Armed chimeric receptors and methods of use thereof

Abstract

Described herein are immune response cells engineered to express cytokines and chimeric receptors. Also described herein are nucleic acids, cells, and methods involving the nucleic acids, the cells.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 378,846, filed on October 7, 2022, U.S. Provisional Application No. 63 / 382,477, filed on November 4, 2022, and U.S. Provisional Application No. 63 / 382,646, filed on November 7, 2022. The disclosure of each of the U.S. Provisional Applications is hereby incorporated by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing that has been submitted via the Patent Center and is hereby incorporated by reference in its entirety. The XML copy was created on XX / XX / 20XX, named XXXXXUS_sequencelisting.xml, and is X,XXX,XXX bytes in size. Background of the Invention

[0005] Cell - based therapy platforms offer promising approaches for treating a variety of diseases. One such promising platform is CAR - T - based therapy in cancer treatment. Given their promise, there is a need to improve cell - based therapies. An active area of exploration is cell - based engineered therapies to produce and / or secrete effector molecules, such as cytokines, that can enhance the efficacy of cell - based therapies, a process known as arming. For example, unarmed CAR - T therapies have poor efficacy against solid tumors, and arming can affect the entire cancer immune cycle and enhance CAR - T activity. However, uncontrolled or unregulated arming strategies can have negative impacts on treatment, such as off - target effects and toxicity to the subject. Therefore, additional methods are needed to control and regulate the arming of cell - based therapies (such as regulating the production and / or secretion of payload effector molecules). Summary of the Invention

[0006] In some embodiments, provided herein is a cell - based therapy platform that involves regulated arming of cell - based therapies, such as regulated secretion of payload effector molecules. In some embodiments, provided herein is also a cell - based combination immunotherapy that involves regulated arming for targeted treatment of cancers (such as ovarian cancer, breast cancer, colon cancer, lung cancer, and pancreatic cancer).

[0007] However, the therapies provided herein can limit armed systemic toxicity. For example, the immunotherapies provided herein can be tumor-specific and effective while limiting systemic toxicity and / or other off-target effects caused by arming. These therapies deliver the protein of interest, such as an immunomodulatory effector molecule, in a regulated manner, including regulating secretion kinetics, cell state specificity, and cell- or tissue-specificity. The design of the delivery vehicle is optimized to improve the overall function of cell-based therapies, such as cancer therapies, including but not limited to optimization of membrane cleavage sites, promoters, linkers, signal peptides, delivery methods, combinations of immunomodulatory effector molecules, regulation, and order.

[0008] Non-limiting examples of effector molecules covered by this disclosure include cytokines, antibodies, chemokines, nucleotides, peptides, enzymes, and oncolytic viruses. For example, cells can be engineered to express and secrete, in a regulated manner, at least one, two, three, or more of the following effector molecules: IL12, IL16, IFN-β, IFN-γ, IL2, IL15, IL7, IL36γ, IL18, IL1β, IL21, OX40-ligand, CD40L, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-TGFβ antibody, anti-TNFR2, MIP1α (CCL3), MIP1β (CCL5), CCL21, CpG oligodeoxynucleotides, and anti-tumor peptides (e.g., antimicrobial peptides with anti-tumor activity, see, e.g., Gaspar, D. et al. Front Microbiol 2013; 4:294; Chu, H. et al. PLoS ONE. 2015; 10(5):e0126390 and website aps.unmc.edu / AP / main.php).

[0009] In various embodiments, provided herein is a polycistronic expression system comprising: (a) an exogenous polynucleotide sequence encoding a first cytokine; (b) an exogenous polynucleotide sequence encoding a second cytokine; and (c) an exogenous polynucleotide sequence encoding an activating chimeric antigen receptor (aCAR), optionally wherein the aCAR comprises: (i) a first antigen-binding domain, (ii) one or more intracellular signaling domains that stimulate an immune response, and (iii) one or more polypeptides selected from the group consisting of a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof; and (d) an exogenous polynucleotide sequence encoding an inhibitory CAR (iCAR), wherein each exogenous polynucleotide sequence comprises a 5' end and a 3' end.

[0010] In various embodiments, provided herein is also a polycistronic expression system comprising: (a) an exogenous polynucleotide sequence encoding a first cytokine; (b) an exogenous polynucleotide sequence encoding a second cytokine; and (c) an exogenous polynucleotide sequence encoding an activating chimeric antigen receptor (aCAR), wherein each exogenous polynucleotide sequence comprises a 5'-end and a 3'-end, and wherein the aCAR comprises: (i) a first antigen-binding domain that binds to a target selected from the group consisting of CEA, CEACAM1, CEACAM5, and CEACAM6, optionally wherein the first antigen-binding domain of the aCAR binds CEACAM5, optionally wherein the first antigen-binding domain of the aCAR comprises the amino acid sequence shown in SEQ ID NO: 381; (ii) one or more intracellular signaling domains that stimulate an immune response; and (iii) one or more polypeptides selected from the group consisting of a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

[0011] In some embodiments, (i) the one or more intracellular signaling domains of the aCAR are selected from the group consisting of: CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, CD97, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, LFA-1, CD7, LIGHT, NKG2C, B7-H3, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, SLAM proteins, activating NK cell receptors, BTLA, Toll ligand receptors, CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a and combinations thereof;and / or (ii) the aCAR comprises a hinge domain selected from the group consisting of: human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular linker, and combinations thereof, and / or the aCAR comprises a transmembrane domain selected from the group consisting of: PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, LIR1, and BTLA, and / or (iv) the aCAR comprises a signal peptide selected from the group consisting of: IgE, IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin precursor, azurin precursor, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa.;

[0012] In some embodiments, the iCAR comprises: (a) a second antigen-binding domain; (b) one or more intracellular signaling domains that inhibit an immune response; and (c) one or more polypeptides selected from the group consisting of: signal peptide, transmembrane domain, hinge domain, spacer region, one or more peptide linkers, and combinations thereof. In some embodiments, the second antigen-binding domain of the iCAR binds VSIG2, optionally wherein: (i) the iCAR comprises an LIR1 intracellular inhibitory domain, optionally wherein the intracellular inhibitory domain comprises the amino acid sequence shown in SEQ ID NO: 387; or (ii) the iCAR comprises an SIRPα intracellular inhibitory domain, optionally wherein the intracellular inhibitory domain comprises the amino acid sequence shown in SEQ ID NO: 385.

[0013] In some embodiments, (i) the iCAR comprises a hinge domain selected from the group consisting of a human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular linker, and combinations thereof, and / or (ii) the iCAR comprises a transmembrane domain selected from the group consisting of PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, LIR1, SIRPα, and BTLA, and / or (iii) the iCAR comprises a signal peptide selected from the group consisting of IgE, IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin precursor, azurin precursor, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa.

[0014] In some embodiments, (i) the exogenous polynucleotide encoding the first cytokine, the exogenous polynucleotide encoding the second cytokine, the exogenous polynucleotide encoding the aCAR, and the exogenous polynucleotide encoding the iCAR are contained in a single expression vector, or (ii) the exogenous polynucleotide encoding the first cytokine, the exogenous polynucleotide encoding the second cytokine, and the exogenous polynucleotide encoding the aCAR are contained in a first expression vector, and the exogenous polynucleotide encoding the iCAR is contained in a second expression vector. In some embodiments, the polycistronic expression system further comprises ribosome skipping sites between each exogenous polynucleotide.

[0015] In some embodiments, at least one of the first cytokine and the second cytokine is a controlled-release cytokine having the following formula:

[0016] S–C–MT or MT–C–S

[0017] Wherein S comprises an effector molecule-secreting moiety; C comprises a protease cleavage site; and MT comprises a cell membrane tethering domain. In some embodiments, (i) the protease cleavage site is cleaved by ADAM10 and / or ADAM17, and / or (ii) the protease cleavage site comprises the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 191, and / or (iii) the cell membrane tethering domain comprises a transmembrane domain selected from the group consisting of: B7-1, PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, and BTLA, optionally wherein the cell membrane tethering domain comprises the B7-1 transmembrane domain, and the B7-1 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 219.

[0018] In some embodiments, (i) the first cytokine is IL15, optionally wherein the IL15 comprises the amino acid sequence shown in SEQ ID NO: 285, or optionally wherein the IL15 is controlled release IL15 (crIL15), and / or (ii) the second cytokine is IL21, optionally wherein the IL21 comprises the amino acid sequence shown in SEQ ID NO: 360, or optionally wherein the IL21 is controlled release IL21 (crIL21), and / or (iii) the first cytokine or the second cytokine comprises the amino acid sequence shown in any one of SEQ ID NOs: 355-359, 361, and 391, and / or (iv) the first cytokine or the second cytokine is encoded by the nucleic acid sequence shown in any one of SEQ ID NOs: 367-372 and 392.

[0019] In various embodiments, provided herein is also a polycistronic expression system comprising: (a) an exogenous polynucleotide sequence encoding a first cytokine; (b) an exogenous polynucleotide sequence encoding a second cytokine; and (c) an exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each exogenous polynucleotide sequence comprises a 5'-end and a 3'-end.

[0020] In various embodiments, provided herein is also an engineered cell comprising the polycistronic expression system provided herein. In some embodiments, the engineered cell is an immune cell, optionally wherein the engineered cell is selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, myeloid cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs) and iPSC-derived cells, optionally wherein the engineered cell is an NK cell.

[0021] In various embodiments, provided herein is also a pharmaceutical composition comprising the engineered cell provided herein and a pharmaceutically acceptable carrier.

[0022] In various embodiments, provided herein is also a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective dose of the engineered cell or pharmaceutical composition of the claims provided herein, optionally wherein: (i) the disease is cancer, and / or (ii) the isolated cells are allogeneic or autologous to the subject.

[0023] In various embodiments, provided herein is also a method of preparing an engineered cell, the method comprising transducing an isolated cell with the polycistronic expression system provided herein, optionally wherein: (i) the isolated cell is an immune cell, and / or (ii) the isolated cell is selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, myeloid cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs) and iPSC-derived cells, optionally wherein the isolated cell is an NK cell.

[0024] In various embodiments, provided herein is also a polycistronic expression system comprising: (a) an exogenous polynucleotide sequence encoding a first cytokine; (b) an exogenous polynucleotide sequence encoding a second cytokine; and (c) an exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each exogenous polynucleotide sequence comprises a 5' end and a 3' end. In certain embodiments, at least one of the first cytokine and the second cytokine is a controlled-release cytokine.

[0025] In certain embodiments, each controlled-release cytokine has the formula:

[0026] S–C–MT or MT–C–S

[0027] Wherein S comprises an effector molecule-secreting moiety; C comprises a protease cleavage site; and MT comprises a cell membrane tethering domain. In certain embodiments, the protease cleavage site is cleaved by ADAM10 and / or ADAM17. In certain embodiments, the protease cleavage site comprises the amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 191. In certain embodiments, the cell membrane tethering domain comprises a transmembrane domain selected from the group consisting of: PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, B7-1, and BTLA. In certain embodiments, the cell membrane tethering domain comprises the B7-1 transmembrane domain, and the B7-1 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 219.

[0028] In certain embodiments, the first cytokine is IL15. In certain embodiments, the IL15 comprises the amino acid sequence shown in SEQ ID NO: 285. In certain embodiments, the IL15 is controlled release IL15 (crIL15). In certain embodiments, the second cytokine is IL21. In certain embodiments, it comprises the amino acid sequence shown in SEQ ID NO: 360. In certain embodiments, the IL21 is controlled release IL21 (crIL21).

[0029] In certain embodiments, the first cytokine or the second cytokine comprises the amino acid sequence shown in any one of SEQ ID NOs: 355 - 359, 361, and 391. In certain embodiments, the first cytokine or the second cytokine is encoded by the nucleic acid sequence shown in any one of SEQ ID NOs: 367 - 372 and 392.

[0030] In certain embodiments, the polycistronic expression system comprises an exogenous polynucleotide sequence encoding an activating chimeric antigen receptor (aCAR) and an exogenous polynucleotide sequence encoding an inhibitory chimeric antigen receptor (iCAR). In certain embodiments, the aCAR comprises: (a) a first antigen-binding domain; (b) one or more intracellular signaling domains that stimulate an immune response; and (c) one or more polypeptides selected from the group consisting of a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof. In certain embodiments, the first antigen-binding domain of the aCAR binds an antigen selected from CEA, CEACAM1, CEACAM5, and CEACAM6. In certain embodiments, the first antigen-binding domain of the aCAR binds CEA, CEACAM1, CEACAM5, and CEACAM6. In certain embodiments, the first antigen-binding domain of the aCAR binds CEACAM5. In certain embodiments, the first antigen-binding domain of the aCAR comprises the amino acid sequence shown in SEQ ID NO: 381.

[0031] In certain embodiments, the one or more intracellular signaling domains of the aCAR are selected from the group consisting of: CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, CD97, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, LFA-1, CD7, LIGHT, NKG2C, B7-H3, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activating NK cell receptor, BTLA, Toll ligand receptor, CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a and combinations thereof. In certain embodiments, the aCAR comprises a hinge domain selected from the group consisting of: human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular linker and combinations thereof.In certain embodiments, the aCAR comprises a transmembrane domain selected from the group consisting of PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, LIR1, and BTLA. In certain embodiments, the aCAR comprises a signal peptide selected from the group consisting of IgE, IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin precursor, azurin precursor, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa.

[0032] In certain embodiments, the aCAR comprises the amino acid sequence shown in any one of SEQ ID NOs: 362-365. In certain embodiments, the aCAR is encoded by the nucleic acid sequence shown in any one of SEQ ID NOs: 373-376.

[0033] In certain embodiments, the iCAR comprises: (a) a second antigen-binding domain; (b) one or more intracellular signaling domains that inhibit an immune response; and (c) one or more polypeptides selected from the group consisting of a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof. In certain embodiments, the second antigen-binding domain of the iCAR binds VSIG2.

[0034] In certain embodiments, the iCAR comprises an LIR1 intracellular inhibitory domain. In certain embodiments, the intracellular inhibitory domain comprises the amino acid sequence shown in SEQ ID NO: 387. In certain embodiments, the iCAR comprises an SIRPα intracellular inhibitory domain. In certain embodiments, the intracellular inhibitory domain comprises the amino acid sequence shown in SEQ ID NO: 385.

[0035] In certain embodiments, the iCAR comprises a hinge domain selected from the group consisting of: human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular linker, and combinations thereof. In certain embodiments, the iCAR comprises a transmembrane domain selected from the group consisting of: PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, LIR1, SIRPα, and BTLA. In certain embodiments, the iCAR comprises a signal peptide selected from the group consisting of: IgE, IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin precursor, azurin precursor, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa.

[0036] In certain embodiments, the iCAR comprises the amino acid sequence shown in SEQ ID NO:366. In certain embodiments, the iCAR is encoded by the nucleic acid sequence shown in SEQ ID NO:377.

[0037] In certain embodiments, the exogenous polynucleotide encoding the first cytokine, the exogenous polynucleotide encoding the second cytokine, the exogenous polynucleotide encoding the aCAR, and the exogenous polynucleotide encoding the iCAR are contained in a single expression vector. In certain embodiments, the exogenous polynucleotide encoding the first cytokine, the exogenous polynucleotide encoding the second cytokine, and the exogenous polynucleotide encoding the aCAR are contained in a first expression vector, and the exogenous polynucleotide encoding the iCAR is contained in a second expression vector. In certain embodiments, each exogenous polynucleotide sequence further comprises a promoter sequence at the 5' end. In certain embodiments, the promoter is a constitutive promoter or an inducible promoter. In certain embodiments, the polycistronic expression system provided herein further comprises a ribosome skipping site between each exogenous polynucleotide.

[0038] In various embodiments, provided herein is also an engineered cell comprising the polycistronic expression system provided herein. In certain embodiments, the engineered cell is an immune cell. In certain embodiments, the engineered cell is selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, myeloid cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs) and iPSC-derived cells. In certain embodiments, the engineered cell is an NK cell.

[0039] In various embodiments, provided herein is also a pharmaceutical composition comprising the engineered cell provided herein and a pharmaceutically acceptable carrier.

[0040] In various embodiments, provided herein is also a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective dose of the engineered cell or pharmaceutical composition provided herein. In certain embodiments, the disease is cancer. In certain embodiments, the isolated cells are allogeneic to the subject. In certain embodiments, the isolated cells are autologous to the subject.

[0041] In various embodiments, provided herein is also a method of preparing an engineered cell, the method comprising transducing isolated cells with the polycistronic expression system provided herein. In certain embodiments, the isolated cells are immune cells. In certain embodiments, the isolated cells are selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, myeloid cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs) and iPSC-derived cells. In certain embodiments, the isolated cells are NK cells.

[0042] In various embodiments, provided herein is also an immune response cell comprising: (a) an exogenous polynucleotide encoding a first cytokine; (b) an exogenous polynucleotide encoding a second cytokine; and (c) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR). BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figures 1A - 1D Shows head-to-head orientation ( Figure 1A ), head-to-tail orientation ( Figure 1B ), tail-to-tail orientation ( Figure 1C) cytokine-CAR bidirectional construct and exemplary anti-GPC3 CAR+IL15 bidirectional construct( Figure 1D ) schematic diagram.

[0044] Figure 2 CAR expression plots (day 7) are provided for cells transduced with lentivirus encoding a CAR+IL15 bidirectional construct and cells transduced with lentivirus encoding only CAR, as evaluated by flow cytometry.

[0045] Figure 3 CAR expression plots (day 7) are provided for cells transduced with retrovirus encoding a CAR+IL15 bidirectional construct and cells transduced with retrovirus encoding only CAR, as evaluated by flow cytometry.

[0046] Figure 4 CAR expression plots (day 15) are provided for cells transduced with lentivirus encoding a CAR+IL15 bidirectional construct and cells transduced with lentivirus encoding only CAR, as evaluated by flow cytometry.

[0047] Figure 5 CAR expression plots (day 15) are provided for cells transduced with retrovirus encoding a CAR+IL15 bidirectional construct and cells transduced with retrovirus encoding only CAR, as evaluated by flow cytometry.

[0048] Figure 6 IL15 levels are provided for NK cells transduced with lentivirus encoding a CAR+IL15 bidirectional construct (“Lenti”) or gamma-retrovirus encoding a CAR+IL15 bidirectional construct (“SinVec”), as evaluated by immunoassay.

[0049] Figure 7 Killing by NK cells transduced with lentivirus encoding only CAR or a CAR+IL15 bidirectional construct is provided, as evaluated by co-culture killing assay.

[0050] Figure 8 Killing by NK cells transduced with gamma-retrovirus encoding only CAR or a CAR+IL15 bidirectional construct is provided, as evaluated by co-culture killing assay.

[0051] Figure 9 Schematic diagrams of bidirectional constructs are shown, which include an IL12 expression cassette having an mRNA instability element in the 3' untranslated region.

[0052] Figure 10Provided are IL12 levels evaluated by immunoassay of NK cells transduced with a bidirectional construct comprising an inducible IL12 expression cassette and an expression cassette encoding a synthetic transcription factor.

[0053] Figure 11 Shown is a schematic diagram of a bidirectional construct encoding an IL15 that is cleavably released.

[0054] Figure 12 Provided is a summary of the IL15 bicistronic constructs and performance tested in functional assays.

[0055] Figure 13A and Figure 13B Provided are expression plots of GPC3 CAR and IL15 of NK cells transduced with SB06251, SB06257, and SB06254 as evaluated by flow cytometry. Two independent replicates are shown ( Figure 13A and Figure 13B ).

[0056] Figure 14A and Figure 14B Provided are secreted IL15 levels as evaluated by immunoassay for NK cells transduced with SB06251, SB06257, and SB06254. Two independent replicates are shown ( Figure 14A and Figure 14B ).

[0057] Figure 15A and Figure 15B Provided is the cell growth of the target cell population after co-culture with NK cells induced with SB06251, SB06257, and SB06254. Two independent replicates are shown ( Figure 15A and Figure 15B ).

[0058] Figure 16 Provided are target cell counts in a continuous killing assay when co-cultured with NK cells transduced with SB06251, SB06257, and SB06254.

[0059] Figure 17A and Figure 17B Provided are expression plots of GPC3 CAR and IL15 of NK cells transduced with SB06252, SB06258, and SB06255 as evaluated by flow cytometry. Two independent replicates are shown ( Figure 17A and Figure 17B ).

[0060] Figure 18A and Figure 18BProvided are the secreted IL15 levels, as evaluated by immunoassay for NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown ( Figure 18A and Figure 18B ).

[0061] Figure 19A and Figure 19B Provided is the cell growth of the target cell population after co-culture with NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown ( Figure 19A and Figure 19B ).

[0062] Figure 20 Provided is the target cell count in a continuous killing assay when co-cultured with NK cells transduced with SB06252, SB06258, and SB06255.

[0063] Figure 21A and Figure 21B Provided are the expression profiles of GPC3 CAR and IL15 of NK cells transduced with the bicistronic constructs SB06261, SB6294, and SB6298, as evaluated by flow cytometry. Two independent replicates are shown ( Figure 21A and Figure 21B ).

[0064] Figure 22A and Figure 22B Provided are the secreted IL15 levels, as evaluated by immunoassay for NK cells transduced with SB06261, SB6294, and SB6298. Two independent replicates are shown ( Figure 22A and Figure 22B ).

[0065] Figure 23A and Figure 23B Provided is the cell growth of the target cell population after co-culture with NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown ( Figure 23A and Figure 23B ).

[0066] Figure 24A and Figure 24B Provided is the characterization of the cleavable IL15 bicistronic constructs SB06691, SB06692, and SB06693. The expression profiles of GPC3 CAR and IL15 of NK cells transduced with SB06691, SB06692, and SB06693, as evaluated by flow cytometry, are shown in Figure 24AShown in. The levels of secreted IL15 in NK cells induced by SB06691, SB06692, and SB06693, as evaluated by immunoassay, are shown in Figure 24B Shown in.

[0067] Figure 25 Schematic diagram showing a bidirectional construct encoding IL12 that can be cleaved and released is presented.

[0068] Figure 26 A dose - response curve of IL12 secretion in NK cells after treatment with glecaprevir (GRZ) is provided.

[0069] Figure 27A and Figure 27B In - vivo mouse data are provided, which demonstrate the IL12 levels in mouse blood after NK cells transduced with SB04599, SB05042, and SB05058. The IL12 levels are shown in Figure 27A Shown in, and the fold change of IL12 is shown in Figure 27B Shown in.

[0070] Figures 28A - 28C Characterization of cells transduced with different constructs expressing GPC3 CAR and IL15 is provided. Figure 28A Flow cytometry plots are shown, which confirm the expression of GPC3 CAR, membrane - bound IL15, and the corresponding copy numbers on NK cells transduced with different GPC3 CAR / IL15 expression constructs. Figure 28B Measurement of secreted IL15 is shown. Figure 28C Cell killing of HepG2, as evaluated by continuous killing assay, is shown.

[0071] Figure 29A and Figure 29B Additional data on continuous killing using transduced NK cells are provided. Figure 29A Continuous killing of HepG2 cells is shown. Figure 29B Continuous killing of HuH - 7 cells is shown.

[0072] Figure 30A and Figure 30B Data on evaluating the function of transduced NK cells using rapid expansion (G - Rex) are provided. Figure 30A Expression of GPC3 CAR, membrane - bound IL15 (mIL15), and secreted IL15 (sIL15) is shown. Figure 30B Continuous killing of transduced NK cells is shown.

[0073] Figure 31 Results from xenograft tumor models measured by bioluminescence imaging are provided, where mice are injected with NK cells.

[0074] Figure 32A and Figure 32B Provide the results and summary of a xenograft tumor model in mice injected with NK cells. Figure 32A Provide the survival curves of mice treated with NK cells. Figure 32B Provide a summary of the median survival period of mice treated with NK cells.

[0075] Figure 33 Provide the results of BLI experiments to evaluate tumor reduction in mice injected with NK cells.

[0076] Figure 34 Provide the quantification of each condition with respect to BLI measurements normalized to day 10.

[0077] Figure 35A and Figure 35B Provide the results from a xenograft tumor (HepG2) mouse model where mice were injected with NK cells three times during the study. Figure 35A Provide the results of mice using BLI imaging. Figure 35B Provide the time course of the fold change of BLI during the study.

[0078] Figure 36A and Figure 36B Provide the fold change of BLI in mice injected with transduced NK cells. Figure 36A Provide the results corresponding to the measurements taken 13 days after tumor implantation. Figure 36B Provide the results corresponding to the measurements taken 20 days after tumor implantation.

[0079] Figure 37A and Figure 37B Provide the results of tumor reduction in the xenograft model. Figure 37A Show a summary of the fold change of BLI in two different in vivo experiments. Figure 37B Show a summary of the normalized average fold change of BLI in two different in vivo experiments, but separate the treatment groups and track the animals individually.

[0080] Figure 38A and Figure 38B Provide the results from a xenograft tumor model where NK cells were injected intratumorally. Figure 38A Provide the measured values of tumor volume. Figure 38B Show the survival curves.

[0081] Figure 39A and Figure 39B Provide the results of the expression of IL12 in the presence or absence of grazoprevir. Figure 39AProvided are measurements of concentration and fold change at 24 hours after induction with grazoprevir. Figure 39B Provided are measurements of concentration and fold change at 72 hours after induction.

[0082] Figure 40 Provided are results from mice injected with NK cells expressing regulated IL12 at different concentrations throughout the experiment.

[0083] Figure 41 Provided are results of expression (GPC3 CAR and IL15) co-transduced into NK cells with IL12 and GPC3 CAR / IL15 constructs.

[0084] Figure 42A and Figure 42B Provided are results of the expression of secreted IL15 and secreted IL12 in the presence or absence of grazoprevir. Figure 42A Provided are measurements of the concentration of secreted IL15. Figure 42B Provided are measurements of the expression of secreted IL12.

[0085] Figure 43 Provided are measurements of secreted IL15 and secreted IL12 of NK cells during serial killing assays.

[0086] Figures 44A - 44D Provided are results of serial killing assays of different co-transductions in NK cells for cell killing of Huh-7 and HepG2 cells. Figure 44A Provided are serial killing results of NK cells co-transduced with SB05042 + SB06258. Figure 44B Provided are serial killing results of NK cells co-transduced with SB05042 + SB06257. Figure 44C Provided are serial killing results of NK cells co-transduced with SB05042 + SB06294. Figure 44D Provided are Figure 44A -combinations of the results in C.

[0087] Figures 45A - 45D Provided are results from the evaluation of clonal selection of NK cells expressing GPC3 CAR. Figure 45A Provided are results per cell copy. Figure 45B Provided are results of GPC3 CAR expression. Figure 45C Provided are results of IL15 expression. Figure 45D Provided is the measurement of secreted IL15.

[0088] Figure 46A and Figure 46BFlow cytometry data of GPC3 CAR and IL15 expression on selected clones transduced with SB06258 are provided. Figure 46A Results of selected clones are provided. Figure 46B Results of selected clones further transduced with SB05042 (IL12) are provided.

[0089] Figures 47A - 47D Data on STAT5 phosphorylation in response to controlled release IL15 (crIL15) are provided. Figure 47A Results of STAT5 phosphorylation in NK cells expressing CAR and the indicated IL15 constructs are provided. Figure 47B Results of STAT5 phosphorylation in CD3+ PBMCs incubated with NK cells expressing CAR and the indicated IL15 constructs are provided. Figure 47C Results of STAT3 and STAT5 phosphorylation in NK cells expressing the indicated IL15 constructs are provided. Figure 47D Surface binding and secretion of IL15 in NK cells transduced with the indicated IL15 constructs are provided.

[0090] Figure 48A and 48B Results of killing of target cells by CAR-NK cells expressing the indicated IL15 constructs are provided. Figure 48A Shows the abundance of target cells over time during incubation with CAR-NK cells expressing the indicated IL15 constructs. Figure 48B Shows the abundance of target cells after 120 hours of incubation with CAR-NK cells expressing the indicated IL15 constructs.

[0091] Figures 49A - 49C Results of killing of tumor cells by CAR-NK cells expressing one or two cytokines are provided. Figure 49A Shows the abundance of tumor cells over time during incubation with CAR-NK cells expressing the indicated cytokines. Figure 49B Shows images of tumor cells incubated with CAR-NK cells expressing the indicated cytokines. Figure 49C Shows the abundance of tumor cells after 120 hours of incubation with CAR-NK cells expressing the indicated cytokines.

[0092] Figure 50A and 50B Shows the analysis results of the optimal distribution between membrane-bound cytokines and soluble cytokines. Figure 50A Shows surface staining of IL15 (vertical axis) and CAR (horizontal axis) in CAR-NK cells expressing the indicated IL15 constructs. Figure 50BShows the expansion (left panel) and viability (right panel) of CAR-NK cells expressing the indicated IL15 constructs.

[0093] Figure 51A and 51B Shows the analysis results of IL15 and IL21 constructs in CAR-NK cells. Figure 51A Shows the expansion of CAR-NK cells expressing the indicated cytokine constructs. Figure 51B Shows the viability of CAR-NK cells expressing the indicated cytokine constructs.

[0094] Figure 52A and 52B Shows the results of the effect of cytokine expression on the survival of CAR-NK cells in the absence of cytokines in the medium. Figure 52A Shows the viability of CAR-NK cells expressing the indicated cytokine constructs. Figure 52B Shows the fold expansion of CAR-NK cells expressing the indicated cytokine constructs.

[0095] Figures 53A - 53C Shows the analysis of CAR NK cell activation in the co-expression of crIL15 and IL21. Figure 53A Shows the flow cytometry analysis of CAR-NK cells activated by the indicated cytokines, as measured by IFNγ (vertical axis) and granzyme B (horizontal axis). Figure 53B Shows Figure 53A Quantification of IFNγ (left panel) and granzyme B (right panel) staining in NK cells as shown in Figure 53C Shows images of target cells after incubation with CAR-NK cells expressing the indicated cytokines.

