Alpha-Beta TCR Mutant Screening With Correct-Folding Phage Display
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Solution Overview
Problem
Existing methods for screening T cell receptor (TCR) mutants face low efficiency and challenges in achieving correct folding and pairing of αβ-TCR heterodimers, which are crucial for effective antitumor therapies.
Innovation Solution
A method involving the construction of a phagemid with integrated ribosomal binding sites and molecular chaperone genes, using A1 and B1 peptides to promote correct pairing and folding of αβ-TCR heterodimers, and employing a phage display system to screen for stable and high-affinity TCR mutants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Volume of moving object
If scTCR is used for display experiments, then molecular weight is reduced and solubility is improved, but stability is extremely poor requiring multiple mutations that increase workload and failure rate
Solution Approach 1:
The TCR is divided into variable regions (Vα, Vβ) and constant regions (Cα, Cβ), with only the variable regions being mutated for affinity improvement while constant regions maintain structural stability. This segmentation allows independent optimization of different functional domains.
Solution Approach 2:
The constant regions are pre-designed with optimized amino acid sequences that ensure correct folding and stable heterodimer formation before display experiments begin. This preliminary structural optimization eliminates the need for stability-enhancing mutations during the screening process.
2Stability of the object's composition
If dsTCR is used for display experiments, then stability is maintained without requiring mutations, but folding and pairing efficiency is extremely low making it difficult to obtain correctly folded and paired αβ-TCR
Solution Approach 1:
The amino acid sequences at the interaction interface between α and β chains are locally optimized to enhance folding efficiency. Specific residues in the constant regions are engineered to promote correct heterodimer formation while maintaining the overall disulfide-linked structure.
Solution Approach 2:
The amino acid sequences of the constant regions are modified to change folding kinetics and pairing efficiency parameters. These sequence optimizations enable dsTCR to fold correctly and pair efficiently without compromising structural stability.
3Productivity
If conventional phage display is used to screen TCR mutants, then screening can be performed, but efficiency is low due to difficulty in achieving correct folding and pairing of αβ-TCR heterodimers
Solution Approach 1:
The constant regions are pre-engineered with optimized amino acid sequences that ensure correct folding and stable heterodimer formation before display experiments begin. This preliminary structural optimization eliminates folding issues during screening.
Solution Approach 2:
The amino acid sequences are optimized to change folding kinetics and pairing efficiency parameters, enabling reliable formation of correct αβ-TCR heterodimers on the phage surface throughout the screening process.
Data Source
AI summary
A method for screening αβ-TCR mutants, the method comprising the construction of a phagemid. In the construction, a gene encoding an A1 polypeptide and a gene encoding B1 polypeptide are inserted into the 3′ ends of genes encoding the constant regions of the α chain and the β chain of TCR respectively, and a ribosome binding site and a molecular chaperone gene are integrated into a phage vector, wherein the amino acid sequence of the A1 polypeptide is as shown in SEQ ID NO: 1 or is represented by the variant of the A1 polypeptide; the amino acid sequence of the B1 polypeptide is as shown in SEQ ID NO: 2 or is represented by the variant of the B1 polypeptide; and the variants at least retain the function of the polypeptides before mutation. The A1 peptide and the B1 peptide are used for promoting the correct pairing and folding of the αβ heterodimer TCR, so that the efficiency of displaying the correctly folded TCR on the surface of a phage is improved. The method can efficiently complete the screening of αβ heterodimer TCR mutants.