Antigen-Specific TCR Generation via Fusion Protein Targeting
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Solution Overview
Problem
Current methods for adoptive T cell therapy face challenges in generating antigen-specific T lymphocytes with high avidity and specificity for tumor antigens, as tumors often have poor immunogenicity and negative T cell selection mechanisms delete auto-antigen-specific T cells, limiting the effectiveness of T cell isolation and expansion for cancer treatment.
Innovation Solution
The method involves expressing a fusion protein comprising an antigen or antigen fragment with an endoplasmic reticulum translocation signal and an endosomal/lysosomal targeting sequence in antigen-presenting cells to efficiently load MHC class II and class I complexes, allowing for the generation of CD4 and CD8 TCRs, and using in vitro RNA-coded proteins to activate and isolate antigen-specific T lymphocytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional T cell isolation methods are used, then T cells can be obtained from patient blood or tumor resections, but the T cells have poor immunogenicity and low avidity for tumor-specific antigens due to negative T cell selection mechanisms
Solution Approach 1:
The patent applies preliminary action by pre-loading antigen-presenting cells with RNA-coded tumor antigens using fusion proteins containing ER-translocation and endosomal/lysosomal targeting sequences before T cell exposure. This pre-preparation ensures optimal antigen presentation capability is established in advance, enabling subsequent high-efficiency activation and expansion of antigen-specific T cells with high avidity
Solution Approach 2:
The patent uses fusion proteins as intermediaries that combine tumor antigens with specific targeting sequences (ER-translocation signal and endosomal/lysosomal targeting sequence). These fusion proteins act as mediators to deliver antigens to the correct cellular compartments for MHC class I and II presentation, thereby enabling effective T cell activation while maintaining high specificity and avidity
2Productivity
If T cells are expanded rapidly to large numbers for adoptive cell therapy, then sufficient therapeutic doses can be obtained, but the risk of attacking normal tissues increases
Solution Approach 1:
The patent applies local quality by using fusion proteins that target antigens to specific cellular compartments (endoplasmic reticulum and endosomes/lysosomes) through specialized sequences. This localized delivery ensures antigens are presented in the correct context to activate only the desired antigen-specific T cell clones with high precision, enabling rapid expansion while maintaining specificity and reducing off-target effects
Solution Approach 2:
The patent performs preliminary action by pre-selecting and activating only antigen-specific T cell clones through controlled exposure to antigen-presenting cells loaded with specific tumor antigens. This pre-selection process ensures that only T cells with the correct specificity are expanded, allowing rapid proliferation while minimizing the risk of attacking normal tissues
3Reliability
If MHC class II presentation is used to activate CD4+ T helper cells, then tumor immunogenicity can be enhanced, but the complexity of achieving both MHC class I and II presentation increases
Solution Approach 1:
The patent applies universality by designing a dual-targeting fusion protein system that can deliver the same antigen to both MHC class I and MHC class II presentation pathways. The fusion protein contains both an ER-translocation signal (for MHC class I) and an endosomal/lysosomal targeting sequence (for MHC class II), enabling a single construct to activate both CD8+ cytotoxic T cells and CD4+ helper T cells, thereby enhancing tumor immunogenicity while simplifying the overall system
Solution Approach 2:
The patent merges multiple functional elements into a single fusion protein construct: the tumor antigen, the ER-translocation signal sequence, and the endosomal/lysosomal targeting sequence are combined into one molecule. This merging allows simultaneous engagement of both MHC class I and MHC class II presentation pathways, enhancing immunogenicity while reducing the complexity of using separate systems
Data Source
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AI summary
The present invention contemplates methods for the generation of human antigen-specific T lymphocytes. The methods employ MHC class-II targeting signals fused to an antigen or fragment thereof to obtain MHC class presentation of RNA coded proteins. Accordingly, the present invention concerns expression vectors comprising MHC class-II targeting signal and at least one antigen or fragment thereof and its use for the in vitro generation of antigen-specific T lymphocytes. T cell clones and T cell receptors (TCRs) specific for tumor antigens or viral antigens are also described.