[0096] Figures 54A - 54D Shows the analysis of CAR-NK cell killing of target cells. Figure 54A Shows the ratio of CAR-NK cell-mediated killing of control cells relative to cells expressing the target after one round of killing. The inset shows two different donors. Figure 54B Shows the ratio of CAR-NK cell-mediated killing of control cells relative to cells expressing the target after multiple rounds of killing. The inset shows two different donors. Figure 54C Shows images of control (red) or target-expressing (green) after incubation with CAR-NK cells expressing the indicated constructs. Figure 54D Shows the results of consecutive killing of target cells by CAR-NK cells.

[0097] Figure 55Shows the continuous killing of target cells by CAR-NK cells expressing the indicated constructs under the inhibition of the culture in the presence of TGFβ.

[0098] Figure 56 Shows the continuous killing of cells expressing an inhibitory CAR (iCAR) target antigen by NK cells expressing an iCAR and an activating CAR (aCAR).

[0099] Figures 57A - 57E Shows in vivo tumor suppression by CAR-NK cells co-expressing crIL15 and IL21. Figure 57A Shows images of tumors in mice treated as indicated at the indicated time points. Figure 57B Shows tumor growth over time in mice treated as indicated. Figure 57C Shows the progression-free survival over time of mice treated with the specified CAR-NK cells. Figure 57D Shows the percentage of survival over time of mice treated with the specified CAR-NK cells. Figure 57E Shows images of tumors in mice treated as indicated 15 days after tumor transplantation (upper panel) and a graph showing the percentage of mice in which tumor reduction was observed compared to untreated controls (lower panel).

[0100] Figures 58A - 58D Shows the persistence of CAR-NK cells expressing crIL15 and IL21 in tumor-bearing mice. Figure 58A Shows the percentage of CD45-expressing cells in peritoneal fluid (left panel) and blood (right panel) 27 days after administration as a percentage of total cells. Figure 58B Shows the staining of human CD45 (vertical axis) and mouse CD45 (horizontal axis) in NK cells expressing the indicated constructs 27 days after administration. Figure 58C Shows the percentage of CD45-expressing cells in peritoneal fluid 70 days after administration as a percentage of total cells. Figure 58D Shows the staining of human CD45 (vertical axis) and mouse CD45 (horizontal axis) in NK cells expressing the indicated constructs 70 days after administration.

[0101] Figure 59A and 59B Details the screening of various IL15 constructs and combinations with IL7 or IL21. Figure 59A Shows the continuous killing of target cells at the indicated effector-to-target ratio (E:T) when incubated with NK cells expressing the indicated constructs. Figure 59B Shows the percentage of NK cells expressing a CAR.

[0102] Figures 60A - 60EThe analysis of IL15 in combination with IL21 or IL7 is described in detail. Figure 60A Shows the serial killing of target cells by NK cells expressing a control or the indicated cytokine construct. Figure 60B Shows the serial killing of target cells by NK cells with a control or the indicated cytokine construct. Figure 60C Shows the serial killing of target cells by NK cells expressing a control or the indicated IL15 construct. Figure 60D Shows the serial killing of target cells by NK cells expressing a control or the indicated cytokine construct. Figure 60E Shows the serial killing of target cells by NK cells expressing a control or the indicated IL15 construct.

[0103] Figures 61A - 61C The construction of proteins containing a recombinant IL15 sushi domain is described in detail. Figure 61A The design of synthetic protein structures is described in detail. Figure 61B Shows the killing of target cells incubated with NK cells expressing a control or the indicated construct. Figure 61C Shows the second round of killing of target cells incubated with NK cells expressing a control or the indicated construct.

[0104] Figures 62A - 62D The production of NK cells engineered to express an inhibitory CAR (iCAR) and a controlled release IL15 (crIL15 or mIL15) is described in detail. Figure 62A The percentage of engineered cells expressing iCAR or crIL15 as measured by flow cytometry is described in detail. Figure 62B Depicts the expression of iCAR or crIL15 in engineered cells as measured by flow cytometry. Figure 62C Depicts the secretion of IL-15 by NK cells engineered to express the indicated construct. Figure 62D Depicts the secretion of IL-21 by NK cells engineered to express the indicated construct. Detailed Description

[0105] In various embodiments, a polycistronic expression system is provided herein. In some embodiments, the polycistronic expression system comprises: (a) an exogenous polynucleotide encoding a first cytokine; (b) an exogenous polynucleotide encoding a second cytokine; and (c) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR). In certain embodiments, the polycistronic expression system comprises an activating CAR (aCAR) and an inhibitory CAR (iCAR).

[0106] In various embodiments, immune response cells are also provided herein that are engineered to have the following:

[0107] (a) An exogenous polynucleotide encoding a first cytokine; (b) An exogenous polynucleotide encoding a second cytokine; and (c) An exogenous polynucleotide encoding a chimeric antigen receptor (CAR).

[0108] The polycistronic expression systems or immune response cells disclosed herein may include an activation control polypeptide. The ACP may include a synthetic transcription factor. A synthetic transcription factor is a non-naturally occurring protein that includes a DNA binding domain and a transcriptional effector domain and is capable of regulating (i.e., activating or inhibiting) transcription by binding to a cognate promoter (ACP-responsive promoter) recognized by the DNA binding domain. In some embodiments, the ACP is a transcriptional repressor. In some embodiments, the ACP is a transcriptional activator.

[0109] Membrane-cleavable chimeric proteins can be engineered such that the secretion of effector molecules can be regulated in a protease-dependent manner. Specifically, membrane-cleavable chimeric proteins can be engineered such that the secretion of effector molecules can be regulated as part of a "membrane-cleavable" system in which the incorporation of a protease cleavage site ("C") and a cell membrane tethering domain ("MT") allows for the protease-dependent regulation of effector molecule secretion. Without wishing to be bound by theory, the components of the membrane-cleavable system present in the membrane-cleavable chimeric protein generally regulate secretion through the following cellular processes:

[0110] - MT: The cell membrane tethering domain contains a transmembrane domain (or transmembrane-intracellular domain) that directs the cellular trafficking of the chimeric protein such that the protein inserts into the cell membrane or otherwise associates with the cell membrane ("tethers").

[0111] - C: After the chimeric protein is expressed and localized to the cell membrane, the protease cleavage site directs the cleavage of the chimeric protein such that the effector molecule is released ("secreted") into the extracellular space. Generally, protease cleavage sites are protease-specific, including sites engineered to be protease-specific. The protease cleavage site can be selected or engineered to achieve optimal protein expression, cell type-specific cleavage, cell state-specific cleavage, and / or cleavage and release of the payload at a desired kinetics (e.g., the ratio of membrane-bound chimeric protein level to secreted chimeric protein level).

[0112] In some aspects, provided herein are membrane-cleavable chimeric proteins (or engineered nucleic acids encoding membrane-cleavable chimeric proteins) having a protein of interest (e.g., any effector molecule described herein), a protease cleavage site, and a cell membrane tethering domain.

[0113] "Effector molecule" refers to a molecule that binds to another molecule and modulates the biological activity of the molecule to which it binds (e.g., a nucleic acid, such as DNA or RNA, or a protein (polypeptide) or peptide). For example, an effector molecule can act as a ligand for enhancing or reducing enzymatic activity, gene expression, or cell signaling. Thus, in some embodiments, effector molecules modulate (activate or inhibit) different immunomodulatory mechanisms. By directly binding to and modulating a molecule, an effector molecule can also indirectly modulate a second downstream molecule.

[0114] Generally, for all membrane-cleavable chimeric proteins described herein, the effector molecule is a cytokine or an active fragment thereof (the secreted effector molecule referred to as "S" in the formula S-C-MT or MT-C-S), which includes a cytokine or an active fragment thereof.

[0115] The term "modulate" encompasses maintaining biological activity, inhibiting (partially or completely) biological activity, and stimulating / activating (partially or completely) biological activity. The term also encompasses reducing or increasing (e.g., enhancing) biological activity. Two different effector molecules are considered to "modulate different tumor-mediated immunosuppressive mechanisms" when the tumor-mediated immunosuppressive mechanism modulated by one effector molecule (e.g., stimulating T cell signaling) is different from the tumor-mediated immunosuppressive mechanism modulated by another effector molecule (e.g., stimulating antigen presentation and / or processing).

[0116] The modulation carried out by an effector molecule can be direct or indirect. Direct modulation occurs when an effector molecule binds to another molecule and modulates the activity of that molecule. Indirect modulation occurs when an effector molecule binds to another molecule, modulates the activity of that molecule, and as a result of that modulation, modulates the activity of yet another molecule (to which the effector molecule does not bind).

[0117] In some embodiments, at least one effector molecule modulates a tumor-mediated immunosuppressive mechanism such that an immune stimulation and / or an anti-tumor immune response (e.g., systemically or in the tumor microenvironment) is increased by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulating the tumor-mediated immunosuppressive mechanism can increase the immune stimulation and / or the anti-tumor immune response by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%. In some embodiments, modulating the tumor-mediated immunosuppressive mechanism increases the immune stimulation and / or the anti-tumor immune response by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20%-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200%. It should be understood that an "increase" in the immune stimulation and / or the anti-tumor immune response, e.g., systemically or in the tumor microenvironment, is relative to the immune stimulation and / or the anti-tumor immune response that would otherwise occur in the absence of the effector molecule.

[0118] In some embodiments, at least one effector molecule modulates a tumor-mediated immunosuppressive mechanism such that an immune stimulation and / or an anti-tumor immune response (e.g., systemically or in the tumor microenvironment) is increased by at least 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 20-fold, 25-fold, 50-fold, or 100-fold). For example, modulating the tumor-mediated immunosuppressive mechanism can increase the immune stimulation and / or the anti-tumor immune response by at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold. In some embodiments, modulating the tumor-mediated immunosuppressive mechanism increases the immune stimulation and / or the anti-tumor immune response by 2-10 fold, 2-20 fold, 2-30 fold, 2-40 fold, 2-50 fold, 2-60 fold, 2-70 fold, 2-80 fold, 2-90 fold, or 2-100 fold.

[0119] Non-limiting examples of immune-stimulating and / or anti-tumor immune mechanisms include T cell signaling, activation and / or recruitment, antigen presentation and / or processing, natural killer cell-mediated cytotoxic signaling, activation and / or recruitment, dendritic cell differentiation and / or maturation, immune cell recruitment, pro-inflammatory macrophage signaling, activation and / or recruitment, matrix degradation, production of immune-stimulating metabolites, stimulator of interferon genes (STING) signaling (which increases IFN secretion and Th1 polarization, promoting the anti-tumor immune response), and / or type I interferon signaling. An effector molecule can stimulate at least one (or more) of the foregoing immune-stimulating mechanisms, thereby increasing the immune-stimulating response. Changes in the foregoing immune-stimulating and / or anti-tumor immune mechanisms can be evaluated, for example, using in vitro assays for T cell proliferation or cytotoxicity, in vitro antigen presentation assays, assays for expression (e.g., of specific markers), and / or assays for cell secretion (e.g., of cytokines).

[0120] In some embodiments, at least one effector molecule modulates a tumor-mediated immunosuppressive mechanism such that the immunosuppressive response (e.g., systemically or in the tumor microenvironment) is reduced by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulating a tumor-mediated immunosuppressive mechanism can reduce the immunosuppressive response by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%. In some embodiments, modulating a tumor-mediated immunosuppressive mechanism reduces the immunosuppressive response by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20%-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200%. It should be understood that a "reduction" in the immunosuppressive response, e.g., systemically or in the tumor microenvironment, is relative to the immunosuppressive response that would otherwise occur in the absence of the effector molecule.

[0121] In some embodiments, at least one effector molecule modulates a tumor-mediated immunosuppressive mechanism such that an immunosuppressive response (e.g., systemically or in a tumor microenvironment) is reduced by at least 2 times (e.g., 2 times, 3 times, 4 times, 5 times, 10 times, 25 times, 20 times, 25 times, 50 times, or 100 times). For example, modulating a tumor-mediated immunosuppressive mechanism may reduce an immunosuppressive response by at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times. In some embodiments, modulating a tumor-mediated immunosuppressive mechanism reduces an immunosuppressive response by 2-10 times, 2-20 times, 2-30 times, 2-40 times, 2-50 times, 2-60 times, 2-70 times, 2-80 times, 2-90 times, or 2-100 times.

[0122] Non-limiting examples of immunosuppressive mechanisms include negative co-stimulatory signaling, pro-apoptotic signaling of cytotoxic cells (e.g., T cells and / or NK cells), T regulatory (Treg) cell signaling, tumor checkpoint molecule production / maintenance, myeloid-derived suppressor cell signaling, activity and / or recruitment, immunosuppressive factor / metabolite production and / or vascular endothelial growth factor signaling. Effector molecules can inhibit at least one (one or more) of the aforementioned immunosuppressive mechanisms, thereby reducing the immunosuppressive response. The changes in the aforementioned immunosuppressive mechanisms can be evaluated, for example, by measuring the increase in T cell proliferation and / or the increase in IFNγ production (negative co-stimulatory signaling, T reg cell signaling and / or MDSC); Annexin V / PI flow staining (pro-apoptotic signaling); flow staining for expression, e.g., PDL1 expression (tumor checkpoint molecule production / maintenance); ELISA, RNA was assayed by qPCR, enzymatic assays, e.g., IDO tryptophan catabolism (immunosuppressive factor / metabolite production); and PI3K, Akt, p38 phosphorylation (VEGF signaling).

[0123] In some embodiments, the effector molecules act additively: the effect of the two effector molecules, for example, can be equal to the sum of the effects of the two effector molecules acting separately. In other embodiments, the effector molecules act synergistically: the effect of the two effector molecules, for example, can be greater than the combined function of the two effector molecules.

[0124] The effector molecule that modulates tumor-mediated immunosuppression mechanisms and / or alters the tumor microenvironment can be any cytokine described herein.

[0125] In some embodiments, at least one effector molecule stimulates immunostimulatory mechanisms in the tumor microenvironment and / or inhibits immunosuppressive mechanisms in the tumor microenvironment.

[0126] In some embodiments, at least one effector molecule (a) stimulates T cell signaling, activity, and / or recruitment, (b) stimulates antigen presentation and / or processing, (c) stimulates natural killer cell-mediated cytotoxic signaling, activity, and / or recruitment, (d) stimulates dendritic cell differentiation and / or maturation, (e) stimulates immune cell recruitment, (f) stimulates pro-inflammatory macrophage signaling, activity, and / or recruitment or inhibits anti-inflammatory macrophage signaling, activity, and / or recruitment, (g) stimulates matrix degradation, (h) stimulates production of immunostimulatory metabolites, (i) stimulates type I interferon signaling, (j) inhibits negative co-stimulatory signaling, (k) inhibits pro-apoptotic signaling of anti-tumor immune cells, (l) inhibits T regulatory (T reg ) cell signaling, activity, and / or recruitment, (m) inhibits tumor checkpoint molecules, (n) stimulates stimulator of interferon genes (STING) signaling, (o) inhibits myeloid-derived suppressor cell signaling, activity, and / or recruitment, (p) degrades immunosuppressive factors / metabolites, (q) inhibits vascular endothelial growth factor signaling, and / or (r) directly kills tumor cells.

[0127] Non-limiting examples of cytokines are listed in Table 1, and specific sequences encoding exemplary effector molecules are listed in Table 2. The effector molecule can be human, such as those listed in Table 1 or Table 2, or a human equivalent of a murine effector molecule listed in Table 1 or Table 2. The effector molecule can be human-derived, such as an endogenous human effector molecule or an effector molecule that has been functionally modified and / or optimized (e.g., codon-optimized for improved expression, modified for improved stability, or modified at its signal sequence) (see below). Various procedures and algorithms for optimizing function are known to those skilled in the art and can be selected based on the desired improvements, such as codon optimization for a particular species (e.g., human, mouse, bacterium, etc.).

[0128] Table 1: Exemplary Effector Molecules

[0129] Effector Name Category Function IFNβ Cytokine T cell response, tumor cell killing IFNγ Cytokine T cell response, tumor cell killing IL12 (e.g., IL12p70 fusion) Cytokine T cells, NK cells IL1-β Cytokine T cells, NK cells IL15 Cytokine Stimulate T cells and NK IL2 Cytokine Stimulate T cells and NK IL21 Cytokine Stimulate T cells IL24 Cytokine Stimulate T cells IL36-γ Cytokine Stimulate T cells IL7 Cytokine Stimulate T cells IL22 Cytokine Stimulate T cells IL18 Cytokine Stimulate T cells

[0130] Table 2: Sequences Encoding Exemplary Effector Molecules

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140] The first engineered nucleic acid can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 309. The first engineered nucleic acid can comprise a nucleotide sequence having the sequence shown in SEQ ID NO: 309.

[0141] The first engineered nucleic acid can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 326. The first engineered nucleic acid can comprise a nucleotide sequence having the sequence shown in SEQ ID NO: 326.

[0142] The first engineered nucleic acid can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 310. The first engineered nucleic acid can comprise a nucleotide sequence having the sequence shown in SEQ ID NO: 310.

[0143] The first engineered nucleic acid can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 327. The first engineered nucleic acid can comprise a nucleotide sequence having the sequence shown in SEQ ID NO: 327.

[0144] The first engineered nucleic acid can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 314. The first engineered nucleic acid can comprise a nucleotide sequence having the sequence shown in SEQ ID NO: 314.

[0145] The first engineered nucleic acid can include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 315. The first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 315.

[0146] The second engineered nucleic acid can include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317. The second engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 317.

[0147] The second engineered nucleic acid can include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 318. The second engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 318.

[0148] (a) The first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 310; and (b) the second engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 317.

[0149] (a) The first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 327; and (b) the second engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 317.

[0150] (a) The first engineered nucleic acid can include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310; and (b) the second engineered nucleic acid can include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.

[0151] (a) The first engineered nucleic acid can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 327; and (b) The second engineered nucleic acid can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 317.

[0152] The immune response cells provided herein can comprise any one of the engineered nucleic acids described herein. The immune response cells provided herein can comprise a combination of any one of the engineered nucleic acids described herein. The immune response cells provided herein can comprise two or more of any one of the engineered nucleic acids described herein.

[0153] The immune response cells provided herein can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 309. The immune response cells provided herein can comprise a nucleotide sequence having the sequence shown in SEQ ID NO: 309.

[0154] The immune response cells provided herein can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 326. The immune response cells provided herein can comprise a nucleotide sequence having the sequence shown in SEQ ID NO: 326.

[0155] The immune response cells provided herein can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 310. The immune response cells provided herein can comprise a nucleotide sequence having the sequence shown in SEQ ID NO: 310.

[0156] The immune response cells provided herein can comprise a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 327. The immune response cells provided herein can comprise a nucleotide sequence having the sequence shown in SEQ ID NO: 327.

[0157] The immune response cells provided herein may include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 314. The immune response cells provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 314.

[0158] The immune response cells provided herein may include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 315. The immune response cells provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 315.

[0159] The immune response cells provided herein may include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317. The immune response cells provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 317.

[0160] The immune response cells provided herein may include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 318. The immune response cells provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 318.

[0161] The immune response cells provided herein may include a first engineered nucleic acid comprising a nucleotide sequence having the sequence shown in SEQ ID NO: 310; and (b) a second engineered nucleic acid comprising a nucleotide sequence having the sequence shown in SEQ ID NO: 317.

[0162] The immune response cells provided herein may include a first engineered nucleic acid comprising a nucleotide sequence having the sequence shown in SEQ ID NO: 327; and (b) a second engineered nucleic acid comprising a nucleotide sequence having the sequence shown in SEQ ID NO: 317.

[0163] The immune response cells provided herein may include a first engineered nucleic acid comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310; and (b) a second engineered nucleic acid comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.

[0164] The immune response cells provided herein may include a first engineered nucleic acid comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 327; and (b) a second engineered nucleic acid comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317.

[0165] The expression vectors provided herein may include any one of the engineered nucleic acids described herein. The expression vectors provided herein may include a combination of any of the engineered nucleic acids described herein. The expression vectors provided herein may include two or more of any of the engineered nucleic acids described herein.

[0166] The expression vectors provided herein may include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 309. The expression vectors provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 309.

[0167] The expression vectors provided herein may include a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 326. The expression vectors provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 326.

[0168] The expression vectors provided herein may include nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 310. The expression vectors provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 310.

[0169] The expression vectors provided herein may include nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 327. The expression vectors provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 327.

[0170] The expression vectors provided herein may include nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 314. The expression vectors provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 314.

[0171] The expression vectors provided herein may include nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 315. The expression vectors provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 315.

[0172] The expression vectors provided herein may include nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 317. The expression vectors provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 317.

[0173] The expression vectors provided herein may include nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 318. The expression vectors provided herein may include a nucleotide sequence having the sequence shown in SEQ ID NO: 318.

[0174] The expression vector provided herein may comprise a first engineered nucleic acid, the first engineered nucleic acid comprising a nucleotide sequence having the sequence shown in SEQ ID NO: 310; and (b) a second engineered nucleic acid, the second engineered nucleic acid comprising a nucleotide sequence having the sequence shown in SEQ ID NO: 317.

[0175] The expression vector provided herein may comprise a first engineered nucleic acid, the first engineered nucleic acid comprising a nucleotide sequence having the sequence shown in SEQ ID NO: 327; and (b) a second engineered nucleic acid, the second engineered nucleic acid comprising a nucleotide sequence having the sequence shown in SEQ ID NO: 317.

[0176] The expression vector provided herein may comprise a first engineered nucleic acid, the first engineered nucleic acid comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 310; and (b) a second engineered nucleic acid, the second engineered nucleic acid comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 317.

[0177] The expression vector provided herein may comprise a first engineered nucleic acid, the first engineered nucleic acid comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 327; and (b) a second engineered nucleic acid, the second engineered nucleic acid comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 317.

[0178] Secretory signals and signal anchors

[0179] One or more effector molecules of the membrane-cleavable chimeric proteins provided herein (e.g., any cytokine described herein) are generally secretable effector molecules having a secretion signal peptide (also referred to as a signal peptide or signal sequence) at the N-terminus of the chimeric protein (e.g., the N-terminus of the effector molecule of S-C-MT), which directs newly synthesized proteins destined for secretion or membrane localization (also referred to as membrane insertion) to the appropriate protein processing pathway. For chimeric proteins having the formula MT-C-S, the membrane tethering domain generally has a signal anchor sequence (e.g., the signal anchor sequence of a type II transmembrane protein), which directs newly synthesized proteins destined for membrane localization to the appropriate protein processing pathway. For chimeric proteins having the formula S-C-MT, a membrane tethering domain (usually without a separate secretion signal peptide) having a reverse signal anchor sequence (e.g., the signal anchor sequence of certain type III transmembrane proteins) can be used, which directs newly synthesized proteins destined for membrane localization to the appropriate protein processing pathway.

[0180] Generally, for all membrane-cleavable chimeric proteins described herein, one or more effector molecules are secretable effector molecules (referred to as "S" in the formulas S-C-MT or MT-C-S). In embodiments having two or more chimeric proteins, each chimeric protein can comprise a secretion signal. In embodiments having two or more chimeric proteins, each chimeric protein can comprise a secretion signal such that each effector molecule can be secreted from the engineered cell after cleavage at the protease cleavage site.

[0181] The secretion signal peptide operably associated with the effector molecule can be a native secretion signal peptide (e.g., a secretion signal peptide normally associated endogenously with a given effector molecule, such as the endogenous secretion signal peptide of a cytokine). The secretion signal peptide operably associated with the effector molecule can be a non-native secretion signal peptide native secretion signal peptide. Non-native secretion signal peptides can promote improved expression and function, such as maintaining secretion in a particular environment (e.g., the tumor microenvironment). Non-limiting examples of non-native secretion signal peptides are shown in Table 3.

[0182] Table 3. Exemplary signal secretion peptides

[0183]

[0184]

[0185]

[0186]

[0187] Protease cleavage site

[0188] Generally, all the membrane-cleavable chimeric proteins described herein contain a protease cleavage site (referred to as "C" in Formula S-C-MT or MT-C-S). Generally speaking, the protease cleavage site can be any amino acid sequence motif that can be cleaved by a protease. Examples of protease cleavage sites include, but are not limited to, type I transmembrane protease cleavage sites, type II transmembrane protease cleavage sites, GPI-anchored protease cleavage sites, ADAM8 protease cleavage sites, ADAM9 protease cleavage sites, ADAM10 protease cleavage sites, ADAM12 protease cleavage sites, ADAM15 protease cleavage sites, ADAM17 protease cleavage sites, ADAM19 protease cleavage sites, ADAM20 protease cleavage sites, ADAM21 protease cleavage sites, ADAM28 protease cleavage sites, ADAM30 protease cleavage sites, ADAM33 protease cleavage sites, BACE1 protease cleavage sites, BACE2 protease cleavage sites, SIP protease cleavage sites, MT1-MMP protease cleavage sites, MT3-MMP protease cleavage sites, MT5-MMP protease cleavage sites, furin protease cleavage sites, PCSK7 protease cleavage sites, proteinase 3 protease cleavage sites, proteinase 3-2 protease cleavage sites, MMP9 protease cleavage sites or NS3 protease cleavage sites.

[0189] An example of a protease cleavage site is the hepatitis C virus (HCV) non-structural protein 3 (NS3) protease cleavage site, including but not limited to the NS3 / NS4A cleavage site, NS4A / NS4B cleavage site, NS4B / NS5A cleavage site, or NS5A / NS5B cleavage site. For a description of representative sequences of NS3 protease and its cleavage sites for various HCV strains, see, for example, Hepatitis C Viruses: Genomes and Molecular Biology (edited by S.L. Tan, Taylor and Francis, 2006), Chapter 6, pages 163-206; the entire text of which is incorporated herein by reference. For example, sequences of the HCV NS4A / 4B protease cleavage site, HCV NS5A / 5B protease cleavage site, C-terminal degron with the NS4A / 4B protease cleavage site, and N-terminal degron with the HCV NS5A / 5B protease cleavage site are provided. Representative NS3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: accession numbers YP_001491553, YP_001469631, YP_001469632, NP_803144, NP_671491, YP_001469634, YP_001469630, YP_001469633, ADA68311, ADA68307, AFP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99041, CBF60982, CBF60817, AHH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063; all of which sequences (sequences entered as of the filing date of this application) are incorporated herein by reference.

[0190] Another example of a protease cleavage site is an ADAM17 specific protease (also referred to as a tumor necrosis factor-α converting enzyme [TACE]) cleavage site. The ADAM17 specific protease cleavage site can be an endogenous sequence of a substrate naturally cleaved by ADAM17. The ADAM17 specific protease cleavage site can be an engineered sequence that can be cleaved by ADAM17. The engineered ADAM17 specific protease cleavage site can be engineered to obtain specific desired properties, including but not limited to the optimal expression of chimeric protein, the specificity to ADAM17, the cleavage rate of ADAM17, the ratio of the level of secreted chimeric protein and the level of membrane-bound chimeric protein, and the cleavage under different cell states. The protease cleavage site can be selected for the specific cleavage of ADAM17. For example, some protease cleavage sites that can be cleaved by ADAM17 can also be cleaved by other ADAM family proteases such as ADAM10. Therefore, the ADAM17 specific protease cleavage site can be selected and / or engineered to reduce or eliminate the cleavage of other proteases such as ADAM10. The protease cleavage site can be selected for the cleavage rate of ADAM17. For example, it may be desirable to select a protease cleavage site that displays a specific cleavage rate of ADAM17 (such as the cleavage kinetics reduced by the endogenous sequence of the substrate naturally cleaved by ADAM17). In such cases, in general, a specific cleavage rate can be selected to regulate the processing rate of the chimeric protein, and then the release / secretion rate of the payload effector molecule is regulated. Therefore, the ADAM17 specific protease cleavage site can be selected and / or engineered so that the sequence exhibits the desired ADAM17 cleavage rate. The protease cleavage site can be selected for both the specific cleavage of ADAM17 and the cleavage rate of ADAM17. Exemplary ADAM17 specific protease cleavage sites (including those sites that exhibit specific specificity and cleavage rate kinetics) are shown in Table 4A below, wherein cleavage sites (P5-P1: N-terminal; P1'-P5': C-terminal) are mentioned. Further details of ADAM17 and ADAM10, including expression and protease cleavage sites, are described in Sharma et al. (J Immunol. 2017 Oct 15, 199(8) 2865-2872), Pham et al. (Anticancer Res. 2017 Oct;37(10):5507-5513), Caescu et al. (Biochem J. 2009 Oct 23;424(1):79-88), and Tucher et al. (J. Proteome Res. 2014, 13, 4, 2205-2214), each of which is incorporated herein by reference for its own purposes.

[0191] Table 4A - Potential ADAM17 Protease Cleavage Site Sequences

[0192]

[0193] In some embodiments, the protease cleavage site comprises a first region having an amino acid sequence of PRAE (SEQ ID NO:176). In some embodiments, the protease cleavage site comprises a second region having an amino acid sequence of KGG (SEQ ID NO:177). In some embodiments, the first region is located at the N-terminus of the second region. In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEX1X2KGG (SEQ ID NO:178), where X1 is A, Y, P, S, or F, and where X2 is V, L, S, I, Y, T, or A. In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEAVKGG (SEQ ID NO:179). In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEALKGG (SEQ ID NO:180). In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEYSKGG (SEQ ID NO:181). In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEPIKGG (SEQ ID NO:182). In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEAYKGG (SEQ ID NO:183). In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAESSKGG (SEQ ID NO:184). In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEFTKGG (SEQ ID NO:185). In some embodiments, the protease cleavage site comprises an amino acid sequence of PRAEAAKGG (SEQ ID NO:186). In some embodiments, the protease cleavage site comprises an amino acid sequence of DEPHYSQRR (SEQ ID NO:187). In some embodiments, the protease cleavage site comprises an amino acid sequence of PPLGPIFNPG (SEQ ID NO:188). In some embodiments, the protease cleavage site comprises an amino acid sequence of PLAQAYRSS (SEQ ID NO:189). In some embodiments, the protease cleavage site comprises an amino acid sequence of TPIDSSFNPD (SEQ ID NO:190). In some embodiments, the protease cleavage site comprises an amino acid sequence of VTPEPIFSLI (SEQ ID NO:191).

