WO2021087561A1 - Cytokine or growth factor fusion proteins - Google Patents

Cytokine or growth factor fusion proteins Download PDF

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Publication number
WO2021087561A1
WO2021087561A1 PCT/AU2020/051199 AU2020051199W WO2021087561A1 WO 2021087561 A1 WO2021087561 A1 WO 2021087561A1 AU 2020051199 W AU2020051199 W AU 2020051199W WO 2021087561 A1 WO2021087561 A1 WO 2021087561A1
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growth factor
fusion protein
seq
cytokine
vegf
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PCT/AU2020/051199
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French (fr)
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Mikaël MARTINO
Jeffrey A. Hubbell
Mayumi Mochizuki
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Monash University
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Priority claimed from AU2019904152A external-priority patent/AU2019904152A0/en
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Publication of WO2021087561A1 publication Critical patent/WO2021087561A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/49—Platelet-derived growth factor [PDGF]
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • the invention relates to cytokine or growth factor fusion proteins and uses thereof.
  • Growth factors and cytokines are signalling molecules that control cell activities in an autocrine, paracrine or endocrine manner. They exert their biological functions by binding to specific receptors and activating associated downstream signalling pathways which in turn, regulate gene transcription in the nucleus and ultimately stimulate a biological response.
  • Growth factors and cytokines affect a wide variety of physiological processes such as cell proliferation, differentiation, apoptosis, immunological or hematopoietic response, morphogenesis, angiogenesis, metabolism, wound healing and maintaining tissue homeostasis in adult organisms.
  • the abnormal production or regulation of growth factors and cytokines can cause various diseases such as cancer, liver fibrosis and bronchopulmonary dysplasia.
  • growth factors were thought to be biological moieties that have a positive effect on cell growth and proliferation while cytokines were typically considered to have an immunological or hematopoietic response.
  • ‘cytokines’ and ‘growth factors’ can have similar functions and therefore, these terms are now used interchangeably.
  • the present invention seeks to provide growth factors and cytokines optimized for delivery and controlled signalling. Summary
  • a first aspect provides a fusion protein comprising a growth factor or cytokine and a syndecan binding peptide.
  • a second aspect provides a nucleic acid molecule encoding the fusion protein of the first aspect.
  • a third aspect provides a vector comprising the nucleic acid molecule of the second aspect.
  • a fourth aspect comprises a cell or a non-human organism transformed or transfected with the nucleic acid molecule of the second aspect or the vector of the third aspect.
  • a fifth aspect provides a method of making the fusion protein of the first aspect, the method comprising culturing the cell of the fourth aspect under conditions to produce the fusion protein and recovering the fusion protein.
  • a sixth aspect provides a fusion protein produced by the method of the fifth aspect.
  • a seventh aspect provides a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect or the cell or non-human organism of the fourth aspect, optionally with one or more excipient and/or carriers.
  • An eighth aspect provides a method of treatment of a condition in which cytokine or growth factor administration is beneficial, the method comprising administering to a subject in need thereof the fusion protein of the first aspect or the sixth aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the cell or non-human organism of the fourth aspect or the pharmaceutical or veterinary composition of the seventh aspect.
  • An alternative form of the eighth aspect provides a composition for treatment of a condition in which in which cytokine or growth factor administration is beneficial, the composition comprising the fusion protein of the first aspect or the sixth aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the cell or non-human organism of the fourth aspect or the pharmaceutical or veterinary composition of the seventh aspect.
  • a further alternative form of the eighth aspect provides use of the fusion protein of the first aspect or the sixth aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the cell or non-human organism of the fourth aspect or the pharmaceutical or veterinary composition of the seventh aspect in the manufacture of a medicament for treating a condition in which cytokine or growth factor administration is beneficial.
  • a ninth aspect provides a fusion protein comprising platelet-derived growth factor-BB (PDGF-BB) and a syndecan binding peptide.
  • PDGF-BB platelet-derived growth factor-BB
  • a tenth aspect provides a method of enhancing tissue regeneration, particularly bone regeneration and/or wound repair comprising administering the fusion protein of the ninth aspect.
  • An alternative form of the tenth aspect provides a fusion protein comprising platelet- derived growth factor-BB (PDGF-BB) and a syndecan binding peptide for use in enhancing tissue regeneration, particularly bone regeneration and/or wound repair.
  • PDGF-BB platelet- derived growth factor-BB
  • syndecan binding peptide for use in enhancing tissue regeneration, particularly bone regeneration and/or wound repair.
  • a further alternative form of the tenth aspect provides use of a fusion protein comprising platelet-derived growth factor-BB (PDGF-BB) and a syndecan binding peptide in the manufacture of a medicament for enhancing tissue regeneration, particularly bone regeneration and/or wound repair.
  • PDGF-BB platelet-derived growth factor-BB
  • syndecan binding peptide in the manufacture of a medicament for enhancing tissue regeneration, particularly bone regeneration and/or wound repair.
  • An eleventh aspect provides a fusion protein comprising vascular endothelial growth factor A (VEGF-A) and a syndecan binding peptide.
  • VEGF-A vascular endothelial growth factor A
  • a twelfth aspect provides a method of treating wounds, burns and muscle, cartilage, tendon and bone disorders comprising administering the fusion protein of the eleventh aspect.
  • An alternative form of the twelfth aspect provides a fusion protein comprising vascular endothelial growth factor A and a syndecan binding peptide for use in treating wounds, burns and muscle, cartilage, tendon and bone disorders.
  • a further alternative form of the twelfth aspect provides use of a fusion protein comprising vascular endothelial growth factor A and a syndecan binding peptide in the manufacture of a medicament for treating wounds, burns and muscle, cartilage, tendon and bone disorders.
  • a thirteenth aspect provides a method of inducing tonic signalling in response to a growth factor or cytokine, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
  • a fourteenth aspect provides a method of reducing growth factor receptor or cytokine receptor internalization and degradation in response to binding by their respective growth factor or cytokine, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
  • a fifteenth aspect provides a method of reducing desensitization to growth factor stimulation, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
  • a sixteenth aspect provides a method of reducing a side effect associated with cytokine or growth factor administration, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
  • the invention is based on the inventors’ finding that fusion proteins of PDGF-BB or VEGF-A with a syndecan binding peptide did not provide the wild type growth factor burst signalling they anticipated, even though PDGF-BB and VEGF-A165 bind sydecans to some extent.
  • the enhanced binding to syndecans provided by the fusion proteins changes growth factor signalling kinetics with receptor and downstream kinase phosphorylation levels being much lower during the first hour post-stimulation.
  • Interactions of growth factors with cell-surface heparan sulfate proteoglycans are well-known in developmental biology to regulate tissue formation by controlling their bioavailability. Sequestration of morphogens by cell-surface heparan sulfate proteoglycans provides a mechanism to locally control the intensity and kinetics of morphogen signalling - a typical example is for FGF-2. Without wishing to be bound by theory, the inventors propose that by enhancing growth factor binding to syndecans and their retention on the cell surface, they can mimic such a mechanism and locally control the intensity and kinetics of cytokine or growth factor signalling.
  • the inventors demonstrate that the tonic signalling generated by syndecan-binding growth factors is specifically due to binding to syndecans via the syndecan binding sequence (evidence provided).
  • Figures 1 a -e provide -
  • ECM extracellular matrix
  • syndecans In some cases (e.g. FGF-2), binding to syndecans facilitates growth factor-growth factor receptor assembly (2’). Growth factor binding to its main receptor (3) and signalling triggers receptor internalization and eventually degradation (4).
  • the wedge represents increasing growth factor availability for growth factor co-receptors and receptors and at the cell surface.
  • (1c) Location of the syndecan-binding domain SB (RKRLQVQLSIRT, known as AG73) in laminin 111.
  • SB was added at the C-terminus of VEGF-A121 to generate VEGF-A-SB.
  • the heparin-binding domain of VEGF- A165 is represented by a light grey oval.
  • SB was added at the C-terminus of PDGF-BB to generate PDGF-BB-SB. (1d, 1e).
  • Growth factors 100 nM were absorbed on enzyme-linked immunosorbent assay (ELISA) plates and further incubated with His-tagged syndecans. Syndecan binding was detected via His-tag and bovine serum albumin (BSA) was used as a control for unspecific binding.
  • Figure 2 provides an SDS-PAGE showing the analysis of recombinant syndecans produced in mouse myeloma cell line.
  • Theoretical molecular weights (grey lines) based on amino-acid sequences are 24.9 kDa for syndecan-1 (S1, Entry identifier P18827), 14.8 kDa for syndecan-2 (S2, Entry identifier P34741), 35.5 kDa for syndecan-3 (S3, Entry identifier 075056) and 14.7 kDa for syndecan-4 (Entry identifier P31431, S4). Because of the polysaccharide chains, syndecans run much higher on SDS-PAGE. Experiment was repeated independently twice with similar results.
  • Figure 3 provides graphs showing binding affinity of growth factor variants to syndecans as determined by ELISA.
  • FIG 4 provides graphs showing binding affinity of VEGF-A121 to syndecans as determined by ELISA.
  • Figure 5 provides graphs showing binding affinity of VEGF-A variants to neuropilin-1 as determined by ELISA.
  • ELISA plates were coated with VEGF-A variants (100 nM) and further incubated with his-tagged neuropilin-1 (100 nM). Bound neuropilin-1 was detected using an antibody against the his-tag.
  • FIG. 6 shows flow cytometry analysis of MSCs and ECs for expression of PDGFR- a and PDGFR-b.
  • PDGFR-b is highly expressed on the surface of MSCs but not on ECs. Experiment was repeated independently three times with similar results.
  • Figure 7 shows flow cytometry analysis of syndecans 1-4 on MSCs and ECs.
  • the graphs show representative flow cytometry histograms. Black dotted curves represent isotype controls, while grey coloured curves represent signals obtained with anti-syndecan antibodies.
  • Syndecans 1-4 are highly expressed on MSCs.
  • Syndecans 2-4 are highly expressed on ECs. Experiment was repeated independently three times with similar results.
  • Figure 8 a-d provide data to show that enhancing growth factor binding to syndecans triggers tonic signalling.
  • 8a Cells were stimulated with growth factor variants (ECs with VEGF proteins at 20 ng ml -1 , MSCs with PDGF-BB proteins at 20 ng ml -1 ) and SB in excess (SB excess , 2 mg ml -1 ).
  • VEGF-A165, PDGF-BB wild-type growth factors
  • PDGF-BB syndecan-binding growth factors
  • Cells were stimulated with growth factors ECs with VEGF proteins, MSCs with PDGF-BB proteins).
  • PDGFR-b present on cell surface as well as the total amount of growth factor receptor (cell surface and intracellular) were measured using flow cytometry using cell surface staining only or both surface and intracellular staining, respectively.
  • MFI mean fluorescence intensities
  • Figure 9 a and b demonstrate signalling of growth factor variants via arrays. MSCs and ECs were stimulated with growth factor variants for 20, 180 and 360 min. Phosphorylation and cleavage of intracellular singling molecules were analysed using an antibody array (chemiluminescence). (9a) Arrays are shown.
  • Extracellular signal-regulated kinase ERK
  • AKT serine/threonine kinase
  • PRAS40 protein kinase B
  • S6RP proline-rich AKT substrate of 40 kDa
  • Bcl-2- associated death promoter Bad
  • GSK ⁇ glycogen synthase kinase 3 beta
  • JNK c-Jun N- terminal kinase
  • SAPK stress-activated kinases
  • ST AT 5' AMP-activated protein kinase alpha
  • mTOR mammalian target of rapamycin
  • HSP27 heat shock protein 27
  • HSP27 serine/threonine kinase p70 S6
  • tumor proteins p53 p53
  • Figure 10 demonstrates binding of PDGF-BB and PDGF-BB-SB to syndecans using immunostaining.
  • Figure 11 demonstrates binding of VEGF-A165 and VEGF-A-SB to syndecans using immunostaining.
  • Figure 12 a- d demonstrate that syndecan-binding growth factors have enhanced morphogenetic capacity.
  • NQEQVSPL alpha-2 plasmin inhibitor
  • M matrix metalloproteinase sensitive sequence derived from type I collagen
  • the heparin-binding domain of VEGF- A165 is represented by a light grey oval.
  • MSCs were stimulated with PDGF-BB variants (20 ng ml -1 ), CX2PI1-8-M-SB in excess (2 mg ml -1 ) or saline control.
  • ECs were stimulated with VEGF-A variants (20 ng ml -1 ), CX2PI1-8-M-SB in excess (2 mg ml -1 ) or saline control.
  • Figure 13 a and b demonstrate the release of growth factor variants from fibrin matrix.
  • Fibrin matrices were made with the various growth factors and incubated in 10 times volume of release buffer that was changed every day.
  • PDGF-BB and VEGF-A165 are quickly released, while a 2 Pli- 8 -M-PDGF-BB, a 2 Pli- 8 -M-PDGF-BB-SB, a 2 Pli- 8 -M-VEGF-A121, CX2PI1-8-M-VEGF-AI65 and CX2PI1-8-M-VEGF-A-SB stay within the matrix, due to the fibrin binding sequence (CX2PI1-8).
  • Figure 14 a-d show syndecan-binding VEGF-A has enhanced capacity to induce EC assembly.
  • 14a Schematic representation of the 3D microfluidic system with nine chambers. Chambers were filled with a growth factor-functionalized fibrin matrix containing ECs and incubated under flow for 4 days. Matrices were functionalized with CX2PI1-8-M-VEGF-A-SB, CX2PI1-8-M-VEGF-AI65, or CX2PI1-8-M-VEGF-AI2I. Unfunctionalized matrix (fibrin only) is represented in white. Empty chambers and media are represented in pink.
  • EC growth supplement (ECGS, 200 mg ml -1 ) was used as a positive control in a separate multichamber system.
  • Representative structure formed at 500 ng ml -1 of VEGF-A variants. Scale bar 100 mGP. Experiment was repeated independently four times with similar results.
  • Figure 15 a and b show dose dependent structure formation induced by syndecan- binding VEGF-A.
  • Figure 16 a-c show syndecan-binding PDGF-BB improves bone regeneration.
  • Critical size calvarial defects (4.5 mm diameter) in mice were treated with PDGF-BB variants (2 mg) delivered via a fibrin matrix. Eight weeks after treatment, bone regeneration was measured by microCT.
  • Figure 17 a-d show syndecan-binding VEGF-A promotes impaired wound healing and angiogenesis.
  • Full-thickness back-skin wounds (6 mm diameter) of diabetic mice (db/db) were treated with VEGF-A variants (200 ng per wound) delivered via a fibrin matrix (a) Representative histology (hematoxylin and eosin staining) after 10 days.
  • Black arrows indicate wound edges; grey arrows indicate tips of epithelium tongue.
  • Scale bar 1 mm.
  • Higher magnification (5 c ) of the granulation tissue is shown on the right taken from the locations enclosed in the black dashed squares in the panels on the left.
  • FIG 18 shows fibrin-binding VEGF-A121 does not promote wound healing in diabetic mice.
  • Full-thickness back-skin wounds (6 mm diameter) of diabetic mice ( db/db ) were treated with VEGF-A121 or CX2PI1-8-M-VEGF-AI2I (200 ng per wound) delivered via a fibrin matrix
  • a) Representative histology (hematoxylin and eosin staining) after 10 days. Black arrows indicate wound edges; grey arrows indicate tips of epithelium tongue. Scale bar 1 mm.
  • Figure 19 a-f show Syndecan-binding growth factors induces less side effects.
  • Statistical comparisons are between CX2PI1-8-M-PDGF-BB and CX2PI1-8-M-PDGF-BB-SB. Two-way ANOVA with Bonferroni post hoc test; numbers and stars indicate P values between groups (*** p ⁇ 0.001).
  • Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.
  • fusion protein is a protein made from a fusion gene, which is created by joining of two or more genes that originally coded for separate polypeptides.
  • polypeptide refers to any sequence of two or more amino acids, regardless of length, post-translation modification, or function. Polypeptides can include natural amino acids and non-natural amino acids. Polypeptides can also be modified in any of a variety of standard chemical ways (e.g., an amino acid can be modified with a protecting group; the carboxy- terminal amino acid can be made into a terminal amide group; the amino-terminal residue can be modified with groups to, e.g., enhance lipophilicity; or the polypeptide can be chemically glycosylated or otherwise modified to increase stability or in vivo half-life).
  • an amino acid can be modified with a protecting group
  • the carboxy- terminal amino acid can be made into a terminal amide group
  • the amino-terminal residue can be modified with groups to, e.g., enhance lipophilicity
  • the polypeptide can be chemically glycosylated or otherwise modified to increase stability or in vivo half-life).
  • Polypeptide modifications can include the attachment of another structure such as a cyclic compound or other molecule to the polypeptide and can also include polypeptides that contain one or more amino acids in an altered configuration (i.e. , R or S; or, L or D).
  • growth factors are proteins that regulate many aspects of cellular function, including survival, proliferation, migration and differentiation. Growth factors determine the fate of cells as they differentiate from being progenitors along either neuronal or glial lineages. In addition, during embryonic development, growth factors are crucial for regulating neuronal survival, determining cell fate and establishing proper connectivity. Growth factors typically act as signalling molecules between cells. They often promote cell differentiation and maturation, which varies between growth factors. For example, platelet- derived growth factor BB (PDGF BB) enhances osteogenic differentiation, while fibroblast growth factors and vascular endothelial growth factors stimulate blood vessel differentiation (angiogenesis).
  • PDGF BB platelet- derived growth factor BB
  • angiogenesis blood vessel differentiation
  • cytokines are loose category of small proteins (-5-20 kDa) that are important in cell signalling. Cytokines have been shown to be involved in autocrine, paracrine and endocrine signalling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally not hormones or growth factors (despite some overlap in the terminology). Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a given cytokine may be produced by more than one type of cell.
  • Cytokines act through receptors and are especially important in the immune system; cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations.
  • growth factors and cytokines contemplated for use in the invention are interleukins including lnterleukin-1 receptor antagonist, Interleukin 2,
  • Interleukin 4 Interleukin 10, Interleukin 16, Interleukin 33, Interleukin 27b
  • C-X-C motif chemokines including C-X-C motif chemokine 9, C-X-C motif chemokine 10, C-X-C motif chemokine 12, C-X-C motif chemokine 19, C-X-C motif chemokine 20, C-X-C motif chemokine 22
  • fibroblast growth factors including Fibroblast Growth Factor-2, Fibroblast Growth Factor-5, Fibroblast Growth Factor-7, Fibroblast Growth Factor-10, Fibroblast Growth Factor- 18
  • vascular endothelial growth factors including Vascular Endothelial Growth Factor-A165, Vascular Endothelial Growth Factor-A121, Vascular Endothelial Growth Factor-B, Vascular Endothelial Growth Factor-C
  • placental growth factors including Placental Growth Factor-1, Placental Growth Factor-2, Placental Growth Factor-4
  • IL-1Ra lnterleukin-1 receptor antagonist
  • IL-4 lnterleukin-4
  • Interleukin- 10 (IL-10):
  • Interleukin- 16 (IL-16):
  • Interleukin-33 (IL-33):
  • Interleukin-35 (IL-27b):
  • CXCL9 C-X-C motif che okine 9
  • CXCL10 C-X-C motif chemokine 10
  • C-X-C motif chemokine 12 (CXCL12):
  • CCL19 C-C motif chemokine 19
  • CCL20 C-C motif chemokine 20
  • CCL22 C-C motif chemokine 22
  • Fibroblast growth factor-2 (FGF-2):
  • Fibroblast growth factor-5 (FGF-5):
  • Fibroblast growth factor-7 (FGF-7):
  • Fibroblast growth factor-10 (FGF-10):
  • Vascular endothelial growth factor-A165 (VEGF-A165):
  • VEGF-A121 Vascular endothelial growth factor-A121
  • VEGF-B Vascular endothelial growth factor-B
  • VEGF-C Vascular endothelial growth factor-C
  • PIGF-1 Placenta growth factor-1
  • PIGF-4 Placenta growth factor-4 (PIGF-4):
  • PDGF-AA Platelet-derived growth factor-A
  • PDGF-BB Platelet-derived growth factor-B
  • PDGF-CC Platelet-derived growth factor-C
  • PDGF-DD Platelet-derived growth factor-D
  • EGF Epidermal growth factor
  • Heparin-binding epidermal growth factor (HB-EGF):
  • Neuroregulin-2 (NRG-2):
  • IGF-I Insulin-like growth factor-l
  • IGF-II Insulin-like growth factor-ll
  • TGF- b1 Transforming growth factor-b ⁇ (TGF- b1):
  • TGF- b2 Transforming growth factor- b2 (TGF- b2):
  • TGF- b3 Transforming growth factor- b3 (TGF- b3):
  • Bone morphogenetic-2 (BMP-2):
  • Bone morphogenetic-7 (BMP-7):
  • Nerve growth factor (NGF):
  • Neurotrophin-3 (NT-3):
  • BDNF Brain-derived neurotrophic factor
  • the growth factor or cytokine binds syndecans.
  • syndecan binding growth factors or cytokines are PDGF-BB and VEGF-A165.
  • the present invention also extends to fusion proteins comprising growth factors or cytokines which are functional homologues or variants of human growth factor or cytokine sequences or those of other animal species.
  • amino acid insertion variants include amino and/or carboxylic terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • Insertion amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletion variants are characterised by the removal of one or more amino acids from the sequence.
  • Substitution amino acid variants are those in which at least one amino acid residue in the sequence has been replaced by another of the twenty primary protein amino acids, or by a non-protein amino acid.
  • substitutions are with conservative amino acids.
  • Chemical modifications of the native carboxyl-terminal sequence include the acetylation of the amino-terminus and/or amidation of the carboxyl-terminus and/or side chain cyclisation of the native carboxyl-terminal sequence.
  • variants of the growth factor or cytokine used in the fusion protein of the invention may comprise one, two, three, four or five insertions, deletions or substitutions compared to the natural growth factor or cytokine sequence provided that the function of the native sequence is retained.
  • amino acids, except for glycine are of the L-absolute configuration. D configuration amino acids may also be used.
  • the growth factor or cytokine used may be modified to improve storage stability, bioactivity, circulating half-life, or for any other purpose using methods available in the art, such as glycosylation, by conjugation to a polymer to increase circulating half-life, by pegylation or other chemical modification.
  • Variants of the human growth factor or cytokine sequences provided as SEQ ID Nos: 1-45 preferably have at least about 80% amino acid sequence identity with the human sequence as disclosed herein (the reference sequence).
  • a variant will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to the reference sequence.
  • a determination of the percent identity of a peptide or protein to a sequence set forth herein may be required. In such cases, the percent identity is measured in terms of the number of residues of the peptide or protein, or a portion of the peptide or protein.
  • a polypeptide of, e.g., 90% identity may also be a portion of a larger polypeptide or protein.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • syndecan binding peptide is a peptide or polypeptide that specifically binds syndecans. “Specifically binds” as used herein means that the peptide or polypeptide has greater binding affinity for syndecans than for other receptors or other heparan sulfate proteoglycans. For example, syndecan binding peptides may bind integrin receptors or other heparan sulfate proteoglycans but bind syndecans with greater binding affinity.
  • Specific binding refers to a molecule that binds to a target with a relatively high affinity compared to non-target tissues, and generally involves a plurality of non-covalent interactions, such as electrostatic interactions, van der Waals interactions, hydrogen bonding, and the like.
  • Specific binding interactions characterize antibody-antigen binding, enzyme-substrate binding, and specifically binding protein-receptor interactions; while such molecules may bind tissues besides their targets from time to time, such binding is said to lack specificity and is not specific binding.
  • Syndecans are type I transmembrane proteins that comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans are present on the surface of various adherent and non-adherent cells, and their domains interact with various soluble and insoluble factors in the ECM. Syndecans can interact with various extracellular ligands through glycosaminoglycan chains attached to the core protein.
  • HSPGs cell surface heparan sulfate proteoglycans
  • Syndecans have numerous ligands, including laminins, which are major glycoproteins in the basement membrane.
  • laminins which are major glycoproteins in the basement membrane.
  • the globular domain of laminin alpha 1 chain binds heparan sulfate in syndecans with high affinity via the sequence RKRLQVQLSIRT (SEQ ID NO: 46) (laminin subunit alpha-12719-2730), commonly known as AG73 and named SB in this study.
  • the syndecan binding peptide is from laminin a1, a2, a3 or a4.
  • syndecan binding peptide is a variant of the SB sequence: Mouse sequence: RKRLQVQLSIRT (SEQ ID NO: 46)
  • Rat sequence RKRLQVQLNIRT (SEQ ID NO: 47)
  • Human sequence RKKLSVELSIRT (SEQ ID NO: 53) or a further variant based on the consensus sequence Po-Po-Po-L-X-X-X-L-X-I-Po-T (SEQ ID NO :54) where Po is a positively charged amino-acid (K or R) and X is a polar amino-acid (Y, C, Q, T, N, or S).
  • syndecan binding peptides that may be used in the invention include, but are not limited to:
  • Human laminin a4 TLFLAHGRLVYM (SEQ ID NO: 55) or a variant based on the consensus sequence: T-L-F-L-A-H-G-Po-L-V-X-M (SEQ ID NO: 56) where Po is a positively charged amino-acid (K or R) an and X is a polar amino-acid (G, Y, C, Q, T, N, or S).
  • KNSFMALYLSKGRLVFALG Human or mouse laminin a3 chain: KNSFMALYLSKGRLVFALG (SEQ ID NO: 57) Human or mouse laminin a3 chain (short sequence): KNSFMALYLSKG (SEQ ID NO: 58) or a variant based on the consensus sequence: Po-N-S-F-M-A-L-Y-L-S-Po-G (SEQ ID NO: 59) where Po is a positively charged amino-acid (K or R) or consensus sequence: Po- N-S-F-M-A-L-Y-L-S-Po-G-Po-L-V-F-A-L-G (SEQ ID NO: 60) where Po is a positively charged amino-acid (K or R).
  • LAIKNDNLVYVY SE ID NO: 61
  • L-A-I-Po-N-D-N-L-V-Y-V-Y SEQ ID NO: 62
  • Po is a positively charged amino-acid (K or R).
  • DVISLYNFKHIY (SEQ ID NO: 63) or a variant based on consensus sequence: D-V-I-S-L-Y-N-F-Po-H-I-Y (SEQ ID NO: 64) where Po is a positively charged amino-acid (K or R).
  • Mouse laminin a1 chain GLIYYVAHQNQM (SEQ ID NO: 65) or a variant based on consensus sequence: G-L-l-Y-Y-Z-A-H-Q-N-Q-M (SEQ ID NO: 66) where Z is a non-polar amino acid (A, V, L, I, P, F, M, W, or G).
  • Human laminin B2 chain or mouse laminin y1 chain KAFDITYVRLKF (SEQ ID NO:
  • Po is a positively charged amino-acid (K or R).
  • DFLAVEMRRGKVAFLWDLG Human or mouse laminin a1 chain: DFLAVEMRRGKVAFLWDLG (SEQ ID NO: 69) or a variant based on consensus sequence: D-F-L-A-Z-E-M-Po-Po-G-Po-V-X/Z-F-L-W-D-L- G (SEQ ID NO: 70) where Po is a positively charged amino-acid (K or R), X is a polar amino-acid (Y, C, Q, T, N, or S), and Z is a non-polar amino acid (A, V, L, I, P, F, M, W, or G).
