Cancer diagnosis and therapy based on mutations in TGF-beta receptors

• US 20020064786A1
• Filed: 06/13/2001
• Published: 05/30/2002
• Est. Priority Date: 04/07/1995
• Status: Active Grant

• Current Assignee: Case Western Reserve University
• Original Assignee: Case Western Reserve University
• Inventors: Willson, James K. V., Markowitz, Sanford D., Brattain, Michael G.
• Granted Patent: US 6,630,326 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function

Abstract

This invention is based on the discovery that the type II TGFβ receptor (RII) is a cancer suppressor gene which is genetically inactivated (mutated) in approximately 25% of colon cancers, including nearly all colon cancers of the class identified as mutator/microsatellite instability/RER. Methods are provided for detecting inactivation of RII for use in cancer diagnosis or prognosis.

Claims

1. A method to aid in predicting prognosis of a cancer patient comprising determining the quantity of functional type II receptor for TGFβ

(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.

2. The method of claim 1, wherein the patient is a colon cancer patient.

3. A method to aid in classifying tumor cell phenotype in a patient comprising determining the presence or absence of functional RII receptor for TGFβ

in tumor tissue from the patient, the absence of functional RII being indicative of carcinoma with replication errors (RER phenotype).

4. The method of claim 3, wherein the tumor tissue is selected from the group consisting of colon cancer, endometrial cancer, ovarian cancer, gastric cancer, pancreatic cancer and other malignancies.

5. A method to aid in classifying tumor cell phenotype comprising determining, in a sample containing cells or extract of cells of the tumor, presence or absence of a nucleotide sequence encoding a mutant form of TGFβ

receptor RII, wherein the mutation is selected from the group consisting of a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at codons 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, and deletion of two A base pairs from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, the presence of the nucleotide sequence encoding a mutant form of TGFβ

receptor RII being indicative of carcinoma with replication errors (RER phenotype).

6. The method of claim 5, wherein presence or absence of a nucleotide sequence encoding a mutant form of TGFβ

receptor RII is determined by detection of binding of antibody specifically immunoreactive with mutant RII to mutant RII protein expressed by the cells from the nucleotide sequence, or detection of DNA or RNA sequences encoding the mutant form of RII.

7. A method to aid in diagnosing cancer in a patient comprising determining the presence or absence of functional RII receptor for TGFβ

in tissue from the patient, the absence of functional RII being indicative of tumor tissue or precancerous lesions in the patient.

8. The method of claim 7, wherein absence of functional RII is determined by detecting a mutant form of RII in tissue from the patient.

9. The method of claim 8, wherein the mutant form of RII is 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 codons 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, and deletion of two A base pairs from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence.

10. A screening method to aid in diagnosing genetic susceptibility to cancer in a patient comprising determining the presence or absence of mutant forms of RII receptor for TGFβ

in a sample from the patient, the presence of mutant forms of RII being indicative of precancerous lesions in the patient.

11. The screening method of claim 10, wherein the mutant forms of RII are 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 codons 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, and deletion of two A base pairs from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence.

12. The screening method of claim 11, wherein the presence of RII mutant forms is detected by immunoassay.

13. The screening method of claim 11, wherein the sample is a biological fluid, selected from the group consisting of serum, plasma, effusions, ascites, urine, stool, cerebrospinal fluid, semen, breast aspirates and fluids of ovarian origin.

16. The heterologous polynucleotide of claim 15, wherein the polynucleotide is RNA.

17. The heterologous polynucleotide of claim 15, wherein the polynucleotide is DNA.

18. The heterologous polynucleotide of claim 17, wherein the polynucleotide is an expression vector which expresses an inactive mutant form of RII.

19. A therapeutic method for treating a colon cancer patient, wherein neoplastic cells of the patient express a mutant form of RII, the mutation being selected from the group consisting of a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at codons 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, and deletion of two A base pairs from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, comprising administering to the patient an immunogenic amount of an immunogenic composition comprising an expression vector encoding mutant TGFβ

receptor RII according to claim 18, the expression vector encoding the same mutant form of RII as the neoplastic cells of the patient.

14. An assay for expression in cells of mutant type II receptor for TGFβ

(RII) comprising determining, in a sample containing cells or cell extracts, presence or absence of a nucleotide sequence encoding a mutant form of TGFβ

receptor RII, wherein the mutation is selected from the group consisting of a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at codons 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, and deletion of two A base pairs from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence.

15. A heterologous polynucleotide comprising a nucleotide sequence encoding a mutant form of TGFβ

receptor RII, wherein the mutation is selected from the group consisting of a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at codons 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, and deletion of two A base pairs from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence.

20. A mutant protein of human TGFβ

receptor RII substantially free of other human proteins, the mutant RII protein having a sequence 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 codons 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, and deletion of two A base pairs from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence.