[0194] In certain embodiments, the cleavage site comprises a linker sequence. The cleavage site can be flanked on the N-terminal side and / or the C-terminal side by the linker sequence. For example but not limited to, the cleavage site can be flanked on the N-terminal side and the C-terminal side by a partial glycine-serine (GS) linker sequence. Upon cleavage, the N-terminal partial GS linker and the C-terminal partial GS linker join to form a GS linker sequence, such as SEQ ID NO: 215.

[0195] In certain embodiments, the cleavage site and the linker comprise the amino acid sequence of SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO 287). An exemplary nucleic acid sequence encoding SEQ ID NO: 287 is TCTGGCGGCGGAGGATCTGGCGGAGGTGGAA GCGGAGTTACACCCGAGCCTATCTTCAGCCTGATCGGAGGCGGTAGCG GAGGCGGAGGAAGTGGTGGCGGATCTCTGCAA (SEQ ID NO: 288). In some embodiments, the nucleic acid encoding SEQ ID NO: 287 can comprise SEQ ID NO: 288, or a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 288.

[0196] In certain embodiments, the protease cleavage site is at the N-terminus of the linker. In certain embodiments, the protease cleavage site and the linker comprise the amino acid sequence of PRAEALKGGSGGGGSGGGGSGGGGSG GGGSGGGSLQ (SEQ ID NO: 289). An exemplary nucleic acid sequence encoding SEQ ID NO: 289 is CCCAGAGCCGAGGCTCTGAAAGGCGGATCAGGCG GCGGTGGTAGTGGAGGCGGAGGCTCAGGCGGCGGAGGTTCCGGAGGT GGCGGTTCCGGCGGAGGATCTCTTCAAT (SEQID NO: 292). In some embodiments, the nucleic acid encoding SEQ ID NO: 289 can comprise SEQ ID NO: 292, or a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 292.

[0197] In some embodiments, the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198), which is a cleavage site native to CD16 and cleavable by ADAM17. In certain embodiments, SEQ ID NO: 198 is included within the linker. In certain embodiments, the linker comprises the amino acid sequence of SGGGGSGGGGSGITQGLAVSTISSFFGGGSGGGGSGGGSLQ (SEQ ID NO 290). An exemplary nucleic acid sequence encoding SEQ ID NO: 290 is AGCGGCGGAGGTGGTAGCGGAGGCGGAGGATCTGGAATTACACAGGG ACTCGCCGTGTCTACAATCTCCAGCTTCTTTGGTGGCGGTAGTGGCGGC GGTGGCAGTGGCGGTGGATCTCTTCAA (SEQ ID NO: 291). In some embodiments, the nucleic acid encoding SEQ ID NO: 290 may comprise SEQ ID NO: 291, or a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ IDNO: 291.

[0198] The protease cleavage site can be the C-terminus of a secretable effector molecule. The protease cleavage site can be located at the N-terminus of a secretable effector molecule. Generally, for all membrane-cleavable chimeric proteins described herein, the protease cleavage site is either: (1) the C-terminus of a secretable effector molecule and the N-terminus of a cell membrane tethering domain (in other words, the protease cleavage site is between the secretable effector molecule and the cell membrane tethering domain); or (2) the N-terminus of a secretable effector molecule and the C-terminus of a cell membrane tethering domain (also between the secretable effector molecule and the cell membrane tethering domain, but with the domain orientation reversed). The protease cleavage site can be linked to the secretable effector molecule by a polypeptide linker (i.e., a polypeptide sequence that is generally not considered part of the effector molecule or the protease cleavage site). The protease cleavage site can be linked to the cell membrane tethering domain by a polypeptide linker (i.e., a polypeptide sequence that is generally not considered part of the cell membrane tethering domain or the protease cleavage site). The polypeptide linker can be any amino acid sequence that links a first polypeptide sequence and a second polypeptide sequence. The polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, the GSG linker (e.g., [GS]4GG[SEQ ID NO:347]), A(EAAAK)3A (SEQ ID NO:348), and the Whitlow linker (e.g., the "KEGS" linker, such as the amino acid sequence KESGSVSSEQLAQFRSLD [SEQ ID NO:349], the eGK linker, such as the amino acid sequence EGKSSGSGSESKST [SEQ ID NO:350], the LR1 linker, such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ [SEQ ID NO:215], the amino acid sequence GSTSGSGKPGSGEGSTKG [SEQ ID NO:395], and the linkers more specifically described in U.S. Patent No. 5,990,275, which is incorporated herein by reference). Additional exemplary polypeptide linkers include SGGGGSGGGGSG (SEQ ID NO:194), TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:196), and GGGSGGGGSGGGSLQ (SEQ ID NO:197). Other polypeptide linkers can be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those of skill in the art.An exemplary nucleic acid sequence encoding SEQ ID NO: 196 is ACCACCACACCAGCTCCTCGGCCACCAACTCCAGCTCCAACAATTGCCAGCCAGCCTCTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCAGGCGGAGCCGTGCATACAAGAGGACTGGATTTCGCCTGCGAC (SEQ ID NO: 337). In certain embodiments, the nucleic acid encoding SEQ ID NO: 196 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 337. Yet other polypeptide linkers include GGSGSGGSGS (SEQ ID NO: 396) and SAGSGSGASGSG (SEQ ID NO: 397).

[0199] In a membrane-cleavable system, after the chimeric protein is expressed and localized to the cell membrane, the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space of the cell.

[0200] Generally, the protease that cleaves the protease cleavage site is a protease specific for the particular protease cleavage site. For example, in the case of proteases of the disintegrin-metalloprotease (“ADAM”) family, the protease that cleaves a particular ADAM protease cleavage site is typically limited to an ADAM protease that specifically recognizes the motif of the particular ADAM protease cleavage site. The protease cleavage site can be selected and / or engineered such that cleavage by undesired proteases is reduced or eliminated. The protease can be membrane-bound or membrane-associated. The protease can be secreted, for example, in a particular cellular environment such as the tumor microenvironment (“TME”).

[0201] The protease that cleaves the protease cleavage site of the chimeric protein can be expressed in the same cells that express the chimeric protein. The protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to the cells that express the chimeric protein. In other words, the cells engineered to express the chimeric protein can endogenously express a protease that is specific for the protease cleavage site present in the chimeric protein. Endogenous expression of the protease refers to both expression under normal steady-state conditions (e.g., cells that are generally considered healthy) and differential expression under non-steady-state conditions (e.g., upregulated expression in tumor cells). The protease cleavage site can be selected based on known proteases endogenously expressed by the desired cell population. In such cases, generally, cleavage of the protease cleavage site (and thus release / secretion of the payload) can be limited to those cells of interest because the cell-restricted protease needs to come into contact with the protease cleavage site of the chimeric protein expressed in the same cell. For example, without wishing to be bound by theory, ADAM17 is thought to be endogenously expressed only in NK cells and T cells. Thus, selection of an ADAM17-specific protease cleavage site can limit cleavage of the protease cleavage site to NK cells and T cells that co-express the chimeric protein. In other instances, the protease cleavage site can be selected for a specific tumor-associated protease known to be expressed in the particular tumor population of interest (e.g., in specific tumor cells engineered to express the chimeric protein). Protease and / or expression databases can be used to select appropriate protease cleavage sites, such as by consulting Oncomine (www.oncomine.org), the European Bioinformatics Institute (www.ebi.ac.uk) (specifically (www.ebi.ac.uk / gxa)), PMAP (www.proteolysis.org), ExPASy Peptide Cutter (ca.expasy.org / tools / peptide cutter), and PMAP.Cut DB (cutdb.burnham.org) for protease cleavage sites cleaved by tumor-associated proteases, each of which is incorporated by reference for all purposes.

[0202] The protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to the cell expressing the chimeric protein. For example, a cell engineered to express a chimeric protein can also be engineered to express a protease that is not normally expressed by the cell, and the protease is specific for the protease cleavage site present in the chimeric protein. A cell engineered to express both the chimeric protein and the protease can be engineered to express each by a separate engineered nucleic acid or by a polycistronic system (polycistronic and multipromoter systems are described in more detail in the section entitled "Polycistronic and Multipromoter Systems" herein). The heterologous protease and its corresponding protease cleavage site can be selected as described above with reference to endogenous proteases.

[0203] The protease that cleaves the protease cleavage site of the chimeric protein can be expressed on a separate cell that is different from the cell expressing the chimeric protein. For example, the protease can generally be expressed in a specific cellular environment, such as the tumor microenvironment. In such cases, generally speaking, the cleavage of the protease cleavage site can be limited to those cellular environments of interest (e.g., the tumor microenvironment) because the environment-restricted protease needs to contact the protease cleavage site. In embodiments having a membrane-cleavable chimeric protein, generally speaking, the secretion of the effector molecule can be limited to those cellular environments of interest (e.g., the tumor microenvironment) because the environment-restricted protease needs to contact the protease cleavage site. The protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to different separate cells. The protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to different separate cells. For example, different separate cells can be engineered to express a protease that is not normally expressed by the different separate cells.

[0204] Proteases include but are not limited to type I transmembrane proteases, type II transmembrane proteases, GPI-anchored proteases, ADAM8 protease, ADAM9 protease, ADAM10 protease, ADAM12 protease, ADAM15 protease, ADAM17 protease, ADAM19 protease, ADAM20 protease, ADAM21 protease, ADAM28 protease, ADAM30 protease, ADAM33 protease, BACE1 protease, BACE2 protease, SIP protease, MT1-MMP protease, MT3-MMP protease, MT5-MMP protease, furin protease, PCSK7 protease, proteinase K protease, proteinase K-2 protease, and MMP9 protease. The protease can be the NS3 protease. The protease can be the ADAM17 protease.

[0205] The protease can be a tumor-associated protease, such as cathepsin, cysteine protease, aspartyl protease, serine protease or metalloprotease. Specific examples of tumor-associated proteases include cathepsin B, cathepsin L, cathepsin S, cathepsin D, cathepsin E, cathepsin A, cathepsin G, thrombin, plasmin, urokinase, tissue plasminogen activator, metalloprotease 1 (MMP1), MMP2, MMP3, MMP4, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP20, MMP21, MMP23, MMP24, MMP25, MMP26, MMP28, ADAM, ADAMTS, CD10 (CALLA) or prostate-specific antigen. The protease can also include, but is not limited to, the proteases listed in Table 4B below. Exemplary homologous protease cleavage sites for certain proteases are also listed in Table 4B.

[0206] Table 4B: Exemplary Proteases with Homologous Cleavage Sites and Inhibitors

