TAPBPR-Stabilized Peptide-Deficient MHC-I Complexes for Multimer Screening
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Solution Overview
Problem
Existing high-throughput screening strategies for peptide-MHC class I multimers are limited by the instability of peptide deficient MHC class I molecules, leading to difficulties in large-scale production due to sample aggregation and high background levels of exchange, which affects the production of peptide-MHC class I multimer complexes.
Innovation Solution
A method involving incubating MHC class I heavy and light chains with placeholder peptides to form a complex, followed by contacting with a Tapasin Binding Protein Related (TAPBPR) chaperone to displace the placeholder peptide, forming stable peptide deficient-MHC class I/chaperone complexes, which can then be used to produce large collections of peptide-MHC class I multimers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If conditional ligands are used to circumvent instability of peptide deficient MHC class I molecules, then large scale production becomes feasible, but sample aggregation and precipitation occur during photolysis/peptide exchange
Solution Approach 1:
The patent introduces a conditional ligand as an intermediary molecule that temporarily occupies the peptide-binding groove of MHC class I molecules. This conditional ligand can be cleaved by UV light or heat to release the placeholder peptide and allow exchange with the peptide of interest. The conditional ligand acts as a mediator that enables stable handling of peptide-deficient MHC molecules during production while facilitating controlled peptide exchange without causing aggregation or precipitation.
Solution Approach 2:
The patent employs parameter changes by using conditional ligands with specific photophysical or thermal properties. The conditional ligand contains a cleavable bond that responds to changes in physical parameters (UV irradiation or temperature increase), triggering the release of the placeholder peptide. This parameter-based control allows precise timing of peptide exchange and maintains molecular stability throughout the process, preventing aggregation and precipitation.
2Productivity
If suboptimal peptides are used in conditional ligand exchange, then exchange efficiency may be compromised, but high background levels of exchange occur
Solution Approach 1:
The patent applies local quality by designing conditional ligands with specific structural features and binding characteristics tailored for particular MHC class I alleles and peptide sequences. The conditional ligand is engineered to have optimal local interactions with the MHC binding groove, ensuring high specificity and low background exchange. This localized optimization of binding properties enables efficient peptide exchange only when intended, reducing spurious background signals.
3Adaptability or versatility
If large collections of peptide-MHC class I multimers are produced for high-throughput screening, then identification capability improves, but production becomes limited by instability of peptide deficient MHC class I molecules
Solution Approach 1:
The patent implements preliminary action by pre-assembling MHC class I molecules with conditional ligands bound before large-scale production and storage. This preliminary conditioning allows the peptide-deficient MHC molecules to be manufactured, purified, and stored in a stable state. The conditional ligand protects the MHC molecule from instability issues until the moment of peptide exchange, enabling large collections of multimers to be prepared in advance for high-throughput screening applications.
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
The method produces stable peptide deficient-MHC class I/chaperone complexes that can be stored for extended periods, enabling the production of up to 10,000 different specificities of peptide-MHC class I multimer complexes for high-throughput screens in disease diagnosis and therapy development.
Implementation Method 1
contacting the placeholder peptide-MHC class I complex with a dipeptide and Tapasin Binding Protein Related (TAPBPR) chaperone, thereby displacing the placeholder peptide from the placeholder peptide-MHC complex and forming the peptide deficient-MHC class I/chaperone complex
Data Source
Figure 1A~1D
Figure 2A~2C
Figure 3
AI summary
Compositions that include stable peptide deficient MHC class I/chaperone complexes and methods of making and using such complexes are provided. In particular embodiments, such peptide deficient MHC class I/chaperone complexes are used to form peptide MHC class I (pMHC-I) multimers useful for high throughput applications, such as, for the detection of antigen specific T cells and characterization of T cell profiles in subjects.