Armed chimeric receptors and methods of use thereof

Abstract

Described herein are immunoresponsive cells engineered to express cytokines and chimeric receptors. Nucleic acids, cells, and methods relating thereto are also described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 378,846, filed October 7, 2022, U.S. Provisional Patent Application No. 63 / 382,477, filed November 4, 2022, and U.S. Provisional Patent Application No. 63 / 382,646, filed November 7, 2022, the disclosures of each of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence Listing This application has been filed via the Patent Center and contains a Sequence Listing which is incorporated herein by reference in its entirety. The XML copy was created in XX / 20XX, is named XXXXXUS_sequencelisting.xml, and is X,XXX,XXX bytes in size. [Background technology]

[0003] background Cell-based therapeutic platforms offer promising avenues for treating various diseases. One such promising platform is CAR-T-based therapy for the treatment of cancer. Given these promises, improvements in cell-based therapies are needed. An active area of ​​exploration is engineering cell-based therapies to produce and / or secrete effector molecules, such as cytokines, that enhance cell-based therapies, a process referred to as armoring. For example, unarmored CAR-T therapies have poor efficacy in solid tumors, and armoring can affect the entire cancer-immune cycle and enhance CAR-T activity. However, uncontrolled or unregulated armoring strategies can have negative effects on treatment, such as off-target effects and toxicity in the subject. Thus, additional methods for controlling and regulating the armoring of cell-based therapies, such as modulating the production and / or secretion of payload effector molecules, are needed. Summary of the Invention

[0004] overview Provided herein, in some embodiments, are cell-based therapeutic platforms that include regulated armoring of cell-based therapies, such as regulated secretion of payload effector molecules. Also provided herein, in some embodiments, are combination cell-based immunotherapies that include regulated armoring for targeted treatment of cancer, such as ovarian cancer, breast cancer, colon cancer, lung cancer, and pancreatic cancer.

[0005] However, the treatments provided herein can limit the systemic toxicity of armoring. For example, the immunotherapies provided herein can be tumor-specific and effective while limiting the systemic toxicity and / or other off-target effects caused by armoring. These treatments deliver proteins of interest, such as immunomodulatory effector molecules, in a regulated manner, including modulation of secretion kinetics, cell state specificity, and cell or tissue specificity. The design of delivery vehicles is optimized to improve overall functionality in cell-based therapies, such as cancer therapy, including, but not limited to, optimizing membrane cleavage sites, promoters, linkers, signal peptides, delivery methods, and the combination, regulation, and order of immunomodulatory effector molecules.

[0006] Non-limiting examples of effector molecules encompassed by the present 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).

[0007] Provided herein, in various embodiments, is a multicistronic 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.

[0008] Also provided herein, in various embodiments, is a multicistronic expression system comprising: (a) an exogenous polynucleotide sequence encoding a first cytokine; (b) an exogenous polynucleotide sequence encoding a second cytokine; and (i) an exogenous polynucleotide sequence encoding an activating chimeric antigen receptor (aCAR), wherein each of the exogenous polynucleotide sequences 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 to CEACAM5, and optionally wherein the first antigen-binding domain of the aCAR comprises the amino acid sequence set forth 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.

[0009] In some embodiments, (i) one or more intracellular signaling domains of the aCAR are selected from the group consisting of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, 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, LIGH T, 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, IL2R beta, IL2R gamma, IL7R alpha, 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, TRA NCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, S and / or (ii) the aCAR is selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, a LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker,and combinations thereof; and / or (iii) the aCAR comprises a transmembrane domain selected from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta 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 an IgE, IL12, IL2, optimized IgE. L2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GRO alpha, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa.

[0010] 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 a signal peptide, a transmembrane domain, a hinge domain, a spacer region, one or more peptide linkers, and combinations thereof. In some embodiments, the second antigen-binding domain of the iCAR binds to VSIG2, optionally in which (i) the iCAR comprises an LIR1 intracellular inhibitory domain, optionally in which the intracellular inhibitory domain comprises the amino acid sequence set forth in SEQ ID NO: 387, or (ii) the iCAR comprises a SIRPα intracellular inhibitory domain, optionally in which the intracellular inhibitory domain comprises the amino acid sequence set forth in SEQ ID NO: 385.

[0011] In some embodiments, (i) the iCAR comprises a hinge domain selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, a LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof, and / or (ii) the iCAR comprises a hinge domain selected from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta 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, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GRO alpha, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa.

[0012] In some embodiments, (i) the exogenous polynucleotide encoding the first cytokine, the exogenous polynucleotide encoding the second cytokine, the exogenous polynucleotide encoding an aCAR, and the exogenous polynucleotide encoding an iCAR are contained within 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 an aCAR are contained within a first expression vector, and the exogenous polynucleotide encoding an iCAR is contained within a second expression vector. In some embodiments, the multicistronic expression system further comprises a ribosome skipping site between each exogenous polynucleotide.

[0013] In some embodiments, at least one of the first and second cytokines has the following formula: SC-MT or MT-CS wherein S comprises a secreted effector molecule, 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 set forth 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-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, and BTLA, and optionally, the cell membrane-tethering domain comprises a B7-1 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 219.

[0014] In some embodiments, (i) the first cytokine is IL15, optionally wherein IL15 comprises the amino acid sequence set forth in SEQ ID NO: 285, or optionally wherein IL15 is controlled-release IL15 (crIL15); and / or (ii) the second cytokine is IL21, optionally wherein IL21 comprises the amino acid sequence set forth in SEQ ID NO: 360, or optionally wherein IL21 is controlled-release IL21 (crIL21); and / or (iii) the first or second cytokine comprises the amino acid sequence set forth in any one of SEQ ID NOs: 355-359, 361, and 391; and / or (iv) the first or second cytokine is encoded by the nucleic acid sequence set forth in any one of SEQ ID NOs: 367-372, and 392.

[0015] Also provided herein, in various embodiments, is a multicistronic 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 of the exogenous polynucleotide sequences comprises a 5' end and a 3' end.

[0016] Also provided herein, in various embodiments, are engineered cells comprising the multicistronic expression systems provided herein. In some embodiments, the engineered cells are immune cells, optionally in which the engineered 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, bone marrow cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs) and iPSC-derived cells, optionally in which the engineered cells are NK cells.

[0017] Also provided herein, in various embodiments, are pharmaceutical compositions comprising the engineered cells provided herein and a pharmaceutically acceptable carrier.

[0018] Also provided herein, in various embodiments, are methods of treating a disease in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of an engineered cell or pharmaceutical composition provided herein, optionally wherein (i) the disease is cancer, and / or (ii) the isolated cells are allogeneic or autologous to the subject.

