CMV-pp65 T-Cell Receptor Pairing for Target-Cell Killing
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Solution Overview
Problem
Existing methods for preparing TCR-T cells against cytomegalovirus (CMV) infection do not adequately address the heterogeneity of TCR recognition, leading to insufficient killing of target cells.
Innovation Solution
The method involves determining TCR pairing sequences through high-throughput sequencing and evaluating gene expression levels to construct TCR-transduced specific T cells using CMV pp65 short peptide antigens, HLA-A24-CMV-pp65 antigen complexes, and 2A peptides for linking TCR chains, with verification of TCR function in vitro.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional TCR screening methods are used, then the process is simple, but the killing effect on target cells is not obvious
Solution Approach 1:
The patent segments the TCR screening process into multiple independent steps: single-cell sequencing to obtain TCR sequences, MHC tetramer sorting to isolate antigen-specific T cells, and functional verification to confirm killing activity. This segmentation allows each step to be optimized independently, resulting in TCRs with significantly enhanced killing effects while maintaining systematic control over the complexity.
Solution Approach 2:
The patent introduces MHC tetramers as an intermediary tool to bridge the gap between TCR sequences and functional verification. The MHC tetramer acts as a selector that specifically binds to T cells expressing the desired TCR, enabling precise isolation and enrichment of antigen-specific T cells before functional testing, thereby improving the reliability of the final TCR product.
2Reliability
If TCR-T cells are prepared without considering TCR recognition heterogeneity, then the preparation process is straightforward, but the antiviral ability is insufficient
Solution Approach 1:
The patent applies local quality by focusing on the specific recognition properties of individual TCRs rather than treating all T cells uniformly. Through single-cell sequencing and MHC tetramer sorting, the method identifies and isolates T cells with specific TCR variants that have optimal recognition of CMV antigens, thereby enhancing antiviral ability while managing preparation complexity through targeted approaches.
Solution Approach 2:
The patent changes key parameters in the TCR preparation process, including using high-throughput sequencing to obtain TCR sequence data, applying MHC tetramer sorting to isolate specific T cell populations, and conducting functional verification to confirm antiviral activity. These parameter changes transform the preparation process from a straightforward but insufficient method to a complex but highly effective protocol.
3Measurement precision
If high-throughput sequencing and MHC tetramer sorting are used to determine TCR pairing sequences, then TCR specificity is enhanced, but the time and resource consumption increase
Solution Approach 1:
The patent performs preliminary actions by conducting single-cell sequencing to obtain TCR sequences and using MHC tetramer sorting to pre-isolate antigen-specific T cells before functional verification. This preliminary characterization and enrichment of TCR sequences reduces the time and resources needed for subsequent functional testing and TCR-T cell construction, as the pool of candidates is already narrowed to high-probability hits.
Solution Approach 2:
The patent replaces traditional mechanical sorting methods with molecular-based identification through sequencing and specific binding through MHC tetramer recognition. This substitution allows for precise identification of TCR sequences and their pairing information without relying on time-consuming functional assays at each sorting step, thereby improving measurement precision while managing time consumption through molecular specificity.
Data Source
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AI summary
The present invention relates to the technical field of T-cell antigen receptors, and in particular to a TCR capable of recognizing and binding to a CMVpp65 antigen complex, a nucleic acid comprising a nucleotide sequence encoding the TCR, a vector containing nucleic acid molecules, a cell transducing the nucleic acid molecules or the vector, a pharmaceutical composition comprising the TCR, the nucleic acid molecules, the vector or the cell as an active component, and a use of the TCR, the nucleic acid molecules, the vector, the cell, and the pharmaceutical composition in the preparation of a drug for treating tumors or viral infections, respectively.