Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Reexamination Certificate
2000-03-06
2003-01-14
Kunz, Gary L. (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
C530S350000, C435S007100, C435S069100, C435S007800, C435S252300, C514S002600, C514S008100
Reexamination Certificate
active
06506884
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns novel nucleic acid sequences, vectors and host cells containing them, amino acid sequences encoded by said sequences, and antibodies reactive with said amino acid sequences, as well as pharmaceutical compositions comprising any of the above. The present invention further concerns methods for screening for candidate activator or deactivators utilizing said amino acid sequences.
BACKGROUND OF THE INVENTION
Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo. DNA sequencing suggests the existence of several molecular species of VEGF. VEGFs are secreted proteins in contrast to other endothelial cell mitogens such as acidic or basic fibroblast growth factors and platelet-derived endothelial cell growth factors. VEGF was found to augment human growth by inducing neovascularization. Thus it was suggested that neutralization of VEGF activity may have clinical application in inhibiting malignant cells-induced angiogenesis, decreasing blood supply to the cancerous tissue, leading eventually to its destruction. VEGF has various other functions on endothelial cells, the most prominent of which is the induction of proliferation and differentiation. It was found to be capable of preventing serum starvation-induced apoptosis and this inhibition may represent a major aspect of the regulatory activity of VEGF on vascular endothelium.
VEGF was also found to be involved in the development and the growth of ovarian corpus luteum (CL), since its development is dependent on the growth of new capillary vessels. It has been reported that Flt-1 receptors which inhibit vascular endothelial growth factor bioactivity, resulted in complete separation of corpus luteum angiogenesis in a rat model of hormonally induced ovulation, indicated that VEGF is essential for CL angiogenesis and may be involved in the control of fertility and treatment of ovarian disorders characterized by hypervascularity and hyperplasia.
The human VEGF gene has been recently assigned to chromosome 6p21.2. cDNA sequence analysis of a variety of human VEGF clones had initially indicated that VEGF may exist as one of four different molecular species, having respectively, 121, 165, 189 and 206 amino acids (VEGF
121
, VEGF
165
, BEGF
206
). Alternative exon splicing of a single VEGF gene is the basis for this molecular heterogeneity, VEGF
165
lacks the residues encoded by exon 6, while VEGF
121
lacks the residues encoded by exons 6 and 7. VEGF
189
has an insertion of 24 amino acids highly enriched in basic residues and VEGF
206
has an additional insertion of 17 amino acids. VEGF
165
is the predominant isoform secreted by a variety of normal and transformed cells. Transcripts encoding VEGF
121
and VEGF
189
are detected in the majority of cells and tissues expressing the VEGF gene. In contrast, VEGF
206
is a very rare form.
Native VEGF is a basic, heparin-binding, homodimeric glycoprotein of 45 kDA. These properties correspond to those of VEGF
165
. VEGF
121
is an acidic polypeptide that fails to bind to heparin. VEGF
189
and VEGF
200
are more basic and bind to heparin with greater affinity than VEGF
165
. VEGF
121
is a freely soluble protein; VEGF
165
is also secreted, although a significant fraction remains bound to the cell surface and the extracellular matrix (ECM). VEGF
189
and VEGF
206
are almost completely sequestered in the ECM, but may be released in a soluble form by heparin or heparinase. Also, these long forms may be released by plasmin following cleavage at the COOH terminus (Ferrara, N.,
European J. of Cancer,
32A(14):2413-2422 (1996)).
GLOSSARY
In the following description and claims use will be made, at times, with a variety of terms, and the meaning of such terms as they should be construed in accordance with the invention is as follows:
“Vascular endothelial growth factor variant (VEGFV) nucleic acid sequence”—the sequence shown in SEQ ID NO: 1, sequences having at least 70% identity to said sequence and fragments of the above sequences of least 20 b.p. long. This sequence is a sequence coding for a novel alternative splice variant of the native VEGF. While the known VEGF peptides include 206, 189, 105 or 121 amino acids, the novel VEGF variant peptide of the invention includes only 141 amino acids, −27 of which being in the signal peptide and 114 being present in the mature protein. According to the terminology used in the publication of Ferrara (supra) this new variant should be termed VEGF
114
.
“Vascular endothelial growth factor variant (VEGFV product)—also referred at times as the “VEGFV protein” or “VEGFV polypeptide”—is an amino acid sequence having the first 141 amino acids of the native VEGF. This naturally occurring sequence is the result of alternative splicing. The amino acid sequence may be a peptide, a protein, as well as peptides or proteins having chemically modified amino acids (see below) such as a glycopeptide or glycoprotein. An example of an VEGFV product is shown in SEQ ID NO: 2. The term also includes analogues of said sequences in which one or more amino acids has been added, deleted, substituted (see below) or chemically modified (see below) as well as fragments of this sequence having at least 10 amino acids.
“Nucleic acid sequence”—a sequence composed of DNA nucleotides, RNA nucleotides or a combination of both types and may includes natural nucleotides, chemically modified nucleotides and synthetic nucleotides.
“Amino acid sequence”—a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified (see below), or composed of synthetic amino acids.
“Fragment of VEGFV product”—a polypeptide which has an amino acid sequence which is the same as part of but not all of the amino acid sequence of the VEGFV product.
“Fragments of VEGFV nucleic acid sequence” a continuous portion, preferably of about 20 nucleic acid sequences of the VEGFV nucleic acid sequence.
“Conservative substitution”—refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. [Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution.
“Non-conservative substitution”—refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gln.
“Chemically modified”—when referring to the product of the invention, means a product (protein) where at least one of its amino acid residues is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art. Among the numerous known modifications typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
“Biologically active”—refers to the VEGFV product having structural, regulatory or biochemical functions of the naturally occurring VEGFV product, for example the same effect on vascular endothelial cells.
“Immunologically active” defines the capability of a natural, recombinant or synthetic VEGFV product, or any fragment thereof, to induce a specific immune response in appropriate animals or
Engel Sharon
Mintz Liat
Savitzky Kinneret
Birch & Stewart Kolasch & Birch, LLP
Compugen Ltd.
Kunz Gary L.
Prasad Sarada C
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