  • Human or mouse laminin a1 chain KEYM G LA I KN D N LVYVYN LG (SEQ ID NO: 71) or a variant based on consensus sequence: Po-E-Y-M-G-L-A-I-Po -N-D-N-L-V-Y-V-Y-N-L-G (SEQ ID NO: 72) where Po is a positively charged amino-acid (K or R).
  • Human laminin a1 chain DFLSIELFRGRVKVMTDLG (SEQ ID NO: 73) or a variant based on consensus sequence: D-F-L-S-I-E-L-F-Po-G-Po-V-Po-V-M-T-D-L-G (SEQ ID NO: 74) where Po is a positively charged amino-acid (K or R).
  • Mouse laminin a3 chain AYYAIFLNKGRLEVHLSSG (SEQ ID NO: 75) or a variant based on consensus sequence: A-Y-Y-A-I-F-L-N-Po-G-Po-L-E-V-H-L-S-S-G (SEQ ID NO: 76) where Po is a positively charged amino-acid (K or R).
  • Mouse laminin a4 chain DFMTLFLAHGRLVFMFNVG (SEQ ID NO: 77) or a variant based on consensus sequence: D-F-M-T-L-F-L-A-H-G-Po-L-V-F-M-F-N-V-G (SEQ ID NO: 78) where Po is a positively charged amino-acid (K or R).
  • Peptide as used herein means any chain of amino acids from 8 to 50 amino acid residues in length, preferably 8 to 40, 8 to 30, 8 to 25, or 8 to 20, or more preferably about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acid residues in length.
  • the syndecan-binding peptide is inserted near the C-terminus or N-terminus of the cytokine or growth factor, at the terminus most remote from the cytokine or growth factor receptor-binding site. Insertion of the syndecan-binding domain at the C-terminus or N-terminus may change the stability of the fusion protein.
  • the cytokine or growth factor may be directly linked to the syndecan binding peptide or indirectly linked by a linker.
  • a linker is present between the cytokine or growth factor and syndecan binding peptide.
  • Suitable linkers comprise Glycine and Serine, for example GGS or SGG or repeats thereof.
  • the linker sequence comprises from about 1 to 20 amino acids, more preferably from about 1 to 16 amino acids.
  • the linker sequence is preferably flexible so as not hold the cytokine or growth factor in a single undesired conformation.
  • the fusion protein as described herein may additionally comprise an N-terminal signal peptide domain, which allows processing, e.g., extracellular secretion, in a suitable host cell.
  • the N-terminal signal peptide domain comprises a protease, e.g., a signal peptidase cleavage site and thus may be removed after or during expression to obtain the mature protein.
  • the fusion protein may comprise comprises a recognition/purification domain, e.g., a Strep-tag domain and/or a poly-His domain, which may be located at the N- terminus or at the C-terminus.
  • a recognition/purification domain e.g., a Strep-tag domain and/or a poly-His domain, which may be located at the N- terminus or at the C-terminus.
  • the fusion protein comprises a human growth factor or cytokine of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked to a syndecan binding peptide of SEQ ID NO: 46-78, either directly or via a linker, preferably comprising GGS or SGG or repeats thereof.
  • the syndecan binding peptide may be at the N or C terminus of the fusion protein.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 46, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1- 45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 46, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 54, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 54, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 56, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 56, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 59, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 59, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 60, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 60, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 62, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1- 45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 62, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 64, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 64, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 66, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 66, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 68, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 68, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 70, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 70, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 72, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 72, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 74, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 74, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 76, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 76, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 78, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 78, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
  • the fusion protein comprises platelet-derived growth factor-BB (PDGF-BB) and a syndecan binding peptide.
  • PDGF-BB platelet-derived growth factor-BB
  • syndecan binding peptide a syndecan binding peptide
  • the fusion protein comprises CX2PI1-8-M-PDGF-BB-SB with the following amino acid sequence:
  • the fusion protein comprises VEGF-A and a syndecan binding peptide.
  • the VEGF-A is VEGF-A, VEGF-A121 or VEGF-A165.
  • the fusion protein comprises CX2PI1-8-M-VEGF-A-SB with the following amino acid sequence:
  • the fusion protein comprises VEGF-A-SB with the following amino acid sequence: ASAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGG CCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENCDKP RR-AGSGGSRKRLQVQLSI RT (SEQ ID NO: 81)
  • preparation of the fusion proteins of the invention can be accomplished by procedures disclosed herein and by recognized recombinant DNA techniques involving, e.g., polymerase chain amplification reactions (PCR), preparation of plasmid DNA, cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of the DNA into a suitable cell, transformation or transfection of a host, culturing of the host.
  • PCR polymerase chain amplification reactions
  • preparation of plasmid DNA e.g., cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of the DNA into a suitable cell, transformation or transfection of a host, culturing of the host.
  • PCR polymerase chain amplification reactions
  • preparation of plasmid DNA e.g., cleavage of DNA with restriction enzymes
  • preparation of oligonucleotides
  • the invention further provides nucleic acid sequences and particularly DNA sequences that encode the present fusion proteins.
  • the DNA sequence is carried by a vector suited for extrachromosomal replication such as a phage, virus, plasmid, phagemid, cosmid, YAC, or episome.
  • a DNA vector that encodes a desired fusion protein can be used to facilitate preparative methods described herein and to obtain significant quantities of the fusion protein.
  • the DNA sequence can be inserted into an appropriate expression vector, i.e. , a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • a variety of host-vector systems may be utilized to express the protein-coding sequence.
  • mammalian cell systems infected with virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA.
  • any one of a number of suitable transcription and translation elements may be used.
  • a preferred DNA vector according to the invention comprises a nucleotide sequence linked by phosphodiester bonds comprising, in a 5' to 3' direction a first cloning site for introduction of a first nucleotide sequence encoding a syndecan binding peptide operatively linked to a sequence encoding a cytokine or growth factor.
  • each of the fusion protein components encoded by the DNA vector be provided in a "cassette" format.
  • cassette is meant that each component can be readily substituted for another component by standard recombinant methods.
  • the fusion proteins described herein are preferably produced by standard recombinant DNA techniques.
  • the resultant hybrid DNA molecule can be expressed in a suitable host cell to produce the fusion protein.
  • the DNA molecules are ligated to each other in a 5' to 3' orientation such that, after ligation, the translational frame of the encoded polypeptides is not altered (i.e. , the DNA molecules are ligated to each other in-frame).
  • the resulting DNA molecules encode an in-frame fusion protein.
  • the components of the fusion protein can be organized in nearly any order provided each can perform its intended function.
  • the gene fusion construct described above can be incorporated into a suitable vector by known means such as by use of restriction enzymes to make cuts in the vector for insertion of the construct followed by ligation.
  • the vector containing the gene construct is then introduced into a suitable host for expression of the fusion protein.
  • Suitable host cells include eukaryotic and prokaryotic cells, preferably those cells that can be easily transformed and exhibit rapid growth in culture medium.
  • preferred hosts cells include prokaryotes such as E. coli, Bacillus subtillus, etc. and eukaryotes such as animal cells and yeast strains, e.g., S. cerevisiae.
  • Mammalian cells are generally preferred, particularly J558, NSO, SP2-0 or CHO.
  • Other suitable hosts include, e.g., insect cells such as Sf9. Conventional culturing conditions are employed. Stable transformed or transfected cell lines can then be selected. Cells expressing fusion proteins according to the invention can be determined by known procedures.
  • Nucleic acid encoding a desired fusion protein can be introduced into a host cell by standard techniques for transfecting cells.
  • transfecting or “transfection” is intended to encompass all conventional techniques for introducing nucleic acid into host cells, including calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, viral transduction and/or integration.
  • the present invention further provides a production process for isolating a fusion protein of interest.
  • a host cell e.g., a yeast, fungus, insect, bacterial or animal cell
  • a nucleic acid encoding the fusion protein operatively linked to a regulatory sequence is grown at production scale in a culture medium.
  • the fusion protein of interest is isolated from harvested host cells or from the culture medium.
  • Standard protein purification techniques can be used to isolate the fusion protein from the medium or from the harvested cells.
  • the purification techniques can be used to express and purify a desired fusion protein on a large-scale (i.e. in at least milligram quantities) from a variety of implementations including roller bottles, spinner flasks, tissue culture plates, bioreactor, or a fermentor.
  • An expressed fusion protein can be isolated and purified by known methods. Typically, the culture medium is centrifuged and then the supernatant is purified by affinity or immunoaffinity chromatography, e.g. Protein-A or Protein-G affinity chromatography or an immunoaffinity protocol comprising use of monoclonal antibodies that bind the expressed fusion protein.
  • affinity or immunoaffinity chromatography e.g. Protein-A or Protein-G affinity chromatography or an immunoaffinity protocol comprising use of monoclonal antibodies that bind the expressed fusion protein.
  • the fusion proteins of the present invention can be separated and purified by appropriate combination of known techniques.
  • methods utilizing solubility such as salt precipitation and solvent precipitation
  • methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis
  • methods utilizing a difference in electrical charge such as ion-exchange column chromatography
  • methods utilizing specific affinity such as affinity chromatograph
  • methods utilizing a difference in hydrophobicity such as reverse- phase high performance liquid chromatograph
  • methods utilizing a difference in isoelectric point such as isoelectric focusing electrophoresis, metal affinity columns such as Ni-NTA.
  • the fusion proteins of the present invention be substantially pure. That is, the fusion proteins have been isolated from cell substituents that naturally accompany it so that the fusion proteins are present preferably in at least 80% or 90% to 95% homogeneity (w/w). Fusion proteins having at least 98 to 99% homogeneity (w/w) are most preferred for many pharmaceutical, clinical and research applications.
  • the fusion protein should be substantially free of contaminants for therapeutic applications.
  • the soluble fusion proteins can be used therapeutically, or in performing in vitro or in vivo assays as disclosed herein. Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis.
  • Fusion proteins according to the invention may be administered in a pharmaceutical composition optionally together with pharmaceutically acceptable carriers or excipients for administration. Fusion proteins according to the invention may be administered in a veterinary composition optionally together with carriers or excipients suitable for administration to animals.
  • the pharmaceutical diluents, excipients, extenders, or carriers are suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • Pharmaceutically acceptable carriers or excipients may be used to deliver embodiments as described herein.
  • Excipient refers to an inert substance used as a diluent or vehicle for a therapeutic agent.
  • Pharmaceutically acceptable carriers are used, in general, with a compound to make the compound useful for a therapy or as a product.
  • a carrier is a material that is combined with the substance for delivery to an animal. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • the carrier is essential for delivery, e.g., to solubilize an insoluble compound for liquid delivery; a buffer for control of the pH of the substance to preserve its activity; or a diluent to prevent loss of the substance in the storage vessel.
  • the carrier is for convenience, e.g., a liquid for more convenient administration.
  • Pharmaceutically acceptable salts of the compounds described herein may be synthesized according to methods known to those skilled in the arts. Pharmaceutically acceptable substances or compositions are highly purified to be free of contaminants, are sterile, and are biocompatible. They further may include a carrier, salt, or excipient suited to administration to a patient. In the case of water as the carrier, the water is highly purified and processed to be free of contaminants, e.g., endotoxins.
  • the deliverable compound may be made in a form suitable for oral, rectal, topical, intravenous injection, intra-articular injection, parenteral administration, intra-nasal, or tracheal administration.
  • Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used. Suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents may be included as carriers, e.g., for pills.
  • an active component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.
  • the compounds can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • the active compounds can also be administered parentally, in sterile liquid dosage forms. Buffers for achieving a physiological pH or osmolarity may also be used.
  • the invention in one aspect relates to the treatment of conditions.
  • the terms “treating”, and “treatment” as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms (prophylaxis) and/or their underlying cause, and improvement or remediation of damage.
  • the present method of "treating" a condition encompasses both prevention of the condition in a predisposed individual, treatment of the condition in a clinically symptomatic individual and treatment of a healthy individual for beneficial effect.
  • “Prophylaxis” or “prophylactic” or “preventative” therapy as used herein includes preventing the condition from occurring or ameliorating the subsequent progression of the condition in a subject that may be predisposed to the condition but has not yet been diagnosed as having it.
  • condition refers to any deviation from normal health and includes a disease, disorder, defect or injury, such as injury caused by trauma, and deterioration due to age, inflammatory, infectious or genetic disorder or due to environment.
  • cytokine or growth factor administration is beneficial
  • conditions that may be treated in accordance with the present invention fall generally into the categories of those in which increased chondrocyte, collagen, proteoglycan, cartilage or muscle mass form or function is desirable.
  • Chondrocytes are the only cells found in cartilage. They produce and maintain the cartilaginous matrix, which consists mainly of Type II collagen, proteoglycans and elastin.
  • Cartilage is a flexible connective tissue found in many areas in the bodies of humans and animals, including joints between bones, rib cage, ear, nose, elbow, knee, ankle, bronchial tubes and intervertebral discs. Unlike other connective tissues, cartilage does not contain blood vessels and thus has limited repair capabilities. Because chondrocytes are bound in lacunae, they cannot migrate to damaged areas. Therefore, if cartilage is damaged, it is difficult and slow to heal.
  • conditions that can be treated include Chondrocyte-Related Conditions that will benefit from repair or new growth of cartilage tissue or chondrocytes. This is not exclusive however and is used descriptively to emphasise the benefit of the presently disclosed methods.
  • Chondrocyte-Related Conditions include joint disorders involving cartilage damage and include cartilage damage caused by tibial plateau decompression.
  • the cause of osteoarthritis is multifactorial and includes body habitus, genetics and hormonal status.
  • the cartilage covering bones (articular cartilage - a subset of hyaline cartilage) is thinned, eventually completely wearing out, resulting in a “bone against bone” joint, reduced motion and pain.
  • Current therapeutic modalities are aimed at reducing pain and increasing joint function.
  • Non-invasive interventions such as exercise and weight loss are the first lines of treatment, followed by anti-inflammatory medications. These latter treatments alleviate the symptoms but do not inhibit the processes that result in the changes characteristic of this disease and may actually accelerate joint destruction. Failure of these treatments usually culminates in surgical intervention (arthroplasty). Joint replacement is extremely successful with respect to restoring patient mobility and decreasing pain.
  • the present invention provides a treatment for osteoarthritis.
  • the cartilage in the knee is frequently damaged, and can be partially repaired through knee cartilage replacement therapy.
  • Costochondritis is an inflammation of cartilage in the ribs, causing chest pain.
  • an asymmetrical compression of an intervertebral disc ruptures the sac-like disc, causing a herniation of its soft content.
  • the hernia often compresses the adjacent nerves and causes back pain.
  • Tumours made up of cartilage tissue can occur.
  • the present invention provides a treatment for each of the conditions above. Any of these conditions can be treated by repairing or growing new cartilage or chondrocytes according to the methods disclosed herein utilising a fusion protein according to the present invention.
  • chondromalacia patella chondromalacia; chondrosarcoma- head and neck; chondrosarcoma; costochondritis; enchondroma; hallux rigidus; hip labral tear; osteochondritis dissecans (OCD); osteochondrodysplasias; perichondritis; polychondritis; or torn meniscus.
  • OCD osteochondritis dissecans
  • the invention provides means to improve the function of existing chondrocytes and cartilage in maintaining a cartilaginous matrix. It also provides means to promote growth of chondrocytes and cartilage and provide a cartilaginous matrix, with or without an implant or prosthesis. In one embodiment the invention provides means to promote cartilage formation or repair in a cellular scaffold or in tissue engineering techniques, for example for cartilage generation or repair to grow new cartilage tissue in tissues including the nose, septum, ear, elbow, knee, ankle and invertebrate discs.
  • the fusion protein is administered with an implant or the like to produce or repair chondrocytes or cartilage tissue that may interact with the implant to treat a condition as disclosed herein.
  • implant refers to the effect in conjunction of components to achieve a desired biological outcome. While not wishing to be bound by theory, when an implant “interacts” with chondrocytes, the effect of the implant in treating the condition is greater than the effect of the implant alone and may be synergistic.
  • the fusion protein is administered in combination with mesenchymal stem cells therapies to enhance repair.
  • the effect of treatment with the fusion protein and stem cells may be more than the additive effect of the separate treatments and may be synergistic.
  • the “desired biological outcome” provided by the invention is preferably cartilage repair and cartilage growth, more preferably removal of the symptoms of osteoarthritis and most preferably treatment and prevention of osteoarthritis.
  • fusion proteins of the invention can be used to promote muscle growth, to improve recovery of muscle from injury, trauma or use, to improve muscle strength, to improve exercise tolerance, to increase the proportion of muscle, to increase muscle mass, decrease muscle wasting, improve muscle repair, or may be useful to treat disorders of muscle including wasting disorders, such as cachexia, and hormonal deficiency, anorexia, AIDS wasting syndrome, sarcopenia, muscular dystrophies, neuromuscular diseases, motor neuron diseases, diseases of the neuromuscular junction, and inflammatory myopathies in a subject in need thereof.
  • wasting disorders such as cachexia, and hormonal deficiency, anorexia, AIDS wasting syndrome, sarcopenia, muscular dystrophies, neuromuscular diseases, motor neuron diseases, diseases of the neuromuscular junction, and inflammatory myopathies in a subject in need thereof.
  • the invention extends to treatment of disorders of muscle and of diseases associated with muscular degeneration characteristics.
  • disorders are various neuromuscular diseases, cardiac insufficiency, weakness of single muscles such as e.g. the constrictor or bladder muscle, hypo- or hypertension caused by problems with the constrictor function of vascular smooth muscle cells, impotence/erectile dysfunction, incontinence, AIDS-related muscular weakness, and general and age-related amyotrophia.
  • disorders of muscle as referred to herein particularly include muscle wasting conditions or disorders in which muscle wasting is one of the primary symptoms.
  • Muscle wasting refers to the progressive loss of muscle mass and/or to the progressive weakening and degeneration of muscles, including the skeletal or voluntary muscles which control movement, cardiac muscles which control the heart, and smooth muscles.
  • the muscle wasting condition or disorder is a chronic muscle wasting condition or disorder.
  • Chronic muscle wasting is defined herein as the chronic (i.e. persisting over a long period of time) progressive loss of muscle mass and/or to the chronic progressive weakening and degeneration of muscle. Chronic muscle wasting may occur as part of the aging process. The loss of muscle mass that occurs during muscle wasting can be characterized by a muscle protein breakdown or degradation, by muscle protein catabolism.
  • Protein catabolism occurs because of an unusually high rate of protein degradation, an unusually low rate of protein synthesis, or a combination of both. Protein catabolism or depletion, whether caused by a high degree of protein degradation or a low degree of protein synthesis, leads to a decrease in muscle mass and to muscle wasting.
  • the term "catabolism” has its commonly known meaning in the art, specifically an energy burning form of metabolism.
  • Muscle wasting can occur as a result of age, a pathology, disease, condition or disorder.
  • the pathology, illness, disease or condition is chronic.
  • the pathology, illness, disease or condition is genetic.
  • the pathology, illness, disease or condition is neurological.
  • the pathology, illness, disease or condition is infectious.
  • the pathologies, diseases, conditions or disorders directly or indirectly produce a wasting (i.e. loss) of muscle mass, that is a muscle wasting disorder.
  • muscle wasting in a subject is a result of the subject having a muscular dystrophy; muscle atrophy; orX-linked spinal-bulbar muscular atrophy (SBMA).
  • the muscular dystrophies are genetic diseases characterized by progressive weakness and degeneration of the skeletal or voluntary muscles that control movement.
  • the muscles of the heart and some other involuntary muscles are also affected in some forms of muscular dystrophy.
  • the major forms of muscular dystrophy are: Duchenne muscular dystrophy, myotonic dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy and Emery- Dreifuss muscular dystrophy.
  • Muscular dystrophy can affect people of all ages. Although some forms first become apparent in infancy or childhood, others may not appear until middle age or later. Duchenne MD is the most common form, typically affecting children. Myotonic dystrophy is the most common of these diseases in adults.
  • Muscle atrophy is characterized by wasting away or diminution of muscle and a decrease in muscle mass.
  • Post-Polio MA is a muscle wasting that occurs as part of the post- polio syndrome (PPS). The atrophy includes weakness, muscle fatigue, and pain.
  • Another type of MA is X-linked spinal-bulbar muscular atrophy (SBMA - also known as Kennedy's Disease). This disease arises from a defect in the androgen receptor gene on the X chromosome, affects only males, and its onset is in adulthood.
  • Sarcopenia is a debilitating disease that afflicts the elderly and chronically ill patients and is characterized by loss of muscle mass and function. Further, increased lean body mass is associated with decreased morbidity and mortality for certain muscle-wasting disorders. In addition, other circumstances and conditions are linked to, and can cause muscle wasting disorders. For example, studies have shown that in severe cases of chronic lower back pain, there is paraspinal muscle wasting.
  • Muscle wasting and other tissue wasting is also associated with advanced age. It is believed that general weakness in old age is due to muscle wasting. As the body ages, an increasing proportion of skeletal muscle is replaced by fibrous tissue. The result is a significant reduction in muscle power, performance and endurance.
  • Injuries or damage to the central nervous system are also associated with muscle wasting and other wasting disorders.
  • Injuries or damage to the CNS can be, for example, caused by diseases, trauma or chemicals. Examples are central nerve injury or damage, peripheral nerve injury or damage and spinal cord injury or damage.
  • CNS damage or injury comprise Alzheimer's diseases (AD); stroke, anger (mood); anorexia, anorexia nervosa, anorexia associated with aging and/or assertiveness (mood).
  • muscle wasting or other tissue wasting may be a result of alcoholism.
  • the wasting disease, disorder or condition being treated is associated with chronic illness
  • This embodiment is directed to treating, in some embodiments, any wasting disorder, which may be reflected in muscle wasting, weight loss, malnutrition, starvation, or any wasting or loss of functioning due to a loss of tissue mass.
  • wasting diseases or disorders such as cachexia, including cachexia caused by malnutrition, tuberculosis, leprosy, diabetes, renal disease, chronic obstructive pulmonary disease (COPD), cancer, end stage renal failure, emphysema, osteomalacia, or cardiomyopathy, may be treated by the methods of this invention
  • wasting is due to infection with enterovirus, Epstein-Barr virus, herpes zoster, HIV, trypanosomes, influenza, coxsackie, rickettsia, trichinella, schistosoma or mycobacteria.
  • Cachexia is weakness and a loss of weight caused by a disease or as a side effect of illness.
  • Cardiac cachexia i.e. a muscle protein wasting of both the cardiac and skeletal muscle, is a characteristic of congestive heart failure.
  • Cancer cachexia is a syndrome that occurs in patients with solid tumours and haematological malignancies and is manifested by weight loss with massive depletion of both adipose tissue and lean muscle mass.
  • Cachexia is also seen in COPD, acquired immunodeficiency syndrome (AIDS), human immunodeficiency virus (HlV)-associated myopathy and/or muscle weakness/wasting is a relatively common clinical manifestation of AIDS.
  • AIDS acquired immunodeficiency syndrome
  • HlV human immunodeficiency virus
  • Individuals with HIV-associated myopathy or muscle weakness or wasting typically experience significant weight loss, generalized or proximal muscle weakness, tenderness, and muscle atrophy.
  • Untreated muscle wasting disorders can have serious health consequences.
  • the changes that occur during muscle wasting can lead to a weakened physical state resulting in poor performance of the body and detrimental health effects.
  • Muscle wasting due to chronic diseases can lead to premature loss of mobility and increase the risk of disease-related morbidity.
  • Muscle wasting due to disuse is an especially serious problem in elderly, who may already suffer from age-related deficits in muscle function and mass, leading to permanent disability and premature death as well as increased bone fracture rate.
  • the inventors propose that the fusion proteins of the invention can be used to prevent, repair and treat muscle wasting or atrophy associated with any of the conditions recited above.
  • the fusion protein is used to treat burns and sepsis.
  • the invention in other aspects also contemplates treating healthy individuals to cause an increase in muscle mass, strength, function or overall physique.
  • increase in muscle mass refers to the presence of a greater amount of muscle after treatment relative to the amount of muscle mass present before the treatment.
  • increase in muscle strength refers to the presence of a muscle with greater force generating capacity after treatment relative to that present before the treatment.
  • the term “increase in muscle function” refers to the presence of muscle with greater variety of function after treatment relative to that present before the treatment.
  • the term “increase in exercise tolerance” refers to the ability to exercise with less rest between exercise after treatment relative to that needed before the treatment.
  • a muscle is a tissue of the body that primarily functions as a source of power.
  • muscles in the body There are three types of muscles in the body: a) skeletal muscle — striated muscle responsible for generating force that is transferred to the skeleton to enable movement, maintenance of posture and breathing; b) cardiac muscle — the heart muscle; and c) smooth muscle — the muscle that is in the walls of arteries and bowel.
  • the methods of the invention are particularly applicable to skeletal muscle but may have some effect on cardiac and or smooth muscle.
  • Reference to skeletal muscle as used herein also includes interactions between bone, muscle and tendons and includes muscle fibres and joints.
  • the fusion proteins of the invention are used to treat conditions such as skin wound healing, (including diabetic wounds and ulcers), skin burns, bone defects and fractures, osteoporosis, osteoarthritis, spinal fusion, ankle fusion, muscle and tendon defects, cartilage defects and degeneration, ischemic tissues (including ischemic limb, ischemic cardiac tissue, and ischemic brain after a stroke).
  • skin wound healing including diabetic wounds and ulcers
  • skin burns including diabetic wounds and ulcers
  • bone defects and fractures including osteoporosis, osteoarthritis, spinal fusion, ankle fusion, muscle and tendon defects, cartilage defects and degeneration
  • ischemic tissues including ischemic limb, ischemic cardiac tissue, and ischemic brain after a stroke.
  • the fusion proteins of the present invention induce tonic signalling in response to a growth factor or cytokine rather than the burst signalling induced in response to the wild type growth factor or cytokine.
  • Tonic signalling refers to controlled slow or graded signalling over a prolonged or sustained period.
  • the induction of tonic signalling rather than burst signalling allows the growth factor or cytokine to exert its effect without associated side effects.
  • the fusion protein comprising VEGF-A and a syndecan binding peptide, particularly a fusion protein of SEQ ID NO: 80 or SEQ ID NO: 81) induces less vascular permeability than VEGF-A alone.
  • the fusion protein comprising PDGF-BB and a syndecan binding peptide, particularly a fusion protein of SEQ ID NO: 79) induces less tumour growth than PDGF-BB alone.
  • Fusion proteins according to the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated and the subject. Fusion proteins may be administered orally, sublingually, buccally, intranasally, by inhalation, transdermally, topically, intra-articularly or parenterally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrathecal, intracranial, injection or infusion techniques.
  • the fusion protein may be administered with or in an implant, medical device or prosthesis.
  • the implant may be a biodegradable implant or slow release depot, or other implant as known to persons skilled in the art. Such embodiment is particularly appropriate for improving muscle growth and strength after muscle trauma or damage.
  • the fusion protein When used to treat burns the fusion protein may be administered orally, topically or parenterally.
  • compositions comprising the fusion protein are to be administered in a therapeutically effective amount.
  • an "effective amount" is a dosage which is sufficient to reduce to achieve a desired biological outcome.
  • the desired biological outcome may be any therapeutic benefit including an increase in muscle mass, an increase in muscle strength, muscle growth, or treatment of burns or wounds. Such improvements may be measured by a variety of methods including those that measure lean and fat body mass (such as duel ray scanning analysis), muscle strength, or the formation of muscle cells.
