MULTIPARTITE SIGNALING PROTEINS AND USES THEREOF
The present disclosure relates to compositions and methods for using cells having chemically-induced fusion protein complexes to spatially and temporally control immune cell signal initiation and downstream responses for treating disease.
1 - 158 . (canceled)
159 . A polynucleotide encoding from 5′ to 3′:
(a) a first polypeptide comprising a first secretion signal, a first multimerization domain, a first transmembrane domain, and an actuator domain;
(b) a second polypeptide comprising a second secretion signal; a binding agent; a second multimerization domain; and a second transmembrane domain.
160 . The polynucleotide of claim 159 , further comprising a viral self-cleaving polypeptide.
161 . The polynucleotide of claim 159 , wherein the viral self-cleaving polypeptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a Theilovirus 2A peptide, an encephalomyocarditis virus 2A peptide, an aphthovirus 2A peptide, a potyvirus 2A peptide, and a cardiovirus 2A peptide.
162 . The polynucleotide of claim 159 , wherein the first and second secretion signals are the same or different.
163 . The polynucleotide of claim 159 , wherein the first and second multimerization domains are the same or different.
164 . The polynucleotide of claim 159 , wherein the multimerization domains of the first and second polypeptides associate with a bridging factor selected from the group consisting of: rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506/cyclosporin A (FKCsA) or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof, AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
165 . The polynucleotide of claim 164 , wherein the first and second multimerization domains are a pair selected from the group consisting of: FKBP and FKBP12-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin, and PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
166 . The polynucleotide of claim 159 , wherein the first multimerization domain comprises a first FKBP polypeptide or variant thereof, and the second multimerization domain comprises a first FRB polypeptide or variant thereof.
167 . The polynucleotide of claim 159 , wherein the first multimerization domain comprises a first FRB polypeptide or variant thereof, and the second multimerization domain comprises a first FKBP polypeptide or variant thereof.
168 . The polynucleotide of claim 159 , wherein the first transmembrane domain is a CD4, CD8 or CD28 transmembrane domain.
169 . The polynucleotide of claim 159 , wherein the second transmembrane domain is a CD4, CD8 or CD28 transmembrane domain.
170 . The polynucleotide of claim 159 , wherein the actuator domain comprises one or a plurality of immunoreceptor tyrosine-based activation motifs (ITAMs).
171 . The polynucleotide of claim 159 , wherein the actuator domain comprises a lymphocyte receptor signaling domain of a polypeptide selected from the group consisting of: CD3ε, CD3δ, CD3ζ, pre-T cell receptor α (pTα), T cell receptor α (TCRα), T cell receptor β (TCRβ), Fc receptor α (FcR α), Fc receptor β (FcR β), Fc receptor γ (FcRγ), natural-killer group 2, member D (NKG2D), CD79A, CD79B, and any combination thereof.
172 . The polynucleotide of claim 159 , wherein the first fusion protein further comprises a different actuator domain, a costimulatory domain, an adhesion factor, or any combination thereof.
173 . The polynucleotide of claim 172 , wherein the costimulatory domain is from a polypeptide selected from the group consisting of: CD27, CD28, CD30, CD40, linker for activation of T-cells (LAT), zeta-chain-associated protein kinase 70 (Zap70), inducible T-cell costimulator (ICOS), DNAX-activation protein 10 (DAP10), 4-1BB, caspase recruitment domain family member 11 (CARD11), herpesvirus entry mediator (HVEM), lymphocyte activating gene 3 (LAG3), signaling lymphocytic activation molecule family member 1 (SLAMF1), lymphocyte-specific protein tyrosine kinase (Lck), fyn oncogene related to src, fgr, yes (Fyn), SH2 domain-containing leukocyte protein of 76 KDa (Slp76), OX40, and any combination thereof.
174 . The polynucleotide of claim 159 , wherein the actuator domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein that is a lymphocyte receptor or signaling domain thereof, a protein comprising a plurality of immunoreceptor tyrosine-based activation motifs (ITAMs), a costimulatory domain, an adhesion factor, or any combination thereof.
175 . The polynucleotide of claim 159 , further encoding a third exogenous polynucleotide encoding a costimulatory factor, an immunomodulatory factor, an agonist for a costimulatory factor, an agonist for an immunomodulatory factor, or any combination thereof.
176 . The polynucleotide of claim 159 , wherein the binding agent specifically binds to a target on a target cell surface.
177 . The polynucleotide of claim 176 , wherein the binding agent comprises an antibody, a single chain variable fragment (scFv), an antigen binding fragment (Fab), a receptor ectodomain, or a ligand.
178 . The polynucleotide of claim 159 , wherein the second polypeptide further comprises a sequence encoding a linker disposed between the binding agent and the second multimerization domain.
179 . The polynucleotide of claim 176 , wherein the domain specifically binds to a target selected from the group consisting of: α-folate receptor, αvβ6 integrin, B cell maturation antigen (BCMA), B7 homolog 3 (B7-H3), B7 homolog 6 (B7-H6), Carbonic anhydrase IX (CAIX), CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, carcinoembryonic antigen (CEA), delta like canonical Notch ligand 4 (DLL4), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor receptor (EGFR), EGFR family including ErbB2 (HER2), EGFR variant III (EGFRvIII), epithelial cellular adhesion molecule (EPCAM), ephrin type-A receptor 2 (EphA2), fibroblast activation protein alpha (FAP), fetal acetylcholine receptor, frizzled class receptor 7 (Fzd7), ganglioside GD2 (GD2), ganglioside GD3 (GD3), Glypican-3 (GPC3), human oncofetal antigen 5T4 (h5T4), interleukin 11 receptor alpha (IL-11R□), interleukin 13 receptor alpha 2 (IL13R-α2), κ light chain, λ light chain, Lewis Y antigen (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen gene family member A1 (MAGE-A1), mesothelin, major histocompatibility complex (MHC) presented peptides, mucin 1, cell surface associated (MUC1), mucin 16, cell surface associated (MUC16), neural cell adhesion molecule (NCAM), NKG2D ligands, Notch1, Notch2/3, New York esophageal squamous cell carcinoma 1 (NY-ESO-1), preferentially expressed antigen of melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), Survivin, tumor associate glycoprotein 72 (TAG-72), telomerase reverse transcriptase (TERT), vascular endothelial growth factor receptor 2 (VEGFR2), and receptor tyrosine kinase-like orphan receptor 1 (ROR1).
180 . The polynucleotide of claim 160 , wherein the first polypeptide comprises the first secretion signal, a FRB T2098L multimerization domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ actuator domain; wherein the viral self-cleaving polypeptide comprises a viral 2A sequence; and wherein the second polypeptide comprises a binding agent, an FKBP12 multimerization domain, and a CD4 transmembrane domain.
181 . The polynucleotide of claim 180 , wherein the binding agent comprises an anti-CD19 scFv domain or an anti-BCMA scFv domain.
182 . The polynucleotide of claim 159 , wherein the second polypeptide further comprises an anchor domain.
183 . The polynucleotide of claim 182 , wherein the anchor domain is a transmembrane domain or a GPI signal sequence.
184 . A vector comprising the polynucleotide of claim 159 .
185 . The vector of claim 184 , wherein the vector is a viral vector, a retroviral vector, or a lentiviral vector.
186 . A non-natural cell comprising the vector of claim 184 .