Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• 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.
• the extracellular matrix 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.
• Fibronectin can also modulate availability of growth factors, such as TGF- ⁇ (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.
• TGF- ⁇ Kerke et al. PLoS One. 2011 ; 6:e28181. Epub 2011 Nov 28.
• 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.
• the size of anastellin makes it poorly suited for administration to subjects.
• Anginex is a 33 amino acid long ⁇ -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 ⁇ -sheet containing anti-angiogenic proteins such as interleukin-8, platelet factor-4, and
• Anginex is not a fibronectin-based molecule, the downstream fibronectin aggregates are obviously non-physiological.
• CLT1 (a cyclic lOmer) and BBK32 (a 22-mer) each initiate fibronectin assembly and have been implicated in accumulation and/or homing to tumors.
• CTL1 is a cyclic decapeptide, it requires additional cyclization steps not required by the, e.g. linear peptide embodiments described herein.
• CLT1 binds fibrin-FN complexes, it is unable to bind fibrin or immobilized fibronectin (Pilch et al. (2006) PNAS 103:2800-4).
• 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.
• 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.
• 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.
• 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.
• the technology described herein relates to an isolated peptide comprising the amino acid sequence of
• 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).
• the peptide comprises the amino acid sequence of
• 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).
• 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
• 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
• 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,
• 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.
• X 3 is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Tr
• 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)
• the technology described herein relates to an isolated peptide comprising the amino acid sequence of ⁇ 1 - ⁇ 2 - ⁇ 3 - ⁇ 4 - ⁇ 5 - ⁇ 6 - ⁇ 7 - ⁇ 8 - ⁇ 1 ⁇ 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,
• 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
• 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
• 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
• 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
• 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).
• 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.
• the peptide comprises the amino acid sequence of Tyr 1 -Thr 2 -Ala 3 -Thr 4 -Ile 5 -Ser 6 (SEQ ID NO: 10).
• 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
• 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.
• the peptide comprises the amino acid sequence of
• 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.
• 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.
• 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.
• 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.
• the peptide comprises the amino acid sequence of Gln 1 -Glu 2 -Phe 3 -Thr 4 -Val 5 -Pro 6 (SEQ ID NO: 18) .
• 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) .
• 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
• 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.
• the isolated peptide comprises 75 or
• 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.
• 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.
• the peptide comprises the amino acid sequence of
• He 1 -Ser 2 -Ile 3 -Asn 4 -Tyr 5 (SEQ ID NO: 23) .
• the peptide comprises the corresponding amino acid sequence of a homolgous fibronectin gene.
• 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.
• the multiple occurrences of the peptides have a physical arrangement selected from the group consisting of: linear, branched; arrayed; multiplexed; cyclized.
• 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.
• 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.
• the peptide comprises a mutation that locks the peptide into a beta strand conformation or otherwise constrained conformation.
• the mutation comprises a mutation selected from the group consisting of double Cys mutations or click chemistry or other crosslinking methodologies.
• 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.
• the peptide comprises at least one peptide bond replacement.
• 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.
• 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), di
• 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.
• the peptide has a modification selected from the non-limiting group consisting of: PEGylation; post-translational derivatizations; glycosylation; hydroxylation;
• 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.
• the peptide comprises a conservative substitution, insertion, or deletion of one or more amino acids.
• the peptide is a fusion peptide.
• the peptide is coupled to a targeting molecule or inserted intrasequence to the targeting molecule in a single or multivalent fashion.
• 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.
• the peptide is coupled to a therapeutic molecule.
• the therapeutic molecule is a chemotherapeutic molecule.
• the therapeutic molecule is a fibrosis treatment molecule.
• the fibrosis treatment molecule is selected from the non-limiting group consisting of: a steroid; a corticosteroid; an anti-inflammatory agent; and an immunosuppressant.
• 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.
• 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.
• 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.
• 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.
• the composition further comprises a second pharmaceutically active agent.
• the composition further comprises a pharmaceutically acceptable carrier.
• 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.
• the subject is in need of treatment for fibrosis.
• the peptide is administered in combination with a fibrosis-treating agent.
• 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; emphylococcus
• the subject is in need of treatment for a proliferative disease.
• the peptide is administered in combination with a chemotherapeutic agent.
• 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.
• 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.
• the peptide is administered in combination with a fibrosis-treating agent.
• 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.
• 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.
• the peptide is administered alone or in combination with a chemotherapeutic agent, cytotoxic agent, pro-apoptotic agent or anti-angiogenic agent.
• the subject is a subject in need of treatment for a proliferative disease.
• 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.
• cancer cancer
• a tumor rheumatoid arthritis
• inflammatory bowel disease e.g., osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre -neoplastic lesions (e.g., adenomatous hyperplasia, prostatic intraepitheli
• 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.
• the peptide is administered in combination with a chemotherapeutic agent.
• the subject is a subject in need of treatment for a proliferative disease.
• 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.
• 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.
• 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.
• 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.
• the site of extracellular matrix production or accumulation is a tumor.
• the site of extracellular matrix production or accumulation is a fibrotic lesion.