  • a typical daily dosage might range from about 1 mg/kg to up to 100 mg/kg or more, depending on the mode of delivery.
  • Dosage levels of the fusion protein could be of the order of about 0.1 mg per day to about 50mg per day or will usually be between about 0.25mg to about 1mg per day.
  • the amount of fusion protein which may be combined with the carrier materials to produce a single dosage will vary, depending upon the subject to be treated and the particular mode of administration.
  • a formulation intended for oral administration to humans may contain about 1mg to 1g of the fusion protein with an appropriate and convenient amount of carrier material, which may vary from about 5 to 95 percent of the total composition.
  • Dosage unit forms will generally contain between from about 0.1 mg to 50mg of active ingredient.
  • Dosage schedules can be adjusted depending on the half-life of the fusion protein, or the severity of the subject’s condition.
  • compositions are administered as a bolus dose, to maximize the circulating levels of peptide for the greatest length of time after the dose.
  • Continuous infusion may also be used after the bolus dose.
  • Subject refers to human and non-human animals.
  • non-human animals includes all vertebrates, e.g., mammals, such as non human primates (particularly higher primates), sheep, horse, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc.
  • the subject is an experimental animal, an animal suitable as a disease model, or in animal husbandry (animals as food source), where methods to increase lean muscle mass will greatly benefit the industry. Additionally, the method is particularly important in race horses.
  • the treatment is for humans, particularly adult humans, children aged 11 to 16 years old, aged 4 to 10 years old, infants of 18 months up to 4 years old, babies up to 18 months old.
  • the treatment may also be used for elderly or infirm humans.
  • the treatments of the present invention are used to supplement alternative treatments for the same condition.
  • the fusion proteins can be used to supplement stem cell therapies for joint and muscle repair.
  • ECs Human umbilical vein endothelial cells
  • MCDB-131 endothelial cell basal medium containing EC supplement solution (Promocell).
  • MSCs were isolated form compact bone of C57BL/6 mice as described previously 51 and expanded until passage three with a-MEM (100 mg ml -1 penicillin/streptomycin, 10% FBS). Cells used for experiments were at passage 3-4.
  • PDGF-BB (SEQ ID NO: 27), PDGF-BB-SB, ⁇ X 2 PI I.8 -M-PDGF- BB (SEQ ID NO:82) and a 2 Pli- 8 -M-PDGF-BB-SB (SEQ ID NO: 79) were cloned into the protein expression vector pcDNA3.1 + (ThermoFisher Scientific) PDGF-BB variants were produced as described in Martino, M.M., et al. (2014) and purified via size exclusion.
  • VEGF- A121 (SEQ ID NO: 20), VEGF-A165 (SEQ ID NO: 19), VEGF-A-SB, a 2 Ph.
  • VEGF-A165 SEQ ID NO: 83
  • 2 Ph. 8 -M-VEGF-A121 SEQ ID NO: 84
  • 2 Ph. 8 -M-VEGF-A-SB SEQ ID NO: 80
  • VEGFs were expressed into E.coli BL21 (Ds3) pLys (Novagen).
  • VEGF-A variants were produced, refolded and purified by size exclusion as described in Traub, S., et al. (2013).
  • SB and CX2PI1-8-M-SB were produced as a glutathione S-transferases (GST) fusion as described in Martino, M.M.
  • GST was cleaved using precision protease (GE Healthcare). SB and CX2PI1-8-M-SB were purified via size exclusion. The concentration of recombinant proteins was determined via A280 and extinction coefficient correction. The proteins were verified as >99% pure by SDS-PAGE and MALDI-TOF.
  • MSCs primary antibodies used were anti- syndecan-1 biotin (Miltenyi Biotech, clone REA104), anti-syndecan-2 PE (Abeam, polyclonal, ab205884), and anti-human syndecan-3 APC (R&D Systems, polyclonal, FAB3539A), and anti-syndecan-4 APC (R&D Systems, AF2918). Biotin was subsequently detected via streptavidin-PE (Biolegend), and anti-syndecan-2 was detected via a secondary goat anti-rabbit H&L Alexa Fluor 488 (Abeam, ab150077).
  • antibodies were anti- syndecan-1 FITC (Miltenyi Biotech, clone 44F9), anti-syndecan-2 PE (Miltenyi Biotech, clone REA468), anti-syndecan-3 APC (R&D Systems, polyclonal) and anti-syndecan-4 APC (R&D Systems, AF2918).
  • the same protocol was used for PDGF-BB receptor detections.
  • Antibodies were anti-mouse CD140b APC (Biolegend, clone APB5) and anti-mouse CD140a Biotin (Biolegend, clone APA5) conjugated to Streptavidin PE (Biolegend) for MSCs, antihuman CD140b PE (Miltenyi, clone REA363) conjugated to Streptavidin APC (Biolegend) and anti-human CD140a FITC (Miltenyi Biotech, clone REA911) for ECs. All antibodies were used at 0.5 mg ml -1 . Cell samples were acquired with a flow cytometer (Cyan, Beckman Coulter) and data were analysed with FlowJo Software (TreeStar).
  • MSCs and ECs were seeded in 6-well plates and cultured until 70-80% confluency. MSCs and ECs were starved 24 h with a-MEM containing 1% FBS and MCDB-131 containing 2% FBS, respectively. Then, cells were stimulated with 20 ng ml -1 of PDGF-BB or PDGF-BB-SB (for MSCs), and with 20 ng m of VEGF-A165, VEGF-A121, or VEGF-A-SB (for ECs). For the conditions with SB in excess, the concentration of SB was 2 mg ml -1 .
  • ELISA Mae PDGF-Rp and Human Phospho-VEGF R2/KDR Duoset, Cell Signalling Technology, R&D Systems
  • MSCs and ECs were seeded in 6-well plates and cultured until 70-80% confluency. MSCs and ECs were starved 24 h with a-MEM containing 1% FBS and MCDB-131 containing 2% FBS, respectively. Then, cells were stimulated with growth factor variants (20 ng ml -1 ) for 10, 180 or 360 min (3 wells per condition and per time points that were further pulled together). Phosphorylation and cleavage of intracellular signalling molecules were detected using an antibody array (PathScan Intracellular Signalling Array, Cell Signalling) according to the manufacturer's instructions. The chemiluminescent signals were detected using ImageQuant LAS 4000 and quantified with ImageQuant TL software (GE Healthcare Life Sciences).
  • MSCs (100 ⁇ 00 cells) were plated on 6-well plates with low serum medium (a-MEM, 100 mg ml -1 penicillin/streptomycin, 2% FBS) for 48 h. Then, cells were treated with PDGF- BB variants (10 ng ml -1 ) for 5, 15, 30 and 60 min. As a negative control (basal level of receptor), cells were treated with PBS. Following stimulation, cells were detached with TrypLE diluted 1:2 in PBS supplemented with 20 mM EDTA.
  • a-MEM low serum medium
  • PDGF- BB variants 10 ng ml -1
  • PBS positive control
  • TrypLE was quenched with medium containing 10% FBS and cells were stained with viability fixable dye (Zombie Aqua, Biolegend) for 10 min at room temperature followed by anti-mouse CD140b APC (Biolegend, clone APB5) in FACS Buffer (PBS with 1 % BSA and 5 mM EDTA) for 30 min on ice.
  • viability fixable dye Zombie Aqua, Biolegend
  • FACS Buffer PBS with 1 % BSA and 5 mM EDTA
  • To assess internalization only cell-surface staining was performed. To assess degradation, surface and internal staining was performed. In that case, cells were fixed and permeabilized (eBioScience FoxP3 fixation and permeabilization kit) for 30 min on ice and stained with anti-mouse CD140b for 30 min on ice.
  • MSCs and ECs were plated on glass slides chambers (m-Slide 8 Well, Ibdid, 10,000 cells per well) coated with a solution of 1% gelatin.
  • Cells were cultured in 400 mI of media (MSCs with a-MEM, 100 mg ml -1 penicillin/streptomycin, 10% FBS; ECs with MCDB-131,
  • Rabbit anti-syndecan-2 (1 mV ml -1 , Abeam, ab205884), rabbit anti- syndecan-4 (1 mV ml -1 , Abeam, ab24511) and goat anti-PDGF-BB (2 mg ml -1 , Abeam, ab111310) or mouse anti-VEGF-A (2 mg ml -1 , Abeam, clone VG-1).
  • Goat anti-rabbit IgG Alexa Fluor 594, 2 mg ml -1 , Abeam, ab150088
  • donkey anti-goat IgG Alexa Fluor 488, 2 mg ml -1 , Abeam, ab150129
  • goat anti-mouse IgG Alexa Fluor 488, 2 mg ml -1 , Abeam, ab150117
  • Fibrin matrices were made as described in Martino, M.M., et al. (2009) with human fibrinogen. Briefly, fibrin matrices were generated with 8 mg ml -1 fibrinogen, 2 U ml -1 human thrombin (Sigma-Aldrich), 4 U ml -1 factor Xllla (Fibrogammin, Behring), 5 mM CaCL, and 500 ng ml -1 of growth factor. Fibrin gels were polymerized at 37 °C for 1 h and transferred in Ultra Low Cluster 24-well plate (Corning Incorporated) containing 500 mI of buffer (20 mM Tris-HCI, 150 mM NaCI, 0.1% BSA, pH 7.4). A control well that served as 100% released control contained only the growth factors in 500 mI of buffer. Each 24 h, buffers were removed, kept at -20 °C and replaced with fresh buffer. For the 100% release control well,
  • MMPs 20 mI of buffer was taken out every day and stored at -20 °C. After 7 days, growth factor cumulative release was quantified using ELISA using the 100% released control as reference (DuoSet, R&D Systems).
  • release buffer contained 0.25 mg ml -1 of recombinant MMP-1 and MMP- 2 (Sigma). MMPs were reconstituted at 50 mg ml -1 in 50 mM Tris, 10 mM CaCL, 150 mM NaCI, 0.05% Brij-35, pH 7.5. Then, MMPs were activated by adding p-amino-phenylmercuric acetate (1 mM) for 1 h at 37 °C and stored at -80 °C until used.
  • Proliferation assays were performed as described in Martino, M.M. & Hubbell, J.A. (2010). Briefly, cells in large culture flasks were starved for 24 h (a-MEM, 100 mg ml -1 penicillin/streptomycin, 2% FBS for MSCs; MCDB-131 , 100 mg ml -1 penicillin/streptomycin, 5% FBS for ECs).
  • cells were seeded on 96-well cell culture plate (3,000 cells per well) with growth factor variants in low serum medium (300 mI of a-MEM, 100 mg ml -1 penicillin/streptomycin, 2% FBS for MSCs; 300 mI of MCDB-131 , 100 mg ml -1 penicillin/streptomycin, 5% FBS for ECs).
  • low serum medium 300 mI of a-MEM, 100 mg ml -1 penicillin/streptomycin, 2% FBS for MSCs
  • 300 mI of MCDB-131 100 mg ml -1 penicillin/streptomycin, 5% FBS for ECs.
  • cell number was quantified using CyQUANT dye (Invitrogen). Florescence intensity was measured with a florescence plate reader. For the 9- and 12-day time points, media was changed once without the addition of growth factors.
  • Cell proliferation was analysed by calculating percentage proliferation increases over basal proliferation (without growth factors
  • a high-throughput multichamber fluidic device was used to compare the effects of VEGF-A variants on endothelial morphogenesis in the 3D environment as described in Bonvin, C., et al. (2010).
  • Fibrin matrices were prepared to have a final concentration of 4 mg ml -1 human fibrinogen (depleted of fibronectin, plasminogen, and von Willebrand factor; Enzyme Research Laboratories), 2 U ml -1 human thrombin (Sigma), 2 U ml -1 factor Xllla (Fibrogammin, Behring), 2.5 mM of CaCh.
  • ECs (75,000 cells per 50 mI of gel) were mixed together with the fibrinogen solution and VEGF-A variants (50, 200, or 500 ng ml -1 ) before matrix polymerization via a thrombin solution of equal volume.
  • the culture medium was MCDB-131 containing 2% FBS, 1.7 mg ml -1 of aprotinin (Roche), 100 mg ml -1 penicillin/streptomycin, and 4 mM L-glutamine added to both the central and outer medium reservoirs.
  • the multichamber filled with fibrin only was incubated with the medium containing 200 mg ml -1 of endothelial cell growth supplement (ECGS) as positive control multichamber.
  • ECGS endothelial cell growth supplement
  • mice C57BL/6, 10-12 wk old were first anesthetized with isoflurane. The top of their head was shaved and a longitudinal incision was performed to reveal the skull. Bone tissue was exposed by retracting the soft tissues. Using a micromotor drill, two craniotomy defects (4.5 mm diameter) were created in the parietal bones of the skull on each side of the sagittal suture line.
  • mice received a subcutaneous injection of Tramdol (100 mg kg -1 ). Experiments were performed in accordance with the Monash University Animal Ethics Committee and the Animal Research Committee of the Research Institute for Microbial Diseases of Osaka University.
  • microCT Microcomputed tomography
  • Skulls were scanned with a microCT 40 (Scanco Medical AG) operated at energy of 70 kVp and intensity of 145 ms. Scans were performed at high-resolution mode resulting in a nominal isotropic resolution of 30 mhi. After reconstruction, a 3D Gaussian filter (sigma 1.2, support 1) was applied to all images. Bone was segmented from background using a global threshold of 22.4% of maximum grey value. Afterwards, cylindrical masks were placed manually at the defects. Bone volume within these masks was calculated using a standardized procedure developed for quantitative bone morphometry as described in Hildebrand, T., (1999). Coverage was calculated on a dorso-ventral projection of the cylindrical area as described in Lutolf, M.P., et al., (2003).
  • the wounds were covered with non-adhering dressing (Adaptic, Johnson & Johnson) and adhesive film dressing (Hydrofilm, Hartmann). After 10 days, animals were euthanized and the wounds were harvested for histological analysis. An area of 8 mm in diameter was excised and wounds were embedded. Histological analysis was performed on serial sections (20 mhi cryosections and 4 mGP paraffin sections) until reaching the central portion of the wound. The extent of re- epithelialization and granulation tissue formation was measured by histomorphometric analysis of tissue sections (H&E stain) using ImageJ software (National Institutes of Health, USA).
  • the distance that the epithelium had traveled across the wound was measured.
  • the muscle edges of the panniculus carnosus were used as indicator for the wound edges and re-epithelialization was calculated as the percentage of the distance of edges of the panniculus carnosus muscle.
  • tissue quantification the area covered by a highly cellular density tissue was determined and normalized with the distance of muscle edges of the panniculus carnosus, in order to obtain the area at the centre of the wound.
  • E.G7-OVA thymoma cells were obtained from American Type Culture Collection (ATCC) and grown in RPMI medium 1640 supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 30 mM Hepes, 50 mM 2-mercaptoethanol, and 0.4 mg/ml G418 antibiotic (Sigma).
  • Tumor cells (10 6 ) were implanted subcutaneously in the back at the level of the junction between the thoracic and lumbar vertebrae of C57BL/6 mice (10 wk old) in 30 pi of PBS. On the same day, mice received PDGF-BB variants delivered in two fibrin matrix on the calvaria (10 mg/matrix).
  • Tumors were measured every day and volumes were calculated as ellipsoids based on three orthogonal measures as described in Julier, Z., et al. (2015). Animal were monitored for 14 days. Animals having a tumor volume > 1000 mm 3 before 14 days were humanly killed. Experiments were performed in accordance with the Cantonal Veterinary Office of Canton de Vaud and the Animal Research Committee of the Research Institute for Microbial Diseases of Osaka University. Plasma concentration of growth factors
  • Calvarial defect and full thickness wounds were created in C57BL/6 mice (10 wk old) as described above. Calvarial defects were directly treated with PDGF-BB variants delivered via a fibrin matrix (10 mV growth factor per matrix). Skin wounds were directly treated with VEGF-A variants delivered via a fibrin matrix as described (5 mV growth factor per matrix). Blood (100 mI) was collected after 6h, 12h and 24h until 7 days by tail bleeding in anticoagulant coated tubes (Eppendorf). Directly after collection samples were centrifuge for 15 min at 1,000 x g. Plasma concentrations of growth factors were detected by ELISA (VEGF and PDGF-BB Quantikine ELISA Kit, R&D Systems). Experiments were performed in accordance with the Animal Research Committee of the Research Institute for Microbial Diseases of Osaka University.
  • Miles assay was performed with 8-10 wk old male BALB/c mice as described in Xu, D., et al (2011). Briefly, the back of the mouse was shaved and divided into four treatment areas. Evan’s blue solution (100 mI, 1% in PBS) was injected intravenously in the tail vein. After 15 min, 20 mI of PBS containing 100 ng of VEGF-A variants were injected intradermally into the back. PBS was applied as a negative control. The mice were sacrificed after 30 min and the back skin was removed, photographed, and the treated area was cut out with a biopsy punch. The Evans’s blue dye was extracted from the tissue with formamide at 55°C for 24 h. The extracted dye was quantified by measuring absorbance at 605 nm and converted to mass using a standard curve. Experiments were performed in accordance with Cantonal Veterinary Office of Canton de Vaud.
  • the model was performed as described in Martino, M.M., (2014) on 8-10 wk old male BALB/c mice. Mice were used and anesthetized with a 200 mI intraperitoneal injection of mixture of dormitore (0.05 mg ml -1 ) and ketamine (12.5 mg ml -1 ). Body temperature was kept at 37 °C by using heating pad with a rectal thermistor. The ventral side of the ear dermis and the cartilage under the tissue was disconnected from dorsal dermis by cutting the ventral skin and the cartilage along through the antihelix pinna. Ventral dermis and cartilage were gently peeled off and completely removed with tweezers.
  • the flap was stabilized on a glass slide by gluing the intact part, and 200 mI TRITC labelled 155 kDa dextran (200 pi, 120 mg ml -1 , Sigma) was injected intravenously via the tail vein. Initial leakage due to the increased blood pressure was washed twice with 5 ml Ringer’s buffer. Isotonic ascorbate ringer (140 mM sodium ascorbate, 25 mM HEPES, 4 mM KCI and 2 mM CaCh, pH 7.5) was applied on the dermis to prevent the photo bleaching and ear was protected with a coverslip.
  • Isotonic ascorbate ringer 140 mM sodium ascorbate, 25 mM HEPES, 4 mM KCI and 2 mM CaCh, pH 7.5
  • I aging was started 30 min after the TRITC dextran injection in order to equilibrate a balanced blood pressure and vascular leakage.
  • Vascular leakage was imaged by using a fluorescence stereomicroscope equipped with a motorized stage (M250 FA, Leica Microsystems CMS GmbH) with 1 x lens providing linear system magnification 7.5-160 x. Images were collected, and the mean intensity was calculated by Leica LASA. In the first 25 min, basal vascular permeability was visualized after applying 50 mI ascorbate ringer.
  • Example 1 Engineering growth factors for enhanced binding to syndecans:
  • Syndecans have numerous ligands, including laminins, which are major glycoproteins in the basement membrane.
  • laminins which are major glycoproteins in the basement membrane.
  • the globular domain of laminin alpha 1 chain binds heparan sulfate in syndecans with high affinity (mM-hM range), via the sequence RKRLQVQLSIRT (laminin subunit alpha-12719-2730), commonly known as AG73 and named SB in this study (Fig. 1 b).
  • the inventors sought to engineer PDGF-BB and VEGF-A to include the SB sequence.
  • the natural heparin-binding domain of PDGF-BB - which is likely the syndecan-binding domain - is implicated in PDGF-BB binding to its receptor.
  • SB was added at the C-terminus of PDGF-BB to generate PDGF-BB-SB and maintain receptor binding.
  • SB was added at the C-terminus of VEGF-A121 which does not contain a syndecan-binding domain (Fig. 1c).
  • VEGF-A121 did not show binding to syndecans (Fig. 4). While VEGF-A165 and PDGF-BB naturally have binding affinity for some syndecans, the engineered growth factors showed stronger binding to all syndecans (syndecans 1-4), with dissociation constant values (KD) decreasing up to 7-fold (Fig. 1e, Table 1). Interestingly, VEGF-A-SB does not bind to neuropilin-1 , a VEGF-A coreceptor that enhances binding of VEGF-A to its main receptor (Fig. 5).
  • ELISA enzyme-linked immunosorbent assay
  • KD Dissociation constants
  • Example 2 Enhancing binding to syndecans triggers tonic signalling
  • MSCs express high levels of PDGF-BB receptor-b (PDGFR-b, Fig. 6), and ECs express high levels of VEGF-A receptor-2 (VEGFR-2).
  • both cell types express syndecans 1-4 (Fig. 7).
  • syndecan-binding growth factors induced lower phosphorylation levels during the first 30 min.
  • phosphorylation levels stayed significantly higher with the syndecan-binding growth factors.
  • phosphorylation levels of AKT and ERK1/2 a similar effect was observed with phosphorylation levels of AKT and ERK1/2 (Fig. 2a, Fig.
  • syndecan-binding growth factors display very different signalling kinetics. Growth factors with enhanced binding to syndecans trigger a relatively low but sustained form of signalling (i.e. tonic signalling). In contrast, wild-type growth factors induce a strong but short form of signalling (i.e. burst signalling) (Fig. 2d).
  • Example 3 Syndecan-binding growth factors have higher morphogenic activity
  • ci 2 Pli- 8 -VEGF-A165 induced the formation of structures to some extent, but c ⁇ Ph-s-VEGF-A-SB and the positive control EC growth supplement (ECGS) promoted significantly more multi-cellular EC structures with capillary-like organization.
  • ECGS positive control EC growth supplement
  • the total number of cells and the total number of structures per volume of matrix were significantly higher with c ⁇ Ph-s-VEGF-A-SB (Fig. 4b-d).
  • the effect of c ⁇ Ph-s-VEGF-A-SB was dose-dependent (Fig. 4d, Fig. S12).
  • PDGF-BB is known to promote bone regeneration to some extent by stimulating MSC and pre-osteoblast migration as well as proliferation.
  • the wild-type growth factor is usually inefficient for regenerating critical-size bone defects.
  • CX 2 PI 1-8 -M-PDGF-BB-SB would be more efficient in promoting bone regeneration than PDGF-BB and CX 2 PI 1-8 -M-PDGF-BB.
  • CX 2 PI 1-8 -M-PDGF-BB significantly enhanced bone regeneration
  • CX 2 PI 1-8 -M-PDGF-BB-SB led to a marked increase of bone tissue deposition compared to CX 2 PI 1-8 -M-PDGF-BB (Fig. 5), yielding coverage at about 95%.
  • Example 5 Syndecan-binding VEGF-A improves diabetic wound healing
  • VEGF-A is known to induce skin wound healing to some extent, but the wild-type growth factor is usually inefficient for promoting wound closure.
  • CX 2 PI 1-8 -M-VEGF-A-SB would be more potent than wild-type and fibrin-binding VEGF-As.
  • db/db diabetic mice
  • Delivering micrograms of wild-type VEGF-A is typically ineffective to promote wound healing in diabetic mice.
  • CX 2 PI 1-8 -M-VEGF-A-SB significantly enhanced wound healing compared to both fibrin only and fibrin-binding VEGF-A treatments (Fig. 6a, b, Fig. S13).
  • angiogenesis is a key step in sustaining newly formed granulation tissue, we compared how it differed between treatments.
  • Immunohistological analysis for CD31 which is highly expressed by ECs, and desmin, which is expressed by smooth muscle cells (SMCs) and pericytes, revealed that angiogenesis within the granulation tissue was much more pronounced and likely more stable when wounds were treated with CX 2 PI 1-8 -M-VEGF-A- SB (Fig. 6c, d).
  • PDGF-BB has received a warning from the USFDA for potentially increasing cancer risk.
  • PDGF-BB variants accelerate tumour growth when delivered at a distant site.
  • Tumor cells were implanted in the back of mice and PDGF-BB variants were delivered on the calvaria in a fibrin matrix.
  • PDGF-BB and CX 2 PI 1-8 -M-PDGF-BB accelerated tumor growth but not CX 2 PI 1-8 -M-PDGF-BB-SB (Fig. 7a).
  • VEGF-A variants plasma concentrations of CX 2 PI 1-8 -M-VEGF-A-SB were significantly lower compared to CX 2 PI 1-8 -M-VEGF-AI2I and CX 2 PI I-8 -M-VEGF-A165, after delivery in skin wounds via a fibrin matrix (Fig. 7b).
  • Vascular permeability is a major problem that emerged in translating VEGF-A to clinical use. Because CX2PI1-8-M-VEGF-A-SB displays slower activation of VEGFR-2 compared to CX2PI1-8-M-VEGF-AI2I and CX 2 PI I-8 -M-VEGF-A165, we hypothesized that syndecan-binding VEGF-A would induce less vascular permeability.
  • a modified Miles assay This assay is a classic method to assess alterations in vessel permeability and to evaluate anti-VEGF-A drugs, as described in Radu, M. & Chernoff, J. (2013).
  • the most common strategy to deliver growth factors at a tissue lesion is to control their release from biomaterials.
  • the growth factor-binding domain acts as a linker between the biomaterial and growth factors, providing a classic controlled release that is ideally driven by the biomaterial degradation.
  • this approach heavily depends on the binding affinity of growth factors with the linker and works only if the biomaterial remains present at the injured site.
  • the delivery of more than one growth factor is often required to obtain good regenerative effects.
  • the tonic signalling generated by syndecan- binding growth factors is specifically due to binding to syndecans via the SB sequence, because saturation of binding sites by using the SB peptide in excess or degradation of syndecans with heparinases abolished tonic signalling as well as cell-surface retention of the growth factors (Fig. 2a, c).
  • experiments done with the SB peptide in excess and with heparinases indicate that fusing VEGF-A121 and PDGF-BB to the SB sequence does not change signalling via changing affinity to growth factor receptors, because - in those conditions - wild-type and syndecan-binding growth factors induce similar signalling.
  • PDGF-BB and VEGF-A165 do not induce a tonic from of signalling, while they bind syndecans to some extent (Fig. 1e, Table 1).
  • SB-fused growth factors to multiple syndecans is likely critical to induce a tonic signalling.
  • the stronger and faster signalling of VEGF-A165 compared to VEGF-A-SB can be attributed to VEGF-A165 binding to neuropilin-1 (Fig. S5) which facilitate VEGF-receptor signalling 35 .
  • syndecan-binding PDGF-BB To assess the effects of syndecan-binding PDGF-BB, we chose MSC colony formation and MSC proliferation, since these cells express high levels of PDGFR-b and syndecans, and because they are critical for bone regeneration.
  • syndecan- binding PDGF-BB has a greater capacity to stimulate MSC colony formation and MSC proliferation (Fig. 3b-d).
  • EC proliferation induced by syndecan-binding VEGF-A Interestingly, the proliferative effect was observed only a late time points. Considering that cells were stimulated only once, these results support that the activity of syndecan-binding growth factors is preserved over time due to their ability to stay bound to cells.
  • the SB sequence alone had no significant effect on MSCs nor ECs (Fig. 3b-d), although it has been reported to have some activity on ECs when coupled to biomaterials. This lack of effect is most likely due to the use of the SB peptide in a soluble form.
  • the SB sequence we utilized (AG73) has been shown to be active in some models at mM concentrations, while, in this study, the concentration of growth factor-fused with SB was in the nM range.
  • PDGF-BB has gained great interest for bone regeneration because it stimulates MSC and pre-osteoblast migration/proliferation.
