Sortase-Mediated CAR Repair for T-Cell Longevity
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Solution Overview
Problem
Chimeric Antigen Receptors (CARs) expressed by cells, such as T-cells, have a limited activity due to the degradation of antigen binding domains over time, necessitating a method to maintain their functionality and longevity.
Innovation Solution
The use of sortase molecules to mediate the transfer of antigen binding domains onto CARs, creating a sortase acceptor member with a sortase acceptor motif, transmembrane domain, and intracellular signaling domain, allowing for the in situ construction and maintenance of CARs on immune effector cells like T-cells, thereby providing a 'safety mechanism' against domain degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CARs are expressed by cells through traditional transduction methods, then the cells can target and kill cancer cells, but the antigen binding domains degrade over time leading to limited activity duration
Solution Approach 1:
The sortase acceptor motif is pre-installed on the CAR membrane anchor sequence before the CAR is expressed on the cell surface. This preliminary preparation allows for subsequent enzymatic attachment of antigen binding domains, ensuring the CAR can be replenished or repaired after degradation occurs, thereby extending the functional duration of CAR-expressing cells
Solution Approach 2:
The system enables self-repair of degraded CARs through the sortase-mediated attachment mechanism. The sortase acceptor motif on the membrane anchor allows new antigen binding domains to be attached to replace degraded ones, creating a self-maintaining system that can sustain CAR functionality over extended periods without requiring complete retransduction
2Stability of the object's composition
If the gene encoding the antigen binding domain is included in the CARX cell genome, then the CAR can be stably expressed, but the antigen binding domain degrades over time due to cell division and membrane protein turnover
Solution Approach 1:
The invention separates the antigen binding domain from the membrane anchor sequence, placing them as distinct components that can be independently managed. The membrane anchor retains the sortase acceptor motif while the antigen binding domain can be supplied extracellularly via sortase-mediated attachment, allowing the system to replace degraded domains without affecting the stable membrane anchor portion
Solution Approach 2:
The system allows for the discarding of degraded antigen binding domains and their recovery through sortase-mediated attachment of new domains. The sortase acceptor motif on the membrane anchor serves as a persistent attachment point that can repeatedly bind new antigen binding domains, enabling continuous recovery of functional CARs despite ongoing degradation
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 ensures the long-term functionality of CARs by preventing the degradation of antigen binding domains, enhancing the persistence and activity of CAR-expressing cells, such as T-cells, in targeting and inactivating or killing cancer cells.
Implementation Method 1
sortase mediated transfer of an antigen binding domain, e.g., a scFv, onto a CAR member having a sortase acceptor motif
Data Source
AI summary
Sortase molecules and methods described herein allow for the construction of a CAR or CAR member, e.g., in situ, on a CARX, e.g., CART, cell. For example, sortase mediated transfer of an antigen binding domain, e.g., a scFv, onto a CAR member having a sortase acceptor motif in place of an antigen binding domain can provide for a complete CAR member on a cell wherein the cell does not comprise nucleic acid that encodes the complete CAR member.