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216] The protease can be any of the following human proteases (the MEROPS peptidase database numbers are provided in parentheses; Rawlings N.D., Morton F.R., Kok C.Y., Kong J. and Barrett A.J. (2008) MEROPS: the peptidase database. Nucleic Acids Res. 36 Database issue, D320 - 325; the said document is incorporated herein by reference for all purposes): pepsin A (MER000885), prochymosin (MER000894), membrane aspartic protease - 2 (MER005870), renin (MER000917), cathepsin D (MER000911), cathepsin E (MER000944), membrane aspartic protease - 1 (MER005534), ne aspartic protease A (MER004981), Mername - AA034 peptidase (MER014038), pepsin A4 (MER037290), pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens) - type putative peptidase (MER107386), ne aspartic protease B pseudogene (MER004982), CYMP g.p. (Homo sapiens) (MER002929), subfamily A1A unassigned peptidase (MER181559), mouse mammary tumor virus retropepsin (MER048030), rabbit endogenous retrovirus endopeptidase (MER043650), S71 - related human endogenous retropepsin (MER001812), RTVL - H - type putative peptidase (MER047117), RTVL - H - type putative peptidase (MER047133), RTVL - H - type putative peptidase (MER047160), RTVL - H - type putative peptidase (MER047206), RTVL - H - type putative peptidase (MER047253), RTVL - H - type putative peptidase (MER047260), RTVL - H - type putative peptidase (MER047291), RTVL - H - type putative peptidase (MER047418), RTVL - H - type putative peptidase (MER047440), RTVL - H - type putative peptidase (MER047479), RTVL - H - type putative peptidase (MER047559), RTVL - H - type putative peptidase (MER047583), RTVL - H - type putative peptidase (MER015446), human endogenous retrovirus retropepsin homolog 1 (MER015479), human endogenous retrovirus retropepsin homolog 2 (MER015481), endogenous retrovirus retropepsin pseudogene 1 (human chromosome 14) (MER029977),Endogenous retrovirus aspartyl protease pseudogene 2 (human chromosome 8) (MER029665), endogenous retrovirus aspartyl protease pseudogene 3 (human chromosome 17) (MER002660), endogenous retrovirus aspartyl protease pseudogene 3 (human chromosome 17) (MER030286), endogenous retrovirus aspartyl protease pseudogene 3 (human chromosome 17) (MER047144), endogenous retrovirus aspartyl protease pseudogene 5 (human chromosome 12) (MER029664), endogenous retrovirus aspartyl protease pseudogene 6 (human chromosome 7) (MER002094), endogenous retrovirus aspartyl protease pseudogene 7 (human chromosome 6) (MER029776), endogenous retrovirus aspartyl protease pseudogene 8 (human chromosome Y) (MER030291), endogenous retrovirus aspartyl protease pseudogene 9 (human chromosome 19) (MER029680), endogenous retrovirus aspartyl protease pseudogene 10 (human chromosome 12) (MER002848), endogenous retrovirus aspartyl protease pseudogene 11 (human chromosome 17) (MER004378), endogenous retrovirus aspartyl protease pseudogene 12 (human chromosome 11) (MER003344), endogenous retrovirus aspartyl protease pseudogene 13 (human chromosome 2 and analogs) (MER029779), endogenous retrovirus aspartyl protease pseudogene 14 (human chromosome 2) (MER029778), endogenous retrovirus aspartyl protease pseudogene 15 (human chromosome 4) (MER047158), endogenous retrovirus aspartyl protease pseudogene 15 (human chromosome 4) (MER047332), endogenous retrovirus aspartyl protease pseudogene 15 (human chromosome 4) (MER003182), endogenous retrovirus aspartyl protease pseudogene 16 (MER047165), endogenous retrovirus aspartyl protease pseudogene 16 (MER047178), endogenous retrovirus aspartyl protease pseudogene 16 (MER047200), endogenous retrovirus aspartyl protease pseudogene 16 (MER047315), endogenous retrovirus aspartyl protease pseudogene 16 (MER047405), endogenous retrovirus aspartyl protease pseudogene 16 (MER030292), endogenous retrovirus aspartyl protease pseudogene 17 (human chromosome 8) (MER005305), endogenous retrovirus aspartyl protease pseudogene 18 (human chromosome 4) (MER030288), endogenous retrovirus aspartyl protease pseudogene 19 (human chromosome 16) (MER001740), endogenous retrovirus aspartyl protease pseudogene 21 (human) (MER047222), endogenous retrovirus aspartyl protease pseudogene 21 (human) (MER047454),Endogenous retrovirus renin-like protease pseudogene 21 (human) (MER047477), Endogenous retrovirus renin-like protease pseudogene 21 (human) (MER004403), Endogenous retrovirus renin-like protease pseudogene 22 (human chromosome X) (MER030287), Subfamily A2A non-peptidase homolog (MER047046), Subfamily A2A non-peptidase homolog (MER047052), Subfamily A2A non-peptidase homolog (MER047076), Subfamily A2A non-peptidase homolog (MER047080), Subfamily A2A non-peptidase homolog (MER047088), Subfamily A2A non-peptidase homolog (MER047089), Subfamily A2A non-peptidase homolog (MER047091), Subfamily A2A non-peptidase homolog (MER047092), Subfamily A2A non-peptidase homolog (MER047093), Subfamily A2A non-peptidase homolog (MER047094), Subfamily A2A non-peptidase homolog (MER047097), Subfamily A2A non-peptidase homolog (MER047099), Subfamily A2A non-peptidase homolog (MER047101), Subfamily A2A non-peptidase homolog (MER047102), Subfamily A2A non-peptidase homolog (MER047107), Subfamily A2A non-peptidase homolog (MER047108), Subfamily A2A non-peptidase homolog (MER047109), Subfamily A2A non-peptidase homolog (MER047110), Subfamily A2A non-peptidase homolog (MER047111), Subfamily A2A non-peptidase homolog (MER047114), Subfamily A2A non-peptidase homolog (MER047118), Subfamily A2A non-peptidase homolog (MER047121), Subfamily A2A non-peptidase homolog (MER047122), Subfamily A2A non-peptidase homolog (MER047126), Subfamily A2A non-peptidase homolog (MER047129), Subfamily A2A non-peptidase homolog (MER047130), Subfamily A2A non-peptidase homolog (MER047134), Subfamily A2A non-peptidase homolog (MER047135), Subfamily A2A non-peptidase homolog (MER047137), Subfamily A2A non-peptidase homolog (MER047140), Subfamily A2A non-peptidase homolog (MER047141), Subfamily A2A non-peptidase homolog (MER047142), Subfamily A2A non-peptidase homolog (MER047148), Subfamily A2A non-peptidase homolog (MER047149), Subfamily A2A non-peptidase homolog (MER047151), Subfamily A2A non-peptidase homolog (MER047154), Subfamily A2A non-peptidase homolog (MER047155)Subfamily A2A non-peptidase homolog (MER047156), Subfamily A2A non-peptidase homolog (MER047157), Subfamily A2A non-peptidase homolog (MER047159), Subfamily A2A non-peptidase homolog (MER047161), Subfamily A2A non-peptidase homolog (MER047163), Subfamily A2A non-peptidase homolog (MER047166), Subfamily A2A non-peptidase homolog (MER047171), Subfamily A2A non-peptidase homolog (MER047173), Subfamily A2A non-peptidase homolog (MER047174), Subfamily A2A non-peptidase homolog (MER047179), Subfamily A2A non-peptidase homolog (MER047183), Subfamily A2A non-peptidase homolog (MER047186), Subfamily A2A non-peptidase homolog (MER047190), Subfamily A2A non-peptidase homolog (MER047191), Subfamily A2A non-peptidase homolog (MER047196), Subfamily A2A non-peptidase homolog (MER047198), Subfamily A2A non-peptidase homolog (MER047199), Subfamily A2A non-peptidase homolog (MER047201), Subfamily A2A non-peptidase homolog (MER047202), Subfamily A2A non-peptidase homolog (MER047203), Subfamily A2A non-peptidase homolog (MER047204), Subfamily A2A non-peptidase homolog (MER047205), Subfamily A2A non-peptidase homolog (MER047207), Subfamily A2A non-peptidase homolog (MER047208), Subfamily A2A non-peptidase homolog (MER047210), Subfamily A2A non-peptidase homolog (MER047211), Subfamily A2A non-peptidase homolog (MER047212), Subfamily A2A non-peptidase homolog (MER047213), Subfamily A2A non-peptidase homolog (MER047215), Subfamily A2A non-peptidase homolog (MER047216), Subfamily A2A non-peptidase homolog (MER047218), Subfamily A2A non-peptidase homolog (MER047219), Subfamily A2A non-peptidase homolog (MER047221), Subfamily A2A non-peptidase homolog (MER047224), Subfamily A2A non-peptidase homolog (MER047225), Subfamily A2A non-peptidase homolog (MER047226), Subfamily A2A non-peptidase homolog (MER047227), Subfamily A2A non-peptidase homolog (MER047230), Subfamily A2A non-peptidase homolog (MER047232), Subfamily A2A non-peptidase homolog (MER047233), Subfamily A2A non-peptidase homolog (MER047234)Subfamily A2A non-peptidase homolog (MER047236), Subfamily A2A non-peptidase homolog (MER047238), Subfamily A2A non-peptidase homolog (MER047239), Subfamily A2A non-peptidase homolog (MER047240), Subfamily A2A non-peptidase homolog (MER047242), Subfamily A2A non-peptidase homolog (MER047243), Subfamily A2A non-peptidase homolog (MER047249), Subfamily A2A non-peptidase homolog (MER047251), Subfamily A2A non-peptidase homolog (MER047252), Subfamily A2A non-peptidase homolog (MER047254), Subfamily A2A non-peptidase homolog (MER047255), Subfamily A2A non-peptidase homolog (MER047263), Subfamily A2A non-peptidase homolog (MER047265), Subfamily A2A non-peptidase homolog (MER047266), Subfamily A2A non-peptidase homolog (MER047267), Subfamily A2A non-peptidase homolog (MER047268), Subfamily A2A non-peptidase homolog (MER047269), Subfamily A2A non-peptidase homolog (MER047272), Subfamily A2A non-peptidase homolog (MER047273), Subfamily A2A non-peptidase homolog (MER047274), Subfamily A2A non-peptidase homolog (MER047275), Subfamily A2A non-peptidase homolog (MER047276), Subfamily A2A non-peptidase homolog (MER047279), Subfamily A2A non-peptidase homolog (MER047280), Subfamily A2A non-peptidase homolog (MER047281), Subfamily A2A non-peptidase homolog (MER047282), Subfamily A2A non-peptidase homolog (MER047284), Subfamily A2A non-peptidase homolog (MER047285), Subfamily A2A non-peptidase homolog (MER047289), Subfamily A2A non-peptidase homolog (MER047290), Subfamily A2A non-peptidase homolog (MER047294), Subfamily A2A non-peptidase homolog (MER047295), Subfamily A2A non-peptidase homolog (MER047298), Subfamily A2A non-peptidase homolog (MER047300), Subfamily A2A non-peptidase homolog (MER047302), Subfamily A2A non-peptidase homolog (MER047304), Subfamily A2A non-peptidase homolog (MER047305), Subfamily A2A non-peptidase homolog (MER047306), Subfamily A2A non-peptidase homolog (MER047307), Subfamily A2A non-peptidase homolog (MER047310), Subfamily A2A non-peptidase homolog (MER047311)Subfamily A2A non-peptidase homolog (MER047314), Subfamily A2A non-peptidase homolog (MER047318), Subfamily A2A non-peptidase homolog (MER047320), Subfamily A2A non-peptidase homolog (MER047321), Subfamily A2A non-peptidase homolog (MER047322), Subfamily A2A non-peptidase homolog (MER047326), Subfamily A2A non-peptidase homolog (MER047327), Subfamily A2A non-peptidase homolog (MER047330), Subfamily A2A non-peptidase homolog (MER047333), Subfamily A2A non-peptidase homolog (MER047362), Subfamily A2A non-peptidase homolog (MER047366), Subfamily A2A non-peptidase homolog (MER047369), Subfamily A2A non-peptidase homolog (MER047370), Subfamily A2A non-peptidase homolog (MER047371), Subfamily A2A non-peptidase homolog (MER047375), Subfamily A2A non-peptidase homolog (MER047376), Subfamily A2A non-peptidase homolog (MER047381), Subfamily A2A non-peptidase homolog (MER047383), Subfamily A2A non-peptidase homolog (MER047384), Subfamily A2A non-peptidase homolog (MER047385), Subfamily A2A non-peptidase homolog (MER047388), Subfamily A2A non-peptidase homolog (MER047389), Subfamily A2A non-peptidase homolog (MER047391), Subfamily A2A non-peptidase homolog (MER047394), Subfamily A2A non-peptidase homolog (MER047396), Subfamily A2A non-peptidase homolog (MER047400), Subfamily A2A non-peptidase homolog (MER047401), Subfamily A2A non-peptidase homolog (MER047403), Subfamily A2A non-peptidase homolog (MER047406), Subfamily A2A non-peptidase homolog (MER047407), Subfamily A2A non-peptidase homolog (MER047410), Subfamily A2A non-peptidase homolog (MER047411), Subfamily A2A non-peptidase homolog (MER047413), Subfamily A2A non-peptidase homolog (MER047414), Subfamily A2A non-peptidase homolog (MER047416), Subfamily A2A non-peptidase homolog (MER047417), Subfamily A2A non-peptidase homolog (MER047420), Subfamily A2A non-peptidase homolog (MER047423), Subfamily A2A non-peptidase homolog (MER047424), Subfamily A2A non-peptidase homolog (MER047428), Subfamily A2A non-peptidase homolog (MER047429)Subfamily A2A non-peptidase homolog (MER047431), Subfamily A2A non-peptidase homolog (MER047434), Subfamily A2A non-peptidase homolog (MER047439), Subfamily A2A non-peptidase homolog (MER047442), Subfamily A2A non-peptidase homolog (MER047445), Subfamily A2A non-peptidase homolog (MER047449), Subfamily A2A non-peptidase homolog (MER047450), Subfamily A2A non-peptidase homolog (MER047452), Subfamily A2A non-peptidase homolog (MER047455), Subfamily A2A non-peptidase homolog (MER047457), Subfamily A2A non-peptidase homolog (MER047458), Subfamily A2A non-peptidase homolog (MER047459), Subfamily A2A non-peptidase homolog (MER047463), Subfamily A2A non-peptidase homolog (MER047468), Subfamily A2A non-peptidase homolog (MER047469), Subfamily A2A non-peptidase homolog (MER047470), Subfamily A2A non-peptidase homolog (MER047476), Subfamily A2A non-peptidase homolog (MER047478), Subfamily A2A non-peptidase homolog (MER047483), Subfamily A2A non-peptidase homolog (MER047488), Subfamily A2A non-peptidase homolog (MER047489), Subfamily A2A non-peptidase homolog (MER047490), Subfamily A2A non-peptidase homolog (MER047493), Subfamily A2A non-peptidase homolog (MER047494), Subfamily A2A non-peptidase homolog (MER047495), Subfamily A2A non-peptidase homolog (MER047496), Subfamily A2A non-peptidase homolog (MER047497), Subfamily A2A non-peptidase homolog (MER047499), Subfamily A2A non-peptidase homolog (MER047502), Subfamily A2A non-peptidase homolog (MER047504), Subfamily A2A non-peptidase homolog (MER047511), Subfamily A2A non-peptidase homolog (MER047513), Subfamily A2A non-peptidase homolog (MER047514), Subfamily A2A non-peptidase homolog (MER047515), Subfamily A2A non-peptidase homolog (MER047516), Subfamily A2A non-peptidase homolog (MER047520), Subfamily A2A non-peptidase homolog (MER047533), Subfamily A2A non-peptidase homolog (MER047537), Subfamily A2A non-peptidase homolog (MER047569), Subfamily A2A non-peptidase homolog (MER047570), Subfamily A2A non-peptidase homolog (MER047584)Subfamily A2A non-peptidase homolog (MER047603), Subfamily A2A non-peptidase homolog (MER047604), Subfamily A2A non-peptidase homolog (MER047606), Subfamily A2A non-peptidase homolog (MER047609), Subfamily A2A non-peptidase homolog (MER047616), Subfamily A2A non-peptidase homolog (MER047619), Subfamily A2A non-peptidase homolog (MER047648), Subfamily A2A non-peptidase homolog (MER047649), Subfamily A2A non-peptidase homolog (MER047662), Subfamily A2A non-peptidase homolog (MER048004), Subfamily A2A non-peptidase homolog (MER048018), Subfamily A2A non-peptidase homolog (MER048019), Subfamily A2A non-peptidase homolog (MER048023), Subfamily A2A non-peptidase homolog (MER048037), Subfamily A2A unassigned peptidase (MER047164), Subfamily A2A unassigned peptidase (MER047231), Subfamily A2A unassigned peptidase (MER047386), Cutinase (MER057097), Presenilin 1 (MER005221), Presenilin 2 (MER005223), IMPAS 1 peptidase (MER019701), IMPAS 1 peptidase (MER184722), IMPAS 4 peptidase (MER019715), IMPAS 2 peptidase (MER019708), IMPAS 5 peptidase (MER019712), IMPAS 3 peptidase (MER019711), Possible family A22 pseudogene (Homo sapiens chromosome 18) (MER029974), Possible family A22 pseudogene (Homo sapiens chromosome 11) (MER023159), Cathepsin V (MER004437), Cathepsin X (MER004508), Cathepsin F (MER004980), Cathepsin L (MER000622), Cathepsin S (MER000633), Cathepsin O (MER001690), Cathepsin K (MER000644), Cathepsin W (MER003756), Cathepsin H (MER000629), Cathepsin B (MER000686), Dipeptidyl peptidase I (MER001937), Bleomycin hydrolase (animal) (MER002481), Tubulointerstitial nephritis antigen (MER016137), Tubulointerstitial nephritis antigen-related protein (MER021799), Cathepsin L-like pseudogene 1 (Homo sapiens) (MER002789), Cathepsin B-like pseudogene (chromosome 4, Homo sapiens) (MER029469)Cathepsin B-like pseudogene (chromosome 1, Homo sapiens) (MER029457), CTSLL2 g.p. (Homo sapiens) (MER005210), CTSLL3 g.p. (Homo sapiens) (MER005209), calpain-1 (MER000770), calpain-2 (MER000964), calpain-3 (MER001446), calpain-9 (MER004042), calpain-8 (MER021474), calpain-15 (MER004745), calpain-5 (MER002939), calpain-11 (MER005844), calpain-12 (MER029889), calpain-10 (MER013510), calpain-13 (MER020139), calpain-14 (MER029744), Mername-AA253 peptidase (MER005537), calpain (calpamodulin) (MER000718), hypothetical protein 940251 (MER003201), ubiquitin hydrolase-L1 (MER000832), ubiquitin hydrolase-L3 (MER000836), ubiquitin hydrolase-BAP1 (MER003989), ubiquitin hydrolase-UCH37 (MER005539), ubiquitin-specific peptidase 5 (MER002066), ubiquitin-specific peptidase 6 (MER000863), ubiquitin-specific peptidase 4 (MER001795), ubiquitin-specific peptidase 8 (MER001884), ubiquitin-specific peptidase 13 (MER002627), ubiquitin-specific peptidase 2 (MER004834), ubiquitin-specific peptidase 11 (MER002693), ubiquitin-specific peptidase 14 (MER002667), ubiquitin-specific peptidase 7 (MER002896), ubiquitin-specific peptidase 9X (MER005877), ubiquitin-specific peptidase 10 (MER004439), ubiquitin-specific peptidase 1 (MER004978), ubiquitin-specific peptidase 12 (MER005454), ubiquitin-specific peptidase 16 (MER005493), ubiquitin-specific peptidase 15 (MER005427), ubiquitin-specific peptidase 17 (MER002900), ubiquitin-specific peptidase 19 (MER005428), ubiquitin-specific peptidase 20 (MER005494), ubiquitin-specific peptidase 3 (MER005513), ubiquitin-specific peptidase 9Y (MER004314), ubiquitin-specific peptidase 18 (MER005641), ubiquitin-specific peptidase 21 (MER006258), ubiquitin-specific peptidase 22 (MER012130), ubiquitin-specific peptidase 33 (MER014335), ubiquitin-specific peptidase 29 (MER012093)Ubiquitin-specific peptidase 25 (MER011115), ubiquitin-specific peptidase 36 (MER014033), ubiquitin-specific peptidase 32 (MER014290), ubiquitin-specific peptidase 26 (Homo sapiens) (MER014292), ubiquitin-specific peptidase 24 (MER005706), ubiquitin-specific peptidase 42 (MER011852), ubiquitin-specific peptidase 46 (MER014629), ubiquitin-specific peptidase 37 (MER014633), ubiquitin-specific peptidase 28 (MER014634), ubiquitin-specific peptidase 47 (MER014636), ubiquitin-specific peptidase 38 (MER014637), ubiquitin-specific peptidase 44 (MER014638), ubiquitin-specific peptidase 50 (MER030315), ubiquitin-specific peptidase 35 (MER014646), ubiquitin-specific peptidase 30 (MER014649), Mername-AA091 peptidase (MER014743), ubiquitin-specific peptidase 45 (MER030314), ubiquitin-specific peptidase 51 (MER014769), ubiquitin-specific peptidase 34 (MER014780), ubiquitin-specific peptidase 48 (MER064620), ubiquitin-specific peptidase 40 (MER015483), ubiquitin-specific peptidase 41 (MER045268), ubiquitin-specific peptidase 31 (MER015493), Mername-AA129 peptidase (MER016485), ubiquitin-specific peptidase 49 (MER016486), Mername-AA187 peptidase (MER052579), USP17-like peptidase (MER030192), ubiquitin-specific peptidase 54 (MER028714), ubiquitin-specific peptidase 53 (MER027329), ubiquitin-specific endopeptidase 39 [misleading] (MER064621), Mername-AA090 peptidase homolog (MER014739), ubiquitin-specific peptidase 43 [misleading] (MER030140), ubiquitin-specific peptidase 52 [misleading] (MER030317), NEK2 pseudogene (MER014736), C19 pseudogene (Homo sapiens: chromosome 5) (MER029972), Mername-AA088 peptidase (MER014750), autophagin-2 (MER013564), autophagin-1 (MER013561), autophagin-3 (MER014316), autophagin-4 (MER064622), Cezanne deubiquitinating peptidase (MER029042), Cezanne-2 peptidase (MER029044), tumor necrosis factor alpha-induced protein 3 (MER029050), trabid peptidase (MER029052)VCIP135 deubiquitinating peptidase (MER152304), otubain-1 (MER029056), otubain-2 (MER029061), CylD protein (MER030104), UfSP1 peptidase (MER042724), UfSP2 peptidase (MER060306), DUBA deubiquitinating enzyme (MER086098), KIAA0459 (Homo sapiens)-like protein (MER122467), Otud1 protein (MER125457), glycosyltransferase 28 domain-containing 1, isoform CRA_c (Homo sapiens)-like (MER123606), hin1L g.p. (Homo sapiens) (MER139816), ataxin-3 (MER099998), ATXN3L putative peptidase (MER115261), Josephin domain-containing 1 (Homo sapiens) (MER125334), Josephin domain-containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), podocin (plant alpha form) (MER044591), podocin (MER001800), glycosylphosphatidylinositol:protein transamidase (MER002479), podocin pseudogene (Homo sapiens) (MER029741), family C13 unassigned peptidase (MER175813), caspase-1 (MER000850), caspase-3 (MER000853), caspase-7 (MER002705), caspase-6 (MER002708), caspase-2 (MER001644), caspase-4 (MER001938), caspase-5 (MER002240), caspase-8 (MER002849), caspase-9 (MER002707), caspase-10 (MER002579), caspase-14 (MER012083), caspase-like (MER019325), Mername-AA143 peptidase (MER021304), Mername-AA186 peptidase (MER020516), putative caspase (Homo sapiens) (MER021463), FLIP protein (MER003026), Mername-AA142 protein (MER021316), caspase-12 pseudogene (Homo sapiens) (MER019698), Mername-AA093 caspase pseudogene (MER014766), subfamily C14A non-peptidase homolog (MER185329), subfamily C14A non-peptidase homolog (MER179956), separase (Homo sapiens type) (MER011775), separase-like pseudogene (MER014797)SENP1 peptidase (MER011012), SENP3 peptidase (MER011019), SENP6 peptidase (MER011109), SENP2 peptidase (MER012183), SENP5 peptidase (MER014032), SENP7 peptidase (MER014095), SENP8 peptidase (MER016161), SENP4 peptidase (MER005557), pyroglutamyl peptidase I (chordate) (MER011032), Mername-AA073 peptidase (MER029978), sonic hedgehog (MER002539), indian hedgehog (MER002538), desert hedgehog (MER012170), dipeptidyl peptidase III (MER004252), Mername-AA164 protein (MER020410), LOC138971g.p. (Homo sapiens) (MER020074), Atp23 peptidase (MER060642), isoprenyl peptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyroglutamyl peptidase II (MER012221), cytoplasmic alanyl aminopeptidase (MER002746), cysteinyl aminopeptidase (MER002060), aminopeptidase B (MER001494), aminopeptidase PILS (MER005331), arginyl aminopeptidase-like 1 (MER012271), leukocyte-derived arginine aminopeptidase (MER002968), aminopeptidase Q (MER052595), aminopeptidase O (MER019730), Tata-binding protein-associated factor (MER026493), angiotensin-converting enzyme peptidase unit 1 (MER004967), angiotensin-converting enzyme peptidase unit 2 (MER001019), angiotensin-converting enzyme-2 (MER011061), Mername-AA153 protein (MER020514), parathion oligopeptidase (MER001737), neurolysin (MER010991), mitochondrial intermediate peptidase (MER003665), Mername-AA154 protein (MER021317), leishmanolysin-2 (MER014492), leishmanolysin-3 (MER180031), matrix metallopeptidase-1 (MER001063), matrix metallopeptidase-8 (MER001084), matrix metallopeptidase-2 (MER001080), matrix metallopeptidase-9 (MER001085), matrix metallopeptidase-3 (MER001068), matrix metallopeptidase-10 (Homo sapiens type) (MER001072), matrix metallopeptidase-11 (MER001075),Matrix metallopeptidase-7 (MER001092), Matrix metallopeptidase-12 (MER001089), Matrix metallopeptidase-13 (MER001411), Membrane-type matrix metallopeptidase-1 (MER001077), Membrane-type matrix metallopeptidase-2 (MER002383), Membrane-type matrix metallopeptidase-3 (MER002384), Membrane-type matrix metallopeptidase-4 (MER002595), Matrix metallopeptidase-20 (MER003021), Matrix metallopeptidase-19 (MER002076), Matrix metallopeptidase-23B (MER004766), Membrane-type matrix metallopeptidase-5 (MER005638), Membrane-type matrix metallopeptidase-6 (MER012071), Matrix metallopeptidase-21 (MER006101), Matrix metallopeptidase-22 (MER014098), Matrix metallopeptidase-26 (MER012072), Matrix metallopeptidase-28 (MER013587), Matrix metallopeptidase-23A (MER037217), Macrophage elastase homolog (chromosome 8, Homo sapiens) (MER030035), Mername-AA156 protein (MER021309), Matrix metallopeptidase-like 1 (MER045280), Subfamily M10A non-peptidase homolog (MER175912), Subfamily M10A non-peptidase homolog (MER187997), Subfamily M10A non-peptidase homolog (MER187998), Subfamily M10A non-peptidase homolog (MER180000), Transmembrane peptidase alpha subunit (MER001111), Transmembrane peptidase beta subunit (MER005213), Procollagen C-proteinase (MER001113), Mammalian tolloid-like 1 protein (MER005124), Mammalian tolloid-like 2 protein (MER005866), ADAMTS9 peptidase (MER012092), ADAMTS14 peptidase (MER016700), ADAMTS15 peptidase (MER017029), ADAMTS16 peptidase (MER015689), ADAMTS17 peptidase (MER016302), ADAMTS18 peptidase (MER016090), ADAMTS19 peptidase (MER015663), ADAM8 peptidase (MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382), ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15 peptidase (MER002386), ADAM17 peptidase (MER003094), ADAM20 peptidase (MER004725), ADAMDEC1 peptidase (MER000743),ADAMTS3 peptidase (MER005100), ADAMTS4 peptidase (MER005101), ADAMTS1 peptidase (MER005546), ADAM28 peptidase (human type) (MER005495), ADAMTS5 peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893), ADAMTS7 peptidase (MER005894), ADAM30 peptidase (MER006268), ADAM21 peptidase (human type) (MER004726), ADAMTS10 peptidase (MER014331), ADAMTS12 peptidase (MER014337), ADAMTS13 peptidase (MER015450), ADAM33 peptidase (MER015143), ovastacin (MER029996), ADAMTS20 peptidase (human type) (MER026906), procollagen I N-peptidase (MER004985), ADAM2 protein (MER003090), ADAM6 protein (MER047044), ADAM7 protein (MER005109), ADAM18 protein (MER012230), ADAM32 protein (MER026938), non-peptidase homolog (human chromosome 4) (MER029973), family M12 non-peptidase homolog (human chromosome 16) (MER047654), family M12 non-peptidase homolog (human chromosome 15) (MER047250), ADAM3B protein (human type) (MER005199), ADAM11 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), protein similar to ADAM21 peptidase proprotein (human) (MER026944), Mername-AA225 peptidase homolog (human) (MER047474), putative ADAM pseudogene (chromosome 4, human) (MER029975), ADAM3Ag.p. (human) (MER005200), ADAM1 g.p. (human) (MER003912), subfamily M12B non-peptidase homolog (MER188210), subfamily M12B non-peptidase homolog (MER188211), subfamily M12B non-peptidase homolog (MER188212), subfamily M12B non-peptidase homolog (MER188220), enkephalinase (MER001050), endothelin converting enzyme 1 (MER001057), endothelin converting enzyme 2 (MER004776), DINE peptidase (MER005197)Enkephalinase-2 (MER013406), Kell blood group protein (MER001054), PHEX peptidase (MER002062), i-AAA peptidase (MER001246), i-AAA peptidase (MER005755), Paraplegin (MER004454), Afg3-like protein 2 (MER005496), Afg3-like protein 1A (MER014306), Coronin-1 (MER002217), Coronin-2 (MER014521), Farnesylated protein convertase 1 (MER002646), Metalloprotease-associated protein-1 (MER030873), Aminopeptidase AMZ2 (MER011907), Aminopeptidase AMZ1 (MER058242), Carboxypeptidase A1 (MER001190), Carboxypeptidase A2 (MER001608), Carboxypeptidase B (MER001194), Carboxypeptidase N (MER001198), Carboxypeptidase E (MER001199), Carboxypeptidase M (MER001205), Carboxypeptidase U (MER001193), Carboxypeptidase A3 (MER001187), Metallocarboxypeptidase D peptidase unit 1 (MER003781), Metallocarboxypeptidase Z (MER003428), Metallocarboxypeptidase D peptidase unit 2 (MER004963), Carboxypeptidase A4 (MER013421), Carboxypeptidase A6 (MER013456), Carboxypeptidase A5 (MER017121), Metallocarboxypeptidase O (MER016044), Cytoplasmic carboxypeptidase-like protein 5 (MER033174), Cytoplasmic carboxypeptidase 3 (MER033176), Cytoplasmic carboxypeptidase 6 (MER033178), Cytoplasmic carboxypeptidase 1 (MER033179), Cytoplasmic carboxypeptidase 2 (MER037713), Metallocarboxypeptidase D non-peptidase unit (MER004964), Adipocyte enhancer-binding protein 1 (MER003889), Carboxypeptidase-like protein X1 (MER013404), Carboxypeptidase-like protein X2 (MER078764), Cytoplasmic carboxypeptidase (MER026952), Family M14 non-peptidase homolog (MER199530), Insulinase (MER001214), Mitochondrial processing peptidase beta subunit (MER004497), Phenelzine lyase (MER003883), Eupitrilysin (MER004877), Mitochondrial processing peptidase non-peptidase alpha subunit (MER001413), Ubiquinol-cytochrome c reductase core protein I (MER003543), Ubiquinol-cytochrome c reductase core protein II (MER003544), Ubiquinol-cytochrome c reductase core domain 2 (MER043998), Insulinase unit 2 (MER046821), Phenelzine lyase unit 2 (MER046874),Insulinase unit 3 (MER078753), mitochondrial processing peptidase subunit alpha unit 2 (MER124489), phenelzine lyase unit 3 (MER142856), LOC133083g.p. (Homo sapiens) (MER021876), subfamily M16B non-peptidase homolog (MER188757), leucyl aminopeptidase (animal) (MER003100), Mername-AA040 peptidase (MER003919), leucyl aminopeptidase-1 (Caenorhabditis elegans type) (MER013416), methionyl aminopeptidase 1 (MER001342), methionyl aminopeptidase 2 (MER001728), aminopeptidase P2 (MER004498), Xaa-Pro dipeptidase (eukaryotic cell) (MER001248), aminopeptidase P1 (MER004321), mitochondrial intermediate peptidase 55 kDa (MER013463), mitochondrial methionyl aminopeptidase (MER014055), Mername-AA020 peptidase homolog (MER010972), proliferation-associated protein 1 (MER005497), chromatin-specific transcription elongation factor 140 kDa subunit (MER026495), proliferation-associated protein 1-like (Homo sapiens chromosome X) (MER029983), Mername-AA226 peptidase homolog (Homo sapiens) (MER056262), Mername-AA227 peptidase homolog (Homo sapiens) (MER047299), subfamily M24A non-peptidase homolog (MER179893), aspartyl aminopeptidase (MER003373), Gly-Xaa carboxypeptidase (MER033182), carnosinase II (MER014551), carnosinase I (MER015142), Mername-AA161 protein (MER021873), aminoacylase (MER001271), glutamate carboxypeptidase II (MER002104), NAALADASE L peptidase (MER005239), glutamate carboxypeptidase III (MER005238), plasma glutamate carboxypeptidase (MER005244), Mername-AA103 peptidase (MER015091), Fxna peptidase (MER029965), transferrin receptor protein (MER002105), transferrin receptor 2 protein (MER005152), glutaminyl cyclase (MER015095), glutamate carboxypeptidase II (Homo sapiens) type non-peptidase homolog (MER026971), nicalin (MER044627), membrane dipeptidase (MER001260), membrane-bound dipeptidase-2 (MER013499), membrane-bound dipeptidase-3 (MER013496), dihydroorotase (MER005767),Dihydropyrimidinase (MER033266), Dihydropyrimidinase-related protein-1 (MER030143), Dihydropyrimidinase-related protein-2 (MER030155), Dihydropyrimidinase-related protein-3 (MER030151), Dihydropyrimidinase-related protein-4 (MER030149), Dihydropyrimidinase-related protein-5 (MER030136), Hypothetical protein-like 5730457F11RIK (MER033184), 1300019j08rik protein (MER033186), Guanine aminohydrolase (MER037714), Kae1 putative peptidase (MER001577), OSGEPL1-like protein (MER013498), S2P peptidase (MER004458), Subfamily M23B non-peptidase homolog (MER199845), Subfamily M23B non-peptidase homolog (MER199846), Subfamily M23B non-peptidase homolog (MER199847), Subfamily M23B non-peptidase homolog (MER137320), Subfamily M23B non-peptidase homolog (MER201557), Subfamily M23B non-peptidase homolog (MER199417), Subfamily M23B non-peptidase homolog (MER199418), Subfamily M23B non-peptidase homolog (MER199419), Subfamily M23B non-peptidase homolog (MER199420), Subfamily M23B non-peptidase homolog (MER175932), Subfamily M23B non-peptidase homolog (MER199665), Poh1 peptidase (MER020382), Jab1 / MPN domain metalloenzyme (MER022057), Mername-AA165 peptidase (MER021865), Brcc36 isopeptidase (MER021890), Histone H2A deubiquitinase MYSM1 (MER021887), AMSH deubiquitin peptidase (MER030146), Putative peptidase (Homo sapiens chromosome 2) (MER029970), Mername-AA168 protein (MER021886), COP9 signalosome subunit 6 (MER030137), 26S proteasome non-ATPase regulatory subunit 7 (MER030134), Eukaryotic translation initiation factor 3 subunit 5 (MER030133), IFP38 peptidase homolog (MER030132), Subfamily M67A non-peptidase homolog (MER191181), Subfamily M67A unassigned peptidase (MER191144), Granzyme B (Homo sapiens type) (MER000168), Testisin (MER005212), Tryptase beta (MER000136), Kallikrein-related peptidase 5 (MER005544), Corin (MER005881),Kallikrein-related peptidase 12 (MER006038), DESC1 peptidase (MER006298), tryptase gamma 1 (MER011036), kallikrein-related peptidase 14 (MER011038), hyaluronan-binding peptidase (MER003612), transmembrane peptidase, serine 4 (MER011104), intestinal serine peptidase (cariogenic animals) (MER016130), adrenal secretory serine peptidase (MER003734), tryptase delta 1 (Homo sapiens) (MER005948), proteolytic enzyme-3 (MER029902), marapsin (MER006119), tryptase-6 (MER006118), ovum protease-1 domain 1 (MER099182), transmembrane peptidase, serine 3 (MER005926), kallikrein-related peptidase 15 (MER000064), Mername-AA031 peptidase (MER014054), TMPRSS13 peptidase (MER014226), Mername-AA038 peptidase (MER062848), Mername-AA204 peptidase (MER029980), cationic trypsin (Homo sapiens type) (MER000020), elastase-2 (MER000118), mannan-binding lectin-associated serine peptidase-3 (MER031968), cathepsin G (MER000082), medullasin (MER000170), granzyme A (MER001379), granzyme M (MER001541), chymotrypsin (Homo sapiens type) (MER000123), tryptase alpha (MER000135), granzyme K (MER001936), granzyme H (MER000166), chymotrypsin B (MER000001), elastase-1 (MER003733), pancreatic endopeptidase E (MER000149), pancreatic elastase II (MER000146), enteropeptidase (MER002068), chymotrypsin C (MER000761), prostasin (MER002460), kallikrein 1 (MER000093), kallikrein-related peptidase 2 (MER000094), kallikrein-related peptidase 3 (MER000115), mesotrypsin (MER000022), complement component C1r-like peptidase (MER016352), complement factor D (MER000130), complement component C1r activator (MER000238), complement component C1s activator (MER000239), complement component C2a (MER000231), complement factor B (MER000229), mannan-binding lectin-associated serine peptidase 1 (MER000244), complement factor I (MER000228),Pancreatic endopeptidase E form B (MER000150), pancreatic elastase IIB (MER000147), coagulation factor XIIa (MER000187), plasma kallikrein (MER000203), coagulation factor Xia (MER000210), coagulation factor IXa (MER000216), coagulation factor Vila (MER000215), coagulation factor Xa (MER000212), thrombin (MER000188), protein C (activated) (MER000222), acrosin (MER000078), hepsin (MER000156), hepatocyte growth factor activator (MER000186), mannan-binding lectin-associated serine peptidase 2 (MER002758), u-plasminogen activator (MER000195), t-plasminogen activator (MER000192), plasmin (MER000175), kallikrein-related peptidase 6 (MER002580), neurotrypsin (MER004171), kallikrein-related peptidase 8 (MER005400), kallikrein-related peptidase 10 (MER003645), epitheliasin (MER003736), kallikrein-related peptidase 4 (MER005266), prosemin (MER004214), chymopasin (MER001503), kallikrein-related peptidase 11 (MER004861), kallikrein-related peptidase 11 (MER216142), trypsin-2 type A (MER000021), HtrA1 peptidase (Homo sapiens type) (MER002577), HtrA2 peptidase (MER208413), HtrA2 peptidase (MER004093), HtrA3 peptidase (MER014795), HtrA4 peptidase (MER016351), Tysnd1 peptidase (MER050461), TMPRSS12 peptidase (MER017085), HAT-like putative peptidase 2 (MER021884), trypsin C (MER021898), kallikrein-related peptidase 7 (MER002001), proteinase (MER003735), kallikrein-related peptidase 13 (MER005269), kallikrein-related peptidase 9 (MER005270), proteinase-2 (MER005278), umbilical vein peptidase (MER005421), LCLP peptidase (MER001900), notochord protein (MER014385), marapsin-2 (MER021929), complement factor D-like putative peptidase (MER056164), ooplasmase-2 (MER022410), HAT-like 4 peptidase (MER044589),Ooplasm enzyme 1 domain 1 (MER022412), Epidermis-specific SP-like putative peptidase (MER029900), Testis serine peptidase 5 (MER029901), Mername-AA258 peptidase (MER000285), Polymerase-IA unit 1 (MER030879), Polymerase-IA unit 2 (MER030880), Testis serine peptidase 2 (human type) (MER033187), Hypothetical spermase-like peptidase (Homo sapiens) (MER033253), HAT-like 5 peptidase (MER028215), Polymerase-3 unit 1 (MER061763), Polymerase-3 unit 2 (MER061748), Peptidase similar to tryptophan / serine protease (MER056263), Polymerase-2 unit 1 (MER061777), Mername-AA123 peptidase (MER021930), HAT-like 2 peptidase (MER099184), hCG2041452-like protein (MER099172), hCG22067 (Homo sapiens) (MER099169), Brain rescue factor-1 (Homo sapiens) (MER098873), hCG2041108 (Homo sapiens) (MER099173), Polymerase-2 unit 2 (MER061760), Polymerase-2 unit 3 (MER065694), Mername-AA201 (peptidase homolog) MER099175, Secreted trypsin-like serine peptidase homolog (MER030000), Polymerase 1A unit 3 (MER029880), Azurocidin (MER000119), Haptoglobin-1 (MER000233), Haptoglobin-related protein (MER000235), Macrophage-stimulating protein (MER001546), Hepatocyte growth factor (MER000185), Protein Z (MER000227), TESP1 protein (MER047214), LOC136242 protein (MER016132), Plasma kallikrein protein 4 (MER016346), PRSS35 protein (MER016350), DKFZp586H2123-like protein (MER066474), Apolipoprotein (MER000183), psi-KLK1 pseudogene (Homo sapiens) (MER033287), Trypsin-like pseudogene I (MER015077), Trypsin-like pseudogene II (MER015078), Trypsin-like pseudogene III (MER015079), Subfamily S1A unassigned peptidase (MER216982), Subfamily S1A unassigned peptidase (MER216148), Amidophosphoribosyltransferase (MER003314), Glutamine-fructose-6-phosphate transaminase 1 (MER003322),Glutamine:fructose-6-phosphate aminotransferase (MER012158), Mername-AA144 protein (MER021319), asparagine synthetase (MER033254), family C44 non-peptidase homologue (MER159286), family C44 unassigned peptidase (MER185625), family C44 unassigned peptidase (MER185626), secernin 1 (MER045376), secernin 2 (MER064573), secernin 3 (MER064582), acid ceramidase precursor (MER100794), N-acylethanolamine acid amidase precursor (MER141667), proteasome catalytic subunit 1 (MER000556), proteasome catalytic subunit 2 (MER002625), proteasome catalytic subunit 3 (MER002149), proteasome catalytic subunit 1i (MER000552), proteasome catalytic subunit 2i (MER001515), proteasome catalytic subunit 3i (MER000555), proteasome catalytic subunit 5t (MER026203), protein serine kinase c17 (MER026497), proteasome subunit α6 (MER000557), proteasome subunit α2 (MER000550), proteasome subunit α4 (MER000554), proteasome subunit α7 (MER033250), proteasome subunit α5 (MER000558), proteasome subunit α1 (MER000549), proteasome subunit 3 (MER000553), proteasome subunit XAPC7 (MER004372), proteasome subunit β3 (MER001710), proteasome subunit β2 (MER002676), proteasome subunit β1 (MER000551), proteasome subunit β4 (MER001711), Mername-AA230 peptidase homologue (Homo sapiens) (MER047329), Mername-AA231 pseudogene (Homo sapiens) (MER047172), Mername-AA232 pseudogene (Homo sapiens) (MER047316), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), taspase-1 (MER016969), γ-glutamyltransferase 5 (mammalian type) (MER001977), γ-glutamyltransferase 1 (mammalian type) (MER001629), γ-glutamyltransferase 2 (Homo sapiens) (MER001976), γ-glutamyltransferase-like protein 4 (MER002721), γ-glutamyltransferase-like protein 3 (MER016970), similar to γ-glutamyltransferase 1 precursor (Homo sapiens) (MER026204)Similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Mername-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyl transpeptidase homolog (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (MER172554), gamma-glutamyl hydrolase (MER002963), guanosine 5''-monophosphate synthase (MER043387), carbamoyl-phosphate synthase (Homo sapiens type) (MER078640), dihydroorotase (N-terminal unit) (Homo sapiens type) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase (MER014802), Mername-AA101 non-peptidase homolog (MER014803), KIAA0361 protein (Homo sapiens type) (MER042827), F1134283 protein (Homo sapiens) (MER044553), non-peptidase homolog chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 non-peptidase homolog (MER177016), family C56 non-peptidase homolog (MER176613), family C56 non-peptidase homolog (MER176918), mucin-like hormone receptor-like 2 containing EGF-like modules (MER037230), CD97 antigen (Homo sapiens type) (MER037286), mucin-like hormone receptor-like 3 containing EGF-like modules (MER037288), mucin-like hormone receptor-like 1 containing EGF-like modules (MER037278), mucin-like hormone receptor-like 4 containing EGF-like modules (MER037294), cadherin EGF LAG seven-pass G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), latrotoxin receptor 2 (MER122199), latrotoxin receptor-1 (MER126380), latrotoxin receptor 3 (MER124612), protocadherin Flamingo2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven transmembrane helix receptor (MER125448), Gpr114 protein (MER159320), GPR126 vascular-inducible G protein-coupled receptor (MER140015), GPR125 (Homo sapiens) type protein (MER159279), GPR116 (Homo sapiens) type G protein-coupled receptor (MER159280)GPR128 (Homo sapiens) type G protein-coupled receptor (MER162015), GPR133 (Homo sapiens) type protein (MER159334), GPR110 G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), unassigned homolog (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD auto-processing protein unit 1 (MER020001), PIDD auto-processing protein unit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 protease (MER001948), furin (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER000377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl peptidase II (MER000355), subfamily S8A non-peptidase homolog (MER201339), subfamily S8A non-peptidase homolog (MER191613), subfamily S8A unassigned peptidase (MER191611), subfamily S8A unassigned peptidase (MER191612), subfamily S8A unassigned peptidase (MER191614), tripeptidyl peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl peptidase IV (eukaryotic cell) (MER000401), amidopeptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl peptidase 8 (MER013484), dipeptidyl peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Mername-AA194 putative peptidase (MER017353), Mername-AA195 putative peptidase (MER017367), Mername-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), hypothetical esterase / lipase / thioesterase (MER047309), protein bat5 (MER037840)Hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DPP10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213), hepatic carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4X-linked (MER033235), neuroligin 4Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homolog (MER212939), family S9 non-peptidase homolog (MER211490), subfamily S9C unassigned peptidase (MER192341), family S9 unassigned peptidase (MER209181), family S9 unassigned peptidase (MER200434), family S9 unassigned peptidase (MER209507), family S9 unassigned peptidase (MER209142), serine carboxypeptidase A neuroligin (MER000430), vitelline carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unassigned peptidase (MER199442), family S15 unassigned peptidase (MER200437), family S15 unassigned peptidase (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl peptidase I (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), protein containing autohydrolase domain 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcription protein (MER199890), mesoderm-specific transcription protein (MER017123),Cytosolic epoxide hydrolase (MER029997), cytosolic epoxide hydrolase (MER213866), similar to hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein 922408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1 interacting factor b (MER210738), glycosylasparaginase precursor (MER003299), isodipeptidase (threonine type) (MER031622), taspase-1 (MER016969), gamma-glutamyltransferase 5 (mammalian type) (MER001977), gamma-glutamyltransferase 1 (mammalian type) (MER001629), gamma-glutamyltransferase 2 (Homo sapiens) (MER001976), gamma-glutamyltransferase-like protein 4 (MER002721), gamma-glutamyltransferase-like protein 3 (MER016970), similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204), similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Mername-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyl transpeptidase homolog (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (MER172554), gamma-glutamyl hydrolase (MER002963), guanosine 5''-monophosphate synthase (MER043387), carbamoyl phosphate synthetase (Homo sapiens type) (MER078640), dihydroorotase (N-terminal unit) (Homo sapiens type) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase (MER014802), Mername-AA101 non-peptidase homolog (MER014803), KIAA0361 protein (Homo sapiens type) (MER042827), F1134283 protein (Homo sapiens) (MER044553), non-peptidase homolog chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 non-peptidase homolog (MER177016), family C56 non-peptidase homolog (MER176613), family C56 non-peptidase homolog (MER176918)EGF-like module-containing mucin-like hormone receptor-like 2 (MER037230), CD97 antigen (human form) (MER037286), EGF-like module-containing mucin-like hormone receptor-like 3 (MER037288), EGF-like module-containing mucin-like hormone receptor-like 1 (MER037278), EGF-like module-containing mucin-like hormone receptor-like 4 (MER037294), cadherin EGF LAG seven-pass G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), latrophilin receptor 2 (MER122199), latrophilin receptor-1 (MER126380), latrophilin receptor 3 (MER124612), protocadherin Flamingo 2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven transmembrane helix receptor (MER125448), Gpr114 protein (MER159320), GPR126 angiogenesis-inducible G protein-coupled receptor (MER140015), GPR125 (Homo sapiens) type protein (MER159279), GPR116 (Homo sapiens) type G protein-coupled receptor (MER159280), GPR128 (Homo sapiens) type G protein-coupled receptor (MER162015), GPR133 (Homo sapiens) type protein (MER159334), GPR110 G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), unassigned homolog (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD autoprocessing protein unit 1 (MER020001), PIDD autoprocessing protein unit 2 (MER063690), MUC1 autocleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 protease (MER001948), furin protease (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER000377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl peptidase II (MER000355),Subfamily S8A non-peptidase homolog (MER201339), subfamily S8A non-peptidase homolog (MER191613), subfamily S8A unassigned peptidase (MER191611), subfamily S8A unassigned peptidase (MER191612), subfamily S8A unassigned peptidase (MER191614), tripeptidyl peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl peptidase IV (eukaryotic cell) (MER000401), acylaminoacyl peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl peptidase 8 (MER013484), dipeptidyl peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Mername-AA194 putative peptidase (MER017353), Mername-AA195 putative peptidase (MER017367), Mername-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), hypothetical esterase / lipase / thioesterase (MER047309), protein bat5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DPP10 (MER005988), protein similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213), hepatic carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4 X-linked (MER033235), neuroligin 4 Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neuroligin 1 (MER033280),Neurexin 2 (MER033283), family S9 non-peptidase homologue (MER212939), family S9 non-peptidase homologue (MER211490), subfamily S9C unassigned peptidase (MER192341), family S9 unassigned peptidase (MER209181), family S9 unassigned peptidase (MER200434), family S9 unassigned peptidase (MER209507), family S9 unassigned peptidase (MER209142), serine carboxypeptidase A (MER000430), vitelline carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unassigned peptidase (MER199442), family S15 unassigned peptidase (MER200437), family S15 unassigned peptidase (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), protein containing autohydrolase domain 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcription protein (MER199890), mesoderm-specific transcription protein (MER017123), cytoplasmic epoxide hydrolase (MER029997), cytoplasmic epoxide hydrolase (MER213866), similar to hypothetical protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), hypothetical protein flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1 interacting factor b (MER210738).