  • PDGF-BB also supports angiogenesis by targeting pericytes and smooth muscle cells. While the potential of PDGF-BB for bone regeneration is supported by several reports, other studies have failed to show significant effects.
  • PDGF-BB is approved as an alternative to bone autograft during ankle and hindfoot fusion surgery. Therefore, we tested whether the tonic signalling triggered by syndecan-binding PDGF-BB could promote superior bone regeneration. As expected, ci 2 Pli.
  • 8-M-PDGF-BB displayed significantly better bone regeneration compared to PDGF-BB, since the release of PDGF-BB was not controlled due to its weak binding to fibrin (Fig. S11). Most likely, slowing the release of wild-type PDGF-BB by delivering it in fibrin functionalized with a growth factor binding-domain linker would have triggered better bone regeneration.
  • VEGF-A is well-known to support the formation of granulation tissue in wounds via neovessel formation.
  • VEGF-A has been widely explored for chronic wounds, the growth factor failed to show effectiveness beyond phase I trials.
  • slowing the release of VEGF-A using the 0 2 Pli- 8 system slightly improved wound healing and angiogenesis, because the growth factor is released in a controlled manner via fibrin degradation and MMPs.
  • PDGF-BB has received a warning from the USFDA for potentially increasing cancer risk. Comparing the tumor growth effect of PDGF-BB variants when delivered at a distant site of implanted tumor cells, we found that wild-type PDGF-BB and fibrin-binding PDGF-BB accelerate tumor growth. In contrast, syndecan-binding PDGF-BB had no effect (Fig. 7a), likely due to the very low diffusion of CX2PI1-8-M-PDGF-BB-SB from the delivery site into the circulatory system (Fig. 7b).
  • VEGF-A is one of the most promising growth factors for induction of therapeutic angiogenesis
  • a major impediment for its application lies in its ability to rapidly induce vascular permeability - a side effect that clearly limits clinical translation 1 ⁇ 4 .
  • a2Pli-8-M-VEGF-A-SB induces much less vascular permeability compared to ci 2 Pli- 8 -M-VEGF-A121 and ci 2 Pli- 8 -M-VEGF-A165, even when not fibrin-associated (Fig. 7c-f).
  • This effect is likely due to the capacity of c ⁇ Ph-s-M-VEGF-A-SB to stay bound to syndecans before reaching its receptor, although c ⁇ Ph-s-M-VEGF-A-SB may also bind other heparan sulfate proteoglycans in the ECM, further delaying signalling of the engineered growth factor to its main receptor.
  • TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine. Sci Rep 5, 8569 (2015).

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Abstract

The invention provides fusion proteins comprising a growth factor or cytokine and a syndecan binding peptide and the use of such fusion proteins in regenerative medicine, particularly in treating skin wounds, including diabetic wounds and ulcers, skin burns, bone defects or fractures, osteoporosis, osteoarthritis, spinal fusion, ankle fusion, muscle or tendon defects, cartilage defects or degeneration, or ischemic tissues, including ischemic limb, ischemic cardiac tissue, and ischemic brain after a stroke. The fusion proteins also reduce side effects associated with growth factor or cytokine administration, and reduce internalisation and desensitisation associated with growth factor administration.

Description

CYTOKINE OR GROWTH FACTOR FUSION PROTEINS
Field
The invention relates to cytokine or growth factor fusion proteins and uses thereof.
Background
All references, including any patents or patent application, cited in this specification are hereby incorporated by reference to enable full understanding of the invention. Nevertheless, such references are not to be read as constituting an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents.
Growth factors and cytokines are signalling molecules that control cell activities in an autocrine, paracrine or endocrine manner. They exert their biological functions by binding to specific receptors and activating associated downstream signalling pathways which in turn, regulate gene transcription in the nucleus and ultimately stimulate a biological response.
Growth factors and cytokines affect a wide variety of physiological processes such as cell proliferation, differentiation, apoptosis, immunological or hematopoietic response, morphogenesis, angiogenesis, metabolism, wound healing and maintaining tissue homeostasis in adult organisms. The abnormal production or regulation of growth factors and cytokines can cause various diseases such as cancer, liver fibrosis and bronchopulmonary dysplasia. Historically, growth factors were thought to be biological moieties that have a positive effect on cell growth and proliferation while cytokines were typically considered to have an immunological or hematopoietic response. However, as different lines of research have converged, it has been found that ‘cytokines’ and ‘growth factors’ can have similar functions and therefore, these terms are now used interchangeably.
Based on their crucial roles, many growth factors and cytokines have been utilized for preclinical and clinical applications. However, most growth factors that have entered clinical trials have failed, or raised major safety concerns, once approved. Safety and cost- effectiveness issues are associated with suboptimal delivery systems, which may lead to uncontrolled signalling.
Therefore, there is a strong need to develop efficient systems to optimize growth factor and cytokine delivery and signalling for a variety of therapeutic applications. The present invention seeks to provide growth factors and cytokines optimized for delivery and controlled signalling. Summary
A first aspect provides a fusion protein comprising a growth factor or cytokine and a syndecan binding peptide.
A second aspect provides a nucleic acid molecule encoding the fusion protein of the first aspect.
A third aspect provides a vector comprising the nucleic acid molecule of the second aspect.
A fourth aspect comprises a cell or a non-human organism transformed or transfected with the nucleic acid molecule of the second aspect or the vector of the third aspect.
A fifth aspect provides a method of making the fusion protein of the first aspect, the method comprising culturing the cell of the fourth aspect under conditions to produce the fusion protein and recovering the fusion protein.
A sixth aspect provides a fusion protein produced by the method of the fifth aspect.
A seventh aspect provides a pharmaceutical or veterinary composition comprising the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect or the cell or non-human organism of the fourth aspect, optionally with one or more excipient and/or carriers.
An eighth aspect provides a method of treatment of a condition in which cytokine or growth factor administration is beneficial, the method comprising administering to a subject in need thereof the fusion protein of the first aspect or the sixth aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the cell or non-human organism of the fourth aspect or the pharmaceutical or veterinary composition of the seventh aspect.
An alternative form of the eighth aspect provides a composition for treatment of a condition in which in which cytokine or growth factor administration is beneficial, the composition comprising the fusion protein of the first aspect or the sixth aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the cell or non-human organism of the fourth aspect or the pharmaceutical or veterinary composition of the seventh aspect.
A further alternative form of the eighth aspect provides use of the fusion protein of the first aspect or the sixth aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the cell or non-human organism of the fourth aspect or the pharmaceutical or veterinary composition of the seventh aspect in the manufacture of a medicament for treating a condition in which cytokine or growth factor administration is beneficial.
A ninth aspect provides a fusion protein comprising platelet-derived growth factor-BB (PDGF-BB) and a syndecan binding peptide.
A tenth aspect provides a method of enhancing tissue regeneration, particularly bone regeneration and/or wound repair comprising administering the fusion protein of the ninth aspect.
An alternative form of the tenth aspect provides a fusion protein comprising platelet- derived growth factor-BB (PDGF-BB) and a syndecan binding peptide for use in enhancing tissue regeneration, particularly bone regeneration and/or wound repair.
A further alternative form of the tenth aspect provides use of a fusion protein comprising platelet-derived growth factor-BB (PDGF-BB) and a syndecan binding peptide in the manufacture of a medicament for enhancing tissue regeneration, particularly bone regeneration and/or wound repair.
An eleventh aspect provides a fusion protein comprising vascular endothelial growth factor A (VEGF-A) and a syndecan binding peptide.
A twelfth aspect provides a method of treating wounds, burns and muscle, cartilage, tendon and bone disorders comprising administering the fusion protein of the eleventh aspect.
An alternative form of the twelfth aspect provides a fusion protein comprising vascular endothelial growth factor A and a syndecan binding peptide for use in treating wounds, burns and muscle, cartilage, tendon and bone disorders.
A further alternative form of the twelfth aspect provides use of a fusion protein comprising vascular endothelial growth factor A and a syndecan binding peptide in the manufacture of a medicament for treating wounds, burns and muscle, cartilage, tendon and bone disorders.
A thirteenth aspect provides a method of inducing tonic signalling in response to a growth factor or cytokine, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
A fourteenth aspect provides a method of reducing growth factor receptor or cytokine receptor internalization and degradation in response to binding by their respective growth factor or cytokine, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
A fifteenth aspect provides a method of reducing desensitization to growth factor stimulation, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide. A sixteenth aspect provides a method of reducing a side effect associated with cytokine or growth factor administration, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
The invention is based on the inventors’ finding that fusion proteins of PDGF-BB or VEGF-A with a syndecan binding peptide did not provide the wild type growth factor burst signalling they anticipated, even though PDGF-BB and VEGF-A165 bind sydecans to some extent.
The enhanced binding to syndecans provided by the fusion proteins changes growth factor signalling kinetics with receptor and downstream kinase phosphorylation levels being much lower during the first hour post-stimulation.
However, to their surprise, the inventors found that receptor and downstream kinase phosphorylation levels increased significantly after the first hour post-stimulation. They propose that this clearly demonstrates that syndecan-binding growth factors induce a tonic form of signalling, which is different from the burst signalling of wild-type growth factors.
They also found that, in line with receptor phosphorylation levels, receptor internalization and degradation were significantly less pronounced after stimulation with syndecan-binding growth factors, indicating that cells are not desensitized to growth factor stimulation.
Interactions of growth factors with cell-surface heparan sulfate proteoglycans are well-known in developmental biology to regulate tissue formation by controlling their bioavailability. Sequestration of morphogens by cell-surface heparan sulfate proteoglycans provides a mechanism to locally control the intensity and kinetics of morphogen signalling - a typical example is for FGF-2. Without wishing to be bound by theory, the inventors propose that by enhancing growth factor binding to syndecans and their retention on the cell surface, they can mimic such a mechanism and locally control the intensity and kinetics of cytokine or growth factor signalling.
To support their theory, the inventors also demonstrated that the tonic signalling generated by syndecan-binding growth factors is specifically due to binding to syndecans via the syndecan binding sequence (evidence provided).
Brief description of the drawings
Embodiments of the invention will now be described by way of example only with reference to the accompanying drawings in which:
Figures 1 a -e provide - (1a) Schematic representation of growth factor (GF) availability to cell-surface growth factor receptors (GFR) depending of growth factor binding to extracellular matrix (ECM) (1) and syndecans (2). In some cases (e.g. FGF-2), binding to syndecans facilitates growth factor-growth factor receptor assembly (2’). Growth factor binding to its main receptor (3) and signalling triggers receptor internalization and eventually degradation (4). The wedge represents increasing growth factor availability for growth factor co-receptors and receptors and at the cell surface. (1b) Location of the syndecan-binding domain SB (RKRLQVQLSIRT, known as AG73) in laminin 111. (1c). SB was added at the C-terminus of VEGF-A121 to generate VEGF-A-SB. The heparin-binding domain of VEGF- A165 is represented by a light grey oval. SB was added at the C-terminus of PDGF-BB to generate PDGF-BB-SB. (1d, 1e). Growth factors (100 nM) were absorbed on enzyme-linked immunosorbent assay (ELISA) plates and further incubated with His-tagged syndecans. Syndecan binding was detected via His-tag and bovine serum albumin (BSA) was used as a control for unspecific binding. Graphs in 1d show absorbance signals with 100 nM of syndecans. Data are means ± SEM (n = 3 independent experiments). For comparisons between PDGF-BB versus PDGF-BB-SB, two-tailed Student’s t-test (***P < 0.001). For comparisons between VEGF-A variants, one-way ANOVA with Bonferroni post hoc test for pair-wise comparisons. Stars above horizontal bars indicate P values between groups as indicated (*** P < 0.001). Graphs in 1e show absorbance signals at increasing concentration of syndecans (5 nM to 1000 nM). Data are means ± SEM (n = 3 independent experiments). The data were fitted by non-linear regression to obtain the binding curves and dissociation constant (KD). KD values and statistics are shown in table 1.
Figure 2 provides an SDS-PAGE showing the analysis of recombinant syndecans produced in mouse myeloma cell line. Theoretical molecular weights (grey lines) based on amino-acid sequences are 24.9 kDa for syndecan-1 (S1, Entry identifier P18827), 14.8 kDa for syndecan-2 (S2, Entry identifier P34741), 35.5 kDa for syndecan-3 (S3, Entry identifier 075056) and 14.7 kDa for syndecan-4 (Entry identifier P31431, S4). Because of the polysaccharide chains, syndecans run much higher on SDS-PAGE. Experiment was repeated independently twice with similar results.
Figure 3 provides graphs showing binding affinity of growth factor variants to syndecans as determined by ELISA. ELISA plates were coated with growth factors variants at 100 nM and further detected with antibodies. No differences in coating efficiency were observed. Data are means ± SEM (n = 3 technical replicates).
Figure 4 provides graphs showing binding affinity of VEGF-A121 to syndecans as determined by ELISA. VEGF-A121 was absorbed on ELISA plates and further incubated with His-tagged syndecans (5 nM to 1000 nM). Syndecan binding was detected via His-tag and bovine serum albumin (BSA) was used as a control for unspecific binding. Data are means ± SEM (n = 3 technical replicates). VEGF-A121 displays very low binding to syndecans, due to the lack of the heparin-biding domain (KD > 1000 nM). Figure 5 provides graphs showing binding affinity of VEGF-A variants to neuropilin-1 as determined by ELISA. ELISA plates were coated with VEGF-A variants (100 nM) and further incubated with his-tagged neuropilin-1 (100 nM). Bound neuropilin-1 was detected using an antibody against the his-tag. VEGF-A-SB cannot bind neuropilin-1, likely because it does not display the KPRR sequence at its C-terminus. These amino acids are naturally displayed in VEGF-A121 and VEGF-A165 at their C-terminus allowing binding to neuropilin- 1. Data are means ± SEM (n = 3 technical replicates).
Figure 6 shows flow cytometry analysis of MSCs and ECs for expression of PDGFR- a and PDGFR-b. PDGFR-b is highly expressed on the surface of MSCs but not on ECs. Experiment was repeated independently three times with similar results.
Figure 7 shows flow cytometry analysis of syndecans 1-4 on MSCs and ECs. The graphs show representative flow cytometry histograms. Black dotted curves represent isotype controls, while grey coloured curves represent signals obtained with anti-syndecan antibodies. Syndecans 1-4 are highly expressed on MSCs. Syndecans 2-4 are highly expressed on ECs. Experiment was repeated independently three times with similar results.
Figure 8 a-d provide data to show that enhancing growth factor binding to syndecans triggers tonic signalling. (8a) Cells were stimulated with growth factor variants (ECs with VEGF proteins at 20 ng ml -1 , MSCs with PDGF-BB proteins at 20 ng ml -1) and SB in excess (SBexcess, 2 mg ml-1). Phosphorylated growth factor receptors (pVEGFR-2 and pPDGFR-b), AKT, and ERK1/2 were quantified using enzyme-linked immunosorbent assays (ELISA). Data are means ± SEM (n = 3 independent experiments). Statistical comparisons are between wild-type growth factors (VEGF-A165, PDGF-BB) and syndecan-binding growth factors. Two-way ANOVA with Bonferroni post hoc test; numbers and stars indicate P values (***P £ 0.001) (8b) Cells were stimulated with growth factors (ECs with VEGF proteins, MSCs with PDGF-BB proteins). The amount of growth factor receptors (VEGFR-2 and
PDGFR-b) present on cell surface as well as the total amount of growth factor receptor (cell surface and intracellular) were measured using flow cytometry using cell surface staining only or both surface and intracellular staining, respectively. To calculate the percentage of receptor internalization and degradation, mean fluorescence intensities (MFI) of stimulated cells were normalized to unstimulated controls for each timepoint. Percentage of receptor internalization or degradation = 100 - 100 x (MF timuiated - MFIUnstained)/(MFIstimuiated - MFlunstained). Data are means ± SEM (n = 6 independent experiments for VEGF-As, n = 9 independent experiments for PDGF-BBs). Two-way ANOVA with Bonferroni post hoc test; numbers and stars indicate P values (***P£ 0.001). (8c) Cells grown on tissue culture wells were treated with saline or with heparinases and further incubated with growth factors (ECs with VEGF proteins 20 ng ml-1, MSCs with PDGF-BB proteins at 20 ng ml-1). Then, cells were fixed, and surface-bound growth factors were detected by on-cell ELISA. Data are means ± SEM (n = 3 independent experiments). Statistical comparisons displayed are between wild-type growth factors (VEGF-A165, PDGF-BB) and syndecan-binding growth factors. Two-way ANOVA with Bonferroni post hoc test; stars indicate P values ( ***p< 0.001). (8d) Schematic representation of the signalling induced by wild-type growth factors and syndecan-binding growth factors. Wild-type growth factors induce strong and rapid signalling (i.e. burst singling), while syndecan-binding growth factors induce a lower but sustained form of signalling (i.e. tonic signalling).
Figure 9 a and b demonstrate signalling of growth factor variants via arrays. MSCs and ECs were stimulated with growth factor variants for 20, 180 and 360 min. Phosphorylation and cleavage of intracellular singling molecules were analysed using an antibody array (chemiluminescence). (9a) Arrays are shown. Extracellular signal-regulated kinase (ERK), serine/threonine kinase (AKT, also referred to as protein kinase B (PKB)), proline-rich AKT substrate of 40 kDa (PRAS40), ribosomal protein S6 (S6RP), Bcl-2- associated death promoter (Bad), glycogen synthase kinase 3 beta (GSK^), c-Jun N- terminal kinase (JNK, also referred to as stress-activated kinases (SAPK)), signal transducer and activator of transcription (ST AT), 5' AMP-activated protein kinase alpha (AMPKa), mammalian target of rapamycin (mTOR), heat shock protein 27 (HSP27), serine/threonine kinase p70 S6 (p70 S6 Kinase), tumor proteins p53 (p53), p38 mitogen-activated protein kinase (p38), poly ADP ribose polymerase (PARP), cysteine-aspartic acid protease 3 (Casapase-3). (9b) Quantification of ERK1/2 (Thr202/Tyr204), AKT (Thr308) and AKT (Ser473) signals (1 array per condition in duplicate).
Figure 10 demonstrates binding of PDGF-BB and PDGF-BB-SB to syndecans using immunostaining. MSCs were cultured on glass slides and incubated with PDGF-BB or PDGF-BB-SB for 10, 180 and 360 min. Then, syndecans 2, 4 and PDGF-BBs were detected by immunostaining. Confirming the high binding of PDGF-BB-SB to syndecans, the engineered growth factor colocalizes with syndecans for an extended period. Scale bar = 20 mGP. Experiment was repeated independently three times with similar results.
Figure 11 demonstrates binding of VEGF-A165 and VEGF-A-SB to syndecans using immunostaining. MSCs were cultured on glass slides and incubated with VEGF-A165 or VEGF-A-SB for 10, 180 and 360 min. Then, syndecans 2, 4 and VEGF-As were detected by immunostaining. Confirming the high binding of VEGF-A-SB to syndecans, the engineered growth factor colocalizes with syndecans for an extended period. Scale bar = 20 mhi. Experiment was repeated independently three times with similar results.
Figure 12 a- d demonstrate that syndecan-binding growth factors have enhanced morphogenetic capacity. (12a) Wild-type growth factors and syndecan-binding growth factors were designed with a fibrin-binding sequence derived from alpha-2 plasmin inhibitor (NQEQVSPL, named CX2PI1-8,) and an intervening matrix metalloproteinase sensitive sequence derived from type I collagen (GPQGIWGQ, named M) to allow covalent binding to fibrin matrix and release via enzymatic cleavage. The heparin-binding domain of VEGF- A165 is represented by a light grey oval. (12b) MSCs were stimulated with PDGF-BB variants (20 ng ml-1), CX2PI1-8-M-SB in excess (2 mg ml-1) or saline control. ECs were stimulated with VEGF-A variants (20 ng ml-1), CX2PI1-8-M-SB in excess (2 mg ml-1) or saline control. The percentage of new cells was measured after 3, 6, 9, and 12 days. Percentage of new cells = (number of cells in saline control group/number of cells in stimulation groups) - 1) x 100. Data are means ± SEM (n = 3 independent experiments). Statistical comparisons shown are between wild-type growth factors (VEGF-A165, PDGF-BB) and syndecan-binding growth factors. Two-way ANOVA with Bonferroni post hoc test; numbers and stars indicate P values ( ***p< 0.001). (12c, 12d) MSC were stimulated with PDGF-BB variants. Graphs in 12c show colony-forming unit-fibroblast (CFU-F) and average size of colonies. Data are means ± SEM (n = 6 independent experiments). One-way ANOVA with Bonferroni post hoc test; stars above horizontal bars indicate P values between groups as indicated (*** P£ 0.001). Representative wells (9 cm2) are shown in 12d. Experiment was repeated independently six times with similar results.
Figure 13 a and b demonstrate the release of growth factor variants from fibrin matrix. Fibrin matrices were made with the various growth factors and incubated in 10 times volume of release buffer that was changed every day. (13a) PDGF-BB and VEGF-A165 are quickly released, while a2Pli-8-M-PDGF-BB, a2Pli-8-M-PDGF-BB-SB, a2Pli-8-M-VEGF-A121, CX2PI1-8-M-VEGF-AI65 and CX2PI1-8-M-VEGF-A-SB stay within the matrix, due to the fibrin binding sequence (CX2PI1-8). Data are means ± SEM (n = 3 independent experiments). (13b) The same experiment was performed with MMP-1 and MMP-2 in the release buffer. Growth factors containing CX2PI1-8-M are gradually released. Data are means ± SEM (n = 3 independent experiments).
Figure 14 a-d show syndecan-binding VEGF-A has enhanced capacity to induce EC assembly. (14a) Schematic representation of the 3D microfluidic system with nine chambers. Chambers were filled with a growth factor-functionalized fibrin matrix containing ECs and incubated under flow for 4 days. Matrices were functionalized with CX2PI1-8-M-VEGF-A-SB, CX2PI1-8-M-VEGF-AI65, or CX2PI1-8-M-VEGF-AI2I. Unfunctionalized matrix (fibrin only) is represented in white. Empty chambers and media are represented in pink. EC growth supplement (ECGS, 200 mg ml-1) was used as a positive control in a separate multichamber system. (14b) Representative structure formed at 500 ng ml-1 of VEGF-A variants. Scale bar = 100 mGP. Experiment was repeated independently four times with similar results. (14c) Quantification of EC number per volume of matrix at 500 ng ml-1 of VEGF-A variants. Data are means ± SEM (n = 4 independent experiments for ECGS and fibrin only, n = 6 independent experiments for VEGF-A variants). One-way ANOVA with Bonferroni post hoc test for pair-wise comparisons; stars above horizontal bars indicate P values between groups as indicated (*** P£ 0.001). (14d) Number of ECs per structure depending on VEGF- A variant concentration (50, 200, and 500 ng ml-1). Data are means ± SEM (n = 4 independent experiments for ECGS, fibrin only and VEGF-A variant concentration at 50 and 200 ng ml-1, n = 6 independent experiments for VEGF-A variants at 500 ng ml-1). One-way ANOVA with Bonferroni post hoc test for pair-wise comparisons; number above horizontal bars indicate P values between groups as indicated.
Figure 15 a and b show dose dependent structure formation induced by syndecan- binding VEGF-A. (15a) Representative structure formed at 50 ng ml-1, 200 ng ml-1, and 500 ng ml-1 of CX2PI1-8-M-VEGF-A-SB. Scale bar = 100 mhi. (15b) 3D reconstruction of a typical lumen-like structure formed at 500 ng ml-1 of CX2PI1-8-M-VEGF-A-SB. Scale bar = 50 mhi. Experiment was repeated independently four times with similar results.
Figure 16 a-c show syndecan-binding PDGF-BB improves bone regeneration. Critical size calvarial defects (4.5 mm diameter) in mice were treated with PDGF-BB variants (2 mg) delivered via a fibrin matrix. Eight weeks after treatment, bone regeneration was measured by microCT. (16a) Representative calvarial reconstructions are shown. Original defect area is shaded with a blue dotted outline. Experiment was repeated independently two times with similar results. (16b, 16c) Quantification of coverage of the defect and volume of new bone formed. Data are means ± SEM (n= 6 defects for fibrin only and PDGF-BB, n = 8 defects for CX2PI1-8-M-PDGF-BB and CX2PI1-8-M-PDGF-BB-SB). One-way ANOVA with Bonferroni post hoc test for pair-wise comparisons; numbers and stars above horizontal bars indicate P values between groups as indicated (*** P£ 0.001).
Figure 17 a-d show syndecan-binding VEGF-A promotes impaired wound healing and angiogenesis. Full-thickness back-skin wounds (6 mm diameter) of diabetic mice (db/db) were treated with VEGF-A variants (200 ng per wound) delivered via a fibrin matrix (a) Representative histology (hematoxylin and eosin staining) after 10 days. Black arrows indicate wound edges; grey arrows indicate tips of epithelium tongue. Scale bar = 1 mm. Higher magnification (5c) of the granulation tissue is shown on the right taken from the locations enclosed in the black dashed squares in the panels on the left. Experiment was repeated independently three times with similar results (b) Granulation tissue area and wound closure were evaluated by histomorphometric analysis of tissue sections. Data are means ± SEM (n = 8 wounds for CX2PII-8-M-VEGF-A165 and CX2PI1-8-M-VEGF-A-SB, n = 10 wounds for fibrin only). One-way ANOVA with Bonferroni post hoc test for pair-wise comparisons; numbers and stars above horizontal bars indicate P values between groups as indicated (*** P£ 0.001). (c) Angiogenesis within the granulation tissue was assessed by staining for ECs (CD31 + cells) and pericytes/SMCs (desmin+ cells) in the wound tissue at 10 days. Representative images that were quantified are shown: E, epidermis; D, dermis; hashed line, basement membrane. Scale bar = 0.2 mm. Experiment was repeated independently three times with similar results (d) Quantification of stained area for CD31 and desmin as well as the overlay. Boxes extend from 25th to 75th percentiles, horizontal bars represent the median and whiskers represent minimum and maximum values (n = 16 area per condition). One-way ANOVA with Bonferroni post hoc test for pair-wise comparisons; numbers and stars above horizontal bars indicate P values between groups as indicated (*** P£ 0.001).
Figure 18 shows fibrin-binding VEGF-A121 does not promote wound healing in diabetic mice. Full-thickness back-skin wounds (6 mm diameter) of diabetic mice ( db/db ) were treated with VEGF-A121 or CX2PI1-8-M-VEGF-AI2I (200 ng per wound) delivered via a fibrin matrix (a) Representative histology (hematoxylin and eosin staining) after 10 days. Black arrows indicate wound edges; grey arrows indicate tips of epithelium tongue. Scale bar = 1 mm. Experiment was repeated independently twice with similar results (b) Granulation tissue area and wound closure were evaluated by histomorphometric analysis of tissue sections. Data are means ± SEM (n = 8 wounds per condition). Statistical comparisons are between fibrin only and other conditions. One-way ANOVA with Bonferroni post hoc test for pair-wise comparisons; NS = non-significant.
