Modified vegf-a with improved angiogenic properties   

This application claims priority to U.S. Provisional Application No. 60/708,226, filed Aug. 15, 2005, the disclosure of which is incorporated herein by reference in its entirety.

The file copy of the sequence listing is submitted on a Compact-Disc-Read Only Memory (CD-ROM). The sequence listing is saved as an ASCII DOS text file named 41018A.txt (118 KB), which was created on Aug. 15, 2006. The contents of the CD-ROM are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present application is directed to materials and methods for promoting angiogenesis.

Angiogenesis is a fundamental process required for normal growth and development of tissues, and involves the proliferation of new capillaries from pre-existing blood vessels. Angiogenesis is not only involved in embryonic development and normal tissue growth, repair, and regeneration, but is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and in repair of wounds and fractures. In addition to angiogenesis which takes place in the healthy individual, angiogenic events are involved in a number of pathological processes, notably tumor growth and metastasis, and other conditions in which blood vessel proliferation, especially of the microvascular system, is increased, such as diabetic retinopathy, psoriasis and arthropathies. Inhibition of angiogenesis is useful in preventing or alleviating these pathological processes.

Because of the crucial role of angiogenesis in so many physiological and pathological processes, factors involved in the control of angiogenesis have been intensively investigated. A number of growth factors have been shown to be involved in the regulation of angiogenesis; these include fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), transforming growth factor α (TGFα), and hepatocyte growth factor (HGF). See for example Folkman et al, “Angiogenesis”, J. Biol. Chem., 1992 267 10931-10934 for a review.

It has been suggested that a particular family of endothelial cell-specific growth factors and their corresponding receptors is primarily responsible for stimulation of endothelial cell growth and differentiation, and for certain functions of the differentiated cells. These factors are members of the PDGF/VEGF family, and appear to act via receptor tyrosine kinases (RTKs).

To date a number of PDGF/VEGF family members have been identified. These include PDGF-A (see e.g., GenBank Acc. No. X06374), PDGF-B (see e.g., GenBank Acc. No. M12783), PDGF-C (Intl. Publ. No. WO 00/18212), PDGF-D (Intl. Publ. No. WO 00/027879), VEGF (also known as VEGF-A or by particular isoform), Placenta growth factor, P1GF (U.S. Pat. No. 5,919,899), VEGF-B (also known as VEGF-related factor (VRF) Intl. Publ. No. PCT/US96/02597 and WO 96/26736), VEGF-C, (U.S. Pat. No. 6,221,839 and WO 98/33917), VEGF-D (also known as c-fos-induced growth factor (FIGF) (U.S. Pat. No. 6,235,713, Intl. Publ. No. WO98/07832), VEGF-E (also known as NZ7 VEGF or OV NZ7; Intl. Publ. No. WO00/025805 and U.S. Patent Publ. No. 2003/0113870), NZ2 VEGF (also known as OV NZ2; see e.g., GenBank Acc. No. S67520), D1701 VEGF-like protein (see e.g., GenBank Acc. No. AF106020; Meyer et al., EMBO J. 18:363-374), and NZ10 VEGF-like protein (described in Intl. Patent Application PCT/US99/25869) [Stacker and Achen, Growth Factors 17:1-11 (1999); Neufeld et al., FASEB J 13:9-22 (1999); Ferrara, J Mol Med 77:527-543 (1999)].

Vascular endothelial growth factors act by binding to receptor tyrosine kinases. Seven receptor tyrosine kinases have been identified, namely Flt-1 (VEGFR-1), KDR/Flk-1 (VEGFR-2), Flt4 (VEGFR-3), PDGFR-α, PDGFR-β, Tie and Tek/Tie-2. All of these have the intrinsic tyrosine kinase activity which is necessary for signal transduction. The essential, specific role in vasculogenesis and angiogenesis of Flt-1, Flk-1, Tie and Tek/Tie-2 has been demonstrated by targeted mutations inactivating these receptors in mouse embryos. Overexpression of either the VEGF/PDGF family of growth factors or VEGF/PDGF receptors can lead to aberrant development of the vasculature system (Saaristo et al., FASEB J. 16:1041-9, 2002; Kubo et al., Proc Natl Acad Sci USA. 99:8868-73, 2002.). The activity of VEGF/VEGFR also promotes angiogenesis of new cells and developing tissue, thereby facilitating the angiogenesis and vascularization of tumor cells.

While the aforementioned VEGF molecules have shown some promise with respect to the development of new blood vessels and other growth factor properties, there remains a need for the development of improved therapeutic approaches for promoting angiogenesis.

SUMMARY

The present invention is directed recombinant polynucleotides and polypeptides of the VEGF PDGF family of growth factors that are modified with flanking sequences to impart improved properties. Polypeptide and polynucleotide materials and methods for stimulation of angiogenesis are among the preferred embodiments of the invention.

The invention addresses existing needs by providing new compounds, compositions of matter, materials, devices, and methods for modulating processes such as angiogenesis, lymphangiogenesis, and wound healing, which have numerous therapeutic and prophylactic applications.