21. An antibody immunospecific for the mutant protein of claim 20.

22. The mutant protein of claim 20, wherein the mutation is a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at codons 1931-1936 of the normal RII sequence.

23. An antibody immunospecific for a cell surface epitope of the mutant protein of claim 22.

24. An immunogenic composition comprising the mutant protein of claim 20.

25. A therapeutic method for treating a colon cancer patient, wherein neoplastic cells of the patient express a mutant form of RII, the mutation being selected from the group consisting of a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at codons 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, and deletion of two A base pairs from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, comprising administering to the patient an immunogenic amount of an immunogenic composition according to claim 24, the mutant protein in the immunogenic composition containing the same RII mutation as the neoplastic cells of the patient.

26. A therapeutic method for treating a colon cancer patient, wherein neoplastic cells of the patient express a mutant form of RII, the mutation being selected from the group consisting of a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at codons 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, and deletion of two A base pairs from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, comprising administering to the patient an effective amount of an antibody according to claim 23, the antibody being specifically immunoreactive with the mutant form of RII expressed by the neoplastic cells of the patient.

30. The method to aid in diagnosing cancer in a patient according to claim 29, wherein detection of said mutant form of RII comprises detecting antibody immunologically reactive with the mutant form of RII in a sample of biological fluid from the patient.

31. The method of claim 30, wherein said antibody immunologically reactive with mutant RII receptor for TGFβ

is also immunologically reactive with wild type RII receptor.

27. A therapeutic method for treating a colon cancer patient, wherein neoplastic cells of the patient express a mutant form of RII, the mutation being selected from the group consisting of a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at codons 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, and deletion of two A base pairs from a ten base pair poly A sequence at codons 709 to 718 of the normal RII sequence, comprising administering to the patient an effective amount of specific activated cytotoxic immune cells, wherein the activated immune cells are specifically immunoreactive with the mutant form of RII expressed by the neoplastic cells of the patient.

28. A therapeutic method for treating a patient having colon cancer of the RER phenotype comprising administering to the patient a gene therapy vector encoding functional TGFβ

receptor RII operably linked to a promoter, wherein said gene therapy vector is expressed in the patient to produce functional RII.

29. A method to aid in diagnosing cancer in a patient comprising detecting a mutant RII receptor for TGFβ

in a sample of biological fluid from the patient.

32. 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 II receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGFβ

-like factors or the growth regulatory gene contains repetitive DNA sequence motifs in the wild-type 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.

33. A method to aid in diagnosing cancer in a patient according to claim 32, wherein the growth regulatory gene encodes a type II receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGFβ

-like factors.

34. A method to aid in diagnosing cancer in a patient according to claim 32, wherein the wild-type growth regulatory gene contains repetitive DNA sequence motifs in the coding region.

35. A therapeutic method for treating a cancer patient, wherein neoplastic cells of the patient express a non-functional mutant form of a growth regulatory gene, wherein the growth regulatory gene encodes a type II receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGFβ

-like factors or the growth regulatory gene contains repetitive DNA sequence motifs in the wild-type coding region, comprising administering to the patient an immunogenic amount of an immunogenic composition comprising the non-functional mutant for of the growth regulatory gene product or an expression vector encoding the same non-functional mutant form of the growth regulatory gene as the neoplastic cells of the patient.

36. A therapeutic method for treating a cancer patient, wherein neoplastic cells of the patient express a non-functional mutant form of a growth regulatory gene, wherein the growth regulatory gene encodes a type II receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGFβ

-like factors or the growth regulatory gene contains repetitive DNA sequence motifs in the wild-type coding region, comprising administering to the patient an effective amount of an antibody, the antibody being specifically immunoreactive with the non-functional mutant form of the growth regulatory gene expressed by the neoplastic cells of the patient.

37. A therapeutic method for treating a cancer patient, wherein neoplastic cells of the patient express a non-functional mutant form of a growth regulatory gene, wherein the growth regulatory gene encodes a type II receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGFβ

-like factors or the growth regulatory gene contains repetitive DNA sequence motifs in the wild-type coding region, comprising administering to the patient an effective amount of specific activated cytotoxic immune cells, wherein the activated immune cells are specifically immunoreactive with the non-functional mutant form of the growth regulatory gene expressed by the neoplastic cells of the patient.

38. A therapeutic method for treating a cancer patient, wherein neoplastic cells of the patient express a non-functional mutant form of a growth regulatory gene, wherein the growth regulatory gene encodes a type II receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGF-like factors or the growth regulatory gene contains repetitive DNA sequence motifs in the wild-type coding region, comprising administering to the patient a gene therapy vector encoding a functional form of the growth regulatory gene expressed by the neoplastic cells of the patient,the gene being operably linked to a promoter, wherein said gene therapy vector is expressed in the patient to produce a functional form of the growth regulatory gene expressed by the neoplastic cells of the patient.