• the site of extracellular matrix production or accumulation is an injury or wound site or blood clot.
• 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.
• 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.
• 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.
• 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.
• Figures 2A-2C depict the multimerization of fibronectin induced by CP1 in a concentration dependent manner.
• 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.
• 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 ⁇ ) 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.
• Figures 3A-3D demostrate that the C-terminal fragment of CP1 retains multimerization activity.
• 750 ⁇ CP1 or varying concentrations of CPB are incubated with FN and analyzed by non-reducing
• 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 ⁇ ) 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.
• 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 ⁇ , 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 ⁇ , 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.
• ANS (50 ⁇ ) and lOFNIII (50 ⁇ ) in the presence or absence of CP1 (50 ⁇ ) 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.
• Figure 5 depicts enhanced adhesion of FN to lung fibroblast cells in the presence of
• Lung fibroblast cells are cultured in the presense of increasing concentrations of CPB (0-200 ⁇ ) with biotinylated FN (10 ng/ ⁇ ) 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.
• DOC deoxycholate
• 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 ⁇ ), CPA (150 ⁇ ), CPB (150 ⁇ ), or CPA (150 ⁇ ) + CPB (150 ⁇ ) 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 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 ⁇ of peptide and refreshed every other day. On Day 4, the media was replaced with fresh peptide solution containing 10 ⁇ Click-iT EdU for a 24 h incubation before labeling with Pacific Blue azide.
• Figure 7 depicts SMD simulations modeling force applied between the RGD loop and the N-terminus predict unfolding of 10FNIII to an intermediate.
• FCell cell traction imparts force
• the domain is predicted to unfold to a kinetic intermediate (right) with an unraveled N-terminus exposing ⁇ -strands A and B (labeled CPl) but not strand E (26).
• 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 ⁇ 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).
• 'Multimer' multimeric FN species
• FIG. 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).
• Figures 9A-9B demonstrate the effect of CPE and 10FNIII on fibronectin
• FIG. 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 ⁇ ). Multimers were induced significantly above background by high concentrations of CPE (150 & 250 ⁇ , *, p ⁇ 0.05, two-tailed; 400 ⁇ : **, p ⁇ 0.01, two-tailed).
• Figure 9B depicts a graph of densitometry of rhodamine fluorescence revealing that recombinant 10FNIII (50-500 ⁇ ) does not induce significant fibronectin multimerization above background (p > 0.05, two-tailed).
• 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 ⁇ CPl was reduced in the presence of 75 ⁇ 10FNIII (**, p ⁇ 0.01, one-tailed).
• the multimerization by CPl (150 ⁇ ) in the presence of 10FNIII (75 ⁇ ) 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 ⁇ ) emission intensities in the presence of buffer, CPl (150 ⁇ ), lOFNIII (50 ⁇ ), anastellin (50 ⁇ ), and a comixture of lOFNIII (50 ⁇ ) and CPl (150 ⁇ ).
• Figure IOC depicts a graph of the CD spectrum of 10 ⁇ lOFNIII.
• Figure 10D depicts a graph of CD spectrum of 30 ⁇ 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 ⁇ ) for samples containing lOFNIII (50 ⁇ ) with or without CPl or CPlscr (150 ⁇ ).
• ANS fluorescence due to anastellin and coniixtures of lOFNIII with CPl or CPlscr are significantly higher than ANS background (*, p ⁇ 0.05, one -tailed).
• Figures 11 A-l IF demonstrate the effect of CPB on fibronectin and lOFNIII.
• Figure 11B depicts a graph of densitometry analysis of CPl or CPB (150-750 ⁇ ) mixtures with rhodamine labeled fibronectin separated by SDS-PAGE showing significant fibronectin multimerization (p ⁇ 0.01, two-tailed).
• CPl induced more multimers at 150 ⁇ than at 500 ⁇ (**, p ⁇ 0.01, one-tailed) and was significantly different from CPB at 150 ⁇ (**, p ⁇ 0.01, two-tailed) but similar to 500-750 ⁇ CPB (p > 0.05, two-tailed).
• Figure 11C depicts a graph of a comparison of ANS (50 ⁇ ) emission intensities in the presence of buffer, CPB (500 ⁇ ), lOFNIII (50 ⁇ ), anastellin (50 ⁇ ), and a comixture of lOFNIII (50 ⁇ ) and CPB (500 ⁇ ).
• 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 ⁇ ) for samples containing lOFNIII (50 ⁇ ) with or without 500 ⁇ CPB or CPB(W6A).
• Figure 1 IF depicts a graph comparison of ThT (20 ⁇ ) fluorescence at 482 nm after a 60 min incubation at 25 °C for lOFNIII (10 ⁇ ) in the presence or absence of 500 ⁇ CPB or CPB(W6A).
• lOFNIII 10 ⁇
• 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).
• Figures 12A-12C demonstrates the effect of CPB on FBG polymerization.
• Figurel2B depicts a graph of time traces of ThT -dependent (20 ⁇ ) fluorescence at 482 nm (25 °C) for buffer, CPB (500 ⁇ ), FBG (10 ⁇ ), and a comixture of FBG (10 ⁇ ) and CPB (500 ⁇ ).