One aspect of the invention is novel compounds, especially proteinaceous compounds, with angiogenic properties. One aspect of the invention is a chimeric construct comprising: an RTK binding domain; at least one heterologous flanking domain or CUB domain; and at least one linkage that connects the RTK binding domain to the at least one flanking domain or CUB domain; wherein the construct and the RTK binding domain bind to the extracellular domain of at least one receptor tyrosine kinase selected from the group consisting of: VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, and PDGFR-beta. Those constructs that bind and stimulate the receptor are useful as receptor agonists. Those constructs that bind, but fail to stimulate the receptor, are useful as receptor antagonists. As described in greater detail below, some embodiments of the invention behave as pro-agonists, insofar as a cleavable CUB domain prevents receptor activation, but under appropriate conditions (e.g., administration or stimulation of a protease to cleave the CUB domain), the CUB domain is cleaved, creating a receptor agonist. The term “heterologous” in the context used above, refers to the flanking/CUB domain being from a different protein than the RTK binding domain. For example, if the at least one flanking domain comprises a VEGF-C pro-peptide, then exemplary heterologous RTK domains include any VEGF/PDGF family member other than VEGF-C (e.g., VEGF-A, -B, or -D; PDGF-A, -B, -C, or -D; or P1GF, but not VEGF-C).

In certain exemplary embodiments, the present invention is a construct comprising a receptor tyrosine kinase RTK binding domain, at least one flanking domain, and at least one linkage that connects the RTK binding domain to the at least one flanking domain. The term “construct” generally refers to a molecule, compound, or composition of matter and is not intended to be limiting as to structure or function. The term “domain” as used herein is descriptive of the fact that a portion of a molecule (that may be less than the whole molecule) may be used, and also is descriptive of the fact that the construct itself has discrete portions that contribute to the overall functionality (biological activity) of the construct. In some embodiments, the RTK binding domain comprises an amino acid sequence that is at least 90% identical to an RTK binding domain amino acid sequence selected from the group consisting of: mammalian VEGF-A, mammalian VEGF-B, mammalian VEGF-C, mammalian VEGF-D, mammalian VEGF-E, P1GF, PDGF-A, PDGF-B, PDGF-C and PDGF-D RTK binding domain amino acid sequences. The construct and the RTK binding domain bind to at least one receptor tyrosine kinase selected from the group consisting of: VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, and PDGFR-beta. In preferred embodiments, the flanking domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: a mammalian VEGF-C amino-terminal propeptide; a mammalian VEGF-C carboxy-terminal propeptide; a mammalian VEGF-D amino-terminal propeptide; a mammalian VEGF-D carboxy-terminal propeptide; a mammalian TGF-β1 LAP peptide; and fragments thereof that are effective to bind extracellular matrix proteins or neuropilin proteins, with the proviso that when the RTK binding domain is at least 90% identical to a VEGF-C RTK binding domain amino acid sequence, the construct contains at least one flanking domain that is not at least 90% identical to a VEGF-C pro-peptide; and when the RTK binding domain is at least 90% identical to a VEGF-D RTK binding domain amino acid sequence, the construct contains at least one flanking domain that is not at least 90% identical to a VEGF-D pro-peptide. In other words, constructs of the invention are heterologous compounds, the domains of which do not come exclusively from VEGF-C or exclusively from VEGF-D.

In the constructs described herein, a linker (or linkers) is used to attach the RTK binding domain(s) to one or more flanking domains (or to further attach flanking domains to each other or to attach optional additional domains such as a heparin binding domain or a CUB domain). In some embodiments, the linker comprises a covalent bond. In related embodiments, the linker comprises a peptide bond.

In some variations, the domains constitute separate and distinct peptides attached by the linker. In other variations, domains are joined by peptide bonds to form a continuous polypeptide chain.

For example, in some embodiments, the construct is a chimeric polypeptide comprising a structure that satisfies the formula Fn-L-RTK-L-Fc, wherein Fn comprises a flanking domain that comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: a mammalian VEGF-C amino-pro-peptide and a mammalian VEGF-D amino-pro-peptide; wherein Fc comprises a flanking domain that comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from a mammalian VEGF-C carboxy-pro-peptide and a mammalian VEGF-D carboxy-pro-peptide; wherein RTK comprises the RTK binding domain; and wherein L comprises the linkage between the flanking domain and the RTK binding domain.

In some particular embodiments, the at least one flanking domain of the polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: the VEGF-C amino-pro-peptide sequence of SEQ ID NO: 46; the VEGF-C carboxy-pro-peptide sequence of SEQ ID NO: 47; the VEGF-D amino-pro-peptide sequence of SEQ ID NO: 48; and the VEGF-D carboxy-pro-peptide sequence of SEQ ID NO: 49.

In some particular embodiments, the chimeric polypeptide that comprises the formula Fn-L-RTK-L-Fc binds VEGFR-1 or VEGFR-2; wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of: amino acids 27 to 147 of the VEGF121 amino acid sequence of SEQ ID NO: 4 or fragment thereof; amino acids 27 to 171 of the VEGF145 amino acid sequence of SEQ ID NO: 5 or fragment thereof; amino acids 27 to 191 of the VEGF165 amino acid sequence of SEQ ID NO: 6 or fragment thereof; amino acids 27 to 215 of the VEGF189 amino acid sequence of SEQ ID NO: 7 or fragment thereof; amino acids 27-232 of the VEGF206 amino acid sequence of SEQ ID NO: 3 or fragment thereof.

In a particularly preferred embodiment the chimeric polypeptide comprises an amino acid sequence that is at least 90% identical to the CAC construct amino acid sequence of SEQ ID NO 27.

