X ₂ 4 (SEQ ID NO: 72).

wherein X ₆ is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val;

wherein X ₇ is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, Asn;

wherein X ₈ is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His;

wherein X ₉ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser;

wherein Xi ₀ is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro;

wherein Xn is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid;

wherein X _i2 is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, Asn, Glu, and any beta-branched amino acid;

wherein X _i3 is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn;

wherein X ₁₄ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin;

wherein X ₁₆ is selected from the group consisting of Pro, Gly, Ala, Ser, Thr, Asp, Glu, Val, He and Leu; wherein X ₁₇ is selected from the group consisting of Thr, Gly, Ala, Ser, Gin, Glu, Asp, Asn, Leu, and Val;

wherein X ₁₉ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr;

wherein X ₂₀ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, or any beta-branched amino acid;

wherein X _2i is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp;

wherein X ₂₃ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; and wherein X ₂₄ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe.

An isolated peptide comprising the amino acid sequence of

Vali-Ser ₂ -Asp ₃ -Val ₄ -X5-X ₆ -X ₇ -X8-X ₉ -Xio-Xii-Xi ₂ -Xi ₃ -Thri ₄ -Xi ₅ -Xi ₆ -Seri ₇ -Xi ₈ -Xi ₉ -X ₂ o-Ser ₂ i-X _{2 2} -X ₂₃ (SEQ ID NO: 39) _;

wherein X ₅ is selected from the group consisting of Pro; Gly; Ala; Leu; Ser; and Val;

wherein X ₆ is selected from the group consisting of Arg; Ser; and Thr;

wherein X ₇ is selected from the group consisting of Asp; Lys; Asn; and His;

wherein X ₈ is selected from the group consisting of Leu; Trp; Ala; Val; He; Phe; Met; Pro; Glu; and Ser;

wherein X ₉ is selected from the group consisting of Glu; Gin; and Pro;

wherein X ₁₀ is selected from the group consisting of Val; Ala; He; Leu; Phe; Tyr; Trp; and Thr; wherein X _n is selected from the group consisting of Val; Trp; He; Leu; Phe; Met; Arg; Ser; Cys; Pro; Thr; Asp; Lys; and any beta-branched amino acid;

wherein X _i2 is selected from the group consisting of Ala; Gly; Ser; Val; Asp; Arg; His; Gin; Trp; Phe; Tyr; Leu; Met; Glu; Lys; Thr; and Asn;

wherein X _i3 is selected from the group consisting of Ala; Val; He; Leu; Phe; Tyr; Trp; Thr; Pro; Glu; and Ser;

wherein X _i5 is selected from the group consisting of Pro; Gly; Ala; Val; He; and Leu;

wherein X _i6 is selected from the group consisting of Thr; Gly; Ala; Ser; and Val;

wherein X _i8 is selected from the group consisting of Leu; Ala; Val; He; Phe; Tyr; Trp; Met; His; and Thr;

wherein X _i9 is selected from the group consisting of Leu; Trp; Val; He; Phe; Pro; Thr; Lys; Met;

Gin; Arg; Ser; He; Tyr; His; Cys; and any beta-branched amino acid;

wherein X ₂₀ is selected from the group consisting of He; Ala; Val; Leu; Phe; Tyr; Trp;

wherein X ₂₂ is selected from the group consisting of Trp; Val; He; Leu; Phe; and Tyr;

wherein X ₂₃ is selected from the group consisting of Asp; Gin; Thr.

or a pharmaceutically acceptable salt; analog; prodrug; derivative; solvate; or functional fragment thereof;

wherein the isolated peptide is not fibronectin; and

wherein the isolated peptide comprises 75 or fewer amino acids.

The isolated peptide of paragraph 3, wherein the peptide comprises the amino acid sequence of Val ₁ -Ser ₂ -Asp ₃ -Val ₄ -Pro ₅ -Arg ₆ -Asp ₇ -Leu ₈ -Glu ₉ -Val ₁ o-Val ₁₁ -Ala ₁₂ -Ala ₁₃ -Thr ₁₄ -Pro ₁₅ -Thr ₁₆ -Ser ₁ 7-Leui ₈ -Leui ₉ -Ile ₂ o-Ser ₂ i-Trp ₂₂ -Asp ₂₃ (SEQ ID NO: 2).

The isolated peptide of paragraph 3, wherein the peptide comprises the amino acid sequence of Cys ₁ -Val ₂ -Ser ₃ -Asp4-Val5-X ₆ -X ₇ -X ₈ -X ₉ -X ₁ o-Xii-Xi ₂ -Xi3-Xi4- Thr ₁₅ -X ₁₆ - Xi ₇ -Ser ₁₈ - X ₁₉ - X ₂₀ -X ₂ i-Ser ₂₂ -X ₂₃ -X ₂₄ (SEQ ID NO: 40);

wherein X ₆ is selected from the group consisting of Pro; Gly; Ala; Leu; Ser; and Val;

wherein X ₇ is selected from the group consisting of Arg; Ser; and Thr;

wherein X ₈ is selected from the group consisting of Asp; Lys; Asn; and His;

wherein X ₉ is selected from the group consisting of Leu; Trp; Ala; Val; He; Phe; Met; Pro; Glu; and Ser;

wherein Xi ₀ is selected from the group consisting of Glu; Gin; and Pro;

wherein Xn is selected from the group consisting of Val; Ala; He; Leu; Phe; Tyr; Trp; and Thr; wherein X _i2 is selected from the group consisting of Val; Trp; He; Leu; Phe; Met; Arg; Ser; Cys; Pro; Thr; Asp; Lys; and any beta-branched amino acid; wherein X ₁₃ is selected from the group consisting of Ala; Gly; Ser; Val; Asp; Arg; His; Gin; Trp; Phe; Tyr; Leu; Met; Glu; Lys; Thr; and Asn;

wherein X ₁₄ is selected from the group consisting of Ala; Val; He; Leu; Phe; Tyr; Trp; Thr; Pro; Glu; and Ser;

wherein X ₁₆ is selected from the group consisting of Pro; Gly; Ala; Val; He; and Leu;

wherein X _i7 is selected from the group consisting of Thr; Gly; Ala; Ser; and Val;

wherein X _i9 is selected from the group consisting of Leu; Ala; Val; He; Phe; Tyr; Trp; Met; His; and Thr;

wherein X ₂ o is selected from the group consisting of Leu; Trp; Val; He; Phe; Pro; Thr; Lys; Met; Gin; Arg; Ser; He; Tyr; His; Cys; and any beta-branched amino acid;

wherein X ₂ i is selected from the group consisting of He; Ala; Val; Leu; Phe; Tyr; and Trp; wherein X ₂ 3 is selected from the group consisting of Trp; Val; He; Leu; Phe; and Tyr; and wherein X ₂₄ is selected from the group consisting of Asp; Gin; and Thr.

The isolated peptide of Paragraph 5, wherein the peptide comprises the amino acid sequence of Cysi-Val ₂ -Ser ₃ -Asp ₄ -Val ₅ -Pro ₆ -Arg ₇ -Asp ₈ -Leu ₉ -Gluio-Valii-Vali ₂ -Alai ₃ -Alai ₄ -Thri ₅ -Proi ₆ -Thri 7-Seri ₈ -Leui9-Leu2o-Ile2i-Ser ₂ 2-Trp23-Asp24 (SEQ ID NO: 3).

An isolated peptide comprising the amino acid sequence of Seri-X ₂ -X ₃ -X ₄ -Ser ₅ -X ₆ -X ₇ (SEQ ID NO: 73) _;

wherein X ₂ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr;

wherein X ₃ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, or any beta-branched amino acid;

wherein X ₄ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp; wherein X ₆ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; wherein X ₇ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe;

or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof;

wherein the isolated peptide is not fibronectin; and

wherein the isolated peptide comprises 75 or fewer amino acids.

The peptide of paragraph 7, wherein the peptide comprises the amino acid sequence of Cysi-Ser ₂ -X3-X4-X5-Ser ₆ -X7-X8 (SEQ ID NO: 74);

wherein X ₃ is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr;

X ₄ is selected from the group consisting of Leu, Trp, Val, He, Phe, Pro, Thr, Lys, Met, Gin, Arg, Ser, He, Tyr, His, Cys, Gly, and any beta-branched amino acid; X ₅ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp;

X ₇ is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala; and

X ₈ is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe.

The isolated peptide of paragraph 7, wherein the peptide comprises the amino acid sequence of Seri-X ₂ -X3-X4-Sers-X6-X ₇ (SEQ ID NO: 12).

wherein X ₂ is selected from the group consisting of Leu; Ala; Val; He; Phe; Tyr; Trp; Met; His; and Thr;

wherein X ₃ is selected from the group consisting of Leu; Trp; Val; He; Phe; Pro; Thr; Lys; Met; Gin; Arg; Ser; He; Tyr; His; Cys; and any beta-branched amino acid;

wherein X ₄ is selected from the group consisting of He; Ala; Val; Leu; Phe; Tyr; and Trp; wherein X ₆ is selected from the group consisting of Trp; Val; He; Leu; Phe; and Tyr;

wherein X ₇ is selected from the group consisting of Asp; Gin; and Thr.

The isolated peptide of Paragraph 9, wherein the peptide comprises the amino acid sequence of Seri-Leu ₂ -Leu ₃ -Ile ₄ -Ser ₅ -Trp ₆ -Asp ₇ (SEQ ID NO: 5) _.

The isolated peptide of Paragraph 9, wherein the peptide comprises the amino acid sequence of Cysi-Ser ₂ -X3-X4-X5-Ser ₆ -X7-X8 (SEQ ID NO: 41) _;

wherein X ₃ is selected from the group consisting of Leu; Ala; Val; He; Phe; Tyr; Trp; Met; His; and Thr;

wherein X ₄ is selected from the group consisting of Leu; Trp; Val; He; Phe; Pro; Thr; Lys; Met; Gin; Arg; Ser; He; Tyr; His; Cys; and any beta-branched amino acid;

wherein X ₅ is selected from the group consisting of He; Ala; Val; Leu; Phe; Tyr; and Trp; wherein X ₇ is selected from the group consisting of Trp; Val; He; Leu; Phe; and Tyr; and wherein X ₈ is selected from the group consisting of Asp; Gin; and Thr.

The isolated peptide of Paragraph 11 , wherein the peptide comprises the amino acid sequence of Cys ₁ -Ser ₂ -Leu ₃ -Leu ₄ -Ile ₅ -Ser ₆ -Trp ₇ -Asp ₈ (SEQ ID NO: 14) _.

An isolated peptide comprising the amino acid sequence of:

Xi-X ₂ -X ₃ -X4-X5- 6- 7- 8- hr ₉ (SEQ ID NO: 75);

wherein is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, and Asn; wherein X ₂ is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His;

wherein X ₃ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser;

wherein X ₄ is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid;

wherein X ₆ is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, Asn, Glu, and any beta-branched amino acid;

wherein X ₇ is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn;

wherein X ₈ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin;

or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof;

wherein the isolated peptide is not fibronectin; and

wherein the isolated peptide comprises 75 or fewer amino acids.

The isolated peptide of paragraph 13, wherein the peptide comprises the amino acid sequence of Tyri-X ₂ -X ₃ -X ₄ -X5-X6-X ₇ -X ₈ -X9- hrio (SEQ ID NO: 76);

wherein X ₂ is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, and Asn; wherein X ₃ is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His;

wherein X ₄ is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser;

wherein X ₅ is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro;

wherein X ₆ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid;

wherein X ₇ is selected from the group consisting of Val, Trp, He, Leu, Phe, Met, Arg, Ser, Cys, Pro, Thr, Asp, Lys, Asn, Glu, and any beta-branched amino acid;

wherein X ₈ is selected from the group consisting of Ala, Gly, Ser, Val, Asp, Arg, His, Gin, Trp, Phe, Tyr, Leu, Met, Glu, Lys, Thr, and Asn; and

wherein X ₉ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin.

