In Vitro Selection of High Affinity T Cell Receptors
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Solution Overview
Problem
Current methods for generating enhanced affinity T cell receptors (TCRs) face limitations in creating high-affinity TCRs specific for non-mutated tumor antigens due to constraints in CDR3 diversity and the risk of reducing specificity when enhancing affinity, particularly for tumor antigens over-expressed as self-proteins.
Innovation Solution
A method involving the differentiation of hematopoietic progenitor cells into DN TCRαβ+ thymocytes by contacting them with stromal cells expressing Delta-like-1 or Delta-like-4 and an MHC molecule, in the presence of a peptide antigen, to select and pair TCRα chains with enhanced affinity TCRβ chains, bypassing negative selection and increasing antigen-specific affinity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If site-directed mutagenesis is used to enhance TCR affinity, then affinity for target peptide/MHC is improved, but CDR3 diversity is limited and specificity may be reduced
Solution Approach 1:
The patent changes the approach from site-directed mutagenesis to in vitro selection, fundamentally altering the methodology parameter. This allows exploration of a vastly expanded sequence space including diverse CDR3 regions while maintaining affinity enhancement goals through positive selection with agonist peptide/MHC complexes
Solution Approach 2:
The system uses self-selection through in vitro thymic differentiation where TCRαβ+ progenitor cells automatically select and pair TCRα chains with complementary TCRβ chains based on their affinity for the peptide/MHC ligand. This self-service mechanism identifies high-affinity combinations without external manipulation, preserving both affinity and diversity
2Reliability
If mutations are introduced in CDR1 and CDR2 regions to enhance MHC contact, then affinity is improved, but overall specificity for cognate peptide decreases
Solution Approach 1:
Instead of directly mutating CDR regions to enhance affinity (conventional approach), the patent inverts the strategy by using in vitro selection with agonist peptide/MHC to indirectly identify and select TCRs with enhanced affinity and maintained specificity. The selection process itself guides which mutations or sequence variations are beneficial
Solution Approach 2:
The patent introduces an intermediary selection process using agonist peptide/MHC complexes during in vitro thymic differentiation. This intermediary mechanism filters and selects TCRs that achieve the desired balance between affinity enhancement and specificity maintenance, avoiding direct manipulation of CDR regions
3Reliability
If high affinity T cells are generated for tumor antigens, then antitumor effect is enhanced, but negative selection in thymus eliminates these T cells
Solution Approach 1:
The patent performs preliminary action by conducting in vitro thymic differentiation and TCR selection before in vivo transplantation. High-affinity TCRαβ+ progenitor cells are pre-selected and expanded ex vivo using agonist peptide/MHC stimulation, ensuring they will survive negative selection barriers and effectively target tumors upon transplantation
Solution Approach 2:
The patent uses an intermediary in vitro selection system that mimics thymic selection but is controllable and manipulatable. This intermediary environment allows enrichment of high-affinity TCRs that would normally be eliminated by negative selection in the natural thymus, bridging the gap between affinity requirements and survival requirements
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 generates TCRs with up to 10-fold higher affinity for tumor antigens while maintaining specificity, potentially overcoming the limitations of existing methods in TCR gene therapy for cancer treatment.
Implementation Method 1
contacting hematopoietic progenitor cells with stromal cells expressing Delta-like-1 or Delta-like-4
Implementation Method 2
select and pair TCRα chains with enhanced affinity TCRβ chains, bypassing negative selection and increasing antigen-specific affinity
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 2
AI summary
The present disclosure provides methods for generating enhanced affinity T cell receptors by agonist selection of hematopoietic progenitor cells expressing an antigen specific TCRα cultured with stromal cells expressing Delta-like-1 or Delta-like-4, compositions prepared from such methods, and uses of thereof.