In other particular embodiments, the chimeric polypeptide that comprises the formula Fn-L-RTK-L-Fc binds VEGFR-1 or VEGFR-2; wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of: amino acids 22 to 188 of the VEGF-B167 amino acid sequence of SEQ ID NO: 44 or fragment thereof; amino acids 22 to 207 of the VEGF-B186 amino acid sequence of SEQ ID NO: 45 or fragment thereof; amino acids 19 to 149 of the P1GF-1 amino acid sequence of SEQ ID NO: 9 or fragment thereof; amino acids 19 to 170 of the P1GF-2 amino acid sequence of SEQ ID NO: 42 or fragment thereof; and amino acids 19 to 221 of the P1GF-3 amino acid sequence of SEQ ID NO: 43 or fragment thereof.

In alternative embodiments, the chimeric polypeptide that comprises the formula Fn-L-RTK-L-Fc binds PDGFR-alpha or PDGFR-beta; wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of: amino acids 87 to 211 of the PDGF-A amino acid sequence of SEQ ID NO: 17 or fragment thereof; amino acids 82 to 190 of the PDGF-B amino acid sequence of SEQ ID NO: 19 or fragment thereof; amino acids 230 to 345 of the PDGF-C amino acid sequence of SEQ ID NO: 21 or fragment thereof; and amino acids 255 to 370 of the PDGF-D amino acid sequence of SEQ ID NO: 24 or fragment thereof.

In still other embodiments, the chimeric polypeptide comprising the formula Fn-L-RTK-L-Fc binds VEGFR-3 or VEGFR-2; wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of: amino acids 103 to 227 of the VEGF-C amino acid sequence of SEQ ID NO: 13 or fragment thereof; and amino acids 93 to 201 of the VEGF-D amino acid sequence of SEQ ID NO: 15 or fragment thereof.

In some embodiments, the chimeric polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of: the CAC construct amino acid sequence of SEQ ID NO 27; the CDD construct amino acid sequence of SEQ ID NO: 37; the CDC construct amino acid sequence of SEQ ID NO: 39; and; the DDC construct amino acid sequence of SEQ ID NO: 41.

In yet another embodiment, another chimeric construct is provided (designated “CUB-VEGF”) which comprises an RTK binding domain, a CUB domain, and at least one linkage that connects the RTK binding domain to the CUB domain, wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: mammalian VEGF-A RTK binding domain amino acid sequences; mammalian VEGF-B RTK binding domain amino acid sequences; mammalian VEGF-C RTK binding domain amino acid sequences; mammalian VEGF-D RTK binding domain amino acid sequences; mammalian VEGF-E RTK binding domain amino acid sequences; mammalian P1GF RTK binding domain amino acid sequences; mammalian PDGF-A RTK binding domain amino acid sequences; and mammalian PDGF-B RTK binding domain amino acid sequences, wherein the CUB domain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of PDGF-C CUB domain amino acid sequences and PDGF-D CUB domain amino acid sequences; and wherein the construct and the RTK binding domain bind to the extracellular domain of at least one receptor tyrosine kinase selected from the group consisting of: VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, and PDGFR-beta. It will be apparent that the construct of this embodiment is not required to have a flanking domain (although constructs of this embodiment optionally further comprise a flanking domain).

In some embodiments, the CUB domain is connected to the N-terminus of the RTK binding domain, while in other embodiments the CUB domain is connected to the C-terminus of the RTK binding domain. In one embodiment, the CUB domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in either SEQ ID NO: 54 or SEQ ID NO: 56.

In particular embodiments, RTK binding domain comprises an amino acid sequence that is at least 90% identical to amino acids 27 to 127 of the VEGF109 amino acid sequence of SEQ ID NO: 52.

In other particular embodiments, the chimeri construct polypeptide comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 57, 59 and 61.

In some embodiments, the CUB domain is attached to the RTK binding domain via a recognition sequence specifically recognized by a proteolytic enzyme such that the proteolytic enzyme if present cleaves at the recognition sequence to remove the CUB domain and produce an activated growth factor.

It is particularly contemplated that any of the constructs and polypeptides of the inventions may be prepared to further include a peptide tag, e.g., a polyhistidine tag. Inclusion of such a tag facilitates purification. In additional embodiments, the constructs or polypeptides may be PEGylated with one or more polyethylene glycol (PEG) moieties.

The constructs and polypeptides of the present invention may advantageously be formulated into compositions wherein such compositions comprise a construct of polypeptide of the invention in a pharmaceutically acceptable carrier, excipient or diluent.

Constructs of the invention comprise polypeptide domains, and are optionally made by recombinant techniques in vitro and/or expressed in vivo. Polynucleotides that comprise nucleotide sequences that encode all (or a portion of) a construct are an additional aspect of the invention. Vectors including expression vectors for in vitro production and gene therapy vectors for in vivo production/expression of constructs, are also an aspect of the invention.

For example, the invention includes polynucleotides comprising a nucleotide sequence that encodes any chimeric polypeptide of the invention, including a chimeric polypeptide of the formula Fn-L-RTK-L-Fc, or a chimeric polypeptide designated “CUB-VEGF,” as discussed above and described in further detail in the description below. In specific embodiments, the polynucleotide further comprises a nucleotide sequence that encodes a signal peptide fused in-frame with the polypeptides described above. The signal peptide facilitates extracellular secretion of the encoded construct when expressed in a suitable host cell.