The isolated peptide of paragraph 13, wherein the peptide comprises the amino acid sequence of:

Xi-X2-X ₃ -X ₄ -X5-X6-X ₇ -X ₈ -Thr ₉ (SEQ ID NO: 15);

wherein Xi is selected from the group consisting of Arg; Ser; and Thr;

wherein X ₂ is selected from the group consisting of Asp; Lys; Asn; and His;

wherein X ₃ is selected from the group consisting of Leu; Trp; Val; Ala; He; Phe; Met; Pro; Glu; and Ser;

wherein X ₄ is selected from the group consisting of Glu;Gln; and Pro; wherein X ₅ is selected from the group consisting of Val; Ala; He; Leu; Phe; Tyr; Trp; and Thr; wherein X ₆ is selected from the group consisting of Val; Trp; He; Leu; Phe; Met; Arg; Ser; Cys; Pro; Thr; Asp; Lys; and any beta-branched amino acid;

wherein X ₇ is selected from the group consisting of Ala; Gly; Ser; Val; Asp; Arg; His; Gin; Trp; Phe; Tyr; Leu; Met; Glu; Lys; Thr; and Asn;

wherein X ₈ is selected from the group consisting of Ala; Val; He; Leu; Phe; Tyr; Trp; Thr; Pro; Glu; and Ser;

or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof;

wherein the isolated peptide is not fibronectin; and

wherein the isolated peptide comprises 75 or fewer amino acids.

The isolated peptide of Paragraph 15, wherein the peptide comprises the amino acid sequence of Argi-Asp ₂ -Leu ₃ -Glu ₄ -Val ₅ -Val ₆ -Ala ₇ -Ala ₈ -Thr ₉ (SEQ ID NO: 7).

The isolated peptide of Paragraph 15, wherein the peptide comprises the amino acid sequence of Tyri-X ₂ -X ₃ -X ₄ -X5-X6-X7-X8-X9- hrio (SEQ ID NO: 26);

wherein X ₂ is selected from the group consisting of Arg; Ser; and Thr;

wherein X ₃ is selected from the group consisting of Asp; Lys; Asn; and His;

wherein X ₄ is selected from the group consisting of Leu; Trp; Ala; Val; He; Phe; Met; Pro; Glu; and Ser;

wherein X ₅ is selected from the group consisting of Glu;Gln; and Pro;

wherein X ₆ is selected from the group consisting of Val; Ala; He; Leu; Phe; Tyr; Trp; and Thr; wherein X ₇ is selected from the group consisting of Val; Trp; He; Leu; Phe; Met; Arg; Ser; Cys; Pro; Thr; Asp; Lys; and any beta-branched amino acid;

wherein X ₈ is selected from the group consisting of Ala; Gly; Ser; Val; Asp; Arg; His; Gin; Trp; Phe; Tyr; Leu; Met; Glu; Lys; Thr; and Asn; and

wherein X ₉ is selected from the group consisting of Ala; Val; He; Leu; Phe; Tyr; Trp; Thr; Pro; Glu; and Ser.

The isolated peptide of Paragraph 17, wherein the peptide comprises the amino acid sequence of Tyr ₁ -Arg ₂ -Asp ₃ -Leu ₄ -Glu ₅ -Val ₆ -Val ₇ -Ala ₈ -Ala ₉ -Thr ₁ o (SEQ ID NO: 8).

An isolated peptide comprising the amino acid sequence of Thr ₁ -Ala ₂ -Thr ₃ -Ile ₄ -Ser ₅ (SEQ ID NO: 9);

or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof;

wherein the isolated peptide is not fibronectin; and

wherein the isolated peptide comprises 75 or fewer amino acids. The isolated peptide of Paragraph 13, wherein the peptide comprises the amino acid sequence of Tyri-Thr ₂ -Ala ₃ -Thr ₄ -nes-Ser ₆ (SEQ ID NO: 10).

An isolated peptide comprising the amino acid sequence of :

Tyr ₁ -X ₂ -Arg ₃ -X ₄ -Thr ₅ -X ₆ -X ₇ -Glu ₈ (SEQ ID NO: 77);

wherein X ₂ is selected from a group consisting of Tyr, Ser, Ala, Val, He, Leu, Phe, and Trp; wherein X ₄ is selected from a group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp;

wherein X ₆ is selected from a group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, His, Thr, and Cys;

wherein X ₇ is selected from the group consisting of Gly, Arg, Glu, Ser, He, Thr, Val, His, Trp, and Cys;

or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof;

wherein the isolated peptide is not fibronectin; and

wherein the isolated peptide comprises 75 or fewer amino acids.

The isolated peptide of paragraph 21, wherein the peptide comprises the amino acid sequence of: Tyri-X ₂ -Arg3-X4-Thr ₅ -X6-X7- Glu ₈ (SEQ ID NO: 16);

wherein X ₂ is selected from a group consisting of Tyr; Ala; Val; He; Leu; Phe; and Trp;

wherein X ₄ is selected from a group consisting of He; Ala; Val; Leu; Phe; Tyr; and Trp;

wherein X ₆ is selected from a group consisting of Tyr; Ala; Val; He; Leu; Phe; Trp; His; Thr; and Cys; and

wherein X ₇ is selected from the group consisting of Gly; Arg; Glu; Trp; and Cys.

The isolated peptide of Paragraph 22, wherein the peptide comprise the amino acid sequence of Tyr ₁ -Tyr ₂ -Arg ₃ -Ile ₄ -Thr ₅ -Tyr ₆ -Gly ₇ -Glu ₈ (SEQ ID NO: 11) _.

An isolated peptide comprising the amino acid sequence of

Glni-Glu ₂ -X ₃ -Thr ₄ -Xs-Pro ₆ (SEQ ID NO: 78);

wherein X ₃ is selected from the group consisiting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, Thr, Pro, and Glu;

wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, Pro, Glu, Lys, Ser, and any beta4)ranched amino acid;

or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof;

wherein the isolated peptide is not fibronectin; and

wherein the isolated peptide comprises 75 or fewer amino acids.

The isolated peptide of paragraph 24, wherein the peptide comprises the amino acid sequence of Tyr ₁ -Gln ₂ -Glu ₃ -X ₄ -Thr ₅ -X ₆ -Pro ₇ (SEQ ID NO: 79); wherein X ₄ is selected from the group consisiting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, Thr, Pro, and Glu; and

wherein X ₆ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Trp, Pro, Glu, Lys, Ser, and any beta-branched amino acid.

The isolated peptide of paragraph 24, wherein the peptide comprises the amino acid sequence of:

Glni-Glu ₂ -X3-Thr ₄ -Xs-Pro ₆ (SEQ ID NO: 17);

wherein X ₃ is selected from the group consisiting of Phe; Ala; Val; He; Leu; Tyr; Trp; Asp; Lys; Arg; and Thr;

wherein X ₅ is selected from the group consisting of Val; Ala; He; Leu; Phe; Tyr; Trp; Pro; Glu; Lys; and Ser;

The isolated peptide of Paragraph 26, wherein the peptide comprises the amino acid sequence of Glni-Glu ₂ -Phe ₃ -Thr ₄ -Val5-Pro6 (SEQ ID NO: 18).

The isolated peptide of Paragraph 26, wherein the peptide comprises the amino acid sequence of Tyri-Gln ₂ -Glu ₃ -X4-Thr ₅ -X6-Pro ₇ (SEQ ID NO: 27);

wherein X ₄ is selected from the group consisiting of Phe; Ala; Val; He; Leu; Tyr; Trp; Asp; Lys; Arg; and Thr; and

wherein X ₆ is selected from the group consisting of Val; Ala; He; Leu; Phe; Tyr; Trp; Pro; Glu; Lys; and Ser.

The isolated peptide of Paragraph 28, wherein the peptide comprises the amino acid sequence of Tyri-Gln ₂ -Glu ₃ -Phe ₄ -Thr ₅ -Val ₆ -Pro ₇ (SEQ ID NO: 19)

An isolated peptide comprising the amino acid sequence of X ₁ -Thr ₂ -X ₃ -Thr ₄ -X5-Tyr ₆ -X ₇ -Val ₈ (SEQ ID NO: 80); wherein Xi is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, and Trp;

wherein X ₃ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, and Gly; wherein X ₅ is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, and Trp; wherein X ₇ is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Ser, Thr, and Gin;

or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof;

wherein the isolated peptide is not fibronectin; and

wherein the isolated peptide comprises 75 or fewer amino acids.

The isolated peptide of paragraph 30, wherein the peptide comprises the amino acid sequence of Xi-Thr ₂ -X3-Thr ₄ -Xs-Tyr ₆ -X ₇ -Valg (SEQ ID NO: 20)

wherein Xj is selected from the group consisting of Tyr; Ala; Val; He; Leu; Phe; and Trp; wherein X ₃ is selected from the group consisting of He ; Ala; Val; Leu; Phe; Tyr; Trp; and Gly; wherein X ₅ is selected from the group consisting of Val; Ala; He; Leu; Phe; Tyr; and Trp; wherein X ₇ is selected from the group consisting of Ala; Val; He; Leu; Phe; Tyr; Trp; Ser; Thr; and Gin.

The isolated peptide of paragraph 31 , wherein the peptide comprises the amino acid sequence of Tyr ₁ -Thr ₂ -Ile ₃ -Thr ₄ -Val ₅ -Tyr ₆ -Ala ₇ -Val ₈ (SEQ ID NO: 21) _.

An isolated peptide comprising the amino acid sequence of Xi-Ser ₂ -X3-Asn ₄ -X ₅ (SEQ ID NO: 81);

wherein is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, Lys, Arg, Asp, Gin, Thr, and Pro;

wherein X ₃ is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, Gly, Gin, Asp, Thr, Ser, Arg, and Asn;

wherein X ₅ is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, Lys, Gin, Ser, Thr, and Pro;

or a pharmaceutically acceptable salt, analog, prodrug, derivative, solvate, or functional fragment thereof;

wherein the isolated peptide is not fibronectin; and

wherein the isolated peptide comprises 75 or fewer amino acids.

The isolated peptide of paragraph 33, wherein the peptide comprises the amino acid sequence of: Xi-Ser ₂ -X3-Asn ₄ -X5 (SEQ ID NO: 22);

wherein Xi is selected from the group consisting of He ; Ala; Val; Leu; Phe; Tyr; Trp; Lys; Thr; and Pro;

wherein X ₃ is selected from the group consisting of He ; Ala; Val; Leu; Phe; Tyr; Trp; Gly; Gin; Asp; Thr; Ser; and Asn;

wherein X ₅ is selected from the group consisting of Tyr; Ala; Val; He; Leu; Phe; Trp; Lys; Gin; Ser; and Pro;

The isolated peptide of paragraph 34, wherein the peptide comprises the amino acid sequence of He ₁ -Ser ₂ -Ile ₃ -Asn ₄ -Tyr ₅ (SEQ ID NO: 23) _.

The peptide of any of paragraphs 1-35, wherein the peptide comprises the corresponding amino acid sequence of a homolgous fibronectin gene.

The peptide of any of paragraphs 1-36, wherein the peptide is comprised by a polypeptide comprising multiple or tandem occurrences of one or more of the peptides of paragraphs 1-36. The peptide of any of paragraphs 1-37, wherein the multiple occurrences of the peptide have a physical arrangement selected from the group consisting of:

linear; branched; arrayed; multiplexed; and cyclized. The peptide of any of paragraphs 1-38, wherein the peptide is comprised by a polypeptide comprises at least two of the peptides linked by peptide bonds, chemical cross linkers, linkers, spacers, or other chemical bonds.

The peptide of any of paragraphs 1-39, wherein the peptide comprises a mutation elongating the loop region defined by Pro ₁₅ -Thr ₁₆ of SEQ ID NO:2 or by Pro ₁₆ -Thr ₁₇ of SEQ ID NO:3.

The peptide of any of paragraphs 1-40, wherein the peptide comprises a mutation that locks the peptide into a beta strand conformation or otherwise constrained conformation.

The peptide of paragraph 41, wherein the mutation comprises a mutation selected from the group consisting of double Cys mutations or click chemistry or other crosslinking

methodologies.

The peptide of any of paragraphs 1-42, wherein the peptide comprises at least one D-amino acid. The peptide of any of paragraphs 1-43, wherein the peptide comprises at least one beta-amino acid.

The peptide of any of paragraphs 1-44, wherein the peptide comprises at least one peptide bond replacement.

The peptide of any of paragraphs 1-45, wherein the peptide comprises at least one peptide bond replacement selected from the group consisting of:

urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid, para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronic ester, olefinic group, and derivatives thereof.

The peptide of any of paragraphs 1-46, wherein the peptide comprises at least one amino aid selected from the group consisting of:

amino acid analogs; chemically modified amino acids; non-natural amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, hydroxyllysine, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3 -mercapto-D -valine), ornithine, citruline, alpha-methyl-alanine, para-benzoylphenylalanine, para-amino

phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine), diaminobutyric acid, 7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine, biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine,

1-amino-l-cyclopentanecarboxylic acid, 1-amino-l-cyclohexanecarboxylic acid, amino-benzoic acid, amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine, nipecotic acid, alpha-amino butyric acid, thienyl-alanine, t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs; azide-modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and derivatives thereof.

