KRAS-Binding T Cells With Endogenous TCR Knockout
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Solution Overview
Problem
Adoptive T cell therapy (ACT) for solid tumors requires optimization due to competition between transgenic and endogenous TCRs for the CD3 pool, leading to decreased surface expression and sensitivity of engineered TCRs.
Innovation Solution
Engineering host cells with a heterologous extracellular binding protein that targets the KRAS G12D mutant peptide, combined with CRISPR-mediated knockout of endogenous TCRs to enhance TCR expression and specificity, using vectors like lentiviral or γ-retroviral vectors to introduce the binding protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If transgenic TCR is introduced into host T cells, then the T cells gain ability to recognize mutant KRAS, but endogenous TCRs compete for CD3 pool and reduce surface expression of engineered TCR
Solution Approach 1:
The patent removes endogenous TCR genes (TRAC and TRBC1/TRBC2) from the host T cell genome using CRISPR-Cas9 technology. This extraction eliminates the competing endogenous TCRs that were consuming CD3 components and reducing the surface expression of the engineered KRAS-specific TCR, thereby resolving the contradiction by eliminating the harmful competition while preserving the desired transgenic TCR expression.
Solution Approach 2:
The patent applies preliminary anti-action by pre-knocking out the endogenous TCR genes before introducing or expressing the transgenic TCR. This preemptive removal prevents the competition for CD3 pool from occurring in the first place, ensuring that the engineered TCR achieves optimal surface expression without interference from endogenous TCRs.
2Reliability
If endogenous TCRs are present in host cells, then T cells maintain normal TCR function, but mispairing with transgenic TCR chains decreases sensitivity to targeted cells
Solution Approach 1:
The patent extracts and removes the endogenous TCR genes (specifically TRAC and TRBC1/TRBC2) from the host T cell genome using CRISPR-Cas9. This elimination prevents mispairing between endogenous TCR chains and transgenic TCR chains, thereby resolving the contradiction by eliminating the source of mispairing while allowing the transgenic TCR to function with full sensitivity and specificity for the mutant KRAS target.
3Manufacturing precision
If CRISPR-mediated knockout of endogenous TCRs is performed, then TCR expression and sensitivity are improved, but genomic mutation is introduced
Solution Approach 1:
The patent uses CRISPR-Cas9 as an intermediary tool to achieve precise genomic editing. The system introduces a controlled, targeted mutation at specific TCR loci (TRAC/TRBC) to knock out endogenous TCR expression. While this does introduce genomic changes, they are precisely targeted and localized only to the necessary TCR genes, minimizing off-target effects and accepting the localized genomic modification as a necessary means to achieve the desired high-level transgenic TCR expression and sensitivity.
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
Improves the sensitivity and specificity of engineered T cells to target tumor cells expressing the KRAS G12D mutant peptide, enhancing in vitro cytotoxicity against solid tumors.
Implementation Method 1
this engineering involves use of a Type V-A CRISPR nuclease alongside compatible gRNAs that resulted in TCR knockout in >90% of human primary T cells
Implementation Method 2
the extracellular binding protein is capable of binding to a peptide:HLA complex, where the peptide comprises a KRAS G12D mutant peptide
Data Source
AI summary
The present disclosure provides methods and compositions for adoptive T cell therapy, particularly extracellular binding proteins targeting KRAS peptides, host cells comprising the binding proteins, and methods of use and manufacture thereof.