T Cell TCR Mispairing via Endogenous Chain Knockout
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Solution Overview
Problem
Current methods for introducing TCR genes into T cells to impart cytotoxic activity against specific antigens face challenges such as mispairing of endogenous and exogenous TCR chains, leading to decreased recognition of the objective antigen and increased recognition of unexpected antigens, and competition between endogenous and exogenous polypeptides in oligomeric proteins, which can result in undesired oligomer formation and decreased function.
Innovation Solution
A cell expressing a non-natural oligomeric protein is created by introducing a gene encoding an exogenous polypeptide corresponding to an endogenous polypeptide, with the expression of the endogenous polypeptide inhibited using RNA interference, ensuring the exogenous polypeptide forms a functional oligomeric protein without competition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a TCR gene composed of an α chain and a β chain recognizing an objective antigen is introduced into a T cell, then the T cell can recognize the objective antigen, but the endogenous TCR α chain and TCR β chain cause mispairing between introduced and endogenous chains, decreasing the number of proper heterodimers and increasing heterodimers recognizing unexpected antigens
Solution Approach 1:
The patent removes the endogenous TCR α chain or β chain from the T cell system through genetic modification (knockout or knockdown), eliminating the source of mispairing. This extraction of the problematic endogenous component allows the introduced TCR chains to form proper heterodimers without competition or mispairing, thereby resolving the contradiction between antigen recognition capability and heterodimer formation accuracy
Solution Approach 2:
The patent performs preliminary genetic modification to delete or suppress endogenous TCR chain expression before introducing the exogenous TCR gene. This preliminary action ensures that when the exogenous TCR chains are introduced, there are no endogenous chains present to cause mispairing, thus guaranteeing proper heterodimer formation and maintaining high antigen recognition specificity
2Manufacturing precision
If a single-stranded TCR or chimeric receptor is introduced into a T cell to avoid mispairing, then mispairing is avoided, but the T cell may recognize two kinds of antigens and safety concerns arise due to the recombinant TCR not being naturally occurring
Solution Approach 1:
The patent extracts/removes one of the TCR chains (α or β) through genetic knockout, creating a T cell that lacks the ability to form endogenous TCR heterodimers. This extraction eliminates the mispairing problem while maintaining natural TCR structure and function, as the introduced chains still form natural αβ heterodimers without the safety concerns of chimeric receptors
Solution Approach 2:
Instead of introducing a modified single-stranded TCR or chimeric receptor that causes safety concerns, the patent inverts the approach by removing one chain entirely and introducing only the necessary exogenous chains. This inversion maintains natural TCR architecture and signaling while achieving the goal of preventing mispairing and ensuring antigen recognition specificity
3Quantity of substance
If an exogenous polypeptide gene is introduced into a cell expressing an oligomeric protein, then the exogenous polypeptide can be produced, but endogenous and exogenous polypeptides compete and undesired oligomers are formed
Solution Approach 1:
The patent removes the endogenous polypeptide from the cell through genetic knockout or knockdown, eliminating the competing component. This extraction ensures that when the exogenous polypeptide is introduced and expressed, it is the only available subunit to form oligomers, thereby preventing undesired heterogeneous oligomer formation and ensuring pure exogenous polypeptide oligomers are produced
Solution Approach 2:
The patent performs preliminary genetic modification to delete or suppress endogenous polypeptide expression before introducing the exogenous polypeptide gene. This preliminary action ensures that the cellular machinery is primed to produce only exogenous polypeptide-containing oligomers, eliminating competition and ensuring high purity of the desired oligomer product
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 results in a high rate of desired oligomeric protein function retention, where the endogenous polypeptide is replaced by the exogenous one, enhancing antigen specificity and reducing side effects, making it useful for cellular medical treatments.
Implementation Method 1
the expression of the endogenous polypeptide is inhibited
Data Source
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AI summary
Disclosed is a cell which can express a non-natural oligomeric protein, which has, introduced therein, a gene encoding an exogenous polypeptide corresponding to at least one endogenous polypeptide constituting a natural oligomeric protein, and in which the expression of the endogenous polypeptide is inhibited.