• Figure 12C depicts a graph of ThT-dependent fluorescence for buffer, CPB(W6A) (500 ⁇ ), FBG (10 ⁇ ), and a comixture of FBG (10 ⁇ ) and CPB(W6A) (500 ⁇ ) over time.
• 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 ⁇ ) 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• compositions, methods, and respective component(s) thereof are 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.
• 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.
• compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
• 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.
• “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.
• 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.
• the terms “treat,” “treatment,” and the like refer to a decrease in severity, indicators, symptoms, and/or markers of fibrotic conditions or proliferative diseases as described 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.
• 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%.
• 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.
• a "subject" as used herein can be a human or an animal.
• 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.
• the subject is a mammal, e.g., a primate, e.g., a human.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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
• RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.
• 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).
• heterologous nucleic acid fragments refers to nucleic acid sequences that are not naturally occurring in that cell.
• vector refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells.
• a vector can be viral or non-viral.
• 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.
• expression vector refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector.
• 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.
• 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.
• replication incompetent when used in reference to a viral vector means the viral vector cannot further replicate and package its genomes.
• 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.
• transduction refers to the use of viral particles or viruses to introduce exogenous nucleic acids into a cell.
• transfection 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.
• exogenous nucleic acids such as the nucleic acid sequences encoding an an isolated peptide as described herein
• 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
• 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.
• proteins 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.
• proteins proteins
• peptide and “polypeptides” are used interchangeably herein when referring to a gene product and fragments thereof.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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
• a fibronectin type III domain can comprise the amino acid sequence of SEQ ID NO: 38.
• 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;
• MERTK MIDI ; MID2; MPL; MYBPC1 ; MYBPC2; MYBPC3; MYBPH; MYBPHL; MYLK;
• 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).
• proteins with a beta sandwich fold e.g. nectin and the immunoglobin superfamily (cll 1960 in the NCBI CDD)
• proteins containing an immunoglobin-like fold e.g. tenascin, titin, etc.
• 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.
• the isolated peptides comprise fragments of human fibronectin which have been identified to have the assembly, multimerization, aggregation and/or polymerization activity.
• 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.
• an isolated peptide as described herein is not fibronectin.
• an isolated peptide as described herein comprises 75 or fewer amino acids.
• 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.
• the isolated peptides comprise fragments of human fibronectin which have been identified to have the assembly, multimerization, aggregation and/or polymerization activity.
• 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.
• an isolated peptide as described herein is not fibronectin.
• an isolated peptide as described herein comprises 75 or fewer amino acids.
• 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,
• 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).
• 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,
• 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
• 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
• the peptide comprises the amino acid sequence of
• 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 - ⁇ 7 - ⁇ 8 - ⁇ 9 - ⁇ 0 - ⁇ - ⁇ 2- ⁇ 3- 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
• 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).
• 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.
• 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.
• 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,
• 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,
• 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.
• 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.
• 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
• 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
• X 2 is selected from the group consisting of Asp, Lys, and Asn
• X 3 is selected from the group consisting of Leu, Trp, Val, He, and Phe
• X 4 is selected from the group consisting of Glu and Gin
• X 6 is selected from the group consisting of Val, Trp, He, Leu, and Phe
• X 7 is selected from the group consisting of Ala and Gly.
• 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).
• 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
• 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).
• 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).
• 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 is not fibronectin
• 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.
• an isolated peptide as described herein can comprise the amino acid sequence of
• 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.
• 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.
• 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.
• an isolated peptide as described herein can comprise the amino acid sequence of:
• 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.
• an isolated peptide as described herein can comprise the amino acid sequence of:
• 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
• 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.
• an isolated peptide as described herein can comprise the amino acid sequence of:
• 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.
• 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.
• 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).
• an isolated peptide as described herein can be 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.
• 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.
• 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.
• 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.
• phosphonates Novel lipophilic alphaacyloxyalkyl ester derivatives of phosphate- or phosphonate containing drugs masking the negative charges of these groups
• an isolated peptide as described herein can be a
• 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.
• 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.
• an isolated peptide as described herein can be in a non-crystalline, i.e. amorphous solid form.
• 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.
• 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.
• 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
• 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).
• reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
• 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.
• an isolated peptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.
• 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.
• 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.
• 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.
• these conserved amino acids and structures are not altered when generating conservatively substituted sequences.
• amino acids found at equivalent positions in other fibronectin polypeptides are substituted.
• 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).
• 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.
• 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).
• 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.
• 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.
• conservative substitutions encompass residue exchanges with those of similar physicochemical properties (i.e. substitution of a hydrophobic residue for another hydrophobic amino acid).
• 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.
• 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.
• the loop comprised by amino acid 15 or 16 of SEQ ID NOs: 1-2 (and the equivalents thereof) can be mutated for altered
• a variant of an isolated peptide as described herein can comprise a mutation affecting the beta turn.
• 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.
• 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.
• a variant of an isolated peptide as described herein can comprise a mutation wherein hydrophobic substitutions are made in the conserved core residue.
• 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.
