Protein proximity assay in formalin fixed paraffin embedded tissue using caged haptens
Inventors
Belosludtsev, Yuri • DeGeer, Traci D. • French, Wendy J. • Hao, Junshan • Kelly, Brian D. • Murillo, Adrian E. • Polaske, Nathan W.
Assignees
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Publication Number
US-12559511-B2
Publication Date
2026-02-24
Expiration Date
Abstract
Disclosed herein are caged haptens and caged hapten-antibody conjugates useful for enabling the detection of targets located proximally to each other in a sample.
Core Innovation
The disclosure describes caged hapten derivatives in which hapten recognition is masked by a caging group and becomes available for anti-hapten antibody binding only after enzymatic cleavage releases an uncaged hapten. The caged hapten is defined by a hapten moiety, a reactive caging group Y, a linker/group X with a repeat portion, an enzyme-substrate moiety R3, and a small alkyl or —O-alkyl group V. Representative hapten classes include nitropyrazole, nitrophenyl, benzofuran, dinitrophenol, biotin, digoxigenin, and fluorescein, and the structures include Formula (IF) and related Formula IV variants.
The disclosure further specifies that Y is selected from carbonyl-reactive, amine-reactive, or thiol-reactive groups, while X comprises between 1 and 30 carbon atoms and includes a repeat group having Formula (IIIB), with d and e independently ranging from 1 to 24, Q being a bond or O, and X terminating in an amine, carbonyl, amide, or imine S. R3 is selected from phosphate, amide, sulfate, and urea, and t is 0 or 1. The described embodiments include caged-hapten conjugates and antibody-linked forms with defined linker Z structures.
The disclosure also describes analyzing samples to determine whether first and second targets are proximal using caged hapten–antibody conjugates and an unmasking enzyme–antibody conjugate. The assay uses a decaging step followed by detection, and generates a signal associated with target proximity rather than separated targets, with hapten-specific detection, HRP amplification, multiplex total-protein readout, and chromogenic and fluorescent readouts.
Claims Coverage
The claims center on a family of caged hapten structures defined by Formula (IF), with inventive features spanning the hapten moiety, the reactive caging group Y, the linker/repeat architecture X, the small alkyl or —O-alkyl group V, the enzyme-substrate moiety R3, and the parameter t. Additional claim features narrow the same framework to specific hapten and caging selections.
Caged hapten formula with defined hapten selection
A caged hapten having Formula (IF) wherein Hapten is selected from nitropyrazole, nitrophenyl, benzofuran, dinitrophenol, biotin, digoxigenin, and fluorescein.
Reactive caging group Y selected by reactive chemistry
Y is selected from a carbonyl-reactive group, an amine-reactive group, or a thiol-reactive group; the carbonyl-reactive group is selected from hydrazine or an amine; the amine-reactive group is selected from NHS ester, sulfo-NHS, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide/oxirane, carbonate, aryl halide, imidoester, and anhydrides; and the thiol-reactive group is selected from haloacetyl, alkyl halide, maleimide, aziridine, acryloyl, vinyl sulfone, and benzoquinone.
Repeat-containing linker/group X with defined termination
X is a group comprising between 1 and 30 carbon atoms and includes a repeat group having Formula (IIIB) wherein d and e are integers independently ranging from 1 to 24; Q is a bond or O; and the group X terminates in one of an amine, a carbonyl, an amide, or an imine S.
Small alkyl/O-alkyl V and enzyme-substrate R3
V is an unsubstituted alkyl group having between 1 and 4 carbon atoms or an unsubstituted —O-alkyl group having between 1 and 4 carbon atoms, and R3 is an enzyme substrate selected from a phosphate group, an amide group, a sulfate group, and a urea group.
Parameter t limited to 0 or 1
t is 0 or 1.
Specific digoxigenin hapten selection
The caged hapten of a dependent claim specifies that Hapten is digoxigenin.
Specific enzyme-substrate selection
The caged hapten of a dependent claim specifies that R3 is a phosphate group.
Caging group Y narrowed to an amine-reactive group
The caged hapten of a dependent claim specifies that Y is an amine-reactive group.
Caged hapten defined by a specified chemical structure
A caged hapten having a specified chemical structure as stated in an additional independent claim, with the structure details not included in the provided record.
The claim set covers a Formula (IF) caged hapten with selectable hapten moieties, reactive caging group chemistry categories, a defined linker/repeat architecture with termination functionality, a selectable small alkyl or —O-alkyl component, and an enzyme-substrate group selected from phosphate, amide, sulfate, or urea, with dependent narrowing to digoxigenin, t = 0, phosphate R3, and amine-reactive Y.
Stated Advantages
Detection of protein target proximity using caged haptens that are unmasked by unmasking enzymes.
The generated unmasked hapten enables signal associated with target proximity rather than separated targets.
Hapten-specific detection with HRP amplification and multiplex total-protein readout.
The disclosure supports multiplex/proximal signal workflows and chromogenic and fluorescent readouts.
Stability and mitigation of false positives via reduced decaging for certain caged hapten structures.
The modular set of caged hapten constructs is intended to control the timing of hapten availability for antibody recognition under enzyme-mediated labeling conditions.
Documented Applications
Analyzing samples to determine whether first and second targets are proximal using caged hapten–antibody conjugates, an unmasking enzyme–antibody conjugate, and subsequent detection.
Detecting proximity of protein targets using unmasking enzymes to decage a hapten and enable hapten-specific detection with HRP amplification and multiplex total-protein readout.
Detection of whether two targets or proteins are in close proximity in formalin-fixed paraffin embedded tissue.
Measuring protein proximity between E-cadherin and beta-catenin.
Measuring protein proximity between PD-1 and PD-L1.
Enzyme-mediated labeling and anti-hapten antibody binding.
FFPE validation with target pairs and staining controls and signal behaviors.
Detection modality comparisons using DAB, silver, and tyramine detection approaches.
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