Aptamers and diagnostic methods for detecting the EGF receptor
The present invention provides aptamers that specifically bind to the EGF receptor in a sample, and diagnostic and analytical methods using those aptamers. In some embodiments, the aptamers include a 3′ cap. In some embodiments, the 3′ cap is an inverted deoxythymidine. In some embodiments the aptamers include a spacer and at least one moiety selected from the group consisting of binding pair member and a detectable label, wherein the spacer is attached to the 5′-end of the aptamer and the moiety is attached the 5′ end of the spacer. In some embodiments the spacer is hexaethylene glycol. In some embodiments, the binding pair member biotin. In some embodiments the detectable label is a fluorophore.
1. A method for determining whether a dosage of a therapeutic molecule that binds the extracellular domain of the Epithelial Growth Factor Receptor (EGFR) is sufficient for the treatment of a cancer in a patient, comprising:
(a) detecting an amount of free EGFR and an amount of total EGFR in a sample from a patient being treated with the therapeutic molecule, wherein the free EGFR is EGFR that is not bound to a therapeutic molecule, and wherein the amount of free EGFR is detected by:
(i) contacting the sample with an aptamer that specifically binds to free EGFR to form an aptamer-EGFR complex, wherein the aptamer comprises the structure defined by SEQ ID No: 55; and
(ii) detecting the amount of the free EGFR in the sample based on the amount of the free EGFR bound by the aptamer; and
(b) identifying the dosage as not sufficient when the proportion of free EGFR to total EGFR in the sample is greater than a predetermined threshold and sufficient when the proportion of free EGFR to total EGFR in the sample is less than the predetermined threshold.
2. The method of claim 1 , wherein the predetermined threshold is about 10%.
3. The method of claim 1 , wherein the patient has an epithelial carcinoma.
4. The method of claim 3 , wherein the epithelial carcinoma is selected from the group consisting of a squamous cell carcinoma, an adenocarcinoma, and a transitional cell carcinoma.
5. The method of claim 1 , wherein the therapeutic molecule is an anti-EGFT receptor antibody.
6. The method of claim 5 , wherein the anti-EGFR receptor antibody is cetuximab panitumumab.
7. The method of claim 1 , further comprising increasing the dose of the therapeutic molecule administered to the patient when the proportion of free EGFR to total EGFR in the sample is greater than the predetermined threshold.
8. The method of claim 1 , further comprising predicting poor carcinoma response to continued administration of the therapeutic molecule when the proportion of free EGFR to total EGFR in the sample is greater than the predetermined threshold.
9. The method of claim 1 , wherein the total amount of EGFR in the sample is detected using an enzyme-linked immunosorbent assay.
10. The method of claim 1 , wherein the aptamer comprises the structure defined by a sequence selected from the group comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.
11. The method of claim 1 , wherein X of SEQ ID NO: 55 is G,C, or A; Y of SEQ ID NO: 55 is G or A and/or each N of SEQ ID NO: 55 is, independently, a 5-position modified 2′-deoxyuridine.
12. The method of claim 1 , wherein each N of SEQ ID NO: 55 is, independently, selected from a 5-benzylaminocarbonyl-2′-deoxyuridine, 5-(N -benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N -tryptaminocarboxyamide)2′-deoxyuridine, 5-(N-[1-( 3 -trimethylamonium) propyl]carboxyamide)-2′-deoxyuridine chloride, 5-(N-naphthylinethylcarboxamide)-2′-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.
13. The method of claim 1 , wherein the aptamer comprises a first binding pair member and is immobilized on a first solid support via a second binding pair member that specifically binds to the first binding pair member.
14. The method of claim 13 , wherein the sample is contacted with the aptamer prior to or after immobilization on the first solid support.
15. The method of claim 13 , wherein the amount of free EGFR is determined by determining the amount of EGFR immobilized on the first solid support.
16. The method of claim 13 , said method further comprising:
(i) attaching a member pair to the EGFR;
(ii) releasing the aptamer-EGFR complex from the solid support;
(iii)immobilizing aptamer-EGFR complex on a second solid support via a fourth binding pair member that specifically binds to the third binding pair member; and
(iv) determining the amount of EGFR immobilized to the second solid support.
17. The method of claim 16 , further comprising labeling the EGFR, with a detectable label either before or after immobilization on the second solid support.
18. The method of claim 16 , wherein the aptamer further comprises a detectable label.
19. The method of claim 16 , wherein the amount of EGFR immobilized on the second solid support is determined by releasing the aptamer from the aptamer-EGFR complex and determining the amount of the released aptamer.
20. The method of claim 1 , wherein the sample is a serum sample.