• 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
• X 5 is selected from the group consisting of Pro, Ala, Ser, and Val
• X 8 is selected from the group consisting of Leu, He, Met, Ala, Pro, Glu, Phe, and Ser
• X 10 is selected from the group consisting of Val, Phe, Leu, Tyr, and Thr
• X 13 is selected from the group consisting of Ala, Thr, Val, He, Pro, Glu, and Ser
• X 18 is selected from the group consisting of Leujle, Phe, Met, Val, His, and Thr
• X 20 is selected from the group consisting of He, Val, and Ala
• X 22 is selected from the group consisting of Trp, Val, He, Leu, Phe, and Tyr.
• 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
• 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
• 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.
• 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,
• 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.
• 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.
• 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.
• 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.
• 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.
• a variant of an isolated peptide as described herein can comprise a mutation modulating the stability of the 10FNIII precursor.
• 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).
• 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.
• 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.
• 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.
• the equivalent mutations in the peptide of SEQ ID NO: 3 can have the same effects.
• 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: 1141-115 ⁇ ) 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.
• 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.
• 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.
• 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).
• 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).
• 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.
• 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
• 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).
• 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).
• 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).
• 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.
• 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).
• a variant of an isolated peptide as described herein can comprise a mutation which preserves the beta bulge.
• 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
• 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.
• 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.
• 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).
• the loop region of the peptides of SEQ ID NOs: 1-3 can be elongated while maintaining the activity of the isolated peptides.
• 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).
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• 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.
• a polypeptide can comprise tandem occurrences of one or more peptides as described herein, e.g.
• 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.
• 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.
• 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.
• 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 disuccudimidyl esters such as 3,3'-dithiobis
• 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.
• 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).
• a lysine residue in a first peptide may be conjugated to a glutamic or aspartic acid residue in a second peptide, respectively, using
• an isolated peptide as described herein can comprise a mutation that locks the peptide into a beta strand conformation or otherwise constrained conformation.
• the mutation is a double Cys mutation.
• 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.
• 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 .
• 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.
• 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).
• 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 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 para-benzoylphenylalanine, para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine), diaminobutyric acid,
• 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.
• an isolated peptide can be modified, e.g. a moiety can be added to one or more of the amino acids comprising the peptide.
• 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.
• 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.
• modifications and/or moieties include PEGylation; post-traslational derivitzations; glycosylation; hydroxylation; methylation; HESylation; ELPylation; lipidation;
• an end-capping modification can comprise acetylation at the N-terminus, N-terminal acylation, and N-terminal formylation.
• an end-capping modification can comprise amidation at the C-terminus, introduction of C-terminal alcohol, aldehyde, ester, and thioester moieties.
• 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".
• an isolated peptide as described herein is coupled to a targeting molecule.
• 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.
• a "targeting molecule" can be any molecule, e.g.
• 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.
• 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.
• 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.
• 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.
• 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.
• the site of extracellular matrix production or accumulation is a tumor.
• the site of extracellular matrix production or accumulation is a fibrotic lesion.
• the site of extracellular matrix production or accumulation is a wound site.
• 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.
• 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.
• the therapeutic molecule can be a chemotherapeutic molecule.
• "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, vinorel
• 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.,
• 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
• biological response modifiers e.g., interferon-; interleukin 2, etc.
• 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.
• CDDP cis-diaminedichioroplatinum II
• Carboplatin Anthracendione
• Mitoxantrone e
• Tamoxifen, etc. Tamoxifen, etc.); androgens (e.g.. testosterone propionate, Fluoxymesterone, etc.); antiandrogens (e.g., Flutaniide); gonadotropiareleasing hormone analogs (e.g., Leuproli.de); and Gleevec.
• androgens e.g.. testosterone propionate, Fluoxymesterone, etc.
• antiandrogens e.g., Flutaniide
• gonadotropiareleasing hormone analogs e.g., Leuproli.de
• Gleevec Gleevec.
• An isolated peptide as described herein can comprise, be coupled to, and/or connected to a detectable tag molecule.
• 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.
• a tag molecule can be detected or imaged in vivo after it is administered to a subject.
• 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.
• 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.
• 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).
• 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.
• Non-limiting examples of fluorescent tag molecules include fluorescein, phycoerytllrin.
• 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)
• N.N,N ⁇ '-tetramethyL-6carboxyrhodaniine TAMRA or T
• 6-carboxy-X-rhodaniine ROX or R
• cyanine dyes e.g. Cy3, Cy5 and Cy7 dyes
• coumarins e.g urnbelS iferone
• benzimide dyes e.g.
• phenanthridine dyes e.g. Texas Red
• ethidium dyes e.g. acridine dyes
• carbazole dyes e.g. phenoxazine dyes
• porphyrin dyes polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5 t etc
• BODIPY dyes quinoline dyes
• commercially available dyes such as the Aie Fiuors.
• 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.
• 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.
• the activity of the isolated peptides described herein can be measured or deteremined by assays to determine their anti-angiogenic or anti-tumorigenic activity.
• 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
• tumorigenic cells e.g. M28 or M6 cells derived from a mammary adenocarcinoma
• the isolated peptide is added to at least one well (e.g. at a concentration of 150 ⁇ ).
