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-12577266-B2
Publication Date
2026-03-17
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 disclosed subject matter defines caged haptens that are designed to be enzymatically unmasked to regenerate native hapten. The invention relates to a compound having Formula (IE) or (IF) in which a Hapten is selected from defined hapten groups and is linked within a structured caged-hapten construct, with reactive group Y selected for carbonyl-reactive, amine-reactive, or thiol-reactive attachment.
The structure includes defined group A having Formula (IIID), group X having Formula (IIIB), a defined group W, and substituent variables R1, V, and R3 constrained by enumerated groups and positioning relationships. The enzyme substrate group R3 is selected from phosphate, ester, sulfate, glycoside, amide, urea, and nitro, and [V]-R3 is positioned para relative to [A] when q is 1, with discrete parameter settings including q=1 and restrictions on s and t.
The description frames this within enzyme-mediated in situ labeling using tyramide signal amplification (TSA), including definitions of biological sample, chromophore, conjugate, detectable moiety, epitopes, and specific binding entities and targets. It further discusses unmasking logic for proximity assays, where an unmasking enzyme-antibody conjugate is used to decage a hapten by enzyme-substrate logic, enabling binding of detectable moieties and resulting chromogenic, fluorescent, or enzyme-linked detection.
Claims Coverage
The independent claims are clm-00001, clm-00007, and clm-00011. Across these three independent claims, the coverage centers on Formula (IE)/(IF) compound structures that incorporate a specified hapten identity, a reactive attachment group Y, defined A/X/W structural subparts with constrained substituent sets, an enzyme substrate group R3 positioned para relative to [A] when q=1, and discrete parameter constraints on q, s, and t.
Formula-defined caged hapten compound with constrained hapten and reactive attachment group
A compound having Formula (IE) or (IF), wherein Hapten is selected from nitropyrazole, benzofuran, oxazole, oxazole sulfonamide, thiazole, thiazole sulfonamide, pyrazole, or digoxigenin; Y is selected from a carbonyl-reactive group, an amine-reactive group, and a thiol-reactive group; A has the structure of Formula (IIID) with defined integer ranges and substituent options; X comprises a repeat group having Formula (IIIB) with defined integer ranges and termination options; W is a 5- or 6-membered aromatic or heterocyclic group; each R1 is selected from enumerated substituents; V is a substituted or unsubstituted alkyl or O-alkyl group; R3 comprises an enzyme substrate selected from phosphate, ester, sulfate, glycoside, amide, urea, or nitro; q is 1; s is 0 or an integer from 1 to 4; t is 0 or 1; and [V]-R3 is positioned para relative to [A].
Defined reactive chemistries and para positioning
A compound having Formula (IE) or (IF) wherein Y includes a carbonyl-reactive group selected from hydrazine or an amine, an amine-reactive group, and a thiol-reactive group; A has the structure of Formula (IIID) with defined integer ranges and substituent options; X comprises a repeat group having Formula (IIIB) with defined termination; W is a 5- or 6-membered aromatic or heterocyclic group with one or two O, N, or S heteroatoms; each R1 is selected from specified substituents; V is a substituted or unsubstituted alkyl or O-alkyl group; R3 is selected from enzyme-substrate functional groups including phosphate and nitro; q is 1; s is 0 or 1 to 4; and t is 0 or 1, with [V]-R3 positioned para relative to [A] when q is 1.
Digoxigenin-containing compound with fixed parameter constraints
A compound having Formula (IE) or (IF) wherein Hapten is digoxigenin, pyrazole, nitropyrazole, benzofuran, oxazole, oxazole sulfonamide, thiazole, or thiazole sulfonamide; Y is selected from a carbonyl-reactive group, an amine-reactive group, and a thiol-reactive group; A is a branched or unbranched unsubstituted aliphatic group having between 1 and 4 carbon atoms; X comprises a repeat group having Formula (IIIB) with specified integer ranges and termination; W is a 6-membered aromatic group or a 5-membered heterocyclic group comprising one or two N, O, or S heteroatoms; each R1 is selected from a specified set; V is a substituted or unsubstituted O-alkyl group; R3 is selected from phosphate, ester, sulfate, glycoside, amide, urea, and nitro; q is 1; s is an integer from 1 to 4; and t is 1, with [V]-R3 positioned para relative to [A] when q is 1.
Across clm-00001, clm-00007, and clm-00011, the independent claims focus on a compound defined by Formula (IE)/(IF) with an enumerated hapten choice, a selected reactive Y group, and a parameterized linker architecture including A, X (repeat unit Formula (IIIB)), W, R1 substituents, V, enzyme-substrate group R3, and discrete values or ranges for q, s, and t, including a para-relative positioning constraint when q is 1.
Stated Advantages
Decaging enables binding of detectable moieties and resulting chromogenic, fluorescent, or enzyme-linked detection.
The document discusses qualitative positive versus negative proximity results.
The document discusses multiplexing with HRP detection systems.
The disclosed subject matter defines caged haptens that are designed to be enzymatically unmasked to regenerate native hapten.
The description frames this within enzyme-mediated in situ labeling using tyramide signal amplification (TSA).
Structural parameters of the scaffolds influence signaling, including linker types such as PEG versus non-PEG linkers.
Caging electronics considerations are discussed based on electron-donating or electron-withdrawing substituent effects tied to hapten pKa.
Documented Applications
Unmasking logic for proximity assays.
Use of an unmasking enzyme-antibody conjugate to decage a hapten by enzyme-substrate logic.
Secondary anti-unmasked hapten antibodies for detectable binding.
Enzyme-mediated in situ labeling using tyramide signal amplification (TSA).
Conjugates where a caged hapten is conjugated to an antibody, represented by Formula (IV).
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