Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-06-21
2003-03-25
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C530S300000, C530S350000, C435S006120
Reexamination Certificate
active
06537804
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of malignant cell proliferation. More particularly, it provides compositions and methods to limit Bcr-Abl oncoprotein-driven malignant cell proliferation. Peptide and protein molecules are provided that inhibit various Bcr-Abl signal transduction pathways, e.g., activation of the Ras protein. Methods for reducing Philadelphia chromosome-positive cells in cell populations, including bone marrow culture, and methods of treating various leukemias are also provided.
BACKGROUND OF THE INVENTION
The Philadelphia chromosome (Ph
1
) is associated with the bulk of chronic myelogenous leukemia (CML) patients (more than 95%), 10-25% of acute lymphocytic leukemia (ALL) patients, and about 2-3% of acute myelogenous leukemias (AML). This abnormal chromosome fuses most of the ABL gene to the 5′ two-thirds of the BCR gene.
A number of different kinds of evidence support the contention that Bcr-Abl oncoproteins, such as p210 and p185 BCR-ABL, are causative factors in these leukemias (Campbell et al., 1991). The malignant activity is due in large part to the Bcr-Abl protein's highly activated protein tyrosine kinase activity and its abnormal interaction with protein substrates (Campbell et al., 1991, Arlinghaus et al., 1990). The Bcr-Abl oncoprotein p210 Bcr-Abl is associated with both CML and ALL, whereas the smaller oncoprotein, p185 BCR-ABL, is associated with ALL patients, although some CML patients also express p185 (Campbell et al., 1991).
Some reports suggest that Bcr-Abl oncoproteins, p210 and p185 BCR-ABL, function at least in part by activating the Ras pathway. The RAS gene is a proto-oncogene involved in controlling normal cell growth. When continuously activated, the Ras protein becomes a potent cancer gene product. Bcr-Abl oncoproteins have been observed by the present inventors and others to perturb normal Ras function (Pendergast et al., 1993).
The mechanism by which Bcr-Abl oncoproteins activate p21 Ras is believed to involves several factors. One event involves the autophosphorylation of the Bcr-Abl oncoprotein on tyrosine residues within the coding sequence of the first Bcr exon (Liu et al., 1993). This finding was unexpected, as it had previously been postulated that Bcr-Abl phosphorylates itself on Abl tyrosines, not Bcr tyrosine residues.
Several adaptor proteins have been implicated in Ras-activation as well.
FIG. 2
lists several such adaptor proteins that contain SH2/SH3 motifs. Such domains have been observed in proteins involved in transmitting growth signals to the nucleus (Pawson et al., 1992).
Grb2 is an adaptor protein that binds to tyrosine phosphorylated receptor proteins. Bcr-Abl induced oncogenesis has also been reported to be mediated by direct interaction with the SH2 domain of Grb2 (Pendergast et al., 1993; Puil et al., 1994). Grb2 also binds mSos1, a GTP exchange factor (see FIG.
3
). The latter activates Ras by forming GTP/Ras. GTP/Ras in turn activates Raf, a serine/threonine protein kinase that activates Mek. Mek is a kinase that phosphorylates and activates MAP kinase. The latter is believed to activate and/or regulate various transcription factors (i.e., c-Jun), resulting in cell growth (FIG.
4
).
Another peptide that has been implicated in the malignant effects of Bcr-Abl involves Shc (Puil et al., 1994). Crkl is another adaptor molecule that forms a protein/protein interaction with Bcr-Abl (Reichman et al., 1992; Ten Hoeve et al., 1993; 1994). Still another adaptor molecule that interacts with Bcr-Abl is p120 Ras Gap (Druker et al. 1992).
Another protein/protein interaction that has been examined in relation to Bcr-Abl induced malignancy concerns the formation of tetramer structures. In Philadelphia chromosome-positive human leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene on chromosome 22, and chimeric Bcr-Abl proteins are produced. The fused Bcr sequences activate the tyrosine kinase, actin-binding, and transforming functions of Abl.
