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
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10620210-B2
Publication Date
2020-04-14
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 a method for determining whether a dosage of a therapeutic molecule that binds the extracellular domain of EGFR is sufficient for the treatment of a cancer in a patient. The method detects an amount of free EGFR and an amount of total EGFR in a sample from a patient being treated with the therapeutic molecule, where free EGFR is EGFR that is not bound to a therapeutic molecule.
Free EGFR is detected by contacting the sample with an aptamer that specifically binds to free EGFR to form an aptamer-EGFR complex. The aptamer comprises the structure defined by SEQ ID No: 55, and the amount of free EGFR is determined based on the amount of free EGFR bound by the aptamer.
The method then determines the proportion of free EGFR to total EGFR in the sample and identifies the dosage as not sufficient when the proportion is greater than a predetermined threshold and as sufficient when the proportion is less than the predetermined threshold. The document further characterizes EGFR aptamer sequence families, provides example constructs, and reports binding and assay performance for measuring free EGFR in patient serum while distinguishing free EGFR from EGFR bound by therapeutic antibody agents.
Claims Coverage
The independent claim covers threshold-based treatment-sufficiency determination by measuring the proportion of free EGFR to total EGFR using an EGFR-specific aptamer of SEQ ID No: 55 and comparing the measured proportion to a predetermined threshold. The claim set refines the aptamer sequence and modifications, and constrains the threshold and assay context in dependent claims.
Aptamer-based detection of free versus total EGFR
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 free EGFR is EGFR that is not bound to a therapeutic molecule, and wherein the amount of free EGFR is detected by 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.
Proportion threshold decision for dosage sufficiency
Detecting the amount of the free EGFR in the sample based on the amount of the free EGFR bound by the aptamer; and 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.
Specific SEQ ID No: 55 sequence and N modification constraints
The aptamer comprises the structure defined by SEQ ID No: 55; wherein X is G, C, or A; Y is G or A; and wherein each N of SEQ ID No: 55 is, independently, a 5-position modified 2′-deoxyuridine.
Permissible identities for the N-modified 2′-deoxyuridine substituents
Each N in SEQ ID No: 55 is independently selected from 5-position modified 2′-deoxyuridine derivatives including benzyl, N-benzyl, isobutyl, tryptamino, trimethylammonium-propyl, naphthylmethyl, and dihydroxypropyl derivatives.
Aptamer immobilized via binding-pair members on a solid support
The aptamer includes a first binding-pair member and is immobilized on a first solid support through a second binding-pair member that specifically binds the first binding-pair member.
Predetermined threshold about 10%
Using a predetermined threshold of about 10%.
Overall, the claim coverage centers on measuring free EGFR using an EGFR-specific aptamer of SEQ ID No: 55, calculating the proportion of free EGFR to total EGFR, and classifying whether the therapeutic dosage is sufficient based on comparison to a predetermined threshold. Dependent claims narrow the SEQ ID No: 55 sequence and modifications, include immobilization via binding-pair members on a solid support, and constrain the threshold and related therapeutic context.
Stated Advantages
Provides a determination of whether a dosage of a therapeutic molecule that binds the extracellular domain of EGFR is sufficient for the treatment of a cancer in a patient.
Enables classification of dosage as not sufficient or sufficient based on the proportion of free EGFR to total EGFR compared to a predetermined threshold.
Documented Applications
Determining whether a dosage of an EGFR-binding therapeutic molecule is sufficient for the treatment of a cancer in a patient by measuring free EGFR and total EGFR in a patient sample.
Serum-based detection and measurement of free EGFR using described aptamer assay approaches and detection routes.
Assessing therapeutic efficacy and prognostic prediction in the context of cancer resistance to EGFR therapy by linking unbound free EGFR proportion to response.
Interested in licensing this patent?