Conditional Ligand Exchange for pMHC Complex Generation
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Solution Overview
Problem
Current methods for generating peptide-MHC complexes are time-consuming and incompatible with high throughput applications due to the instability of MHC molecules without peptide ligands, leading to the selection of false positive immune cells specific for conditional ligands rather than the intended peptide.
Innovation Solution
The use of weakly immunogenic conditional peptides with specific amino acid sequences for generating pMHC complexes by conditional ligand exchange, allowing for the selection of immune cells expressing antigen-binding proteins with reduced false positives through sequential exposure to different pMHC complexes generated by conditional ligand exchange.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional in vitro refolding and purification steps are used to generate pMHC complexes, then the complexes can be produced, but the process becomes time-consuming and incompatible with high throughput applications
Solution Approach 1:
The MHC molecule is pre-loaded with a conditional peptide ligand that is stable under storage conditions but can be exchanged under defined stimulus conditions. This preliminary preparation eliminates the need for time-consuming in vitro refolding and purification steps during actual pMHC complex generation, enabling high throughput applications while maintaining complex reliability.
Solution Approach 2:
The patent utilizes conditional peptide ligands that change their binding properties based on environmental parameters (e.g., pH, temperature, or chemical stimuli). The conditional ligand is stable under storage conditions but undergoes exchange when exposed to defined stimulus conditions, allowing rapid generation of pMHC complexes without traditional refolding procedures.
2Productivity
If conditional peptide ligands are used for MHC molecules, then fast and easy production of numerous different pMHC complexes is achieved, but the risk of selecting false positive immune cells increases
Solution Approach 1:
The patent introduces a control pMHC complex as an intermediary element in the selection process. This control complex, generated using the same conditional ligand exchange method, serves as a reference to distinguish between specific and non-specific binding. By comparing immune cell responses against both the test and control pMHC complexes, false positive selections are significantly reduced while maintaining the speed advantage of conditional ligand exchange.
Solution Approach 2:
The method incorporates a feedback mechanism through the control pMHC complex that provides information about non-specific binding. The control complex allows researchers to identify and exclude false positive immune cell selections, thereby improving selection accuracy while maintaining the rapid production capability of conditional ligand exchange.
3Measurement precision
If weakly immunogenic conditional peptides are used, then false positive immune cell selection is reduced, but the binding strength and stability of pMHC complexes must be maintained
Solution Approach 1:
The patent applies different properties to different parts of the peptide-MHC system. The conditional peptide ligand is designed with specific local characteristics: it binds strongly to the MHC molecule under storage conditions but can be exchanged under defined stimulus conditions. This local quality differentiation allows the peptide to maintain binding strength when needed while enabling controlled exchange to prevent false positive selections.
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 reduces the selection of false positive immune cells, making the method more accurate, cost-effective, faster, and suitable for high throughput applications while maintaining strong binding and stability of pMHC complexes.
Implementation Method 1
Recently, conditional peptide ligands for MHC molecules, which degrade upon exposure to a defined stimulus, were developed. By addition of a peptide of interest the conditional ligand can be replaced. This conditional ligand exchange allows fast and easy production of numerous different pMHC complexes
Implementation Method 2
These conditional peptides provide inter alia for one or more of the following advantages: (ii) strong binding between conditional peptide and MHC molecule, (iv) stable conditional peptide:MHC complexes in absence of the defined stimulus
Implementation Method 3
These conditional peptides provide inter alia for one or more of the following advantages: (iii) high refolding yield of conditional peptide:MHC complexes
Data Source
AI summary
The present invention relates to a method for selecting an immune cell expressing on its surface an antigen-binding protein specifically binding to a complex of a peptide A (PA) and a Major Histocompatibility Complex (MHC) molecule, comprising the steps of (i) providing a plurality of immune cells expressing different antigen-binding proteins; (ii) contacting the plurality of immune cells with a first composition comprising a complex 1A, comprising a MHC molecule 1 (M1) and a peptide A (PA), and a complex 1X, comprising M1 and a peptide B (PB); (iii) contacting the plurality of immune cells with a second composition comprising a complex 2A, comprising a MHC molecule 2 (M2) and PA, and a complex 2X, comprising M2 and a peptide C (PC); (iv) selecting from the plurality of immune cells a cell expressing an antigen binding protein that specifically binds to complex 1A, wherein complexes 1X and 2X, but not 1A and 2A, dissociate upon stimulation with a defined chemical or physical stimulus; and wherein the amino acid sequences of PB and PC differ in at least one amino acid. The invention further relates to an immune cell selected by the method according to the invention, a method of treatment using said immune cell, a kit for selecting a cell expressing on its surface an antigen-binding protein, and a peptide suitable for use in the method according to the invention.