Ubiquitination-Lacking CAR With Lysine-to-Arginine Mutation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
CAR-T therapy for solid tumors is limited by the poor proliferation capacity of CAR-T cells, which can be attributed to inefficient expansion and consistent activation in patients, contrasting with the successful treatment of hematological tumors.
Innovation Solution
A ubiquitination-lacking chimeric antigen receptor (CAR) is designed with mutated lysine sites changed to arginine, specifically for the 41BB intracellular costimulatory domain, enhancing proliferation and metabolic dependency on oxidative phosphorylation, leading to increased central memory T cell differentiation and enhanced tumor killing activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If co-expression of cytokines (IL-7, IL-15, IL-18) is used to promote T cell stemness and proliferative capacity, then the proliferative capacity of CAR-T cells is improved, but the construction difficulty increases and conversion efficiency decreases
Solution Approach 1:
The invention extracts and removes the ubiquitination modification sites (lysine residues) from the CAR intracellular domain, specifically targeting the costimulatory signaling region and CD3ζ intracellular region. By mutating lysine residues to arginine, the patent eliminates the ubiquitination modification pathway that leads to CAR degradation, thereby improving CAR-T cell proliferative capacity without requiring complex cytokine co-expression systems
Solution Approach 2:
The invention changes the chemical parameter of the CAR protein by mutating lysine residues (which are ubiquitination sites) to arginine residues. This parameter change prevents ubiquitination modification, stabilizes the CAR protein, and enhances its proliferative capacity while simplifying the overall construct design compared to cytokine co-expression approaches
2Reliability
If co-expression of cytokines is used to enhance CAR-T cell proliferative capacity, then the anti-tumor activity is improved, but the large-scale industrial production becomes limited
Solution Approach 1:
The invention removes the requirement for complex cytokine co-expression systems by directly modifying the CAR structure itself. By eliminating ubiquitination sites through lysine-to-arginine mutations in the intracellular domain, the patent achieves stable CAR-T cell proliferation and anti-tumor activity that is compatible with large-scale industrial production processes
Solution Approach 2:
The invention creates a simplified version of the CAR structure that copies only the essential functional elements while removing the complex cytokine co-expression requirements. The ubiquitination-lacking CAR design maintains anti-tumor efficacy through structural modification alone, making it suitable for standardized manufacturing
3Productivity
If conventional CAR structure is used, then the CAR-T cells can be produced, but the CAR-T cells cannot expand efficiently and consistently in patients
Solution Approach 1:
The invention changes the stability parameter of the CAR protein by mutating lysine residues to arginine in the intracellular domain, preventing ubiquitination-mediated degradation. This parameter change ensures consistent and efficient expansion of CAR-T cells in patients by stabilizing the CAR structure throughout the therapeutic process
Solution Approach 2:
The invention performs preliminary modification of the CAR structure by removing ubiquitination sites before the CAR-T cells are administered to patients. This preliminary action prevents subsequent degradation and ensures consistent expansion and activation of CAR-T cells during treatment
Data Source
AI summary
The present invention provides a chimeric antigen receptor, which includes: an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence. The extracellular domain includes an antigen recognition region; the intracellular domain includes a costimulatory signaling region and a CD3ζ intracellular region that are connected in sequence, to form a costimulatory signaling region-CD3ζ intracellular region; and the costimulatory signaling region-CD3ζ intracellular region is a polypeptide formed by mutation of lysine in a wild-type costimulatory signaling region-CD3ζ intracellular region into arginine. The present invention provides a method for optimization and modification of CAR-T, in which all lysine sites in an intracellular segment of CAR are mutated into arginine, thereby blocking ubiquitination modification of the CAR after antigen challenge. This strategy is applicable to different CARs and changing different intracellular costimulatory domains, and in particular provides a solution to the problem of poor proliferation of CAR-T in solid tumors.