• additional doses of the isolated peptide can be added to additional samples over the duration of the experiment.
• resazurin an indicator of metabolic capacity
• Percent viability can be calculated by the turnover of resazurin during the 4h incubation.
• 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).
• 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.
• 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.
• 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.
• polypeptides comprising a fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet, e.g. fibronectin
• 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.
• 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.
• 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.
• 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.
• 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.
• fibrotic conditions include, but are not limited to pulmonary fibrosis; scarring; scarring of the skin; trauma; a wound; chronic wounds (e.g.
• 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.
• 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.
• 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.
• 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.
• the subject is in need of treatment for fibrosis.
• the peptide is administered in combination with a fibrosis-treating agent.
• 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.
• corneal defects corneal ulceration; corneal wounds; diabetic ulcer; ulcer; sepsis; arthritis; idiopathic pulmonary fibrosis; cystic fibrosis; cirrhosis; endomyocardial fibrosis; mediastinal fibrosis; myelofibrosis; retroperitoneal fibrosis;
• 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.
• 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.
• 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.
• 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.
• angiogenesis can be associated with a proliferative disease, e.g. cancer, obesity, macular degeneration, and blindness.
• a proliferative disease e.g. cancer, obesity, macular degeneration, and blindness.
• 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.
• 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.
• 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.
• 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.
• these anti-tumor drugs e.g. cilengitde
• 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.
• 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.
• 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.
• connective tissue examples 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.
• 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.
• the subject in need of inhibition of angiogenesis is a subject in need of treatment for a proliferative disease.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 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.
• 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.
• the dosage ranges are typically from O.OOlmg/kg body weight to 5 g/kg body weight.
• 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.
• 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.
• the dose range is from 5 ⁇ g/kg body weight to 30 ⁇ g/kg body weight.
• the dose range will be titrated to maintain serum levels between 5 ⁇ g/mL and 30
• compositions comprising an isolated peptide as described herein are suitably administered to the patient at one time or over a series of treatments.
• a series of treatments for purposes herein, a
• terapéuticaally 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.
• the doses are given once a day, or multiple times a day, for example but not limited to three times a day.
• 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.
• compositions containing at least one agent can be conventionally administered in a unit dose.
• 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.
• 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.
• the invention described herein consists essentially of one or more of the isolated peptides described herein as an active ingredient.
• the invention described herein is directed to a composition comprising a nucleic acid encoding an isolated peptide as described herein.
• the nucleic acid comprises RNA or DNA.
• the nucleic acid comprises an mRNA.
• 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).
• 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.
• inducers have been developed that can be included in vectors to be used in some embodiments of the present invention described herein.
• Yamavera 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.)).
• 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.
• recombinant vector is meant a vector that includes a heterologous nucleic acid sequence, or "transgene” that is capable of expression in vivo.
• 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.
• 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)
• 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.
• Adeno-associated viruses 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
• avipox vectors e.g. see WO 91/12882; WO 89/03429; and WO 92/03545; which are incorporated by reference herein in their entireties).
• Vectors can be packaged and/or delivered using liposomes (e.g., see U.S. Pat. Nos.
• 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.
• the composition described herein is a pharmaceutical composition.
• the composition can be administered in a therapeutically effective amount in admixture with pharmaceutical carriers.
• pharmaceutically acceptable 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.
• compositions that contains active ingredients dissolved or dispersed therein are 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.
• compositions comprising an isolated peptide as described herein can comprise a second pharmaceutically active agent.
• a second pharmaceutically active agent can be a fibrosis treatment or a chemotherapeutic, examples of which are described above herein.
• 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).
• topical formulations contain the active ingredient in a concentration range of 0.1 to 100 mg/ml, in admixture with a pharmaceutically acceptable carrier.
• 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.
• 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.
• additional conventional pharmaceutical preparations such as tablets, granules, powders, capsules, and sprays may be preferentially required.
• further conventional additives such as binding-agents, wetting agents, propellants, preservatives, lubricants, and stabilizers may also be required.
• 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.
• ophthalmic isolated peptide compositions are used to treat fibrotic disorders of the eye, e.g.corneal ulceration.
• an isolated peptide as described herein is administered in an aqueous solution.
• an isolated peptide as described herein is administered in an aqueous buffer with a pH of approximately 7.4.
• 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.
• the pharmaceutically acceptable formulation used to administer the active compound provides sustained delivery, such as "slow release" of the active compound to a subject.
• 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.
• 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.
• 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.
• compositions can be formulated as a sustained release composition.
• sustained-release means or delivery devices 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.
• sustained-release matrix 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).
• 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.
• 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.
• an isolated peptide as described herein, or a nucleic acid encoding the peptide can be administered in a pharamaceutical composition comprising liposomes.
• 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.
• liposomal composition not generally found in nature includes phospholipids other than naturally-derived phosphatidylcholine.
• Neutral lipid vesicle compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
• DPPC dipalmitoyl phosphatidylcholine
• Anionic lipid vesicle compositions generally are formed from dimyristoyl phosphatidylglycerol.