Figure 19 a-f show Syndecan-binding growth factors induces less side effects. (19a) E.G7 tumor cells (106 cells) were injected subcutaneously in the back of mice and growth factors (10 mg per matrix) were delivered on the calvaria with a fibrin matrix. Tumor growth was monitored for 14 days. Data are means ± SEM (n = 6 mice per condition). Statistical comparisons are between CX2PI1-8-M-PDGF-BB and CX2PI1-8-M-PDGF-BB-SB. Two-way ANOVA with Bonferroni post hoc test; numbers and stars indicate P values between groups (*** p < 0.001). (19b) PDGF-BB and VEGF-A variants (10 mg per matrix) were delivered via fibrin in calvarial defects and skin wounds, respectively. Plasma levels of growth factors were measured for 7 days. Data are means ± SEM (n = 4 mice per condition). Two-way ANOVA with Bonferroni post hoc test; numbers and stars indicate P values between groups (*** p < 0.001). (19c, 19d) Mice were injected with Evans blue dye i.v., and 100 ng of VEGF- A variants were injected intradermally in the back. After 15 min, the back skin was collected to observe Evans blue leakage (c) Representative Evans blue leakage induced by the VEGF-A variants. Scale bar = 0.5 cm. Experiment was repeated independently two times with similar results (d) Mass of Evans blue extracted from the leakage area. Data are means ± SEM (n = 10 mice injected with the four conditions). Statistical comparisons are between saline and other conditions. One-way ANOVA with Bonferroni post hoc test for pair wise comparisons; numbers and stars indicate P values between saline and the VEGF-A groups (*** P£ 0.001). NS = non-significant. (19e, 19f) Mice were injected with fluorescent- dextran i.v., and VEGF-A variants were applied on ear skin flaps that were imaged for 25 min. (e) Representative images of the microvasculature at different time points. Scale bar = 0.2 mm. Experiment was repeated independently two times with similar results. (19f) Measurement of vascular permeability as maximum fluorescence at 25 min and microvasculature leakage rate calculated by taking the slope of the linear fit of polynomial release curve. Data are means ± SEM (n = 4 mice per condition). One-tailed Mann-Whitney test (numbers indicate P values).
Detailed description
As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein.
Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.
Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. It is understood that wherever embodiments are described herein with the language "comprising," otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are also provided.
Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.
As used herein, a "fusion protein” is a protein made from a fusion gene, which is created by joining of two or more genes that originally coded for separate polypeptides.
As used herein "polypeptide" refers to any sequence of two or more amino acids, regardless of length, post-translation modification, or function. Polypeptides can include natural amino acids and non-natural amino acids. Polypeptides can also be modified in any of a variety of standard chemical ways (e.g., an amino acid can be modified with a protecting group; the carboxy- terminal amino acid can be made into a terminal amide group; the amino-terminal residue can be modified with groups to, e.g., enhance lipophilicity; or the polypeptide can be chemically glycosylated or otherwise modified to increase stability or in vivo half-life). Polypeptide modifications can include the attachment of another structure such as a cyclic compound or other molecule to the polypeptide and can also include polypeptides that contain one or more amino acids in an altered configuration (i.e. , R or S; or, L or D).
As used herein, “growth factors” are proteins that regulate many aspects of cellular function, including survival, proliferation, migration and differentiation. Growth factors determine the fate of cells as they differentiate from being progenitors along either neuronal or glial lineages. In addition, during embryonic development, growth factors are crucial for regulating neuronal survival, determining cell fate and establishing proper connectivity. Growth factors typically act as signalling molecules between cells. They often promote cell differentiation and maturation, which varies between growth factors. For example, platelet- derived growth factor BB (PDGF BB) enhances osteogenic differentiation, while fibroblast growth factors and vascular endothelial growth factors stimulate blood vessel differentiation (angiogenesis).
As used herein, “cytokines” are loose category of small proteins (-5-20 kDa) that are important in cell signalling. Cytokines have been shown to be involved in autocrine, paracrine and endocrine signalling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally not hormones or growth factors (despite some overlap in the terminology). Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a given cytokine may be produced by more than one type of cell.
Cytokines act through receptors and are especially important in the immune system; cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations.
They are important in health and disease, specifically in host responses to infection, immune responses, inflammation, trauma, sepsis, cancer, and reproduction
Illustrative examples of growth factors and cytokines contemplated for use in the invention are interleukins including lnterleukin-1 receptor antagonist, Interleukin 2,
Interleukin 4, Interleukin 10, Interleukin 16, Interleukin 33, Interleukin 27b; C-X-C motif chemokines including C-X-C motif chemokine 9, C-X-C motif chemokine 10, C-X-C motif chemokine 12, C-X-C motif chemokine 19, C-X-C motif chemokine 20, C-X-C motif chemokine 22; fibroblast growth factors, including Fibroblast Growth Factor-2, Fibroblast Growth Factor-5, Fibroblast Growth Factor-7, Fibroblast Growth Factor-10, Fibroblast Growth Factor- 18; vascular endothelial growth factors including Vascular Endothelial Growth Factor-A165, Vascular Endothelial Growth Factor-A121, Vascular Endothelial Growth Factor-B, Vascular Endothelial Growth Factor-C; placental growth factors including Placental Growth Factor-1, Placental Growth Factor-2, Placental Growth Factor-4; platelet derived growth factors including Platelet Derived Growth Factor-A, Platelet Derived Growth Factor-B, Platelet Derived Growth Factor-C, Platelet Derived Growth Factor-D; epidermal growth factors including Epidermal Growth Factor, Heparin-Binding Epidermal Growth Factor; Amphiregulin; Epiregulin; Neuriregulin-2; insulin-like growth factors including Insulin-like Growth Factor-I, Insulin-like Growth Factor-ll; transforming growth factors including Transforming growth factor-bΐ, Transforming growth factory, Transforming growth factor- b3; bone morphogenic proteins including Bone Morphogenetic Protein-2, Bone Morphogenetic Protein-7; Nerve Growth Factor; Neurotrophin-3; Brain-derived Neurotrophic Factor and Protein Wnt-3a.
Illustrative human amino acid sequences for these cytokines and growth factors are provided below: lnterleukin-1 receptor antagonist (IL-1Ra):
RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DVVPIEPHALFLGIH GGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLC TAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO: 1) lnterleukin-2 (IL-2):
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEEL KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS IISTLT (SEQ ID NO: 2) lnterleukin-4 (IL-4):
HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTR CLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYS KCSS (SEQ ID NO: 3)
Interleukin- 10 (IL-10):
SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYL GCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAV EQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 4)
Interleukin- 16 (IL-16):
SAASASAASDVSVESTAEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLTINRIFKGAASE QSETVQPGDEILQLGGTAMQGLTRFEAWNIIKALPDGPVTIVIRRKSLQSKETTAAGDS (SEQ ID NO: 5)
Interleukin-33 (IL-33):
AFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDK VLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFF VLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET (SEQ ID NO: 6)
Interleukin-35 (IL-27b):
PPEGVRLSPLAERQLQVQWEPPGSWPFPEI FSLKYWI RYKRQGAARFHRVGPI EATSFILR A V R P R A R YY V Q VA AQ D LT D YG E LS D WS LP AT AT M S L (SEQ ID NO: 7)
C-X-C motif che okine 9 (CXCL9):
TPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELI KKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTT (SEQ ID NO: 8)
C-X-C motif chemokine 10 (CXCL10):
VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLL KAVSKERSKRSP (SEQ ID NO: 9) C-X-C motif chemokine 12 (CXCL12):
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYL EKALNKRFKM (SEQ ID NO: 10)
C-C motif chemokine 19 (CCL19):
GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERII QRLQRTSAKMKRRSS (SEQ ID NO: 11)
C-C motif chemokine 20 (CCL20):
ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYIVRLLS KKVKNM (SEQ ID NO: 12)
C-C motif chemokine 22 (CCL22):
GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPW VKMILNKLSQ (SEQ ID NO: 13)
Fibroblast growth factor-2 (FGF-2):
PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEER GVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRT GQYKLGSKTGPGQKAILFLPMSAKS (SEQ ID NO: 14)
Fibroblast growth factor-5 (FGF-5):
FAVSQGIVGIRGVFSNKFLAMSKKGKLHASAKFTDDCKFRERFQENSYNTYASAIHRTEKT GREWYVALNKRGKAKRGCSPRVKPQHISTHFLPRFKQSEQPELSFTVTVPEKKKPPSPIKP KIPLSAPRKNTNSVKYRLKFRFG (SEQ ID NO: 15)
Fibroblast growth factor-7 (FGF-7):
KNNYNIMEIRTVAVGIVAIKGVESEFYLAMNKEGKLYAKKECNEDCNFKELILENHYNTYASA KWTHNGGEMFVALNQKGIPVRGKKTKKEQKTAHFLPMAIT (SEQ ID NO: 16)
Fibroblast growth factor-10 (FGF-10):
QALGQDMVSPEATNSSSSSFSSPSSAGRHVRSYNHLQGDVRWRKLFSFTKYFLKIEKNGK VSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENG YNTYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMVVHS (SEQ ID NO: 17) Fibroblast growth factor-18 (FGF-18):
EENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLV ETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGW YVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKRSRRIRPTHPA (SEQ ID NO: 18)
Vascular endothelial growth factor-A165 (VEGF-A165):
APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCC NDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGK GKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCS CKNTDSRCKARQLELNERTCRCDKPRR (SEQ ID NO: 19)
Vascular endothelial growth factor-A121 (VEGF-A121):
MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLV DIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMS FLQHNKCECRPKKDRARQEKCDKPRR (SEQ ID NO: 20)
Vascular endothelial growth factor-B (VEGF-B):
PVSQPDAPGHQRKVVSWIDVYTRATCQPREVVVPLTVELMGTVAKQLVPSCVTVQRCGG CCPDDGLECVPTGQHQVRMQILMIRYPSSQLGEMSLEEHSQCECRPKKKDSAVKPDRAAT PHHRPQPRSVPGWDSAPGAPSPADITHPTPAPGPSAHAAPSTTSALTPGPAAAAADAAAS SVAKGGA (SEQ ID NO: 21)
Vascular endothelial growth factor-C (VEGF-C):
AHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGL QCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLDVYRQVHSIIRR (SEQ ID NO: 22)
Placenta growth factor-1 (PIGF-1):
MPVMRLFPCFLQLLAGLALPAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLV DVVSEYPSEVEHMFSPSCVSLLRCTGCCGDENLHCVPVETANVTMQLLKIRSGDRPSYVE LTFSQHVRCECRPLREKMKPERCGDAVPRR (SEQ ID NO: 23) Placenta growth factor-2 (PIGF-2):
MPVMRLFPCFLQLLAGLALPAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLV DVVSEYPSEVEHMFSPSCVSLLRCTGCCGDENLHCVPVETANVTMQLLKIRSGDRPSYVE LTFSQHVRCECRPLREKMKPERRRPKGRGKRRREKQRPTDCHLCGDAVPRR (SEQ ID NO: 24)
Placenta growth factor-4 (PIGF-4):
MPVMRLFPCFLQLLAGLALPAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLV DVVSEYPSEVEHMFSPSCVSLLRCTGCCGDENLHCVPVETANVTMQLLKIRSGDRPSYVE LTFSQHVRCECRHSPGRQSPDMPGDFRADAPSFLPPRRSLPMLFRMEWGCALTGSQSAV WPSSPVPEEIPRMHPGRNGKKQQRKPLREKMKPERRRPKGRGKRRREKQRPTDCHLCG DAVPRR (SEQ ID NO: 25)
Platelet-derived growth factor-A (PDGF-AA):
SIEEAVPAVCKTRTVIYEIPRSQVDPTSANFLIWPPCVEVKRCTGCCNTSSVKCQPSRVHHR SVKVAKVEYVRKKPKLKEVQVRLEEHLECACATTSLNPDYREEDTGRPRESGKKRKRKRL KPT (SEQ ID NO: 26)
Platelet-derived growth factor-B (PDGF-BB):
SLGSLTIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNVQCRP TQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVT (SEQ ID NO: 27)
Platelet-derived growth factor-C (PDGF-CC):
ESNLSSKFQFSSNKEQNGVQDPQHERIITVSTNGSIHSPRFPHTYPRNTVLVWRLVAVEEN
VWIQLTFDERFGLEDPEDDICKYDFVEVEEPSDGTILGRWCGSGTVPGKQISKGNQIRIRFV
SDEYFPSEPGFCIHYNIVMPQFTEAVSPSVLPPSALPLDLLNNAITAFSTLEDLIRYLEPERW
QLDLEDLYRPTWQLLGKAFVFGRKSRVVDLNLLTEEVRLYSCTPRNFSVSIREELKRTDTIF
WPGCLLVKRCGGNCACCLHNCNECQCVPSKVTKKYHEVLQLRPKTGVRGLHKSLTDVAL
EHHEECDCVCRGSTGG (SEQ ID NO: 28)
Platelet-derived growth factor-D (PDGF-DD):
RDTSATPQSASIKALRNANLRRDESNHLTDLYRRDETIQVKGNGYVQSPRFPNSYPRNLLL
TWRLHSQENTRIQLVFDNQFGLEEAENDICRYDFVEVEDISETSTIIRGRWCGHKEVPPRIK
SRTNQIKITFKSDDYFVAKPGFKIYYSLLEDFQPAAASETNWESVTSSISGVSYNSPSVTDPT
LIADALDKKIAEFDTVEDLLKYFNPESWQEDLENMYLDTPRYRGRSYHDRKSKVDLDRLND DAKRYSCTPRNYSVNIREELKLANVVFFPRCLLVQRCGGNCGCGTVNWRSCTCNSGKTVK KYHEVLQFEPGHIKRRGRAKTMALVDIQLDHHERCDCICSSRPPR (SEQ ID NO: 29)
Epidermal growth factor (EGF):
NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR (SEQ ID NO: 30)
Heparin-binding epidermal growth factor (HB-EGF):
DLQEADLDLLRVTLSSKPQALATPNKEEHGKRKKKGKGLGKKRDPCLRKYKDFCIHGECKY VKELRAPSCICHPGYHGERCHGLSL (SEQ ID NO: 31)
A phiregulin (Areg):
SVRVEQVVKPPQNKTESENTSDKPKRKKKGGKNGKNRRNRKKKNPCNAEFQNFCIHGEC KYIEHLEAVTCKCQQEYFGERCGEKSMK (SEQ ID NO: 32)
Epiregulin (Ereg):
VSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFL (SEQ ID NO: 33)
Neuroregulin-2 (NRG-2):
CYSPSLKSVQDQAYKAPVVVEGKVQGLVPAGGSSSNSTREPPASGRVALVKVLDKWPLRS GGLQREQVISVGSCVPLERNQRYIFFLEPTEQPLVFKTAFAPLDTNGKNLKKEVGKI LCTDC ATRPKLKKMKSQTGQVGEKQSLKCEAAAGNPQPSYRWFKDGKELNRSRDIRIKYGNGRK NSRLQFNKVKVEDAGEYVCEAENILGKDTVRGRLYVNSVSTTLSSWSGHARKCNETAKSY CVNGGVCYYIEGINQLSCKCPNGFFGQRCLEKLPLRLYMPDPKQKAEELYQK (SEQ ID NO: 34)
Insulin-like growth factor-l (IGF-I):
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMY CAPLKPAKSA (SEQ ID NO: 35)
Insulin-like growth factor-ll (IGF-II):
AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCA TPAKSE (SEQ ID NO: 36) Transforming growth factor-bΐ (TGF- b1):
ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKV LALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 37)
Transforming growth factor- b2 (TGF- b2):
ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSR VLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS (SEQ ID NO: 38)
Transforming growth factor- b3 (TGF- b3):
ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKVWHEPKGYYANFCSGPCPYLRSADTTHST VLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS (SEQ ID NO: 39)
Bone morphogenetic-2 (BMP-2):
QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTN HAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR (SEQ ID NO: 40)
Bone morphogenetic-7 (BMP-7):
STGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAP EGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSS NVILKKYRNMVVRACGCH (SEQ ID NO: 41)
Nerve growth factor (NGF):
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDP NPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (SEQ ID NO: 42)
Neurotrophin-3 (NT-3):
YAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEAR PVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT (SEQ ID NO: 43) Brain-derived neurotrophic factor (BDNF):
HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNP MGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR (SEQ ID NO: 44)
Protein Wnt-3a (Wnt3a):
SYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQF
RGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRH
QGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMH
LKCKCHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRPR
YTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNAR
AERRREKCRCVFHWCCYVSCQECTRVYDVHTCK (SEQ ID NO: 45)
In one embodiment, the growth factor or cytokine binds syndecans. Examples of syndecan binding growth factors or cytokines are PDGF-BB and VEGF-A165.
The present invention also extends to fusion proteins comprising growth factors or cytokines which are functional homologues or variants of human growth factor or cytokine sequences or those of other animal species.
These functional homologues or variants may be derived by insertion, deletion or substitution of amino acids in, or chemical modification of, the native carboxyl-terminal sequence. Amino acid insertion variants include amino and/or carboxylic terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertion amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletion variants are characterised by the removal of one or more amino acids from the sequence. Substitution amino acid variants are those in which at least one amino acid residue in the sequence has been replaced by another of the twenty primary protein amino acids, or by a non-protein amino acid. In one embodiment substitutions are with conservative amino acids. Chemical modifications of the native carboxyl-terminal sequence include the acetylation of the amino-terminus and/or amidation of the carboxyl-terminus and/or side chain cyclisation of the native carboxyl-terminal sequence.
In one embodiment variants of the growth factor or cytokine used in the fusion protein of the invention may comprise one, two, three, four or five insertions, deletions or substitutions compared to the natural growth factor or cytokine sequence provided that the function of the native sequence is retained. In one embodiment amino acids, except for glycine, are of the L-absolute configuration. D configuration amino acids may also be used.
Persons skilled in the art will appreciate that the growth factor or cytokine used may be modified to improve storage stability, bioactivity, circulating half-life, or for any other purpose using methods available in the art, such as glycosylation, by conjugation to a polymer to increase circulating half-life, by pegylation or other chemical modification.
For example, it may be desirable to introduce modification to improve storage stability or to improve bioavailability.
Variants of the human growth factor or cytokine sequences provided as SEQ ID Nos: 1-45 preferably have at least about 80% amino acid sequence identity with the human sequence as disclosed herein (the reference sequence). Ordinarily, a variant will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to the reference sequence.
In some cases, a determination of the percent identity of a peptide or protein to a sequence set forth herein may be required. In such cases, the percent identity is measured in terms of the number of residues of the peptide or protein, or a portion of the peptide or protein. A polypeptide of, e.g., 90% identity, may also be a portion of a larger polypeptide or protein.
“Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
As used herein a “syndecan binding peptide” is a peptide or polypeptide that specifically binds syndecans. “Specifically binds” as used herein means that the peptide or polypeptide has greater binding affinity for syndecans than for other receptors or other heparan sulfate proteoglycans. For example, syndecan binding peptides may bind integrin receptors or other heparan sulfate proteoglycans but bind syndecans with greater binding affinity. Specific binding, as that term is commonly used in the biological arts, refers to a molecule that binds to a target with a relatively high affinity compared to non-target tissues, and generally involves a plurality of non-covalent interactions, such as electrostatic interactions, van der Waals interactions, hydrogen bonding, and the like. Specific binding interactions characterize antibody-antigen binding, enzyme-substrate binding, and specifically binding protein-receptor interactions; while such molecules may bind tissues besides their targets from time to time, such binding is said to lack specificity and is not specific binding.
Syndecans are type I transmembrane proteins that comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans are present on the surface of various adherent and non-adherent cells, and their domains interact with various soluble and insoluble factors in the ECM. Syndecans can interact with various extracellular ligands through glycosaminoglycan chains attached to the core protein.
Syndecans have numerous ligands, including laminins, which are major glycoproteins in the basement membrane. In particular, the globular domain of laminin alpha 1 chain binds heparan sulfate in syndecans with high affinity via the sequence RKRLQVQLSIRT (SEQ ID NO: 46) (laminin subunit alpha-12719-2730), commonly known as AG73 and named SB in this study.
In one embodiment, the syndecan binding peptide is from laminin a1, a2, a3 or a4.
In one embodiment the syndecan binding peptide is a variant of the SB sequence: Mouse sequence: RKRLQVQLSIRT (SEQ ID NO: 46)
Rat sequence: RKRLQVQLNIRT (SEQ ID NO: 47)
Horse sequence: RKRLSVQLSLRT (SEQ ID NO: 48)
Cat sequence: KRLQVQLNIRT (SEQ ID NO: 49)
Dog sequence: KRLSVQLSIRT (SEQ ID NO: 50)
Pig sequence: RRRLSVQLSIRT (SEQ ID NO: 51)
Monkey sequence: RKRLSVELSIRT (SEQ ID NO: 52)
Human sequence: RKKLSVELSIRT (SEQ ID NO: 53) or a further variant based on the consensus sequence Po-Po-Po-L-X-X-X-L-X-I-Po-T (SEQ ID NO :54) where Po is a positively charged amino-acid (K or R) and X is a polar amino-acid (Y, C, Q, T, N, or S).
Other syndecan binding peptides that may be used in the invention include, but are not limited to:
Human laminin a4: TLFLAHGRLVYM (SEQ ID NO: 55) or a variant based on the consensus sequence: T-L-F-L-A-H-G-Po-L-V-X-M (SEQ ID NO: 56) where Po is a positively charged amino-acid (K or R) an and X is a polar amino-acid (G, Y, C, Q, T, N, or S). Human or mouse laminin a3 chain: KNSFMALYLSKGRLVFALG (SEQ ID NO: 57) Human or mouse laminin a3 chain (short sequence): KNSFMALYLSKG (SEQ ID NO: 58) or a variant based on the consensus sequence: Po-N-S-F-M-A-L-Y-L-S-Po-G (SEQ ID NO: 59) where Po is a positively charged amino-acid (K or R) or consensus sequence: Po- N-S-F-M-A-L-Y-L-S-Po-G-Po-L-V-F-A-L-G (SEQ ID NO: 60) where Po is a positively charged amino-acid (K or R).
Human or mouse laminin a4 chain: LAIKNDNLVYVY (SE ID NO: 61) or a variant based on consensus sequence: L-A-I-Po-N-D-N-L-V-Y-V-Y (SEQ ID NO: 62) where Po is a positively charged amino-acid (K or R).
Human or Mouse laminin a4 chain: DVISLYNFKHIY (SEQ ID NO: 63) or a variant based on consensus sequence: D-V-I-S-L-Y-N-F-Po-H-I-Y (SEQ ID NO: 64) where Po is a positively charged amino-acid (K or R).
Mouse laminin a1 chain: GLIYYVAHQNQM (SEQ ID NO: 65) or a variant based on consensus sequence: G-L-l-Y-Y-Z-A-H-Q-N-Q-M (SEQ ID NO: 66) where Z is a non-polar amino acid (A, V, L, I, P, F, M, W, or G).
Human laminin B2 chain or mouse laminin y1 chain: KAFDITYVRLKF (SEQ ID NO:
67) or a variant based on consensus sequence: Po-A-F-D-I-T-Y-V-Po-L-Po-F (SEQ ID NO:
68) where Po is a positively charged amino-acid (K or R).
Human or mouse laminin a1 chain: DFLAVEMRRGKVAFLWDLG (SEQ ID NO: 69) or a variant based on consensus sequence: D-F-L-A-Z-E-M-Po-Po-G-Po-V-X/Z-F-L-W-D-L- G (SEQ ID NO: 70) where Po is a positively charged amino-acid (K or R), X is a polar amino-acid (Y, C, Q, T, N, or S), and Z is a non-polar amino acid (A, V, L, I, P, F, M, W, or G).
Human or mouse laminin a1 chain: KEYM G LA I KN D N LVYVYN LG (SEQ ID NO: 71) or a variant based on consensus sequence: Po-E-Y-M-G-L-A-I-Po -N-D-N-L-V-Y-V-Y-N-L-G (SEQ ID NO: 72) where Po is a positively charged amino-acid (K or R).
Human laminin a1 chain: DFLSIELFRGRVKVMTDLG (SEQ ID NO: 73) or a variant based on consensus sequence: D-F-L-S-I-E-L-F-Po-G-Po-V-Po-V-M-T-D-L-G (SEQ ID NO: 74) where Po is a positively charged amino-acid (K or R).
Mouse laminin a3 chain: AYYAIFLNKGRLEVHLSSG (SEQ ID NO: 75) or a variant based on consensus sequence: A-Y-Y-A-I-F-L-N-Po-G-Po-L-E-V-H-L-S-S-G (SEQ ID NO: 76) where Po is a positively charged amino-acid (K or R).
Mouse laminin a4 chain: DFMTLFLAHGRLVFMFNVG (SEQ ID NO: 77) or a variant based on consensus sequence: D-F-M-T-L-F-L-A-H-G-Po-L-V-F-M-F-N-V-G (SEQ ID NO: 78) where Po is a positively charged amino-acid (K or R). “Peptide” as used herein means any chain of amino acids from 8 to 50 amino acid residues in length, preferably 8 to 40, 8 to 30, 8 to 25, or 8 to 20, or more preferably about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acid residues in length.
In the fusion proteins of the invention, the syndecan-binding peptide is inserted near the C-terminus or N-terminus of the cytokine or growth factor, at the terminus most remote from the cytokine or growth factor receptor-binding site. Insertion of the syndecan-binding domain at the C-terminus or N-terminus may change the stability of the fusion protein.
In the fusion proteins of the invention, the cytokine or growth factor may be directly linked to the syndecan binding peptide or indirectly linked by a linker. In an embodiment a linker is present between the cytokine or growth factor and syndecan binding peptide. Suitable linkers comprise Glycine and Serine, for example GGS or SGG or repeats thereof.
Preferably the linker sequence comprises from about 1 to 20 amino acids, more preferably from about 1 to 16 amino acids. The linker sequence is preferably flexible so as not hold the cytokine or growth factor in a single undesired conformation.
The fusion protein as described herein may additionally comprise an N-terminal signal peptide domain, which allows processing, e.g., extracellular secretion, in a suitable host cell. Preferably, the N-terminal signal peptide domain comprises a protease, e.g., a signal peptidase cleavage site and thus may be removed after or during expression to obtain the mature protein.
Further, the fusion protein may comprise comprises a recognition/purification domain, e.g., a Strep-tag domain and/or a poly-His domain, which may be located at the N- terminus or at the C-terminus.
In a particular embodiment the fusion protein comprises a human growth factor or cytokine of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked to a syndecan binding peptide of SEQ ID NO: 46-78, either directly or via a linker, preferably comprising GGS or SGG or repeats thereof. The syndecan binding peptide may be at the N or C terminus of the fusion protein.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 46, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1- 45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 46, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 54, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS. In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 54, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 56, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 56, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 59, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 59, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 60, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 60, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 62, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1- 45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 62, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 64, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 64, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 66, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS. In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 66, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 68, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 68, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 70, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 70, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 72, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 72, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 74, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 74, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 76, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 76, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its C terminus to SEQ ID NO: 78, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS. In a particular embodiment the fusion protein comprises any one of SEQ ID NO: 1-45 or a variant thereof having at least 80% sequence identify thereto, linked at its N terminus to SEQ ID NO: 78, optionally via a linker such as SGG or GGS or SGGSGG or GGSGGS.
In a particular aspect the fusion protein comprises platelet-derived growth factor-BB (PDGF-BB) and a syndecan binding peptide.