[0019] Also provided herein, in various embodiments, are methods of producing engineered cells, the methods comprising transducing an isolated cell with a multicistronic 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 a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a bone marrow cell, a macrophage, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, and an induced pluripotent stem cell (iPSC) and an iPSC-derived cell, optionally wherein the isolated cell is an NK cell.

[0020] Also provided herein, in various embodiments, is a multicistronic 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 of the exogenous polynucleotide sequences comprises a 5' end and a 3' end. In certain embodiments, at least one of the first and second cytokines is a controlled-release cytokine.

[0021] In certain embodiments, each controlled-release cytokine has the following formula: SC-MT or MT-CS wherein S comprises a secreted effector molecule, 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 set forth 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-beta, CD8, CD28, CD3 zeta 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 a B7-1 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 219.

[0022] In certain embodiments, the first cytokine is IL15. In certain embodiments, the IL15 comprises the amino acid sequence set forth 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 set forth in SEQ ID NO: 360. In certain embodiments, the IL21 is controlled-release IL21 (crIL21).

[0023] In certain embodiments, the first or second cytokine comprises an amino acid sequence set forth in any one of SEQ ID NOs: 355-359, 361, and 391. In certain embodiments, the first or second cytokine is encoded by a nucleic acid sequence set forth in any one of SEQ ID NOs: 367-372, and 392.

[0024] In certain embodiments, the multicistronic expression comprises an exogenous polynucleotide sequence encoding an activating CAR (aCAR) and an exogenous polynucleotide sequence encoding an inhibitory CAR (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 to an antigen selected from CEA, CEACAM1, CEACAM5, and CEACAM6. In certain embodiments, the first antigen-binding domain of the aCAR binds to CEA, CEACAM1, CEACAM5, and CEACAM6. In certain embodiments, the first antigen-binding domain of the aCAR binds to CEACAM5. In certain embodiments, the first antigen-binding domain of the aCAR comprises the amino acid sequence set forth in SEQ ID NO: 381.

[0025] In certain embodiments, one or more intracellular signaling domains of an aCAR are selected from the group consisting of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, 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, 2B 4, 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, I L2R beta, IL2R gamma, IL7R alpha, ITGA4, VLAl, 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), 2B 4), 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 a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, an LNGFR hinge, an LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof. In certain embodiments, the aCAR comprises a transmembrane domain selected from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta 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, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GRO alpha, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa.

[0026] In certain embodiments, the aCAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 362-365. In certain embodiments, the aCAR is encoded by the nucleic acid sequence set forth in any one of SEQ ID NOs: 373-376.

[0027] 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 to VSIG2.

[0028] In certain embodiments, the iCAR comprises an LIR1 intracellular inhibitory domain. In certain embodiments, the intracellular inhibitory domain comprises the amino acid sequence set forth in SEQ ID NO: 387. In certain embodiments, the iCAR comprises a SIRPα intracellular inhibitory domain. In certain embodiments, the intracellular inhibitory domain comprises the amino acid sequence set forth in SEQ ID NO: 385.

[0029] In certain embodiments, the iCAR comprises a hinge domain selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, an LNGFR hinge, an LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof. In certain embodiments, the iCAR comprises a transmembrane domain selected from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta 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, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GRO alpha, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa.

[0030] In certain embodiments, the iCAR comprises the amino acid sequence set forth in SEQ ID NO: 366. In certain embodiments, the iCAR is encoded by the nucleic acid sequence set forth in SEQ ID NO: 377.

[0031] In certain embodiments, the exogenous polynucleotide encoding the first cytokine, the exogenous polynucleotide encoding the second cytokine, the exogenous polynucleotide encoding aCAR, and the exogenous polynucleotide encoding iCAR are contained within 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 aCAR are contained within a first expression vector, and the exogenous polynucleotide encoding iCAR is contained within a second expression vector. In certain embodiments, each exogenous polynucleotide sequence further comprises a promoter sequence at its 5' end. In certain embodiments, the promoter is a constitutive promoter or an inducible promoter. In certain embodiments, the multicistronic expression system provided herein further comprises a ribosome skipping site between each exogenous polynucleotide.

[0032] Also provided herein, in various embodiments, are engineered cells comprising the multicistronic expression systems provided herein. In some embodiments, the engineered cells are immune cells. In certain embodiments, the engineered 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, bone marrow cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs) and iPSC-derived cells. In some embodiments, the engineered cells are NK cells.

[0033] Also provided herein, in various embodiments, are pharmaceutical compositions comprising the engineered cells provided herein and a pharmaceutically acceptable carrier.

[0034] Also provided herein, in various embodiments, are methods of treating a disease in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of an engineered cell or pharmaceutical composition provided herein. In certain embodiments, the disease is cancer. In certain embodiments, the isolated cells are allogeneic with respect to the subject. In certain embodiments, the isolated cells are autologous with respect to the subject.

[0035] Also provided herein, in various embodiments, are methods of producing engineered cells, the methods comprising transducing an isolated cell with a multicistronic expression system provided herein. In certain embodiments, the isolated cell is an immune cell. In certain embodiments, the isolated cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a bone marrow cell, a macrophage, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, and an induced pluripotent stem cell (iPSC) and an iPSC-derived cell. In certain embodiments, the isolated cell is an NK cell.

[0036] Also provided herein, in various embodiments, is an immunoresponsive 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 explanation of the drawings]