The invention also includes an expression vector comprising a nucleotide sequence that encodes any chimeric polypeptide of the invention, including a chimeric polypeptide of the formula Fn-L-RTK-L-Fc, or a chimeric polypeptide designated “CUB-VEGF,” operably linked to an expression control sequence or promoter sequence. In some variations, a tissue-specific promoter is used to make a polynucleotide that encodes a construct and that is preferentially expressed in one or a few tissues of an organism; such as skin (for wound healing), muscle (for ischemia), endothelial cells, neurons, or other tissues. In certain embodiments, the promoter sequence is a skin-specific promoter selected from the group consisting of K14, K5, K6, K16 and alpha 1(I) collagen promoter. In other embodiments the promoter is an endothelial cell specific promoter. The expression vector may be any vector used for the expression of a nucleic acid and may for example, be selected from the group consisting of replication deficient adenoviral vectors, adeno-associated viral vectors, and lentivirus vectors. The polynucleotides and vectors of the invention may be formulated as compositions in which the polynucleotides or the vector is presented in a pharmaceutically acceptable carrier, excipient or diluent.

Other aspects of the invention include host cells that have been transformed or transfected with a polynucleotide or vector of the invention. In some variations, the cells are any prokaryotic or eukaryotic cell that can be manipulated (e.g., through transformation or transfection) to express polypeptide constructs of the invention. In some variations, the cells are suitable for ex vivo transfection/transformation and reinplantation into a host organism. For example, in one variation, the host cells are mammalian endothelial cells or mammalian endothelial precursor cells. In another variation, the cells are muscle or neuronal cells or precursors.

Other aspects of the invention are directed to methods of modulating the growth of mammalian endothelial cells or mammalian endothelial precursor cells, using constructs of the invention or using polynucleotides/vectors that encode the constructs. An exemplary method comprises contacting the cells with a composition comprising one or more of the following: a polypeptide construct comprising an RTK binding domain, at least one flanking domain, and at least one linkage that connects the RTK binding domain to the at least one flanking domain; a chimeric polypeptide comprising the formula Fn-L-RTK-L-Fc; a polynucleotide that encodes such a chimeric polypeptide; an expression vector containing such a polynucleotide operatively linked to an expression control sequence; and a cell transformed or transfected with such a polynucleotide or such a vector that expresses the polypeptide construct. In certain embodiments, the contacting comprises administering the composition to a mammalian subject in an amount effective to modulate endothelial cell growth in vivo. In particular embodiments, the mammalian subject is human.

Also contemplated as part of the invention is a method of modulating angiogenesis in a mammalian subject comprising administering to a mammalian subject in need of modulation of angiogenesis a composition, in an amount effective to modulate angiogenesis, comprising one or more of the following: any construct of the invention; a polynucleotide that encodes such a construct; an expression vector containing such a polynucleotide operatively linked to an expression control sequence; and a cell transformed or transfected with such a polynucleotide or such a vector that expresses the polypeptide construct.

Other embodiments of the invention are directed to methods of modulating lymphangiogenesis in a mammalian subject comprising administering to a mammalian subject in need of modulation of lymphangiogenesis a composition in an amount effective to modulate lymphangiogenesis, comprising one or more of the following: any construct of the invention; a polynucleotide that encodes such a construct; an expression vector containing such a polynucleotide operatively linked to an expression control sequence; and a cell transformed or transfected with such a polynucleotide or such a vector that expresses the polypeptide construct.

Also contemplated herein is a method of improving the healing of a skin graft or skin flap to underlying tissue of a mammalian subject, comprising contacting skin graft or skin flap tissue or underlying tissue with a composition comprising a healing agent that is present in said composition in an amount effective to reduce edema or increase perfusion at the skin graft or skin flap, thereby improving the healing of the skin graft or skin flap; wherein the healing agent comprises one or more of the following: any construct of the invention; a polynucleotide that encodes such a construct; an expression vector containing such a polynucleotide operatively linked to an expression control sequence; and a cell transformed or transfected with such a polynucleotide or such a vector that expresses the polypeptide construct.

Also contemplated is an improvement in a medical device for improving circulation, wound healing, or blood flow, comprising coating or impregnating the device with a composition comprising one or more of the following: any construct of the invention; a polynucleotide that encodes such a construct; an expression vector containing such a polynucleotide operatively linked to an expression control sequence; and a cell transformed or transfected with such a polynucleotide or such a vector that expresses the polypeptide construct.

Other aspects of the invention include a patch comprising a pad material having an upper surface and lower surface, an adhesive on the lower surface, and a therapeutic composition, wherein the composition comprises one or more of the following: any construct of the invention; a polynucleotide that encodes such a construct; an expression vector containing such a polynucleotide operatively linked to an expression control sequence; and a cell transformed or transfected with such a polynucleotide or such a vector that expresses the polypeptide construct.

Yet another aspect of the invention is a surgical suturing thread impregnated with a composition, wherein the composition comprises one or more of the following: any construct of the invention; a polynucleotide that encodes such a construct; an expression vector containing such a polynucleotide operatively linked to an expression control sequence; and a cell transformed or transfected with such a polynucleotide or such a vector that expresses the polypeptide construct.

Another aspect of the invention includes methods and compositions for antagonizing a receptor for a PDGF/VEGF family growth factor, the method comprising providing a construct of the invention which inhibits the binding of a growth factor to its respective receptor. In some embodiments, the invention includes methods for antagonizing in a cell at least one receptor selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-α and PDGFR-β, wherein the method comprises administering to the cell an agent selected from the group consisting of a CUB-VEGF construct; a dimer comprising two chimeric polypeptides of the invention, a polynucleotide that encodes such a construct; an expression vector containing such a polynucleotide operatively linked to an expression control sequence; and a cell transformed or transfected with such a polynucleotide or such a vector that expresses the polypeptide construct.