[0217] The enzymatic activity of a protease can be modulated. For example, certain proteases can be inactivated in the presence or absence of a specific reagent (e.g., a reagent that binds to the protease, such as a specific small molecule inhibitor). Such proteases can be referred to as "repressible proteases". Exemplary inhibitors of certain proteases are listed in Table 4B. For example, the NS3 protease can be repressed by protease inhibitors including but not limited to: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sofosbuvir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxilaprevir. In another example, protease enzymatic activity can be modulated by regulating the expression of the protease itself, such as engineering a cell to express the protease using an inducible promoter system (e.g., the Tet On / Off system) or a cell-specific promoter (promoters useful for expressing heterologous proteases are described in more detail in the section entitled "Promoters" herein). A protease can also contain a degron, such as any degron described herein, and can be regulated using any degron system described herein.

[0218] The enzymatic activity of a protease can also be modulated by selecting a specific protease cleavage site. For example, a protease cleavage site can be selected and / or engineered such that the sequence exhibits a desired rate of cleavage by the desired protease, such as reduced cleavage kinetics relative to the endogenous sequence of a substrate that is naturally cleaved by the desired protease. As another example, a protease cleavage site can be selected and / or engineered such that the sequence exhibits a desired cleavage rate in a cell state-specific manner. For example, various cell states (e.g., after cell signaling such as immune cell activation) can affect the expression and / or localization of certain proteases. As an illustrative example, it is known that the protein level and localization of ADAM17 are affected by signaling, such as through the protein kinase C (PKC) signaling pathway (e.g., activation by the PKC activator phorbol-12-myristate-13-acetate [PMA]). Thus, a protease cleavage site can be selected and / or engineered such that cleavage of the protease cleavage site and subsequent release of effector molecules can be increased or decreased as needed, depending on the protease properties (e.g., expression and / or localization) in a particular cell state. As another example, a protease cleavage site (especially in combination with a specific membrane tethering domain) can be selected and / or engineered to achieve optimal protein expression of a chimeric protein.

[0219] Cell membrane tethering domain

[0220] The membrane-cleavable chimeric proteins provided herein include a cell membrane tethering domain (referred to as "MT" in the formula S-C-MT or MT-C-S). Generally speaking, the cell membrane tethering domain can be any amino acid sequence motif capable of directing the chimeric protein to localize to the cell membrane of the cell expressing the chimeric protein (e.g., inserted into the cell membrane) or otherwise associate with it. The cell membrane tethering domain can be a transmembrane-intracellular domain. The cell membrane tethering domain can be a transmembrane domain. The cell membrane tethering domain can be a complete membrane protein domain (e.g., a transmembrane domain). The cell membrane tethering domain can be derived from a type I, type II, or type III transmembrane protein. The cell membrane tethering domain can include a post-translational modification tag or a motif capable of modifying the chimeric protein by post-translational modification to include a post-translational modification tag, wherein the post-translational modification tag permits attachment to the cell membrane. Examples of post-translational modification tags include, but are not limited to, lipid anchor domains (e.g., GPI lipid anchor, myristoylation tag, or palmitoylation tag). Examples of cell membrane tethering domains include, but are not limited to, transmembrane-intracellular domains and / or transmembrane domains derived from PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA. The cell membrane tethering domain can be a cell surface receptor or a cell membrane-binding portion thereof. Sequences of exemplary cell membrane tethering domains are provided in Table 4C.

[0221] Table 4C.

[0222]

[0223] Generally, for all membrane-cleavable chimeric proteins described herein, the cell membrane tethering domain is any one of the following: (1) the C-terminus of the protease cleavage site and the N-terminus of any intracellular domain (if present) (in other words, the cell membrane tethering domain is between the protease cleavage site and the intracellular domain (if present)); or (2) the N-terminus of the protease cleavage site and the C-terminus of any intracellular domain (if present) (also between the protease cleavage site and the intracellular domain (if present), but with the domain orientation reversed). In embodiments characterized by a degron associated with the chimeric protein, the degron domain is a terminal cytoplasmic targeting domain, particularly relative to the cell membrane tether (in other words, the cell membrane tethering domain is located between the protease cleavage site and the degron). The cell membrane tethering domain can be linked to the protease cleavage site by a polypeptide linker, i.e., a polypeptide sequence that is not generally considered to be part of the cell membrane tethering domain or the protease cleavage site. The cell membrane tethering domain can be linked to the intracellular domain (if present) by a polypeptide linker (i.e., a polypeptide sequence that is not generally considered to be part of the cell membrane tethering domain or the intracellular domain). The cell membrane tethering domain can be linked to the degron (if present) by a polypeptide linker (i.e., a polypeptide sequence that is not generally considered to be part of the cell membrane tethering domain or the degron). The polypeptide linker can be any amino acid sequence that links a first polypeptide sequence and a second polypeptide sequence. The polypeptide linker can be a flexible linker (e.g., the Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, the GSG linker (e.g., [GS]4GG[SEQ ID NO:347]), A(EAAAK)3A (SEQ ID NO:348), and the Whitlow linker (e.g., the "KEGS" linker, such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO:349), the eGK linker, such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO:350), the LR1 linker, such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO:215), and the linkers more particularly described in U.S. Patent No. 5,990,275, which is incorporated herein by reference). Additional polypeptide linkers include SEQ ID NO:194, SEQ ID NO:196, and SEQ ID NO:197. Other polypeptide linkers can be selected according to desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those of skill in the art.

[0224] Generally speaking, the cell membrane tethering domain is oriented such that the secreted effector molecule and the protease cleavage site are exposed extracellularly after insertion into or attachment to the cell membrane, so that the protease cleavage site can be cleaved by its corresponding protease and the effector molecule is released ("secreted") into the extracellular space.

[0225] Degron systems and domains

[0226] In some embodiments, any protein described herein can include a degron domain, including but not limited to cytokines, CARs, proteases, transcription factors, components of a promoter or promoter system (e.g., ACP), and / or any membrane-cleavable chimeric protein described herein. Generally, a degron domain can be any amino acid sequence motif capable of directing regulated degradation (such as regulated degradation via the ubiquitin-mediated pathway). In the presence of an immunomodulatory drug (IMiD), the degron domain directs the degradation of the degron-fusion protein via ubiquitin-mediated degradation.

[0227] The degron domain can be a CRBN polypeptide substrate domain capable of binding to cereblon (CRBN) in response to an immunomodulatory drug (IMiD), and the immunomodulatory drugs include but are not limited to IKZF1, IKZF3, CK1a, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827 or fragments thereof that can undergo drug-induced binding to CRBN. The CRBN polypeptide substrate domain can be a chimeric fusion product of a native CRBN polypeptide sequence, such as an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLR HIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 175). The degron domain and specifically the CRBN degron system are described in more detail in International Application Publication No. WO2019 / 089592A1, which is incorporated herein by reference for all purposes.Other examples of degron domains include, but are not limited to, the HCV NS4 degron PEST (two copies of residues 277 - 307 of human IκBα; SEQ ID NO:161), GRR (residues 352 - 408 of human p105; SEQ ID NO:162), DRR (residues 210 - 295 of yeast Cdc34; SEQ ID NO:163), SNS (tandem repeat of SP2 and NB (SP2 - NB - SP2 of influenza A virus or influenza B virus; e.g., SEQ ID NO:164), RPB (four copies of residues 1688 - 1702 of yeast RPB; SEQ ID NO:165), Spmix (tandem repeat of SP1 and SP2 (SP2 - SP1 - SP2 - SP1 - SP2 of influenza A virus M2 protein; SEQ ID NO:166), NS2 (three copies of residues 79 - 93 of influenza A virus NS protein; SEQ ID NO:167), ODC (residues 106 - 142 of ornithine decarboxylase; SEQ ID NO:168), Nek2A, mouse ODC (residues 422–461; SEQ ID NO:169), mouse ODC_DA (residues 422 - 461 of mODC, containing D433A and D434A point mutations), APC / C degron, COP1 E3 ligase - binding degron motif, CRL4 - Cdt2 - binding PIP degron, actinfilin - binding degron, KEAP1 - binding degron, KLHL2 and KLHL3 - binding degron, MDM2 - binding motif, N - degron, hydroxyproline modification in hypoxia signaling, plant hormone - dependent SCF - LRR - binding degron, SCF ubiquitin ligase - binding phospho - degron, plant hormone - dependent SCF - LRR - binding degron, DSGxxS phospho - dependent degron (SEQ ID NO:345), Siah - binding motif, SPOP SBC docking motif, or PCNA - binding PIP box.

[0228] The regulated degradation can be drug-induced. The drug capable of mediating / regulating the degradation can be a small molecule compound. The drug capable of mediating / regulating the degradation can include "immunomodulatory drugs" (IMiDs). Generally, as used herein, IMiDs refer to a class of small molecule immunomodulatory drugs containing an imide group. Cereblon (CRBN) is a known IMiD target, and the binding of an IMiD to CRBN or the CRBN polypeptide substrate domain alters the substrate specificity of the CRBN E3 ubiquitin ligase complex, resulting in the degradation of proteins having a CRBN polypeptide substrate domain (e.g., any of the secreted effector molecules or other proteins of interest described herein). Examples of imide-containing IMiDs for a degron having a CRBN polypeptide substrate domain include, but are not limited to, thalidomide, lenalidomide, or pomalidomide. IMiDs can be FDA-approved drugs.

[0229] The proteins described herein can contain a degron domain (e.g., referred to as "D" in the formula S-C-MT-D or D-MT-C-S of the membrane-cleavable chimeric proteins described herein). In the absence of an IMiD, degron / ubiquitin-mediated degradation of the chimeric protein does not occur. After the chimeric protein is expressed and localized to the cell membrane, the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released ("secreted") into the extracellular space. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of the chimeric protein, resulting in reduced or eliminated secretion of the effector molecule. Generally, for a membrane-cleavable chimeric protein fused to a degron domain, the degron domain is a terminal cytoplasmic targeting domain, particularly relative to the cell membrane tethering domain, such as the most C-terminal domain in the formula S-C-MT-D or the most N-terminal domain in the formula D-MT-C-S. The degron domain can be linked to the cell membrane tethering domain by a polypeptide linker (i.e., a polypeptide sequence that is generally not considered part of the cell membrane tethering domain or the degron domain). The polypeptide linker can be any amino acid sequence that links a first polypeptide sequence and a second polypeptide sequence. The polypeptide linker can be a flexible linker (e.g., the Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, the GSG linker (e.g., [GS]4GG[SEQ ID NO:347]), A(EAAAK)3A (SEQ ID NO:348), and the Whitlow linker (e.g., the "KEGS" linker, such as the amino acid sequence KESGSVSSEQLAQFRSLD [SEQ ID NO:349], the eGK linker, such as the amino acid sequence EGKSSGSGSESKST [SEQ ID NO:350], the LR1 linker, such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ [SEQ ID NO:215], and the linkers more specifically described in U.S. Patent No. 5,990,275, which is incorporated herein by reference). Additional polypeptide linkers include SEQ ID NO:194, SEQ ID NO:196, and SEQ ID NO:197. Other polypeptide linkers can be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those of skill in the art. Generally, the degron is oriented relative to the cell membrane tethering domain such that the degron is exposed to the cytoplasm after localization to the cell membrane, such that the degron domain can mediate degradation (e.g., exposure to the cytoplasm and cytosol) and can mediate ubiquitin-mediated degradation.

[0230] For a degron-fusion protein, the degron domain can be located at the N-terminus or C-terminus of the protein of interest, e.g., an effector molecule. The degron domain can be linked to the protein of interest via a polypeptide linker, i.e., a polypeptide sequence that is not normally considered part of the protein of interest or the degron domain. The polypeptide linker can be any amino acid sequence that links a first polypeptide sequence and a second polypeptide sequence. The polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, the GSG linker (e.g., [GS]4GG [SEQ ID NO:347]), A(EAAAK)3A (SEQ ID NO:348), and the Whitlow linker (e.g., the "KEGS" linker, such as the amino acid sequence KESGSVSSEQLAQFRSLD [SEQ ID NO:349], the eGK linker, such as the amino acid sequence EGKSSGSGSESKST [SEQ ID NO:350], the LR1 linker, such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ [SEQ ID NO:215], and the linkers more particularly described in U.S. Patent No. 5,990,275, which is incorporated herein by reference). Additional polypeptide linkers include SEQ ID NO:194, SEQ ID NO:196, and SEQ ID NO:197. Other polypeptide linkers can be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those of skill in the art. The polypeptide linker can be cleavable, e.g., any protease cleavage site described herein.

[0231] Engineered nucleic acid

[0232] Provided herein are engineered nucleic acids (e.g., expression cassettes) encoding at least one protein of the present disclosure, such as a cytokine, a CAR, an ACP, and / or a membrane-cleavable chimeric protein having the formula S-C-MT or MT-C-S described herein. Also provided are engineered nucleic acids (e.g., expression cassettes) encoding two or more proteins, such as two or more of a cytokine, a CAR, an ACP, and / or a membrane-cleavable chimeric protein having the formula S-C-MT or MT-C-S described herein.

[0233] In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine, CAR, ACP, and / or a membrane-cleavable chimeric protein, oriented from the N-terminus to the C-terminus, having the formula: S-C-MT or MT-C-S. S refers to a secretable effector molecule. C refers to a protease cleavage site. MT refers to a cell membrane tethering domain. The promoter is operably linked to the exogenous polynucleotide sequence, and the S-C-MT or MT-C-S is configured to be expressed as a single polypeptide.

[0234] In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine. In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a CAR. In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein having a protein of interest (e.g., any effector molecule described herein). The promoter is operably linked to the exogenous polynucleotide sequence and the membrane-cleavable chimeric protein is configured to be expressed as a single polypeptide.

[0235] In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a combination of a cytokine, CAR, ACP, and / or a membrane-cleavable chimeric protein described herein. In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine and a CAR. In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine and an ACP.

[0236] In certain embodiments described herein, the engineered nucleic acid encodes two or more expression cassettes, each expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a cytokine, CAR, ACP, and / or a membrane-cleavable chimeric protein described herein. In certain embodiments described herein, the engineered nucleic acid encodes two or more expression cassettes, each expression cassette containing a promoter, and each expression cassette separately encoding an exogenous polynucleotide sequence encoding a cytokine and a CAR. In certain embodiments described herein, the engineered nucleic acid encodes two or more expression cassettes, each expression cassette containing a promoter, and each expression cassette separately encoding an exogenous polynucleotide sequence encoding a cytokine and an ACP. In certain embodiments, the two or more expression cassettes are oriented in a head-to-tail directionality. In certain embodiments, the two or more expression cassettes are oriented in a head-to-head directionality. In certain embodiments, the two or more expression cassettes are oriented in a tail-to-tail directionality. In some cases, each expression cassette contains its own promoter to drive the expression of the polynucleotide sequence encoding the cytokine and / or CAR. In certain embodiments, the cytokine and CAR are organized as follows: 5'-cytokine-CAR-3' or 5'-CAR-cytokine-3'.

[0237] "Engineered nucleic acid" is a nucleic acid that does not exist in nature. However, it should be understood that although an engineered nucleic acid as a whole is not naturally occurring, it can include nucleotide sequences that exist in nature. In some embodiments, the engineered nucleic acid contains nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, the engineered nucleic acid includes murine nucleotide sequences, bacterial nucleotide sequences, human nucleotide sequences, and / or viral nucleotide sequences. The term "engineered nucleic acid" includes recombinant nucleic acids and synthetic nucleic acids. "Recombinant nucleic acid" refers to a molecule constructed by ligating nucleic acid molecules and that can, in some embodiments, replicate in a living cell. "Synthetic nucleic acid" refers to a molecule that is amplified or chemically synthesized or synthesized by other means. Synthetic nucleic acids include those that have been chemically modified or otherwise modified but can base pair with naturally occurring nucleic acid molecules. Modifications include, but are not limited to, one or more modified internucleotide linkages and unnatural nucleic acids. Modifications are described in more detail in U.S. Patent No. 6,673,611 and U.S. Application Publication 2004 / 0019001, each of which is incorporated herein by reference in its entirety. Modified internucleotide linkages can be phosphorothioate or phosphorodithioate linkages. Unnatural nucleic acids can be locked nucleic acid (LNA), peptide nucleic acid (PNA), glycol nucleic acid (GNA), phosphorodiamidate morpholino oligomers (PMO or "morpholino"), and threose nucleic acid (TNA). Unnatural nucleic acids are described in further detail in International Application WO 1998 / 039352, U.S. Application Publication No. 2013 / 0156849, and U.S. Patents Nos. 6,670,461; 5,539,082; 5,185,444, each of which is incorporated herein by reference in its entirety. Recombinant nucleic acids and synthetic nucleic acids also include those molecules produced by replication of any of the foregoing. The engineered nucleic acids of the present disclosure can be encoded by a single molecule (e.g., included in the same plasmid or other vector) or by multiple different molecules (e.g., multiple different independently replicating molecules). Engineered nucleic acids can be isolated nucleic acids. Isolated nucleic acids include, but are not limited to, cDNA polynucleotides, RNA polynucleotides, RNAi oligonucleotides (e.g., siRNA, miRNA, antisense oligonucleotides, shRNA, etc.), mRNA polynucleotides, circular plasmids, linear DNA fragments, vectors, microcircles, ssDNA, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), as well as oligonucleotides.

[0238] The engineered nucleic acids of the present disclosure can be generated using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, GIBSON cloning is used to generate the engineered nucleic acid constructs (see, e.g., Gibson, D.G. et al. Nature Methods, 343 - 345, 2009; and Gibson, D.G. et al. Nature Methods, 901 - 903, 2010, each of which is incorporated herein by reference). GIBSON typically uses three enzymatic activities in a single - tube reaction: 5' exonuclease, the Y - extension activity of DNA polymerase, and DNA ligase activity. The 5' exonuclease activity back - cuts the 5' - end sequence and exposes the complementary sequence for annealing. Then the polymerase activity fills in the gaps on the annealed region. Subsequently, the DNA ligase seals the nicks and covalently links the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, thus obtaining a higher percentage of correct assembly. In some embodiments, Cloning (Clontech) is used to generate the engineered nucleic acid constructs.

[0239] Promoter

[0240] Generally, in all embodiments described herein, the engineered nucleic acids encoding the proteins herein (e.g., cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins described herein) encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding the protein. In some embodiments, the engineered nucleic acids (e.g., engineered nucleic acids containing the expression cassette) comprise a promoter operably linked to a nucleotide sequence (e.g., exogenous polynucleotide sequence) encoding at least 2 different proteins. For example, the engineered nucleic acids can comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 different proteins. In some embodiments, the engineered nucleic acids comprise a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different proteins. In some embodiments, the engineered nucleic acids (e.g., engineered nucleic acids containing the expression cassette) comprise a promoter operably linked to a nucleotide sequence (e.g., exogenous polynucleotide sequence) encoding at least 2 cytokines. For example, the engineered nucleic acids can comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 cytokines. In some embodiments, the engineered nucleic acids comprise a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cytokines. In some embodiments, the engineered nucleic acids (e.g., engineered nucleic acids containing the expression cassette) comprise a promoter operably linked to a nucleotide sequence (e.g., exogenous polynucleotide sequence) encoding at least 2 membrane-cleavable chimeric proteins. For example, the engineered nucleic acids can comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 membrane-cleavable chimeric proteins. In some embodiments, the engineered nucleic acids comprise a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more membrane-cleavable chimeric proteins.

[0241] "Promoter" refers to the control region of a nucleic acid sequence, in which region the initiation and transcription rate of the remainder of the nucleic acid sequence are controlled. A promoter can also contain sub-regions to which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. A promoter can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or transcription of the nucleic acid sequence it regulates. In the present context, a promoter is considered to be "operably linked" when it is in the correct functional position and orientation relative to the nucleic acid sequence that it regulates to control ("drive") the initiation of transcription and / or the expression of said sequence.

[0242] A promoter can be a promoter that is naturally associated with a gene or sequence, such as can be obtained by isolating the 5' non-coding sequence located upstream of the coding fragment of a given gene or sequence. Such a promoter can be referred to as "endogenous". In some embodiments, the coding nucleic acid sequence can be under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the coding sequence in its natural environment. Such promoters can include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring", for example promoters or enhancers that contain different elements of altered transcriptional regulatory regions and / or mutations by genetic engineering methods known in the art. In addition to nucleic acid sequences that are synthetically produced for promoters and enhancers, sequences can also be produced using recombinant cloning and / or nucleic acid amplification techniques, including polymerase chain reaction (PCR) (see, for example, U.S. Patent No. 4,683,202 and U.S. Patent No. 5,928,906).

[0243] The promoter of an engineered nucleic acid can be an "inducible promoter", which refers to a promoter characterized by regulating (e.g., initiating or activating) transcriptional activity in the presence of, affected by, or contacted by a signal. The signal can be an endogenous or normal exogenous condition (e.g., light), a compound (e.g., a chemical or non-chemical compound), or a protein (e.g., a cytokine), which contacts the inducible promoter in a certain way, thereby actively regulating the transcriptional activity of the inducible promoter. The activation of transcription can involve directly acting on the promoter to drive transcription or indirectly acting on the promoter by inactivating a repressor that blocks the promoter from driving transcription. Conversely, transcriptional inactivation can involve directly acting on the promoter to block transcription or indirectly acting on the promoter by activating a repressor that then acts on the promoter.

[0244] A promoter is "responsive" or "regulated" by a local tumor state (e.g., inflammation or hypoxia) or a signal if transcription from the promoter is activated, inactivated, increased, or decreased in the presence of the state or signal. In some embodiments, the promoter contains a response element. A "response element" is a short DNA sequence within the promoter region that binds a specific molecule (e.g., a transcription factor) and regulates gene expression from the promoter. Response elements that can be used according to the present disclosure include, but are not limited to, the phloretin - regulatable control element (PEACE), zinc - finger DNA - binding domain (DBD), interferon - γ - activated sequence (GAS) (Decker, T. et al., Journal of Interferon and Cytokine Research (JInterferon CytokineRes). March 1997; 17(3):121 - 34, which is incorporated by reference), interferon - stimulated response element (ISRE) (Han, K. J. et al., Journal of Biological Chemistry (J Biol Chem) April 9, 2004; 279(15):15652 - 61, which is incorporated by reference), NF - κB response element (Wang, V. et al., Cell Reports. 2012; 2(4):824 - 839, which is incorporated by reference), and STAT3 response element (Zhang, D. et al., Journal of Biological Chemistry 1996; 271:9503 - 9509, which is incorporated by reference). Other response elements are encompassed herein. A response element can also contain tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase the sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2X, 3X, 4X, 5X, etc. to denote the number of repeats present.

[0245] Non - limiting examples of responsive promoters (also referred to as "inducible promoters") (e.g., TGF - β - responsive promoters) are listed in Table 5A, which shows the design of the promoter and transcription factor, and shows the effect of the inducing molecule on the transcription factor (TF) and transgenic transcription (T) (B, binding; D, dissociation; n.d., not determined) (A, activation; DA, inactivation; DR, derepression) (see Horner, M. and Weber, W., FEBS Letters 586(2012)20784 - 2096m, and the references cited therein). Non - limiting examples of components of inducible promoters are presented in Table 5B.

[0246] Table 5A. Examples of Responsive Promoters

[0247]

[0248]

[0249] Table 5B: Exemplary Components of Inducible Promoters

[0250]

[0251]

[0252] Non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, elongation factor 1-α (EF1a) promoter, elongation factor (EFS) promoter, MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), phosphoglycerate kinase (PGK) promoter, spleen focus-forming virus (SFFV) promoter, simian virus 40 (SV40) promoter, and ubiquitin C (UbC) promoter (see Table 5C).

[0253] Table 5C. Exemplary Constitutive Promoters

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] The promoter can be a tissue-specific promoter. Generally, a tissue-specific promoter directs the transcription of nucleic acids (e.g., engineered nucleic acids encoding the proteins herein (e.g., cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins described herein)), such that expression is limited to a particular cell type, organelle, or tissue. Tissue-specific promoters include, but are not limited to, albumin (liver-specific, Pinkert et al., (1987)), lymphocyte-specific promoters (Calame and Eaton, 1988), specific promoters of the T cell receptor (Winoto and Baltimore, (1989)), and immunoglobulins (Banerji et al., (1983); Queen and Baltimore, 1983), neuron-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, 1989), pancreas-specific promoters (Edlund et al., (1985)), or mammary gland-specific promoters (whey promoter, U.S. Patent No. 4,873,316 and European Application Publication No. 264,166), and developmentally regulated promoters such as the murine hox promoter (Kessel and Gruss, Science 249:374-379 (1990)) or the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546 (1989)), the entire contents of each of these references being incorporated herein by reference. The promoter can be constitutive in the corresponding particular cell type, organelle, or tissue. Tissue-specific promoters and / or regulatory elements can also include the promoter from the liver-type fatty acid binding (FAB) protein gene that is specific for colon epithelial cells; the insulin gene that is specific for pancreatic cells; the transthyretin, alpha1-antitrypsin, type 1 plasminogen activator inhibitor (PAI-I), apolipoprotein AI, and LDL receptor genes that are specific for liver cells; the myelin basic protein (MBP) gene that is specific for oligodendrocytes; the glial fibrillary acidic protein (GFAP) gene that is specific for glial cells; OPSIN that specifically targets the eye; and the neuron-specific enolase (NSE) promoter that is specific for nerve cells. Examples of tissue-specific promoters include, but are not limited to, the creatine kinase promoter for directing expression in muscle and heart tissues and the immunoglobulin heavy or light chain promoter for expression in B cells. Other tissue-specific promoters include the human smooth muscle alpha-actin promoter.Exemplary tissue-specific expression elements of the liver include, but are not limited to, the HMG-COA reductase promoter, sterol regulatory element 1, phosphoenolpyruvate carboxykinase (PEPCK) promoter, human C-reactive protein (CRP) promoter, human glucokinase promoter, cholesterol L 7-α hydroxylase (CYP-7) promoter, β-galactoside α-2,6 sialyltransferase promoter, insulin-like growth factor binding protein (IGFBP-I) promoter, aldolase B promoter, human transferrin promoter, and type I collagen promoter. Exemplary tissue-specific expression elements of the prostate include, but are not limited to, the prostate acid phosphatase (PAP) promoter, prostate secretory protein 94 (PSP 94) promoter, prostate-specific antigen complex promoter, and human glandular kallikrein gene promoter (hgt-1). Exemplary tissue-specific expression elements of gastric tissue include, but are not limited to, the human H+ / K+-ATPase α-subunit promoter. Exemplary tissue-specific expression elements of the pancreas include, but are not limited to, the pancreatitis-associated protein promoter (PAP), elastase 1 transcriptional enhancer, pancreas-specific amylase and elastase enhancer promoter, and pancreatic cholesterol esterase gene promoter. Exemplary tissue-specific expression elements of the endometrium include, but are not limited to, the uteroglobin promoter. Exemplary tissue-specific expression elements of adrenal cells include, but are not limited to, the cholesterol side-chain cleavage (SCC) promoter. Exemplary tissue-specific expression elements of the general nervous system include, but are not limited to, the γ-γ enolase (neuron-specific enolase, NSE) promoter. Exemplary tissue-specific expression elements of the brain include, but are not limited to, the neurofilament heavy chain (NF-H) promoter. Exemplary tissue-specific expression elements of lymphocytes include, but are not limited to, the human CGL-1 / granzyme B promoter, terminal deoxynucleotidyl transferase (TdT), λ5, VpreB, and lck (lymphocyte-specific tyrosine protein kinase p561ck) promoter, the human CD2 promoter and its 3’ transcriptional enhancer, and the human NK and T cell-specific activation (NKG5) promoter. Exemplary tissue-specific expression elements of the colon include, but are not limited to, the pp60c-src tyrosine kinase promoter, organ-specific neoantigen (OSN) promoter, and colon-specific antigen-P promoter. Tissue-specific expression elements of mammary cells include, for example but not limited to, the human α-lactalbumin promoter. Exemplary tissue-specific expression elements of the lung include, but are not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) gene promoter.