[0037] [Figure 1A] Figure 1A shows a schematic diagram of the cytokine-CAR bidirectional construct arranged in a head-to-head orientation. [Figure 1B] Figure 1B shows a schematic diagram of the cytokine-CAR bidirectional construct arranged in a head-to-tail orientation. [Figure 1C] Figure 1C shows a schematic diagram of the cytokine-CAR bidirectional construct arranged in a tail-to-tail orientation. [Figure 1D] Figure ID shows a schematic of an exemplary anti-GPC3 CAR+IL15 bidirectional construct. [Figure 2] Figure 2 provides a CAR expression plot (day 7) assessed by flow cytometry for cells transduced with lentivirus encoding the CAR+IL15 bidirectional construct, and cells transduced with lentivirus encoding CAR only. [Figure 3] Figure 3 provides a CAR expression plot (day 7) assessed by flow cytometry for cells transduced with retrovirus encoding the CAR+IL15 bidirectional construct, and cells transduced with retrovirus encoding only the CAR. [Figure 4] Figure 4 provides a CAR expression plot (day 15) assessed by flow cytometry for cells transduced with lentivirus encoding the CAR+IL15 bidirectional construct and cells transduced with lentivirus encoding only CAR. [Figure 5] Figure 5 provides a CAR expression plot (day 15) assessed by flow cytometry for cells transduced with retrovirus encoding the CAR+IL15 bidirectional construct and cells transduced with retrovirus encoding only the CAR. [Figure 6] Figure 6 provides IL15 levels assessed by immunoassay in NK cells transduced with a lentivirus encoding a CAR+IL15 bidirectional construct ("Lenti") or a gamma-retrovirus encoding a CAR+IL15 bidirectional construct ("SinVec"). [Figure 7] Figure 7 provides killing by NK cells transduced with lentivirus encoding CAR only or CAR+IL15 bidirectional constructs, assessed by co-culture killing assay. [Figure 8] Figure 8 provides killing by NK cells transduced with gamma-retrovirus encoding CAR only or CAR+IL15 bidirectional constructs as assessed by co-culture killing assay. [Figure 9] FIG. 9 shows a schematic diagram of a bidirectionally oriented construct containing an IL12 expression cassette with an mRNA destabilizing element in the 3′ untranslated region. [Figure 10] FIG. 10 provides IL12 levels assessed by immunoassay of NK cells transduced with a bidirectional construct containing an inducible IL12 expression cassette and an expression cassette encoding a synthetic transcription factor. [Figure 11] FIG. 11 shows a schematic diagram of the bidirectional construct encoding cleaved-release IL15. [Figure 12] FIG. 12 provides an overview of the IL15 bicistronic constructs tested and their performance in functional assays. [Figure 13A] Figure 13A provides expression plots for GPC3 CAR and IL15 as assessed by flow cytometry of NK cells transduced with SB06251, SB06257, and SB06254. Two independent replicates are shown (Figure 13A and Figure 13B). [Figure 13B] Figure 13B provides expression plots for GPC3 CAR and IL15 as assessed by flow cytometry of NK cells transduced with SB06251, SB06257, and SB06254. Two independent replicates are shown (Figure 13A and Figure 13B). [Figure 14] Figures 14A and 14B provide secreted IL15 levels assessed by immunoassay of NK cells transduced with SB06251, SB06257, and SB06254. Two independent replicates are shown (Figures 14A and 14B). [Figure 15] Figures 15A and 15B provide cell proliferation of target cell populations after co-culture with NK cells transduced with SB06251, SB06257, and SB06254. Two independent replicates are shown (Figures 15A and 15B). [Figure 16]FIG. 16 provides the target cell numbers in a serial killing assay when SB06251, SB06257, and SB06254 were co-cultured with transduced NK cells. [Figure 17A] Figure 17A provides expression plots for GPC3 CAR and IL15 as assessed by flow cytometry of NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (Figure 17A and Figure 17B). [Figure 17B] Figure 17B provides expression plots for GPC3 CAR and IL15 as assessed by flow cytometry of NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (Figure 17A and Figure 17B). [Figure 18] Figures 18A and 18B provide secreted IL15 levels assessed by immunoassay of NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (Figures 18A and 18B). [Figure 19] Figures 19A and 19B provide cell proliferation of target cell populations after co-culture with NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (Figures 19A and 19B). [Figure 20] Figure 20 provides the target cell numbers in a serial killing assay when SB06252, SB06258, and SB06255 were co-cultured with transduced NK cells. [Figure 21A] Figure 21A provides expression plots for GPC3 CAR and IL15 as assessed by flow cytometry of NK cells transduced with bicistronic constructs SB06261, SB6294, and SB6298. Two independent replicates are shown (Figure 21A and Figure 21B). [Figure 21B]Figure 21B provides expression plots for GPC3 CAR and IL15 as assessed by flow cytometry of NK cells transduced with bicistronic constructs SB06261, SB6294, and SB6298. Two independent replicates are shown (Figure 21A and Figure 21B). [Figure 22] Figures 22A and 22B provide secreted IL15 levels assessed by immunoassay of NK cells transduced with SB06261, SB6294, and SB6298. Two independent replicates are shown (Figures 22A and 22B). [Figure 23] Figures 23A and 23B provide cell proliferation of target cell populations after co-culture with NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (Figures 23A and 23B). [Figure 24A] Figure 24A provides characterization of the cleaved-release IL15 bicistronic constructs SB06691, SB06692, and SB06693. Expression plots for GPC3 CAR and IL15, assessed by flow cytometry of NK cells transduced with SB06691, SB06692, and SB06693, are shown in Figure 24A. [Figure 24B] Figure 24B provides characterization of the cleaved-release IL15 bicistronic constructs SB06691, SB06692, and SB06693. Secreted IL15 levels assessed by immunoassay of NK cells transduced with SB06691, SB06692, and SB06693 are shown in Figure 24B. [Figure 25] FIG. 25 shows a schematic diagram of the bidirectional construct encoding cleaved-release IL12. [Figure 26] FIG. 26 provides a dose response curve of IL12 secretion for NK cells after treatment with grazoprevir (GRZ). [Figure 27]Figures 27A and 27B provide in vivo mouse data showing IL12 levels in mouse blood after injection of NK cells transduced with SB04599, SB05042, and SB05058. IL12 levels are shown in Figure 27A, and the fold change in IL12 is shown in Figure 27B. [Figure 28A] Figure 28A provides a characterization of cells transduced with various constructs expressing GPC3 CAR and IL15. Flow cytometry plots showing the expression of GPC3 CAR, membrane-bound IL15, and the copy number of each on NK cells transduced with various GPC3 CAR / IL15 expression constructs are shown. [Figure 28B] Figure 28B provides characterization of cells transduced with various constructs expressing GPC3 CAR and IL15. Measurement of secreted IL-15 is shown. [Figure 28C] Figure 28C provides characterization of cells transduced with various constructs expressing GPC3 CAR and IL15. HepG2 cell killing assessed by serial killing assay is shown. [Figure 29A] Figure 29A provides