In a preferred embodiment the fusion protein comprises PDGF-BB-SB with the following amino acid sequence:
SLGSLAAAEPAVIAECKTRTEVFQISRNLIDRTNANFLVWPPCVEVQRCSGCCNNRN VQCRASQVQMRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETIVTPRPVT- AGSGGSRKRLQVQLSIRT (SEQ ID NO: 85)
In a preferred embodiment the fusion protein comprises CX2PI1-8-M-PDGF-BB-SB with the following amino acid sequence:
NQEQVSPL-GPQGIWGQ-
SLGSLAAAEPAVIAECKTRTEVFQISRNLIDRTNANFLVWPPCVEVQRCSGCCNNRN VQCRASQVQMRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETIVTPRPVT- AGSGGSRKRLQVQLSIRT (SEQ ID NO: 79)
In a particular aspect the fusion protein comprises VEGF-A and a syndecan binding peptide. In a preferred embodiment the VEGF-A is VEGF-A, VEGF-A121 or VEGF-A165.
In a preferred embodiment the fusion protein comprises CX2PI1-8-M-VEGF-A-SB with the following amino acid sequence:
NQEQVSPL-GPQGIWGQ-
ASAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLM RCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEN CDKPRR-AGSGGSRK RLQVQLSIRT (SEQ ID NO: 80)
In a preferred embodiment the fusion protein comprises VEGF-A-SB with the following amino acid sequence: ASAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGG CCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENCDKP RR-AGSGGSRKRLQVQLSI RT (SEQ ID NO: 81)
In general, preparation of the fusion proteins of the invention can be accomplished by procedures disclosed herein and by recognized recombinant DNA techniques involving, e.g., polymerase chain amplification reactions (PCR), preparation of plasmid DNA, cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of the DNA into a suitable cell, transformation or transfection of a host, culturing of the host. Additionally, the fusion proteins can be isolated and purified using chaotropic agents and well known electrophoretic, centrifugation and chromatographic methods.
The invention further provides nucleic acid sequences and particularly DNA sequences that encode the present fusion proteins. Preferably, the DNA sequence is carried by a vector suited for extrachromosomal replication such as a phage, virus, plasmid, phagemid, cosmid, YAC, or episome. In particular, a DNA vector that encodes a desired fusion protein can be used to facilitate preparative methods described herein and to obtain significant quantities of the fusion protein. The DNA sequence can be inserted into an appropriate expression vector, i.e. , a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. A variety of host-vector systems may be utilized to express the protein-coding sequence. These include mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
In general, a preferred DNA vector according to the invention comprises a nucleotide sequence linked by phosphodiester bonds comprising, in a 5' to 3' direction a first cloning site for introduction of a first nucleotide sequence encoding a syndecan binding peptide operatively linked to a sequence encoding a cytokine or growth factor.
In most instances, it will be preferred that each of the fusion protein components encoded by the DNA vector be provided in a "cassette" format. By the term "cassette" is meant that each component can be readily substituted for another component by standard recombinant methods.
The fusion proteins described herein are preferably produced by standard recombinant DNA techniques. The resultant hybrid DNA molecule can be expressed in a suitable host cell to produce the fusion protein. The DNA molecules are ligated to each other in a 5' to 3' orientation such that, after ligation, the translational frame of the encoded polypeptides is not altered (i.e. , the DNA molecules are ligated to each other in-frame). The resulting DNA molecules encode an in-frame fusion protein.
The components of the fusion protein can be organized in nearly any order provided each can perform its intended function.
A number of strategies can be employed to express the fusion proteins of the invention. For example, the gene fusion construct described above can be incorporated into a suitable vector by known means such as by use of restriction enzymes to make cuts in the vector for insertion of the construct followed by ligation. The vector containing the gene construct is then introduced into a suitable host for expression of the fusion protein.
Selection of suitable vectors can be made empirically based on factors relating to the cloning protocol. For example, the vector should be compatible with, and have the proper replicon for the host that is being employed. Further the vector must be able to accommodate the DNA sequence coding for the fusion protein that is to be expressed. Suitable host cells include eukaryotic and prokaryotic cells, preferably those cells that can be easily transformed and exhibit rapid growth in culture medium. Specifically, preferred hosts cells include prokaryotes such as E. coli, Bacillus subtillus, etc. and eukaryotes such as animal cells and yeast strains, e.g., S. cerevisiae. Mammalian cells are generally preferred, particularly J558, NSO, SP2-0 or CHO. Other suitable hosts include, e.g., insect cells such as Sf9. Conventional culturing conditions are employed. Stable transformed or transfected cell lines can then be selected. Cells expressing fusion proteins according to the invention can be determined by known procedures.
Nucleic acid encoding a desired fusion protein can be introduced into a host cell by standard techniques for transfecting cells. The term "transfecting" or "transfection" is intended to encompass all conventional techniques for introducing nucleic acid into host cells, including calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, viral transduction and/or integration.
The present invention further provides a production process for isolating a fusion protein of interest. In the process, a host cell (e.g., a yeast, fungus, insect, bacterial or animal cell), into which has been introduced a nucleic acid encoding the fusion protein operatively linked to a regulatory sequence, is grown at production scale in a culture medium. Subsequently, the fusion protein of interest is isolated from harvested host cells or from the culture medium. Standard protein purification techniques can be used to isolate the fusion protein from the medium or from the harvested cells. In particular, the purification techniques can be used to express and purify a desired fusion protein on a large-scale (i.e. in at least milligram quantities) from a variety of implementations including roller bottles, spinner flasks, tissue culture plates, bioreactor, or a fermentor.
An expressed fusion protein can be isolated and purified by known methods. Typically, the culture medium is centrifuged and then the supernatant is purified by affinity or immunoaffinity chromatography, e.g. Protein-A or Protein-G affinity chromatography or an immunoaffinity protocol comprising use of monoclonal antibodies that bind the expressed fusion protein. The fusion proteins of the present invention can be separated and purified by appropriate combination of known techniques. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electrical charge such as ion-exchange column chromatography, methods utilizing specific affinity such as affinity chromatograph, methods utilizing a difference in hydrophobicity such as reverse- phase high performance liquid chromatograph and methods utilizing a difference in isoelectric point, such as isoelectric focusing electrophoresis, metal affinity columns such as Ni-NTA.
It is preferred that the fusion proteins of the present invention be substantially pure. That is, the fusion proteins have been isolated from cell substituents that naturally accompany it so that the fusion proteins are present preferably in at least 80% or 90% to 95% homogeneity (w/w). Fusion proteins having at least 98 to 99% homogeneity (w/w) are most preferred for many pharmaceutical, clinical and research applications. Once substantially purified the fusion protein should be substantially free of contaminants for therapeutic applications. Once purified partially or to substantial purity, the soluble fusion proteins can be used therapeutically, or in performing in vitro or in vivo assays as disclosed herein. Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis.
Fusion proteins according to the invention may be administered in a pharmaceutical composition optionally together with pharmaceutically acceptable carriers or excipients for administration. Fusion proteins according to the invention may be administered in a veterinary composition optionally together with carriers or excipients suitable for administration to animals.
The pharmaceutical diluents, excipients, extenders, or carriers (termed herein as a pharmaceutically acceptable carrier, or a carrier) are suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. Pharmaceutically acceptable carriers or excipients may be used to deliver embodiments as described herein. Excipient refers to an inert substance used as a diluent or vehicle for a therapeutic agent. Pharmaceutically acceptable carriers are used, in general, with a compound to make the compound useful for a therapy or as a product. In general, for any substance, a carrier is a material that is combined with the substance for delivery to an animal. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. In some cases, the carrier is essential for delivery, e.g., to solubilize an insoluble compound for liquid delivery; a buffer for control of the pH of the substance to preserve its activity; or a diluent to prevent loss of the substance in the storage vessel. In other cases, however, the carrier is for convenience, e.g., a liquid for more convenient administration. Pharmaceutically acceptable salts of the compounds described herein may be synthesized according to methods known to those skilled in the arts. Pharmaceutically acceptable substances or compositions are highly purified to be free of contaminants, are sterile, and are biocompatible. They further may include a carrier, salt, or excipient suited to administration to a patient. In the case of water as the carrier, the water is highly purified and processed to be free of contaminants, e.g., endotoxins.
The deliverable compound may be made in a form suitable for oral, rectal, topical, intravenous injection, intra-articular injection, parenteral administration, intra-nasal, or tracheal administration. Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used. Suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents may be included as carriers, e.g., for pills. For instance, an active component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. The compounds can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. The active compounds can also be administered parentally, in sterile liquid dosage forms. Buffers for achieving a physiological pH or osmolarity may also be used.
The invention in one aspect relates to the treatment of conditions. The terms "treating”, and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms (prophylaxis) and/or their underlying cause, and improvement or remediation of damage. Thus, for example, the present method of "treating" a condition encompasses both prevention of the condition in a predisposed individual, treatment of the condition in a clinically symptomatic individual and treatment of a healthy individual for beneficial effect. “Prophylaxis” or “prophylactic” or “preventative” therapy as used herein includes preventing the condition from occurring or ameliorating the subsequent progression of the condition in a subject that may be predisposed to the condition but has not yet been diagnosed as having it.
As used herein, “condition” refers to any deviation from normal health and includes a disease, disorder, defect or injury, such as injury caused by trauma, and deterioration due to age, inflammatory, infectious or genetic disorder or due to environment.
Conditions in which cytokine or growth factor administration is beneficial are disclosed in the prior art. Of those, conditions that may be treated in accordance with the present invention fall generally into the categories of those in which increased chondrocyte, collagen, proteoglycan, cartilage or muscle mass form or function is desirable.
Chondrocytes are the only cells found in cartilage. They produce and maintain the cartilaginous matrix, which consists mainly of Type II collagen, proteoglycans and elastin.
Cartilage is a flexible connective tissue found in many areas in the bodies of humans and animals, including joints between bones, rib cage, ear, nose, elbow, knee, ankle, bronchial tubes and intervertebral discs. Unlike other connective tissues, cartilage does not contain blood vessels and thus has limited repair capabilities. Because chondrocytes are bound in lacunae, they cannot migrate to damaged areas. Therefore, if cartilage is damaged, it is difficult and slow to heal.
For the purpose of the present disclosure, conditions that can be treated include Chondrocyte-Related Conditions that will benefit from repair or new growth of cartilage tissue or chondrocytes. This is not exclusive however and is used descriptively to emphasise the benefit of the presently disclosed methods.
Chondrocyte-Related Conditions include joint disorders involving cartilage damage and include cartilage damage caused by tibial plateau decompression.
The cause of osteoarthritis is multifactorial and includes body habitus, genetics and hormonal status.
In osteoarthritis, the cartilage covering bones (articular cartilage - a subset of hyaline cartilage) is thinned, eventually completely wearing out, resulting in a “bone against bone” joint, reduced motion and pain. Current therapeutic modalities are aimed at reducing pain and increasing joint function. Non-invasive interventions such as exercise and weight loss are the first lines of treatment, followed by anti-inflammatory medications. These latter treatments alleviate the symptoms but do not inhibit the processes that result in the changes characteristic of this disease and may actually accelerate joint destruction. Failure of these treatments usually culminates in surgical intervention (arthroplasty). Joint replacement is extremely successful with respect to restoring patient mobility and decreasing pain. However, failure as a result of osteolysis and aseptic loosening due to effects of wear debris or biomechanically-related bone loss limit the lifetime of these implants necessitating higher- risk revision surgery at the expense of increased patient morbidity and failure rate. The present invention provides a treatment for osteoarthritis.
In traumatic rupture or detachment, the cartilage in the knee is frequently damaged, and can be partially repaired through knee cartilage replacement therapy.
In achondroplasia, reduced proliferation of chondrocytes in the epiphyseal plate of long bones during infancy and childhood results in dwarfism.
Costochondritis is an inflammation of cartilage in the ribs, causing chest pain.
In spinal disc herniation, an asymmetrical compression of an intervertebral disc ruptures the sac-like disc, causing a herniation of its soft content. The hernia often compresses the adjacent nerves and causes back pain.
In relapsing polychondritis, a destruction, probably autoimmune, of cartilage, especially of the nose and ears, causes disfiguration. Death occurs by suffocation as the larynx loses its rigidity and collapses.
Tumours made up of cartilage tissue, either benign or malignant, can occur.
The present invention provides a treatment for each of the conditions above. Any of these conditions can be treated by repairing or growing new cartilage or chondrocytes according to the methods disclosed herein utilising a fusion protein according to the present invention.
Other conditions that may be treated in accordance with the invention include: chondromalacia patella; chondromalacia; chondrosarcoma- head and neck; chondrosarcoma; costochondritis; enchondroma; hallux rigidus; hip labral tear; osteochondritis dissecans (OCD); osteochondrodysplasias; perichondritis; polychondritis; or torn meniscus.
The invention provides means to improve the function of existing chondrocytes and cartilage in maintaining a cartilaginous matrix. It also provides means to promote growth of chondrocytes and cartilage and provide a cartilaginous matrix, with or without an implant or prosthesis. In one embodiment the invention provides means to promote cartilage formation or repair in a cellular scaffold or in tissue engineering techniques, for example for cartilage generation or repair to grow new cartilage tissue in tissues including the nose, septum, ear, elbow, knee, ankle and invertebrate discs.
In one aspect the fusion protein is administered with an implant or the like to produce or repair chondrocytes or cartilage tissue that may interact with the implant to treat a condition as disclosed herein. As used herein, “interact” refers to the effect in conjunction of components to achieve a desired biological outcome. While not wishing to be bound by theory, when an implant “interacts” with chondrocytes, the effect of the implant in treating the condition is greater than the effect of the implant alone and may be synergistic.
In one aspect the fusion protein is administered in combination with mesenchymal stem cells therapies to enhance repair. The effect of treatment with the fusion protein and stem cells may be more than the additive effect of the separate treatments and may be synergistic.
In this embodiment, the “desired biological outcome” provided by the invention is preferably cartilage repair and cartilage growth, more preferably removal of the symptoms of osteoarthritis and most preferably treatment and prevention of osteoarthritis.
It is also contemplated that fusion proteins of the invention can be used to promote muscle growth, to improve recovery of muscle from injury, trauma or use, to improve muscle strength, to improve exercise tolerance, to increase the proportion of muscle, to increase muscle mass, decrease muscle wasting, improve muscle repair, or may be useful to treat disorders of muscle including wasting disorders, such as cachexia, and hormonal deficiency, anorexia, AIDS wasting syndrome, sarcopenia, muscular dystrophies, neuromuscular diseases, motor neuron diseases, diseases of the neuromuscular junction, and inflammatory myopathies in a subject in need thereof.
The invention extends to treatment of disorders of muscle and of diseases associated with muscular degeneration characteristics. Non-limiting examples of such disorders are various neuromuscular diseases, cardiac insufficiency, weakness of single muscles such as e.g. the constrictor or bladder muscle, hypo- or hypertension caused by problems with the constrictor function of vascular smooth muscle cells, impotence/erectile dysfunction, incontinence, AIDS-related muscular weakness, and general and age-related amyotrophia.
Disorders of muscle as referred to herein particularly include muscle wasting conditions or disorders in which muscle wasting is one of the primary symptoms.
“Muscle wasting" refers to the progressive loss of muscle mass and/or to the progressive weakening and degeneration of muscles, including the skeletal or voluntary muscles which control movement, cardiac muscles which control the heart, and smooth muscles. In one embodiment, the muscle wasting condition or disorder is a chronic muscle wasting condition or disorder. "Chronic muscle wasting" is defined herein as the chronic (i.e. persisting over a long period of time) progressive loss of muscle mass and/or to the chronic progressive weakening and degeneration of muscle. Chronic muscle wasting may occur as part of the aging process. The loss of muscle mass that occurs during muscle wasting can be characterized by a muscle protein breakdown or degradation, by muscle protein catabolism. Protein catabolism occurs because of an unusually high rate of protein degradation, an unusually low rate of protein synthesis, or a combination of both. Protein catabolism or depletion, whether caused by a high degree of protein degradation or a low degree of protein synthesis, leads to a decrease in muscle mass and to muscle wasting. The term "catabolism" has its commonly known meaning in the art, specifically an energy burning form of metabolism.
Muscle wasting can occur as a result of age, a pathology, disease, condition or disorder. In one embodiment, the pathology, illness, disease or condition is chronic. In another embodiment, the pathology, illness, disease or condition is genetic. In another embodiment, the pathology, illness, disease or condition is neurological. In another embodiment, the pathology, illness, disease or condition is infectious. As described herein, the pathologies, diseases, conditions or disorders directly or indirectly produce a wasting (i.e. loss) of muscle mass, that is a muscle wasting disorder.
Also contemplated is the treatment of neuromuscular diseases which are aligned with joint or skeletal deformities. In one embodiment, muscle wasting in a subject is a result of the subject having a muscular dystrophy; muscle atrophy; orX-linked spinal-bulbar muscular atrophy (SBMA).
The muscular dystrophies are genetic diseases characterized by progressive weakness and degeneration of the skeletal or voluntary muscles that control movement. The muscles of the heart and some other involuntary muscles are also affected in some forms of muscular dystrophy. The major forms of muscular dystrophy (MD) are: Duchenne muscular dystrophy, myotonic dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy and Emery- Dreifuss muscular dystrophy.
Muscular dystrophy can affect people of all ages. Although some forms first become apparent in infancy or childhood, others may not appear until middle age or later. Duchenne MD is the most common form, typically affecting children. Myotonic dystrophy is the most common of these diseases in adults.
Muscle atrophy (MA) is characterized by wasting away or diminution of muscle and a decrease in muscle mass. For example, Post-Polio MA is a muscle wasting that occurs as part of the post- polio syndrome (PPS). The atrophy includes weakness, muscle fatigue, and pain. Another type of MA is X-linked spinal-bulbar muscular atrophy (SBMA - also known as Kennedy's Disease). This disease arises from a defect in the androgen receptor gene on the X chromosome, affects only males, and its onset is in adulthood.
Sarcopenia is a debilitating disease that afflicts the elderly and chronically ill patients and is characterized by loss of muscle mass and function. Further, increased lean body mass is associated with decreased morbidity and mortality for certain muscle-wasting disorders. In addition, other circumstances and conditions are linked to, and can cause muscle wasting disorders. For example, studies have shown that in severe cases of chronic lower back pain, there is paraspinal muscle wasting.
Muscle wasting and other tissue wasting is also associated with advanced age. It is believed that general weakness in old age is due to muscle wasting. As the body ages, an increasing proportion of skeletal muscle is replaced by fibrous tissue. The result is a significant reduction in muscle power, performance and endurance.
Long term hospitalization due to illness or injury, or disuse deconditioning that occurs, for example, when a limb is immobilized, can also lead to muscle wasting, or wasting of other tissue. Studies have shown that in patients suffering injuries, chronic illnesses, burns, trauma or cancer, who are hospitalized for long periods of time, there is a long-lasting unilateral muscle wasting, and a decrease in body mass.
Injuries or damage to the central nervous system (CNS) are also associated with muscle wasting and other wasting disorders. Injuries or damage to the CNS can be, for example, caused by diseases, trauma or chemicals. Examples are central nerve injury or damage, peripheral nerve injury or damage and spinal cord injury or damage. In one embodiment CNS damage or injury comprise Alzheimer's diseases (AD); stroke, anger (mood); anorexia, anorexia nervosa, anorexia associated with aging and/or assertiveness (mood).
In another embodiment, muscle wasting or other tissue wasting (e.g. tendons or ligaments) may be a result of alcoholism.
In one embodiment, the wasting disease, disorder or condition being treated is associated with chronic illness
This embodiment is directed to treating, in some embodiments, any wasting disorder, which may be reflected in muscle wasting, weight loss, malnutrition, starvation, or any wasting or loss of functioning due to a loss of tissue mass.
In some embodiments, wasting diseases or disorders, such as cachexia, including cachexia caused by malnutrition, tuberculosis, leprosy, diabetes, renal disease, chronic obstructive pulmonary disease (COPD), cancer, end stage renal failure, emphysema, osteomalacia, or cardiomyopathy, may be treated by the methods of this invention In some embodiments, wasting is due to infection with enterovirus, Epstein-Barr virus, herpes zoster, HIV, trypanosomes, influenza, coxsackie, rickettsia, trichinella, schistosoma or mycobacteria.
Cachexia is weakness and a loss of weight caused by a disease or as a side effect of illness. Cardiac cachexia, i.e. a muscle protein wasting of both the cardiac and skeletal muscle, is a characteristic of congestive heart failure. Cancer cachexia is a syndrome that occurs in patients with solid tumours and haematological malignancies and is manifested by weight loss with massive depletion of both adipose tissue and lean muscle mass.
Cachexia is also seen in COPD, acquired immunodeficiency syndrome (AIDS), human immunodeficiency virus (HlV)-associated myopathy and/or muscle weakness/wasting is a relatively common clinical manifestation of AIDS. Individuals with HIV-associated myopathy or muscle weakness or wasting typically experience significant weight loss, generalized or proximal muscle weakness, tenderness, and muscle atrophy.
Untreated muscle wasting disorders can have serious health consequences. The changes that occur during muscle wasting can lead to a weakened physical state resulting in poor performance of the body and detrimental health effects.
Thus, muscle atrophy can seriously limit the rehabilitation of patients after immobilizations. Muscle wasting due to chronic diseases can lead to premature loss of mobility and increase the risk of disease-related morbidity. Muscle wasting due to disuse is an especially serious problem in elderly, who may already suffer from age-related deficits in muscle function and mass, leading to permanent disability and premature death as well as increased bone fracture rate. Despite the clinical importance of the condition few treatments exist to prevent or reverse the condition. The inventors propose that the fusion proteins of the invention can be used to prevent, repair and treat muscle wasting or atrophy associated with any of the conditions recited above.
In a preferred embodiment the fusion protein is used to treat burns and sepsis.
The invention in other aspects also contemplates treating healthy individuals to cause an increase in muscle mass, strength, function or overall physique.
The term "increase in muscle mass" refers to the presence of a greater amount of muscle after treatment relative to the amount of muscle mass present before the treatment.
The term "increase in muscle strength" refers to the presence of a muscle with greater force generating capacity after treatment relative to that present before the treatment.
The term "increase in muscle function" refers to the presence of muscle with greater variety of function after treatment relative to that present before the treatment. The term "increase in exercise tolerance" refers to the ability to exercise with less rest between exercise after treatment relative to that needed before the treatment.
A muscle is a tissue of the body that primarily functions as a source of power. There are three types of muscles in the body: a) skeletal muscle — striated muscle responsible for generating force that is transferred to the skeleton to enable movement, maintenance of posture and breathing; b) cardiac muscle — the heart muscle; and c) smooth muscle — the muscle that is in the walls of arteries and bowel. The methods of the invention are particularly applicable to skeletal muscle but may have some effect on cardiac and or smooth muscle. Reference to skeletal muscle as used herein also includes interactions between bone, muscle and tendons and includes muscle fibres and joints.
In a preferred embodiment the fusion proteins of the invention are used to treat conditions such as skin wound healing, (including diabetic wounds and ulcers), skin burns, bone defects and fractures, osteoporosis, osteoarthritis, spinal fusion, ankle fusion, muscle and tendon defects, cartilage defects and degeneration, ischemic tissues (including ischemic limb, ischemic cardiac tissue, and ischemic brain after a stroke).
The fusion proteins of the present invention induce tonic signalling in response to a growth factor or cytokine rather than the burst signalling induced in response to the wild type growth factor or cytokine.
Tonic signalling refers to controlled slow or graded signalling over a prolonged or sustained period.
In one embodiment the induction of tonic signalling rather than burst signalling allows the growth factor or cytokine to exert its effect without associated side effects.
In one embodiment the fusion protein comprising VEGF-A and a syndecan binding peptide, particularly a fusion protein of SEQ ID NO: 80 or SEQ ID NO: 81) induces less vascular permeability than VEGF-A alone.
In one embodiment the fusion protein comprising PDGF-BB and a syndecan binding peptide, particularly a fusion protein of SEQ ID NO: 79) induces less tumour growth than PDGF-BB alone.
Fusion proteins according to the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated and the subject. Fusion proteins may be administered orally, sublingually, buccally, intranasally, by inhalation, transdermally, topically, intra-articularly or parenterally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrathecal, intracranial, injection or infusion techniques. In one embodiment the fusion protein may be administered with or in an implant, medical device or prosthesis. The implant may be a biodegradable implant or slow release depot, or other implant as known to persons skilled in the art. Such embodiment is particularly appropriate for improving muscle growth and strength after muscle trauma or damage.
When used to treat burns the fusion protein may be administered orally, topically or parenterally.
Compositions comprising the fusion protein are to be administered in a therapeutically effective amount. As used herein, an "effective amount" is a dosage which is sufficient to reduce to achieve a desired biological outcome. The desired biological outcome may be any therapeutic benefit including an increase in muscle mass, an increase in muscle strength, muscle growth, or treatment of burns or wounds. Such improvements may be measured by a variety of methods including those that measure lean and fat body mass (such as duel ray scanning analysis), muscle strength, or the formation of muscle cells.
A typical daily dosage might range from about 1 mg/kg to up to 100 mg/kg or more, depending on the mode of delivery.
Dosage levels of the fusion protein could be of the order of about 0.1 mg per day to about 50mg per day or will usually be between about 0.25mg to about 1mg per day. The amount of fusion protein which may be combined with the carrier materials to produce a single dosage will vary, depending upon the subject to be treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain about 1mg to 1g of the fusion protein with an appropriate and convenient amount of carrier material, which may vary from about 5 to 95 percent of the total composition. Dosage unit forms will generally contain between from about 0.1 mg to 50mg of active ingredient.
It will be understood, however, that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
Dosage schedules can be adjusted depending on the half-life of the fusion protein, or the severity of the subject’s condition.
Generally, the compositions are administered as a bolus dose, to maximize the circulating levels of peptide for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
The treatments of the present invention are suitable for subjects in need thereof. “Subject,” as used herein, refers to human and non-human animals. The term “non-human animals” includes all vertebrates, e.g., mammals, such as non human primates (particularly higher primates), sheep, horse, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In one embodiment, the subject is an experimental animal, an animal suitable as a disease model, or in animal husbandry (animals as food source), where methods to increase lean muscle mass will greatly benefit the industry. Additionally, the method is particularly important in race horses.
In one embodiment the treatment is for humans, particularly adult humans, children aged 11 to 16 years old, aged 4 to 10 years old, infants of 18 months up to 4 years old, babies up to 18 months old. The treatment may also be used for elderly or infirm humans.
In one embodiment the treatments of the present invention are used to supplement alternative treatments for the same condition. For example, the fusion proteins can be used to supplement stem cell therapies for joint and muscle repair.
Examples
The invention described herein will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure and are not intended to limit the invention in any way.
Methods:
Primary cells
Human umbilical vein endothelial cells (ECs, Promocell) were expanded until passage three with endothelial cell basal medium (MCDB-131) containing EC supplement solution (Promocell). MSCs were isolated form compact bone of C57BL/6 mice as described previously51 and expanded until passage three with a-MEM (100 mg ml-1 penicillin/streptomycin, 10% FBS). Cells used for experiments were at passage 3-4.