The peptide of any of paragraphs 1-47, wherein the peptide has a modification selected from the group consisting of:

PEGylation; post-translational derivitzations; glycosylation; hydroxylation; methylation; HESylation; ELPylation; lipidation; acetylation; amidation; biotinylation; end-capping modifications; cyano groups; phosphorylation; cyclization; or other conjugation moieties. The peptide of any of paragraphs 1-48, comprising a conservative substitution of one or more amino acids.

The peptide of any of paragraphs 1-49, comprising a deletion or insertion of one or more amino acids.

The peptide of any of paragraphs 1-50, wherein the peptide is a fusion peptide.

The peptide of any of paragraphs 1-51 wherein the peptide is coupled to a targeting molecule. The peptide of paragraph 52, wherein the peptide is coupled to or comprises a tag molecule selected from the group consisting of:

a detectable agent; a contrast agent; electron dense material; magnetic resonance imaging agents; radioactive molecule; non-radioactive detectable agents; a dye; a radioactive dye; a fluorescent molecule; 19 F; 2 H; 13 C; 15 N; an isotope; paramagnetic contrasting agents; compounds that enhances magnetic resonance imaging (MRI); radiopharmaceuticals; radionuclides; or a combination

The peptide of any of paragraphs 1-53, wherein the peptide is coupled to a therapeutic molecule. The peptide of paragraph 54, wherein the therapeutic molecule is a chemotherapeutic molecule. The peptide of paragraph 54, wherein the therapeutic molecule is a fibrosis treatment molecule. The peptide of paragraph 56, wherein the fibrosis treatment molecule is selected from the group consisting of:

a steroid; a corticosteroid; an anti-inflammatory agent; and an immunosuppressant.

An isolated nucleic acid encoding any of the peptides of paragraphs 1-57.

An expression vector comprising the isolated nucleic acid of paragraph 58.

A composition consisting essentially of one or more peptides, nucleic acids, or vectors of any of paragraphs 1-59 as an active ingredient.

The pharmaceutical composition of paragraph 60, further comprising a second pharmaceutically active agent.

A pharmaceutical composition or medicament of paragraphs 61-61 further comprising a pharmaceutically acceptable carrier. 63. A method of promoting the production or maintenance of the extracellular matrix in a subject in need thereof, the method comprising administering a peptide, nucleic acid, or composition of any of paragraphs 1-62.

64. The method of paragraph 63, wherein the subject is in need of treatment for fibrosis.

65. The method of any of paragraphs 63-64, wherein the peptide is administered in combination with a fibrosis-treating agent.

66. The method of any of paragraphs 63-65, wherein the subject suffers from a condition selected from the group consisting of:

pulmonary fibrosis; scarring; scarring of the skin; trauma; a wound; corneal defects; corneal ulceration; diabetic ulcer; ulcer; sepsis; arthritis; idiopathic pulmonary fibrosis; cystic fibrosis; cirrhosis; endomyocardial fibrosis; mediastinal fibrosis; myelofibrosis; retroperitoneal fibrosis; progressive massive fibrosis; nephrogenic systemic fibrosis; Crohn's disease; keloid;

scleroderma; systemic sclerosis; arthroiibrosis; adhesive capsulitis; lung fibrosis; liver fibrosis; kidney fibrosis; heart fibrosis; vascular fibrosis; skin fibrosis; eye fibrosis; bone marrow fibrosis; asthama; sarcoidosis; COPD; emphysema; nschistomasomiasis; cholangitis; diabetic nephropathy; lupus nephritis; postangioplasty aterial restenosis; atherosclerosis; burn scarring; hypertrophic scarring; nephrogenic fibrosing dermatopathy; postcataract surgery; proliferative vitreoretinopathy; Peyronie's disease; Duputren's contracture; dermatomyositis; and graft versus host disease.

67. The method of paragraph 63, wherein the subject is a subject in need of treatment for a

proliferative disease.

68. The method of any of paragraph 63 or paragraph 67, wherein the peptide is administered in combination with a chemotherapeutic agent.

69. The method of any of paragraph 63 or paragraphs 67-68, wherein the proliferative disease is selected from the group consisting of:

cancer; a tumor; rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre -neoplastic lesions (e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia , carcinoma, oral hairy leukoplakia, and psoriasis.

70. A method of inhibiting fibrosis in a subject in need thereof, the method comprising

administering a peptide, nucleic acid, or composition of any of Paragraphs 1-50.

71. The method of Paragraph 58, wherein the peptide is administered in combination with a

fibrosis-treating agent.

72. The method of any of Paragraphs 58-59, wherein the fibrosis is a result of a condition selected from the group consisting of:

pulmonary fibrosis; scarring; scarring of the skin; trauma; a wound; corneal defects; corneal ulceration; diabetic ulcer; ulcer; sepsis; arthritis; idiopathic pulmonary fibrosis; cystic fibrosis; cirrhosis; endomyocardial fibrosis; mediastinal fibrosis; myelofibrosis; retroperitoneal fibrosis; progressive massive fibrosis; nephrogenic systemic fibrosis; Crohn's disease; keloid;

scleroderma; systemic sclerosis; arthrofibrosis; adhesive capsulitis; lung fibrosis; liver fibrosis; kidney fibrosis; heart fibrosis; vascular fibrosis; skin fibrosis; eye fibrosis; bone marrow fibrosis; asthama; sarcoidosis; COPD; emphysema; nschistomasomiasis; cholangitis; diabetic nephropathy; lupus nephritis; postangioplasty aterial restenosis; atherosclerosis; burn scarring; hypertrophic scarring; nephrogenic fibrosing dermatopathy; postcataract surgery; proliferative vitreoretinopathy; Peyronie's disease; Duputren's contracture; dermatomyositis; and graft versus host disease.

73. A method of inhibiting cellular growth in a subject in need thereof, the method comprising administering a peptide, nucleic acid, or composition of any of paragraphs 1-62.

74. The method of paragraph 73, wherein the peptide is administered in combination with a

chemotherapeutic agent.

75. The method of any of paragraphs 73-74, wherein the subject is a subject in need of treatment for a proliferative disease.

76. The method of any of paragraphs 73-75, wherein the proliferative disease is selected from the group consisting of:

cancer; a tumor; rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibrornas, vascular occlusion, restenosis, atherosclerosis, pre -neoplastic lesions (e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia), carcinoma, oral hairy leukoplakia, and psoriasis.

77. A method of inhibiting angiogenesis in a subject in need thereof, the method comprising

administering a peptide, nucleic acid, or composition of any of paragraphs 1-62.

78. The method of paragraph 77, wherein the peptide is administered in combination with a

chemotherapeutic agent.

79. The method of any of paragraphs 77-78, wherein the subject is a subject in need of treatment for a proliferative disease.

80. The method of any of paragraphs 77-79, wherein the angiogenesis is associated with a condition selected from the group consisting of:

cancer; a tumor; rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibrornas, vascular occlusion, restenosis, atherosclerosis, pre -neoplastic lesions (e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia), carcinoma, oral hairy leukoplakia, psoriasis, obesity, macular degeneration, and blindness. 81. A method of increasing the strength of bone, tendon, ligaments, cartilage, or connective tissue wherein the method comprises administering a peptide, nucleic acid, or composition of any of paragraphs 1-62.

82. A method of promoting wound heling wherein the method comprises administering a peptide, nucleic acid, or composition of any of paragraphs 1-62.

83. A method of targeting therapeutic agents or imaging molecules to sites of extracellular matrix production or accumulation wherein the method comprises administering to the subject a peptide, nucleic acid, or composition of paragraphs 1-62, wherein the peptide is coupled to a therapeutic agent or imaging molecule.

84. The method of paragraph 83, wherein the site of extracellular matrix production or

accumulation is a tumor.

85. The method of paragraph 83, wherein the site of extracellular matrix production or

accumulation is a fibrotic lesion.

86. The method of paragraph 83, wherein the site of extracellular matrix production or

accumulation is a wound site.

87. A method of inducing the polymerization of a polypeptide comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet, wherein the method comprises contacting at least two polypeptide molecules with a peptide, nucleic acid, or composition of paragraphs 1-62.

88. The method of paragraph 87, wherein the polypeptide comprising a comprising a

fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet is selected from the group consisting of:

fibronectin and fibrinogen.

89. Use of a composition of any of paragraphs 60-62, for preventing, treating and/or ameliorating fibrosis.

90. Use of a composition of any of paragraphs 60-62, for preventing, treating and/or ameliorating a proliferative disease.

91. Use of a composition of any of paragraphs 60-62, for promoting wound healing.

92. Use of a composition of any of paragraphs 60-62, for increasing the strength of bone, tendon, ligaments, cartilage, or connective tissue.

EXAMPLES

EXAMPLE 1

[00183] The extracellular matrix (ECM) provides chemical and mechanical cues that support cell survival, adhesion, and growth. Fibronectin matrix assembly is an important cellular process during embryogenesis and wound healing where a fibronectin ECM is deposited by cells. The fibronectin ECM is a provisional scaffold that is required for the assembly of other matrix components found in the more mature ECM, like collagen [1]. The ability to induce fibronectin polymerization with a short peptide can greatly enhance the rate at which scaffolds integrate into the target site as well as augment their mechanical properties through the highly stretchable character of fibronectin [2,3,4]. One can envision a simple application of this peptide as stimulating local fibronectin matrix assembly, one of the initial steps of wound healing, at the site of injury when delivered by diffusion, for example, from a peptide-soaked dressing. Such a peptide will not only accelerate wound healing by accelerating the deposition of the fibronectin matrix but also incorporate any material conjugated to the peptide into the endogenous matrix for matrix-targeting applications.

[00184] Described herein is a 23 amino acid long peptide, CP1

(VSDVPRDLEVVAATPTSLLISWD) (SEQ ID NO: 2), which induces fibronectin aggregation. CP1 was designed from an unfolding kinetic intermediate of the cell-binding module of fibronectin predicted by unfolding simulations [5] (Figure 1A). Control peptides were derived from the beta sheet designations within CP1 to include CPA (YRDLEVVAAT) (SEQ ID NO: 8) and CPB (SLLISWD) (SEQ ID NO: 5), both of which are subsequences of CP1 (Figure IB).

[00185] The ability of CP1 to induce fibronectin aggregation was examined by SDS-PAGE analysis and an aggregation assay by turbidity (Figures 2A-2C). The aggregation was studied by incubating fibronectin with various concentrations of CP1 and monitoring an increase in absorbance at 450 nm (Figure 2C). It was demonstrated with a scrambled peptide that the induced FN multimerization is sequence specific for CP1.

[00186] The peptides described herein induce fibronection polymerization and assembly both in vitro and in cell culture. Furthermore, the peptides described herein demonstrate anti-tumorigenic properties.

[00187] A subsequence of CP1, CPB, was demonstrated to multimerize fibronectin and fibrinogen where CPB multimerized fibronectin at a lower activity level than CP1 for a given concentration (Figures 3A-3D). The ability of the peptides described herein to induce aggregation of high molecular weight FN aggregates was determined by SDS-PAGE analysis. Figures 3A-3D demostrate that the C-terminal fragment of CP1 retains multimerization activity. Figure 3A depicts the results of the quantitative analysis by densitometry for multimers analyzed by non-reducing SDS-PAGE. Comparison of the peptides at 750 μΜ show that CPB retains the multimerization activity of CP1 at high concentrations. Error bars represent one standard deviation of the mean Rhod-FN intensity as determined by densitometric analysis of reaction mixtures analyzed by non-reducing SDS-PAGE (n=2 independent gels not shown). In Figure 3B, 750 μΜ CP1 or varying concentrations of CPB (50-750 μΜ) are incubated with FN and analyzed by non-reducing

SDS-PAGE. Figure 3C compares the multimer density as a function of CPB or CP1 concentration illustrating the enhanced activity of CP1. Figure 3D shows the aggregation of 5 mg/ml fibrinogen, a protein containing beta sheet structure, in the presence of increasing concentrations of CPB (0-500 μΜ) as monitored by turbidity for 2 h at 37 °C. The turbidity illustrates the action of CPB on other proteins containing domains with beta sheet content.

