Ert2 mutants, inducible cell death systems, and uses thereof

An inducible cell death system comprising a first polypeptide and a second polypeptide monomer, wherein the first and the second polypeptide monomers each comprise a ligand binding domain and a cell death inducing domain, wherein the first and the second polypeptide monomers are configured to oligomerize upon contact with a ligand of the ligand binding domain, thereby generating a cell-death inducing signal in a cell in which the first and the second polypeptide monomers are expressed, and wherein

• the ligand binding domain comprises a modified estrogen receptor ligand binding domain (ER-LBD) comprising an amino acid sequence corresponding to a hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO:
• 1), wherein the modified ER-LBD comprises:
• (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and
• (b) additional amino acid substitutions, wherein the additional amino acid substitutions comprise, with reference to SEQ ID NO:
• 1:
• (i) an L384M substitution, an L391V substitution, a N413D substitution, an M421L substitution, a S463P substitution, and a H524L substitution,
• (ii) an L391V substitution, a N413D substitution, a Q414E substitution, a S463P substitution, and a H524F substitution,
• (iii) an L354I substitution, a L391V substitution, a N413D substitution, a Q414E substitution, a M421L substitution, a M517A substitution, and a H524F substitution, or
• (iv) an L354I substitution, a L391V substitution, a L409V substitution, a N413D substitution, a Q414E substitution, and a H524L substitution.

A modified estrogen receptor ligand binding domain (ER-LBD) corresponding to a hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO:

• 1), wherein:
• a. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an N413D substitution, an S463P substitution, an L354I substitution, an L384M substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• b. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an N413D substitution, an S463P substitution, an M421L substitution, an L354I substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• c. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an L409V substitution, an N413D substitution, an S463P substitution, an M421L substitution, an L384M substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• d. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an L391V substitution, an L409V substitution, an Q414E substitution, an N413D substitution, an S463P substitution, an M517A substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• e. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an L391V substitution, an L409V substitution, an N413D substitution, an S463P substitution, an M517A substitution, an M421L substitution, an L354I substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• f. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an L391V substitution, an Q414E substitution, an N413D substitution, an S463P substitution, an M421L substitution, an L354I substitution, an L384M substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• g. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an L391V substitution, an L409V substitution, an N413D substitution, an S463P substitution, an M517A substitution, an M421L substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• h. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an L391V substitution, an L409V substitution, an Q414E substitution, an N413D substitution, an S463P substitution, an L354I substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• i. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an L391V substitution, an L409V substitution, an N413D substitution, an S463P substitution, an M421L substitution, an L354I substitution, an L384M substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• j. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an L391V substitution, an Q414E substitution, an N413D substitution, an S463P substitution, an M517A substitution, an M421L substitution, an L354I substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• k. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an L391V substitution, an N413D substitution, an S463P substitution, an M517A substitution, an L384M substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• l. the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:
• 1;
• and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions comprise:
• an L391V substitution, an L409V substitution, an N413D substitution, an S463P substitution, an M517A substitution, and an H524L substitution, with reference to SEQ ID NO:
• 1;
• or
• m. the modified ER-LBD comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to any one of SEQ ID NO:
• 90, SEQ ID NO:
• 91, SEQ ID NO:
• 92, SEQ ID NO:
• 93, SEQ ID NO:
• 94, SEQ ID NO:
• 95, SEQ ID NO:
• 96, SEQ ID NO:
• 97, SEQ ID NO:
• 98, SEQ ID NO:
• 99, SEQ ID NO:
• 100, SEQ ID NO:
• 101, SEQ ID NO:
• 103, SEQ ID NO:
• 104, SEQ ID NO:
• 105, SEQ ID NO:
• 106, SEQ ID NO:
• 107, SEQ ID NO:
• 108, SEQ ID NO:
• 109, SEQ ID NO:
• 110, SEQ ID NO:
• 111, SEQ ID NO:
• 112, SEQ ID NO:
• 113, and SEQ ID NO:
• 114,
• optionally wherein the modified ER-LBD has greater sensitivity and/or selectivity to a non-endogenous ligand as compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:
• 2, or as compared to an endogenous ligand as a result of the one or more additional amino acid substitutions, optionally wherein the non-endogenous ligand is selected from the group consisting of:
• 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, tamoxifen, and endoxifen, optionally wherein the endogenous ligand is estradiol, optionally wherein the modified ER-LBD further comprises a V595A amino acid substitution.