additional data on sequential killing using transduced NK cells. Serial killing of HepG2 cells is shown. [Figure 29B] Figure 29B provides additional data on serial killing using transduced NK cells. Serial killing of HuH-7 cells is shown. [Figure 30A] Figure 30A provides data assessing the function of transduced NK cells using rapid expansion (G-Rex). Expression of GPC3 CAR, membrane-bound IL15 (mIL15), and secreted IL15 (sIL15) is shown. [Figure 30B] Figure 30B provides data assessing the function of transduced NK cells using rapid expansion (G-Rex). Serial killing of transduced NK cells is shown. [Figure 31] FIG. 31 provides results from a xenograft tumor model measured by bioluminescence imaging, in which mice are injected with NK cells. [Figure 32] Figures 32A and 32B provide results and summaries of xenograft tumor models in mice injected with NK cells. Figure 32A provides survival curves for mice treated with NK cells. Figure 32B provides a summary of median survival for mice treated with NK cells. [Figure 33] FIG. 33 provides the results of a BLI experiment to assess tumor reduction in mice injected with NK cells. [Figure 34] FIG. 34 provides quantification of each condition in terms of BLI measurements normalized to day 10. [Figure 35] Figures 35A and 35B provide results over the course of a study in a xenograft tumor (HepG2) mouse model, where mice were injected with NK cells three times. Figure 35A provides results from mice imaged using BLI. Figure 35B provides a time course of BLI fold change over the course of the study. [Figure 36] Figures 36A and 36B provide BLI fold changes in mice injected with transduced NK cells. Figure 36A provides results corresponding to measurements performed 13 days after tumor implantation. Figure 36B provides results corresponding to measurements performed 20 days after tumor implantation. [Figure 37A] Figure 37A provides the results of tumor reduction in a xenograft model, showing a summary of the BLI fold change in two different in vivo experiments. [Figure 37B] Figure 37B provides the results of tumor reduction in a xenograft model. A summary of the normalized mean BLI fold change is shown for two different in vivo experiments. Treatment groups were separated and animals were followed individually. [Figure 38] Figures 38A and 38B provide the results of a xenograft tumor model when NK cells were injected intratumorally. Figure 38A provides measurements of tumor volume. Figure 38B shows survival curves. [Figure 39]Figures 39A and 39B provide the results of IL12 expression in the presence or absence of grazoprevir. Figure 39A provides measurements of concentration and fold change 24 hours after induction with grazoprevir. Figure 39B provides measurements of concentration and fold change 72 hours after induction. [Figure 40] FIG. 40 provides the results of mice injected with NK cells expressing IL12 at different concentrations and controlled throughout the experiment. [Figure 41] Figure 41 provides the results of expression (GPC3 CAR and IL15) of IL12 and GPC3 CAR / IL15 constructs co-transduced into NK cells. [Figure 42] Figures 42A and 42B provide the results of secreted IL15 and secreted IL12 expression in the presence or absence of grazoprevir. Figure 42A provides measurements of secreted IL15 concentrations. Figure 42B provides measurements of secreted IL12 expression. [Figure 43] Figure 43 provides measurements of secreted IL15 and IL12 from NK cells during a sequential killing assay. [Figure 44A] Figure 44A provides the results of a serial killing assay for different co-transduction in NK cells on cell killing of Huh-7 cells and HepG2 cells. The serial killing results of NK cells co-transduced with SB05042+SB06258 are provided. [Figure 44B] Figure 44B provides the results of a serial killing assay for different co-transduction in NK cells on cell killing of Huh-7 cells and HepG2 cells. The serial killing results of NK cells co-transduced with SB05042+SB06257 are provided. [Figure 44C] Figure 44C provides the results of a serial killing assay for different co-transduction in NK cells on cell killing of Huh-7 cells and HepG2 cells. The serial killing results of NK cells co-transduced with SB05042+SB06294 are provided. [Figure 44D]Figure 44D provides the results of a sequential killing assay for different co-transduction in NK cells on cell killing of Huh-7 cells and HepG2 cells. A combination of the results of Figures 44A-C is provided. [Figure 45] Figures 45A-45D provide results from an assessment of clonal selection of NK cells expressing GPC3 CAR. Figure 45A provides the copy number results per cell. Figure 45B provides the results of GCP3 CAR expression. Figure 45C provides the results of IL15 expression. Figure 45D provides measurements of secreted IL15. [Figure 46A] Figure 46A provides flow cytometry data of GPC3 CAR and IL15 expression on selected clones transduced with SB06258. Results for selected clones are provided. [Figure 46B] Figure 46B provides flow cytometry data of GPC3 CAR and IL15 expression on select clones transduced with SB06258. Results are provided for select clones further transduced with SB05042 (IL12). [Figure 47A] Figure 47A provides data on STAT5 phosphorylation in response to controlled-release IL15 (crIL15). Results of STAT5 phosphorylation in NK cells expressing CAR and the indicated IL15 constructs are provided. [Figure 47B] Figure 47B provides data on STAT5 phosphorylation in response to controlled-release IL15 (crIL15). Results of STAT5 phosphorylation in CD3+ PBMCs incubated with CAR and NK cells expressing the indicated IL15 constructs are provided. [Figure 47C] Figure 47C provides data on STAT5 phosphorylation in response to controlled-release IL15 (crIL15). Results of STAT3 and STAT5 phosphorylation in NK cells expressing the indicated IL15 constructs are provided. [Figure 47D]Figure 47D provides data on STAT5 phosphorylation in response to controlled-release IL15 (crIL15), surface association, and secretion of IL15 in NK cells transduced with the indicated IL15 constructs. [Figure 48] Figures 48A and 48B provide the results of target cell killing by CAR-NK cells expressing the designated IL15 constructs. Figure 48A shows the abundance of target cells over time during incubation with CAR-NK cells expressing the designated IL15 constructs. Figure 48B shows the abundance of target cells after 120 hours of incubation with CAR-NK cells expressing the designated IL15 constructs. [Figure 49] Figures 49A-49C provide the results of tumor cell killing by CAR-NK cells expressing one or two cytokines. Figure 49A shows tumor cell abundance 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 tumor cell abundance after 120 hours of incubation with CAR-NK cells expressing the indicated cytokines. [Figure 50] Figures 50A and 50B show the results of an analysis of optimal distribution between membrane-bound 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 50B shows the proliferation (left panel) and viability (right panel) of CAR-NK cells expressing the indicated IL15 constructs. [Figure 51] Figures 51A and 51B show the results of an analysis of IL15 and IL21 constructs in CAR-NK cells. Figure 