Disulfide Trap MHC Class I Molecules for Stable Peptide Presentation
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Solution Overview
Problem
Current MHC-based technologies face limitations in efficiently expressing and stabilizing peptide-MHC complexes, particularly with low-affinity peptides, which affects their ability to activate T cells and is crucial for effective antiviral and antitumor immunity.
Innovation Solution
The development of disulfide trap molecules, where an antigen peptide is covalently attached to an MHC class I heavy chain via a disulfide bond, enhancing the association of peptides with the MHC and preventing exogenous peptide binding, thereby improving the stability and refractoriness of peptide-MHC complexes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If conventional non-covalent peptide-MHC complexes are used, then the complexes can be formed and presented, but the stability and surface expression are insufficient, especially for low-affinity peptides
Solution Approach 1:
The patent introduces a cysteine residue at the C-terminus of the peptide and a cysteine mutation in the MHC class I heavy chain (e.g., at position 80, 84, or 86) to enable disulfide bond formation. This preliminary structural modification ensures that when the peptide binds to the MHC, a covalent disulfide linkage is automatically formed, locking the peptide in place and preventing dissociation. This resolves the stability issue by transforming the transient non-covalent interaction into a permanent covalent bond.
Solution Approach 2:
The patent creates a composite structure by combining the peptide, MHC class I heavy chain, and β2-microglobulin into a single covalently linked complex through disulfide bonds. This composite approach integrates multiple components (peptide-MHC-β2m) with strong covalent linkages, enhancing the overall structural integrity and surface expression efficiency compared to conventional non-covalent assemblies.
2Reliability
If conventional peptide-MHC complexes are used, then T cell activation can occur, but exogenous competitor peptides can displace the original peptides, reducing response reliability
Solution Approach 1:
The disulfide bond is pre-engineered into the peptide-MHC complex structure through cysteine incorporation. This preliminary covalent linkage prevents exogenous competitor peptides from displacing the original peptide, as the disulfide bond creates a kinetically stable complex that resists peptide exchange. This ensures reliable and specific T cell responses without interference from competing peptides.
3Stability of the object's composition
If covalent disulfide bonds are introduced to stabilize peptide-MHC complexes, then stability improves, but the complexity of molecule construction increases
Solution Approach 1:
Instead of modifying the entire peptide-MHC complex structure, the patent applies a localized solution by introducing single cysteine residues at specific positions: the C-terminus of the peptide and a specific position in the MHC heavy chain (80, 84, or 86). This localized modification approach minimizes construction complexity while achieving the desired global stability enhancement through disulfide bond formation.
4Reliability
If conventional soluble recombinant class I heavy chains are used for tetramer production, then tetramers can be made, but stability is limited by peptide-MHC affinity
Solution Approach 1:
The patent pre-engineers disulfide bond capability into the class I heavy chain construct by introducing cysteine mutations at positions 80, 84, or 86. When these engineered heavy chains are refolded with peptides containing C-terminal cysteines, automatic disulfide bond formation occurs, creating extremely stable peptide-MHC complexes that serve as the foundation for stable tetramer reagents, overcoming the affinity limitation of conventional approaches.
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 significantly enhances the surface expression and stability of peptide-MHC complexes, even with low-affinity peptides, effectively preventing competitor peptide binding and improving T cell activation, making it suitable for vaccines and diagnostic reagents.
Implementation Method 1
The covalent linkage between the antigen peptide and the MHC class I heavy chain comprises a disulfide bond
Data Source
AI summary
A disulfide trap, comprising an antigen peptide covalently attached to an MHC class I heavy chain molecule by a disulfide bond extending between two cysteines, is disclosed. In some configurations, a disulfide trap, such as a disulfide trap single chain trimer (dtSCT), can comprise a single contiguous polypeptide chain. Upon synthesis in a cell, a disulfide trap oxidizes properly in the ER, and can be recognized by T cells. In some configurations, a peptide moiety of a disulfide trap is not displaced by high-affinity competitor peptides, even if the peptide binds the heavy chain relatively weakly. In various configurations, a disulfide trap can be used for vaccination, to elicit CD8 T cells, and in multivalent MHC/peptide reagents for the enumeration and tracking of T cells. Also disclosed are nucleic acids comprising a sequence encoding a disulfide trap. Such nucleic acids, which can be DNA vectors, can be used as vaccines.