SH2 domain-containing peptides

Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives

Reexamination Certificate

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Reexamination Certificate

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06326482

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides which are characterized by the presence of novel SH2-containing proteins (Nsp's).
BACKGROUND OF THE INVENTION
Interactions between ligands and the cognate cell surface receptors are critical for a variety of biological processes including maintenance of cellular and organism homeostasis, development, and tumorigenesis. Many of these ligands can activate multiple independent pathways and the strength of the activation of different pathways can be modulated by the presence or absence of signals generated by other receptors, Hostamsigil, et al.,
Proc. Natl. Acad. Sci. USA
91: 4854-58 (1994); Kanety et al.,
J. Biol. Chem
. 270: 23780-84 (1995); Luttrell et al.,
J. Biol. Chem
. 272: 4637-44 (1997). Adaptor molecules may be critical in integrating multiple signaling cascades and in determining the cell type specific response to extracellular stimuli. These adaptor proteins have no apparent catalytic activity. Rather, they contain one or more domains that mediate protein-protein or protein-lipid interactions. The most common conserved interaction domains in these adaptor molecules are Src homology (SH2), SH3, phosphotyrosine binding (PTB) and pleckstrin homology domains. [Reviewed in Pawson and Scott,
Science
278: 2075-80 (1997)].
Signals generated by growth factors such as epidermal growth factor (EGF) or insulin growth factor-1 (IGF-1) through receptor tyrosine kinases (RTK) or by extracellular matrix components acting through the integrin receptors can induce cytoskeletal changes, Leventhal, et al.,
J. Biol. Chem
. 272: 5214-18 (1997); Ojaniemi & Vuori,
J. Biol. Chem
. 272: 2443-47 (1996). There are also indications that RTKs can modulate intogrin signals and vice versa, Doerr & Jones,
J. Biol. Chem
. 271: 2443-47 (1996); Jones et al.,
Proc. Natl. Acad. Sci. USA
93: 2482-87 (1996); Knight et al.,
J. Biol. Chem
. 270: 10199-203 (1995); Matsumoto et al.,
Cancer Metas. Rev
. 14: 205-17 (1995). However the details of how RTKs signal to the cytoskelelal components have not been fully resolved. Further, while some adaptor proteins have a limited pattern of expression [Liu & Roth,
Proc. Natl. Acad. Sci. USA
92: 10287-91 (1995), Nakamura et al.,
Oncogene
13: 1111-21 (1996)], many are ubiquitously expressed [Araki et al.,
Diabetes
42: 1041-54 (1993): Frantz et al.,
J. Biol. Chem
. 272: 2659-67 (1997)]. Thus, it is not clear how biologically relevant outputs are modulated as cells differentiate.
Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis). In a cancerous state a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
Alteration of gene expression is intimately related to the uncontrolled cell growth and de-differentiation which are a common feature of all cancers. The genomes of certain well studied tumors have been found to show decreased expression of recessive genes, usually referred to as tumor suppression genes, which would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes, such as oncogenes, that act to promote malignant growth. Each of these genetic changes appears to be responsible for importing some of the traits that, in aggregate, represent the full neoplastic phenotype (Hunter,
Cell
64: 1129 [1991]; Bishop,
Cell
64: 235-248 [1991]).
A well known mechanism of gene (eg. oncogene) overexpression in cancer cells is gene amplification. This is a process where in the chromosome of the ancestral cell multiple copies of a particular gene are produced. The process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome (Alitalo et al.,
Adv. Cancer Res
. 47: 235-281 [1986]). It is believed that the overexpression of the gene parallels gene amplification, ie. is proportionate to the number of copies made.
Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer. For example, it has been found that the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2), which encodes a 185-kd transmembrane glycoprotein receptor (p185
HER2
; HER2) related to the epidermal growth factor receptor EGFR), is overexpressed in about 25% to 30% of human breast cancer (Slamon et al.,
Science
235: 177-182 [1987];Slamon et al.,
Science
244: 707-712 [1989]).
It has been reported that gene amplification of a proto-oncogene is an event typically involved in the more malignant forms of cancer, and could act as a predictor of clinical outcome (Schwab et al.,
Genes Chromosomes Cancer
1, 181-193 [1990]; Alitalo et al., supra). Thus, erbB2 overexpression is commonly regarded as a predictor of a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (Slamon et al., [1987] and [1989], supra; Ravdin and Chamness,
Gene
159: 19-27 [1995]; and Hynes and Stern,
Biochem Biophys Acta
1198: 165-184 [1994]), and has been linked to sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines (Baselga et al.,
Oncology
11 (3 Suppl 1): 43-48 [1997]). However, despite the association of erbB2 overexpression with poor prognosis, the odds of HER2-positive patients responding clinically to treatment with taxanes were greater than three times those of HER2-negative patients. A recombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (a humanized version of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or Herceptin*) has been clinically active in patients with ErbB2-overexpressing metastatic breast cancers that had received extensive prior anticancer therapy. (Baseiga et al.,
J. Clin. Oncol
. 14: 737-744 [1996]).
SUMMARY OF THE INVENTION
Applicants have identified cDNA clone (DNA30676, DNA40575, DNA61601)(SEQ ID NO:s 2, 4 & 6, respectively) that encodes a novel polypeptide, designated in the present application as “Nsp1, Nsp2 & Nsp3” (SEQ ID NOS: 1, 3 and 6), respectively.
In one embodiment, the invention provides an isolated nucleic acid molecule having at least about 80% sequence identity to (a) a DNA molecule encoding a polypeptide comprising the sequence of amino acids 1 to 576 of
FIG. 1
(SEQ ID NO: 1), amino acids 1 to 501 of
FIG. 2
(SEQ ID NO: 3) or amino acids 1 to 703 of
FIG. 3
(SEQ ID NO: 5); or (b) the complement of the DNA molecule of (a). The sequence identity preferably is about 85%, more preferably about 90%, most preferably about 95%. In one aspect, the isolated nucleic acid has at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% sequence identity with a polypeptide having amino acid residues 1 to 576 of
FIG. 1
(SEQ ID NO: 1), 1 to 501 of
FIG. 2
, (SEQ ID NO: 3), and 1 to 703 of
FIG. 3
(SEQ ID NO: 5). Preferably, the greatest degree of identity occurs in the serine/proline rich domain (ie., amino acid residues 145-299 of SEQ ID NO: 1, amino acid residues 28-210 of SEQ ID NO: 3 and amino acids 181-415 of SEQ ID NO: 5). Alternatively, the greatest degree of identity occurs in the SH2 domain (i.e., amino acid residues 1-118 of SEQ ID NO: 1 and amino acid residues 50-166 of SEQ ID

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