Granzyme B Reporter APCs for High-Throughput Epitope Discovery

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Solution Overview

Problem

Identifying target antigens specific to T cells, such as cytotoxic T cells, is challenging due to the rarity and complexity of their interactions with antigen-presenting cells, leading to low throughput and biased discovery of epitopes.

Innovation Solution

A system comprising antigen-presenting cells (APCs) engineered with exogenous nucleic acids encoding candidate antigens, a molecular reporter of Granzyme B activity, and an inhibitor of CAD-mediated DNA degradation, allowing for high-throughput detection and isolation of antigens recognized by cytotoxic lymphocytes.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional methods are used to identify target antigens specific to T cells, then the discovery process can be performed, but the throughput is low and the results are biased

Engineering Contradiction:

Improvethroughput of epitope discoveryVSAvoidaccuracy and unbiased nature of epitope identification

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The invention segments the epitope discovery process into distinct functional modules: (1) APCs engineered with candidate antigen libraries, (2) molecular reporters for detecting T cell recognition events, and (3) systematic screening protocols. This segmentation enables high-throughput processing while maintaining measurement accuracy through standardized detection methods.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention introduces molecular reporters as intermediaries that mediate between T cell antigen recognition events and detectable signals. These reporters (e.g., fluorescent proteins, enzymatic reporters) translate rare biological interactions into measurable outputs, enabling high-throughput screening without compromising the accuracy of epitope identification.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If methods are simplified to increase throughput, then productivity improves, but measurement precision and reliability of antigen identification deteriorate

Engineering Contradiction:

Improvescreening capacity and throughputVSAvoidreliability of antigen recognition detection

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The invention incorporates feedback mechanisms through molecular reporters that provide real-time signals of T cell recognition events. This feedback loop allows for immediate detection and recording of positive interactions, enabling high-throughput screening while maintaining reliability through systematic verification of recognition events.

Inventive Principle:

Principle #23Feedback

Solution Approach 2:

The invention replaces manual or low-throughput mechanical detection methods with molecular reporter systems that automatically detect and signal T cell antigen recognition events. This substitution enables high-throughput screening while maintaining reliability through objective, standardized detection readouts.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Measurement precision

If complex detection systems are used to improve measurement precision, then epitope identification accuracy improves, but device complexity and operational difficulty increase

Engineering Contradiction:

Improveaccuracy of antigen recognition detectionVSAvoidcomplexity of detection system

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The invention employs molecular reporters that produce detectable color changes or fluorescent signals upon T cell recognition events. This approach achieves high measurement precision through optical detection methods while keeping the system relatively simple and amenable to high-throughput processing.

Inventive Principle:

Principle #32Color changes

4Measurement precision

If rare T cell interactions are detected using conventional methods, then specific epitopes can be identified, but the process is time-consuming and has low throughput

Engineering Contradiction:

Improveability to detect rare specific interactionsVSAvoidtime required for epitope discovery

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The invention performs preliminary actions by pre-engineering APCs with candidate antigen libraries and equipping them with molecular reporters before screening. This preparation enables rapid detection of rare T cell interactions during the actual screening process, significantly reducing the time required for epitope discovery while maintaining the ability to detect specific rare events.

Inventive Principle:

Principle #10Preliminary action

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

Enables sensitive and robust identification of antigens recognized by cytotoxic T cells, overcoming low throughput and bias in epitope discovery, and facilitating the recovery of presenting APCs and antigens driving the interaction.

Implementation Method 1

a molecular reporter of Granzyme B (GzB) activity; comprising a fusion polypeptide comprising a GzB cleavage site (VGPD, SEQ ID NO:1) linked to a detection molecule

Methodology Applied

Scientific EffectGranzyme B cleavage: Enzyme