Humanized SynNotch Receptors With Augmented Transactivation Domains
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Solution Overview
Problem
First-generation SynNotch receptors have inefficiencies in expression and weak transcriptional activation capabilities, limiting their therapeutic potential due to the large size of the Notch negative regulatory region and reliance on standard proteolysis, which affects their ability to induce effective cell signaling and gene expression.
Innovation Solution
Development of chimeric antigen receptors (SynNotch receptors) with an intracellular domain comprising two or more human or humanized transcriptional regulators, optionally incorporating a CD3ζ activation domain, to enhance transcriptional activation and signaling, and lacking the LIN-12-Notch repeat and heterodimerization domain.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the Notch negative regulatory region (NRR) is included in SynNotch receptors, then the receptor can perform standard two-step proteolysis, but the receptor size increases making expression less efficient and exceeding vector capacity
Solution Approach 1:
The patent extracts and removes the Notch negative regulatory region (NRR) from the SynNotch receptor construct. By taking out this large domain that spans approximately 160 amino acids, the invention reduces receptor size and improves expression efficiency while maintaining the essential ligand-induced proteolysis function through alternative design elements.
Solution Approach 2:
The patent segments the SynNotch receptor into functional modules, separating the proteolysis function from the NRR. The receptor is designed with distinct domains including the extracellular ligand-binding domain, transmembrane domain, and intracellular domain, with the proteolysis function achieved through engineered cleavage sites rather than the full NRR structure.
2Reliability
If first-generation SynNotch receptors use standard two-step proteolysis, then the receptor can be activated by ligand binding, but the transcriptional activation capacity remains weak and insufficient for therapeutic effect
Solution Approach 1:
The patent merges multiple transcriptional activation domains into the SynNotch intracellular domain to create a composite activation module. By combining TADs from different sources (such as VP64, p65, Rta, Vp16) with the Notch intracellular domain, the invention achieves synergistic enhancement of transcriptional activation capacity while maintaining ligand-induced activation through the preserved proteolysis mechanism.
Solution Approach 2:
The patent creates a composite intracellular domain by fusing multiple transcriptional activation domains with the Notch intracellular domain. This composite structure integrates the ligand-induced release mechanism of Notch with the potent transcriptional activation capabilities of heterologous TADs, producing a receptor with both reliable activation and strong transcriptional output.
Data Source
AI summary
The present disclosure relates generally to chimeric antigen receptors (e.g., the synthetic Notch receptors disclosed herein) that contain two or more human or humanized transcriptional regulators to augment their function to regulate cell transcription. The disclosure also provides compositions and methods useful for producing such receptors, nucleic acids encoding same, host cells genetically modified with the nucleic acids, as well as methods for modulating an activity of a cell and/or for the treatment of various health conditions or diseases, such as cancers.