Intein-Based CAR Sorting for Modular Polypeptide Assembly
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Solution Overview
Problem
Existing cell-based immunotherapies face challenges in stably integrating large genetic constructs into T cells, leading to low transduction efficiency and gene expression, which limits the effectiveness of cancer treatment with minimal toxicity.
Innovation Solution
Utilizing orthogonal split-intein tags for post-translational trans-splicing of polypeptide subunits to generate functional chimeric antigen receptors (CARs) and other transmembrane constructs, combined with ER retention motifs and drug-regulated degradation systems, enabling selective enrichment and surface expression of CARs through magnetic bead-based sorting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If large recombinant constructs are delivered and integrated into T cells to target multiple antigens, then cancer eradication potency is improved, but transduction efficiency and gene expression decrease
Solution Approach 1:
The patent divides large recombinant constructs into smaller modular components (e.g., separate CAR constructs targeting different antigens). Each module can be independently transduced into T cells using viral vectors, avoiding the packaging size limits that restrict single large construct delivery. After individual integration, the modular constructs are brought together through protein-protein interactions or trans-splicing mechanisms to form functional multi-antigen targeted receptors on the T cell surface.
Solution Approach 2:
The patent employs preliminary transduction of T cells with multiple separate viral vectors, each carrying a portion of the multi-antigen targeting construct. This preliminary action allows efficient integration of smaller genetic elements before the final functional assembly occurs through post-translational mechanisms such as intein-mediated trans-splicing or affinity-based complex formation, thereby achieving high transduction efficiency while maintaining the ability to target multiple antigens.
2Quantity of substance
If viral vector insert size approaches packaging limit, then more genetic information can be transferred, but viral titer decreases
Solution Approach 1:
The patent segments the total genetic information required for multi-antigen targeting into multiple smaller viral vector inserts, each well below the packaging limit. This allows each vector to be produced at high titer while collectively delivering the complete multi-antigen targeting capability. The segmented genetic elements are designed to function cooperatively once inside the T cell.
Solution Approach 2:
The patent designs viral vectors with universal modular elements (promoters, polyA signals, selection markers) that can accommodate different antigen-targeting coding sequences. This multi-functionality allows the same vector backbone to be used for delivering multiple different antigen-specific CAR modules, maximizing genetic information transfer efficiency while maintaining high viral titer through standardized production protocols.
3Productivity
If multiple viral vectors are used to deliver modular subunits, then transduction efficiency improves, but system complexity increases
Solution Approach 1:
The patent merges the functions of multiple viral vectors through the use of complementary modular subunits that self-assemble or interact through designed protein-protein interaction domains. The complexity of delivering multiple vectors is compensated by the simplicity of the interaction mechanism that brings them together functionally, creating a unified multi-antigen targeting system from separate delivery vehicles.
Solution Approach 2:
The patent employs intermediary elements such as intein tags, affinity tags, or protein interaction domains that mediate the assembly of separately transduced modular subunits. These intermediaries simplify the overall system by providing a standardized interface mechanism that automatically brings together the correct subunits after independent transduction, reducing the complexity of coordinating multiple vector deliveries.
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
Enhances the efficiency of genetic information transfer and surface expression of CARs, allowing for potent cancer eradication with minimal toxicity and off-target activity, and enables selective isolation of cells with correct trans-splicing.
Implementation Method 1
orthogonal split-intein tags are used to post-translationally trans-splice polypeptide subunits to generate functional, mature CARs or other transmembrane constructs
Implementation Method 2
magnetic bead-based sorting (MACS) targeting a specific affinity tag enables selective enrichment of cells that have correctly trans-spliced the mature affinity-tagged polypeptide product
Implementation Method 3
the ER retention motif retains the transmembrane domain containing construct inside the cell in the non-spliced state
Data Source
AI summary
The present application is directed to multiplex intein-based methods and compositions for the generation engineered cells expressing modular polypeptides, for example CARs and CCRs.