• Another type of liposomal composition is formed from
• phosphatidylcholine such as, for example, soybean PC, and egg PC.
• PC phosphatidylcholine
• 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.
• 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.
• Non-limiting examples of liposome compositions include those described U.S. Pat.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• a sugar e.g., sucrose
• 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.
• 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,
• 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.
• 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).
• X 5 is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val;
• X 6 is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, and Asn;
• X 7 is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His;
• X 8 is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser;
• X 9 is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro;
• Xi 0 is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid;
• 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;
• 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;
• X 13 is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin;
• 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;
• X 18 is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr;
• 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;
• X 20 is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp;
• X 22 is selected from the group consisting of Trp, Val, He, Leu, Phe, Tyr, and Ala;
• X 23 is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe;
• the isolated peptide is not fibronectin
• 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).
• X 6 is selected from the group consisting of Pro, Gly, Ala, Leu, Ser, and Val;
• X 7 is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, Asn;
• X 8 is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His;
• X 9 is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser;
• Xi 0 is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro;
• Xn is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid;
• 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;
• 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;
• X 14 is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin;
• 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;
• X 19 is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr;
• 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;
• X 2i is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, and Trp;
• 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.
• X 5 is selected from the group consisting of Pro; Gly; Ala; Leu; Ser; and Val;
• X 6 is selected from the group consisting of Arg; Ser; and Thr;
• X 7 is selected from the group consisting of Asp; Lys; Asn; and His;
• X 8 is selected from the group consisting of Leu; Trp; Ala; Val; He; Phe; Met; Pro; Glu; and Ser;
• X 9 is selected from the group consisting of Glu; Gin; and Pro;
• 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;
• 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;
• X i3 is selected from the group consisting of Ala; Val; He; Leu; Phe; Tyr; Trp; Thr; Pro; Glu; and Ser;
• X i5 is selected from the group consisting of Pro; Gly; Ala; Val; He; and Leu;
• X i6 is selected from the group consisting of Thr; Gly; Ala; Ser; and Val;
• X i8 is selected from the group consisting of Leu; Ala; Val; He; Phe; Tyr; Trp; Met; His; and Thr;
• X i9 is selected from the group consisting of Leu; Trp; Val; He; Phe; Pro; Thr; Lys; Met;
• X 20 is selected from the group consisting of He; Ala; Val; Leu; Phe; Tyr; Trp;
• X 22 is selected from the group consisting of Trp; Val; He; Leu; Phe; and Tyr;
• X 23 is selected from the group consisting of Asp; Gin; Thr.
• the isolated peptide is not fibronectin
• the isolated peptide comprises 75 or fewer amino acids.
• X 6 is selected from the group consisting of Pro; Gly; Ala; Leu; Ser; and Val;
• X 7 is selected from the group consisting of Arg; Ser; and Thr;
• X 8 is selected from the group consisting of Asp; Lys; Asn; and His;
• X 9 is selected from the group consisting of Leu; Trp; Ala; Val; He; Phe; Met; Pro; Glu; and Ser;
• Xi 0 is selected from the group consisting of Glu; Gin; and Pro;
• 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;
• X 14 is selected from the group consisting of Ala; Val; He; Leu; Phe; Tyr; Trp; Thr; Pro; Glu; and Ser;
• X 16 is selected from the group consisting of Pro; Gly; Ala; Val; He; and Leu;
• X i7 is selected from the group consisting of Thr; Gly; Ala; Ser; and Val;
• X i9 is selected from the group consisting of Leu; Ala; Val; He; Phe; Tyr; Trp; Met; His; and Thr;
• 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;
• 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).
• X 2 is selected from the group consisting of Leu, Ala, Val, He, Phe, Tyr, Trp, Met, His, and Thr;
• 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;
• 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;
• the isolated peptide is not fibronectin
• the isolated peptide comprises 75 or fewer amino acids.
• 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;
• X 8 is selected from the group consisting of Asp, Glu, Gin, Thr, Asn, Val, Ser, Ala, He, Leu, Arg, and Phe.
• X 2 is selected from the group consisting of Leu; Ala; Val; He; Phe; Tyr; Trp; Met; His; and Thr;
• 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;
• 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;
• X 7 is selected from the group consisting of Asp; Gin; and Thr.
• 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; 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) .
• X 2 is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His;
• X 3 is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser;
• 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;
• 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;
• 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;
• X 8 is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin;
• the isolated peptide is not fibronectin
• the isolated peptide comprises 75 or fewer amino acids.
• X 2 is selected from the group consisting of Arg, Lys, Ser, Thr, Val, Pro, and Asn;
• X 3 is selected from the group consisting of Asp, Glu, Lys, Arg, Ser, Asn, He, Gly, Gin, Pro, and His;
• X 4 is selected from the group consisting of Leu, Trp, Ala, Val, He, Phe, Met, Pro, Glu, Asn, and Ser;
• X 5 is selected from the group consisting of Glu, Asp, His, Gin, Phe, Ser, Thr, Arg, Ala, Lys, and Pro;
• X 6 is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, Ser, Trp, Thr, and any beta-branched amino acid;
• 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;
• 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
• X 9 is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Thr, Pro, Glu, Ser, Asn, and Gin.
