HLA Class I Negative Cells for Specific TCR Activation
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Solution Overview
Problem
Current methods for isolating and evaluating neoantigen-specific T cells for cancer immunotherapy are hindered by the lack of a high-throughput system to identify and validate patient-specific HLA molecules, leading to time-consuming HLA cloning and promiscuous TCR activation due to native HLA expression on stimulator cells.
Innovation Solution
The development of HLA class I negative cells, such as HEK293T cells, using CRISPR/Cas9-mediated gene disruption to eliminate HLA-A, HLA-B, and HLA-C expression, allowing for the introduction of patient-specific exogenous HLA genes via lentivirus vectors, enabling efficient cloning and expression of tumor-associated antigens for TCR therapy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native HLA expression is present on stimulator cells, then TCR activation can occur, but promiscuous TCR activation and cross-reactivity occur reducing specificity
Solution Approach 1:
The patent removes endogenous HLA class I expression from HEK293T stimulator cells using CRISPR/Cas9-mediated gene disruption. This extraction of the problematic native HLA expression eliminates the source of promiscuous TCR activation, allowing only the introduced patient-specific HLA alleles to activate TCRs, thereby improving activation specificity
Solution Approach 2:
The patent introduces patient-specific HLA class I alleles as exogenous constructs into HLA-negative stimulator cells. These intermediary HLA molecules serve as specific mediators that bridge the TCR and its cognate antigen, enabling selective TCR activation only for the patient's tumor-associated antigens without cross-reactivity to other HLA alleles
2Measurement precision
If HLA cloning and validation is performed using traditional methods, then patient-specific HLA molecules can be identified, but the process is time-consuming and lacks high-throughput capability
Solution Approach 1:
The patent performs preliminary CRISPR/Cas9-mediated disruption of endogenous HLA class I genes in HEK293T cells before introducing patient-specific HLA alleles. This preliminary action creates a clean background that eliminates the need for time-consuming validation steps to ensure specificity, as the stimulator cells are guaranteed to express only the introduced HLA molecules
Solution Approach 2:
The patent changes the HLA expression status of stimulator cells from HLA-positive to HLA-negative using CRISPR/Cas9 gene editing. This parameter change fundamentally alters the cloning and validation process, enabling high-throughput generation of patient-specific stimulator cells without traditional time-consuming validation steps
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
This approach streamlines the production of patient-specific stimulator cells with minimal cross-reactivity, facilitating the identification and validation of tumor-specific antigens and their corresponding TCRs, thereby enhancing the efficiency of cancer immunotherapy.
Implementation Method 1
said cells produced by CRISPR/Cas9-mediated gene disruption targeted to a consensus sequence share between HLA-A, HLA-B, and HLA-C
Data Source
AI summary
The present disclosure provides methods of generating HLA class-I null cells that can be used for expression of exogenous HLA genes and presentation of antigens, such tumor neoantigens. Method for using HLA class-I null cells from selecting, stimulating and propagating immune effector cells are also provided.