[00188] Figure 4A denotes the circular dichroism spectrum of 10FNIII showing significant beta strand content. Figure 4B represents the circular dichroism spectrum of CP1 with a typical random coil signature. Figure 4C shows that 10FNIII exposes hydrophobic sites as shown by the increase in ANS fluorescence over background. Addition of CP1 in the presence of 10FNIII further increases the exposure of hydrophobic sites as shown by the additional increase in ANS fluorescence. Without meaning to be limiting, this offers a mechanism by which CP1 drives fibronectin aggregation.

[00189] The assembly of extracellular matrix by lung fibroblasts in the presence of the peptides described herein was examined. Enhanced adhesion of FN to lung fibroblast cells in the presence of CPB was demonstrated. Lung fibroblast cells are cultured in the presense of increasing concentrations of CPB (0-200 μΜ) with biotinylated FN (10 ng/μΐ) for 36 h. Cells are washed and lysed in deoxycholate (DOC) buffer. DOC soluble fractions are separated by reducing SDS-PAGE and analyzed by Western blot (actin serves as the loading control). DOC soluble fractions show that increasing concentrations of CPB enhanced binding of biotinylated FN to the cell surface.

[00190] Figures 6A-6D demonstrate that the C-terminus of CP1 reduces the viability and cell growth of mammary adenocarcinoma cell lines. Weakly tumorigenic (M28) or tumorigenic (M6) cells are plated at low confluency in a 96-well plate and allowed to spread overnight. Anastellin (30 μΜ), CPA (150 μΜ), CPB (150 μΜ), or CPA (150 μΜ) + CPB (150 μΜ) (suspended in 25 mM HEPES, pH 7.1) are added in media the next day and refreshed on Day 3. On Day 6, resazurin, an indicator of metabolic capacity, was added. Percent viability (normalized against the control sample) was calculated by the turnover of resazurin during the 4h incubation for both M28 (Figure 6A) and M6 (Figure 6B) cells. Error bars represent standard error of the mean (n > 3 samples). Figure 6C shows the results of an EdU-based growth assay analyzed by FACS (10000 events). M6 cells are plated at low density in a 24-well plate and allowed to spread overnight before inoculation with 0, 50, or 200 μΜ of peptide and refreshed every other day. On Day 4, the media was replaced with fresh peptide solution containing 10 μΜ Click-iT EdU for a 24 h incubation before labeling with Pacific Blue azide. Cell growth decreases for carcinoma cells treated with CPB, but not for CPA nor CPE (data not shown). Figure 6D quantifies the decrease in cell growth of carcinoma cells after a three day incubation with increasing CPB concentrations. Error bars represent standard error of the mean across two independent experiments.

[00191] The data presented herein demonstrate that 1) CP1 induces fibronectin (FN) multimerization in a sequence specific and concentration dependent manner; 2) a 7-mer derived from CP1 also induces FN multimerization; 3) the peptides described herein have anti-tumorigenic properties; and 4) a 7-mer derived from CP1 is capable of inducing multimerization of other beta strand containing proteins, such as fibrinogen

[00192] The effects of the peptides described herein on cells in culture can be tested for effects on fibronectin matrix assembly and incorporation of the peptides into the matrix, cell growth (toxicity), survival, adhesion, and migration in vitro. Animal models can be used to look for the effect of the peptides described herein in vivo on tumor growth and survival. Subsequences of CPl can also be tested as inhibitors of fibronectin assembly.

[00193] The ability to design an ECM-based therapeutic is attractive given that the ECM controls cell survival and growth. In addition, fibronectin is a natural reservoir for growth factors and cell signaling molecules. The integration of CPl and other peptides described herein into the matrix during fibronectin polymerization can make them superb candidates to target drug therapies to the matrix. For example, liposomes encapsulating drugs that modulate cell growth can be decorated on the surface with CPl peptides that will target the drugs to the fibronectin matrix.

[00194] The integration of CPl and other peptides described herein may span beyond fibronectin to interact with other proteins with domain(s) with a fibronectin type III fold. The fibronectin type III domain is found in -2% of all animal proteins [7]. If so, CPl will enable the ability to control (by induction or inhibition) the polymerization of more than one type of matrix to enhance scaffold integration into even more physiological cell-derived matrices. This may enable more physiologically relevant responses that improve cellular integration and downstream outside-in cellular signaling induced by the scaffold.

[00195] The relatively short sequence of the peptides described herein can convey three major benefits. More specifically, the peptides described herein are cost-effective, amenable to large scale production, and candidates for further modification. To date, the shortest fibronectin-derived peptides that can induce fibronectin aggregation is 75 amino acids long. The peptides described herein can be less than one-third the length of anastellin. The shorter length makes peptide synthesis an acceptable method of production, whereas anastellin is expressed in E. coli, and this process requires lengthy steps of cloning, bacterial cell culture, and protein purification [6] . This ease of synthesis is not only cost-effective, but also easily scalable to industrial volumes. The peptides described herein can have anastellin-like effects (such as anti-angiogenic and anti-tumorigenic properties) and are potential small molecule replacements for anastellin. The short sequences of the peptides described herein can also make them more amenable to crosslinking technologies, which are not as easily implemented in proteins of longer sequence given potential sites of cross-reaction. Chemically bio-orthogonal moieties can be easily incorporated within the peptides described herein during peptide synthesis to present multiple orthogonal chemical handles for downstream crosslinking steps.

[00196] References

1. Sottile J, Hocking DC (2002) Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions. Molecular biology of the cell 13: 3546-3559.

2. Gildner CD, Lerner AL, Hocking DC (2004) Fibronectin matrix polymerization increases tensile strength of model tissue. American journal of physiology Heart and circulatory physiology 287: H46-53. 3. Hocking DC, Sottile J, Langenbach KJ (2000) Stimulation of integrin-mediated cell contractility by fibronectin polymerization. The Journal of biological chemistry 275: 10673-10682.

4. Ohashi T, Kiehart DP, Erickson HP (1999) Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 96: 2153-2158.

5. Gee EPS, Ingber DE, Stultz CM (2008) Fibronectin Unfolding Revisited: Modeling Cell

Traction-Mediated Unfolding of the Tenth Type-Ill Repeat. PLoS ONE 3: e2373.

6. Morla A, Zhang Z, Ruoslahti E (1994) Superfibronectin is a functionally distinct form of fibronectin. Nature 367: 193-196.

7. Bork P, Doolittle RF (1992) Proposed acquisition of an animal protein domain by bacteria.

Proceedings of the National Academy of Sciences of the United States of America 89: 8990-8994.

8. Ohashi T, Erickson HP (2005) Domain unfolding plays a role in superfibronectin formation. The Journal of biological chemistry 280: 39143-39151.

9. Briknarova K, Akerman ME, Hoyt DW, Ruoslahti E, Ely KR (2003) Anastellin, an FN3 fragment with fibronectin polymerization activity, resembles amyloid fibril precursors. Journal of molecular biology 332: 205-215.

10. Ambesi A, Klein RM, Pumiglia KM, McKeown-Longo PJ (2005) Anastellin, a fragment of the first type III repeat of fibronectin, inhibits extracellular signal-regulated kinase and causes G(l) arrest in human microvessel endothelial cells. Cancer research 65: 148-156.

11. Yi M, Ruoslahti E (2001) A fibronectin fragment inhibits tumor growth, angiogenesis, and metastasis. Proceedings of the National Academy of Sciences of the United States of America 98: 620-624.

12. Neskey DM, Ambesi A, Pumiglia KM, McKeown-Longo PJ (2008) Endostatin and anastellin inhibit distinct aspects of the angiogenic process. Journal of experimental & clinical cancer research : CR 27: 61.

13. Akerman ME, Pilch J, Peters D, Ruoslahti E (2005) Angiostatic peptides use plasma fibronectin to home to angiogenic vasculature. Proceedings of the National Academy of Sciences of the United States of America 102: 2040-2045.

14. Mayo KH, Ilyina E, Park H (1996) A recipe for designing water-soluble, beta-sheet-forming peptides. Protein science : a publication of the Protein Society 5: 1301-1315.

15. Rexeisen EL, Fan W, Pangburn TO, Taribagil RR, Bates FS, et al. (2010) Self-assembly of fibronectin mimetic peptide-amphiphile nanofibers. Langmuir : the ACS journal of surfaces and colloids 26: 1953-1959.

16. Colombi M, Zoppi N, De Petro G, Marchina E, Gardella R, et al. (2003) Matrix assembly induction and cell migration and invasion inhibition by a 13-amino acid fibronectin peptide. The Journal of biological chemistry 278: 14346-14355. 17. Zhong C, Chrzanowska-Wodnicka M, Brown J, Shaub A, Belkin AM, et al. (1998) Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly. The Journal of cell biology 141 : 539-551.

[00197] EXAMPLE 2: SLLISWD (SEQ ID NO: 5) multimerization sequence in the fibronectin lOFNIII domain identified by steered molecular dynamics initiates fibrillogenesis

[00198] Fibronectin (FN) assembly into insoluble extracellular matrix fibrils is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by

cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (lOFNIII) domains. These forces unfold lOFNIII to expose cryptic FN assembly sites; however, a specific sequence had not been previously identified in lOFNIII.

[00199] It is demonstrated herein that a twenty-three residue cryptic peptide 1 (CPl) induces a concentration-dependent increase in FN multimerization, which is mediated by interactions with lOFNIII that expose hydrophobic surfaces capable of supporting 8-anilino-l-napthalenesulfonic acid (ANS) binding. Localization of multimerization activity to the C-terminus of CPl led to the discovery of a minimal seven amino acid 'multimerization sequence' (SLLISWD) (SEQ ID NO: 5), which induces polymerization of FN and the clot-forming protein fibrinogen. A point mutation at Trp6 annihilates its ability to multimerize FN and simultaneously reduces the exposure of hydrophobic sites for ANS binding and β-structure formation detected by Thioflavin T in mixtures with lOFNIII. Described herein is a model for cell-mediated fibrillogenesis whereby cell traction force applied to RGD initiates a cascade of intermolecular exchange starting with the unfolding of lOFNIII to expose the multimerization sequence, which interacts with strand B of another lOFNIII domain via a Trp-mediated β-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.

[00200] The extracellular matrix (ECM) regulates cell and tissue development by presenting solid-phase adhesion sites for growth factors and cells within its constituent macromolecules (1). While some cell binding and recognition sites are present on solvent-exposed surfaces, other 'cryptic sites' are buried within the native structure only to be activated upon solvent exposure resulting from structural changes in the molecule. For example, proteolytic cleavage of ECM proteins fibronectin (FN), collagen, perlecan, fibulin, and thrombospondin unmasks cryptic sites with important pro-angiogenic and anti-angiogenic functions (2-6). Additionally, FN contains mechanically regulated cryptic ECM assembly sites exposed by cell traction force that stretch the molecule (7). The assembly of FN into insoluble fibrils within the ECM is essential to many processes including embryogenesis (8), wound healing (9), atherosclerosis (10), angiogenesis (11), and cancer (12). However, the steps by which cell traction induces FN fibril formation remain unknown.

[00201] FN, a large dimeric glycoprotein structured by repeating units of domains FNI, FNII, and FNIII, supports the adhesion of many cell types via binding to cell-surface transmembrane integrin receptors (13). FN assembly is primarily mediated by the α5β1 integrin (14), which binds the RGD sequence in the tenth FN type III (10FNIII) domain (15). FN fibrillogenesis also requires cell-mediated traction forces generated by a contractile actin cytoskeleton that transmits force onto their FN adhesions (16). This mechanical force is a key regulator of FN fibrillogenesis that exposes buried cryptic assembly sites within FN. Physical stretching of immobilized FN by 30-35% exposes a cryptic FN binding site for the 70 kD fragment, which contains the N-terminal assembly domain (1-5FNI) required for fibrillogenesis (7, 17). Cell traction force stretches FN leading to conformational changes in the molecule that include domain unfolding of FNIII repeats (18-20), which are not stabilized by internal disulfide bonds unlike the FNI and FNII repeats. Interestingly, cryptic FN assembly sites have been identified primarily within the FNIII domains (21), including the RGD-displaying 10FNIII domain.

