Notch receptors with hinge domain
Inventors
Roybal, Kole T. • Liu, Raymond • Zhu, Iowis
Assignees
University of California San Francisco UCSF • University of California San Diego UCSD
Publication Number
US-11202801-B2
Publication Date
2021-12-21
Expiration Date
2040-09-23
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Abstract
The present disclosure generally relates to, inter alia, a new class of receptors engineered to modulate transcriptional regulation in a ligand-dependent manner. Particularly, the new receptors, even though derived from Notch, do not require the Notch negative regulatory regions previously believed to be essential for the functioning of the receptors. In addition, the new receptors described herein incorporate an extracellular oligomerization domain to promote oligomer formation of the chimeric receptors. 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 such as cancers.
Core Innovation
The invention provides a new class of engineered chimeric receptors that modulate transcriptional regulation in a ligand-dependent manner. Specifically, these receptors, termed Hinge-Notch receptors, are derived from Notch yet do not require the Notch negative regulatory regions (NRR) previously thought essential for Notch receptor function. Instead, the Hinge-Notch receptors incorporate an extracellular oligomerization (hinge) domain, such as one derived from CD8α, that promotes the formation of oligomeric forms (e.g., dimers or trimers) through intermolecular disulfide bonding.
The chimeric receptors feature, from N-terminus to C-terminus: an extracellular ligand-binding domain with affinity to a selected ligand, a hinge domain capable of oligomer formation, a transmembrane domain with one or more ligand-inducible proteolytic cleavage sites, and an intracellular domain with a transcriptional regulator. Ligand binding triggers proteolytic cleavage at a site positioned between the transcriptional regulator and the hinge domain, resulting in release of the transcriptional regulator that modulates a custom transcriptional program in the cell.
This approach addresses limitations of previous engineered receptors (such as SynNotch), which required the large Notch negative regulatory region and were less efficient in expression and less modular. Notably, the new design, which omits the Notch NRR and enables a smaller genetic construct, facilitates use in vectors with limited cargo capacity and enables inclusion of additional genetic elements. The invention is intended to provide enhanced regulation of gene expression in engineered cellular therapies, overcoming problems of off-target activity and rigid sensitivity of prior systems.
Claims Coverage
The patent claims cover a chimeric polypeptide receptor class with specific structural and functional features, as well as recombinant nucleic acids and cells comprising such receptors. There is one independent claim (claim 1) in the provided claim set, from which dependent claims specify further embodiments.
Chimeric polypeptide receptor structure omitting Notch LNR/HD domains
A chimeric polypeptide comprising, in order from N-terminus to C-terminus: - An extracellular ligand-binding domain capable of binding a selected ligand. - A hinge domain from CD8α. - A transmembrane domain from a Notch receptor, including one or more ligand-inducible proteolytic cleavage sites. - An intracellular domain comprising a transcriptional regulator. Upon binding of the selected ligand to the extracellular ligand-binding domain, cleavage occurs at a ligand-inducible proteolytic cleavage site between the transcriptional regulator and the hinge domain, releasing the transcriptional regulator. The chimeric polypeptide does not comprise a LIN-12-Notch repeat (LNR) and a heterodimerization domain (HD) of a Notch receptor.
Recombinant nucleic acid encoding the chimeric polypeptide
A recombinant nucleic acid comprising a nucleotide sequence encoding the above-described chimeric polypeptide.
Recombinant cell expressing the chimeric polypeptide
A recombinant cell comprising the described chimeric polypeptide. The polypeptide can be expressed from a recombinant nucleic acid introduced into the cell. Embodiments cover mammalian cells, including immune cells, neurons, epithelial cells, endothelial cells, stem cells, and specifically, B cells, monocytes, natural killer cells, T cells (including CD4+ and CD8+ subsets), and related immune cells.
The claim coverage defines a class of engineered chimeric receptors constructed without Notch LNR/HD domains, their encoding nucleic acids, and host cells expressing them, enabling ligand-triggered transcriptional regulation without reliance on the canonical Notch extracellular regulatory structure.
Stated Advantages
The chimeric polypeptide receptors are smaller in size by omitting the Notch regulatory regions, enabling use of gene delivery vectors with limited capacity and facilitating inclusion of additional genetic elements.
The receptors exhibit easier and more efficient expression than conventional SynNotch receptors, allowing higher surface expression and improved transduction efficiency in primary human T cells.
The new class of receptors provides improved ligand-induced signaling with higher rates of induced signal and lower background ('noise') compared to existing SynNotch receptors.
The platform is modular, allowing rapid and versatile customization of receptor domains for engineering additional Notch receptors with distinct functions.
Hinge-Notch receptors are compatible with ligand-binding domains beyond single chain antibodies (scFvs), expanding the range of targetable diseases and tissues.
The system permits amplified activation under specific cellular and environmental contexts, enabling enhanced and tunable expression of therapeutic payloads by engineered cells.
The chimeric polypeptide receptors provide transcriptional regulation that is responsive to the degree of T cell activation, offering additional flexibility for modulating T cell responses.
Documented Applications
Use in modulating cell activities and gene expression in engineered cell therapies to address off-target activity, on-target off-tumor effects, and lack of controllability of conventional CAR-T approaches.
Treatment of various health conditions or diseases, such as cancers, including solid tumors, soft tissue tumors, metastatic lesions, blood cancers (leukemia, lymphoma, myeloma), and non-tumor cancers.
Engineering immune cells (such as T lymphocytes) to express the chimeric polypeptide receptors for enhanced discrimination and elimination of tumors.
Use in modulating cell signaling and activity for applications including control of autoimmunity and tissue regeneration.
Pharmaceutical compositions comprising recombinant nucleic acids or recombinant cells for therapeutic administration in individuals for modulation of target cell activity or treatment of disease.
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