51A shows the proliferation of CAR-NK cells expressing the indicated cytokine constructs. Figure 51B shows the viability of CAR-NK cells expressing the indicated cytokine constructs. [Figure 52]Figures 52A and 52B show the results of the effect of cytokine expression on CAR-NK cell survival 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. [Figure 53] Figures 53A-C show an analysis of CAR NK cell activation with co-expression of crIL15 and IL21. Figure 53A shows flow cytometry analysis of activation of CAR-NK cells expressing the indicated cytokines, as measured by IFNγ (vertical axis) and granzyme B (horizontal axis). Figure 53B shows quantification of IFNγ (left panel) and granzyme B (right panel) staining in NK cells shown in Figure 53A. Figure 53C shows images of target cells after incubation with CAR-NK cells expressing the indicated cytokines. [Figure 54A] Figure 54A shows an analysis of target cell killing by CAR-NK cells. The ratio of CAR-NK cell-mediated killing of control cells to target-expressing cells after one round of killing is shown. Panels show two separate donors. [Figure 54B] Figure 54B shows an analysis of CAR-NK cell target cell killing. The ratio of CAR-NK cell-mediated killing of control cells to target-expressing cells after multiple rounds of killing is shown. Panels show two separate donors. [Figure 54C] Figure 54C shows an analysis of target cell killing by CAR-NK cells. Images of control cells (red) or target-expressing cells (green) are shown after incubation with CAR-NK cells expressing the indicated constructs. [Figure 54D] Figure 54D shows an analysis of target cell killing of CAR-NK cells. Shown are the results of sequential killing of CAR-NK cells killing target cells. [Figure 55] Figure 55 shows sequential killing of target cells by CAR-NK cells expressing the indicated constructs under restraint by culture in the presence of TGFβ. [Figure 56]Figure 56 shows sequential killing of cells expressing an inhibitory CAR (iCAR) target antigen by NK cells expressing an iCAR and an activating CAR (aCAR). [Figure 57A] Figure 57A shows in vivo tumor suppression by CAR-NK cells co-expressing crIL15 and IL21. Images of tumors at the indicated time points in mice treated as indicated are shown. [Figure 57B] Figure 57B shows in vivo tumor suppression by CAR-NK cells co-expressing crIL15 and IL21. Tumor growth over time is shown in mice treated as indicated. [Figure 57C] Figure 57C shows in vivo tumor suppression by CAR-NK cells co-expressing crIL15 and IL21. Progression-free survival over time is shown with mice treated with the indicated CAR-NK cells. [Figure 57D] Figure 57D shows in vivo tumor suppression by CAR-NK cells co-expressing crIL15 and IL21. Survival over time is shown with mice treated with the indicated CAR-NK cells. [Figure 57E] Figure 57E shows in vivo tumor suppression by CAR-NK cells co-expressing crIL15 and IL21. Images of tumors in mice treated as indicated 15 days after tumor engraftment are shown (upper panel). The graph shows the percentage of mice in which tumor reduction was observed compared to untreated controls (lower panel). [Figure 58A] Figure 58A shows the persistence of CAR-NK cells expressing crIL15 and IL21 in tumor-bearing mice. The percentage of CD45-expressing cells as a percentage of the total is shown in the peritoneal fluid (left panel) and blood (right panel) 27 days after administration. [Figure 58B] Figure 58B shows the persistence of CAR-NK cells expressing crIL15 and IL21 in tumor-bearing mice. Staining of human CD45 (vertical axis) and mouse CD45 (horizontal axis) is shown on NK cells expressing the indicated constructs 27 days after administration. [Figure 58C]Figure 58C shows the persistence of CAR-NK cells expressing crIL15 and IL21 in tumor-bearing mice. The percentage of CD45-expressing cells as a percentage of the total is shown in the peritoneal fluid 70 days after administration. [Figure 58D] Figure 58D shows the persistence of CAR-NK cells expressing crIL15 and IL21 in tumor-bearing mice. Staining of human CD45 (vertical axis) and mouse CD45 (horizontal axis) is shown on NK cells expressing the indicated constructs 70 days after administration. [Figure 59] Figures 59A and 59B show detailed screening of various IL15 constructs and combinations with IL7 or IL21. Figure 59A shows sequential killing of target cells incubated with NK cells expressing the indicated constructs at the indicated effector-to-target ratio (E:T). Figure 59B shows the percentage of NK cells expressing CAR. [Figure 60A] Figure 60A details the analysis of IL15 with IL21 or IL7. Sequential killing of target cells by NK cells expressing control or the indicated cytokine constructs is shown. [Figure 60B] Figure 60B details the analysis of IL15 with IL21 or IL7. Sequential killing of target cells by NK cells expressing control or the indicated cytokine constructs is shown. [Figure 60C] Figure 60C details the analysis of IL15 with IL21 or IL7, showing sequential killing of target cells by NK cells expressing control or the indicated IL15 constructs. [Figure 60D] Figure 60D details the analysis of IL15 with IL21 or IL7. Sequential killing of target cells by NK cells expressing control or the indicated cytokine constructs is shown. [Figure 60E] Figure 60E details the analysis of IL15 with IL21 or IL7. Sequential killing of target cells by NK cells expressing control or the indicated IL15 constructs is shown. [Figure 61]Figures 61A-C detail the construction of recombinant IL15 sushi domain-containing proteins. Figure 61A details the design of the synthetic protein construct. Figure 61B shows killing of target cells incubated with NK cells expressing a control or the indicated construct. Figure 61C shows a second round of killing of target cells incubated with NK cells expressing a control or the indicated construct. [Figure 62A] Figure 62A details the generation of NK cells engineered to express an inhibitory CAR (iCAR) and controlled-release IL15 (crIL15 or mIL15). Figure 62A details the percentage of engineered cells expressing iCAR or crIL15, as measured by flow cytometry. [Figure 62B] Figure 62B details the generation of NK cells engineered to express an inhibitory CAR (iCAR) and controlled-release IL15 (crIL15 or mIL15). Expression of iCAR or crIL15 in the engineered cells is shown as measured by flow cytometry. [Figure 62C] Figure 62C details the generation of NK cells engineered to express an inhibitory CAR (iCAR) and controlled-release IL15 (crIL15 or mIL15). Secretion of IL-15 by NK cells engineered to express the indicated constructs is shown. [Figure 62D] Figure 62D details the generation of NK cells engineered to express an inhibitory CAR (iCAR) and controlled-release IL15 (crIL15 or mIL15). Secretion of IL-21 by NK cells engineered to express the indicated constructs is shown. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description In various embodiments, a multicistronic expression system is provided herein. In some embodiments, the multicistronic 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 multicistronic expression system comprises an activating CAR (aCAR) and an inhibitory CAR (iCAR).

