Engineered HLA-Null Cell Line for High-Throughput TCR Off-Target Screening
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for assessing TCR cross-reactivity and alloreactivity in T cell therapies are low-throughput, costly, and often inaccurate, limiting the identification of safe and effective TCR-based immunotherapies for solid tumors.
Innovation Solution
A cell line, ACDC, is developed with inactivated endogenous HLA alleles and equipped with a polynucleotide encoding a fluorescent marker under STAT control and an IL-2 receptor, enabling high-throughput screening of TCR antigen libraries through CRISPR-targeted mutagenesis and functional screening, using deep sequencing and computational methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional methods (co-culture with primary cells or iPSCs) are used to assess TCR cross-reactivity, then safety screening can be performed, but the process is time-consuming and expensive
Solution Approach 1:
The patent creates a pre-engineered cell line with inactivated endogenous HLA alleles and introduced fluorescent markers under STAT control, along with IL-2 receptors. This preliminary preparation allows TCR cross-reactivity screening to be performed rapidly by simply adding peptide-MHC complexes and observing fluorescent signals, eliminating the need for time-consuming co-culture experiments with primary cells or iPSCs while maintaining reliable safety assessment
Solution Approach 2:
The patent creates a simplified copy or model system - the engineered cell line that mimics the essential features needed for TCR screening (HLA presentation, STAT signaling) but with inactivated endogenous HLA to eliminate background noise. This model system enables rapid high-throughput screening without requiring complex primary cell co-cultures
2Measurement precision
If peptide antigen scanning is used for TCR cross-reactivity profiling, then off-target prediction can be performed, but it remains low-throughput due to requirement of individual synthetic peptides per well
Solution Approach 1:
The patent combines multiple screening capabilities into a single engineered cell line system. The cell line integrates HLA presentation, STAT signaling with fluorescent reporters, and IL-2 receptor signaling, allowing simultaneous assessment of TCR binding and functional activation in one system rather than requiring separate assays with individual peptides in each well
Solution Approach 2:
The engineered cell line serves multiple functions: it presents peptide-MHC complexes, detects TCR binding through fluorescent markers under STAT control, and assesses functional activation via IL-2 receptor signaling. This multi-functional system enables high-throughput screening without sacrificing measurement precision
3Reliability
If EBV-immortalised B-cell lymphoblastoid cell lines are used for alloreactivity screening, then HLA allele recognition can be assessed, but background activation is high due to EBV-reactivity in T cell donors
Solution Approach 1:
The patent extracts or removes the problematic element - endogenous HLA alleles that cause background activation through EBV-reactivity. By inactivating endogenous HLA class I and class II alleles in the engineered cell line, the system eliminates the harmful background noise while retaining the ability to present exogenous peptide-MHC complexes for specific TCR alloreactivity assessment
Solution Approach 2:
The patent converts the potential harm of HLA expression (which causes background activation) into a benefit by selectively inactivating endogenous HLA alleles while preserving the ability to express exogenous peptide-MHC complexes. This creates a clean background system where only specific TCR-alloreactive interactions are detected, improving assessment reliability
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 high-throughput identification of TCR off-targets and alloreactivity, accelerating the development of safe and effective TCR-based immunotherapies by providing large datasets for machine learning predictions and discarding unsafe TCRs during the engineering process.
Implementation Method 1
a polynucleotide encoding a first fluorescent marker under control of at least one STAT response element
Data Source
AI summary
The present invention relates to a cell line wherein the endogenous class I and/or class II HLA alleles are inactivated, the cell line further comprising (a) a polynucleotide encoding a first fluorescent marker under control of at least one STAT response element, and (b) an interleukin 2 (IL-2) receptor. The invention further relates to the use of said cell line for the identification of antigenic peptide and/or the identification of alloreactive T cell receptors.