[0263] In some embodiments, the promoters of the present disclosure are regulated by signals within the tumor microenvironment. A tumor microenvironment is considered to regulate a promoter if, in the presence of a tumor microenvironment, the activity of the promoter is increased or decreased by at least 10% relative to the activity of the promoter in the absence of a tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% relative to the activity of the promoter in the absence of a tumor microenvironment. For example, the activity of the promoter is increased or decreased by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20%-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200% relative to the activity of the promoter in the absence of a tumor microenvironment.

[0264] In some embodiments, the activity of the promoter is increased or decreased by at least 2-fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100-fold) relative to the activity of the promoter in the absence of a tumor microenvironment. For example, the activity of the promoter is increased or decreased by at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold relative to the activity of the promoter in the absence of a tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by 2 to 10-fold, 2 to 20-fold, 2 to 30-fold, 2 to 40-fold, 2 to 50-fold, 2 to 60-fold, 2 to 70-fold, 2 to 80-fold, 2 to 90-fold, or 2 to 100-fold relative to the activity of the promoter in the absence of a tumor microenvironment.

[0265] In some embodiments, the promoters of the present disclosure are activated under hypoxic conditions. "Hypoxic conditions" are conditions in which the body or a region of the body lacks an adequate supply of oxygen at the tissue level. Hypoxic conditions can lead to inflammation (e.g., increased levels of inflammatory cytokines under hypoxic conditions). In some embodiments, a promoter activated under hypoxic conditions is operably linked to a nucleotide encoding a protein, the chimeric protein reducing the expression of inflammatory cytokine activity, thereby reducing inflammation caused by hypoxic conditions. In some embodiments, a promoter activated under hypoxic conditions comprises a hypoxia response element (HRE). A "hypoxia response element (HRE)" is a response element that responds to hypoxia-inducible factor (HIF). In some embodiments, the HRE comprises the consensus motif NCGTG (where N is A or G).

[0266] Conditional activation control polypeptide (ACP) promoter system

[0267] In some embodiments, a synthetic promoter is a promoter system that includes a conditional activation control polypeptide (ACP) binding domain sequence and a promoter sequence. Such systems are also referred to herein as "ACP-responsive promoters." Generally, an ACP promoter system includes a first expression cassette encoding a conditional activation control polypeptide (ACP) and a second expression cassette encoding an ACP-responsive promoter operably linked to an exogenous polynucleotide sequence, such as an exogenous polynucleotide sequence encoding a cytokine as described herein, including a membrane-cleavable chimeric protein version of the cytokine or any other protein of interest (e.g., a protease or a CAR). In some embodiments, the first and second expression cassettes are each encoded by a separate engineered nucleic acid. In other embodiments, the first and second expression cassettes are encoded by the same engineered nucleic acid. The ACP-responsive promoter can be operably linked to a nucleotide sequence encoding a single protein of interest or multiple proteins of interest. In some embodiments, the synthetic promoter comprises the nucleic acid sequence of AATTAACGGGTTTCGTAACAATCGCATGAGGATTCGCAACGCCTTTGAAGCAGTCGACGCCGAAGTCCCGTCTCAGTAAAGGTTGAAGCAGTCGACGCCGAAGAATCGGACTGCCTTCGTATGAAGCAGTCGACGCCGAAGGTATCAGTCGCCTCGGAATGAAGCAGTCGACGCCGAAGATTCGTAAGAGGCTCACTCTCCCTTACACGGAGTGGATAACTAGTTCTAGAGGGTATATAATGGGGGCCAACGCGTACCGGTGTC (SEQ ID NO:298). In some embodiments, the synthetic promoter comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:298.In some embodiments, the synthetic promoter comprises the nucleic acid sequence of CGGGTTTCGTAACAATCGCATGAGGATTCGCAACGCCTTCGGCGTAGCCGATGTCGCGCTCCCGTCTCAGTAAAGGTCGGCGTAGCCGATGTCGCGCAATCGGACTGCCTTCGTACGGCGTAGCCGATGTCGCGCGTATCAGTCGCCTCGGAACGGCGTAGCCGATGTCGCGCATTCGTAAGAGGCTCACTCTCCCTTACACGGAGTGGATAACTAGTTCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO:299). In some embodiments, the synthetic promoter comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:299.

[0268] The promoter of the ACP promoter system (e.g., the promoter driving ACP expression or the promoter sequence of the ACP-responsive promoter) can include any of the promoter sequences described herein (see "Promoters" above). The ACP-responsive promoter can be derived from minP, NFkB-responsive element, CREB-responsive element, NFAT-responsive element, SRF-responsive element 1, SRF-responsive element 2, AP1-responsive element, TCF-LEF-responsive element promoter fusion, hypoxia-responsive element, SMAD-binding element, STAT3-binding site, minCMV, YB_TATA, minTK, inducer molecule-responsive promoter, and tandem repeats thereof. In some embodiments, the ACP-responsive promoter comprises a minimal promoter.

[0269] In some embodiments, the ACP binding domain comprises one or more zinc finger binding sites. In some embodiments, the ACP-responsive promoter comprises a minimal promoter and the ACP binding domain comprises one or more zinc finger binding sites. The ACP binding domain can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more zinc finger binding sites. In some embodiments, the transcription factor is a zinc finger-containing transcription factor. In some embodiments, the zinc finger-containing transcription factor is a synthetic transcription factor. In some embodiments, the ACP binding domain comprises one or more zinc finger binding sites and the ACP has a DNA-binding zinc finger protein domain (ZF protein domain). In some embodiments, the ACP has a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ACP binding domain comprises one or more zinc finger binding sites and the ACP has a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the ZF protein domain is modular in design and is composed of a zinc finger array (ZFA). The zinc finger array contains a plurality of zinc finger protein motifs linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA that is specific for any desired nucleic acid sequence, such as a ZFA that has a desired specificity for an ACP binding domain with a particular zinc finger binding site composition and / or configuration. The ZF motifs can be directly adjacent to each other or separated by a flexible linker sequence. In some embodiments, the ZFA is an array, string or chain of tandemly arranged ZF motifs. The ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 zinc finger motifs. The ZFA can have 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10 or 5-15 zinc finger motifs. The ZF protein domain can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more ZFAs. The ZF domain can have 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10 or 5-15 ZFAs. In some embodiments, the ZF protein domain contains one to ten ZFAs.In some embodiments, the ZF protein domain comprises at least one ZFA. In some embodiments, the ZF protein domain comprises at least two ZFAs. In some embodiments, the ZF protein domain comprises at least three ZFAs. In some embodiments, the ZF protein domain comprises at least four ZFAs. In some embodiments, the ZF protein domain comprises at least five ZFAs. In some embodiments, the ZF protein domain comprises at least ten ZFAs.

[0270] In some embodiments, the DNA binding domain comprises a tetracycline (or derivative thereof) repressor (TetR) domain.

[0271] The ACP may further comprise an effector domain, such as a transcriptional effector domain. For example, the transcriptional effector domain may be an effector domain or an activation domain of a transcription factor. The transcription factor activation domain is also referred to as a transactivation domain and serves as a scaffold domain for proteins, such as transcriptional co-regulators for activating or repressing gene transcription. Any suitable transcriptional effector domain may be used in the ACP, including but not limited to the herpes simplex virus protein 16 (VP16) activation domain; an activation domain consisting of four tandem copies of VP16, i.e., the VP64 activation domain; the p65 activation domain of NFκB; the Epstein-Barr virus R transactivator (Rta) activation domain; a triple activator comprising the VP64, p65, and Rta activation domains, which is referred to as the VPR activation domain; the histone acetyltransferase (HAT) core domain of human E1A-associated protein p300, which is referred to as the p300 HAT core activation domain; the Krüppel-associated box (KRAB) repression domain; the repressor element silencing transcription factor (REST) repression domain; the WRPW motif (SEQ ID NO: 346) of the hairy-related basic helix-loop-helix repressor protein, which is referred to as the WRPW repression domain (SEQ ID NO: 346); the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and the HP1α chromodomain repression domain or any combination thereof.

[0272] In some embodiments, the effector domain is a transcriptional effector domain selected from the following: herpes simplex virus protein 16 (VP16) activation domain; an activation domain consisting of four tandem copies of VP16, i.e., the VP64 activation domain; the p65 activation domain of NFκB; Epstein-Barr virus R transactivator (Rta) activation domain; a triple activator containing the VP64, p65, and Rta activation domains, and the triple activator is called the VPR activation domain; the histone acetyltransferase (HAT) core domain of human E1A-associated protein p300, called the p300 HAT core activation domain; Krüppel-associated box (KRAB) repression domain; repressor element silencing transcription factor (REST) repression domain; the WRPW motif (SEQ ID NO: 346) of the hairy-related basic helix-loop-helix repressor protein, and the motif is called the WRPW repression domain (SEQ ID NO: 346); DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and the HP1α chromosomal shadow repression domain.

[0273] In some embodiments, the ACP is a small molecule (e.g., drug)-inducible polypeptide. For example, in some embodiments, the ACP can be induced by tetracycline (or its derivative) and contains a TetR domain and a VP16 effector domain. In some embodiments, the ACP includes an estrogen receptor variant, such as ERT2, and can be controlled by tamoxifen-mediated nuclear localization and regulated by tamoxifen or its metabolites (such as 4-hydroxy-tamoxifen [4-OHT], N-desmethyl tamoxifen, tamoxifen-N-oxide, or indoxifen). In some embodiments, the ACP contains a nuclear localization signal (NLS). In certain embodiments, the NLS contains the amino acid sequence of MPKKKRKV (SEQ ID NO: 296). Exemplary nucleic acid sequences encoding SEQ ID NO: 296 are ATGCCCAAGAAGAAGCGGAAGGTT (SEQ ID NO: 297) or ATGCCCAAGAAAAAGCGGAAGGTG (SEQ ID NO: 340). In some embodiments, the nucleic acid sequence encoding SEQ ID NO: 296 can contain SEQ ID NO: 297 or SEQ ID NO: 340, or contain a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 297 or SEQ ID NO: 340.

[0274] In some embodiments, the ACP is a small molecule (e.g., drug)-inducible polypeptide that includes a repressor protease and one or more cognate cleavage sites of the repressor protease. In some embodiments, the repressor protease is active (cleaves the cognate cleavage site) in the absence of a specific reagent and inactive (does not cleave the cognate cleavage site) in the presence of the specific reagent. In some embodiments, the specific reagent is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given repressor protease of the present disclosure. The repressor protease can be any protease described herein that can be inactivated by the presence or absence of a specific reagent (for exemplary repressor proteases, cognate cleavage sites, and protease inhibitors, see "Protease Cleavage Sites" above).

[0275] In some embodiments, the ACP has a degron domain (for exemplary degron sequences, see "Degron Systems and Domains"). The degron domain can be in any order or position relative to the individual domains of the ACP. For example, the degron domain can be the N-terminus of the repressor protease, the C-terminus of the repressor protease, the N-terminus of the ZF protein domain, the C-terminus of the ZF protein domain, the N-terminus of the effector domain, or the C-terminus of the effector domain.

[0276] Exemplary sequences of the ACPs of the present disclosure and components of the exemplary ACPs are provided in Table 5D. In some embodiments, the nucleic acid can comprise the sequences in Table 5D or a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequences in Table 5D.

[0277] Table 5D.

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287] Polycistronic and multi-promoter systems

[0288] In some embodiments, the engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) is configured to produce multiple proteins (e.g., cytokines, CARs, ACPs, membrane-cleavable chimeric proteins, and / or combinations thereof). For example, the nucleic acid can be configured to produce 2 - 20 different proteins.In some embodiments, the nucleic acid is configured to produce 2 - 20, 2 - 19, 2 - 18, 2 - 17, 2 - 16, 2 - 15, 2 - 14, 2 - 13, 2 - 12, 2 - 11, 2 - 10, 2 - 9, 2 - 8, 2 - 7, 2 - 6, 2 - 5, 2 - 4, 2 - 3, 3 - 20, 3 - 19, 3 - 18, 3 - 17, 3 - 16, 3 - 15, 3 - 14, 3 - 13, 3 - 12, 3 - 11, 3 - 10, 3 - 9, 3 - 8, 3 - 7, 3 - 6, 3 - 5, 3 - 4, 4 - 20, 4 - 19, 4 - 18, 4 - 17, 4 - 16, 4 - 15, 4 - 14, 4 - 13, 4 - 12, 4 - 11, 4 - 10, 4 - 9, 4 - 8, 4 - 7, 4 - 6, 4 - 5, 5 - 20, 5 - 19, 5 - 18, 5 - 17, 5 - 16, 5 - 15, 5 - 14, 5 - 13, 5 - 12, 5 - 11, 5 - 10, 5 - 9, 5 - 8, 5 - 7, 5 - 6, 6 - 20, 6 - 19, 6 - 18, 6 - 17, 6 - 16, 6 - 15, 6 - 14, 6 - 13, 6 - 12, 6 - 11, 6 - 10, 6 - 9, 6 - 8, 6 - 7, 7 - 20, 7 - 19, 7 - 18, 7 - 17, 7 - 16, 7 - 15, 7 - 14, 7 - 13, 7 - 12, 7 - 11, 7 - 10, 7 - 9, 7 - 8, 8 - 20, 8 - 19, 8 - 18, 8 - 17, 8 - 16, 8 - 15, 8 - 14, 8 - 13, 8 - 12, 8 - 11, 8 - 10, 8 - 9, 9 - 20, 9 - 19, 9 - 18, 9 - 17, 9 - 16, 9 - 15, 9 - 14, 9 - 13, 9 - 12, 9 - 11, 9 - 10, 10 - 20, 10 - 19, 10 - 18, 10 - 17, 10 - 16, 10 - 15, 10 - 14, 10 - 13, 10 - 12, 10 - 11, 11 - 20, 11 - 19, 11 - 18, 11 - 17, 11 - 16, 11 - 15, 11 - 14, 11 - 13, 11 - 12, 12 - 20, 12 - 19, 12 - 18, 12 - 17, 12 - 16, 12 - 15, 12 - 14, 12 - 13, 13 - 20, 13 - 19, 13 - 18, 13 - 17, 13 - 16, 13 - 15, 13 - 14, 14 - 20, 14 - 19, 14 - 18, 14 - 17, 14 - 16, 14 - 15, 15 - 20, 15 - 19, 15 - 18, 15 - 17, 15 - 16, 16 - 20, 16 - 19, 16 - 18, 16 - 17, 17 - 20, 17 - 19, 17 - 18, 18 - 20, 18 - 19 or 19 - 20 proteins. In some embodiments, the nucleic acid is configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 proteins.

[0289] In some embodiments, the engineered nucleic acid can be polycistronic, i.e., more than one separate polypeptide (e.g., multiple proteins such as cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins as described herein) can be produced from a single mRNA transcript. In some embodiments, the polycistronic engineered nucleic acids of the present disclosure can be configured to encode cytokines, CARs, and membrane-cleavable chimeric proteins as described herein. For example, the polycistronic engineered nucleic acids of the present disclosure can be configured to encode cytokines, aCARs, and membrane-cleavable chimeric proteins as described herein. For example, the polycistronic engineered nucleic acids of the present disclosure can be configured to encode cytokines, aCARs, iCARs, and membrane-cleavable chimeric proteins as described herein.

[0290] By using various linkers, engineered nucleic acids can be polycistronic. For example, a polynucleotide sequence encoding a first protein can be linked to a nucleotide sequence encoding a second protein, such as in a 5' to 3' orientation of first gene:linker:second gene. The linker can encode a 2A ribosome skipping element, such as T2A. Other 2A ribosome skipping elements include, but are not limited to, E2A, P2A, and F2A. The 2A ribosome skipping element allows for the production of separate polypeptides encoded by the first gene and the second gene during translation. The linker can encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, where the cleavable linker polypeptide is cleaved after expression, thereby producing separate polypeptides encoded by the first gene and the second gene. The cleavable linker can include polypeptide sequences that further facilitate cleavage, such as such flexible linkers (e.g., Gly-Ser-Gly sequences). In some embodiments, the engineered nucleic acids disclosed herein comprise an E2A / T2A ribosome skipping element. In certain embodiments, the E2A / T2A ribosome skipping element comprises the amino acid sequence of GSGQCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO:281). An exemplary nucleic acid encoding SEQ ID NO:281 is GGTAGCGGCCAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAAAACCCTGGACCT (SEQ ID NO:282). In certain embodiments, the nucleic acid encoding SEQ ID NO:281 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:282. In some embodiments, the engineered nucleic acids disclosed herein comprise an E2A / T2A ribosome skipping element. In certain embodiments, the E2A / T2A ribosome skipping element comprises the amino acid sequence of QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO:283). An exemplary nucleic acid encoding SEQ ID NO:283 is CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAAAACCCTGGACCT (SEQ ID NO:284).In some embodiments, the nucleic acid encoding SEQ ID NO:283 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:284.

[0291] Other suitable linkers comprising 2A ribosomal skip elements are shown in the table below.

[0292]

[0293] The linker can encode an internal ribosome entry site (IRES) such that separate polypeptides encoded by the first and second genes are produced during translation. The linker can encode a splice acceptor, such as a viral splice acceptor.

[0294] The linker can be a combination of linkers, such as a furin-2A linker, which can produce separate polypeptides by 2A ribosomal skipping followed by further cleavage of the furin site to allow complete removal of the 2A residue. In some embodiments, the combination of linkers can include a furin sequence, a flexible linker, and a 2A linker. Thus, in some embodiments, the linker is a furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linker of the present disclosure is a furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0295] Generally, a polycistronic system can use any number of linkers or linker combinations to express any number of genes or portions thereof (e.g., an engineered nucleic acid can encode a first protein, a second protein, and a third protein, each separated by a linker, such that separate polypeptides encoded by the first, second, and third proteins are produced).

[0296] An engineered nucleic acid can express genes from multiple ORFs using multiple promoters, i.e., more than one separate mRNA transcript can be produced from a single engineered nucleic acid. For example, a first promoter can be operably linked to a polynucleotide sequence encoding a first protein, and a second promoter can be operably linked to a polynucleotide sequence encoding a second protein. Generally, any number of promoters can be used to express any number of proteins. In some embodiments, at least one ORF expressed by multiple promoters can be polycistronic.

[0297] Expression cassettes encoded on the same engineered nucleic acid can be oriented in any manner suitable for the expression of the encoded exogenous polynucleotide sequences. Expression cassettes encoded on the same engineered nucleic acid can be oriented in the same direction, i.e., transcription of the individual expression cassettes proceeds in the same direction. Constructs oriented in the same directionality can be organized in a head-to-tail fashion, meaning that the 5' end (head) of the first gene is adjacent to the 3' end (tail) of the upstream gene. Expression cassettes encoded on the same engineered nucleic acid can be oriented in opposite directions, i.e., transcription of the individual expression cassettes proceeds in opposite directions (also referred to herein as "bidirectional"). Expression cassettes encoded on the same engineered nucleic acid oriented in opposite directions can be oriented in a "head-to-head" directionality. As used herein, head-to-head means that the 5' end (head) of the first gene of the bidirectional construct is adjacent to the 5' end (head) of the upstream gene of the bidirectional construct. Expression cassettes encoded on the same engineered nucleic acid oriented in opposite directions can be oriented in a "tail-to-tail" directionality. As used herein, tail-to-tail means that the 3' end (tail) of the first gene of the bidirectional construct is adjacent to the 3' end (tail) of the upstream gene of the bidirectional construct. For example, but not limited to, FIG. 1 schematically depicts cytokine-CAR bidirectional constructs in head-to-head directionality ( Figure 1A ), head-to-tail directionality ( Figure 1B ), and tail-to-tail directionality ( Figure 1C ).

[0298] As used herein, "linker" can refer to a polypeptide that links a first polypeptide sequence and a second polypeptide sequence, the above-mentioned polycistronic linker, or another promoter operably linked to the above-mentioned additional ORF.

[0299] The exogenous polynucleotide sequence encoded by the expression cassette can include a 3' untranslated region (UTR) that contains an mRNA destabilization element operably linked to the exogenous polynucleotide sequence (e.g., an exogenous polynucleotide sequence encoding a cytokine (e.g., IL12 or IL12p70)). In some embodiments, the mRNA destabilization element comprises an AU-rich element and / or a stem-loop destabilization element (SLDE). In some embodiments, the mRNA destabilizing element comprises an AU-rich element. In some embodiments, the AU-rich element comprises at least two overlapping motifs of the sequence ATTTA (SEQ ID NO:209). In some embodiments, the AU-rich element comprises ATTTATTTATTTATTTATTTA (SEQ ID NO:210). In some embodiments, the mRNA destabilization element comprises a stem-loop destabilization element (SLDE). In some embodiments, the SLDE comprises CTGTTTAATATTTAAACAG (SEQ ID NO:211). In some embodiments, the mRNA destabilization element comprises at least one AU-rich element and at least one SLDE. As used herein, "AuSLDE" refers to an AU-rich element operably linked to a stem-loop destabilization element (SLDE). An exemplary AuSLDE sequence comprises ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO:212). In some embodiments, the mRNA destabilization element comprises 2X AuSLDE. An exemplary AuSLDE sequence is provided as ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAGtgcggtaagcATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ IDNO:213).

[0300] In some embodiments, the engineered nucleic acids described herein include insulator sequences. Such insulator sequences are used to prevent improper interactions between adjacent regions of the construct. In some embodiments, the insulator sequence comprises the nucleic acid sequence of ACAATGGCTGGCCCATAGTAAATGCCGTGTTAGTGTGTTAGTTGCTGTTCTTCCACGTCAGAAGAGGCACAGACAAATTACCACCAGGTGGCGCTCAGAGTCTGCGGAGGCATCACAACAGCCCTGAATTTGAATCCTGCTCTGCCACTGCCTAGTTGAGACCTTTTACTACCTGACTAGCTGAGACATTTACGACATTTACTGGCTCTAGGACTCATTTTATTCATTTCATTACTTTTTTTTTCTTTGAGACGGAATCTCGCTCT (SEQ ID NO:300). In some embodiments, the insulator sequence comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:300.

[0301] Engineered cell

[0302] The present disclosure provides engineered immune response cells and methods of generating engineered immune response cells that produce the proteins described herein (e.g., cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins described herein). Generally, the engineered immune response cells of the present disclosure can be engineered to express the proteins provided herein, such as cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins having the formula S-C-MT or MT-C-S described herein. For example, immune response cells can be engineered to express the cytokines, CARs, and membrane-cleavable chimeric proteins described herein. For example, immune response cells can be engineered to express the cytokines, aCARs, and membrane-cleavable chimeric proteins described herein. For example, immune response cells can be engineered to express the cytokines, aCARs, iCARs, and membrane-cleavable chimeric proteins described herein. These cells are referred to herein as "engineered cells." These cells, which typically contain engineered nucleic acids, do not exist in nature. In some embodiments, cells are engineered to include a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a protein, such as a cytokine, CAR, ACP, and / or membrane-cleavable chimeric protein. Engineered cells can contain engineered nucleic acids integrated into the cell genome. Engineered cells can contain engineered nucleic acids capable of being expressed without integration into the cell genome, e.g., nucleic acids engineered with a transient expression system such as a plasmid or mRNA.

[0303] The present disclosure also encompasses additive and synergistic effects between proteins and the engineered cells that produce them. In some embodiments, the cells are engineered to produce at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) proteins, such as at least each of cytokines, CARs, ACPs, and membrane-cleavable chimeric proteins. In some embodiments, the cells are engineered to produce the cytokines, CARs, and membrane-cleavable chimeric proteins described herein. In some embodiments, the cells are engineered to produce the cytokines, aCARs, and membrane-cleavable chimeric proteins described herein. In some embodiments, the cells are engineered to produce the cytokines, aCARs, iCARs, and membrane-cleavable chimeric proteins described herein. Generally, the immune response cells provided herein are engineered to produce at least one membrane-cleavable chimeric protein having a cytokine effector molecule, a CAR, and an ACP that are not naturally produced by the cells. Generally, the immune response cells provided herein are engineered to produce at least two cytokines, wherein at least one is a membrane-cleavable chimeric protein having a cytokine effector molecule, a CAR, and an ACP. In some embodiments, the immune response cells provided herein are engineered to produce at least two cytokines, wherein at least one is a membrane-cleavable chimeric protein having a cytokine effector molecule and a CAR. In some embodiments, the immune response cells provided herein are engineered to produce at least two cytokines, wherein at least one is a membrane-cleavable chimeric protein having a cytokine effector molecule and two CARs. In some embodiments, the immune response cells provided herein are engineered to produce at least two cytokines, wherein at least one is a membrane-cleavable chimeric protein having a cytokine effector molecule, an aCAR, and an iCAR. In some embodiments, for an aCAR, such effector molecules can, for example, complement the function of effector molecules naturally produced by the cells.

[0304] In some embodiments, cells (e.g., immune cells) can be engineered to produce multiple proteins. For example, cells can be engineered to produce 2 - 20 different proteins, such as 2 - 20 different membrane-cleavable chimeric proteins. In some embodiments, cells (e.g., immune response cells) are engineered to produce at least 4 different proteins that are exogenous to the cell. In some embodiments, cells (e.g., immune response cells) are engineered to produce 4 different proteins that are exogenous to the cell.In some embodiments, cells are engineered to produce 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19 or 19-20 proteins. In some embodiments, cells can be engineered to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 proteins.

[0305] In some embodiments, the engineered cell comprises one or more engineered nucleic acids that encode a promoter operably linked to a nucleotide sequence encoding a protein (e.g., an expression cassette). In some embodiments, the cell can be engineered to include multiple engineered nucleic acids, such as at least two engineered nucleic acids, each encoding a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2, or 3) proteins. For example, the cell can be engineered to contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 engineered nucleic acids, each encoding a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2, or 3) proteins. In some embodiments, the cell can be engineered to contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more engineered nucleic acids, each encoding a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2, or 3) proteins. The engineered cell can comprise an engineered nucleic acid that encodes at least one of the above-described linkers, such as a polypeptide that links a first polypeptide sequence and a second polypeptide sequence, one or more of the above-described polycistronic linkers, one or more additional promoters operably linked to additional ORFs, or combinations thereof.

[0306] In some embodiments, a cell (e.g., an immune cell) can be engineered to express a protease. In some embodiments, the cell is engineered to express a protease that is heterologous to the cell. In some embodiments, the cell is engineered to express a protease that is heterologous to a cell expressing a chimeric protein, such as a heterologous protease that cleaves the protease cleavage site of a membrane-cleavable chimeric protein. In some embodiments, the engineered cell comprises one or more engineered nucleic acids that encode a promoter operably linked to a nucleotide sequence encoding a protease (such as a heterologous protease). Proteases and protease cleavage sites are described in more detail in the section herein titled "Protease Cleavage Sites". In other embodiments, the cell is not engineered to express a heterologous protease that cleaves the protease cleavage site of a membrane-cleavable chimeric protein. In such embodiments, the cell endogenously expresses a protease that cleaves the protease cleavage site of the membrane-cleavable chimeric protein.

[0307] The present disclosure also provides engineered cells that are engineered to produce multiple proteins, at least two of which include effector molecules that modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) protein includes an effector molecule that stimulates at least one immunostimulatory mechanism in the tumor microenvironment or inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) protein includes an effector molecule that inhibits at least one immunosuppressive mechanism in the tumor microenvironment, and at least one protein (e.g., 1, 2, 3, 4, 5, or more) inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In yet other embodiments, at least two (e.g., 2, 3, 4, 5, or more) proteins are effector molecules, each of which stimulates at least one immunostimulatory mechanism in the tumor microenvironment. In yet other embodiments, at least two (e.g., 1, 2, 3, 4, 5, or more) proteins are effector molecules, each of which inhibits at least one immunosuppressive mechanism in the tumor microenvironment.

[0308] In some embodiments, cells (e.g., immune cells) are engineered to produce at least one protein comprising effector molecules that stimulate T cell or NK cell signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that stimulate antigen presentation and / or processing. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that stimulate natural killer cell-mediated cytotoxic signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that stimulate dendritic cell differentiation and / or maturation. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that stimulate immune cell recruitment. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that stimulate M1 macrophage signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that stimulate Th1 polarization. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that stimulate matrix degradation. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that stimulate the production of immunostimulatory metabolites. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that stimulate type I interferon signaling. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that inhibit negative co-stimulatory signaling. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that inhibit pro-apoptotic signaling (e.g., via TRAIL) of anti-tumor immune cells. In some embodiments, cells are engineered to produce at least one protein comprising effector molecules that inhibit regulatory T (T reg)At least one protein of an effector molecule of cell signaling, activity, and / or recruitment. In some embodiments, the cell is engineered to produce at least one protein of an effector molecule that includes an inhibitory tumor checkpoint molecule. In some embodiments, the cell is engineered to produce at least one protein of an effector molecule that includes activation of stimulator of interferon genes (STING) signaling. In some embodiments, the cell is engineered to produce at least one protein of an effector molecule that includes inhibition of myeloid-derived suppressor cell signaling, activity, and / or recruitment. In some embodiments, the cell is engineered to produce at least one protein of an effector molecule that includes degradation of immunosuppressive factors / metabolites. In some embodiments, the cell is engineered to produce at least one protein of an effector molecule that includes inhibition of vascular endothelial growth factor signaling. In some embodiments, the cell is engineered to produce at least one protein of an effector molecule that directly kills tumor cells (e.g., granzyme, perforin, oncolytic virus, cytolytic peptides and enzymes, anti-tumor antibodies, such as those that trigger ADCC).

[0309] In some embodiments, at least one protein that includes an effector molecule that stimulates T cell signaling, activity, and / or recruitment, stimulates antigen presentation and / or processing, stimulates natural killer cell-mediated cytotoxic signaling, activity, and / or recruitment, stimulates dendritic cell differentiation and / or maturation, stimulates immune cell recruitment, stimulates macrophage signaling, stimulates matrix degradation, stimulates production of immunostimulatory metabolites, or stimulates type I interferon signaling; and at least one protein that includes an effector molecule that inhibits negative co-stimulatory signaling, inhibits pro-apoptotic signaling of anti-tumor immune cells, inhibits regulatory T (Treg) cell signaling, activity, and / or recruitment, inhibits tumor checkpoint molecules, activates stimulator of interferon genes protein (STING) signaling, inhibits myeloid-derived suppressor cell signaling, activity, and / or recruitment, degrades immunosuppressive factors / metabolites, inhibits vascular endothelial growth factor signaling, or directly kills tumor cells.