[0039] Also provided herein, in various embodiments, are immunoresponsive cells engineered to have: (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).

[0040] The multicistronic expression system or immunoresponsive cell 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 contains a DNA binding domain and a transcription effector domain and can regulate (i.e., activate or repress) transcription through binding to a cognate promoter recognized by the DNA binding domain (ACP-responsive promoter). In some embodiments, the ACP is a transcription repressor. In some embodiments, the ACP is a transcription activator.

[0041] Membrane-cleavable chimeric proteins can be engineered so that secretion of effector molecules can be regulated in a protease-dependent manner. Specifically, membrane-cleavable chimeric proteins can be engineered so that secretion of effector molecules can be regulated as part of a "membrane-cleavable" system, where the incorporation of a protease cleavage site ("C") and a cell membrane anchoring domain ("MT") allows for regulated secretion of the effector molecule in a protease-dependent manner. 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: - MT: Cell membrane anchoring domain comprises a transmembrane domain (or transmembrane-intracellular domain) that directs the cellular transport of the chimeric protein so that the protein is inserted into or associated with (anchored to) the cell membrane. - C: Following expression and localization of the chimeric protein in the cell membrane, the protease cleavage site directs cleavage of the chimeric protein, allowing the effector molecule to be released (secreted) into the extracellular space. Generally, the protease cleavage site is 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 desired kinetics of payload cleavage and release (e.g., ratio of membrane-bound to secreted chimeric protein levels).

[0042] In some aspects, provided herein are membrane-cleavable chimeric proteins (or engineered nucleic acids encoding membrane-cleavable chimeric proteins) that comprise a protein of interest (e.g., any of the effector molecules described herein), a protease cleavage site, and a cell membrane anchoring domain.

[0043] An "effector molecule" refers to a molecule (e.g., a nucleic acid, such as DNA or RNA, or a protein (polypeptide) or peptide) that binds to another molecule and modulates the biological activity of the molecule to which it binds. For example, an effector molecule may act as a ligand to increase or decrease enzyme activity, gene expression, or cell signaling. Thus, in some embodiments, an effector molecule modulates (activates or inhibits) a different immune regulatory mechanism. By directly binding to and modulating a molecule, an effector molecule may also indirectly modulate a second, downstream molecule.

[0044] Generally, for all membrane-cleavable chimeric proteins described herein, the effector molecule is a cytokine or an active fragment thereof that is a secreted effector molecule (referred to as "S" in the formula SC-MT or MT-CS), including a cytokine or an active fragment thereof.

[0045] The term "modulate" encompasses maintaining biological activity, inhibiting biological activity (partially or completely), and stimulating / activating biological activity (partially or completely). The term also encompasses decreasing or increasing (e.g., enhancing) biological activity. Two different effector molecules are considered to "modulate different tumor-mediated immunosuppressive mechanisms" if one effector molecule modulates a different tumor-mediated immunosuppressive mechanism (e.g., stimulating T cell signaling) than the tumor-mediated immunosuppressive mechanism modulated by the other effector molecule (e.g., stimulating antigen presentation and / or processing).

[0046] Modulation 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 and modulates the activity of that molecule, which in turn modulates the activity of yet another molecule (to which the effector molecule is not bound).

[0047] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in an increase in immunostimulatory and / or anti-tumor immune response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in an increase in immunostimulatory and / or 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%, or at least 100%. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a 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% increase in immunostimulatory and / or anti-tumor immune responses. It should be understood that an "increase" in an immunostimulatory and / or anti-tumor immune response relates to an immunostimulatory and / or anti-tumor immune response that would otherwise occur in the absence of the effector molecule, e.g., systemically or in the tumor microenvironment.

[0048] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in an at least 2-fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100-fold) increase in immunostimulatory and / or anti-tumor immune response (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in an 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 increase in immunostimulatory and / or anti-tumor immune response. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100-fold increase in immunostimulatory and / or anti-tumor immune response.

[0049] Non-limiting examples of immunostimulatory and / or antitumor immune mechanisms include T cell signaling, activity, and / or recruitment, antigen presentation and / or processing, natural killer cell-mediated cytotoxic signaling, activity, and / or recruitment, dendritic cell differentiation and / or maturation, immune cell recruitment, proinflammatory macrophage signaling, activity, and / or recruitment, stromal degradation, production of immunostimulatory metabolites, stimulator of interferon genes (STING) signaling (which increases the secretion of IFN and Th1-polarizing cytokines, promoting antitumor immune responses), and / or type I interferon signaling. Effector molecules can stimulate at least one (or more) of the aforementioned immunostimulatory mechanisms, thus resulting in an increase in the immunostimulatory response. Changes in the aforementioned immunostimulatory and / or antitumor immune mechanisms can be assessed, for example, using in vitro assays for T cell proliferation or cytotoxicity, in vitro antigen presentation assays, expression assays (e.g., of specific markers), and / or cellular secretion assays (e.g., of cytokines).

[0050] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in a decrease in the immunosuppressive response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in a decrease in 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%, or at least 100%. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a 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% reduction in the immunosuppressive response. It should be understood that a "reduction" in the immunosuppressive response is relative to the immunosuppressive response that would otherwise occur in the absence of effector molecules, e.g., systemically or in the tumor microenvironment.

[0051] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in at least a 2-fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100-fold) decrease in the immunosuppressive response (e.g., systemically or in the tumor microenvironment). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in at least a 3-fold, at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, or at least a 100-fold decrease in the immunosuppressive response. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100-fold decrease in the immunosuppressive response.

[0052] Non-limiting examples of immunosuppressive mechanisms include negative costimulatory signaling, pro-apoptotic signaling of cytotoxic cells (e.g., T cells and / or NK cells), T regulatory (Treg) cell signaling, production / maintenance of tumor checkpoint molecules, myeloid-derived suppressor cell signaling, activity, and / or recruitment, immunosuppressive factor / metabolite production, and / or vascular endothelial growth factor signaling. An effector molecule can inhibit at least one (or more) of the aforementioned immunosuppressive mechanisms, thus resulting in a decrease in the immunosuppressive response. Alterations in the aforementioned immunosuppressive mechanisms can be, for example, an increase in T cell proliferation and / or an increase in IFNγ production (negative costimulatory signaling, T reg cell signaling, and / or MDSCs); Annexin V / PI flow staining (pro-apoptotic signaling); flow staining for expression, e.g., PDL1 expression (production / maintenance of tumor checkpoint molecules); RNA via ELISA, LUMINEX®, qPCR, enzymatic assays, e.g., IDO tryptophan catabolism (immunosuppressant / metabolite production); and assaying for phosphorylation of PI3K, Akt, p38 (VEGF signaling).

[0053] In some embodiments, effector molecules function additively: the effect of two effector molecules can, for example, be equal to the sum of the effects of the two effector molecules functioning separately. In other embodiments, effector molecules function synergistically: the effect of two effector molecules can, for example, be greater than the combined function of the two effector molecules.

[0054] The effector molecule that modulates tumor-mediated immunosuppressive mechanisms and / or modifies the tumor microenvironment may be any of the cytokines described herein.

