Aptamers and diagnostic methods for detecting the EGF receptor
Inventors
Bock, Chris • Ayers, Deborah • Nikrad, Malti P. • Gawande, Bharat Nathu • Bertino, Jennifer C. • Sun, Weimin • Strom, Charles M. • Park, Noh Jin
Assignees
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Publication Number
US-9651556-B2
Publication Date
2017-05-16
Expiration Date
Abstract
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.
Core Innovation
The invention relates to aptamers that specifically bind free Epidermal Growth Factor Receptor (EGFR) and to methods for determining the amount of free EGFR in a sample. Free EGFR is defined as EGFR that is not bound to a therapeutic molecule if present in the sample. The method contacts the sample with an aptamer comprising the structure defined by SEQ ID NO:55 to form an aptamer-EGFR complex.
After forming the aptamer-EGFR complex, the invention detects the amount of the free EGFR bound by the aptamer and determines the amount of free EGFR in the sample based on the detected amount. The disclosed aptamer structure includes a 3′ cap such as inverted deoxythymidine, a 5′ spacer such as hexaethylene glycol, a binding pair member such as biotin with streptavidin, and detectable label components such as fluorophores.
The aptamer consensus sequence for SEQ ID NO:55 is described as XGANNGNNYGANNCNN, with X = G/C/A, Y = G/A, and N as 5-position modified 2′-deoxyuridines. The partial content also reports affinity data for specific SEQ ID NO aptamers and interference results showing that an aptamer/SOMAmer signal decreases dose-dependently with cetuximab/panitumumab while ELISA remains relatively unaffected.
Claims Coverage
The partial content identifies one independent claim directed to determining the amount of free EGFR using an aptamer defined by SEQ ID NO:55. Dependent claims refine the assay workflow, aptamer chemistry constraints, detection and determination modalities, and therapeutic/patient context.
Determining free EGFR using an SEQ ID NO:55 aptamer
Contacting a sample comprising EGFR with an aptamer that specifically binds to free EGFR under conditions that allow binding of the aptamer to the free EGFR in the sample to form an aptamer-EGFR complex, wherein free EGFR is EGFR not bound to a therapeutic molecule if present, and wherein the aptamer comprises the structure defined by SEQ ID NO:55.
Detecting and determining free EGFR from aptamer binding amount
Detecting the amount of the free EGFR bound by the aptamer; and determining the amount of free EGFR in the sample based on the amount of the free EGFR bound by the aptamer.
Positional constraints within SEQ ID NO:55
In SEQ ID NO:55, X is G, C, or A, Y is G or A and/or each N of SEQ ID NO:55 is, independently, a 5-position modified 2′-deoxyuridine.
Allowed N-modified 5-position 2′-deoxyuridine substituent set
Each N of SEQ ID NO:55 is independently selected from substituted 2′-deoxyuridine 5-position derivatives including named substituent variants as specified in the partial content.
Multi-solid-support dual capture workflow using third and fourth binding pair members
Attaching a third binding pair member to an aptamer-EGFR complex, releasing the aptamer-EGFR complex from a solid support, immobilizing the aptamer-EGFR complex on a second solid support using a fourth binding pair member that binds the third member, and determining the amount of EGFR immobilized on the second solid support.
Measuring total EGFR by ELISA
Detecting the total amount of EGFR in a sample using an enzyme-linked immunosorbent assay (ELISA).
Patient receiving cetuximab or panitumumab
The patient is receiving cetuximab or panitumumab.
Overall, the claim coverage centers on using an aptamer defined by SEQ ID NO:55 to bind and quantify free EGFR, with dependent claim refinements adding structural constraints for SEQ ID NO:55, a multi-binding-pair solid-support workflow, alternative detection of total EGFR by ELISA, and specifying cetuximab/panitumumab context.
Stated Advantages
Detection of the drug-unbound (free) EGFR fraction, supported by aptamer/SOMAmer signal decreasing dose-dependently with cetuximab/panitumumab while ELISA remains relatively unaffected.
Documented Applications
Diagnostic assays to quantify free EGFR in patient samples for monitoring resistance to cetuximab/panitumumab.
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