Receptors with heterologous transmembrane domain

Patent metadata

Field	Value
Publication number	US-2022348677-A1
Application number	US-202017763116-A
Country	US
Kind code	A1
Filing date	Sep 23, 2020
Priority date	Sep 24, 2019
Publication date	Nov 3, 2022
Grant date	—

Abstract

The present disclosure generally relates to, among other things, a new class of receptors engineered to modulate transcriptional regulation in a ligand-dependent manner. In particular, the new receptors contain a heterologous transmembrane domain comprising at least one γ-secretase site. 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.

First claim

What is claimed is:

1 . A chimeric polypeptide comprising, from N-terminus to C-terminus:
a) an extracellular binding domain having a binding affinity for a selected ligand; b) a linking sequence having:
(i) at least about 80% sequence identity to a Notch juxtamembrane domain (JMD);
(ii) at least about 80% sequence identity to a Notch JMD wherein the LIN-12-Notch repeat (LNR) and/or a heterodimerization domain (HD) of a Notch receptor has been deleted;
(iii) at least about 80% sequence identity to a polypeptide hinge domain;
(iv) at least about 80% sequence identity to a ROBO1 JMD including at least one fibronectin repeat; or
(v) a polypeptide having about 2 to about 40 amino acids;
c) a transmembrane domain having at least about 80% sequence identity to the transmembrane domain of a Type 1 transmembrane receptor and comprising one or more ligand-inducible proteolytic cleavage sites; and d) an intracellular domain comprising a transcriptional regulator, wherein binding of the selected ligand to the extracellular binding domain induces cleavage at the ligand-inducible proteolytic cleavage site between the transcriptional regulator and the linking sequence, and wherein
(i) when the linking sequence has at least about 80% sequence identity to a Notch JMD, or a Notch JMD LNR and/or an HD of a Notch receptor has been deleted, the TMD is heterologous to the linking sequence; and
(ii) when the linking sequence does not have at least about 80% sequence identity to a Notch JMD or a Notch JMD wherein the LNR and/or an HD of a Notch receptor has been deleted, the transmembrane domain is not a Notch1 transmembrane domain.

2 . The chimeric polypeptide of claim 1 , wherein the transmembrane domain further comprises a stop-transfer sequence (STS).

3 . The chimeric polypeptide of any one of claims 1 to 2 , wherein the TMD comprises a polypeptide sequence having at least 80% sequence identity to a transmembrane domain from a Type 1 transmembrane receptor and comprises a γ-secretase cleavage site.

4 . The chimeric polypeptide of any one of claims 1 to 3 , wherein the TMD comprises a polypeptide sequence having at least 90% sequence identity to a transmembrane domain from a Type 1 transmembrane receptor and comprises a γ-secretase cleavage site.

5 . The chimeric polypeptide of any one of claims 1 to 4 , wherein the TMD comprises a polypeptide sequence having at least 95% sequence identity to a transmembrane domain from a Type 1 transmembrane receptor and comprises a γ-secretase cleavage site.

6 . The chimeric polypeptide of any one of claims 1 to 5 , wherein the Type 1 transmembrane receptor is selected from the group consisting of CLSTN1, CLSTN2, APLP1, APLP2, LRP8, APP, BTC, TGBR3, SPN, CD44, CSF1R, CXCL16, CX3CL1, DCC, DLL1, DSG2, DAG1, CDH1, EPCAM, EPHA4, EPHB2, EFNB1, EFNB2, ErbB4, GHR, HLA-A, IFNAR2, IL1R1, IL1R2, IL6R, INSR, ERN1, ERN2, JAG2, KCNE1, KCNE2, KCNE3, KCNE4, KL, CHL1, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBO1, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DCT, VASN, FLT1, CDH5, PKHD1, NECTIN1, PCDHGC3, NRG1, LRP1B, CDH2, NRG2, PTPRK, SCN2B, Nradd, and PTPRM, and comprises a γ-secretase cleavage site.

7 . The chimeric polypeptide of any one of claims 1 to 6 , wherein the extracellular domain comprises an antigen-binding moiety capable of binding to a ligand on the surface of a cell.

8 . The chimeric polypeptide of any one of claims 1 to 6 , wherein the cell is a pathogen.

9 . The chimeric polypeptide of any one of claims 1 to 8 , wherein the ligand comprises a protein or a carbohydrate.

10 . The chimeric polypeptide of any one of claims 1 to 9 , wherein the ligand is selected from the group consisting of CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, and MAGE, alkaline phosphatase, placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), and signal regulatory protein α (SIRPα).

11 . The chimeric polypeptide of any one of claims 1 to 10 , wherein the ligand is selected from cell surface receptors, adhesion proteins, integrins, mucins, lectins, tumor-associated antigens, and tumor-specific antigens.

12 . The chimeric polypeptide of any one of claims 1 to 11 wherein the ligand is a tumor-associated antigen or a tumor-specific associated antigen.

13 . The chimeric polypeptide of any one of claims 1 to 12 , wherein the extracellular binding domain comprises the ligand-binding portion of a receptor.

14 . The chimeric polypeptide of any one of claims 7 to 13 , wherein the antigen-binding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, or a minibody, a F(ab′) 2 fragment, a Fab fragment, a single chain variable fragment (scFv), a single domain antibody (sdAb), or a functional fragment thereof.

15 . The chimeric polypeptide of claim 14 , wherein the antigen-binding moiety comprises an scFv.

16 . The chimeric polypeptide of any one of claims 7 to 15 , wherein the antigen-binding moiety is 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-11Ra, 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.

17 . The chimeric polypeptide of claim 16 , wherein the tumor-associated antigen is CD19, CEA, HER2, MUC1, CD20, BCMA, ALPPL2, or EGFR.

18 . The chimeric polypeptide of claim 17 , wherein the tumor-associated antigen is CD19.

19 . The chimeric polypeptide of any one of claims 1 to 18 , wherein the ligand-inducible proteolytic cleavage site is a γ-secretase cleavage site.

20 . The chimeric polypeptide of any one of claims 1 to 19 , wherein the transcriptional regulator comprises a transcriptional activator, or a transcriptional repressor.

21 . The chimeric polypeptide of any one of claims 1 to 20 , wherein the ICD comprises a nuclear localization sequence and a transcriptional regulator sequence selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP1-VP16.

22 . The chimeric polypeptide of any one of claims 1 to 21 , further comprising a signal sequence, a detectable label, a tumor-specific cleavage site, a disease-specific cleavage site, or a combination thereof.

23 . The chimeric polypeptide of any one of claims 2 to 22 , wherein the STS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 96, 135, 136, or 137.

24 . The chimeric polypeptide of any one of claims 1 to 23 , wherein the linking sequence comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOS: 97-99 a

Assignees

• Univ California

Inventors

• Roybal Kole T
• Liu Raymond
• Zhu Iowis

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• C12N2740/16043

  viral genome or elements thereof as genetic vector · CPC title

• C07K2319/71

  containing domain for transcriptional activaation, e.g. VP16 · CPC title

• C07K2319/50

  containing protease site · CPC title

• C07K14/4705

  stimulating, promoting or activating activity · CPC title