CAR Hinge Region Cysteine Substitution for Surface Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current chimeric antigen receptors (CARs) face challenges in achieving efficient surface expression and functionality in lymphocytes due to unpaired cysteine residues in the CD8 alpha-chain hinge region, leading to unwanted covalent bonds that hinder antigen-specific killing efficiency.
Innovation Solution
A modified hinge region of the human CD8 alpha-chain is used, where cysteine residues are replaced with serine or deleted, optimizing the linker region to prevent unproductive covalent linkages, thereby enhancing surface expression and antigen-specific killing potency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If unmodified CD8 hinge region is used in CAR constructs, then the structure is simpler and easier to manufacture, but unpaired cysteine residues form unwanted covalent bonds that reduce surface expression and functionality
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the CD8 hinge region, specifically replacing cysteine residues with serine residues. This chemical substitution eliminates the unwanted covalent bonding capability while preserving the structural and functional properties of the hinge region, thereby resolving the contradiction between ease of manufacture and reliability of CAR constructs
Solution Approach 2:
The patent converts the harmful effect of cysteine residues (forming unwanted covalent bonds that reduce functionality) into a benefit by systematically replacing them with serine residues. This transformation eliminates the harmful covalent bonding while maintaining the beneficial structural flexibility and spacing functions of the hinge region, improving both surface expression and antigen-specific killing efficiency
2Strength
If cysteine residues are present in the hinge region, then the structural integrity is maintained, but unpaired cysteines form unwanted covalent bonds that reduce killing efficiency
Solution Approach 1:
The patent changes the chemical parameter of the hinge region by substituting cysteine with serine at specific positions. This maintains the structural integrity and flexibility provided by the hinge region while eliminating the unwanted covalent bonding that reduces killing efficiency, thus resolving the contradiction between structural strength and functional productivity
Solution Approach 2:
The patent applies local quality changes by modifying only specific cysteine residues within the hinge region rather than the entire structure. The serine substitutions are strategically placed to eliminate unwanted covalent bonding while preserving the overall structural integrity and functional properties of the hinge region, thereby maintaining structural strength while improving killing efficiency
Data Source
AI summary
The present invention relates to multi-functional proteins which comprise (i) a signal peptide, (ii) a target specific recognition domain, (iii) a linker region, connecting domain (ii) and domain (iv) which comprises a specific modified hinge region of the human CD8 alpha-chain, and (iv) an effector domain. The present invention furthermore relates to nucleic acids encoding the proteins, expression constructs for expressing the protein in a host cell and host cells. The proteins of the invention are chimeric antigen receptors with an optimized linker or hinge region that are suitable for generating target-specific effector cells, for use as a medicament, in particular in the treatment of cancer and in adoptive, target-cell specific immunotherapy.