Recombinant growth factor and SB sequence
Sequences encoding PDGF-BB (SEQ ID NO: 27), PDGF-BB-SB, <X2PII.8-M-PDGF- BB (SEQ ID NO:82) and a2Pli-8-M-PDGF-BB-SB (SEQ ID NO: 79) were cloned into the protein expression vector pcDNA3.1+ (ThermoFisher Scientific) PDGF-BB variants were produced as described in Martino, M.M., et al. (2014) and purified via size exclusion. VEGF- A121 (SEQ ID NO: 20), VEGF-A165 (SEQ ID NO: 19), VEGF-A-SB, a2Ph.8-M-VEGF-A165 (SEQ ID NO: 83), a2Ph.8-M-VEGF-A121 (SEQ ID NO: 84) and a2Ph.8-M-VEGF-A-SB (SEQ ID NO: 80) were cloned into the protein expression vector pRSET (Invitrogen). VEGFs were expressed into E.coli BL21 (Ds3) pLys (Novagen). VEGF-A variants were produced, refolded and purified by size exclusion as described in Traub, S., et al. (2013). SB and CX2PI1-8-M-SB were produced as a glutathione S-transferases (GST) fusion as described in Martino, M.M.
& Hubbell, J.A. (2010). GST was cleaved using precision protease (GE Healthcare). SB and CX2PI1-8-M-SB were purified via size exclusion. The concentration of recombinant proteins was determined via A280 and extinction coefficient correction. The proteins were verified as >99% pure by SDS-PAGE and MALDI-TOF.
(X2PI1-8-M -PDGF-BB NQEQVSPL-GPQGIWGQ-
SLGSLAAAEPAVIAECKTRTEVFQISRNLIDRTNANFLVWPPCVEVQRCSGCCNNRN VQCRASQVQMRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETIVTPRPVT (SEQ ID NO: 82) a2Pli-8-M-VEGF-A165
NQEQVSPL-GPQGIWGQ-
ASAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLM RCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEN PCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR (SEQ ID NO: 83)
(X2PI1-8-M-VEGF-AI2I
NQEQVSPL-GPQGIWGQ-
ASAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLM RCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEN CDKPRR (SEQ ID NO: 84)
Syndecan and PDGF-BB receptor detection by flow cytometry
To detect syndecans on MSCs and ECs, cells were trypsinized and resuspended in PBS containing Zombie Aqua (Biolegend) for 10 min at room temperature. Then, cells were washed with PBS and stained at 4 °C for 30 min with a single primary antibody diluted in flow cytometry buffer (PBS with 1% BSA). For MSCs, primary antibodies used were anti- syndecan-1 biotin (Miltenyi Biotech, clone REA104), anti-syndecan-2 PE (Abeam, polyclonal, ab205884), and anti-human syndecan-3 APC (R&D Systems, polyclonal, FAB3539A), and anti-syndecan-4 APC (R&D Systems, AF2918). Biotin was subsequently detected via streptavidin-PE (Biolegend), and anti-syndecan-2 was detected via a secondary goat anti-rabbit H&L Alexa Fluor 488 (Abeam, ab150077). For ECs, antibodies were anti- syndecan-1 FITC (Miltenyi Biotech, clone 44F9), anti-syndecan-2 PE (Miltenyi Biotech, clone REA468), anti-syndecan-3 APC (R&D Systems, polyclonal) and anti-syndecan-4 APC (R&D Systems, AF2918). The same protocol was used for PDGF-BB receptor detections. Antibodies were anti-mouse CD140b APC (Biolegend, clone APB5) and anti-mouse CD140a Biotin (Biolegend, clone APA5) conjugated to Streptavidin PE (Biolegend) for MSCs, antihuman CD140b PE (Miltenyi, clone REA363) conjugated to Streptavidin APC (Biolegend) and anti-human CD140a FITC (Miltenyi Biotech, clone REA911) for ECs. All antibodies were used at 0.5 mg ml-1. Cell samples were acquired with a flow cytometer (Cyan, Beckman Coulter) and data were analysed with FlowJo Software (TreeStar).
Syndecan and neuropilin-1 binding assay
ELISA plates were coated with 100 nM of growth factor variants (in PBS, 1 h at 37°C), and blocked with 2% BSA in PBS containing 0.05% Tween-20 (PBS-T) for 1 h at RT. Then, wells were washed with PBS-T and further incubated with recombinant syndecans or neuropilin-1 (R&D Systems) containing a His-tag for 1 h (in PBS-T with 0.1% BSA). Bound syndecans or neuropilin-1 were detected using an antibody against His-tag (in PBS-T with 0.1% BSA). The data were fitted by non-linear regression to obtain the dissociation constant (KD) using A450 nm = Bmax*[ECM protein]/(KD + [ECM protein]).
Growth factor receptor and kinase phosphorylation assay
MSCs and ECs were seeded in 6-well plates and cultured until 70-80% confluency. MSCs and ECs were starved 24 h with a-MEM containing 1% FBS and MCDB-131 containing 2% FBS, respectively. Then, cells were stimulated with 20 ng ml-1 of PDGF-BB or PDGF-BB-SB (for MSCs), and with 20 ng m of VEGF-A165, VEGF-A121, or VEGF-A-SB (for ECs). For the conditions with SB in excess, the concentration of SB was 2 mg ml-1. Phosphorylation of growth factor receptors, AKT and ERK1/2 was quantified using ELISA (Mouse PDGF-Rp and Human Phospho-VEGF R2/KDR Duoset, Cell Signalling Technology, R&D Systems)58. Briefly ELISA plates were coated with a capture antibody and incubated with cell lysates. The phosphorylation states were detected with an anti-phosphotyrosine antibody and normalized to a standard according to manufacturer instructions.
Intracellular signalling array
MSCs and ECs were seeded in 6-well plates and cultured until 70-80% confluency. MSCs and ECs were starved 24 h with a-MEM containing 1% FBS and MCDB-131 containing 2% FBS, respectively. Then, cells were stimulated with growth factor variants (20 ng ml-1) for 10, 180 or 360 min (3 wells per condition and per time points that were further pulled together). Phosphorylation and cleavage of intracellular signalling molecules were detected using an antibody array (PathScan Intracellular Signalling Array, Cell Signalling) according to the manufacturer's instructions. The chemiluminescent signals were detected using ImageQuant LAS 4000 and quantified with ImageQuant TL software (GE Healthcare Life Sciences).
Growth factor receptor internalization and degradation
MSCs (100Ό00 cells) were plated on 6-well plates with low serum medium (a-MEM, 100 mg ml-1 penicillin/streptomycin, 2% FBS) for 48 h. Then, cells were treated with PDGF- BB variants (10 ng ml-1) for 5, 15, 30 and 60 min. As a negative control (basal level of receptor), cells were treated with PBS. Following stimulation, cells were detached with TrypLE diluted 1:2 in PBS supplemented with 20 mM EDTA. TrypLE was quenched with medium containing 10% FBS and cells were stained with viability fixable dye (Zombie Aqua, Biolegend) for 10 min at room temperature followed by anti-mouse CD140b APC (Biolegend, clone APB5) in FACS Buffer (PBS with 1 % BSA and 5 mM EDTA) for 30 min on ice. To assess internalization, only cell-surface staining was performed. To assess degradation, surface and internal staining was performed. In that case, cells were fixed and permeabilized (eBioScience FoxP3 fixation and permeabilization kit) for 30 min on ice and stained with anti-mouse CD140b for 30 min on ice. For ECs the same experiment was performed, but cells (100,000) were plated with MCDB-131 (100 mg ml-1 penicillin/streptomycin, 5% FBS) and stained with anti-human CD309 PE (Biolegend, clone A16085H). Cell samples were acquired with a flow cytometer (Cyan, Beckman Coulter) and data were analysed with FlowJo Software (TreeStar). To calculate the percentage of receptor internalization and degradation, mean fluorescence intensities (MFI) were normalized to PBS controls for each timepoint. Value = 100 — 100 X (MFI experimental — MFIunstained)/(M FIunstimulated — MFIunstained)·
Growth factor co-localization with syndecans
MSCs and ECs were plated on glass slides chambers (m-Slide 8 Well, Ibdid, 10,000 cells per well) coated with a solution of 1% gelatin. Cells were cultured in 400 mI of media (MSCs with a-MEM, 100 mg ml-1 penicillin/streptomycin, 10% FBS; ECs with MCDB-131,
100 mg ml-1 penicillin/streptomycin, 10% FBS) for 48 h. Then, media was changed to contain 1% FBS and growth factors (100 ng ml-1 of PDGF-BB variants for MSCs and 100 ng ml-1 of VEGF-A variants for ECs). Cells were incubated for 10, 180 and 360 min, washed once with PBS and fixed with 2% paraformaldehyde for 20 min at room temperature. Then, cells were washed 3 times with PBS and incubated over night at 4 °C with a solution of antibodies in PBS containing 1% BSA. Rabbit anti-syndecan-2 (1 mV ml-1, Abeam, ab205884), rabbit anti- syndecan-4 (1 mV ml-1, Abeam, ab24511) and goat anti-PDGF-BB (2 mg ml-1, Abeam, ab111310) or mouse anti-VEGF-A (2 mg ml-1, Abeam, clone VG-1). After three washes with PBS, cells were incubated with secondary antibodies (in PBS with 1% BSA and 1 mg ml-1 DAPI) for 1 h at room temperature. Goat anti-rabbit IgG (Alexa Fluor 594, 2 mg ml-1, Abeam, ab150088), donkey anti-goat IgG (Alexa Fluor 488, 2 mg ml-1, Abeam, ab150129) or goat anti-mouse IgG (Alexa Fluor 488, 2 mg ml-1, Abeam, ab150117). Then, cells were washed three times with excess of PBS and the glass slides were mounted for imaging.
Growth factor release from fibrin matrix
Fibrin matrices were made as described in Martino, M.M., et al. (2009) with human fibrinogen. Briefly, fibrin matrices were generated with 8 mg ml-1 fibrinogen, 2 U ml-1 human thrombin (Sigma-Aldrich), 4 U ml-1 factor Xllla (Fibrogammin, Behring), 5 mM CaCL, and 500 ng ml-1 of growth factor. Fibrin gels were polymerized at 37 °C for 1 h and transferred in Ultra Low Cluster 24-well plate (Corning Incorporated) containing 500 mI of buffer (20 mM Tris-HCI, 150 mM NaCI, 0.1% BSA, pH 7.4). A control well that served as 100% released control contained only the growth factors in 500 mI of buffer. Each 24 h, buffers were removed, kept at -20 °C and replaced with fresh buffer. For the 100% release control well,
20 mI of buffer was taken out every day and stored at -20 °C. After 7 days, growth factor cumulative release was quantified using ELISA using the 100% released control as reference (DuoSet, R&D Systems). For release assay with MMPs, the same method was used except that the release buffer contained 0.25 mg ml-1 of recombinant MMP-1 and MMP- 2 (Sigma). MMPs were reconstituted at 50 mg ml-1 in 50 mM Tris, 10 mM CaCL, 150 mM NaCI, 0.05% Brij-35, pH 7.5. Then, MMPs were activated by adding p-amino-phenylmercuric acetate (1 mM) for 1 h at 37 °C and stored at -80 °C until used.
Proliferation assay
Proliferation assays were performed as described in Martino, M.M. & Hubbell, J.A. (2010). Briefly, cells in large culture flasks were starved for 24 h (a-MEM, 100 mg ml-1 penicillin/streptomycin, 2% FBS for MSCs; MCDB-131 , 100 mg ml-1 penicillin/streptomycin, 5% FBS for ECs). Then, cells were seeded on 96-well cell culture plate (3,000 cells per well) with growth factor variants in low serum medium (300 mI of a-MEM, 100 mg ml-1 penicillin/streptomycin, 2% FBS for MSCs; 300 mI of MCDB-131 , 100 mg ml-1 penicillin/streptomycin, 5% FBS for ECs). After 3, 6, 9, and 12 days, cell number was quantified using CyQUANT dye (Invitrogen). Florescence intensity was measured with a florescence plate reader. For the 9- and 12-day time points, media was changed once without the addition of growth factors. Cell proliferation was analysed by calculating percentage proliferation increases over basal proliferation (without growth factors) using the equation (cell number in saline control group/cell number in stimulation groups) - 1) x 100.
Colony formation assay
Primary cells isolated from mouse compact bone as described in Martino, M.M., et al. (2016) were seeded in 6-well plates (100 bone-derived cells) and cultured in medium (a- MEM, 100 mg ml-1 penicillin/streptomycin, 2% FBS) with or without 10 ng ml-1 of PDGF-BB variants for 5 days. Medium was changed once without additional growth factors and cultured for a further 5 days. Then, cells were washed with PBS and stained with crystal violet solution (0.5% in methanol). The number of colony (more than 50 cells, determined by light microscopy) was counted and their size was measured using ImageJ software.
3D flow multichamber assay
A high-throughput multichamber fluidic device was used to compare the effects of VEGF-A variants on endothelial morphogenesis in the 3D environment as described in Bonvin, C., et al. (2010). Fibrin matrices were prepared to have a final concentration of 4 mg ml-1 human fibrinogen (depleted of fibronectin, plasminogen, and von Willebrand factor; Enzyme Research Laboratories), 2 U ml-1 human thrombin (Sigma), 2 U ml-1 factor Xllla (Fibrogammin, Behring), 2.5 mM of CaCh. ECs (75,000 cells per 50 mI of gel) were mixed together with the fibrinogen solution and VEGF-A variants (50, 200, or 500 ng ml-1) before matrix polymerization via a thrombin solution of equal volume. The culture medium was MCDB-131 containing 2% FBS, 1.7 mg ml-1 of aprotinin (Roche), 100 mg ml-1 penicillin/streptomycin, and 4 mM L-glutamine added to both the central and outer medium reservoirs. The multichamber filled with fibrin only was incubated with the medium containing 200 mg ml-1 of endothelial cell growth supplement (ECGS) as positive control multichamber. Four chambers out of nine in each multichamber were used and fibrin matrix containing the VEGF-A variants (except ECGS positive control) were applied randomly in the same multichamber. The next day, flow was initiated by removing medium from the central reservoir and a pump was used to allow removal of excess media from the inlet, keeping the pressure head constant across all chambers and allowing homogeneous radial flow. The medium was changed at day 2 and ECs in fibrin matrix were fixed with 4% of formaldehyde at day 4. Matrix were washed 3 times and stained with Hochest 33342 (Invitrogen) and Alexa Fluor 488 Phalloidin (Invitrogen). Confocal imaging was performed with a Zeiss LSM 700 confocal microscope (Carl Zeiss AG). Data were quantified by MetaMorph software (Molecular Devices).
Bone regeneration model
Mice (C57BL/6, 10-12 wk old) were first anesthetized with isoflurane. The top of their head was shaved and a longitudinal incision was performed to reveal the skull. Bone tissue was exposed by retracting the soft tissues. Using a micromotor drill, two craniotomy defects (4.5 mm diameter) were created in the parietal bones of the skull on each side of the sagittal suture line. The defects were washed with saline and covered with a fibrin matrix polymerized atop the dura (40 mI per defect, 14 mg ml-1 fibrinogen (Enzyme Research Laboratories), 2 U ml-1 of thrombin (Sigma-Aldrich), 5 mM CaCh, 25 mg ml-1 aprotinin (Roche), 2 pg of PDGF-BB variants). Then, the soft tissue was closed with stitches. As an analgesic, mice received a subcutaneous injection of Tramdol (100 mg kg-1). Experiments were performed in accordance with the Monash University Animal Ethics Committee and the Animal Research Committee of the Research Institute for Microbial Diseases of Osaka University.
Microcomputed tomography (microCT)
Skulls were scanned with a microCT 40 (Scanco Medical AG) operated at energy of 70 kVp and intensity of 145 ms. Scans were performed at high-resolution mode resulting in a nominal isotropic resolution of 30 mhi. After reconstruction, a 3D Gaussian filter (sigma 1.2, support 1) was applied to all images. Bone was segmented from background using a global threshold of 22.4% of maximum grey value. Afterwards, cylindrical masks were placed manually at the defects. Bone volume within these masks was calculated using a standardized procedure developed for quantitative bone morphometry as described in Hildebrand, T., (1999). Coverage was calculated on a dorso-ventral projection of the cylindrical area as described in Lutolf, M.P., et al., (2003).
Impaired skin would healing model
C57BLKS/J-m/Lepr db ( db/db ) 14-16 wk old male mice backs were shaved and four full-thickness punch-biopsy wounds (6 mm in diameter) were created as described in Martino, M.M., et al. (2009). Directly after, fibrin gels (80 mI of 8 mg ml-1 fibrinogen, 2 U ml-1 of thrombin, 5 U ml-1 of factor XI 11 a, 5 mM CaCh, 200 ng of VEGF-A variants) were applied and polymerized on the wounds. To avoid drying of the gels, the wounds were covered with non-adhering dressing (Adaptic, Johnson & Johnson) and adhesive film dressing (Hydrofilm, Hartmann). After 10 days, animals were euthanized and the wounds were harvested for histological analysis. An area of 8 mm in diameter was excised and wounds were embedded. Histological analysis was performed on serial sections (20 mhi cryosections and 4 mGP paraffin sections) until reaching the central portion of the wound. The extent of re- epithelialization and granulation tissue formation was measured by histomorphometric analysis of tissue sections (H&E stain) using ImageJ software (National Institutes of Health, USA). For analysis of re-epithelialization, the distance that the epithelium had traveled across the wound was measured. The muscle edges of the panniculus carnosus were used as indicator for the wound edges and re-epithelialization was calculated as the percentage of the distance of edges of the panniculus carnosus muscle. For granulation tissue quantification, the area covered by a highly cellular density tissue was determined and normalized with the distance of muscle edges of the panniculus carnosus, in order to obtain the area at the centre of the wound. Cryosections were fixed with acetone and blocked with 10% goat serum and further incubated overnight at 4 °C with primary antibodies against CD31 (clone MEC 13.3, BD Pharmingen) and desmin (clone D33, Abeam). Alexa Fluor 488- conjugated anti-mouse and Alexa Fluor 594-conjugated anti-rat (Invitrogen) were used as secondary antibodies. Association area was determined by measuring the area positive for desmin within the dilated area positive for CD31 (dilate iteration = 5), using ImageJ software. Experiments were performed in accordance with the Cantonal Veterinary Office of Canton de Vaud and the Monash University Animal Ethics Committee.
Tumor growth model
E.G7-OVA thymoma cells were obtained from American Type Culture Collection (ATCC) and grown in RPMI medium 1640 supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 30 mM Hepes, 50 mM 2-mercaptoethanol, and 0.4 mg/ml G418 antibiotic (Sigma). Tumor cells (106) were implanted subcutaneously in the back at the level of the junction between the thoracic and lumbar vertebrae of C57BL/6 mice (10 wk old) in 30 pi of PBS. On the same day, mice received PDGF-BB variants delivered in two fibrin matrix on the calvaria (10 mg/matrix). Tumors were measured every day and volumes were calculated as ellipsoids based on three orthogonal measures as described in Julier, Z., et al. (2015). Animal were monitored for 14 days. Animals having a tumor volume > 1000 mm3 before 14 days were humanly killed. Experiments were performed in accordance with the Cantonal Veterinary Office of Canton de Vaud and the Animal Research Committee of the Research Institute for Microbial Diseases of Osaka University. Plasma concentration of growth factors
Calvarial defect and full thickness wounds were created in C57BL/6 mice (10 wk old) as described above. Calvarial defects were directly treated with PDGF-BB variants delivered via a fibrin matrix (10 mV growth factor per matrix). Skin wounds were directly treated with VEGF-A variants delivered via a fibrin matrix as described (5 mV growth factor per matrix). Blood (100 mI) was collected after 6h, 12h and 24h until 7 days by tail bleeding in anticoagulant coated tubes (Eppendorf). Directly after collection samples were centrifuge for 15 min at 1,000 x g. Plasma concentrations of growth factors were detected by ELISA (VEGF and PDGF-BB Quantikine ELISA Kit, R&D Systems). Experiments were performed in accordance with the Animal Research Committee of the Research Institute for Microbial Diseases of Osaka University.
Miles assay
Miles assay was performed with 8-10 wk old male BALB/c mice as described in Xu, D., et al (2011). Briefly, the back of the mouse was shaved and divided into four treatment areas. Evan’s blue solution (100 mI, 1% in PBS) was injected intravenously in the tail vein. After 15 min, 20 mI of PBS containing 100 ng of VEGF-A variants were injected intradermally into the back. PBS was applied as a negative control. The mice were sacrificed after 30 min and the back skin was removed, photographed, and the treated area was cut out with a biopsy punch. The Evans’s blue dye was extracted from the tissue with formamide at 55°C for 24 h. The extracted dye was quantified by measuring absorbance at 605 nm and converted to mass using a standard curve. Experiments were performed in accordance with Cantonal Veterinary Office of Canton de Vaud.
Microvascular permeability assay
The model was performed as described in Martino, M.M., (2014) on 8-10 wk old male BALB/c mice. Mice were used and anesthetized with a 200 mI intraperitoneal injection of mixture of dormitore (0.05 mg ml-1) and ketamine (12.5 mg ml-1). Body temperature was kept at 37 °C by using heating pad with a rectal thermistor. The ventral side of the ear dermis and the cartilage under the tissue was disconnected from dorsal dermis by cutting the ventral skin and the cartilage along through the antihelix pinna. Ventral dermis and cartilage were gently peeled off and completely removed with tweezers. The flap was stabilized on a glass slide by gluing the intact part, and 200 mI TRITC labelled 155 kDa dextran (200 pi, 120 mg ml-1, Sigma) was injected intravenously via the tail vein. Initial leakage due to the increased blood pressure was washed twice with 5 ml Ringer’s buffer. Isotonic ascorbate ringer (140 mM sodium ascorbate, 25 mM HEPES, 4 mM KCI and 2 mM CaCh, pH 7.5) was applied on the dermis to prevent the photo bleaching and ear was protected with a coverslip. I aging was started 30 min after the TRITC dextran injection in order to equilibrate a balanced blood pressure and vascular leakage. Vascular leakage was imaged by using a fluorescence stereomicroscope equipped with a motorized stage (M250 FA, Leica Microsystems CMS GmbH) with 1 x lens providing linear system magnification 7.5-160 x. Images were collected, and the mean intensity was calculated by Leica LASA. In the first 25 min, basal vascular permeability was visualized after applying 50 mI ascorbate ringer. Ears were then washed with Ringer’s buffer for 1 min and 10 pg ml-1 of VEGF-A variants in 50 mI ascorbate solution was applied on the top of the ear dermis and imaging was continued for an additional 25 min. Mean fluorescence intensity data were transferred to a Matlab algorithm and the best-fit line of each polynomial curve was calculated. The maximum leakage (maximum mean fluorescent intensity minus background) and the maximum slope of the best fit (Mean Fluorescent Intensity/sec) was calculated. Each position was normalized to its negative control (basal permeability). For each ear, 6 to 8 positions were chosen and the average of leakage was calculated. Experiments were performed in accordance with the Cantonal Veterinary Office of Canton de Vaud.
Results:
Example 1 - Engineering growth factors for enhanced binding to syndecans:
Syndecans have numerous ligands, including laminins, which are major glycoproteins in the basement membrane. In particular, the globular domain of laminin alpha 1 chain binds heparan sulfate in syndecans with high affinity (mM-hM range), via the sequence RKRLQVQLSIRT (laminin subunit alpha-12719-2730), commonly known as AG73 and named SB in this study (Fig. 1 b). The inventors sought to engineer PDGF-BB and VEGF-A to include the SB sequence. The natural heparin-binding domain of PDGF-BB - which is likely the syndecan-binding domain - is implicated in PDGF-BB binding to its receptor. Thus, SB was added at the C-terminus of PDGF-BB to generate PDGF-BB-SB and maintain receptor binding. To generate VEGF-A-SB, SB was added at the C-terminus of VEGF-A121 which does not contain a syndecan-binding domain (Fig. 1c).
After having recombinantly produced the wild-type and engineered growth factors, we compared their binding affinity to recombinant syndecans having polysaccharide chains (Fig. 2) using an enzyme-linked immunosorbent assay (ELISA)-based approach described above (Fig. 1d, e. Fig. 3). VEGF-A121 did not show binding to syndecans (Fig. 4). While VEGF-A165 and PDGF-BB naturally have binding affinity for some syndecans, the engineered growth factors showed stronger binding to all syndecans (syndecans 1-4), with dissociation constant values (KD) decreasing up to 7-fold (Fig. 1e, Table 1). Interestingly, VEGF-A-SB does not bind to neuropilin-1 , a VEGF-A coreceptor that enhances binding of VEGF-A to its main receptor (Fig. 5).
Table 1 | Affinity of wild-type versus SB-fused growth factors for syndecans 1-4 measured by enzyme-linked immunosorbent assay.
Growth factors Syndecan 1 KD Syndecan 2 KD Syndecan 3 KD Syndecan 4 KD
PDGF-BB 151 ± 39 134 ± 25 122 ± 25 141 ± 25 PDGF-BB-SB 25 ± 3*** 30 ± 3*** 19 ± 2*** 27 ± 3*** VEGF-A121 >1000 >1000 >1000 >1000 VEGF-A 165 102 ± 15 97 ± 10 113 ± 20 272 ± 33 VEGF-A-SB 26 ± 2*** 48 ± 5(0007) 38 ± 5*** 94 ± 13***
Dissociation constants (KD) in nM are shown. Data are means ± SEM (n = 3 independent experiments). KD values of syndecan-binding growth factors are all significantly lower compared to wild-type growth factors (PDGF-BB and VEGF-A165). Two-tailed Student’s t- test ( ***P £ 0.001 , otherwise indicated).
Example 2 - Enhancing binding to syndecans triggers tonic signalling
To compare signalling triggered by wild-type growth factors and syndecan-binding growth factors, we focused on growth factor receptor and downstream kinase (AKT and ERK) phosphorylation in mesenchymal stem cells (MSCs) and endothelial cells (ECs).
MSCs express high levels of PDGF-BB receptor-b (PDGFR-b, Fig. 6), and ECs express high levels of VEGF-A receptor-2 (VEGFR-2). In addition, both cell types express syndecans 1-4 (Fig. 7). Compared to wild-type growth factors, which induced very rapid and strong phosphorylation of their receptors, syndecan-binding growth factors induced lower phosphorylation levels during the first 30 min. However, while phosphorylation rapidly decreased following wild-type growth factor stimulation, phosphorylation levels stayed significantly higher with the syndecan-binding growth factors. In line with growth factor receptor phosphorylation, a similar effect was observed with phosphorylation levels of AKT and ERK1/2 (Fig. 2a, Fig. S8). To confirm that differences in signalling kinetics were due to the SB sequence, we performed the same experiment by co-stimulating cells with excess of SB peptide to saturate binding sites on cell surface. In the presence of SB peptide in excess, the signalling kinetics of syndecan-binding growth factors were very similar to wild-type growth factors, demonstrating that the SB sequence is responsible for the slower but sustained signalling (Fig. 2a). Rapid signalling often results in the internalization and eventually degradation of growth factor receptors, referred to as desensitization. Therefore, we investigated whether syndecan-binding growth factors trigger less internalization/degradation by measuring the amount of growth factor internalized and degraded, using flow cytometry. We found that wild-type growth factors rapidly induce receptor internalization and degradation, while syndecan-binding growth factors trigger significantly less internalization and degradation (Fig. 2b). This observation prompted us to investigate to what extent syndecan-binding growth factors stayed bound to cells. Using an on-cell ELISA approach, we found that only 5% of wild-type growth factors are detected on a cell surface monolayer after 3 h (Fig. 2c).