• Xi is selected from the group consisting of Arg; Ser; and Thr;
• X 2 is selected from the group consisting of Asp; Lys; Asn; and His;
• X 3 is selected from the group consisting of Leu; Trp; Val; Ala; He; Phe; Met; Pro; Glu; and Ser;
• X 4 is selected from the group consisting of Glu;Gln; and Pro
• X 5 is selected from the group consisting of Val; Ala; He; Leu; Phe; Tyr; Trp; and Thr
• 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;
• 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;
• X 8 is selected from the group consisting of Ala; Val; He; Leu; Phe; Tyr; Trp; Thr; Pro; Glu; and Ser;
• the isolated peptide is not fibronectin
• the isolated peptide comprises 75 or fewer amino acids.
• X 2 is selected from the group consisting of Arg; Ser; and Thr;
• X 3 is selected from the group consisting of Asp; Lys; Asn; and His;
• X 4 is selected from the group consisting of Leu; Trp; Ala; Val; He; Phe; Met; Pro; Glu; and Ser;
• X 5 is selected from the group consisting of Glu;Gln; and Pro;
• 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;
• 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
• X 9 is selected from the group consisting of Ala; Val; He; Leu; Phe; Tyr; Trp; Thr; Pro; Glu; and Ser.
• the isolated peptide is not fibronectin
• the isolated peptide comprises 75 or fewer amino acids.
• 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;
• X 6 is selected from a group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, His, Thr, and Cys;
• X 7 is selected from the group consisting of Gly, Arg, Glu, Ser, He, Thr, Val, His, Trp, and Cys;
• the isolated peptide is not fibronectin
• the isolated peptide comprises 75 or fewer amino acids.
• X 2 is selected from a group consisting of Tyr; Ala; Val; He; Leu; Phe; and Trp;
• X 4 is selected from a group consisting of He; Ala; Val; Leu; Phe; Tyr; and Trp;
• X 6 is selected from a group consisting of Tyr; Ala; Val; He; Leu; Phe; Trp; His; Thr; and Cys; and
• X 7 is selected from the group consisting of Gly; Arg; Glu; Trp; and Cys.
• X 3 is selected from the group consisiting of Phe, Ala, Val, He, Leu, Tyr, Trp, Asp, Lys, Arg, Thr, Pro, and Glu;
• 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;
• the isolated peptide is not fibronectin
• the isolated peptide comprises 75 or fewer amino acids.
• 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.
• X 3 is selected from the group consisiting of Phe; Ala; Val; He; Leu; Tyr; Trp; Asp; Lys; Arg; and Thr;
• X 5 is selected from the group consisting of Val; Ala; He; Leu; Phe; Tyr; Trp; Pro; Glu; Lys; and Ser;
• X 4 is selected from the group consisiting of Phe; Ala; Val; He; Leu; Tyr; Trp; Asp; Lys; Arg; and Thr; and
• X 6 is selected from the group consisting of Val; Ala; He; Leu; Phe; Tyr; Trp; Pro; Glu; Lys; and Ser.
• 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;
• X 3 is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, and Gly;
• X 5 is selected from the group consisting of Val, Ala, He, Leu, Phe, Tyr, and Trp;
• X 7 is selected from the group consisting of Ala, Val, He, Leu, Phe, Tyr, Trp, Ser, Thr, and Gin;
• the isolated peptide is not fibronectin
• the isolated peptide comprises 75 or fewer amino acids.
• 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) .
• X 3 is selected from the group consisting of He, Ala, Val, Leu, Phe, Tyr, Trp, Gly, Gin, Asp, Thr, Ser, Arg, and Asn;
• X 5 is selected from the group consisting of Tyr, Ala, Val, He, Leu, Phe, Trp, Lys, Gin, Ser, Thr, and Pro;
• the isolated peptide is not fibronectin
• the isolated peptide comprises 75 or fewer amino acids.
• Xi is selected from the group consisting of He ; Ala; Val; Leu; Phe; Tyr; Trp; Lys; Thr; and Pro;
• X 3 is selected from the group consisting of He ; Ala; Val; Leu; Phe; Tyr; Trp; Gly; Gin; Asp; Thr; Ser; and Asn;
• X 5 is selected from the group consisting of Tyr; Ala; Val; He; Leu; Phe; Trp; Lys; Gin; Ser; and Pro;
• 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.
• 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 mutation comprises a mutation selected from the group consisting of double Cys mutations or click chemistry or other crosslinking
• 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.
• amino acid analogs 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
• 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.
• 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
• MRI magnetic resonance imaging
• radiopharmaceuticals radionuclides
• 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 expression vector comprising the isolated nucleic acid of paragraph 58.
• composition consisting essentially of one or more peptides, nucleic acids, or vectors of any of paragraphs 1-59 as an active ingredient.
• 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.
• 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.
• 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.
• pre -neoplastic lesions e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia , carcinoma, oral hairy leukoplakia, and psoriasis.
• 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.
