Glycosylated MHC-I Complexes for T Cell Receptor Identification
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Solution Overview
Problem
The preparation of properly conformed MHC-I molecules via in vitro refolding of inclusion bodies expressed in E. coli is laborious and inefficient, and these molecules lack the functionally relevant post-translational glycosylation necessary for proper immune surveillance, limiting their ability to identify high-affinity T cell and natural killer cell receptors.
Innovation Solution
The use of mammalian expression systems to produce glycosylated peptide receptive MHC-I complexes, which include an MHC-I heavy chain glycosylated at native positions like N86, along with a TAPBPR chaperone and leucine zipper domains, allows for efficient production of high-affinity peptide-MHC multimers that can be used for T cell and NK cell staining and drug delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If in vitro refolding of inclusion bodies expressed in E. coli is used to produce MHC-I molecules, then production cost is reduced, but manufacturing precision and functional reliability deteriorate due to lack of proper glycosylation and conformation
Solution Approach 1:
The patent uses a heterodimeric leucine zipper system as an intermediary mechanism to enable co-expression and co-purification of MHC-I heavy chain and beta-2 microglobulin in E. coli. The leucine zipper domains facilitate proper folding and assembly of the MHC-I complex, acting as a mediator that bridges the gap between simple bacterial expression and complex eukaryotic protein assembly requirements.
Solution Approach 2:
The patent changes the expression system from traditional separate expression to a co-expression system using heterodimeric leucine zippers. This parameter change enables simultaneous production of properly conformed MHC-I molecules with improved manufacturing precision while maintaining the benefits of bacterial expression systems.
2Loss of time
If in vitro refolding method is used to produce MHC-I molecules, then production time is reduced, but productivity deteriorates due to laborious multi-step process and low refolding yield
Solution Approach 1:
The patent performs preliminary action by co-expressing MHC-I heavy chain and beta-2 microglobulin with heterodimeric leucine zippers in E. coli, which facilitates proper folding and assembly during the expression process itself. This preliminary folding action eliminates the need for subsequent laborious in vitro refolding steps, thereby improving both production time and refolding yield.
Solution Approach 2:
The patent extracts the complex refolding process from the production workflow by implementing co-expression with heterodimeric leucine zippers that enable proper folding in vivo. This removes the bottleneck of multi-step refolding operations, significantly improving productivity.
3Ease of operation
If E. coli expression system is used to produce MHC-I molecules, then ease of operation is improved, but measurement precision deteriorates because T cell receptors cannot be properly identified due to missing glycosylation
Solution Approach 1:
The patent introduces heterodimeric leucine zipper domains as intermediaries that mediate proper assembly and conformation of MHC-I molecules in E. coli. This intermediary system enables the bacterial expression system to produce functionally accurate MHC-I complexes that can properly interact with T cell receptors, thereby maintaining ease of operation while improving measurement precision.
4Device complexity
If traditional refolding methods are used to produce MHC-I tetramers, then device complexity is reduced, but reliability deteriorates due to improper conformation and lack of glycosylation
Solution Approach 1:
The patent performs preliminary folding and assembly actions during the co-expression process in E. coli using heterodimeric leucine zippers. This preliminary conformational establishment ensures proper MHC-I structure is formed in vivo, eliminating the need for complex in vitro refolding procedures and improving reliability of the final product.
Solution Approach 2:
The patent merges the expression and folding processes into a single co-expression step using heterodimeric leucine zippers. This combination simplifies the production process while ensuring proper conformation, thereby reducing device complexity without compromising reliability.
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 enables the efficient production of stable, glycosylated MHC-I tetramers that can identify high-affinity T cell and natural killer cell receptors, overcoming the limitations of traditional methods by providing native, peptide-receptive complexes with correct peptide specificity and improved antigen presentation.
Implementation Method 1
produced in mammalian expression systems (e.g., CHO and HEK cells) that allow for the glycosylation of the complexes at one or more native amino acid positions (e.g., at the conserved N86 in HLA-A, HLA-B, and HLA-C)
Data Source
AI summary
Disclosed herein are novel glycosylated peptide receptive MHC-I complexes that allow for efficient production of glycosylated MHC-I multimers. Such glycosylated peptide receptive MHC-I complexes include a single-chain MHC-I construct and are produced in mammalian expression systems (e.g., CHO and HEK cells) that allow for the glycosylation of the complexes at one or more native positions. Multimers (e.g., tetramers) produced from the glycosylated peptide receptive MHC-I complexes provided herein advantageously allow for the identification of high-affinity T cell and natural killer cell receptors previously unidentified using traditional unglycosylated MHC tetramers.