Dual Chimeric Protein CAR Design to Reduce Tonic Signaling
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Solution Overview
Problem
Chimeric antigen receptors (CARs) face issues such as misfolding, aggregation, and tonic signaling due to unstable antigen binding units, leading to cell exhaustion and reduced therapeutic efficacy.
Innovation Solution
A dual chimeric protein system is introduced, where the first protein is synthesized in the ER and lacks a signaling domain, while the second protein is synthesized in the cytosol and anchored to the membrane, dimerizing only at the plasma membrane to form a functional CAR, thereby reducing unwanted signaling and improving cell persistence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a conventional CAR construct is used, then the CAR can be synthesized in the ER and transported to the cell membrane, but the CAR may misfold, aggregate, or recognize internal epitopes causing tonic signaling and cell exhaustion
Solution Approach 1:
The CAR construct is divided into two separate proteins: a first chimeric protein with an antigen binding unit and transmembrane domain but no signaling domain, and a second chimeric protein with a signaling domain and dimerization domain. This segmentation prevents misfolding and aggregation in the ER while allowing functional assembly at the cell membrane.
Solution Approach 2:
A dimerization domain acts as an intermediary between the two chimeric proteins, enabling them to assemble into a functional CAR complex only at the cell membrane. This intermediary mechanism ensures proper folding and prevents unwanted intracellular interactions.
2Duration of action of moving object
If the CAR is constitutively activated to maintain therapeutic activity, then the cell can sustain effector function, but the cell may be exhausted before a therapeutic effect is achieved
Solution Approach 1:
By separating the signaling domain into a second chimeric protein that only assembles with the first protein at the cell membrane, the system eliminates constitutive activation while maintaining the ability to respond to target antigens. This segmentation ensures signaling occurs only when needed, preventing cell exhaustion.
3Object-generated harmful factors
If the antigen binding unit is stabilized to prevent aggregation, then tonic signaling is reduced, but the CAR may still recognize internal epitopes in intracellular compartments
Solution Approach 1:
The first chimeric protein is designed without a signaling domain, preventing it from causing non-specific signaling even if it binds internal epitopes in intracellular compartments. The signaling function is isolated to the second protein, which only becomes active at the cell membrane upon proper assembly.
Solution Approach 2:
The signaling domain is extracted from the first chimeric protein and placed in the second chimeric protein. This extraction ensures that signaling can only occur when the complete complex is assembled at the cell membrane, eliminating unwanted intracellular signaling while maintaining target specificity.
Data Source
AI summary
Provided herein are cells expressing a first and a second chimeric protein in the cell membrane. Such cells can solve expression problems of CAR constructs caused by these CAR construct's ability to recognize unwanted internal epitopes or exhibiting unwanted signalling due to misfolding/scFv aggregation. The first chimeric protein comprises an extracellular antigen binding unit and an intracellular dimerization domain. The second chimeric protein comprises a lipid anchoring domain, an intracellular dimerization domain and a signaling domain. Accordingly, the expression of the two proteins allows them to translocate to the cell membrane without much interference and subsequently gain signaling capacity when colocalized at the cell membrane.