Humanized notch receptors with hinge domain
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.
1 . A chimeric polypeptide comprising, from N-terminus to C-terminus:
a) an extracellular ligand-binding domain having a binding affinity for a ligand;
b) a hinge domain capable of promoting oligomer formation of the chimeric polypeptide via intermolecular disulfide bonding;
c) a transmembrane domain comprising one or more ligand-inducible proteolytic cleavage sites; and
d) an intracellular domain comprising a human or humanized transcriptional effector,
wherein the transcriptional effector comprises: (i) a DNA-binding domain and (ii) an effector domain through which the transcriptional effector exerts its effect, wherein
(i) the DNA-binding domain comprises the amino acid sequence of SEQ ID NO: 52; and
(ii) the effector domain comprises the amino acid sequence of SEQ ID NO: 54;
wherein binding of the ligand 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,
and wherein the chimeric polypeptide does not comprise a LIN-12-Notch repeat (LNR) and/or a heterodimerization domain (HD) of a Notch receptor.
2 . The chimeric polypeptide of claim 1 , wherein the transmembrane domain further comprises a stop-transfer-sequence.
3 . The chimeric polypeptide of claim 1 , wherein the extracellular ligand-binding domain comprises an antigen-binding moiety capable of binding to a ligand on the surface of a cell, wherein the cell is a pathogen, a human cell, or a human tumor cell.
4 . The chimeric polypeptide of claim 1 , wherein the ligand:
(i) comprises a protein or a carbohydrate;
(ii) is selected from cell surface receptors, adhesion proteins, integrins, mucins, lectins, tumor associated antigens, and tumor-specific antigens; and/or
(iii) is a tumor-associated antigen or a tumor-specific antigen.
5 . The chimeric polypeptide of claim 1 , wherein the extracellular ligand-binding domain comprises the ligand-binding portion of a receptor.
6 . The chimeric polypeptide of claim 3 , wherein the antigen-binding moiety:
(i) is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, a minibody, an F(ab′)2 fragment, an F(ab) v fragment, a single chain variable fragment (scFv), a single domain antibody (sdAb), and a functional fragment thereof; and/or
(ii) specifically binds to a tumor-associated antigen selected from the group consisting of CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRVIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2/neu), IGLL1, IL-11Rα, KIT (CD117), MUC1, NCAM, PAP, PDGFR-β, PRSS21, PSCA, PSMA, ROR1, SIRPα, SSEA-4, TAG72, TEM1/CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Axl.
7 . The chimeric polypeptide of claim 1 , wherein the one or more ligand-inducible proteolytic cleavage sites comprises a γ-secretase cleavage site.
8 . The chimeric polypeptide of claim 1 , wherein the hinge domain is derived from a CD8α hinge domain or a functional variant of any thereof comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 15.
9 . The chimeric polypeptide of claim 2 , wherein the stop-transfer-sequence comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOS: 31-51.
10 . The chimeric polypeptide of claim 1 , wherein the transmembrane domain comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOS: 21-30.
11 . The chimeric polypeptide of claim 1 , wherein the domains recited in (a) through (d) are derived from human polypeptides and are substantially non-immunogenic in a human subject.
12 . A recombinant nucleic acid comprising a nucleotide sequence encoding a chimeric polypeptide according claim 1 .
13 . A recombinant cell comprising:
a) a chimeric polypeptide according to claim 1 ; and/or
b) a recombinant nucleic acid comprising a nucleotide sequence encoding the chimeric polypeptide of (a).
14 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more of the following:
a) a chimeric polypeptide according to claim 1 ;
b) a recombinant nucleic acid comprising a nucleotide sequence encoding the chimeric polypeptide of (a); and
c) a recombinant cell comprising the chimeric polypeptide of (a) and/or the recombinant nucleic acid of (b).
15 . A method for making a recombinant cell, the method comprising:
a) providing a cell capable of protein expression; and
b) contacting the provided cell with a recombinant nucleic acid of claim 12 .
16 . A method of modulating an activity of a cell, inhibiting a target cancer cell, or treating a health condition in a subject in need thereof, the method comprising administering:
a) a chimeric polypeptide according to claim 1 ;
b) a recombinant nucleic acid comprising a nucleotide sequence encoding the chimeric polypeptide of (a);
c) a recombinant cell comprising the chimeric polypeptide of (a) and/or the recombinant nucleic acid of (b); or
d) a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more of the following:
(i) the chimeric polypeptide of (a),
(ii) the recombinant nucleic acid of (b), and
(iii) the recombinant cell of (c).
17 . The chimeric polypeptide of claim 8 , wherein the hinge domain comprises the amino acid sequence of SEQ ID NO: 15.
18 . The chimeric polypeptide of claim 9 , wherein the stop-transfer-sequence comprises the amino acid sequence of SEQ ID NO: 34.
19 . The chimeric polypeptide of claim 10 , wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 21.
20 . A chimeric polypeptide comprising, from N-terminus to C-terminus:
a) an extracellular ligand-binding domain having a binding affinity for a ligand;
b) a hinge domain comprising the amino acid sequence of SEQ ID NO: 15;
c) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 21 and a stop-transfer-sequence comprising the amino acid sequence of any one of SEQ ID NO: 34; and
d) an intracellular domain comprising a human or humanized transcriptional effector comprising (i) a DNA-binding domain comprising the amino acid sequence of SEQ ID NO: 52, N-terminally linked to (ii) an effector domain comprising the amino acid sequence of SEQ ID NO: 54,
wherein binding of the ligand 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,
wherein the chimeric polypeptide does not comprise a LIN-12-Notch repeat (LNR) and/or a heterodimerization domain (HD) of a Notch receptor.
21 . The chimeric polypeptide of claim 1 , wherein the chimeric polypeptide does not comprise a LIN-12-Notch repeat (LNR) nor a heterodimerization domain (HD) of a Notch receptor.