Activation of the Abl transforming function is believed to require two distinct domains of Bcr: domain 1 (Bcr amino acids 1 to 63) and domain 2 (Bcr amino acids 176-242) (McWhirter et al., 1993). Domain 1 of Bcr has been shown to form a homotetramer (McWhirter et al., 1993). The Bcr-Abl tetramer activates its inherent Abl tyrosine kinase activity, its actin binding function, and its cellular transformation function (McWhirter et al., 1993). Disruption of the coiled coil by insertional mutagenesis inactivates the oligomerization function and the ability of Bcr-Abl to transform Rat-1 fibroblasts.
Despite the description of certain events and molecules that are believed to be involved in Bcr/Abl function and pathologies associated with the activities of its gene product, comprehensive strategies for controlling Bcr/Abl and, e.g., its activation of the Ras oncogene, have not been developed. Thus, a need continues to exist in the scientific and medical arts for approaches that target effectively and specifically inhibit Bcr/Abl. Such techniques would provide new therapies for inhibiting Philadelphia chromosome-positive cells in tissues, such as in bone marrow.
SUMMARY OF THE INVENTION
The present invention overcomes certain of the limitations of the prior art by defining specific peptide sequences from Bcr-Abl that inhibit Bcr-Abl function and activation. The peptides and compositions of the invention are thus useful in methods for inhibiting Bcr-Abl, for purging bone marrow of Philadelphia chromosome-positive cells in bone marrow samples and for treating various leukemias, including chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL) and acute myelogenous leukemias (AML).
The peptides of the present invention are those comprising a sequence based upon a segment of a Bcr-Abl amino acid sequence that includes at least one of a combination of several tyrosine residues found by the present inventors to be important in Bcr-Abl function. These are termed herein the “tyrosine-containing peptides”. Generally, the compositions and methods of the invention require that at least one tyrosine-containing peptide be present.
The amino acid sequence of the first exon of p160 Bcr is given in SEQ ID NO:1. Tyrosines are present at residues 58, 70, 177, 231, 246, 276, 279, 283, 316, 328 and 360. The particularly important tyrosines in the context of the present invention are tyrosines at positions 177, 283 and 360, and also tyrosine 328.
The present invention thus provides purified peptides and polypeptides, of between about 4 and about 500 amino acids in length, that have or comprise a contiguous amino acid sequence from the Bcr-Abl protein (of SEQ ID NO:1), which sequence includes or surrounds at least one of tyrosine 177, tyrosine 283, tyrosine 360, or even tyrosine 328, which peptides or polypeptides become phosphorylated on tyrosine 177, 283, 360 and/or 328 upon contact with active Bcr-Abl.
These peptides are thus characterized as being substrates for the tyrosine phosphorylating activity of Bcr-Abl. The peptides are also characterized as being capable of effectively competing with Bcr as a substrate for Bcr-Abl, and being capable of reducing the Bcr-Abl-mediated tyrosine phosphorylation of Bcr in an intact cell that contains Bcr-Abl.
Exemplary useful peptides including tyrosine 177 are those comprising the sequence of SEQ ID NO:8 (positions 164 to 181 of SEQ ID NO:1). Exemplary useful peptides including tyrosine 283 are those comprising the sequence of SEQ ID NO:11 (positions 255 to 293 of SEQ ID NO:1). It is currently preferred that the peptide include the tyrosine in a generally central region, rather than at the extreme termini of the peptide.
Although understanding the mechanism of action of any given peptide is not necessary in order to practice the invention, it should be noted that peptides containing tyrosine 177 or tyrosine 283 likely function by inhibiting the oncogenic effects of Bcr-Abl. This is believed to be achieved by the peptides competing with other substrates, particularly Bcr,
Arlinghaus Ralph B.
Liu Jiaxin
Lopez-Berestein Gabriel
Lu Dai
Wu Yun
Board of Regents The University of Texas Systems
Fulbright & Jaworski
McGarry Sean
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