In other embodiments, the invention includes a method for blocking PDGF-D binding to PDGFR-α, but not to PDGFR-β in a cell where both PDGFR-α and PDGFR-β are present, the method comprising administering to the cell an agent selected from the group selected from the group consisting of a CUB-VEGF construct; a dimer comprising two chimeric polypeptides of the invention, a polynucleotide that encodes such a construct; an expression vector containing such a polynucleotide operatively linked to an expression control sequence; and a cell transformed or transfected with such a polynucleotide or such a vector that expresses the polypeptide construct.

Yet another aspect of the invention includes a method for modulating activities of a receptor of a cell which receptor specifically binds to and is activated by a growth factor selected from the group consisting PDGF-A, PDGF-B, the VEGF-homology domain of PDGF-C, VEGF, VEGF-B, VEGF-C and P1GF, the method comprising administering to the cell an agent selected from the group consisting of a CUB-VEGF construct; a dimer comprising two chimeric polypeptides of the invention, a polynucleotide that encodes such a construct; an expression vector containing such a polynucleotide operatively linked to an expression control sequence; and a cell transformed or transfected with such a polynucleotide or such a vector that expresses the polypeptide construct; and providing the cell with a proteolytic enzyme, whereby the growth factor is activated.

Yet another aspect of the invention includes the discovery that fully-processed PDGF-D binds to and activates both PDGFR-α and PDGFR-β. Accordingly, in one embodiment, the present invention provides fully-processed PDGF-D molecules, pharmaceutical compositions comprising the same, and the use thereof for regulating PDGFR-α.

Additional aspects of the invention are defined or summarized in the following numbered paragraphs:

1. A construct comprising:

an RTK binding domain, at least one flanking domain, and at least one linkage that connects the RTK binding domain to the at least one flanking domain;

wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: mammalian VEGF-A RTK binding domain amino acid sequences; mammalian VEGF-B RTK binding domain amino acid sequences; mammalian VEGF-C RTK binding domain amino acid sequences; mammalian VEGF-D RTK binding domain amino acid sequences; mammalian VEGF-E RTK binding domain amino acid sequences; mammalian P1GF RTK binding domain amino acid sequences; mammalian PDGF-A RTK binding domain amino acid sequences; mammalian PDGF-B RTK binding domain amino acid sequences; mammalian PDGF-C RTK binding domain amino acid sequences; and mammalian PDGF-D RTK binding domain amino acid sequences;

wherein the construct and the RTK binding domain bind to the extracellular domain of at least one receptor tyrosine kinase selected from the group consisting of: VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, and PDGFR-beta; and

wherein the at least one flanking domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: a mammalian VEGF-C amino-terminal propeptide; a mammalian VEGF-C carboxy-terminal propeptide; a mammalian VEGF-D amino-terminal propeptide; a mammalian VEGF-D carboxy-terminal propeptide; a mammalian TGF-β1 LAP peptide; and fragments thereof that are effective to bind extracellular matrix proteins or neuropilin proteins;

with the proviso that when the RTK binding domain is at least 90% identical to a VEGF-C RTK binding domain amino acid sequence, the at least one flanking domain is not at least 90% identical to a VEGF-C pro-peptide; and when the RTK binding domain is at least 90% identical to a VEGF-D RTK binding domain amino acid sequence, the at least one flanking domain is not at least 90% identical to a VEGF-D pro-peptide.

2. A construct according to paragraph 1, wherein at least one flanking domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: a mammalian VEGF-C amino-pro-peptide; a mammalian VEGF-C carboxy-pro-peptide; a mammalian VEGF-D amino-pro-peptide; and a mammalian VEGF-D carboxy-pro-peptide.

3. A construct according to paragraph 1 or paragraph 2, further comprising a heparin binding domain connected to the construct by a linkage.

4. A construct according to any one of paragraphs 1-3, further comprising a CUB domain connected to the construct by a linkage.

5. A construct according to any one of paragraphs 1-4, wherein each of the at least one linkage comprises a peptide bond, whereby the RTK binding domain and the at least one flanking domain comprises a chimeric polypeptide.

6. A construct according to paragraph 5, wherein the chimeric polypeptide further comprises a signal peptide.

7. The construct of paragraph 5 or 6, wherein the chimeric polypeptide further comprises a peptide tag.

8. A construct according to any one of paragraphs 5-7, wherein the chimeric polypeptide comprises at least two flanking domains.

9. A construct according to any one of paragraphs 5-8, wherein the chimeric polypeptide satisfies the formula:

Fn-L-RTK-L-Fc,

wherein Fn comprises a flanking domain that comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: a mammalian VEGF-C amino-pro-peptide and a mammalian VEGF-D amino-pro-peptide;

wherein Fc comprises a flanking domain that comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from a mammalian VEGF-C carboxy-pro-peptide and a mammalian VEGF-D carboxy-pro-peptide;

wherein RTK comprises the RTK binding domain; and

wherein L comprises the linkage between the flanking domain and the RTK binding domain.

10. A construct according to any one of paragraphs 5-9, wherein the at least one flanking domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of:

the VEGF-C amino-pro-peptide sequence of SEQ ID NO: 46; the VEGF-C carboxy-pro-peptide sequence of SEQ ID NO: 47;

the VEGF-D amino-pro-peptide sequence of SEQ ID NO: 48; and

the VEGF-D carboxy-pro-peptide sequence of SEQ ID NO: 49.