[00202] While the RGD cell-binding sequence and neighboring synergy site are required for FN assembly by the α5β1 integrin (14, 22), how 10FNIII transduces force applied at its RGD loop to initiate fiber assembly remains unclear. Both single molecule atomic force microscopy experiments and steered molecular dynamics (SMD) simulations suggest that 10FNIII is one of the mechanically weakest FNIII domains (19, 23). Studies with thermally unfolded 10FNIII have identified cryptic assembly sites in the domain that support FN multimerization in vitro in a manner that does not require its C-terminal residues, and furthermore this unfolded 10FNIII domain inhibits FN incorporation into

fibroblast-deposited ECM (24). Additionally, point mutations P5A and P25A in the N-terminus of 10FNIII partially destabilize the module to an intermediate structure prone to self-aggregation (25). Therefore, it is feasible that under physiological conditions, cell-generated mechanical forces applied at the RGD loop of FN could unfold 10FNIII to expose cryptic assembly sites that initiate fibrillogenesis.

[00203] 10FNIII unfolding was previously approached from a physiological perspective by using SMD simulations to look at its unfolding due to pulling at its RGD loop when anchored at the N-terminus and found that the domain unfolds along a single pathway (26). The unfolding response due to pulling at the RGD loop differed from standard models of force application directed through the termini that resulted in multiple unfolding pathways for 10FNIII (26-28). These simulations predicted 10FNIII to unfold to a partially unfolded kinetic intermediate with solvent exposed N-terminal A and B β-strands in response to pulling at its physiological integrin-binding site. As described herein, it was tested whether this predicted exposed region contributes cryptic assembly sites and the minimal peptide sequence within the 10FNIII domain that is sufficient to induce FN self-assembly was identified.

[00204] EXPERIMENTAL PROCEDURES

[00205] Peptide synthesis and purification - All peptides were synthesized by the Tufts

University Core Facility (Boston, MA). Peptides were acetylated at the N-terminus and amidated at the C-terminus. A control peptide with the same sequence composition as cryptic peptide 1 (CP1) was generated as a scrambled sequence designated as CPlscr (DSALRSPVWIVTDSAEVPVLTLD) (SEQ ID NO: 29). Peptide sequences CPA, CPE, and CPB(W6A) contain an additional N-terminal Tyr not found in the 10FNII sequence to enable spectrophotometric concentration determination of the peptides in solution (Table 1). Peptide concentrations were determined from absorbance by using published calculated molar extinction coefficients (29): CP1, scrCPl, CPB, ε ₂ so = 5630 M-lcm-1 ; and CPA, CPB(W6A), CPE: ε ₂₇₈ = 1295 M ^{' 1} . Peptides were purified and analyzed by RP-HPLC on C18 columns (Agilent Technologies). Peptide molecular weight was confirmed by MALDI-TOF MS intact mass determination with a 4800 MALDI TOF/TOF mass spectrometer (Applied Biosystems).

[00206] Cloning and purification of recombinant 10FNIII - The recombinant 10FNIII domain was constructed with a C-terminal six-histidine affinity tag (SEQ ID NO: 69) by modifying the pETCH-GST-8-11FNIII-His6 vector (30) ("His6" disclosed as SEQ ID NO: 69). The 10FNIII domain in FN encoding amino acids 1416-1509 (Val-Ser-Asp-Val-...Asn-Tyr-Arg-Thr) (residues 1-4 and 91-94 of SEQ ID NO: 38) was amplified by PCR from the pETCH-GST-8-11FNIII-His6 ("His6" disclosed as SEQ ID NO: 69) construct using two primers: 5'-

CTTTAAGAAGGAGATATACATATGGTTTCTGATGTTCCGAGGGACCTG-3' (SEQ ID NO: 30) and 5 ' -GCTTAATGATGATGGTGGTGGTGTGTTCGGTAATT AATGGAAATTGGCTTGC-3 ' (SEQ ID NO: 31). The protein insert was confirmed by DNA sequencing. Protein expression was induced in the BL21 (DE3) strain of E. coli with 1 mM isopropyl β-D-l-thiogalactopyranoside at 37 °C for 6 h. Protein affinity purification was performed using HisPur Cobalt Resin (Thermo Scientific). Proteins were eluted in 50 mM sodium phosphate, 300 mM sodium chloride, and 150 mM imidazole (pH 7.4). The purified proteins were desalted on a Sephadex G-25 pre-packed PD-10 desalting column (GE Healthcare) equilibrated with Dulbecco's PBS (Life Technologies). Proteins were eluted with PBS and concentrated with an Amicon 3 kD filter (Millipore). The purity of protein was analyzed by SDS-PAGE and visualized with SYPRO™ Ruby protein gel stain (Lonza) photographed on a FluorChem M imager with a SYPRO Ruby filter (ProteinSimple). 10FNIII protein concentration was determined from absorbance at 280 nm using published calculated molar extinction coefficients (29) (ε ₂ so = 14,440 M ^{' 1} ).

[00207] Multimerization assay - Rhodamine labeled bovine plasma FN lyophilized powder

(Cytoskeleton) was solubilized in ultrapure water (Milli-Q) at room temperature for 15 min prior to spectrophotometric concentration determination (ε 565 = 70,000 M ^' ^rn ^"1 ). Peptide stock solutions were prepared in 50 mM Tris'HCl (pH 7.4) and various concentrations of CP1 (0-500 μΜ) or CPB (0-750 μΜ) were incubated with rhodamine labeled FN (0.3 mg/ml) for 16 h at 37 °C. Following incubation, samples were analyzed by non-reducing SDS-PAGE analysis. Samples were prepared in NuPAGE lithium dodecyl sulfate non-reducing sample buffer and resolved on 3-8% Tris-Acetate SDS-PAGE gels (Life Technologies). Fluorescent gel images were scanned on a Typhoon FLA 9000 with a RITC filter (GE Healthcare), and densitometric analysis was performed using ImageJ (National Institutes of Health). Band intensities for each molecular weight species (reduced arms, disulfide -bonded dimer, and high molecular weight multimers) were assigned using reduced and non-reduced FN control samples. The percentage of multimers was calculated from the ratio of multimer intensity to the sum of intensities for the reduced arms, dimer, and multimers. Final sample preparations of rhodamine labeled FN contain reduced arms (54 ± 2%) as the lyophilized stock contains β-mercaptoethanol (BME) leading to ~ 2.5 mM BME in prepared reaction samples.

[00208] Turbidity assay - The multimerization of human plasma FN (BD Biosciences) or plasminogen, von Willebrand Factor, and FN depleted human fibrinogen (FBG) (Enzyme Research Laboratories) by peptide was determined by measuring optical density at 590 nm using an Agilent Cary 300 BioMelt UV-Vis spectrophotometer (Agilent Technologies) in 10 mm pathlength quartz cuvettes (Starna Cells). FN and FBG protein concentrations were determined by absorbance at 280 nm (FN, ε ₂ so = 563,200 M-lcm-1 ; FBG, ε ₂₈ o = 498,300 M ^{' 1} ). Peptides CP1 or CPB were prepared in 50 mM Tris'HCl (pH 7.4). After equilibrating peptides to 25 °C for 2 min, turbidity of each peptide sample was monitored for 15 min after which FN or FBG was added to each cuvette at a final concentration of 0.2 mg/ml or 5 mg/ml, respectively. The change in optical density was continuously recorded for 2 h following the addition of FN or FBG. Absorbances are reported relative to the absorbance measured at the time of FN or FBG addition.

[00209] CD spectroscopy - Peptide stock solutions were prepared in 10 mM sodium phosphate buffer (pH 7.4). All CD measurements were collected using a 1 mm pathlength quartz cuvette (Starna Cells) at 25 °C from 260 nm to 190 nm on a JASCO J-720 spectropolarimeter equipped with a JASCO PT-423S Peltier temperature controller. Data were collected in continuous scanning mode, and the spectra were averaged over 4 consecutive accumulations (scan speed 20 nm/min; data pitch 0.5 nm; response time 4 s; bandwidth 1.0 nm; sensitivity 100 mdeg). A baseline measurement for buffer alone was subtracted from each spectrum. Data were expressed as mean residue ellipticity ([Θ], deg cm ² dmol ^"1 ) as calculated using the relation

₌ 9 _&bs MEW ₍ where Bobs is the measured signal in millidegrees, MRW is the mean residue weight (i.e. the molecular weight divided by the number of residues), c is the concentration of the molecule in mg/ml, and 1 is the optical pathlength of the sample cell in cm.

[00210] 8-anilino-l -napthalenesulfonic acid (ANS) binding fluorescence assay - Stock solutions of 100 μΜ ANS (Sigma- Aldrich) were prepared in PBS or 100 mM sodium phosphate buffer (pH 6.8) and filtered prior to spectrophotometric concentration determination (ε ₃ 5 ₀ = 4,950 M ^' ^rn ^"1 ) (31).

Anastellin lyophilized powder (Sigma- Aldrich) was solubilized in PBS at room temperature prior to spectrophotometric concentration determination (ε ₂ 8ο = 16,960 M ^"1 cm ^"1 ) (29). Samples were prepared in PBS with 50 μΜ ANS in the presence or absence of 50 μΜ anastellin or 50 μΜ lOFNIII with or without peptide. Emission spectra were collected at 25 °C at 5 nm intervals (I s integration time) over a range from 380 to 650 nm with an excitation wavelength of 360 nm on a PTI QuantaMaster™ 40 spectrofluorometer with excitation and emission slit widths of 5 nm. A baseline measurement for buffer alone was subtracted from each spectrum. The maximum fluorescence of each emission spectrum was averaged across independent experiments. Relative fluorescence was calculated for each sample by subtracting the ANS fluorescence in the presence of buffer and normalizing to the fluorescence measured for ANS bound to anastellin.

[00211] Thioflavin T (ThT) binding fluorescence assay - A 1 mM stock solution of ThT

(Sigma-Aldrich) was prepared in 50 mM Tris-HCl (pH 7.4) and filtered prior to dilution into ethanol for spectrophotometric concentration determination (ε ₄₁₆ = 26,620 M ^"1 cm ^"1 ) (32). Samples were prepared in 50 mM Tris-HCl (pH 7.4) with 20 μΜ ThT in the presence or absence of 10 μΜ lOFNIII or FBG and/or 500 μΜ CPB or CPB(W6A). Fluorescence emission at 482 nm was collected at 25 °C for 60 min (1 s integration time) with an excitation wavelength of 450 nm on a PTI QuantaMaster 40

spectrofluorometer with excitation and emission slit widths of 5 nm.

[00212] Statistical analysis - Values are expressed as means + S.E. calculated across independent samples; p values were derived from Student's t test.

[00213] RESULTS

[00214] Unfolded N-terminal sequence of predicted lOFNIII intermediate initiates FN multimerization - SMD simulations modeling the unfolding pathway of lOFNIII under tension between its N-terminus and the physiological integrin-binding RGD loop predicted an unfolded intermediate with an unraveled N-terminus through the second 13-strand (26) (Fig. 7). Because this kinetic intermediate is sampled for extended simulation times while under tension, it was hypothesized by the inventors that such a partially unfolded structure might expose cryptic assembly sites that trigger FN assembly. This hypothesis was tested as described herein by evaluating the multimerization capacity of sequences representing the exposed twenty-three amino acids at the N-terminus named CP1 and shorter regions encompassing beta-strand sequences from the native lOFNIII structure (Table 1).

[00215] Anastellin, a C-terminal fragment of 1FNIII that interacts with FN to induce the formation of disulfide-stabilized supramolecular FN known as superfibronectin (33), was used as a positive control. Non-reducing SDS-PAGE analysis shows that supramolecular FN multimers migrate as a distribution of discrete bands of molecular weight larger than the disulfide -bonded molecular FN depending on the number of integrated molecules (Fig. 8). Incubation of anastellin (150 μΜ) with rhodamine labeled FN at a concentration similar to that of plasma FN in blood (34) (220: 1 molar ratio of peptide to FN) led to 22 ± 4% of total FN (distributed as reduced FN arms, disulfide -bonded dimer, and multimers) forming high molecular weight multimers when analyzed by densitometry following separation by non-reducing SDS-PAGE (Fig. 8). Interestingly, incubation of labeled FN with the CP1 peptide at the same concentration led to a 38% increase in multimer formation over anastellin (Fig. 8). Addition of reducing agent to the CP1 -induced multimers prior to SDS-PAGE analysis collapsed the higher order bands suggesting multimer stabilization by disulfide bonds (data not shown). In contrast, the scrambled CPl sequence (represented by peptide CPlscr) did not support FN multimerization when added at the same concentration (Fig. 8). The CPl -induced multimerization effect was also observed with unlabeled FN, where the degree of aggregation was monitored in a turbidity assay (Fig. 2C). While CPl showed no multimerization in the absence of FN even at high peptide concentration, turbidity increased only when FN was added to CPl (molar ratio of CPl to FN of 22:1 and 330:1 for CPl concentrations of 10 μΜ and 150 μΜ, respectively) (Fig. 2C) demonstrating that FN multimerization kinetics depended on CPl concentration (Fig. 2C).

