Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-06-13
2003-10-07
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C530S350000
Reexamination Certificate
active
06630326
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is concerned with diagnostic methods which assist in classification of tumors by phenotype and with therapeutic intervention tailored to particular tumor phenotypes. In particular this invention concerns mutant forms of type II receptors (RII) for transforming growth factor beta (TGF&bgr;) which inactivate growth suppression by TGF&bgr;, methods for detecting inactivation of TGF&bgr; RII receptor and therapeutic methods for restoring tumor suppression by restoring RII faction.
2. Review of Related Art
TGF&bgr; inhibits growth of multiple epithelial cell types, and loss of this negative regulation is thought to contribute to tumor development (Roberts, et al., 1990, In
Peptide Growth Factors and Their Receptors. Handbook of Experimental Pharmacology,
A. Roberts and M. Sporn, Eds., (Springer-Verlag, Heidelberg) pp. 419-472; Massagué, J., 1990,
Annu. Rev. Cell Biol.,
6:597; Moses, et al., 1990,
Cell,
63:247; Filmus, et al., 1993,
Curr. Opin. Oncol.,
5:123; Markowitz, S., et al., 1994,
J. Clin. Invest.,
93:1005; Wu, S., et al., 1992,
J. Cell. Biol.,
116:187; Wu, S., et al., 1993,
Cell Growth Differ.,
4:115; Park, J., et al., 1994,
Proc. Natl. Acad. Sci. U.S.A.,
91:8772; Manning, et al., 1991,
Oncogene,
6:1471; Hoosein, N., et al., 1989,
Exp. Cell Res.,
181:442; Geiser, et al., 1992,
J. Biol. Chem.,
267:2588). Previous studies have demonstrated that TGF&bgr; suppresses growth of certain cancer cell lines, that antisense inhibition of TGF&bgr; enhances the tumorigenicity of weakly tumorigenic cancer cell lines, and that certain tumor cells can become unresponsive to TGF&bgr; (Markowitz, S., et al., 1994,
J. Clin. Invest.,
93:1005; Wu, S., et al., 1992,
J. Cell. Biol.,
116:187; Wu, S., et al., 1993,
Cell Growth Differ.,
4:115; Park, J., et al., 1994,
Proc. Natl. Acad. Sci. U.S.A.,
91:8772; Manning, et al., 1991,
Oncogene,
6:1471; Hoosein, N., et al., 1989,
Exp. Cell Res.,
181:442; Geiser, et al., 1992,
J. Biol. Chem.,
267:2588).
The TGF&bgr; growth inhibitory signal is transduced through two receptors, type I (RI) and type II (RII) which function as a heteromeric complex (Lin, et al., 1992,
Cell,
68:775; Moustakas, A., et al., 1993,
J. Biol. Chem.,
268:22215; Wrana, J., et al., 1992,
Cell,
71:1003).
SUMMARY OF THE INVENTION
It is an object of this invention to provide a method for diagnosis or prognosis of cancer by detection of the absence of functional TGF&bgr; receptor RII in cells of a patient.
It is another object of this invention to provide a nucleotide sequence encoding a mutant form of RII, to provide mutant RII protein and to provide antibodies specifically immunoreactive with mutant RII.
It is yet another object of this invention to provide therapeutic methods for treating tumors having inactive RII by replacement gene therapy.
It is still another object of this invention to provide immunogenic compositions which elicit antibodies specifically reactive with cells expressing mutant forms of RII.
These and other objects are provided by one or more of the following embodiments.
In one embodiment, this invention provides a diagnostic method to aid in predicting prognosis of a colon or gastric cancer patient comprising determining the quantity of functional type II receptor for TGF&bgr; (RII) in cells from tumor tissue of the patient, and comparing the quantity of RII in tumor cells to the quantity of RII in non-neoplastic cells of the patient, reduced RII in the tumor cells being indicative of altered prognosis. In a particular embodiment, this invention provides a screening method to aid in classifying tumor cell phenotype in a patient comprising determining the presence or absence of functional RII receptor for TGF&bgr; in tumor tissue from the patient, the absence of functional RII being indicative of carcinoma with replication errors (RER phenotype). The screening method is preferably applied to tumor tissue from endometrial cancer, ovarian cancer, gastric cancer, or pancreatic cancer, most preferably to colon cancer.
In another embodiment, this invention provides a method to aid in diagnosing cancer in a patient comprising detecting, in the patient, a non-functional mutant form of a growth regulatory gene, wherein the growth regulatory gene encodes a type receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGF&bgr;-like factors or the wild-type of the growth regulatory gene contains repetitive DNA sequence motifs in the coding region, presence of the non-functional mutant form of the growth-regulatory gene being indicative of tumor tissue or precancerous lesions in the patient. In a particular embodiment, this invention provides a screening method to aid in diagnosing cancer in a patient comprising determining the presence or absence of functional RII receptor for TGF&bgr; in tissue from the patient, the absence of functional RII being indicative of tumor tissue or precancerous lesions in the patient. In a particular embodiment, the screening method comprises determining the presence or absence of mutant forms of RII receptor for TGF&bgr;, preferably mutant forms of RII encoded by RII cDNA altered by a mutation selected from the group consisting of a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at nucleotides 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at nucleotides 709 to 718 of the normal RII sequence, deletion of two A base pairs from a ten base pair poly A sequence at nucleotides 709 to 718 of the normal RII sequence, and addition of one or two A base pairs into the ten base pair poly A sequence at nucleotides 709-718 of the normal RII sequence. RII mutations arising from addition or deletion of one or two A bases within the poly A sequence at nucleotides 709-718 encode truncated RII related proteins that contain the RII ligand binding domain but lack the RII membrane anchoring domain so that they will be secreted from the cell into the blood.
Mutant RII may be detected by assays performed on samples from the patient which may be samples from tumor or normal tissue or samples of biological fluids, including serum, plasma, effusions, ascites, urine, stool, cerebrospinal fluid, semen, breast aspirates and fluids of ovarian origin. Suitable assays include immunoassay using antibodies reactive with mutant RII or by assays sensitive to DNA or RNA sequence. Truncated secreted mutant RII proteins may be detected by their ability to bind to TGF&bgr;. Mutant RII proteins may be detected by TGF&bgr; binding to soluble proteins or abnormally sized proteins or reaction of such a soluble protein with antibody specific for the external domain of wild type RII. Alternatively, the presence of mutant RII in a patient may be detected by immunoassays which detect, in the patient, production of an antibody response aimed against either the RII mutation or, due to breaking of immune tolerance, against other epitopes on the native RII protein.
In yet another embodiment, this invention provides a method to aid in diagnosing cancer in a patient comprising detecting a mutant RII receptor for TGF&bgr; in a sample of biological fluid from the patient. The mutant form of RII may be detected directly or it may be detected indirectly by detecting antibody immunologically reactive with the mutant form of RII in a sample of biological fluid from the patient. The antibody immunologically reactive with mutant RII receptor for TGF&bgr; may also be immunologically reactive with wild type RII receptor.
In another embodiment, this invention provides a heterologous polynucleotide, either DNA or RNA, comprising a nucleotide sequence encoding a mutant form of TGF&bgr; receptor RII, the mutation being addition or deletion of one or two adenosine base-pairs from a ten base-pair poly-adenosine sequence at nucleotides 709 to 718 of the normal RII sequence, or preferably, a two base-pair insertion of GT which occurs in a six base pair repeat sequence GTGT
Brattain Michael G.
Markowitz Sanford D.
Willson James K. V.
Brusca John S.
Case Western Reserve University
Ropes & Gray LLP
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