Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-02-26
2004-03-16
Saoud, Christine J. (Department: 1647)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C530S399000, C435S069100
Reexamination Certificate
active
06706871
ABSTRACT:
FIELD OF THE INVENTION
Background
Growth factors, such as fibroblast growth factor (FGF), are ligands for receptor tyrosine kinases (RTK) and are involved in stimulating cellular proliferation and migration. As a result, growth factor and RTK antagonists may be involved in conditions involving uncontrolled growth and vascularization.
Sprouty was initially identified as an antagonist of FGF signaling in the developmental pathway of Drosophila tracheal branching morphogenesis. An FGF family member, Branchless, is the critical determinant of tracheal branching pattern through inducing cell migration which results in branching morphogenesis. It was determined that Branchless induces expression of a potent negative signal, Sprouty, that inhibits branching morphogenesis and was found to act as an negative regulator of tracheal development. Loss of function mutations in the Sprouty gene led to increased tracheal branching while gain of function mutations severely blocked tracheal development. Overexpression of Branchless overrode the Sprouty loss of function mutation indicating that Sprouty's antagonist activity was specific for the FGF activity in this developmental pathway (Hacohen et al. (1998), Cell, 92: 253-263; Metzger et al. (1999), Science, 284:1635-1639).
Currently, there are three known human homologs of the Drosophila Sprouty protein denoted human Sprouty-1, Sprouty-2 and Sprouty-3 (genes denoted h-Spryl, h-Spry2, h-Spry3). H-Spry-2 encodes an approximately 35 Kd protein which contains a cysteine-rich domain. This domain is highly conserved among the human homologs (Hacohen et al. (1998), Cell, 92: 253-263).
The Sprouty mouse homolog (sprouty-2, encoded by gene mSpry-2) also was found to regulate bronchial branching patterns in the developing lung. In this system, mSpry-2 inhibited FGF10-induced bronchial branching. The mSpry-2 amino acid sequence is 97% homologous to h-Spry2, indicating that this protein may be highly conserved among different species (Tefft et al. (1999), Curr Biol, 9: 219-222). Another mouse homolog, sprouty-4, has also been identified (De Maximy et al. (1999), Mech. Dev. 81, 213-216).
Other evidence indicates that Sprouty may act as a general inhibitor of RTK activity. In addition to its inhibitory effects on FGF signaling, Sprouty expression was shown to antagonize epidermal growth factor (EGF) action in the developing Drosophila eye imaginal disc, larval peripheral nervous system, embryonic central nervous system, developing wing and developing ovary (Casci et al. (1999), Cell, 96: 655-665, Kramer et al. (1999), Development, 126: 2515-2525). Sprouty also inhibits the action of an unrelated RRX, Torso, which initiates the development of terminal structures in the developing Drosophila embryo (Casci et al. (1999), Cell, 96: 655-665).
Sprouty is an intracellular protein which associates with the plasma membrane through its cysteine-rich domain. Sprouty acts within the mitogen activated protein (MAP) kinase signaling cascade, and its inhibitory action has been mapped to be downsteam of the RTK and upstream of ras. This suggests that Sprouty may be binding to adaptor proteins, such as Drk (the Drosophila homolog of Grb2) and Gap1, within the MAP kinase signaling cascade (Casci et al. (1999), Cell, 96: 655-665).
The autonomous manner in which Sprouty works in the eye is different from the non-autonomous manner in which it acts in the tracheal system. It has been speculated that Sprouty may not only act intracellularly to repress signal transduction but may also act on a second signaling relay to repress cell fate in the neighbors of Sprouty-expressing cells (Placzek et al. (1999), Current Biol., 9: R506-510).
Many growth factors, such as FGF, EGF, platelet-derived growth factor and insulin-like growth factor, elicit their response through RTKs. Activation of the RTK initiates signaling through the MAP kinase cascade. As a result, the MAP kinase signaling pathway is involved in many cellular actions including proliferation, migration, angiogenesis, and organogenesis.
Thus, there is a need for growth factor and MAP kinase signaling antagonists, such as Sprouty family members, which may be useful in modulating cell growth, migration and vascularization.
SUMMARY OF THE INVENTION
The compositions of the present invention include novel isolated polypeptides, in particular, novel human growth factor antagonist proteins and active variants thereof isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.
The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
A nucleotide sequence encoding a growth factor antagonist protein designated FGFAn-Hy is set forth in SEQ ID NO: 1, and its deduced amino acid sequence is set forth in SEQ ID NO: 2. This growth factor antagonist protein is believed to play a role in the mitogen activated protein (MAP) kinase signaling pathway involving receptor tyrosine kinases (RTKs) and ras. The polypeptide set out in SEQ ID NO: 2 is 300 amino acids in length, and appears to have no readily identifiable signal sequence, indicating that it is most likely an intracellular protein like the Drosophila growth factor antagonist protein Sprouty. The polypeptide of SEQ ID NO: 2 displays amino acid homology with the Drosophila Sprouty, as well as with mammalian family members human Sprouty-1, -2 and -3 and mouse Sprouty-2 and -4. An alignment of FGFAn-Hy and other members of the sprouty family is shown in FIG.
1
. FGFAn-Hy is most closely related to mouse sprouty-4 (92% amino acid sequence identity). Additional family members can be identified using SEQ ID NO: 1 as a molecular probe.
The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a portion thereof corresponding to the full length or mature protein. The isolated polynucleotides of the invention further include, but are not limited to, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; a polynucleotide comprising the full length protein coding sequence of SEQ ID NO: 1; and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NO: 1. The polynucleotides of the present invention also include, but are not limited to, polynucleotides that encode polypeptides with growth factor antagonist activity and that hybridize under stringent hybridization conditions to the complement of (a) the nucleotide sequence of SEQ ID NO: 1, or (b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2; a polynucleotide which is an allelic variant of any polynucleotide recited above; a polynucleotide which encodes a species homolog of any of the proteins recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide having an amino acid sequence of SEQ ID NO: 2. The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.
The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a portion thereof corresponding to the full length or mature protein. Polypeptides of the invention also include polypeptides with growth factor antagonist activity that are encoded by (a) polynucleotides set out in SEQ ID NO: 1; or (b) polynucleotides that hybr
Ballinger Dennis G.
Montgomery Julie Reeder
Nuvelo, Inc.
Polizotto Renée S.
Saoud Christine J.
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