Protein payload release
Described herein are chimeric proteins, specifically membrane-cleavable chimeric systems. Also described herein are nucleic acids, cells, and methods directed to the same.
1 . A membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:
S-C-MT or MT-C-S
wherein
S comprises a secretable effector molecule,
C comprises a protease cleavage site, and
MT comprises a cell-membrane tethering domain,
wherein S-C-MT or MT-C-S is configured to be expressed as a single polypeptide, and
wherein the protease cleavage site comprises
the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181); or
the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182); or
the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183); or
the amino acid sequence of PRAESSKGG (SEQ ID NO: 184); or
the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185); or
the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187); or
the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188); or
the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189); or
the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190); or
the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191).
2 . The membrane-cleavable chimeric protein of claim 1 , wherein the secretable effector molecule comprises a signal peptide or signal-anchor sequence.
3 . The membrane-cleavable chimeric protein of claim 1 , wherein the secretable effector molecule is selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of a cytokine, a chemokine, a homing molecule, a growth factor, a co-activation molecule, a tumor microenvironment modifier, a ligand, an antibody, a peptide, and an enzyme.
4 . The membrane-cleavable chimeric protein of claim 1 , wherein the secretable effector molecule comprises IL-15, IL-12, or an IL-12p70 fusion protein.
5 . The membrane-cleavable chimeric protein of claim 1 , wherein the protease cleavage site is cleavable by a protease selected from the group consisting of: a Type 1 transmembrane protease, a Type II transmembrane protease, a GPI anchored protease, an ADAM8 protease, an ADAM9 protease, an ADAM10 protease, an ADAM12 protease, an ADAM15 protease, an ADAM17 protease, an ADAM19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease, a BACE1 protease, a BACE2 protease, a SIP protease, an MT1-MMP protease, an MT3-MMP protease, an MT5-MMP protease, a furin protease, a PCSK7 protease, a matriptase protease, a matriptase-2 protease, an MMP9 protease, and an NS3 protease.
6 . The membrane-cleavable chimeric protein of claim 1 , wherein the protease cleavage site is cleavable by an ADAM17 protease.
7 . The membrane-cleavable chimeric protein of claim 1 , wherein the cell-membrane tethering domain comprises a transmembrane-intracellular domain or a transmembrane domain.
8 . The membrane-cleavable chimeric protein of claim 1 , wherein the cell-membrane tethering domain comprises a post-translational modification tag, or a motif capable of post-translational modification to modify the chimeric protein to include a post-translational modification tag, wherein the post-translational modification tag is capable of association with a cell membrane.
9 . The membrane-cleavable chimeric protein of claim 1 , wherein:
a) when expressed in a cell, the secretable effector molecule is tethered to a cell membrane of the cell; and/or
b) when expressed in a cell expressing a protease capable of cleaving the protease cleavage site, the secretable effector molecule is released from the cell membrane; and/or
c) the protease is an ADAM17 protease.
10 . An engineered nucleic acid comprising an expression cassette comprising a promoter and an exogenous polynucleotide sequence encoding the membrane-cleavable chimeric protein of claim 1 .
11 . An expression vector comprising the engineered nucleic acid of claim 10 .
12 . An isolated cell comprising the membrane-cleavable chimeric protein of claim 1 .
13 . The isolated cell of claim 12 , wherein the cell is selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.
14 . The isolated cell of claim 12 , wherein the cell further comprises a protease capable of cleaving the protease cleavage site.
15 . The isolated cell of claim 12 , wherein the cell further comprises an antigen-recognizing receptor.
16 . A composition comprising the membrane-cleavable chimeric protein of any one of claim 1 and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.
17 . A method of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of the isolated cell of claim 12 .
18 . A method of inducing release of a membrane-tethered effector molecule, comprising:
a) providing the isolated cell of claim 12 ; and
b) culturing the cell under conditions suitable for expression of a protease and the membrane-cleavable chimeric protein,
wherein upon expression, the membrane-cleavable chimeric protein is tethered to the cell membrane of the cell, and
wherein, upon expression, the protease cleaves the cognate membrane-bound protease cleavage site of the membrane-cleavable chimeric protein, thereby releasing the secretable effector molecule from the cell membrane.
19 . The membrane-cleavable chimeric protein of claim 2 , wherein the signal peptide comprises a native signal peptide native to the secretable effector molecule or the signal peptide comprises a non-native signal peptide or the signal-anchor sequence comprises a non-native signal-anchor sequence non-native to the secretable effector molecule.
20 . The membrane-cleavable chimeric protein of claim 19 , wherein the non-native signal peptide or the non-native signal-anchor sequence is selected from the group consisting of IL-12, IL-2, trypsinogen-2, Gaussia luciferase, CF5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFβ, osteoprotegerin, serpin E1, GROα, CXCL12, IL-21, CD8, NKG2D, TNFR2, and GM-CSF.
21 . The membrane-cleavable chimeric protein of claim 3 , wherein
the cytokine is selected from the group consisting of IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, an IL-12p70 fusion protein, IL-15, IL-17A, IL-18, IL-21, IL-22, Type I interferons, Interferon-gamma, and TNFα;
the chemokine is selected from the group consisting of CCL21α, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1;
the homing molecule is selected from the group consisting of anti-integrin α4β7;
anti-MAdCAM; SDF1; and MMP-2;
the growth factor is selected from the group consisting of FLT3L and GM-CSF;
the co-activation molecule is selected from the group consisting of 4-1BBL and CD40L;
the tumor microenvironment modifier is selected from the group consisting of adenosine deaminase, a TGFβ inhibitor, an immune checkpoint inhibitor, a VEGF inhibitor, and HPGE2;
the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody; or
the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof.
22 . The membrane-cleavable chimeric protein of claim 21 , wherein the TGFβ inhibitor is selected from the group consisting of an anti-TGFβ peptide, an anti-TGFβ antibody, a TGFβ-TRAP, and a combination thereof.
23 . The membrane-cleavable chimeric protein of claim 7 , wherein the transmembrane-intracellular domain and/or transmembrane domain is derived from PDGFR-β, CD8, CD28, CD3 ζ-chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA.
24 . The membrane-cleavable chimeric protein of claim 7 , wherein the cell-membrane tethering domain comprises a cell-surface receptor or a cell-membrane-bound portion of the cell-surface receptor.
25 . The membrane-cleavable chimeric protein of claim 8 , wherein the post-translation modification tag comprises a lipid-anchor domain.
26 . The membrane-cleavable chimeric protein of claim 25 , wherein the lipid-anchor domain is selected from the group consisting of a GPI lipid-anchor, a myristoylation tag, and a palmitoylation tag.
27 . The engineered nucleic acid of claim 10 , wherein the promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-specific promoter, and a synthetic promoter.
28 . The isolated cell of claim 14 , wherein the protease is an endogenous protease.
29 . The isolated cell of claim 28 , wherein the protease is an ADAM17 protease.
30 . The isolated cell of claim 15 , wherein the antigen-recognizing receptor is a CAR.