[00216] The multimerization effect of the CPl sequence in 10FNIII is cryptic - To test our predictions for the cryptic assembly sequence motivated by the SMD calculated intermediate, the multimerization capacity of sequence CPl was compared to that of beta-strand E, which shares a face with CPl in intact 10FNIII but is not solvent exposed in the proposed kinetic intermediate (Fig. 7) (26). Addition of the CPE peptide (50 to 400 μΜ) to rhodamine labeled FN induced minimal FN

multimerization in the range of 8 ± l to l0 ± l , respectively (Fig. 9A). While multimerization was statistically significant for CPE concentrations > 150 μΜ (p < 0.05, two-tailed), the percentage of multimers initiated by CPE is significantly less (approximately one -third) than that initiated by 150 μΜ CPl (p < 0.01, one -tailed). Moreover, it was found that the multimerization activity of CPl is cryptic within 10FNIII as addition of natively folded 10FNIII, which contains the CPl sequence in its N-terminus, at concentrations between 50-500 μΜ to rhodamine labeled FN showed background levels of multimer formation (p > 0.05, two-tailed) (Fig. 9B).

[00217] CPl interacts with 10FNIII to expose buried hydrophobic surfaces - Since the CPl sequence corresponds to 24% of the 10FNIII sequence at the N-terminus, it was investigated whether CPl -initiated multimerization of FN involved interactions with 10FNIII. The presence of 10FNIII in mixtures of CPl (both at large molar excess to rhodamine labeled FN) reduced the percentage of observed multimers detected by SDS-PAGE (Fig. 10A). For a fixed concentration of CPl (150 μΜ), adding 10FNIII at half the molar concentration of CPl (yielding a molar ratio of 110: 1 of 10FNIII to FN) attenuated, but did not abolish, multimer formation by nearly one-third (Fig. 10A). The control sample of 10FNIII (75 μΜ) in the absence of CPl was not significantly different from background (p > 0.05, two-tailed). Increasing the molar ratio of 10FNIII to CPl to 1:1 did not lead to further reduction in multimer formation (p > 0.05, two-tailed). These results suggest that CPl interacts with 10FNIII.

[00218] The interactions of CPl with 10FNIII were further investigated. Because 10FNIII is stabilized by a highly conserved buried hydrophobic core, peptide-induced disruptions of the FNIII fold may yield solvent exposed hydrophobic surfaces. ANS is a fluorescent dye that probes exposed hydrophobic surfaces whereby formation of noncovalent interactions between ANS and hydrophobic cavities leads to enhanced and blue-shifted fluorescence (35). Since anastellin presents hydrophobic binding pockets for ANS (36), mixtures of ANS with anastellin confirm an increase in fluorescence (Fig. 10B). Addition of ANS to 10FNIII produced an enhanced and blue-shifted fluorescence above background as well (Fig. 10B) suggesting that ANS is binding to hydrophobic pockets within 10FNIII. CD spectroscopy studies confirmed that 10FNIII is natively folded (Fig. IOC), as indicated by the presence of negative signal at 190 nm and positive signals at 203 nm and 225 nm characteristic of beta-sheets and turns (37). The observed ANS results for natively folded 10FNIII are consistent with published data describing conformational flexibility in 10FNIII in solution (38). It was found herein that ANS does not interact with CPl (Fig. 10B) despite the CPl sequence being unstructured, as shown by CD spectroscopy (Fig. 10D). However, ANS showed a strong interaction when CPl (150 μΜ) was added to 10FNIII at a molar ratio of 3: 1 (Fig. 10B), as demonstrated by the enhanced blue-shifted fluorescence peak relative to the spectrum observed for either 10FNIII or CPl (Fig. 10B). Moreover, weaker enhancement in ANS fluorescence by CPlscr in the presence of 10FNIII was observed (Fig. 10E). These collective data show that CPl interacts with 10FNIII in a manner that induces the formation of solvent exposed hydrophobic cavities that are accessible for ANS binding.

[00219] A 7 amino acid sequence in CPl is sufficient to initiate FN multimerization - It was explored whether 13 -strands A and B contribute to the multimerization activity of CPl by testing short peptides encompassing these sequences, CPA and CPB, respectively, against CPl at high concentration (500 μΜ). SDS-PAGE analysis revealed that multimers were induced by high concentrations of CPl as well as CPB, but not by CPA (Fig. 11 A). Co-administration of CPA and CPB did not significantly enhance the degree of multimerization due to CPB alone (p = 0.05, two-tailed) (Fig. 11 A). The degree of multimerization by CPl is concentration-dependent in the range of 10-500 μΜ with maximal activity at 150 μΜ (30 ± 1%, Fig. 1 IB). CPB, which is 30% the length of CPl, required a higher concentration (500 μΜ) to achieve similar multimerization as 150 μΜ CPl (Fig. 11B). These results clearly show that the seven amino acid sequence of CPB - SLLISWD - (SEQ ID NO: 5) found at the C-terminus of CPl is sufficient to initiate FN multimerization.

[00220] SLLISWD (SEQ ID NO: 5) multimerization sequence interacts with 10FNIII to expose hydrophobic pockets and form beta-structure - It was evaluated whether CPB interacts with 10FNIII to induce exposure of hydrophobic surfaces in a similar manner to CPl. Addition of CPB at ten-fold molar excess to 10FNIII (500 and 50 μΜ, respectively) enhanced ANS fluorescence above that exhibited by CPB and significantly above that for 10FNIII alone (p < 0.05, one-tailed) (Figs. 11C, 11D). The CD spectrum of CPB also confirmed its random-coil like behavior in solution (Fig. 1 IE). These results demonstrate that the multimerization sequence is also capable of interacting with 10FNIII to form solvent exposed hydrophobic surfaces that support ANS binding.

[00221] The formation of superfibronectin and 9FNIII multimers has been proposed to follow a polymerization mechanism of intermolecular beta-strand exchange where unfolded intermediates present exposed beta-sheet elements for multimerization (37, 39). Such a mechanism may be relevant to CPB-induced FN multimerization as the SLLISWD sequence (SEQ ID NO: 5) has potential for beta-sheet interactions with the parent domain given that its sequence is derived from the second beta-strand of 10FNIII. ThT is a benzothiazole dye that undergoes a spectral shift leading to enhanced fluorescence when bound to beta-sheet rich structures and has been used to detect self-assembling beta-rich fibrils formed by amyloidogenic precursors, immunoglobulin light chain, and 9FNIII (32, 37, 40). ThT fluorescence is similar to background in the presence of 10FNIII (p > 0.05, two-tailed) (Fig. 5F) but significant in the presence of CPB (p < 0.05, two-tailed). However, addition of CPB to 10FNIII (50: 1 molar concentration of peptide to 10FNIII) led to an 87% increase in fluorescence over that measured for CPB alone (p < 0.05, one-tailed) or 10FNIII alone (p < 0.01, one-tailed) (Fig. 11F). Furthermore, the ThT fluorescence profile showed a time-dependent increase following 10FNIII addition to CPB suggesting that CPB interactions with 10FNIII resulted in the formation of beta-contacts.

[00222] A key Trp residue is required for multimerization sequence activity - Residue Trp22 in the second N-terminal beta-strand of 10FNIII is a conserved residue found in all FNIII domains that is critical to the stabilization of their hydrophobic cores (23, 41, 42). This residue corresponds to the largest hydrophobic side-chain within the multimerization sequence at position 6. An alanine point mutation at Trp6 in the multimerization sequence (yielding peptide CPB(W6A)) abolished the sequence's ability to multimerize FN as detected by SDS-PAGE (p > 0.05, two-tailed) (Fig. 11 A), thus emphasizing the key role that Trp6 plays in FN multimerization. Additionally the W6A mutation reduced the ability of the sequence in mixtures with 10FNIII to support ANS binding (Fig. 11D) and eliminated ThT-dependent fluorescence (Fig. 11F).

[00223] CPB also initiates FBG multimerization - Assembly of the plasma protein FBG, which is the main ECM protein involved in blood clot formation, has been shown to require the assembly of FN (43). Anastellin not only polymerizes FN, but also FBG (2). Similar to anastellin, addition of CPB to FBG (5 mg/ml) led to an increase in spectrophotometric absorbance over time indicative of FBG polymerization in a peptide concentration dependent manner (Fig. 12A). CPB -induced FBG multimerization was observed using molar concentrations of peptide to FBG at 33:1 and 50: 1 (CPB at 500 μΜ and 750 μΜ, respectively). In addition, a time-dependent increase in ThT fluorescence is observed in mixtures of CPB and FBG (50: 1 molar concentrations of peptide to FBG) (Fig. 12B), but not for comixtures of CPB(W6A) and FBG (Fig. 12C) or isolated samples of CPB, CPB(W6A), or FBG alone (Figs. 12B, 12C) indicating that the CPB-induced FBG assemblies form beta-containing structures.

[00224] DISCUSSION

[00225] Described herein is the identification of a seven amino acid 'multimerization sequence'

- SLLISWD - (SEQ ID NO: 5) within the cell-binding domain of FN that is sufficient to initiate polymerization of added FN or FBG. A twenty-three amino acid peptide mimic designed from this unfolded terminus is shown to be a cryptic sequence in 10FNIII that initiates FN multimerization as its scrambled sequence, the unexposed beta-strand E, and natively folded lOFNIII are unable to initiate similar multimerization results. It is demonstrated herein that the multimerization process can involve interactions of this twenty-three amino acid sequence with lOFNIII, which leads to solvent exposure of hydrophobic surfaces. Further analysis of the CPl sequence led to the discovery of a seven amino acid multimerization sequence with a key Trp6 residue localized to its C-terminus that is sufficient to initiate FN multimerization. This is the first study that identified a specific sequence in lOFNIII that induces FN multimerization.

[00226] The peptides presented in this study possess similar multimerization capabilities as anastellin, a 75 amino acid fragment of 1FNIII (2, 33). Comparison at a peptide to FN molar ratio of 220: 1, CPl remarkably induces a higher percentage of FN multimers than anastellin as detected by SDS-PAGE despite being less than one third the length of anastellin. The shorter seven amino acid multimerization sequence within CPl requires a three-fold higher molar ratio of peptide to FN to induce a similar degree of multimerization as the twenty-three amino acid CPl sequence (733: 1 versus 220: 1, respectively). Without wishing to be bound by theory, these observations indicate a significantly stronger FN binding affinity for CPl, which can provide additional favorable contacts to position the multimerization sequence to interact with FN. Like anastellin, the multimerization sequence also initiates FBG assembly. It should be noted, however, that the peptides described in this study do differ from anastellin in a few significant ways. First, comparison of the SLLISWD (SEQ ID NO: 5) and anastellin sequences shows a ten-fold difference in length. Such short peptides are easily synthesized and readily amenable to chemical modification enabling the incorporation of bioorthogonal handles. This flexibility is beneficial to the development of multifunctional 'smart' probes with ECM-forming activity relevant for applications addressing diseases of ECM remodeling, such as abnormal wound healing, fibrosis, or cancer, for example. Second, the multimerization sequence is derived from the mechanically weak lOFNIII domain that is recognized by integrins at the RGD loop. As lOFNIII provides the direct point of contact on FN for cell surface integrin receptors, this sequence may play a potential role in the mechanical process of cell-mediated FN fibrillogenesis. Third, CPl and anastellin are complementary structural counterparts in homologous FNIII structures. Anastellin is a N- terminal deletion fragment of lOFNIII lacking the N-terminus that includes strands A and B (44), which is the region spanning CPl in lOFNIII. Despite differences in β strand coverage of the FNIII structure, both systems display hydrophobic surfaces to solvent. The multimerization sequence is unstructured and exposes hydrophobic binding surfaces upon interaction with lOFNIII whereas anastellin possesses exposed hydrophobic binding pockets for ANS in its residual structure (36, 44).

