Multi-chain CARs for ROR1 targeting via segmented assembly
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Solution Overview
Problem
Current chimeric antigen receptors (CARs) for cancer immunotherapy, particularly targeting ROR1, face challenges in optimizing CAR architecture and reducing adverse effects like cytokine-release syndrome, with variability in performance and specificity towards ROR1-positive cells.
Innovation Solution
Development of multi-chain CARs with specific scFv extracellular domains, derived from ROR1-specific antibodies, that assemble in a flexible architecture similar to natural receptors, comprising transmembrane polypeptides from FcεRI, with signal transducing and co-stimulatory domains, to enhance specificity and reduce background activation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If single-chain CAR architecture is used, then simplicity of structure is achieved, but signal transduction efficiency and specificity are insufficient
Solution Approach 1:
The CAR is divided into multiple separate polypeptide chains (alpha, beta, gamma) instead of a single chain. The alpha chain contains the extracellular ligand-binding domain, the beta chain contains the transmembrane domain and co-stimulatory domain, and the gamma chain contains the signal transducing domain. This segmentation allows each component to be optimized independently while assembling into a functional complex that enhances signal transduction efficiency and specificity.
2Measurement precision
If CAR architecture is optimized for specificity, then background activation is reduced, but manufacturing complexity increases
Solution Approach 1:
By separating the CAR into distinct chains with specific functions (ligand binding, co-stimulation, signal transduction), the architecture achieves high specificity through modular design. Each chain can be independently optimized for its specific function while the overall assembly maintains controlled activation thresholds, reducing background activation.
Solution Approach 2:
Different regions of the CAR complex have specialized functions optimized for their specific roles. The extracellular domain is optimized for antigen binding specificity, the transmembrane domain for stable positioning, the co-stimulatory domain for regulated activation, and the signal transducing domain for efficient signal propagation. This local optimization achieves high specificity without requiring uniform complexity throughout the entire structure.
3Reliability
If multi-chain CAR architecture is implemented, then signal transduction and specificity are improved, but manufacturing and assembly difficulty increase
Solution Approach 1:
The multi-chain architecture allows each polypeptide to be expressed separately from its own gene sequence, enabling independent optimization of expression levels and folding for each component. The separate chains can be manufactured independently and then assemble through their transmembrane and extracellular domains, facilitating production while maintaining the functional advantages of the multi-chain structure.
Solution Approach 2:
The multi-chain CAR components are designed to self-assemble into the functional complex through their inherent structural properties. The transmembrane domains and extracellular regions are configured to spontaneously associate in the correct orientation and stoichiometry within the cell membrane, reducing the need for complex post-manufacturing assembly procedures while ensuring proper functional configuration.
Data Source
AI summary
The present invention relates to a new generation of chimeric antigen receptors (CAR) referred to as multi-chain CARs, which are made specific to the antigen ROR1. Such CARs aim to redirect immune cell specificity and reactivity toward malignant cells expressing the tumor antigen ROR1. The alpha, beta and gamma polypeptides composing these CARs are designed to assemble in juxtamembrane position, which forms flexible architecture closer to natural receptors, that confers optimal signal transduction. The invention encompasses the polynucleotides, vectors encoding said multi-chain CAR and the isolated cells expressing them at their surface, in particularly for their use in immunotherapy. The invention opens the way to efficient adoptive immunotherapy strategies for treating cancer, especially chronic lymphocytic leukemia or solid tumors.