[0310] In some embodiments, immune response cells are engineered to produce at least one effector molecule cytokine selected from IL7, IL15, IL12, IL12p70 fusion protein, IL18, and IL21. In some embodiments, immune response cells are engineered to produce at least two effector molecule cytokines selected from IL7, IL15, IL12, IL12p70 fusion protein, IL18, and IL21. In some embodiments, immune response cells are engineered to produce at least two effector molecule cytokines selected from IL7, IL15, IL12, IL12p70 fusion protein, IL18, and IL21. In some embodiments, immune response cells are engineered to produce at least the effector molecule cytokines IL15 and IL12p70 fusion protein. In some embodiments, immune response cells are engineered to produce at least one membrane-cleavable chimeric protein comprising an effector molecule cytokine selected from IL15, IL12, IL12p70 fusion protein, IL18, and IL21. In some embodiments, immune response cells are engineered to produce at least two membrane-cleavable chimeric proteins comprising an effector molecule cytokine selected from IL15, IL12, IL12p70 fusion protein, IL18, and IL21. In some embodiments, immune response cells are engineered to produce at least one membrane-cleavable chimeric protein comprising an effector molecule cytokine selected from IL7, IL15, IL12, IL12p70 fusion protein, IL18, and IL21 and an additional effector molecule cytokine selected from IL7, IL15, IL12, IL12p70 fusion protein, IL18, and IL21. In certain embodiments, immune response cells are engineered to produce two cytokines, IL15 and IL21. In certain embodiments, at least one of the two cytokines is a membrane-cleavable chimeric protein.

[0311] In certain embodiments, IL15 comprises the amino acid sequence of NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:285). An exemplary nucleic acid sequence encoding SEQ ID NO:285 is AATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGAGCGACGTGCACCCTAGCTGTAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAAAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAATGTGACCGAGTCCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAATATCAAAGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACAAGC (SEQ ID NO:286). In certain embodiments, the nucleic acid encoding SEQ ID NO:285 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:286. In certain embodiments, IL15 comprises an mIgGKVII leader sequence. In certain embodiments, IL15 comprises the amino acid sequence of METDTLLLWVLLLWVPGSTGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:357).An exemplary nucleic acid sequence encoding SEQ ID NO:357 is ATGGAAACCGACACACTGCTGCTGTGGGTGCTGCTTCTTTGGGTGCCCGGCTCTACAGGCAACTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGAGCGACGTGCACCCTAGCTGTAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAAAACCTGATCATCCTGGCTAACAACAGCCTGAGCAGCAACGGCAATGTGACCGAGTCCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAATATCAAAGAGTTCCTCCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO:369). In certain embodiments, the nucleic acid encoding SEQ ID NO:357 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:369.

[0312] As used herein, the terms "membrane-cleavable", "controlled release", and "calibrated release" are used interchangeably. In certain embodiments, IL15 is membrane-cleavable. In certain embodiments, the IL15 is controlled release IL15 (crIL15). In certain embodiments, crIL15 comprises a B7-1 transmembrane domain. In certain embodiments, the B7-1 transmembrane domain comprises the amino acid sequence of SEQ ID NO219. In certain embodiments, crIL15 comprises a "slow" protease cleavage site that comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO:191). In certain embodiments, crIL15 that comprises a "slow" protease cleavage site comprises the amino acid sequence of MDWTWILFLVAAATRVHSYPYDVPDYAGGG GSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO:355).An exemplary nucleic acid sequence encoding SEQ ID NO:355 is ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTTATCCATATGATGTTCCAGATTATGCTGGCGGAGGCGGTTCTAATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCATCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTTCAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAATTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG(SEQ ID NO:367).

[0313] In some embodiments, crIL15 comprising a "slow" protease cleavage site also comprises a furin protease cleavage site. CrIL15 comprising a "slow" protease cleavage site and a furin protease cleavage site may comprise the amino acid sequence of MDWTWILFLVAAATRVHSYPYDVPDYAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPVRRKR (SEQ ID NO: 415).An exemplary nucleic acid sequence encoding SEQ ID NO: 415 is ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTTATCCATATGATGTTCCAGATTATGCTGGCGGAGGCGGTTCTAATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCATCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGTGACCCCTGAGCCTATCTTCAGCCTGATCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAATTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTGAGAAGAAAACGC (SEQ ID NO: 416). In certain embodiments, the nucleic acid encoding SEQ ID NO: 355 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 367. In certain embodiments, crIL15 comprises a "rapid" protease cleavage site that comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180).In certain embodiments, the crIL15 comprising a "rapid" protease cleavage site comprises the amino acid sequence of MDWTWILFLVAAATRVHSYPYDVPDYAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSPRAEALKGGSGGGGSGGGGSGGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 356).An exemplary nucleic acid sequence encoding SEQ ID NO:356 is ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTTATCCATATGATGTTCCAGATTATGCTGGCGGAGGCGGTTCTAATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCACCCAGAGCCGAGGCTCTGAAAGGCGGATCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAATTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG(SEQ ID NO:368). In certain embodiments, the nucleic acid encoding SEQ ID NO:356 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:368.

[0314] In certain embodiments, crIL15 comprises the amino acid sequence of MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 410). An exemplary nucleic acid sequence encoding SEQ ID NO: 410 is ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTAATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCA (SEQ ID NO: 411).

[0315] In certain embodiments, crIL15 comprises a sushi domain. In certain embodiments, crIL15 comprises an IgE leader sequence. In certain embodiments, crIL15 comprises a sushi domain and an IgE leader sequence. In certain embodiments, crIL15 comprises the amino acid sequence of MDWTWILFLVAAATRVHSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO:361).An exemplary nucleic acid sequence encoding SEQ ID NO:361 is AACGGGCCGCACAGATTCTCTTCTCAGCCGTTCGTTTCTCCGCCGCTCTCTGCATCTAGGGGCGAAGCAGTAGGTCAGGCAGCAGATCACGAAGATGCCGTTCACGGAGATCAGTGTGATGGCCCAGCTAGGCAGCAGTTGCAGAGATCCGCCACCACTTCCTCCGCCTCCGCTACCGCCTCCGATCAGGCTGAAGATAGGCTCGGGTGTAACTCCGCTTCCACCTCCGCCAGATCCTCCGCCGCCAGAGCTTGTGTTGATGAACATCTGCACGATGTGCACGAAGCTCTGCAGGAACTCTTTGATATTCTTCTCTTCCAGTTCCTCGCACTCTTTGCAGCCGGACTCGGTCACATTGCCGTTGCTGCTCAGGCTGTCGTTGGCCAGGATGATCAGGTTTTCCACGGTGTCGTGGATGCTGGCGTCGCCGCTTTCCAGGCTGATCACTTGCAGTTCCAGCAGAAAGCACTTCATGGCGGTCACTTTACAGCTAGGGTGCACGTCGCTCTCGGTGTACAGTGTGGCGTCGATGTGCATGCTCTGGATCAGGTCCTCGATCTTCTTCAGGTCGCTGATCACGTTGACCCAATTCTGCAGAGATCCTCCGCCTCCGCTTCCACCGCCAGAACCTCCGCCGCCAGATCCGCCGCTTCTGATACACTTCAGGCTAGGTGTGGTCCAGTGGGCCACATTGGTGGCCTTGTTCAGCACACACTCGGTCAGGCTGCTGGTGCCGGCCTTTCTCTTGAAGCCGCTGTTGCAGATGTACCGCTCTCTGCTGTACAGGCTGTAGCTCTTGACCCAGATGTCGGCGTGTTCCACGCTCATAGGTGGAGGACAGGTGATGCTGTGCACTCTTGTGGCAGCGGCCACCAGAAACAGGATCCAGGTCCAGTCCAT(SEQ ID NO:372).In certain embodiments, the nucleic acid encoding SEQ ID NO:361 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:372.

[0316] In certain embodiments, the chimeric IL15 comprises a sushi domain. In certain embodiments, the chimeric IL15 comprises an IgE leader sequence. In certain embodiments, the chimeric IL15 comprises a sushi domain and an IgE leader sequence. In certain embodiments, the chimeric IL15 comprises the amino acid sequence of MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGSGGGGSGGGSGGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO:391). Encoding SEQ IDThe exemplary nucleic acid sequence of NO:391 is ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTAATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGAGCGACGTGCACCCTAGCTGTAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAAAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAATGTGACCGAGTCCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAATATCAAAGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCAGCGGCGGATCTGGCGGCGGAGGTTCTGGCGGTGGAAGCGGAGGCGGAGGATCTCTCCAGATCACATGCCCTCCACCTATGAGCGTGGAACACGCCGACATCTGGGTCAAGAGCTACAGCCTGTACAGCAGAGAGCGGTACATCTGCAACAGCGGCTTCAAGAGAAAGGCCGGCACAAGCAGCCTGACCGAGTGCGTGCTGAACAAGGCCACAAATGTGGCCCACTGGACCACACCTAGCCTGAAGTGCATCAGAGCAGCAGCTATCGAGGTGATGTATCCTCCGCCCTACCTGGATAATGAAAAGAGTAATGGGACTATCATTCATGTAAAAGGGAAGCATCTTTGTCCTTCTCCCCTTTTCCCCGGTCCGTCTAAACCTTTCTGGGTGCTCGTGGTTGTTGGCGGAGTGCTGGCCTGTTACTCTCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTCAAGTCCAGACAGACACCTCCTCTGGCCAGCGTGGAAATGGAAGCCATGGAAGCTCTGCCTGTGACCTGGGGCACCAGCTCCAGAGATGAGGACCTGGAAAACTGCTCCCACCACCTGTAA(SEQ IDNO: 392). In certain embodiments, the nucleic acid encoding SEQ ID NO: 391 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 392.

[0317] In certain embodiments, IL15 is membrane-bound IL15 (mbIL15). In certain embodiments, mbIL15 comprises the amino acid sequence of MDWTWILFLVAAATRVHSYPYDVPDYAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO:358). An exemplary nucleic acid sequence encoding SEQ ID NO:358 is ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTTATCCATATGATGTTCCAGATTATGCTGGCGGAGGCGGTTCTAATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAACAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCATCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAATTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO:370).In certain embodiments, the nucleic acid encoding SEQ ID NO:358 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:370.

[0318] In certain embodiments, IL21 comprises the amino acid sequence of QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO:360). An exemplary nucleic acid sequence encoding SEQ ID NO:360 is CAGGGCCAAGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCTGAGTTTCTGCCCGCTCCTGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGTCCATCAAGAAGCTGAAGCGGAAGCCTCCTAGCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGTCCTAGCTGCGACAGCTACGAGAAGAAGCCTCCAAAAGAGTTTCTCGAGCGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGTCCAGCAGGACACACGGCAGCGAGGATTCT (SEQ ID NO:386). In certain embodiments, the nucleic acid encoding SEQ ID NO:360 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:386.

[0319] In certain embodiments, IL21 comprises a codon-optimized IL21 leader sequence. In certain embodiments, IL21 comprises the amino acid sequence of MERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO:359). An exemplary nucleic acid sequence encoding SEQ ID NO:359 is ATGGAACGGATCGTGATCTGCCTGATGGTCATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCTCTCAGGGCCAAGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCTGAGTTTCTGCCCGCTCCTGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGTCCATCAAGAAGCTGAAGCGGAAGCCTCCTAGCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGTCCTAGCTGCGACAGCTACGAGAAGAAGCCTCCAAAAGAGTTTCTCGAGCGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGTCCAGCAGGACACACGGCAGCGAGGATTCT (SEQ ID NO:371). In certain embodiments, the nucleic acid encoding SEQ ID NO:359 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:371.Another exemplary nucleic acid sequence encoding SEQ ID NO:359 is ATGGAACGGATCGTGATCTGCCTGATGGTCATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCTCTCAGGGCCAAGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCTGAGTTCCTGCCTGCTCCTGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGTCCATCAAGAAGCTGAAGCGGAAGCCTCCTAGCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGTCCTAGCTGCGACAGCTACGAGAAGAAGCCTCCAAAAGAGTTCCTGGAACGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCAGAACCCACGGCAGCGAGGACTCC(SEQ ID NO:412). In some embodiments, IL21 comprises a furin cleavage site. In certain embodiments, IL21 comprises the amino acid sequence of MERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSRRKR(SEQ ID NO:413).An exemplary nucleic acid sequence encoding SEQ ID NO: 413 is ATGGAACGGATCGTGATCTGCCTGATGGTCATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCTCTCAGGGCCAAGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCTGAGTTCCTGCCTGCTCCTGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGTCCATCAAGAAGCTGAAGCGGAAGCCTCCTAGCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGTCCTAGCTGCGACAGCTACGAGAAGAAGCCTCCAAAAGAGTTCCTGGAACGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCAGAACCCACGGCAGCGAGGACTCCAGAAGAAAACGC (SEQ ID NO: 414).

[0320] In certain embodiments, IL7 comprises the amino acid sequence of DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO:394). An exemplary nucleic acid sequence encoding SEQ ID NO:394 is GACTGTGATATCGAGGGCAAAGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAAATCGGCAGCAACTGCCTGAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAAGAAGGCATGTTCCTGTTCAGAGCCGCCAGAAAGCTGCGGCAGTTCCTGAAGATGAACAGCACCGGCGACTTCGACCTGCATCTGCTGAAAGTGTCTGAGGGCACCACCATCCTGCTGAATTGCACCGGCCAAGTGAAGGGCAGAAAGCCTGCTGCTCTGGGAGAAGCCCAGCCTACCAAGAGCCTGGAAGAGAACAAGTCCCTGAAAGAGCAGAAGAAGCTGAACGACCTCTGCTTCCTGAAGCGGCTGCTGCAAGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAAGAGCAC (SEQ ID NO:393). In certain embodiments, the nucleic acid encoding SEQ ID NO:394 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:393.

[0321]

[0322] Generally, cells (e.g., immune cells or stem cells) are engineered to produce two or more cytokines, including at least one cytokine in the form of a membrane-cleavable chimeric protein (e.g., "S" in the formula S-C-MT or MT-C-S).

[0323] In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secreted effector molecule (e.g., "S" in the formula S-C-MT or MT-C-S) is IL15, IL12, an IL12p70 fusion protein, IL18, or IL21.

[0324] In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secreted effector molecule (e.g., "S" in the formula S-C-MT or MT-C-S) is IL15. In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secreted effector molecule is IL15, and the cells are further engineered to produce one or more additional cytokines. In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secreted effector molecule is IL15, and the cells are further engineered to produce IL12, an IL12p70 fusion protein, IL18, or IL21. In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secreted effector molecule is IL15, and the cells are further engineered to produce IL12. In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secreted effector molecule is IL15, and the cells are further engineered to produce an IL12p70 fusion protein.

[0325] In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secretable effector molecule (e.g., "S" in the formula S-C-MT or MT-C-S) is IL15, and the cells are further engineered to produce one or more additional membrane-cleavable chimeric proteins. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secretable effector molecule (e.g., "S" in the formula S-C-MT or MT-C-S) is IL15, and the cells are further engineered to produce one or more additional membrane-cleavable chimeric proteins comprising IL12, IL12p70 fusion protein, IL18, and IL21. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secretable effector molecule (e.g., "S" in the formula S-C-MT or MT-C-S) is IL15, and the cells are further engineered to produce an additional membrane-cleavable chimeric protein comprising IL12p70. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secretable effector molecule (e.g., "S" in the formula S-C-MT or MT-C-S) is IL15, and the cells are further engineered to produce IL21.

[0326] In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secretable effector molecule is IL12p70. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secretable effector molecule is IL12p70, and the cells are further engineered to produce one or more additional cytokines. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secretable effector molecule is IL12p70, and the cells are further engineered to produce IL15, IL18, or IL21. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secretable effector molecule is IL12p70, and the cells are further engineered to produce IL15.

[0327] In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secreted effector molecule (e.g., "S" in the formula S-C-MT or MT-C-S) is IL12p70, and the cells are further engineered to produce one or more additional membrane-cleavable chimeric proteins. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secreted effector molecule (e.g., "S" in the formula S-C-MT or MT-C-S) is IL12p70, and the cells are further engineered to produce one or more additional membrane-cleavable chimeric proteins comprising IL15, IL18, and IL21. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, wherein the secreted effector molecule (e.g., "S" in the formula S-C-MT or MT-C-S) is IL12p70, and the cells are further engineered to produce an additional membrane-cleavable chimeric protein comprising IL15.

[0328] The cells can further be engineered to express additional proteins other than the cytokines and / or membrane-cleavable chimeric proteins having the formula S-C-MT or MT-C-S described herein. As provided herein, immune-responsive cells can be engineered to express a chimeric antigen receptor (CAR). As provided herein, immune-responsive cells can be engineered to express a chimeric antigen receptor (CAR) that binds to GPC3. As provided herein, immune-responsive cells can be engineered to express a chimeric antigen receptor (CAR) that binds to a target selected from the group consisting of CEA, CEACAM1, CEACAM5, and CEACAM6. CEACAM5. In certain embodiments, the CAR binds to CEACAM5. In some embodiments, the chimeric antigen receptor (CAR) (e.g., that binds to CEACAM5) is an activating CAR (aCAR). In some embodiments, immune-responsive cells are engineered to further express a second CAR. The second CAR can be an inactivating CAR (iCAR). The iCAR can be an iCAR that binds to V-set and immunoglobulin domain-containing protein 2 (UniProt accession number Q96IQ7, "VSIG2").

[0329] Also as provided herein, immune-responsive cells are engineered to express an ACP comprising a synthetic transcription factor.

[0330] The CAR may include an antigen-binding domain, such as an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). The antigen recognition receptor may include an scFv. The scFv may include a heavy-chain variable domain (VH) and a light-chain variable domain (VL) that may be separated by a peptide linker. For example, the scFv may include the structure VH-L-VL or VL-L-VH, where VH is the heavy-chain variable domain, L is the peptide linker, and VL is the light-chain variable domain. In certain embodiments, the peptide linker is a gly-ser linker. In certain embodiments, the peptide linker is a (GGGGS)3 linker (SEQ ID NO:223) comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO:223). Exemplary nucleic acid sequences encoding SEQ ID NO:223 are GGCGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGATCT (SEQ ID NO:224) or GGCGGCGGAGGAAGCGGAGGCGGAGGATCCGGTGGTGGTGGATCT (SEQ ID NO:332). In certain embodiments, the nucleic acid sequence encoding SEQ ID NO:223 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:224 or SEQ ID NO:332. In some embodiments, the VH and VL of the aCAR are separated by a peptide linker having the sequence SEQ ID NO:223. In some embodiments, the VH and VL of the iCAR are separated by a peptide linker having the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:395). An exemplary nucleic acid sequence encoding GSTSGSGKPGSGEGSTKG (SEQ ID NO:395) is GGCAGCACAAGCGGCTCTGGAAAACCTGGATCTGGCGAGGGCTCTACCAAGGGC (SEQ ID NO:404).

[0331] A CAR can have one or more intracellular signaling domains. In some embodiments, an activating CAR (aCAR) can activate immune cells and contains an intracellular signaling domain such as the intracellular signaling domain of CD3ζ chain, the intracellular signaling domain of CD97, the intracellular signaling domain of CD11a - CD18, the intracellular signaling domain of CD2, the intracellular signaling domain of ICOS, the intracellular signaling domain of CD27, the intracellular signaling domain of CD154, the intracellular signaling domain of CD8, the intracellular signaling domain of OX40, the intracellular signaling domain of 4 - 1BB, the intracellular signaling domain of CD28, the intracellular signaling domain of ZAP40, the intracellular signaling domain of CD30, the intracellular signaling domain of GITR, the intracellular signaling domain of HVEM, the intracellular signaling domain of DAP10, the intracellular signaling domain of DAP12, the intracellular signaling domain of MyD88, the intracellular signaling domain of 2B4, the intracellular signaling domain of CD16a, the intracellular signaling domain of DNAM - 1, the intracellular signaling domain of KIR2DS1, the intracellular signaling domain of KIR3DS1, the intracellular signaling domain of NKp44, the intracellular signaling domain of NKp46, the intracellular signaling domain of FceRlg, the intracellular signaling domain of NKG2D, the intracellular signaling domain of EAT - 2, fragments thereof, combinations thereof, or combinations of fragments thereof. In some embodiments, the aCAR contains the intracellular signaling domain of CD28. In some embodiments, the aCAR contains the intracellular signaling domain of CD3ζ. In some embodiments, the aCAR contains both the CD28 ICD and the CD3ζ ICD. In certain embodiments, the CD28 ICD contains SEQ ID NO:267, and the CD3ζ ICD contains SEQ ID NO:277. In some embodiments, an inhibitory CAR (iCAR) can inhibit immune cells and contains the intracellular signaling domain of SIRPα or LIR1. In certain embodiments, the iCAR contains the SIRPα ICD, optionally having the sequence SEQ ID NO:385. In some embodiments, the intracellular signaling domain contains a sequence from Table 6A.

[0332] Table 6A.

[0333]

[0334]

[0335]

[0336] In some embodiments, the CAR may also include a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be flexible enough to allow the antigen-binding domain to orient in different directions, thereby facilitating antigen recognition. In some embodiments, the spacer region may be a hinge from a human protein. For example, the hinge may be a human Ig (immunoglobulin) hinge, including but not limited to IgG4 hinge, IgG2 hinge, CD8a hinge, or IgD hinge. In some embodiments, the spacer region may include an IgG4 hinge, IgG2 hinge, IgD hinge, CD28 hinge, KIR2DS2 hinge, LNGFR hinge, or PDGFR-β extracellular linker. In some embodiments, the spacer region comprises a sequence from Table 6B.

[0337] Table 6B. Exemplary hinge and spacer sequences

[0338]

[0339]

[0340]

[0341]

[0342] The CAR may have a transmembrane domain, such as a CD8 transmembrane domain, CD28 transmembrane domain, CD3ζ chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, 2B4 transmembrane domain, BTLA transmembrane domain, OX40 transmembrane domain, DAP10 transmembrane domain, DAP12 transmembrane domain, CD16a transmembrane domain, DNAM-1 transmembrane domain, KIR2DS1 transmembrane domain, KIR3DS1 transmembrane domain, NKp44 transmembrane domain, NKp46 transmembrane domain, FceRlg transmembrane domain, NKG2D transmembrane domain, SIRPα transmembrane domain, fragments thereof, combinations thereof, or combinations of fragments thereof. The CAR may have a spacer region between the antigen-binding domain and the transmembrane domain. Exemplary transmembrane domain sequences are provided in Table 6C. In certain embodiments, the iCAR comprises a SIRPα transmembrane domain, optionally wherein the SIRPα transmembrane domain comprises SEQ ID NO:383. In certain embodiments, the aCAR comprises a CD28 transmembrane domain.

[0343] Table 6C.

[0344]

[0345] In some embodiments, the aCAR antigen-binding domain binds to GPC3. In some embodiments, the aCAR antigen-binding domain that binds to GPC3 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises: heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and heavy chain complementarity determining region 3 (CDR-H3), the CDR-H1 has the amino acid sequence of KNAMN (SEQ ID NO:199), the CDR-H2 has the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO:200), the CDR-H3 has the amino acid sequence of GNSFAY (SEQ ID NO:201), and wherein the VL comprises: light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3), the CDR-L1 has the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO:202), the CDR-L2 has the amino acid sequence of WASSRES (SEQ ID NO:203), the CDR-L3 has the amino acid sequence of QQYYNYPLT (SEQ ID NO:204). In some embodiments, the antigen-binding domain that binds to GPC3 comprises heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO:199). In some embodiments, the antigen-binding domain that binds to GPC3 comprises heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO:200). In some embodiments, the antigen-binding domain that binds to GPC3 comprises heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO:201). In some embodiments, the antigen-binding domain that binds to GPC3 comprises light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO:202). In some embodiments, the antigen-binding domain that binds to GPC3 comprises light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO:203). In some embodiments, the antigen-binding domain that binds to GPC3 comprises light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO:204).

[0346] In some embodiments, the antigen-binding domain that binds to GPC3 comprises a VH region, the amino acid sequence of which has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTNNYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFAYWGQGTLVTVSA (SEQ ID NO: 205) or EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVTVSA (SEQ ID NO: 206).An exemplary nucleic acid sequence encoding SEQ ID NO: 206 is GAAGTGCAGCTGGTGGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAACAAGAACGCCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGACGGATCCGGAACAAGACCAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGCCAGGTTCACCATCTCCAGAGATGACAGCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAAACCGAGGACACCGCCGTGTACTATTGCGTGGCCGGCAATAGCTTTGCCTACTGGGGACAGGGCACCCTGGTTACAGTTTCTGCT (SEQ ID NO: 222) or GAAGTGCAGCTGGTTGAATCAGGTGGCGGCCTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACAAGAACGCCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGACGGATCCGGAACAAGACCAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGCCAGATTCACCATCAGCCGGGACGACAGCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAAACCGAGGACACCGCCGTGTATTATTGCGTGGCCGGCAACAGCTTTGCCTACTGGGGACAGGGAACCCTGGTCACCGTGTCTGCC (SEQ ID NO: 330). In certain embodiments, the nucleic acid encoding SEQ ID NO: 206 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 222 or SEQ ID NO: 330.

[0347] In some embodiments, the antigen-binding domain that binds to GPC3 includes a VL region, the amino acid sequence of which has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASSRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK (SEQ ID NO: 207) or DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIK (SEQ ID NO: 208). An exemplary nucleic acid sequence encoding SEQ ID NO: 208 is GACATCGTGATGACACAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAGAAAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTATTGGGCCAGCTCCAGAGAAAGCGGCGTGCCCGATAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTACTACAACTACCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAAATCAAA (SEQNO: 221) or GACATCGTGATGACACAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAGAAAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTATTGGGCCAGCTCCAGAGAAAGCGGCGTGCCCGATAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACGTGGCCGTGTATTACTGCCAGCAGTACTACAACTACCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 333) or GACATCGTGATGACACAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAGAAAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTATTGGGCCAGCTCCAGAGAAAGCGGCGTGCCCGATAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACGTGGCCGTGTATTACTGCCAGCAGTACTACAACTACCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAAATCAAG (SEQ ID NO: 336). In certain embodiments, the nucleic acid encoding SEQ ID NO: 208 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 221 or SEQ ID NO: 336.

[0348] In some embodiments, the aCAR antigen-binding domain binds to a target selected from CEA, CEACAM1, CEACAM5, and CEACAM6. In some embodiments, the aCAR antigen-binding domain binds to CEACAM5. In some embodiments, the antigen-binding domain that binds to CEACAM5 comprises an scFv, and the amino acid sequence of the scFv has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKGGSGSGGSGSGGSGSEVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS (SEQ ID NO: 381).An exemplary nucleic acid sequence encoding SEQ ID NO:381 is GACATCCAGCTGACACAGAGCCCTAGCAGCCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGACGTGGGCACAAGCGTGGCATGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTGGACCAGCACCAGACACACAGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCTTCACCATAAGCAGCCTGCAGCCTGAGGATATCGCCACCTACTACTGCCAGCAGTACAGCCTGTACAGAAGCTTCGGCCAGGGCACCAAGGTGGAAATCAAAGGCGGATCTGGAAGCGGCGGTTCTGGATCTGGTGGAAGCGGATCTGAGGTGCAGCTGGTGGAATCTGGTGGCGGAGTTGTGCAGCCTGGCAGATCTCTGAGACTGAGCTGTAGCGCCAGCGGCTTCGATTTCACCACCTACTGGATGAGCTGGGTCCGACAGGCCCCTGGCAAAGGACTGGAATGGATCGGCGAGATTCACCCCGACAGCAGCACCATCAATTACGCCCCTAGCCTGAAGGACCGGTTCACCATCTCCAGAGACAACGCCAAGAATACCCTGTTCCTGCAGATGGACAGCCTCCGGCCTGAAGATACCGGCGTGTACTTTTGCGCCAGCCTGTATTTCGGCTTCCCTTGGTTTGCCTACTGGGGCCAGGGAACACCTGTGACCGTTAGCTCT(SEQ ID NO:380). In certain embodiments, the nucleic acid encoding SEQ ID NO:381 comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:380.

[0349] In some embodiments, the antigen-binding domain that binds to CEACAM5 includes a VH region. In some embodiments, the amino acid sequence of the VH region has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS (SEQ ID NO:425). In some embodiments, the VH region has the amino acid sequence EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS (SEQ ID NO:425). In some embodiments, the antigen-binding domain that binds to CEACAM5 includes a VL region. In some embodiments, the amino acid sequence of the VL region has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of DIQMTQTTSSLSASLGDRVTISCRTSQDIGNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKSLPRTFGGGTKLEI (SEQ ID NO:424). In some embodiments, the VL has the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRTSQDIGNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKSLPRTFGGGTKLEI (SEQ ID NO:424).In some embodiments, the amino acid sequence of the VL region has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK (SEQ ID NO: 426). In some embodiments, the VL region has the amino acid sequence DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK (SEQ ID NO: 426). In some embodiments, the antigen-binding domain that binds to CEACAM5 comprises a VH region and a VL region. In some embodiments, the antigen-binding domain that binds to CEACAM5 comprises CDR-H1, CDR-H2 and CDR-H3 from the VH region and CDR-L1, CDR-L2 and CDR-L3 from the VL region, wherein the VH region comprises the sequence EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS (SEQ ID NO: 425), and the VL region comprises the sequence DIQMTQTTSSLSASLGDRVTISCRTSQDIGNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKSLPRTFGGGTKLEI (SEQ ID NO: 424) or DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK (SEQ ID NO: 426).

[0350] Other antigen-binding domains that can be used for aCAR are described in Table 6D below.

