Humanized notch receptors with hinge domain

Inventors

Roybal, Kole T. • Liu, Raymond

Assignees

University of California San Diego UCSD

Abstract

The present disclosure generally relates to, inter alia, a new class of chimeric Notch receptors containing a fully humanized transcriptional effector, engineered to modulate gene expression and cellular activities in a ligand-dependent manner. The new chimeric Notch receptors surprisingly retain the ability to transduce signals in response to ligand binding despite that the Notch extracellular subunit (NEC), which includes the negative regulatory region (NRR) previously believed to be essential for the functioning of Notch receptors is completely absent. In addition, the new receptors described herein incorporate an extracellular oligomerization domain to promote oligomer formation of the chimeric receptors. Also provided are compositions and methods useful for producing such receptors, nucleic acids encoding same, engineered 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 diseases such as cancers.

Core Innovation

The invention provides embodiments in which a heterologous gene product is secreted, cell-surface, intracellular, or multiple products are co-modulated in connection with an HingeNotch/synthetic transcriptional effector system. The disclosed chimeric polypeptide comprises an extracellular ligand-binding domain, a hinge domain that promotes oligomer formation via intermolecular disulfide bonding, a transmembrane domain comprising one or more ligand-inducible proteolytic cleavage sites, and an intracellular domain comprising a human or humanized transcriptional effector with a DNA-binding domain and an effector domain.

Ligand binding to the extracellular ligand-binding domain results in cleavage at the one or more ligand-inducible proteolytic cleavage sites between the transcriptional effector and the hinge domain. This cleavage separates the transcriptional effector from the hinge region and enables the effector domain to exert its effect. The chimeric polypeptide is engineered not to comprise a LIN-12-Notch repeat (LNR) and/or a heterodimerization domain (HD) of a Notch receptor.

The disclosed systems are described for transcriptional outputs linked to immune and stem cell differentiation modulation, including IFNγ and IL-2, and for receptor activity in immune cells such as T cells. Embodiments report improved expression compared with first-generation SynNotch receptors, and the systems are described as exhibiting switch-like behavior in which transcription responds to T cell activation independently of ligand binding.

Claims Coverage

The consolidated independent claims cover two main embodiments of a ligand-binding, hinge-mediated, proteolysis-triggered transcriptional effector chimeric polypeptide. Across the claims, four inventive feature sets are explicitly recited: the defined chimeric polypeptide architecture, the transcriptional effector sequence composition, the ligand-inducible cleavage arrangement producing separation from the hinge, and the exclusion of LIN-12-Notch repeat and/or Notch heterodimerization domain.

Overall, the independent claims define a chimeric polypeptide in which ligand binding triggers cleavage between an intracellular transcriptional effector and a hinge domain, where the transcriptional effector contains a SEQ ID NO: 52 DNA-binding domain and a SEQ ID NO: 54 effector domain, hinge/transmembrane are specified by SEQ IDs, and the polypeptide excludes LIN-12-Notch repeat (LNR) and/or Notch heterodimerization domain (HD).

Stated Advantages

Documented Applications