[00227] FN multimerization has been identified as a hydrophobic process where cells convert

FN into deoxycholate-insoluble fibers stabilized by noncovalent interactions (45). Stretching FN fibers induces conformational changes including exposure of hydrophobic surfaces and unraveling of secondary structure (20, 46). Long stretches of hydrophobic residues can form beta-sheet fibril assemblies as demonstrated by the use of hydrophobic sequences derived from the amyloidogenic beta-Alzheimer peptide to engineer fibril assembly into a 14 amino acid peptide (47). CPl encompasses the most hydrophobic region of lOFNIII (26) including beta-strands A and B, which possess 56% and 57% hydrophobic character, respectively. The identified multimerization sequence corresponds to one of the most hydrophobic beta-strands in lOFNIII. It is clear that sequence specificity is important to the assembly process as strand A shows similar hydrophobic character but lacks the ability to initiate FN multimerization. Moreover, a point mutation of the largest hydrophobic residue (W6A) in the multimerization sequence reduced its ability to expose hydrophobic surfaces and form beta-structure when mixed with lOFNIII and more importantly annihilated its ability to initiate FN multimerization. This Trp6 residue corresponds to the single conserved Trp residue in strand B found in all FNIII domains that is important for stabilization of the fold's hydrophobic core (23, 41, 42). In lOFNIII, randomized mutation of this conserved Trp22 residue in a yeast two-hybrid system severely destabilized complementation of a bisected lOFNIII to form a stable lOFNIII structure (48). It is possible that this Trp6 residue in the multimerization sequence may play a role as a substitute for Trp22 to stabilize an exposed hydrophobic core of a partially unfolded lOFNIII intermediate that subsequently propagates FN self-assembly.

[00228] FN assembly, in the context of superfibronectin and 9FNIII multimer formation, has been hypothesized to follow a process of intermolecular beta-strand exchange, where beta-strands from one partially unfolded domain complements a similarly unfolded intermediate of another domain to form a stable polymeric structure (37, 39, 44). FN assembly initiated by other peptide sequences in addition to anastellin, including peptides anginex, CLT1, and BBK32 has also been described to follow a beta-structure mediated process (49-51). While these inducing sequences are not native to FN, they may share a common mechanism for assembly involving hydrophobic interactions and beta-sheet formation. BBK32, derived from a surface-expressed lipoprotein, shows weak sequence homology to regions present in anastellin, specifically to the C/C loop and the F strand in 1FNIII (51). Anginex is a 33 amino acid peptide designed to mimic β-sheets (49), and CLT1 is a 10-mer identified by phage display that possesses a hydrophobic N-terminus and hydrophilic C-terminus proposed to form β-sheets similar to amyloidogenic fibrils (50). Without wishing to be bound by theory, since the multimerization sequence possesses similar properties to anastellin and these other peptides capable of initiating FN assembly, β-strand exchange may be a potential mechanism for FN assembly initiated by the multimerization sequence.

[00229] A recent study with mutant FN containing a single cysteine point mutation within the E strand in lOFNIII at position Ala 1472 showed that integration into growing fibrils by cell-mediated stretching led to minimal labeling by a fluorescein -conjugated maleimide in solvent (20). These experimental observations do not exclude the model described herein given that this A1472C mutation in the E strand does not probe unfolding within the proposed region of interest at the N-terminus. The results reported here agree that the sequence of strand E does not efficiently initiate FN assembly. [00230] The model proposed herein for cell-mediated FN fibrillogenesis stipulates that the

N-terminus of lOFNIII unfolds. However, it is known that cell adhesion to RGD on FN is enhanced by a synergy site in the N-terminal neighboring domain 9FNIII for integrins alpha5betal and alphallbbeta3, but not alphaVbeta3 (55-57). In particular, FN assembly by alpha5betal, but not by alphaVbeta3, requires the synergy site (58). Since alpha5betal is the main receptor that mediates FN assembly (14) and increasing the spacing between the PHSRN (SEQ ID NO: 70) and RGD sites significantly reduces cell adhesion (59, 60), one would suspect that N-terminal unfolding of lOFNIII would prohibit enhanced adhesion and subsequent fibrillogenesis. However, it has been shown that activation of the alpha5betal integrin overcomes the requirement of the synergy site for improved adhesion to the RGD sequence and FN assembly (57, 58). Therefore, the proposed model of unfolding of the N-terminus in lOFNIII, and thus separating the synergy site and the RGD loop, is feasible to describe cell-mediated FN

fibrillogenesis once the alpha5betal integrin is bound to FN and activated.

[00231] Described herein is the identification of a minimal multimerization sequence from the lOFNIII domain in FN that initiates assembly of FN and FBG, proteins that are important in promoting tissue repair. While there have been other identified peptide sequences capable of inducing FN assembly (49-51), this is the first sequence that is identified in lOFNIII, the mechanically weak domain that contains the initial integrin binding site that enables transmission of tensional force onto FN. The identification of a cryptic multimerization sequence in lOFNIII provides new insight into the initial steps in the physiological driven process of cell-mediated FN fibrillogenesis as well as a new motif for initiating FN assembly to stimulate ECM formation.

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55. Bowditch, R. D., Hariharan, M., Tominna, E. F., Smith, J. W., Yamada, K. M., Getzoff, E. D., and Ginsberg, M. H. (1994) Identification of a novel integrin binding site in fibronectin. Differential utilization by beta 3 integrins. J. Biol. Chem. 269, 10856-10863

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Pro-His-Ser-Arg-Asn (SEQ ID NO: 70) in human fibronectin enhances cell-adhesive function. J. Biol. Chem. 269, 24756-24761

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61. Gao, M., Craig, D., Lequin, O., Campbell, I. D., Vogel, V., and Schulten, K. (2003) Structure and functional significance of mechanically unfolded fibronectin type III1 intermediates. Proc. Natl. Acad. Sci. U. S. A. 100, 14784-14789

[00233] The abbreviations used are: ECM, extracellular matrix; FN, fibronectin; 10FNIII, tenth

FN type III; SMD, steered molecular dynamics; CP1, cryptic peptide 1; CPlscr, scrambled cryptic peptide 1; BME, β-mercaptoethanol; FBG, fibrinogen; ANS, 8-anilino-l-napthalenesulfonic acid; ThT, Thioflavin T

[00234] Table 1

Summary of characteristics for derived peptide mimics including sequence, corresponding 10FNIII -strand(s), and residue indices in FN. CP1 VSDVPRDLEVVAATPTSLLISWD 2 A, B 1416-1438

CPA YRDLEVVAAT 8 A 1421-1429

CPB SLLISWD 5 B 1432-1438

CPE YTATIS 10 E 1471-1475

EXAMPLE 3

[00235] CPB was demonstrated to bind to FN by comparing the fluorescence of rhodamine labeled FN, unlabeled FN, and unlabeled FN incubated with FITC labeled CPB (500 μΜ) separated by non-reducing SDS-PAGE analysis (Figure 13). Addition of FITC labeled CPB to unlabeled FN enables visualization of the protein.

[00236] Ansastellin and CPB, but not mutant CPB (W6A), induced the increased accumulation of FN into the deoxycholate insoluble pool by lung fibroblast cells (Figure 14).

[00237] Accumulation of peptide in mammary tumors was demonstrated in vivo following IV injection of FN multimers initiated by CPB peptide. Figure 15 demonstrates the accumulation of the CPB peptide in 4T1 mammary tumors implanted orthotopically in the mammary fat pad of mice following intravenous injection of pre-formed multimers generated by the pre -incubation of FITC labeled CPB with unlabeled FN. Average fluorescence signal represented as percent injected dose per gram ( ID/g) of homogenized tumor tissue (n=2) shows significant fluorescence above untreated samples (*, p < 0.05, two-tailed).

[00238] SEQ ID NO: 1

Vali-Ser ₂ -Asp ₃ -Val ₄ -X5-Arg ₆ -X ₇ -Leu ₈ -X ₉ -Valio-Xii-Xi ₂ -Alai ₃ - Thri ₄ -Xi ₅ - Thr^-Sern- Leui ₈ -

Xi9-Ile ₂ o-Ser2i-X22-Asp23

[00239] SEQ ID NO: 2

Vali-Ser ₂ -Asp ₃ - Val ₄ -Pro ₅ -Arg ₆ -Asp ₇ -Leu ₈ -Glu ₉ -Valio -Valii-Alai ₂ -Alai ₃ -Thri ₄ -Proi ₅

-Thri6-Seri7-Leui8-Leui ₉ - Ile ₂ o-Ser2i-Trp22-Asp23

[00240] SEQ ID NO: 3

Cysi-Val ₂ -Ser ₃ - Asp ₄ -Val ₅ -Pro ₆ -Arg ₇ -Asp ₈ - Leu ₉ -Gluio-Valii-Vali ₂ -Alai ₃ -Alai ₄ -Thri ₅ - Proi ₆ -Thri ₇ -

Seris- Leui ₉ -Leu ₂ o-Ile2i-Ser ₂ 2-Trp23-Asp2 ₄

[00241] SEQ ID NO: 4

Seri-Leu ₂ -Leu ₃ -Ile ₄ -Ser ₅ -X ₆ -Asp ₇

[00242] SEQ ID NO: 5

Seri-Leu ₂ -Leu ₃ -Ile ₄ -Ser ₅ -Trp ₆ -Asp ₇ [00243] SEQ ID NO: 6

Argi-X ₂ -X3-X4-Vals-X ₆ -X7-Alag-Thr ₉

[00244] SEQ ID NO: 7

Argi-Asp ₂ - Leu ₃ -Glu ₄ -Val ₅ -Val ₆ -Ala ₇ -Ala ₈ -Thr ₉

[00245] SEQ ID NO: 8

Tyr ₁ -Arg ₂ -Asp ₃ -Leu ₄ - Glu ₅ -Val ₆ - Val ₇ -Ala ₈ -Ala ₉ -Thr ₁₀

[00246] SEQ ID NO: 9

Thr _! -Ala ₂ -Thr ₃ -Ile ₄ -Ser ₅

[00247] SEQ ID NO: 10

Tyri-Thr ₂ -Ala ₃ -Thr ₄ -Ile ₅ -Ser ₆

[00248] SEQ ID NO: 11

Tyr ₁ -Tyr ₂ -Arg ₃ -Ile ₄ -Thr ₅ -Tyr ₆ -Gly ₇ -Glu ₈

[00249] SEQ ID NO: 12

[00250] SEQ ID NO: 13

Cysi-Ser ₂ -X ₃ -X ₄ -X ₅ -Ser ₆ -X7-X ₈

[00251] SEQ ID NO: 14

Cys ₁ -Ser ₂ -Leu ₃ -Leu ₄ -Ile ₅ -Ser ₆ -Trp ₇ -Asp ₈

[00252] SEQ ID NO: 15

X _l -X ₂ ~^3~-^4~-^-5~-^-6~-^7~-^-8~Ttir9

[00253] SEQ ID NO: 16

Tyri-X ₂ -Arg ₃ -X ₄ -Thr ₅ -X ₆ -X ₇ -Glu ₈

[00254] SEQ ID NO: 17

Gln ₁ -Glu ₂ -X ₃ -Thr ₄ -X5-Pro ₆

[00255] SEQ ID NO: 18

Glni-Glu ₂ -Phe ₃ -Thr ₄ -Val ₅ -Pro ₆

[00256] SEQ ID NO: 19

Tyr ₁ -Gln ₂ -Glu ₃ -Phe ₄ -Thr ₅ -Val ₆ -Pro ₇

[00257] SEQ ID NO: 20

X ₁ -Thr ₂ -X ₃ -Thr ₄ -X ₅ -Tyr ₆ -X7-Val ₈

[00258] SEQ ID NO: 21

Tyri-Thr ₂ -Ile ₃ -Thr ₄ -Val ₅ -Tyr ₆ -Ala ₇ -Val ₈

[00259] SEQ ID NO: 22

[00260] SEQ ID NO: 23

He _! -Ser ₂ -Ile ₃ - Asn ₄ -Tyr ₅ [00261] SEQ ID NO: 24

Val ₁ -Ser ₂ -Asp3-Val4-Xs-X ₆ -X7-X8-X9-Xio-Xii-Xi2-Xi3- Thr ₁₄ -X ₁₅ - Xi ₆ -Ser ₁₇ - X ₁₈ -

Xl9 ^" X20-Ser ₂ l-X22-X23

[00262] SEQ ID NO: 25

Cys ₁ -Val ₂ -Ser ₃ -Asp4-Val5-X ₆ -X ₇ -X ₈ -X ₉ -X ₁ o-Xii-Xi2-Xi3-Xi4- Thr ₁₅ -X ₁₆ - Xi ₇ -Ser ₁₈ - X ₁₉ -

