Peptide-Receptive MHC-I Complexes for Stable High-Throughput Multimers
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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 and high background levels of exchange due to sample aggregation and precipitation during photolysis/peptide exchange steps.
Innovation Solution
The development of stable peptide-receptive MHC-I complexes, formed by incubating MHC class I heavy chains, β2-microglobulin, and placeholder peptides, which are capable of accepting a peptide of interest, each peptide receptive MHC-I complex comprising an MHC class I heavy chain and a peptide of interest, each peptide receptive MHC-I complex comprising an β2-microglobulin, and placeholder peptides, which are capable of accepting a peptide of interest, each peptide receptive MHC-I complex comprising an α2-microglobulin, and placeholder peptides, which are capable of accepting a peptide of interest, each peptide receptive MHC-I complex comprising an MHC class I heavy chain and an β2-microglobulin, the method comprising: a) incubating a plurality of MHC class I heavy chains, a plurality of β2-microglobulins, and a plurality of placeholder peptides under conditions to form placeholder peptide-MHC class I complexes; and b) contacting these complexes with chaperones and peptides of interest to create peptide receptive MHC-I complexes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conditional ligands are used to produce peptide-deficient MHC class I molecules, then large scale production is enabled, but sample aggregation and precipitation occur during photolysis/peptide exchange steps
Solution Approach 1:
The patent introduces a conditional ligand as an intermediary molecule that temporarily occupies the MHC class I binding groove. This conditional ligand can be cleaved by UV irradiation or temperature increase, allowing controlled release and subsequent peptide exchange. The conditional ligand acts as a mediator that enables large-scale production of peptide-deficient MHC class I molecules while maintaining sample stability during storage, and allows controlled exchange without aggregation or precipitation issues.
2Ease of manufacture
If photolysis/peptide exchange steps are performed, then peptide-MHC class I complexes can be generated, but high background levels of exchange occur
Solution Approach 1:
The patent employs parameter changes by using different activation conditions (UV wavelength, temperature) to control the cleavage of conditional ligands. By carefully selecting and controlling these parameters, the patent achieves specific peptide exchange while minimizing background exchange. The conditional ligand is designed to be stable under storage conditions but undergoes controlled cleavage under specific activation parameters, thereby improving exchange specificity.
3Productivity
If peptide-deficient MHC class I molecules are produced, then high-throughput screening is enabled, but instability and aggregation occur
Solution Approach 1:
The patent applies preliminary action by pre-loading MHC class I molecules with conditional ligands under controlled conditions before large-scale production. This preliminary loading step ensures proper folding and stability of the MHC class I molecules. The conditional ligands are designed to maintain molecular stability during storage and handling, preventing aggregation and precipitation that would otherwise occur with peptide-deficient molecules. This preliminary stabilization enables subsequent 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
Enables the production of stable peptide-MHC class I multimer complexes that can be stored for long periods and used in high-throughput screens for immune profiling and disease diagnosis, reducing sample aggregation and background exchange issues.
Implementation Method 1
contacting the p*MHC-I complexes with a plurality of dipeptides and chaperones, thereby creating the plurality of peptide receptive MHC-I complexes
Data Source
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AI summary
Compositions that include peptide receptive MHC class I complexes and methods of making and using such complexes are provided. In particular embodiments, such peptide receptive MHC class I 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.