[0055] In some embodiments, at least one of the effector molecules stimulates immune stimulatory mechanisms in the tumor microenvironment and / or inhibits immune suppressive mechanisms in the tumor microenvironment.

[0056] In some embodiments, at least one of the effector molecules (a) stimulates T cell signaling, activity, and / or recruitment; (b) stimulates antigen presentation and / or processing; (c) stimulates natural killer cell-mediated cytotoxicity 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 stromal degradation; (h) stimulates immunostimulatory metabolite production; (i) stimulates type I interferon signaling; (j) inhibits negative costimulatory signaling; (k) inhibits pro-apoptotic signaling of anti-tumor immune cells; (l) inhibits regulatory T (T reg (m) inhibit cell signaling, activity, and / or recruitment; (n) inhibit tumor checkpoint molecules; (o) stimulate stimulator of interferon genes (STING) signaling; (o) inhibit myeloid-derived suppressor cell signaling, activity, and / or recruitment; (p) degrade immunosuppressive factors / metabolites; (q) inhibit vascular endothelial growth factor signaling; and / or (r) directly kill tumor cells.

[0057] Non-limiting examples of cytokines are listed in Table 1, and specific sequences encoding exemplary effector molecules are listed in Table 2. The effector molecules can be human, such as those listed in Table 1 or Table 2, or the human equivalent of a mouse effector molecule listed in Table 1 or Table 2. The effector molecule can be human-derived, but is, for example, an endogenous human effector molecule or an effector molecule that has been modified and / or optimized for function, such as optimized for improved expression, modified for improved stability, or codon-modified in its signal sequence (see below). Various programs and algorithms for optimizing function are known to those of skill in the art and can be selected based on the desired improvement, such as codon optimization for a particular species (e.g., human, mouse, bacterial, etc.).

[0058] Table 1. Exemplary effector molecules TIFF2025533842000002.tif72159

[0059] Table 2. Exemplary effector molecule-encoding sequences TIFF2025533842000003.tif243166TIFF2025533842000004.tif253166TIFF2025533842000005.tif253166 TIFF2025533842000006.tif254166TIFF2025533842000007.tif244166TIFF2025533842000008.tif234166

[0060] The first engineered nucleic acid may 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 may comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:309.

[0061] The first engineered nucleic acid may 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 may comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:326.

[0062] The first engineered nucleic acid may 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 may comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:310.

[0063] The first engineered nucleic acid may 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 may comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:327.

[0064] The first engineered nucleic acid may 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 may comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:314.

[0065] The first engineered nucleic acid may 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: 315. The first engineered nucleic acid may comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:315.

[0066] The second engineered nucleic acid may 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. The second engineered nucleic acid may comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:317.

[0067] The second engineered nucleic acid may 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: 318. The second engineered nucleic acid may comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:318.

[0068] The first engineered nucleic acid may comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 310, and (b) the second engineered nucleic acid may comprise a nucleotide sequence set forth in SEQ ID NO: 317.

[0069] The first engineered nucleic acid may comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 327, and (b) the second engineered nucleic acid may comprise a nucleotide sequence set forth in SEQ ID NO: 317.

[0070] The first engineered nucleic acid may 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; and (b) the second engineered nucleic acid may 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.

[0071] The first engineered nucleic acid may 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 may 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.

[0072] The immunoresponsive cells provided herein can comprise any one of the engineered nucleic acids described herein. The immunoresponsive cells provided herein can comprise any one combination of the engineered nucleic acids described herein. The immunoresponsive cells provided herein can comprise two or more of any one of the engineered nucleic acids described herein.

[0073] The immunoresponsive 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 immunoresponsive cells provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 309.

[0074] The immunoresponsive 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 immunoresponsive cells provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 326.

[0075] The immunoresponsive 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 immunoresponsive cells provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 310.

[0076] The immunoresponsive 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 immunoresponsive cells provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:327.

[0077] The immunoresponsive 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: 314. The immunoresponsive cells provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 314.

[0078] The immunoresponsive 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: 315. The immunoresponsive cells provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 315.

[0079] The immunoresponsive 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: 317. The immunoresponsive cells provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:317.

[0080] The immunoresponsive 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: 318. The immunoresponsive cells provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO:318.

[0081] The immunoresponsive cells provided herein can comprise (a) a first engineered nucleic acid comprising a nucleotide sequence having the sequence set forth in SEQ ID NO: 310, and (b) a second engineered nucleic acid having the sequence set forth in SEQ ID NO: 317.

[0082] The immunoresponsive cells provided herein can comprise (a) a first engineered nucleic acid comprising a nucleotide sequence having the sequence set forth in SEQ ID NO: 327, and (b) a second engineered nucleic acid having the sequence set forth in SEQ ID NO: 317.

[0083] The immunoresponsive cells provided herein can comprise (b) 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.

[0084] The immunoresponsive cells provided herein can comprise (b) 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.

[0085] The expression vectors provided herein can contain any one of the engineered nucleic acids described herein. The expression vectors provided herein can contain any one combination of the engineered nucleic acids described herein. The expression vectors provided herein can contain two or more of any one of the engineered nucleic acids described herein.

[0086] The expression vectors 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 expression vectors provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 309.

[0087] The expression vectors 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 expression vectors provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 326.

[0088] The expression vectors 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 expression vectors provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 310.

[0089] The expression vectors 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 expression vectors provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 327.

[0090] The expression vectors 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: 314. The expression vectors provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 314.

[0091] The expression vectors 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: 315. The expression vectors provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 315.

[0092] The expression vectors 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: 317. The expression vectors provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 317.

[0093] The expression vectors 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: 318. The expression vectors provided herein can comprise a nucleotide sequence having the sequence set forth in SEQ ID NO: 318.

[0094] The expression vectors provided herein can include (a) a first engineered nucleic acid comprising a nucleotide sequence having the sequence set forth in SEQ ID NO:310, and (b) a second engineered nucleic acid comprising a nucleotide sequence having the sequence set forth in SEQ ID NO:317.

[0095] The expression vectors provided herein can include (a) a first engineered nucleic acid comprising a nucleotide sequence having the sequence set forth in SEQ ID NO:327, and (b) a second engineered nucleic acid comprising a nucleotide sequence having the sequence set forth in SEQ ID NO:317.

[0096] The expression vectors provided herein may comprise (b) 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.

[0097] The expression vectors provided herein can comprise (b) 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.