11. A construct according to any one of paragraphs 5-10,

wherein the chimeric polypeptide binds VEGFR-1 or VEGFR-2;

wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of:

(a) amino acids 27 to 147 of the VEGF121 amino acid sequence of SEQ ID NO: 4;

(b) amino acids 27 to 171 of the VEGF145 amino acid sequence of SEQ ID NO: 5;

(c) amino acids 27 to 191 of the VEGF165 amino acid sequence of SEQ ID NO: 6;

(d) amino acids 27 to 215 of the VEGF189 amino acid sequence of SEQ ID NO: 7;

(e) amino acids 27-232 of the VEGF206 amino acid sequence of SEQ ID NO: 3; and

(f) fragments of (a)-(e) that bind to VEGFR-1 or VEGFR-2.

12. A construct according to any one of paragraphs 1-11, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO 27.

13. A construct according to any one of paragraphs 5-10,

wherein the chimeric polypeptide binds VEGFR-1 or VEGFR-2; and

wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of:

(a) amino acids 22 to 188 of the VEGF-B167 amino acid sequence of SEQ ID NO: 44;

(b) amino acids 22 to 207 of the VEGF-B 186 amino acid sequence of SEQ ID NO: 45;

(c) amino acids 19 to 149 of the P1GF-1 amino acid sequence of SEQ ID NO: 9;

(d) amino acids 19 to 170 of the P1GF-2 amino acid sequence of SEQ ID NO: 42;

(e) amino acids 19 to 221 of the P1GF-3 amino acid sequence of SEQ ID NO: 43; and

(f) fragments of (a)-(e) that bind to VEGFR-1 or VEGFR-2.

14. A construct according to any one of paragraphs 5-10,

wherein the chimeric polypeptide binds PDGFR-alpha or PDGFR-beta; and

wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of:

(a) amino acids 87 to 211 of the PDGF-A amino acid sequence of SEQ ID NO: 17;

(b) amino acids 82 to 190 of the PDGF-B amino acid sequence of SEQ ID NO: 19;

(c) amino acids 230 to 345 of the PDGF-C amino acid sequence of SEQ ID NO: 21;

(d) amino acids 255 to 370 of the PDGF-D amino acid sequence of SEQ ID NO: 24; and

(e) fragments of (a)-(d) that bind to PDGFR-alpha or PDGFR-beta.

15. A construct according to any one of paragraphs 5-10,

wherein the chimeric polypeptide binds VEGFR-3 or VEGFR-2; and

wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of:

(a) amino acids 103 to 227 of the VEGF-C amino acid sequence of SEQ ID NO: 13;

(b) amino acids 93 to 201 of the VEGF-D amino acid sequence of SEQ ID NO: 15; and

(c) fragments of (a)-(b) that bind to VEGFR-3 or VEGFR-2.

16. A construct according to paragraph 15, comprising an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of:

(a) the CDD construct amino acid sequence of SEQ ID NO 37;

(b) the CDC construct amino acid sequence of SEQ ID NO: 39; and,

(c) the DDC construct amino acid sequence of SEQ ID NO: 41.

17. A composition comprising the construct of any one of paragraphs 1-16 in a pharmaceutically acceptable carrier.

18. A polynucleotide comprising a nucleotide sequence that encodes the construct of any one of paragraphs 1-16, wherein the construct comprises a polypeptide.

19. A polynucleotide according to paragraph 18, wherein the polynucleotide further comprises a nucleotide sequence that encodes a signal peptide fused in-frame with the polypeptide.

20. A polynucleotide according to paragraph 18 or 19, further comprising a promoter sequence that promotes expression of the polynucleotide in a mammalian cell.

21. A polynucleotide according to paragraph 20, wherein the promoter sequence comprises a skin-specific promoter.

22. A polynucleotide according to paragraph 21 wherein the promoter is selected from the group consisting of K14, K5, K6, K16 and alpha 1(I) collagen promoter.

23. A polynucleotide according to paragraph 20, wherein the promoter is an endothelial cell specific promoter.

24. A vector comprising the polynucleotide of any one of paragraphs 18-23.

25. An expression vector comprising the polynucleotide of any one of paragraphs 18-23 operably linked to an expression control sequence.

26. An expression vector of paragraph 25, wherein the expression control sequence comprises an endothelial cell specific promoter.

27. A vector of any one of paragraphs 24-26, selected from the group consisting of replication deficient adenoviral vectors, adeno-associated viral vectors, and lentivirus vectors.

28. A composition comprising the polynucleotide of any one of paragraphs 18-23 and a pharmaceutically acceptable carrier, diluent or excipient.

29. A composition comprising the vector of any one of paragraphs 24-27 and a pharmaceutically acceptable carrier, diluent or excipient.

30. A host cell transformed or transfected with the polynucleotide of any one of paragraphs 18-23.

31. A host cell transformed or transfected with the vector of any one of paragraphs 24-27.

32. A host cell according to paragraph 31 that expresses the polypeptide encoded by the polynucleotide.

33. A host cell according to any one of paragraphs 30-32 that comprises a mammalian endothelial cell or endothelial precursor cell.

34. A method of modulating the growth of mammalian endothelial cells or mammalian endothelial precursor cells, comprising contacting the cells with a composition comprising a member selected from the group consisting of:

(a) the construct of any one of paragraphs 1-16 and 44-54;

(b) a polynucleotide that encodes (a);

(c) an expression vector containing (b) operatively linked to an expression control sequence; and

(d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a).