In contrast, almost 40% of syndecan-binding growth factors were detected on cell surfaces after 3 hr. To demonstrate that binding of the syndecan-binding growth factors is dependent on functional syndecans, we repeated the experiment with cells treated with heparinases, which are enzymes that degrade negatively charged polysaccharide chains in syndecans. With non-functional syndecans, both wild-type and syndecan-binding growth factors were not able to stay bound to cells, further indicating that the SB sequence binds heparan sulfate chains in syndecans (Fig. 2c). Ability of syndecan-binding growth factors to stay bound to the cell surface and colocalize with syndecans was also verified by immunostaining (Fig. S9, Fig. S10). Overall, we found that syndecan-binding growth factors display very different signalling kinetics. Growth factors with enhanced binding to syndecans trigger a relatively low but sustained form of signalling (i.e. tonic signalling). In contrast, wild-type growth factors induce a strong but short form of signalling (i.e. burst signalling) (Fig. 2d).
Example 3 - Syndecan-binding growth factors have higher morphogenic activity
Because wild-type and syndecan-binding growth factors have very distinct signalling kinetics, we explored whether enhanced syndecan binding affects morphogenesis. We also reengineered wild-type and syndecan-binding growth factors with a fibrin-binding site based on residues 1-8 of the coagulation factor Xllla transglutaminase substrate alpha-2 plasmin inhibitor (CX2PI1-8)37 and an intervening matrix metalloproteinase (MMP, M) cleavage site at their N-terminus, anticipating their use in 3D cell culture models and in vivo (Fig. 3a). Fibrin was used as an ECM model, allowing growth factors with 02PII-8-M to be crosslinked into the matrix and released by MMPs due to the inserted MMP-cleavage site (Fig. S11).
As a first assay, we explored MSC and EC proliferation following a single stimulation with growth factor variants. Interestingly, wild-type growth factors induced more proliferation during the first 3 days, but syndecan-binding growth factors ultimately induced significantly more proliferation after 6 days and displayed prolonged activity until 12 days (Fig. 3b). Moreover, demonstrating that the SB sequence was responsible for the superior proliferative effect, the improved proliferation induced by syndecan-binding growth factors was abolished in the presence of a saturating concentration of SB peptide (SB in excess, Fig. 3b). Since PDGF-BB is known to enhance MSC colony formation, we compared CX2PI1-8-M-PDGF-BB and CX2PI1-8-M-PDGF-BB-SB with a colony formation assay. Significantly more and larger colonies were formed with CX2PI1-8-M-PDGF-BB-SB, compared to CX2PI1-8-M-PDGF-BB (Fig. 3c, d).
Then, we thought to compare the activity of the VEGF-A variants for EC assembly, using a high-throughput assay that we have developed to compare the angiogenic effect of growth factors in a 3D environment mimicking interstitial flow. Fibrin matrices containing the various c^Ph-s-VEGFs and ECs were polymerized into independent flow chamber microwells and cultured under flow for 4 days (Fig. 4a). No EC structures were observed with fibrin only and CI2PI1-8-VEGF-AI21, which does not bind syndecans, since ECs stayed round and did not form multi-cellular structures. ci2Pli-8-VEGF-A165 induced the formation of structures to some extent, but c^Ph-s-VEGF-A-SB and the positive control EC growth supplement (ECGS) promoted significantly more multi-cellular EC structures with capillary-like organization. Compared to ci2Pli-8-VEGF-A165, the total number of cells and the total number of structures per volume of matrix were significantly higher with c^Ph-s-VEGF-A-SB (Fig. 4b-d). In addition, the effect of c^Ph-s-VEGF-A-SB was dose-dependent (Fig. 4d, Fig. S12).
Example 4 - Syndecan-binding PDGF-BB promotes superior bone regeneration
PDGF-BB is known to promote bone regeneration to some extent by stimulating MSC and pre-osteoblast migration as well as proliferation. However, the wild-type growth factor is usually inefficient for regenerating critical-size bone defects. In this context, we explored whether CX2PI1-8-M-PDGF-BB-SB would be more efficient in promoting bone regeneration than PDGF-BB and CX2PI1-8-M-PDGF-BB. As a relevant model to determine regenerative potential, we used the critical-size calvarial defect in the mouse described in Spicer, P.P., et al. (2012). Since delivering micrograms of wild-type PDGF-BB is usually insufficient to regenerate such calvarial defects, we tested the regenerative effect of only 1 mg of PDGF-BB (not retained in the matrix), CX2PI1-8-M-PDGF-BB (retained in the matrix), and CX2PI1-8-M-PDGF-BB-SB (retained in the matrix and tonic signalling) delivered via a fibrin matrix. Eight weeks after treatment, bone regeneration (characterized by mineralized bone tissue deposition and coverage of the defects) was analysed using microcomputed tomography (pCT). Wild-type PDGF-BB did not significantly increase bone regeneration, when compared to defects treated with fibrin only. In contrast, CX2PI1-8-M-PDGF-BB significantly enhanced bone regeneration, and CX2PI1-8-M-PDGF-BB-SB led to a marked increase of bone tissue deposition compared to CX2PI1-8-M-PDGF-BB (Fig. 5), yielding coverage at about 95%.
Example 5 - Syndecan-binding VEGF-A improves diabetic wound healing
Similarly, to PDGF-BB for bone regeneration, VEGF-A is known to induce skin wound healing to some extent, but the wild-type growth factor is usually inefficient for promoting wound closure. Thus, we explored whether CX2PI1-8-M-VEGF-A-SB would be more potent than wild-type and fibrin-binding VEGF-As. We used full-thickness wound healing in diabetic ( db/db ) mice, since it is a well-established and relevant experimental model of impaired wound healing. Delivering micrograms of wild-type VEGF-A is typically ineffective to promote wound healing in diabetic mice. Therefore, we tested the regenerative effect of 0.2 pg of either wild-type VEGF-As (not retained in fibrin matrix), a2Pli-8-M-VEGF-A121, CX2PI1-8-M-VEGF-AI65 (both retained in fibrin matrix) and CX2PI1-8-M-VEGF-A-SB (retained in fibrin matrix and tonic signalling) delivered via a fibrin matrix. Low doses of wild-type and fibrin-binding VEGF-As did not significantly enhance wound healing compared to fibrin alone-treated wounds as indicated by either amount of granulation tissue or extent of wound closure (re-epithelialization). In contrast, CX2PI1-8-M-VEGF-A-SB significantly enhanced wound healing compared to both fibrin only and fibrin-binding VEGF-A treatments (Fig. 6a, b, Fig. S13). Since angiogenesis is a key step in sustaining newly formed granulation tissue, we compared how it differed between treatments. Immunohistological analysis for CD31 , which is highly expressed by ECs, and desmin, which is expressed by smooth muscle cells (SMCs) and pericytes, revealed that angiogenesis within the granulation tissue was much more pronounced and likely more stable when wounds were treated with CX2PI1-8-M-VEGF-A- SB (Fig. 6c, d).
Example 6 - Syndecan-binding growth factors induce less side effects
PDGF-BB has received a warning from the USFDA for potentially increasing cancer risk. Thus, we tested to which extent PDGF-BB variants accelerate tumour growth when delivered at a distant site. Tumor cells were implanted in the back of mice and PDGF-BB variants were delivered on the calvaria in a fibrin matrix. PDGF-BB and CX2PI1-8-M-PDGF-BB accelerated tumor growth but not CX2PI1-8-M-PDGF-BB-SB (Fig. 7a). To assess if difference in tumor growth between CX2PI1-8-M-PDGF-BB and CX2PI1-8-M-PDGF-BB-SB was linked to diffusion of the growth factors out of the delivery site, we measured plasma concentrations of the PDGF-BB variants following delivery in calvarial defects. Plasma concentrations of CX2PI1-8-M-PDGF-BB-SB were significantly lower compared to CX2PI1-8-M-PDGF-BB (Fig. 7b). Similarly, for VEGF-A variants, plasma concentrations of CX2PI1-8-M-VEGF-A-SB were significantly lower compared to CX2PI1-8-M-VEGF-AI2I and CX2PII-8-M-VEGF-A165, after delivery in skin wounds via a fibrin matrix (Fig. 7b).
Vascular permeability is a major problem that emerged in translating VEGF-A to clinical use. Because CX2PI1-8-M-VEGF-A-SB displays slower activation of VEGFR-2 compared to CX2PI1-8-M-VEGF-AI2I and CX2PII-8-M-VEGF-A165, we hypothesized that syndecan-binding VEGF-A would induce less vascular permeability. As a first model, we performed a modified Miles assay. This assay is a classic method to assess alterations in vessel permeability and to evaluate anti-VEGF-A drugs, as described in Radu, M. & Chernoff, J. (2013). We injected the soluble (i.e., not fibrin-bound) VEGF-A variants intradermally in mice containing Evans Blue in their vascular system, and the vascular leakage was visualized by a blue stain under the skin. Quantification of leaked dye in the presence of a2Pli-8-M-VEGF-A165 and a2Pli-8-M-VEGF-A121 showed a significant increase of leaking compared to saline control. In contrast, CX2PI1-8-M-VEGF-A-SB induced much less vascular permeability (Fig. 7c, d).
Next, to confirm the lower induction of vessel permeability by CX2PI1-8-M-VEGF-A-SB, we used a sensitive intravital microvessel permeability model as described in Martino, M.M., et al. (2014) and Kilarski, W.W., et al. (2013). In this model, factors are applied on a mouse ear flap, and microvascular permeability is measured by imaging leakage of fluorescent dextran. First, basal permeability was measured by exposing the ear flap to saline to confirm that the majority of vessels were functional with minimal permeability during imaging. Then, VEGF-A variants (10 pg ml 1) were applied on the flap while measuring induced dextran leakage. Interestingly, the rate of leakage due to application of CX2PI1-8-M-VEGF-A-SB was only 30% of that induced by application of wild-type CX2PII-8-M-VEGF-A165 (Fig. 7e,f).
Discussion
Growth factors are essential for tissue regeneration and thus have great potential in regenerative medicine.
The most common strategy to deliver growth factors at a tissue lesion is to control their release from biomaterials. For example, we have taken approaches where we functionalized hydrogels with growth factor-binding domains derived from ECM proteins such as fibronectin, fibrinogen and more recently laminin to retain growth factors in hydrogels. With these strategies, the growth factor-binding domain acts as a linker between the biomaterial and growth factors, providing a classic controlled release that is ideally driven by the biomaterial degradation. However, this approach heavily depends on the binding affinity of growth factors with the linker and works only if the biomaterial remains present at the injured site. In addition, there is no control over growth factor signalling once it is released. As a result, the delivery of more than one growth factor is often required to obtain good regenerative effects. For instance, we have shown that delivering both VEGF-A and PDGF-BB in combination with fibrin functionalized with a growth factor binding-domain derived from laminin improves diabetic wound healing in the mouse. Nevertheless, such a system is somehow complicated since three recombinant molecules are delivered - the two growth factors and the growth factor-binding domain linker. From the point of view of cost- effectiveness and regulatory path, regenerative strategies based on multiple recombinant proteins are particularly challenging and greatly increase the possibility of adverse effects, which is a main issue with growth factor-based therapeutics. Indeed, likely due to these concerns, no regenerative therapies involving the use of multiple growth factors have been approved yet. Therefore, systems allowing the use of a single growth factor therapeutic delivered locally are preferred, while systems based on bioengineered growth factors may also face new challenges since the delivered proteins are genetically modified. To improve the regenerative activity of growth factors, we reasoned to have not only classic controlled release form biomaterials, but both controlled release and controlled signalling via providing binding to cell-surface syndecans.
Most importantly, we found that enhancing binding to syndecans changes growth factor signalling kinetics with receptor and downstream kinase phosphorylation levels being much lower during the first hour post-stimulation but significantly higher afterwards. This clearly demonstrates that syndecan-binding growth factors induce a tonic form of signalling, which is different from the burst signalling of wild-type growth factors. In line with receptor phosphorylation levels, receptor internalization and degradation were significantly less pronounced after stimulation with syndecan-binding growth factors, indicating that cells are not desensitized to growth factor stimulation. This type of tonic signalling is known in the context of immunity, but such a signalling form is still not well described in the context of growth factor receptors and tissue morphogenesis. Nevertheless, interactions of growth factors with cell-surface heparan sulfate proteoglycans are well-known in developmental biology to regulate tissue formation by controlling their bioavailability. Sequestration of morphogens by cell-surface heparan sulfate proteoglycans provides a mechanism to locally control the intensity and kinetics of morphogen signalling - a typical example is for FGF-2. Here, by enhancing growth factor binding to syndecan and their retention on the cell surface, we could mimic such a mechanism.
Importantly, we have demonstrated that the tonic signalling generated by syndecan- binding growth factors is specifically due to binding to syndecans via the SB sequence, because saturation of binding sites by using the SB peptide in excess or degradation of syndecans with heparinases abolished tonic signalling as well as cell-surface retention of the growth factors (Fig. 2a, c). In addition, experiments done with the SB peptide in excess and with heparinases indicate that fusing VEGF-A121 and PDGF-BB to the SB sequence does not change signalling via changing affinity to growth factor receptors, because - in those conditions - wild-type and syndecan-binding growth factors induce similar signalling. Interestingly, PDGF-BB and VEGF-A165 do not induce a tonic from of signalling, while they bind syndecans to some extent (Fig. 1e, Table 1). Thus, the higher affinity and specificity of SB-fused growth factors to multiple syndecans is likely critical to induce a tonic signalling. In addition, the stronger and faster signalling of VEGF-A165 compared to VEGF-A-SB can be attributed to VEGF-A165 binding to neuropilin-1 (Fig. S5) which facilitate VEGF-receptor signalling35.
To assess the effects of syndecan-binding PDGF-BB, we chose MSC colony formation and MSC proliferation, since these cells express high levels of PDGFR-b and syndecans, and because they are critical for bone regeneration. We found that syndecan- binding PDGF-BB has a greater capacity to stimulate MSC colony formation and MSC proliferation (Fig. 3b-d). The same effect was observed with EC proliferation induced by syndecan-binding VEGF-A. Interestingly, the proliferative effect was observed only a late time points. Considering that cells were stimulated only once, these results support that the activity of syndecan-binding growth factors is preserved over time due to their ability to stay bound to cells. Notably, the SB sequence alone had no significant effect on MSCs nor ECs (Fig. 3b-d), although it has been reported to have some activity on ECs when coupled to biomaterials. This lack of effect is most likely due to the use of the SB peptide in a soluble form. In addition, the SB sequence we utilized (AG73) has been shown to be active in some models at mM concentrations, while, in this study, the concentration of growth factor-fused with SB was in the nM range. As a more relevant in vitro model to assess the angiogenic activity of syndecan-binding VEGF-A, we chose a high-throughput angiogenesis assay in a 3D environment mimicking interstitial flow, as flow provides an important biophysical cue that affects growth factor-driven capillary formation. Interestingly, 02Pli-8-M-VEGF-A-SB displayed a much stronger effect compared to ci2Pli-8-M-VEGF-A121 and c^Ph-s-M-VEGF- A165 (Fig. 4a-d). These results demonstrate again that a tonic form of signalling provided by syndecan-binding VEGF-A is critical in promoting vascular formation.
To compare the effect of having a typical growth factor controlled release from biomaterials to both controlled release and tonic signalling, we chose to deliver growth factors via a fibrin hydrogel in models of tissue repair and regeneration. To retain growth factors in fibrin, we did not use a growth factor- binding domain linker derived from ECM proteins such as fibronectin or laminin, but a simpler and more robust system allowing direct covalent coupling of growth factor to fibrin via FXIIIa-mediated transglutamination. Compared to the linker systems, this system has the advantage of being independent of the binding affinity of the growth factor variants to the linker and provided nearly 100% coupling to the fibrin hydrogel (Fig. S11). From a therapeutic point of view, it is also more relevant to deliver only one recombinant molecule (the engineered growth factor) instead of two (the engineered growth factor and the linker).
PDGF-BB has gained great interest for bone regeneration because it stimulates MSC and pre-osteoblast migration/proliferation. In addition, PDGF-BB also supports angiogenesis by targeting pericytes and smooth muscle cells. While the potential of PDGF-BB for bone regeneration is supported by several reports, other studies have failed to show significant effects. Yet, PDGF-BB is approved as an alternative to bone autograft during ankle and hindfoot fusion surgery. Therefore, we tested whether the tonic signalling triggered by syndecan-binding PDGF-BB could promote superior bone regeneration. As expected, ci2Pli. 8-M-PDGF-BB displayed significantly better bone regeneration compared to PDGF-BB, since the release of PDGF-BB was not controlled due to its weak binding to fibrin (Fig. S11). Most likely, slowing the release of wild-type PDGF-BB by delivering it in fibrin functionalized with a growth factor binding-domain linker would have triggered better bone regeneration.
However, it would have been at best equivalent to 02Pli-8-M-PDGF-BB, since the only advantage of both 02Pli-8 and growth factor binding-domain linker systems is to sequester growth factors in hydrogels. In contrast, we could demonstrate that c^Ph-s-M-PDGF-BB-SB, which is sequestered in the matrix and triggers a tonic form of signalling, promotes superior bone regeneration compared to c^Ph-s-M-PDGF-BB, which is only retained in the matrix (Fig. 5). This enhanced regenerative effect is thus attributable to both the controlled release of 02Pli-8-M-PDGF-BB-SB from the matrix and the tonic form of signalling triggered via binding to syndecans.
Next, we chose an impaired skin wound healing in diabetic mice to test syndecan- binding VEGF-A, since VEGF-A is well-known to support the formation of granulation tissue in wounds via neovessel formation. Nevertheless, while VEGF-A has been widely explored for chronic wounds, the growth factor failed to show effectiveness beyond phase I trials. As expected, slowing the release of VEGF-A using the 02Pli-8 system slightly improved wound healing and angiogenesis, because the growth factor is released in a controlled manner via fibrin degradation and MMPs. Slowing the release of wild-type VEGF-A by delivering it in fibrin functionalized with a growth factor-binding domain linker would likely have triggered wound healing to some extent, but it would have been at best similar to c^Ph-s-M-VEGF-A, since the main function of growth factor binding-domain linkers is to sequester growth factors. In contrast, providing both sequestration and a tonic form of signalling with ctePh-s- M-VEGF-A-SB led to significantly faster wound healing and greater angiogenesis (Fig. 6). Overall, our results in bone and skin models indicate that not only controlled release of growth factors via biomaterials is important but triggering a tonic form of signalling is critical to induce tissue healing.
Side effects of growth factors is a major concern for their application in the clinic. For instance, PDGF-BB has received a warning from the USFDA for potentially increasing cancer risk. Comparing the tumor growth effect of PDGF-BB variants when delivered at a distant site of implanted tumor cells, we found that wild-type PDGF-BB and fibrin-binding PDGF-BB accelerate tumor growth. In contrast, syndecan-binding PDGF-BB had no effect (Fig. 7a), likely due to the very low diffusion of CX2PI1-8-M-PDGF-BB-SB from the delivery site into the circulatory system (Fig. 7b). While VEGF-A is one of the most promising growth factors for induction of therapeutic angiogenesis, a major impediment for its application lies in its ability to rapidly induce vascular permeability - a side effect that clearly limits clinical translation1·4. Here we found that a2Pli-8-M-VEGF-A-SB induces much less vascular permeability compared to ci2Pli-8-M-VEGF-A121 and ci2Pli-8-M-VEGF-A165, even when not fibrin-associated (Fig. 7c-f). This effect is likely due to the capacity of c^Ph-s-M-VEGF-A-SB to stay bound to syndecans before reaching its receptor, although c^Ph-s-M-VEGF-A-SB may also bind other heparan sulfate proteoglycans in the ECM, further delaying signalling of the engineered growth factor to its main receptor.
In conclusion, we found that engineering growth factors for enhanced binding to the cell surface via syndecans promotes superior morphogenesis by triggering a tonic form of signalling. In addition to the ECM, we have demonstrated that growth factor binding to the cell surface is a key parameter to consider when engineering these molecules for regenerative medicine applications. Our findings support that controlling growth factor signalling via binding to syndecans is a promising strategy to enhance their regenerative efficacy. We believe this approach will also be applicable to other protein-based biologies such as cytokines and chemokines for many other therapeutic applications.
References
1. Martino, M.M., et al. Growth factors engineered for super-affinity to the extracellular matrix enhance tissue healing. Science 343, 885-888 (2014).
2. Martino, M.M. & Hubbell, J.A. The 12th-14th type III repeats of fibronectin function as a highly promiscuous growth factor-binding domain. FASEB J 24, 4711-4721 (2010).
3. Bonvin, C., Overney, J., Shieh, A.C., Dixon, J.B. & Swartz, M.A. A multichamber fluidic device for 3D cultures under interstitial flow with live imaging: development, characterization, and applications. Biotechnol Bioeng 105, 982-991 (2010).
4. Spicer, P.P., et al. Evaluation of bone regeneration using the rat critical size calvarial defect. Nat Protoc 7, 1918-1929 (2012).
5. Radu, M. & Chernoff, J. An in vivo assay to test blood vessel permeability. J Vis Exp, e50062 (2013).
6. Kilarski, W.W., et al. Intravital immunofluorescence for visualizing the microcirculatory and immune microenvironments in the mouse ear dermis. PLoS One 8, e57135 (2013).
7. Traub, S., et al. The promotion of endothelial cell attachment and spreading using FNIII10 fused to VEGF-A165. Biomaterials 34, 5958-5968 (2013).
8. Martino, M.M., et al. Engineering the growth factor microenvironment with fibronectin domains to promote wound and bone tissue healing. Sci Trans! Med 3, 100ra189 (2011).
9. Martino, M.M., et al. Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability. Biomaterials 30, 1089- 1097 (2009).
10. Hildebrand, T., Laib, A., Muller, R., Dequeker, J. & Ruegsegger, P. Direct three- dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 14, 1167-1174 (1999).
11. Lutolf, M.P., et al. Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nature biotechnology 21 , 513-518 (2003).
12. Julier, Z., Martino, M.M., de Titta, A., Jeanbart, L. & Hubbell, J.A. The TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine. Sci Rep 5, 8569 (2015).

Claims

Claims
1. A fusion protein comprising a growth factor or cytokine and a syndecan binding peptide.
2. The fusion protein of claim 1, in which the growth factor or cytokine is selected from the group consisting of interleukins including lnterleukin-1 receptor antagonist, Interleukin 2, Interleukin 4, Interleukin 10, Interleukin 16, Interleukin 33, Interleukin 27b; C-X-C motif chemokines including C-X-C motif chemokine 9, C-X-C motif chemokine 10, C-X-C motif chemokine 12, C-X-C motif chemokine 19, C-X-C motif chemokine 20, C-X-C motif chemokine 22; fibroblast growth factors, including Fibroblast Growth Factor-2, Fibroblast Growth Factor-5, Fibroblast Growth Factor-7, Fibroblast Growth Factor-10, Fibroblast Growth Factor- 18; vascular endothelial growth factors including Vascular Endothelial Growth Factor-A165, Vascular Endothelial Growth Factor-A121, Vascular Endothelial Growth Factor-B, Vascular Endothelial Growth Factor-C; placental growth factors including Placental Growth Factor-1, Placental Growth Factor-2, Placental Growth Factor-4; platelet derived growth factors including Platelet Derived Growth Factor-A, Platelet Derived Growth Factor-B, Platelet Derived Growth Factor-C, Platelet Derived Growth Factor-D; epidermal growth factors including Epidermal Growth Factor, Heparin-Binding Epidermal Growth Factor; Amphiregulin; Epiregulin; Neuriregulin-2; insulin-like growth factors including Insulin-like Growth Factor-I, Insulin-like Growth Factor-ll; transforming growth factors including Transforming growth factor-bΐ, Transforming growth factory, Transforming growth factor- b3; bone morphogenic proteins including Bone Morphogenic Protein-2, Bone Morphogenic Protein-7; Nerve Growth Factor; Neurotrophin-3; Brain-derived Neurotrophic Factor and Protein Wnt-3a or any one of the amino acid sequences provided as SEQ ID NO: 1-45.
3. The fusion protein of claim 1 or claim 2, in which the syndecan binding peptide has an amino acid sequence selected from any one of SEQ ID NO: 46-78.
4. The fusion protein of claim 1 having an amino acid sequence selected from any one of SEQ ID NO: 79, 80, 81 or 85 or comprising an amino acid sequence selected from any one of SEQ ID NO: 1-45 linked directly or indirectly via a linker to any one of the amino acid sequences provided as SEQ ID NO: 46-78.
5. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 4.
6. A vector comprising the nucleic acid molecule of claim 5.
7. A cell or a non-human organism transformed or transfected with the nucleic acid molecule of claim 5 or the vector of claim 6.
8. A method of making the fusion protein of any one of claims 1 to 4, the method comprising culturing the cell of claim 7 under conditions to produce the fusion protein and recovering the fusion protein.
9. A fusion protein produced by the method of claim 8.
10. A pharmaceutical or veterinary composition comprising the fusion protein of any one of claims 1 to 4, the nucleic acid molecule of claim 5, the vector of claim 6 or the cell or non human organism of claim 7, optionally with one or more excipient and/or carriers.
11. A method of treatment of a condition in which cytokine or growth factor administration is beneficial, the method comprising administering to a subject in need thereof the fusion protein of any one of claims 1 to 4, the nucleic acid molecule of claim 5, the vector of claim 6, the cell or non-human organism of claim 7 or the pharmaceutical or veterinary composition of claim 10.
12. The method of claim 11 , in which the condition is a skin wound, including diabetic wounds and ulcers, a skin burn, a bone defect or fracture, osteoporosis, osteoarthritis, spinal fusion, ankle fusion, a muscle or tendon defect, a cartilage defect or degeneration, or ischemic tissues, including ischemic limb, ischemic cardiac tissue, and ischemic brain after a stroke.
13. A method of inducing tonic signalling in response to a growth factor or cytokine, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
14. A method of reducing growth factor receptor or cytokine receptor internalization and degradation in response to binding by their respective growth factor or cytokine, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
15. A method of reducing desensitization to growth factor stimulation, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
16. A method of reducing a side effect associated with cytokine or growth factor administration, the method comprising providing the growth factor or cytokine as a fusion protein comprising the growth factor or cytokine and a syndecan binding peptide.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019094938A2 (en) * 2017-11-13 2019-05-16 The University Of Chicago Methods and compositions for the treatment of wounds

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019094938A2 (en) * 2017-11-13 2019-05-16 The University Of Chicago Methods and compositions for the treatment of wounds

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ISHIHARA J. ET AL.: "Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing", NATURE COMMUNICATIONS, vol. 9, no. 2163, 4 June 2018 (2018-06-04), XP055568823, Retrieved from the Internet <URL:https://www.nature.com/articles/s41467-018-04525-w.pdf> DOI: 10/1038/s41467-018-04525-w *
MIE M. ET AL.: "Construction of a bFGF-tethered multi-functional extracellular matrix protein through coiled-coil structures for neurite outgrowth induction", BIOMEDICAL MATERIALS, vol. 9, no. 1, 16 December 2013 (2013-12-16), pages 015004, XP020257548, Retrieved from the Internet <URL:https://iopscience.iop.org/article/l0.1088/1748-6041/9/1/015004/pdf> DOI: 10.1088/1748-6041/9/1 /015004 *
MOCHIZUKI M . ET AL.: "Growth factors with enhanced syndecan binding generate tonic signaling and promote tissue healing", NATURE BIOMEDICAL ENGINEERING, vol. 4, April 2020 (2020-04-01), pages 463 - 475, XP037092296, DOI: 10.1038/s41551-019-0469-1 *

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