X20 ^" X21"Ser ₂ 2-X23 ^" X24

[00263] SEQ ID NO: 26

Tyri-X ₂ -X3-X4-X5-X6-X7-X8-X9"Thrio

[00264] SEQ ID NO: 27

Tyr ₁ -Gln ₂ -Glu ₃ -X ₄ -Thr ₅ -X ₆ -Pro ₇

[00265] SEQ ID NO: 82 CPB(W6A)

Tyrl-Ser ₂ -Leu ₃ -Leu ₄ -Ile ₅ -Ser ₆ -Ala ₇ -Asps ₈

[00266] SEQ ID NO: 32 Human Fibronectin Isoform 1 NCBI Ref: NP_997647

1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvavsqskp gcydngkhyq 61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi

121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck

181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy

241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp

301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc

361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc

421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri

481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm

541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq

601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip

661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp

721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl

781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg

841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg

901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt

961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp

1021 raqitgyrlt vgltrrgqpr qynvgpsvsk yplrnlqpas eytvslvaik gnqespkatg

1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv

1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt

1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis

1261 dtiipevpql tdlsfvditd ssiglrwtpl nsstiigyri tvvaagegip ifedfvdssv

1321 gyytvtglep gidydisvit linggesapt tltqqtavpp ptdlrftnig pdtmrvtwap 1381 ppsidltnfl vryspvknee dvaelsisps dnavvltnll pgteyvvsvs svyeqhestp

1441 lrgrqktgld sptgidfsdi tansftvhwi apratitgyr irhhpehfsg rpredrvphs

1501 rnsitltnlt pgteyvvsiv alngreespl ligqqstvsd vprdlevvaa tptslliswd

1561 apavtvryyr itygetggns pvqeftvpgs kstatisglk pgvdytitvy avtgrgdspa

1621 sskpisinyr teidkpsqmq vtdvqdnsis vkwlpssspv tgyrvtttpk ngpgptktkt

1681 agpdqtemti eglqptveyv vsvyaqnpsg esqplvqtav tnidrpkgla ftdvdvdsik

1741 iawespqgqv sryrvtyssp edgihelfpa pdgeedtael qglrpgseyt vsvvalhddm

1801 esqpligtqs taipaptdlk ftqvtptsls aqwtppnvql tgyrvrvtpk ektgpmkein

1861 lapdsssvvv sglmvatkye vsvyalkdtl tsrpaqgvvt tlenvspprr arvtdatett

1921 itiswrtkte titgfqvdav pangqtpiqr tikpdvrsyt itglqpgtdy kiylytlndn

1981 arsspvvida staidapsnl rflattpnsl lvswqpprar itgyiikyek pgspprevvp

2041 rprpgvteat itglepgtey tiyvialknn qksepligrk ktdelpqlvt lphpnlhgpe

2101 ildvpstvqk tpfvthpgyd tgngiqlpgt sgqqpsvgqq mifeehgfrr ttppttatpi

2161 rhrprpyppn vgeeiqighi predvdyhly phgpglnpna stgqealsqt tiswapfqdt

2221 seyiischpv gtdeeplqfr vpgtstsatl tgltrgatyn iivealkdqq rhkvreevvt

2281 vgnsvnegln qptddscfdp ytvshyavgd ewermsesgf kllcqclgfg sghfrcdssr

2341 wchdngvnyk igekwdrqge ngqmmsctcl gngkgefkcd pheatcyddg ktyhvgeqwq 2401 keylgaicsc tcfggqrgwr cdncrrpgge pspegttgqs ynqysqryhq rtntnvncpi

2461 ecfmpldvqa dredsre

267] SEQ ID NO: 33 Human Fibronectin Isoform 4 NCBI Ref : NF _. 997643..1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvavsqskp gcydngkhyq

61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi

121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck

181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy

241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp

301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc

361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc

421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri

481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm 541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq

601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip

661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp

721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl

781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg

841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg

901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt

961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp 1021 raqitgyrlt vgltrrgqpr qynvgpsvsk yplrnlqpas eytvslvaik gnqespkatg

1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv

1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt

1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis

1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd

1321 navvltnllp gteyvvsvss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia

1381 pratitgyri rhhpehfsgr predrvphsr nsitltnltp gteyvvsiva lngreespll

1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk

1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvqdnsisv

1561 kwlpssspvt gyrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge

1621 sqplvqtavt nidrpkglaf tdvdvdsiki awespqgqvs ryrvtysspe dgihelfpap

1681 dgeedtaelq glrpgseytv svvalhddme sqpligtqst aipaptdlkf tqvtptslsa

1741 qwtppnvqlt gyrvrvtpke ktgpmkeinl apdsssvvvs glmvatkyev svyalkdtlt

1801 srpaqgvvtt lenvspprra rvtdatetti tiswrtktet itgfqvdavp angqtpiqrt

1861 ikpdvrsyti tglqpgtdyk iylytlndna rsspvvidas taidapsnlr flattpnsll

1921 vswqpprari tgyiikyekp gspprevvpr prpgvteati tglepgteyt iyvialknnq

1981 ksepligrkk tvqktpfvth pgydtgngiq lpgtsgqqps vgqqmifeeh gfrrttpptt

2041 atpirhrprp yppnvgqeal sqttiswapf qdtseyiisc hpvgtdeepl qfrvpgtsts

2101 atltgltrga tyniivealk dqqrhkvree vvtvgnsvne glnqptddsc fdpytvshya

2161 vgdewermse sgfkllcqcl gfgsghfrcd ssrwchdngv nykigekwdr qgengqmmsc 2221 tclgngkgef kcdpheatcy ddgktyhvge qwqkeylgai csctcfggqr gwrcdncrrp

2281 ggepspegtt gqsynqysqr yhqrtntnvn cpiecfmpld vqadredsre

268] SEQ ID NO: 34 Human Fibronectin Isoform 5 NCBI Ref : NP_997641.1

1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvavsqskp gcydngkhyq 61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi

121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck

181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy

241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp

301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc

361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc

421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri

481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm 541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq

601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip

661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp

721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl

781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg 841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg

901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt

961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp

1021 raqitgyrlt vgltrrgqpr qynvgpsvsk yplrnlqpas eytvslvaik gnqespkatg

1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv

1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt

1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis

1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd

1321 navvltnllp gteyvvsvss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia

1381 pratitgyri rhhpehfsgr predrvphsr nsitltnltp gteyvvsiva lngreespll

1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk

1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvqdnsisv

1561 kwlpssspvt gyrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge

1621 sqplvqtavt tipaptdlkf tqvtptslsa qwtppnvqlt gyrvrvtpke ktgpmkeinl

1681 apdsssvvvs glmvatkyev svyalkdtlt srpaqgvvtt lenvspprra rvtdatetti

1741 tiswrtktet itgfqvdavp angqtpiqrt ikpdvrsyti tglqpgtdyk iylytlndna

1801 rsspvvidas taidapsnlr flattpnsll vswqpprari tgyiikyekp gspprevvpr

1861 prpgvteati tglepgteyt iyvialknnq ksepligrkk tdelpqlvtl phpnlhgpei

1921 ldvpstvqkt pfvthpgydt gngiqlpgts gqqpsvgqqm ifeehgfrrt tppttatpir

1981 hrprpyppnv geeiqighip redvdyhlyp hgpglnpnas tgqealsqtt iswapfqdts

2041 eyiischpvg tdeeplqfrv pgtstsatlt gltrgatyni ivealkdqqr hkvreevvtv

2101 gnsvneglnq ptddscfdpy tvshyavgde wermsesgfk llcqclgfgs ghfrcdssrw

2161 chdngvnyki gekwdrqgen gqmmsctclg ngkgefkcdp heatcyddgk tyhvgeqwqk 2221 eylgaicsct cfggqrgwrc dncrrpggep spegttgqsy nqysqryhqr tntnvncpie

2281 cfmpldvqad redsre

269] SEQ ID NO: 35 Human Fibronectin Isoform 6 NCBI Ref: NP

1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvavsqskp gcydngkhyq 61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi

121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck

181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy

241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp

301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc 361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc

421 hfpfiynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri 481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm 541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq 601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip 661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp 721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl

781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg

841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg

901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt

961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp

1021 raqitgyrlt vgltrrgqpr qynvgpsvsk yplrnlqpas eytvslvaik gnqespkatg

1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv

1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt

1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis

1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd

1321 navvltnllp gteyvvsvss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia

1381 pratitgyri rhhpehfsgr predrvphsr nsitltnltp gteyvvsiva lngreespll

1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk

1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvqdnsisv

1561 kwlpssspvt gyrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge

1621 sqplvqtavt tipaptdlkf tqvtptslsa qwtppnvqlt gyrvrvtpke ktgpmkeinl

1681 apdsssvvvs glmvatkyev svyalkdtlt srpaqgvvtt lenvspprra rvtdatetti

1741 tiswrtktet itgfqvdavp angqtpiqrt ikpdvrsyti tglqpgtdyk iylytlndna

1801 rsspvvidas taidapsnlr flattpnsll vswqpprari tgyiikyekp gspprevvpr

1861 prpgvteati tglepgteyt iyvialknnq ksepligrkk tgqealsqtt iswapfqdts

1921 eyiischpvg tdeeplqfrv pgtstsatlt gltrgatyni ivealkdqqr hkvreevvtv

1981 gnsvneglnq ptddscfdpy tvshyavgde wermsesgfk llcqclgfgs ghfrcdssrw

2041 chdngvnyki gekwdrqgen gqmmsctclg ngkgefkcdp heatcyddgk tyhvgeqwqk 2101 eylgaicsct cfggqrgwrc dncrrpggep spegttgqsy nqysqryhqr tntnvncpie

2161 cfmpldvqad redsre

270] SEQ ID NO: 36 Human Fibronectin Isoform 7 NCBI Ref: NP_4 ^'

1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvavsqskp gcydngkhyq 61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi

121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck

181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy

241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp

301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc

361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc

421 hfpfiynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri

481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm

541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq 601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpvsi pprnlgy

271] SEQ ID NO: 37 Human Fibronectin Isoform 3 NCBI Ref: NP_002017.1

1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvavsqskp gcydngkhyq

61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi

121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck

181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy

241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp

301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc

361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc

421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri

481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm

541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq

601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip

661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp

721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl

781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg

841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg

901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt

961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp

1021 raqitgyrlt vgltrrgqpr qynvgpsvsk yplrnlqpas eytvslvaik gnqespkatg

1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv

1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt

1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis

1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd

1321 navvltnllp gteyvvsvss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia

1381 pratitgyri rhhpehfsgr predrvphsr nsitltnltp gteyvvsiva lngreespll

1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk

1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvqdnsisv

1561 kwlpssspvt gyrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge

1621 sqplvqtavt nidrpkglaf tdvdvdsiki awespqgqvs ryrvtysspe dgihelfpap

1681 dgeedtaelq glrpgseytv svvalhddme sqpligtqst aipaptdlkf tqvtptslsa

1741 qwtppnvqlt gyrvrvtpke ktgpmkeinl apdsssvvvs glmvatkyev svyalkdtlt

1801 srpaqgvvtt lenvspprra rvtdatetti tiswrtktet itgfqvdavp angqtpiqrt

1861 ikpdvrsyti tglqpgtdyk iylytlndna rsspvvidas taidapsnlr flattpnsll

1921 vswqpprari tgyiikyekp gspprevvpr prpgvteati tglepgteyt iyvialknnq

1981 ksepligrkk tdelpqlvtl phpnlhgpei ldvpstvqkt pfvthpgydt gngiqlpgts

2041 gqqpsvgqqm ifeehgfrrt tppttatpir hrprpyppnv gqealsqtti swapfqdtse 2101 yiischpvgt deeplqfrvp gtstsatltg ltrgatynii vealkdqqrh kvreevvtvg

2161 nsvneglnqp tddscfdpyt vshyavgdew ermsesgfkl Icqclgfgsg hfrcdssrwc

2221 hdngvnykig ekwdrqgeng qmmsctclgn gkgefkcdph eatcyddgkt yhvgeqwqke

2281 ylgaicsctc fggqrgwrcd ncrrpggeps pegttgqsyn qysqryhqrt ntnvncpiec

2341 fmpldvqadr edsre

[00272] SEQ ID NO: 38 Human Tenth Repeat of Fibronectin Type III amino acid sequence

VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATIS GLKP GVDYTITVYAVTGRGDSPASSKPISINYRT