35. A method of paragraph 34, wherein the contacting comprises administering the composition to a mammalian subject in an amount effective to modulate endothelial cell growth in vivo.

36. A method of paragraph 35, wherein the mammalian subject is a human.

37. A method of modulating angiogenesis in a mammalian subject comprising administering to a mammalian subject in need of modulation of angiogenesis a composition comprising a member selected from the group consisting of:

(a) the construct of any one of paragraphs 1-16 and 43-54;

(b) a polynucleotide that encodes (a);

(c) an expression vector containing (b) operatively linked to an expression control sequence; and

(d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a);

wherein the composition is administered in an amount effective to modulate angiogenesis.

38. A method of modulating lymphangiogenesis in a mammalian subject comprising administering to a mammalian subject in need of modulation of lymphangiogenesis a composition comprising a member selected from the group consisting of:

(a) the construct of any one of paragraphs 1-16 and 43-54;

(b) a polynucleotide that encodes (a);

(c) an expression vector containing (b) operatively linked to an expression control sequence; and

(d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a);

wherein the composition is administered in an amount effective to modulate lymphangiogenesis.

39. A method of improving the healing of a skin graft or skin flap to underlying tissue of a mammalian subject, comprising:

contacting skin graft or skin flap tissue or underlying tissue with a composition comprising a healing agent that is present in said composition in an amount effective to reduce edema or increase perfusion at the skin graft or skin flap, thereby improving the healing of the skin graft or skin flap;

wherein the healing agent is selected from the group consisting of:

(a) the construct of any one of paragraphs 1-16 and 43-54;

(b) a polynucleotide that encodes (a);

(c) an expression vector containing (b) operatively linked to an expression control sequence; and

(d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a).

40. An improvement in a medical device for improving circulation, wound healing, or blood flow, comprising coating or impregnating the device with a composition comprising an angiogenic agent selected from the group consisting of:

(a) the construct of any one of paragraphs 1-16 and 43-54;

(b) a polynucleotide that encodes (a);

(c) an expression vector containing (b) operatively linked to an expression control sequence; and

(d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a).

41. A patch comprising a pad material having an upper surface and lower surface, an adhesive on the lower surface, and a therapeutic composition,

wherein the composition comprises a healing agent selected from the group consisting of

(a) the construct of any one of paragraphs 1-16 and 43-54;

(b) a polynucleotide that encodes (a);

(c) an expression vector containing (b) operatively linked to an expression control sequence; and

(d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a).

42. A surgical suturing thread coated or impregnated with a composition,

wherein the composition comprises a healing agent selected from the group consisting of:

(a) the construct of any one of paragraphs 1-16 and 43-54;

(b) a polynucleotide that encodes (a);

(c) an expression vector containing (b) operatively linked to an expression control sequence; and

(d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a).

43. A construct comprising:

an RTK binding domain, a CUB domain, and at least one linkage that connects the RTK binding domain to the CUB domain;

wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: mammalian VEGF-A RTK binding domain amino acid sequences; mammalian VEGF-B RTK binding domain amino acid sequences; mammalian VEGF-C RTK binding domain amino acid sequences; mammalian VEGF-D RTK binding domain amino acid sequences; mammalian VEGF-E RTK binding domain amino acid sequences; mammalian P1GF RTK binding domain amino acid sequences; mammalian PDGF-A RTK binding domain amino acid sequences; and mammalian PDGF-B RTK binding domain amino acid sequences;

wherein the CUB domain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of PDGF-C CUB domain amino acid sequences and PDGF-D CUB domain amino acid sequences; and

wherein the construct and the RTK binding domain bind to the extracellular domain of at least one receptor tyrosine kinase selected from the group consisting of: VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, and PDGFR-beta.

44. The construct according to paragraph 43, wherein the CUB domain is connected to the N-terminus of the RTK binding domain.

45. The construct according to paragraph 43, wherein the CUB domain is connected to the C-terminus of the RTK binding domain.

46. The construct according to any one of paragraphs 43-45, wherein the CUB domain comprises an amino acid sequence set forth in SEQ ID NO: 53.

47. The construct according to any one of paragraphs 43-45, wherein the CUB domain comprises an amino acid sequence set forth in SEQ ID NO: 55.

48. The construct according to any one of paragraphs 1-11 and 43-47, wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to amino acids 27 to 127 of the VEGF109 amino acid sequence of SEQ ID NO: 52:

49. The construct according to any one of paragraphs 43-48, further comprising a heparin binding domain connected to the construct by a linkage.

50. A construct according to any one of paragraphs 43-49, wherein the linkage comprises a peptide bond, whereby the RTK binding domain and the CUB domain comprise a chimeric polypeptide.

51. The construct according to paragraph 50, wherein the chimeric polypeptide further comprises a signal peptide.

52. The construct of paragraph 51, wherein the chimeric polypeptide further comprises a peptide tag.

53. The construct according to paragraph 48, wherein the construct comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 57, 59, and 61.

54. The construct according to paragraphs 43-53, wherein the CUB domain is connected to the growth factor via a recognition sequence specifically recognized by a proteolytic enzyme such that the proteolytic enzyme if present cleaves at the recognition sequence to remove the CUB domain and produce an activated growth factor.

55. A dimer comprising two construct polypeptides of paragraphs 5-15 and 49-54.

56. The dimer of paragraph 54, which is a homodimer.

57. The dimer of paragraph 54, which is a heterodimer.

58. A method for antagonizing in a cell at least one receptor selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-α and PDGFR-β, wherein the method comprises administering to the cell an agent selected from the group consisting of:

(a) the construct of any one of paragraphs 43-54;

(b) the dimer of any one of claims 55-57;

(c) a polynucleotide that encodes (a);

(d) an expression vector containing (b) operatively linked to an expression control sequence; and

(e) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a).