• a method of inhibiting cellular growth in a subject in need thereof comprising administering a peptide, nucleic acid, or composition of any of paragraphs 1-62.
• 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.
• pre -neoplastic lesions e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia
• carcinoma oral hairy leukoplakia
• psoriasis e.g., adenomatous hyperplasia, prostatic intraepithelial neoplasia
• 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.
• a method of promoting wound heling wherein the method comprises administering a peptide, nucleic acid, or composition of any of paragraphs 1-62.
• a method of targeting therapeutic agents or imaging molecules to sites of extracellular matrix production or accumulation comprising 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.
• 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 comprising contacting at least two polypeptide molecules with a peptide, nucleic acid, or composition of paragraphs 1-62.
• fibronectin-like type III domain or a domain comprising a fibronectin type III fold or beta sheet is selected from the group consisting of:
• composition of any of paragraphs 60-62 for preventing, treating and/or ameliorating a proliferative disease.
• the extracellular matrix 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].
• 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.
• 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).
• 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.
• FIG. 3A-3D 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 ⁇ show that CPB retains the multimerization activity of CP1 at high concentrations.
• 750 ⁇ CP1 or varying concentrations of CPB 50-750 ⁇ are incubated with FN and analyzed by non-reducing
• 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 ⁇ ) 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.
• 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.
• 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 ⁇ ), CPA (150 ⁇ ), CPB (150 ⁇ ), or CPA (150 ⁇ ) + CPB (150 ⁇ ) 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 ⁇ of peptide and refreshed every other day. On Day 4, the media was replaced with fresh peptide solution containing 10 ⁇ Click-iT EdU for a 24 h incubation before labeling with Pacific Blue azide.
• 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.
• fibronectin is a natural reservoir for growth factors and cell signaling molecules.
• CPl and other peptides described herein into the matrix during fibronectin polymerization can make them superb candidates to target drug therapies to the matrix.
• 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.
• 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.
• 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.
• EXAMPLE 2 SLLISWD (SEQ ID NO: 5) multimerization sequence in the fibronectin lOFNIII domain identified by steered molecular dynamics initiates fibrillogenesis
• Fibronectin (FN) assembly into insoluble extracellular matrix fibrils is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by
• CPl twenty-three residue cryptic peptide 1
• ANS 8-anilino-l-napthalenesulfonic acid
• a point mutation at Trp6 annihilates its ability to multimerize FN and simultaneously reduces the exposure of hydrophobic sites for ANS binding and ⁇ -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 ⁇ -strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.
• the extracellular matrix 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).
• ECM extracellular matrix
• 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.
• 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 ⁇ 5 ⁇ 1 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.
• 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 ⁇ -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.
• 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).
• 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'-
• 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.
• 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 ([ ⁇ ], deg cm 2 dmol "1 ) as calculated using the relation
• Samples were prepared in PBS with 50 ⁇ ANS in the presence or absence of 50 ⁇ anastellin or 50 ⁇ 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 QuantaMasterTM 40 spectrofluorometer with excitation and emission slit widths of 5 nm.
• ThT Thioflavin T binding fluorescence assay - A 1 mM stock solution of ThT
• 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).
• 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).
• 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).
• the degree of multimerization by CPl is concentration-dependent in the range of 10-500 ⁇ with maximal activity at 150 ⁇ (30 ⁇ 1%, Fig. 1 IB).
• CPB which is 30% the length of CPl, required a higher concentration (500 ⁇ ) to achieve similar multimerization as 150 ⁇ CPl (Fig. 11B).
• 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).
• Trp2 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 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).
• 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).
• addition of CPB to FBG 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 ⁇ and 750 ⁇ , respectively).
• SLLISWD - 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.
• 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).
• 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.
• 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 ⁇ 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).
• 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.
• 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).
• 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.
• 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 ⁇ -sheets (49)
• CLT1 is a 10-mer identified by phage display that possesses a hydrophobic N-terminus and hydrophilic C-terminus proposed to form ⁇ -sheets similar to amyloidogenic fibrils (50).
• ⁇ -strand exchange may be a potential mechanism for FN assembly initiated by the multimerization sequence.
• N-terminus of lOFNIII unfolds.
• 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).
• FN assembly by alpha5betal, but not by alphaVbeta3, requires the synergy site (58).
• 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.
• 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
• 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.
• Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly. J. Cell Biol. 141, 539-551
• Angiostatic peptides use plasma fibronectin to home to angiogenic vasculature. Proc. Natl. Acad. Sci. U. S. A. 102, 2040-2045
• ECM extracellular matrix
• FN fibronectin
• 10FNIII tenth
• FN type III SMD, steered molecular dynamics
• CP1 cryptic peptide 1
• CPlscr scrambled cryptic peptide 1
• BME ⁇ -mercaptoethanol
• FBG fibrinogen
• ANS 8-anilino-l-napthalenesulfonic acid
• ThT Thioflavin T
• 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 ⁇ ) separated by non-reducing SDS-PAGE analysis ( Figure 13). Addition of FITC labeled CPB to unlabeled FN enables visualization of the protein.
• FIG. 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.

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