Shared Neoantigen-Reactive TCR Screening With NFAT Functional Validation
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Solution Overview
Problem
Existing methods fail to provide an integrated workflow that seamlessly combines neoantigen discovery, TCR prioritization, and functional validation, often prioritizing sequence identification while insufficiently emphasizing functional testing.
Innovation Solution
A comprehensive method involving steps such as isolating PBMCs, next-generation sequencing, bioinformatic analysis, peptide stimulation, T cell screening, and functional validation using a Nuclear Factor of Activated T cells (NFAT) system to identify and validate shared neoantigen-reactive T cell receptors (TCRs).
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high-throughput sequencing and computational tools are used to identify neoantigen candidates, then neoantigen discovery capability is improved, but functional validation is insufficient
Solution Approach 1:
The patent performs preliminary sequencing and computational analysis to identify neoantigen candidates before functional validation. This preliminary action enables efficient screening of multiple candidates, after which selected candidates undergo rigorous functional validation through T cell activation assays and cytotoxicity tests, thus resolving the contradiction between high productivity in discovery and reliability in validation
Solution Approach 2:
The patent implements a feedback loop where computational predictions of neoantigen candidates are tested through functional validation assays. The results of functional validation feed back into refining the computational models and selection criteria, improving both the productivity of candidate identification and the reliability of functional outcomes through iterative optimization
2Productivity
If TCR sequences are prioritized for identification, then sequence identification efficiency is improved, but functional testing is insufficient
Solution Approach 1:
The patent segments the TCR identification process into distinct phases: high-throughput sequencing for rapid TCR sequence identification, followed by separate functional validation phases including T cell activation assays and target cell recognition tests. This segmentation allows efficient sequence identification while maintaining rigorous functional testing standards for selected candidates
Solution Approach 2:
The patent performs partial functional testing on a selected subset of TCR sequences rather than all identified sequences. By using computational filtering and prioritization criteria, the patent applies excessive action in sequence identification to generate many candidates, then applies appropriate functional testing to the most promising subset, balancing productivity and measurement precision
3Reliability
If comprehensive workflow integrating neoantigen discovery, TCR prioritization, and functional validation is implemented, then treatment reliability is improved, but process complexity increases
Solution Approach 1:
The patent merges multiple previously separate processes (neoantigen discovery, TCR sequencing, computational prioritization, and functional validation) into a single integrated workflow. This merging ensures that each step builds upon the previous steps with standardized protocols and data formats, improving treatment reliability while managing complexity through unified process architecture
Solution Approach 2:
The patent develops universal protocols and standardized assays that can be applied across different neoantigen candidates and patient samples. The functional validation assays and TCR selection criteria are designed to be universally applicable, reducing workflow complexity through standardization while maintaining high treatment reliability across diverse applications
Data Source
AI summary
A method for identifying a shared neoantigen-reactive T cell receptor comprising steps performed in the following specific order: (A) collecting and processing sample of a subject with a cancer; (B) obtaining a shared neoantigen by filtered the mutation sequences based on a collection of 67 off-the-shelf peptides; (C) synthesizing a long peptide corresponding to a panel of shared neoantigen and its corresponding of wild type peptides; (D) stimulating the PBMCs with the long synthetic peptides to obtain a stimulated PBMC; (E) screening the stimulated PBMC based on response of T cells is measured by interferon-γ secretion to mutant peptides and wild type peptides; (F) isolating a neoantigen-specific T cell from the screened stimulated PBMC to identify a clonotype-purified cell; (G) identifying a TCR candidate for shared neoantigen; and (H) evaluating antigenic specificity of the TCR candidate for shared neoantigen to identify a shared neoantigen-reactive TCR.