Ligand for vascular endothelial growth factor receptor

Details Ligand for vascular endothelial growth factor receptor Ligand for vascular endothelial growth factor receptor

2001-02-02

2004-05-11

Ulm, John (Department: 1646)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Amino acid sequence disclosed in whole or in part; or...

C424S184100, C530S300000, C530S326000, C530S345000

Reexamination Certificate

active

06733755

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to compositions comprised of a peptide ligand or derivatives thereof (hereinafter, “Ligand”) that are capable of specifically binding to the high affinity receptor-1 of vascular endothelial growth factor (hereinafter, “VEGF”) and structure similar receptors. The invention further describes the Ligand that is capable of inhibiting angiogenesis induced by VEGF. The invention further describes compositions comprised of a Ligand, or its complex with a carrier associated with a biological agent. The invention further relates to a method of inhibiting angiogenesis by using a Ligand or its complex with a carrier alone or associated with a biological agent. The invention further relates to a method for targeting of a biological agent to a predetermined compartment by associating the agent with a Ligand or its complex with a carrier. These compositions are well suited for use as therapeutic and diagnostic agents for the pathologies that are associated with an increased level of VEGF receptors, and as vehicles for delivering biologically active and diagnostic agents to the sites in which VEGF receptor levels are increased.
BACKGROUND OF THE INVENTION
The growth of new blood vessels from existing endothelium (angiogenesis) is tightly controlled in healthy adults by opposing effects of positive and negative regulations. Under certain pathological conditions, including proliferative retinopathies, rheumatiod arthritis, psoriasis and cancer, positive regulations prevail and angiogenesis contributes to disease progression (Folkman (1995)
Nature Medicine
1:27-31; Achen and Stacker (1998)
Int.J. Exp. Pathol.
79:255-265). In cancer, the notion that angiogenesis represents the rate limiting step of tumor growth and metastasis, (Folkman (1971)
New Engl. J. Med.
285:1182-1186) is now supported by considerable experimental evidence (reviewed in Aznavoorian et al. (1993)
Cancer
71:1368-1383; Bidfer and Ellis (1994)
Cell
79:185-188; Plate and Warnke (1997)
J. Neurooncol
35:365-372; de Jong et al. (1998)
J. Pathol
184:44-52).
A number of angiogenic growth factors have been described to date among which vascular endothelial growth factor (VEGF) appears to play a key role in the regulation of vasculogenesis and angiogenesis as a highly specific mitogen for endothelial cells (Brown et al., (1997) Control of Angiogenesis (Goldberg and Rosen, eds) Birkhauser, Basel, pp233-269; Martiny-Broun and Marme(1995)
Current Opin. in Biotech.
6:675-680; Ferrara and Davis-Smyth (1997)
Endocrine Reviews
18:4-25).
VEGF is a glycosylated, disulfide-linked homodimeric protein consisting of two 23 kD subunits. Four different monomeric isoforms of VEGF exist ranging in size from 121 to 206 residues in humans (VEGF
121
, VEGF
165
, VEGF
189
and VEGF
206
). Transcripts encoding the three shorter forms are detected in the majority of tumor cells and tumor tissue expressing VEGF gene. The isoforms result from different splicing events, and all variants share the same 115 N-terminal as well as six C-terminal residues and have a leader sequence to leave the cells. VEGF
165
is the dominant isoform, while VEGF206 has so far only been identified in human fetal liver cDNA library VEGF
165
and VEGF
189
bind heparin with high affinity, and are sequestered to the cell surface or within the extracellular matrix bound to proteoglycans, while VEGF
121
does not bind heparin and is thus freely diffusible. Plasmiin cleavage of VEGF
165
generates a 110-residue long N-terminal fragment (the receptor-binding domain) that no longer binds heparin but is equipotent to VEGF
121
in its ability to induce endothelial cell proliferation.
VEGF is expressed in embryonic tissues (Breier et al., (1992)
Development