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359] In some embodiments, the antigen-binding domain of iCAR binds to VSIG2. In some embodiments, the antigen-binding domain that binds to VSIG2 comprises a scFv, and the amino acid sequence of the scFv has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of EVQMVESGGDLVKPGGSLKLSCAASGFTFSNSGMSWVRQTPDKRLEWVASISDGGLYTHYPDSVKGRFTISRDNGKSTLYLQMSSLRSEDTAIYYCARQGVRPFFDYWGQGTTLTVSSGSTSGSGKPGSGEGSTKGDIQMTQSPASLSASVGETVTMTCRASENIYSYLAWYQQKQGKSPQLLVFNAETLPEGVPSRFSGTGSGTHFSLRINSLQPEDFGSYYCQHHYVIPWTFGGGTKLEIK (SEQ ID NO:379).An exemplary nucleic acid sequence encoding SEQ ID NO:379 is GAGGTGCAGATGGTTGAGTCTGGCGGCGATCTGGTTAAGCCTGGCGGAAGCCTGAAGCTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAATAGCGGCATGAGCTGGGTCCGACAGACCCCTGACAAGAGACTGGAATGGGTCGCCAGCATCTCTGACGGCGGCCTGTACACACACTACCCCGATTCTGTGAAGGGCAGATTCACCATCAGCAGAGACAACGGCAAGAGCACCCTGTACCTGCAGATGAGCAGCCTGAGAAGCGAGGACACCGCCATCTACTACTGCGCCAGACAGGGCGTCAGACCCTTCTTCGATTATTGGGGCCAGGGCACCACACTGACCGTGTCATCTGGCAGCACAAGCGGCTCTGGAAAACCTGGATCTGGCGAGGGCTCTACCAAGGGCGACATCCAGATGACACAGTCTCCAGCCAGCCTGTCTGCCTCTGTGGGAGAGACAGTGACCATGACCTGTCGGGCCAGCGAGAACATCTACAGCTACCTGGCCTGGTATCAGCAGAAGCAGGGCAAGTCTCCTCAGCTGCTGGTGTTCAACGCCGAGACACTGCCTGAAGGCGTGCCCAGCAGATTTTCTGGAACAGGCAGCGGCACCCACTTCAGCCTGAGAATCAATAGCCTGCAGCCTGAGGACTTCGGCAGCTACTACTGCCAGCACCACTACGTGATCCCTTGGACCTTTGGCGGAGGCACCAAGCTGGAAATCAAG(SEQ ID NO:378).

[0360]

[0361]

[0362]

[0363] In the case where the immune response cell comprises ACP, the ACP of the immune response cell described herein comprises a synthetic transcription factor. A synthetic transcription factor is a non-naturally occurring protein that comprises a DNA-binding domain and a transcriptional effector domain and is capable of regulating (i.e., activating or repressing) transcription by binding to a cognate promoter recognized by the DNA-binding domain. In some embodiments, the ACP is a transcriptional repressor. In some embodiments, the ACP is a transcriptional activator.

[0364] Engineered cell types

[0365] The present disclosure also provides engineered immune response cells. Immune response cells can be engineered to comprise any of the engineered nucleic acids described herein (e.g., any of the engineered nucleic acids encoding cytokines, membrane-cleavable chimeric proteins, and / or CARs described herein). Cells can be engineered to have any of the characteristics of any of the engineered cells described herein. In a particular aspect, the present disclosure provides cells engineered to produce two cytokines and a CAR, wherein at least one cytokine is a membrane-cleavable chimeric protein having the formula S-C-MT or MT-C-S described herein. The present disclosure also provides cells engineered to produce two cytokines and an aCAR and an iCAR, wherein at least one cytokine is a membrane-cleavable chimeric protein having the formula S-C-MT or MT-C-S described herein.

[0366] Engineered immune response cells include, but are not limited to: T cells, CD8+ T cells, CD4+ T cells, γ-δ T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In certain embodiments, the immune response cell is an NK cell or a T cell. In some embodiments, the immune response cell is an NK cell.

[0367] Cells can be engineered to produce the proteins described herein using methods known to those of skill in the art. For example, cells can be transduced to engineer the tumor. In one embodiment, the cells are transduced with a virus.

[0368] In a specific embodiment, oncolytic viruses are used to transduce cells. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indiana vesiculovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic Maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic measles virus, oncolytic lentivirus, oncolytic replicating retrovirus, oncolytic rhabdovirus, oncolytic Seneca Valley virus, oncolytic Sindbis virus, and any variants or derivatives thereof.

[0369] Viruses, including any of the oncolytic viruses described herein, can be recombinant viruses encoding one or more transgenes, such as any of the engineered nucleic acids described herein, wherein the one or more transgenes encode one or more proteins. Viruses, including any of the oncolytic viruses described herein, can be recombinant viruses encoding one or more transgenes, such as any of the engineered nucleic acids described herein, wherein the one or more transgenes encode one or more of two or more proteins.

[0370] Engineered bacterial cells are also provided herein. The bacterial cells can be engineered to contain any of the engineered nucleic acids described herein. The bacterial cells can be engineered to have any of the characteristics of any of the engineered cells described herein. In specific aspects, bacterial cells are provided that are engineered to produce two or more proteins described herein. The bacterial cells can be engineered to produce one or more mammalian-derived proteins. The bacterial cells can be engineered to produce two or more mammalian-derived proteins. Examples of bacterial cells include, but are not limited to, Clostridium beijerinckii, Clostridium sporogenes, Clostridium novyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella choleraesuis.

[0371] The engineered cells can be human cells. The engineered cells can be human primary cells. The engineered primary cells can be tumor-infiltrating primary cells. The engineered primary cells can be primary T cells. The engineered primary cells can be hematopoietic stem cells (HSCs). The engineered primary cells can be natural killer (NK) cells. The engineered primary cells can be any somatic cells. The engineered primary cells can be MSCs. Human cells (e.g., immune cells) can be engineered to contain any of the engineered nucleic acids described herein. Human cells (e.g., immune cells) can be engineered to have any of the characteristics of any of the engineered cells described herein. In certain aspects, provided herein are human cells (e.g., immune cells) engineered to produce one or more of the proteins described herein. In certain aspects, provided herein are human cells (e.g., immune cells) engineered to produce two or more of the proteins described herein.

[0372] The engineered cells can be isolated from a subject (autologous), such as a subject known or suspected of having cancer. Methods of cell isolation are known to those of skill in the art and include, but are not limited to, sorting techniques based on cell surface marker expression, such as FACS sorting, positive isolation techniques and negative isolation, magnetic separation, and combinations thereof.

[0373] For a subject to be treated, the engineered cells can be allogeneic. The allogeneically modified cells can be HLA-matched to the subject to be treated. The engineered cells can be cultured cells, such as cells cultured ex vivo. The engineered cells can be cells cultured ex vivo, such as primary cells isolated from a subject. The cultured cells can be cultured with one or more cytokines.

[0374] Also provided herein are methods of culturing the engineered cells of the present disclosure. Methods of culturing the engineered cells described herein are known. Those of skill in the art will recognize that the culture conditions will depend on the particular target engineered cell. Those of skill in the art will recognize that the culture conditions will depend on the particular downstream use of the engineered cells, e.g., the particular culture conditions for subsequent administration of the engineered cells to a subject.

[0375] Methods of engineering cells

[0376] Also provided herein are compositions and methods for engineering immune response cells to produce one or more proteins of interest (e.g., cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins having the formula S-C-MT or MT-C-S described herein).

[0377] Generally, cells are engineered to produce a protein of interest by introducing (i.e., delivering) a polynucleotide encoding one or more proteins or effector molecules of interest (e.g., chimeric proteins as described herein, including proteins or effector molecules of interest) into the cytoplasm and / or nucleus of the cell. For example, the polynucleotide encoding one or more chimeric proteins can be any engineered nucleic acid encoding a cytokine, CAR, or a membrane-cleavable chimeric protein having the formula S-C-MT or MT-C-S as described herein. Delivery methods include, but are not limited to, virus-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and deformation of the cell membrane by physical means. Those skilled in the art will understand that the choice of delivery method can depend on the specific cell type to be engineered.

[0378] Virus-mediated delivery

[0379] Virus vector-based delivery platforms can be used to engineer cells. Generally speaking, virus vector-based delivery platforms engineer cells by introducing (i.e., delivering) into host cells. For example, virus vector-based delivery platforms can engineer cells by introducing any engineered nucleic acid as described herein (e.g., any exogenous polynucleotide sequence encoding a cytokine, CAR, ACP, and / or a membrane-cleavable chimeric protein having the formula S-C-MT or MT-C-S as described herein, and / or any expression cassette containing a promoter and an exogenous polynucleotide sequence encoding a protein, with the protein oriented from the N-terminus to the C-terminus). Virus vector-based delivery platforms can be nucleic acids, and thus, engineered nucleic acids can also encompass nucleic acids of engineered viral origin. Such nucleic acids of engineered viral origin can also be referred to as recombinant viruses or engineered viruses.

[0380] Virus vector-based delivery platforms can encode more than one engineered nucleic acid, gene, or transgene within the same nucleic acid. For example, engineered virus-derived nucleic acids (e.g., recombinant viruses or engineered viruses) can encode one or more transgenes, including but not limited to any engineered nucleic acid encoding one or more proteins as described herein. One or more transgenes encoding one or more chimeric proteins can be configured to express one or more proteins and / or other proteins of interest. In addition to one or more transgenes (e.g., transgenes encoding one or more proteins and / or other proteins of interest), virus vector-based delivery platforms can also encode one or more genes, such as viral genes required for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.), referred to as cis-acting elements or genes.

[0381] Viral vector-based delivery platforms can contain more than one viral vector, such as separate viral vectors encoding the engineered nucleic acids, genes, or transgenes described herein and referred to as trans-acting elements or genes. For example, in addition to a vector encoding one or more proteins and / or other proteins of interest, a helper virus-dependent viral vector-based delivery platform can also provide additional genes required for viral infectivity and / or viral production on one or more additional separate vectors. One viral vector can deliver more than one engineered nucleic acid, such as a vector delivering an engineered nucleic acid configured to produce two or more proteins and / or other proteins of interest. More than one viral vector can deliver more than one engineered nucleic acid, such as more than one vector delivering one or more engineered nucleic acids configured to produce one or more proteins and / or other proteins of interest. The number of viral vectors used can depend on the packaging capacity of the viral vector-based vaccine platform described above, and one of ordinary skill in the art can select an appropriate number of viral vectors.

[0382] Generally, any viral vector-based system can be used to generate molecules in vitro, such as the proteins, effector molecules, and / or other proteins of interest described herein, or for in vivo and ex vivo gene therapy procedures, for example, for in vivo delivery of engineered nucleic acids encoding one or more proteins and / or other proteins of interest. Selection of an appropriate viral vector-based system will depend on a variety of factors, such as cargo / payload size, immunogenicity of the viral system, target cells, gene expression strength and duration, and other factors known to one of ordinary skill in the art.

[0383] Virus vector-based delivery platforms can be RNA-based viruses or DNA-based viruses. Exemplary virus vector-based delivery platforms include, but are not limited to, herpes simplex virus, adenovirus, measles virus, influenza virus, Indiana vesiculovirus, Newcastle disease virus, vaccinia virus, poliovirus, myxoma virus, reovirus, mumps virus, Maraba virus, rabies virus, rotavirus, hepatitis virus, rubella virus, dengue virus, chikungunya virus, respiratory syncytial virus, lymphocytic choriomeningitis virus, measles virus, lentivirus, replication-competent retrovirus, rhabdovirus, Seneca Valley virus, Sindbis virus, and any variants or derivatives thereof.Other exemplary virus vector-based delivery platforms have been described in the art, such as vaccinia virus, fowlpox virus, self-replicating alphaviruses, Maraba virus, adenoviruses (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629) or lentiviruses, including but not limited to second-generation, third-generation or hybrid second-generation / third-generation lentiviruses and any passaged recombinant lentiviruses designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuman et al., Lentiviral vectors: basic to translational, The Biochemical Journal (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucleic Acids Research (2015) 43(1):682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72(12):9873-9880).

[0384] One or more sequences targeting subcellular compartments may precede these sequences. When introduced (i.e., delivered) into a host cell, the infected cell (i.e., the engineered cell) can express the protein and / or other proteins of interest. Vaccinia vectors and methods useful for vaccination regimens are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus Calmette-Guérin). BCG vectors are described in Stover et al., (Nature 351:456-460 (1991)). According to the description herein, a variety of other vectors (e.g., Salmonella typhi vectors, etc.) useful for introducing (i.e., delivering) engineered nucleic acids will be apparent to those skilled in the art.

[0385] Virus vector-based delivery platforms can be cell-targeting viruses, herein referred to as oncolytic viruses. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indiana vesiculovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic Maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic measles virus, oncolytic lentivirus, oncolytic replication-competent retrovirus, oncolytic rhabdovirus, oncolytic Seneca Valley virus, oncolytic Sindbis virus, and any variants or derivatives thereof. Any of the oncolytic viruses described herein can be a recombinant oncolytic virus that contains one or more transgenes (e.g., engineered nucleic acids) encoding one or more proteins and / or other proteins of interest. The transgene encoding one or more proteins and / or other proteins of interest can be configured to express the protein and / or other proteins of interest.

[0386] Virus vector-based delivery platforms can be retrovirus-based. Generally, retroviral vectors contain cis-acting long terminal repeats with a packaging capacity of up to 6-10 kb of foreign sequences. The minimal cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate one or more engineered nucleic acids (e.g., transgenes encoding one or more proteins and / or other proteins of interest) into target cells to provide permanent transgene expression. Retrovirus-based delivery systems include, but are not limited to, those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., Journal of Virology 66:2731-2739 (1992); Johann et al., Journal of Virology 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., Journal of Virology 63:2374-2378 (1989); Miller et al., Journal of Virology 65:2220-2224 (1991); PCT / US94 / 05700). Other retroviral systems include the Phoenix retroviral system.

[0387] Virus vector-based delivery platforms can be lentivirus-based. Generally, lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing high viral titers. Lentivirus-based delivery platforms can be HIV-based, such as the ViraPower system (ThermoFisher) or the pLenti system (CellBiolabs). Lentivirus-based delivery platforms can be SIV- or FIV-based. Other exemplary lentivirus-based delivery platforms are described in more detail in U.S. Patent Nos. 7,311,907, 7,262,049, 7,250,299, 7,226,780, 7,220,578, 7,211,247, 7,160,721, 7,078,031, 7,070,993, 7,056,699, 6,955,919, each of which is hereby incorporated by reference for all purposes.

[0388] Viral vector-based delivery platforms can be adenovirus-based. Generally, adenovirus-based vectors can have very high transduction efficiency in many cell types, do not require cell division, achieve high titers and expression levels, and can be produced in large quantities in a relatively simple system. Generally, adenoviruses can be used for transient expression of transgenes in infected cells because adenoviruses generally do not integrate into the host genome. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., Hum Gene Ther 5:1088 1097, 1999; WO 94 / 12649; WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655, each of which is incorporated herein by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Patent Nos. 5,585,362, 6,083,716, 7,371,570, 7,348,178, 7,323,177, 7,319,033, 7,318,919 and 7,306,793 and International Patent Application WO96 / 13597, each of which is incorporated herein by reference for all purposes.

[0389] Viral vector-based delivery platforms can be adeno-associated virus (AAV)-based. Adeno-associated virus (“AAV”) vectors can be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). AAV systems can be used to produce proteins of interest, such as the proteins and / or effector molecules described herein, in vitro, or for in vivo and ex vivo gene therapy procedures, such as for in vivo delivery of engineered nucleic acids encoding one or more proteins and / or other proteins of interest (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent Nos. 4,797,368; 5,436,146; 6,632,670; 6,642,051; 7,078,387; 7,314,912; 6,498,244; 7,906,111; U.S. Patent Publications US2003-0138772, US 2007 / 0036760, and US2009 / 0197338; Gao et al., Journal of Virology, 78(12):6381-6388 (June 2004); Gao et al., Proceedings of the National Academy of Sciences of the United States of America, 100(10):6081-6086 (May 13, 2003); and International Patent Applications WO 2010 / 138263 and WO93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, Journal of Clinical Investigation (J.Clin.Invest.) 94:1351 (1994), each of which is incorporated herein by reference for all purposes. Exemplary methods for constructing recombinant AAV vectors are described in more detail in the following documents: U.S. Patent No. 5,173,414; Tratschin et al., Molecular and Cellular Biology (Mol.Cell.Biol.) 5:3251-3260 (1985); Tratschin et al., Molecular and Cellular Biology 4:2072-2081 (1984); Hermonat & Muzyczka, Proceedings of the National Academy of Sciences of the United States of America 81:6466-6470 (1984); and Samuiski et al., Journal of Virology 63:03822-3828 (1989), each of which is incorporated herein by reference for all purposes. Generally, AAV-based vectors contain a capsid protein having an amino acid sequence corresponding to any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, and variants thereof. In a specific example, the AAV-based vector has a capsid protein having an amino acid sequence corresponding to AAV2.In a specific example, the AAV-based vector has a capsid protein having an amino acid sequence corresponding to AAV8.

[0390] The AAV vector can be engineered to have any exogenous polynucleotide sequence encoding a protein described herein, such as a cytokine, CAR, ACP, and / or a membrane-cleavable chimeric protein having the formula: S-C-MT or MT-C-S described herein.

[0391] The virus vector-based delivery platform can be a virus-like particle (VLP) platform. Generally, VLPs are constructed by producing viral structural proteins and purifying the resulting viral particles. Then, after purification, the cargo / payload (e.g., any engineered nucleic acid described herein) is encapsulated ex vivo within the purified particles. Thus, the production of VLPs keeps the nucleic acid encoding the viral structural proteins separate from the nucleic acid encoding the cargo / payload. The viral structural proteins used for VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems. Methods known to those skilled in the art can be used to denature and reform the purified viral particles in the presence of the desired cargo to produce VLPs. The production of VLPs is described in more detail in Seow et al. (Molecular Therapy, May 2009; 17(5):767-777), which is incorporated herein by reference for all purposes.

[0392] The virus vector-based delivery platform can be engineered to target (i.e., infect) a range of cells, a narrow subset of target cells, or a specific cell. Generally, the envelope protein selected for the virus vector-based delivery platform will determine the viral tropism. The virus used in the virus vector-based delivery platform can be pseudotyped to target specific target cells. The virus vector-based delivery platform can be pantropic and infect a range of cells. For example, the delivery platform based on a pantropic virus vector can include a VSV-G envelope. The virus vector-based delivery platform can be amphotropic and infect mammalian cells. Thus, those skilled in the art can select an appropriate tropism, pseudotype, and / or envelope protein to target the desired cell type.

[0393] Lipid-based delivery systems

[0394] Engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) can be introduced into cells using lipid-mediated delivery systems. Generally, lipid-mediated delivery systems use structures composed of an outer lipid membrane encapsulating an inner compartment. Examples of lipid-based structures include, but are not limited to, lipid-based nanoparticles, liposomes, micelles, exosomes, vesicles, extracellular vesicles, cells, or tissues. Lipid-based delivery systems can deliver cargo / payloads (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0395] Lipid-based nanoparticles can include, but are not limited to, unilamellar liposomes, multilamellar liposomes, and lipid formulations. As used herein, "liposome" is a general term that encompasses in vitro formulations of lipid agents formed by encapsulating a desired cargo (e.g., an engineered nucleic acid, such as any of the engineered nucleic acids described herein) within a lipid shell or lipid aggregate. Liposomes can be characterized as having a vesicular structure with a bilayer membrane, typically containing phospholipids, and an internal medium that typically contains an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar sheets, etc. Liposomes can be unilamellar liposomes. Liposomes can be multilamellar liposomes. Liposomes can be multivesicular liposomes. Liposomes can be positively charged, negatively charged, or uncharged. In certain embodiments, the liposome charge is neutral. Liposomes can be formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipids is generally guided by considering the desired purpose, e.g., criteria for in vivo delivery such as liposome size, acid instability, and stability of the liposome in the bloodstream. A variety of methods can be used to prepare liposomes, as described, for example, in Szokan et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Patent Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each of which is incorporated herein by reference for all purposes.

[0396] Multilamellar liposomes are formed spontaneously when a lipid containing a phospholipid is suspended in an excess of an aqueous solution such that multiple lipid layers are separated by an aqueous medium. After the lipid components undergo self - rearrangement, water and dissolved solutes are trapped in the enclosed structures between the lipid bilayers. Desired cargo (e.g., polypeptides, nucleic acids, small molecule drugs, engineered nucleic acids such as any of the engineered nucleic acids described herein, viral vectors, virus - based delivery systems, etc.) can be encapsulated within the aqueous interior of the liposome, associated with the liposome via a linking molecule that is connected to both the liposome and the polypeptide / nucleic acid, dispersed within the lipid bilayer of the liposome, trapped within the liposome, complexed with the liposome, or otherwise associated with the liposome such that it can be delivered to a target entity. Lipophilic molecules or molecules having a lipophilic region can also be dissolved within or associated with the lipid bilayer.

[0397] Liposomes used according to this embodiment can be prepared by different methods as known to those of ordinary skill in the art. The preparation of liposomes is described in more detail in WO 2016 / 201323, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; each of which is incorporated herein by reference for all purposes.

[0398] Liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patent Nos. 5,962,016, 5,030,453, 6,680,068; U.S. Application 2004 / 0208921, and International Patent Applications WO03 / 015757A1, WO04029213A2, and WO02 / 100435A1, each of which is hereby incorporated by reference in its entirety.

[0399] Lipid - mediated gene delivery methods are described, for example, in WO 96 / 18372; WO 93 / 24640; Mannino and Gould - Fogerite, BioTechniques 6(7):682 - 691 (1988); U.S. Patent No. 5,279,833, Rose U.S. Patent No. 5,279,833; WO91 / 06309; and Felgner et al., Proceedings of the National Academy of Sciences of the United States of America 84:7413 - 7414 (1987), each of which is incorporated herein by reference for all purposes.

[0400] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment upon fusion of multivesicular bodies with the plasma membrane. Exosomes are in the size range between 30 nm and 100 nm in diameter. Their surface consists of a lipid bilayer of the plasma membrane from the donor cell, and they contain the cytoplasm from the cell that produces the exosomes and display membrane proteins from the parental cell on their surface. Exosomes for delivering nucleic acids are known to those skilled in the art, for example, the exosomes described in more detail in U.S. Patent No. 9,889,210, which is incorporated herein by reference for all purposes.

[0401] As used herein, the term “extracellular vesicle” or “EV” refers to a cell-derived vesicle that contains a membrane enclosing an internal space. Generally, extracellular vesicles include all membrane-bound vesicles that are smaller in diameter than the cells from which they are derived. Typically, extracellular vesicles range in diameter from 20 nm to 1000 nm and can contain various macromolecular cargoes that are within the internal space, displayed on the outer surface of the extracellular vesicle, and / or spanning the membrane. The cargo can include nucleic acids (e.g., any engineered nucleic acid described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example and not limitation, extracellular vesicles include apoptotic bodies, cell debris, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with an alkaline solution), vesicularized organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0402] As used herein, the term “exosome” refers to a small (between 20 - 300 nm in diameter, more preferably between 40 - 200 nm in diameter) cell-derived vesicle that contains a membrane enclosing an internal s...

Claims

1. A polycistronic expression system, comprising: (a) an exogenous polynucleotide sequence encoding a first cytokine; (b) an exogenous polynucleotide sequence encoding a second cytokine; and (c) an exogenous polynucleotide sequence encoding an activating chimeric antigen receptor (aCAR), optionally wherein the aCAR comprises: (i) a first antigen-binding domain, (ii) one or more intracellular signaling domains that stimulate an immune response, and (iii) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof; and (d) an exogenous polynucleotide sequence encoding an inhibitory CAR (iCAR), wherein each exogenous polynucleotide sequence comprises a 5' end and a 3' end.

2. A polycistronic expression system, comprising: (a) an exogenous polynucleotide sequence encoding a first cytokine; (b) an exogenous polynucleotide sequence encoding a second cytokine; and (c) an exogenous polynucleotide sequence encoding an activating chimeric antigen receptor (aCAR), wherein each exogenous polynucleotide sequence comprises a 5' end and a 3' end, and wherein the aCAR comprises: (i) a first antigen-binding domain that binds to a target selected from: CEA, CEACAM1, CEACAM5, and CEACAM6, optionally wherein the first antigen-binding domain of the aCAR binds CEACAM5, optionally wherein the first antigen-binding domain of the aCAR comprises the amino acid sequence shown in SEQ ID NO: 381; (ii) one or more intracellular signaling domains that stimulate an immune response; and (iii) one or more polypeptides selected from the group consisting of: a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

3. The polycistronic expression system according to any one of the preceding claims, wherein: (i) The one or more intracellular signaling domains of the aCAR are selected from the group consisting of: CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, CD97, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, LFA-1, CD7, LIGHT, NKG2C, B7-H3, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activating NK cell receptor, BTLA, Toll ligand receptor, CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a and combinations thereof, and / or (ii) The aCAR comprises a hinge domain selected from the group consisting of: human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular linker and combinations thereof, and / or (iii) The aCAR comprises a transmembrane domain selected from the group consisting of PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, LIR1, and BTLA, and / or (iv) The aCAR comprises a signal peptide selected from the group consisting of IgE, IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin precursor, azurin precursor, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa.

4. The polycistronic expression system according to claim 1 or 3, wherein the iCAR comprises: (a) A second antigen-binding domain; (b) One or more intracellular signaling domains that inhibit the immune response; and (c) One or more polypeptides selected from the group consisting of a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof.

5. The polycistronic expression system according to claim 4, wherein the second antigen-binding domain of the iCAR binds to VSIG2, optionally wherein: (i) The iCAR comprises an LIR1 intracellular inhibitory domain, optionally wherein the intracellular inhibitory domain comprises the amino acid sequence shown in SEQ ID NO: 387, or (ii) The iCAR comprises an SIRPα intracellular inhibitory domain, optionally wherein the intracellular inhibitory domain comprises the amino acid sequence shown in SEQ ID NO:

385.

6. The polycistronic expression system according to claim 4 or 5, wherein: (i) The iCAR comprises a hinge domain selected from the group consisting of human Ig (immunoglobulin) hinge, IgG4 hinge, IgG2 hinge, CD8a hinge or IgD hinge, KIR2DS2 hinge, LNGFR hinge, LIR1 hinge, PDGFR-β extracellular linker, and combinations thereof, and / or (ii) The iCAR comprises a transmembrane domain selected from the group consisting of PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, LIR1, SIRPα, and BTLA, and / or (iii) The iCAR comprises a signal peptide selected from the group consisting of IgE, IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin precursor, azurin precursor, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa.

7. The polycistronic expression system according to any one of the preceding claims, wherein: (i) The exogenous polynucleotide encoding the first cytokine, the exogenous polynucleotide encoding the second cytokine, the exogenous polynucleotide encoding the aCAR, and the exogenous polynucleotide encoding the iCAR are contained in a single expression vector, or (ii) The exogenous polynucleotide encoding the first cytokine, the exogenous polynucleotide encoding the second cytokine, and the exogenous polynucleotide encoding the aCAR are contained in a first expression vector, and the exogenous polynucleotide encoding the iCAR is contained in a second expression vector.

8. The polycistronic expression system according to any one of the preceding claims, which further comprises a ribosome skipping site between each exogenous polynucleotide.

9. The polycistronic expression system according to any one of claims 1 to 8, wherein at least one of the first cytokine and the second cytokine is a controlled release cytokine having the following 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.

10. The polycistronic expression system according to claim 9, wherein: (i) The protease cleavage site is cleaved by ADAM10 and / or ADAM17, and / or (ii) The protease cleavage site comprises the amino acid sequence shown in SEQ ID NO:180 or SEQ ID NO:191, and / or (iii) The cell membrane tethering domain comprises a transmembrane domain selected from the group consisting of B7-1, PDGFR-β, CD8, CD28, CD3ζ chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, and BTLA, optionally wherein the cell membrane tethering domain comprises a B7-1 transmembrane domain, and the B7-1 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

219.

11. The polycistronic expression system according to any one of the preceding claims, wherein: (i) The first cytokine is IL15, optionally wherein the IL15 comprises the amino acid sequence shown in SEQ ID NO: 285, or optionally wherein the IL15 is controlled release IL15 (crIL15), and / or (ii) The second cytokine is IL21, optionally wherein the IL21 comprises the amino acid sequence shown in SEQ ID NO: 360, or optionally wherein the IL21 is controlled release IL21 (crIL21), and / or (iii) The first cytokine or the second cytokine comprises the amino acid sequence shown in any one of SEQ ID NO: 355 - 359, 361, and 391, and / or (iv) The first cytokine or the second cytokine is encoded by the nucleic acid sequence shown in any one of SEQ ID NO: 367 - 372 and 392.

12. A polycistronic expression system, comprising: (a) An exogenous polynucleotide sequence encoding a first cytokine; (b) An exogenous polynucleotide sequence encoding a second cytokine; and (c) An exogenous polynucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each exogenous polynucleotide sequence comprises a 5'-end and a 3'-end.

13. An engineered cell comprising the polycistronic expression system according to any one of claims 1 to 12.

14. The engineered cell according to claim 13, wherein the engineered cell is an immune cell, optionally wherein the engineered cell is selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, myeloid cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs) and iPSC-derived cells, optionally wherein the engineered cell is an NK cell.

15. A pharmaceutical composition comprising the engineered cell according to claim 13 or 14, and a pharmaceutically acceptable carrier.

16. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective dose of the engineered cell according to claim 13 or 14 or the pharmaceutical composition according to claim 15, optionally wherein: (i) The disease is cancer, and / or (ii) The isolated cells are allogeneic or autologous to the subject.

17. A method of preparing an engineered cell, the method comprising transducing isolated cells with the polycistronic expression system according to any one of claims 1 to 12, optionally wherein: (i) The isolated cells are immune cells, and / or (ii) The isolated cells are selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, myeloid cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs) and iPSC-derived cells, optionally wherein the isolated cells are NK cells.

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