Engineered TCRs for CMV pp65 Peptide Recognition
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Solution Overview
Problem
Current T-cell receptors (TCRs) face challenges in terms of weak binding affinity and low expression levels, making them less effective as therapeutics for targeting cytomegalovirus (CMV)-infected cells, particularly due to difficulties in engineering and expressing stable, high-affinity TCRs that can recognize CMV-specific peptide-MHC complexes.
Innovation Solution
Engineered TCRs with specific amino acid sequences for the alpha and beta chains, optimized for high affinity and stability, are developed, including fusion with antibody Fc domains and modifications to enhance expression and binding to CMV-specific peptide-MHC complexes, such as NLVPMVATV in complex with HLA-A2*02:01, and incorporation of disulfide bonds for improved stability and expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional TCRs are used, then they can recognize peptide-MHC complexes, but they exhibit weak binding affinity and low expression levels
Solution Approach 1:
The patent applies parameter changes by systematically mutating amino acid residues in the CDR3 loops of the TCR to optimize binding affinity. Specific substitutions (e.g., L100Q, N101E in CDR3α; Y100L, G101S in CDR3β) were introduced to enhance peptide-MHC complex recognition while maintaining expression levels
Solution Approach 2:
The patent creates composite TCR structures by combining engineered variable domains with optimized constant domains. The chimeric TCR constructs integrate human and murine domain elements (e.g., hVα-mCα, hVβ-mCβ) to achieve both high affinity and stable expression
2Strength
If TCR affinity is increased through engineering, then binding strength improves, but structural stability and expression may be compromised
Solution Approach 1:
The patent applies local quality by making targeted amino acid substitutions specifically in the CDR3 loops where peptide contact occurs, while leaving the framework regions and constant domains unchanged to maintain overall structural stability. This localized engineering approach enhances binding without compromising global protein folding
Solution Approach 2:
The patent incorporates disulfide bonds and optimized framework structures in advance to prevent aggregation and misfolding that might result from high-affinity CDR3 mutations. These pre-built structural safeguards ensure proper folding and stability of the engineered high-affinity TCRs
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 engineered TCRs demonstrate significantly increased affinity for CMV-specific peptide-MHC complexes, achieving nanomolar binding levels and high-level production, enabling effective detection and targeting of CMV-infected cells, with potential applications in therapeutic and diagnostic contexts.
Implementation Method 1
Engineered TCRs with specific amino acid sequences for the alpha and beta chains, optimized for high affinity and stability, are developed, including fusion with antibody Fc domains and modifications to enhance expression and binding to CMV-specific peptide-MHC complexes
Data Source
AI summary
Provided herein are engineered T-cell receptors (TCRs) having nanomolar affinity for the immuno-dominant pp65 peptide residing between residues 495-503 (NLV) in complex with HLA-A2*02:01. The TCRs may be membrane-hound TCRs, soluble TCRs, chimeric TCRs, or chimeric antigen receptors. Also provided are methods of using the engineered TCRs to treat diseases, monitor disease progression, monitor vaccine efficacy, and detecting NLV/A2 presentation on the surface of cells.