(Camb.) 114:521), macrophages, proliferating epidermal keratinocytes during wound healing (Brown et al., (1992)
J. Ex. Med.
176:1375-9) and may be responsible for tissue edema associated with inflammation (Ferrara and Davis-Smyth (1997)
Endocrine Reviews
18:4-25). In situ hybridization studies have demonstrated high VEGF expression in a number of human tumors including glioblastoma, ovarian tumors, carcinoma, hemangioblastoma, brain neoplasms and Kaposi's sarcoma (Plate et al., (1992)
Nature
359:845-848; Zebrowski et al., (1999)
Ann. Surg. Oncol.
6:373-378). High levels of VEGF were also observed in hypoxia-induced angiogenesis (Shweiki etal., (1992)
Nature
359:843-845).
The biological function of VEGF is mediated through binding to two high affinity receptors which are selectively expressed on endothelial cells during embryogenesis (Millauer et al., (1993)
Cell
72:835-838) and VEGF related pathologies (tumor formation). VEGF receptors include the human kinase domain receptor (KDR), described in U.S. Pat. No. 5,712,380; its murine analog flk-1, sequenced by Mallhews (1991)
Proc. Natl. Acad. Sci USA,
88:9026-9030; U.S. Pat. No. 5,270,458 and the Fsm—like tyrosine kinase (Flt-1) (Shibuya et al., (1990) Oncogene 5:519-524). All of them are class III tyrosine kinases (Vaisman et al., 1990:
J. Biol Chem.
265, 19461-19466; Kaipainen et al., (1993)
J. Exp. Med.
178:2077-2088). Studies in mice have shown that the expression of KDR reaches the highest levels during embryonic vasculogenesis and angiogenesis (Millauer et al., 1993
Cell
72:835-838). In contrast, only low levels of mRNA for Flt-1 were found during fetal growth and moderate levels during organogenesis, but high levels in newborn mice (Peters et al., 1993
Proc. Natl. Aca. Sci. U.S.A
90(16):7533-7). Experiments with knockout mice deficient in either receptor revealed that KDR is essential for the development of endothelial cells, whereas Flt-1 is necessary for the organization of embryonic vasculature (Fong et al. 1995
Dev. Dyn.
203(1):80-92; Shalaby et al., 1995
Nature
376 (6535:62-6). KDR and Flt-1, each ~1300 amino acid residues long, are composed of 7 extracellular Ig-like domains containing the ligand-binding region, a single short membrane-spanning sequence, and an intracellular region containing tyrosine kinase domains. The amino acid sequences of KDR and Flt-1 are ~45% identical to each other. Flt-1 has the higher affinity for VEGF (K
D
=10-20 pM) compared to 75-125 pM for the KDR receptor. VEGF binding to KDR but not Flt-1 elicits an efficient (ED50~0.1-1 ng/ml) DNA synthetic and chemotactic endothelial cell response. Activation of Flt-1 receptor by VEGF might modulate the interaction of endothelial cells with each other or the basement membrane on which they reside.
The Flt-1 receptor MRNA can be spliced to generate forms encoding either the full-length membrane-spanning receptor or a soluble form, denoted sFlt-1. Pure sFlt-1 retains its specific high affinity binding for VEGF and fully inhibits VEGF-stimulated endothelial cell mitogenesis by dominant negative mechanism.
Like other growth factor transmembrane tyrosine kinase receptors, VEGF receptors presumably undergo ligand-induced dimerization, that triggers signal transduction by promoting either autophosphorylation or transphosphorylation specific downstream signal transduction protein mediators.
To gain a better understanding of the biological activity of VEGF the analysis of structure/activity relationships was performed using site-directed mutagenesis and epitope mapping of neutralizing monoclonal antibodies (Keyt et al., (1996)
J. Biol. Chem.
271:5638-5646). Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64 and Glu67, were associated with Flt-1 binding. The three-dimensional structure of the receptor-binding domain of VEGF (residues 8-109) showed that these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer (Wiesmann et al., (1997)
Cell
91:695-704). Mutations within the KDR site had minimal effect on Flt-1 binding, suggesting that receptors have different binding sites on VEGF which may serve to dimerize tyrosine kinase receptors resulting in initiation of an

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