59. A method for blocking PDGF-D binding to PDGFR-α, but not to PDGFR-β in a cell where both PDGFR-α and PDGFR-β are present, the method comprising administering to the cell an agent selected from the group consisting of:

(a) the construct of any one of paragraphs 43-54;

(b) the dimer of any one of claims 55-57;

(c) a polynucleotide that encodes (a);

(d) an expression vector containing (b) operatively linked to an expression control sequence; and

(e) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a).

60. A method for modulating activities of a receptor of a cell which receptor specifically binds to and is activated by a growth factor selected from the group consisting PDGF-A, PDGF-B, the VEGF-homology domain of PDGF-C, VEGF, VEGF-B, VEGF-C and P1GF, the method comprising administering to the cell an agent selected from the group consisting of:

(a) the construct of any one of claims 43-54;

(b) the dimer of any one of claims 55-57;

(c) a polynucleotide that encodes (a);

(b) an expression vector containing (c) operatively linked to an expression control sequence; and

(e) a cell transformed or transfected with (c) or (d) and that expresses the polypeptide of (a); and

providing the cell with a proteolytic enzyme, whereby the growth factor is activated.

61. An isolated and fully-processed PDGF-D polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 22.

62. The isolated polypeptide of paragraph 61, consisting of the amino acid sequence set forth SEQ ID NO: 22.

63. A pharmaceutical composition comprising the polypeptide of paragraph 61 and a pharmaceutically acceptable excipient.

64. An isolated polynucleotide that encodes the polypeptide of paragraph 61.

65. An expression vector comprising the polynucleotide of paragraph 64 operably linked to a promoter.

66. The expression vector of paragraph 65, wherein the promoter is a tissue- or cell type-specific promoter.

67. A cell comprising the expression vector of paragraph 65 or paragraph 66.

68. A method for stimulating phosphorylation of PDGFR-α of a cell, the method comprising administering to the cell the isolated PDGF-D polypeptide of paragraph 61, wherein the polypeptide binds to PDGFR-α.

69. The method of paragraph 68, wherein the PDGFR-α of the cell is activated.

70. The method of c paragraph 68, wherein the phosphorylation of both PDGFR-α and PDGFR-β of the cell are stimulated.

71. The method of paragraph 70, wherein both PDGFR-α and PDGFR-β of the cell are activated.

The foregoing summary is not intended to define every aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document.

In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations defined by specific paragraphs above. For example, certain aspects of the invention that are described as a genus, and it should be understood that every member of a genus is, individually, an aspect of the invention. Also, aspects described as a genus or selecting a member of a genus, should be understood to embrace combinations of two or more members of the genus. Although the applicant(s) invented the full scope of the invention described herein, the applicants do not intend to claim subject matter described in the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings from part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

FIG. 1A: Schematic depiction of a chimeric polypeptide wherein one flanking domain (F) is positioned amino-terminal to the receptor tyrosine kinase (RTK) binding domain.

FIG. 1B: Schematic depiction of a chimeric polypeptide wherein one flanking domain is positioned carboxy-terminal to the RTK binding domain.

FIG. 1C: Schematic depiction of a chimeric polypeptide wherein two flanking domains (F1, F2) are positioned with a central RTK binding domain.

FIG. 1D: Schematic depiction of a chimeric polypeptide wherein two flanking domains are both positioned amino-terminal to the RTK binding domain.

FIG. 1E: Schematic depiction of a chimeric polypeptide wherein two flanking domains are both positioned carboxy-terminal to the RTK binding domain.

FIG. 1F: Schematic depiction of a chimeric polypeptide wherein two RTK binding domains are positioned with a central flanking domain.

FIG. 1G: Schematic depiction of a chimeric polypeptide wherein three flanking domains are alternating with two RTK binding domains.

FIGS. 2A-2C: Photograph depicting the effects of adenoviral vectors that code for either VEGF-CAC (FIG. 2A), VEGF-A165 (FIG. 2B) or control (LacZ, FIG. 2C) on the blood vasculature of mouse skin.

DETAILED DESCRIPTION

The invention includes new materials (e.g., biomolecules, compositions, medical devices) and methods and medical uses for modulating angiogenic processes as well as modulating the growth and maturation of progenitor cells.

A. Chimeric Molecules of the Present Invention

One aspect of the invention are constructs (e.g., molecules or compounds) comprised of a receptor tyrosine kinase receptor (RTK) binding domain attached to at least one flanking (F) domain that confer novel biological properties to the constructs, compared to the properties of the RTK domain alone. The attachment is generically referred to as a linkage (L) and can be as simple as a peptide bond or oligopeptide that link the domains to form a single, chimeric polypeptide chain, or can be more complex structures described herein. Because the constructs comprise at least one RTK and one flanking sequence that do not originate from the same gene but have been recombined, the constructs are referred to herein as “chimeric” constructs or “chimeric” polypeptides. Chimeric polypeptide constructs of the invention can have a variety of structures, as depicted by the following schematic formulae:

F-L-RTK (one flanking domain positioned amino terminal to the RTK binding domain)

RTK-L-F (one flanking domain positioned carboxy-terminal to the RTK binding domain)

F1-L1-RTK-L2-F2 (two flanking domains with a central RTK binding domain)