[0098] Secretion signals and signal anchors One or more effector molecules of the membrane-cleavable chimeric proteins provided herein (e.g., any of the cytokines described herein) can generally be secreted effector molecules having a secretory 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 an effector molecule for SC-MT) that directs newly synthesized proteins destined for secretion or membrane localization (also referred to as membrane insertion) into the appropriate protein processing pathway. For chimeric proteins having the formula MT-CS, the membrane-tethering domain generally has a signal anchor sequence (e.g., the signal anchor sequence of a type II transmembrane protein) that directs newly synthesized proteins destined for membrane localization into the appropriate protein processing pathway. For chimeric proteins with the formula SC-MT, membrane-tethering domains with reverse signal-anchor sequences (e.g., the signal-anchor sequences of certain type III transmembrane proteins) can be used, generally without a separate secretory signal peptide, thereby directing newly synthesized proteins destined for membrane localization into appropriate protein processing pathways.

[0099] Generally, for all membrane-cleavable chimeric proteins described herein, one or more effector molecules are secretable effector molecules (referred to as "S" in the formula SC-MT or MT-CS). In embodiments involving two or more chimeric proteins, each chimeric protein may contain a secretion signal. In embodiments involving two or more chimeric proteins, each chimeric protein may contain a secretion signal such that each effector molecule is capable of secretion from the engineered cell after cleavage of the protease cleavage site.

[0100] The secretory signal peptide operably associated with the effector molecule can be a native secretory signal peptide (e.g., a secretory signal peptide typically endogenously associated with a given effector molecule, such as the endogenous secretory signal peptide of a cytokine). The secretory signal peptide operably associated with the effector molecule can be a non-native secretory signal peptide, a native secretory signal peptide. The non-native secretory signal peptide can facilitate improved expression and function, such as sustained secretion, in a particular environment, such as a tumor microenvironment. Non-limiting examples of non-native secretory signal peptides are shown in Table 3.

[0101] Table 3. Exemplary signal secretion peptides TIFF2025533842000009.tif235165TIFF2025533842000010.tif245165

[0102] Protease cleavage site Generally, all membrane-cleavable chimeric proteins described herein contain a protease cleavage site (designated "C" in the formula SC-MT or MT-CS). Generally, the protease cleavage site can be any amino acid sequence motif that can be cleaved by a protease. Examples of protease cleavage sites include type 1 transmembrane protease cleavage site, type II transmembrane protease cleavage site, GPI-anchored protease cleavage site, ADAM8 protease cleavage site, ADAM9 protease cleavage site, ADAM10 protease cleavage site, ADAM12 protease cleavage site, ADAM15 protease cleavage site, ADAM17 protease cleavage site, ADAM19 protease cleavage site, ADAM20 protease cleavage site, ADAM21 protease cleavage site, ADAM28 protease cleavage site, ADAM30 protease cleavage site, ADAM33 Protease cleavage sites include, but are not limited to, a BACE1 protease cleavage site, a BACE2 protease cleavage site, a SIP protease cleavage site, an MT1-MMP protease cleavage site, an MT3-MMP protease cleavage site, an MT5-MMP protease cleavage site, a furin protease cleavage site, a PCSK7 protease cleavage site, a matriptase protease cleavage site, a matriptase-2 protease cleavage site, an MMP9 protease cleavage site, or an NS3 protease cleavage site.

[0103] One example of a protease cleavage site is the hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease cleavage site, including, but not limited to, the NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B cleavage site. For a description of representative sequences of NS3 proteases and their cleavage sites for various strains of HCV, see, for example, Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor & Francis, 2006), Chapter 6, pp. 163-206; the entire contents of which are incorporated herein by reference. For example, the sequences of the HCV NS4A / 4B protease cleavage site, the HCV NS5A / 5B protease cleavage site, the C-terminal degron with the NS4A / 4B protease cleavage site, and the 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, e.g., 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, A See HH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063; all of the sequences (as entered by the filing date of this application) are incorporated herein by reference.

[0104] Another example of a protease cleavage site is an ADAM17-specific protease (also referred to as tumor necrosis factor alpha-converting enzyme [TACE]) cleavage site. The ADAM17-specific protease cleavage site may be an endogenous sequence of a substrate naturally cleaved by ADAM17. The ADAM17-specific protease cleavage site may be an engineered sequence capable of being cleaved by ADAM17. The engineered ADAM17-specific protease cleavage site may be engineered for specific desired properties, including, but not limited to, optimal expression of the chimeric protein, specificity for ADAM17, cleavage rate by ADAM17, the ratio of secreted and membrane-bound chimeric protein levels, and cleavage in different cellular conditions. The protease cleavage site may be selected for specific cleavage by ADAM17. For example, a specific protease cleavage site capable of being cleaved by ADAM17 may also be cleaved by additional ADAM family proteases, such as ADAM10. Therefore, ADAM17-specific protease cleavage sites can be selected and / or engineered to reduce or eliminate cleavage by other proteases, such as ADAM10. Protease cleavage sites can be selected for the cleavage rate by ADAM17. For example, it may be desirable to select a protease cleavage site that exhibits a specific cleavage rate by ADAM17, such as reduced cleavage kinetics with respect to the endogenous sequence of the substrate that is naturally cleaved by ADAM17. In such cases, a specific cleavage rate can generally be selected to regulate the processing rate of the chimeric protein, which in turn regulates the release / secretion rate of the payload effector molecule. Thus, ADAM17-specific protease cleavage sites can be selected and / or engineered so that the sequence exhibits a desired cleavage rate by ADAM17. Protease cleavage sites can be selected for both specific cleavage by ADAM17 and the cleavage rate by ADAM17. Exemplary ADAM17-specific protease cleavage sites, including those exhibiting particular specificity and cleavage rate kinetics, are shown below in Table 4A by reference to the cleavage site (P5-P1: N-terminus; P1'-P5': C-terminus).Further details of ADAM17 and ADAM10, including expression and protease cleavage sites, are described in Sharma, et al. (J Immunol October 15, 2017, 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 the purpose.

[0105] Table 4A: Potential ADAM17 protease cleavage site sequences TIFF2025533842000011.tif85164

[0106] In some embodiments, the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located N-terminal to the second region. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), where X1 is A, Y, P, S, or F and X2 is V, L, S, I, Y, T, or A. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186). In some embodiments, the protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187). In some embodiments, the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188). In some embodiments, the protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189). In some embodiments, the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190). In some embodiments, the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191).

[0107] In certain embodiments, the cleavage site comprises a linker sequence. The cleavage site may be flanked on the N-terminus and / or C-terminus by a linker sequence. For example, but not limited to, the cleavage site may be flanked on both the N-terminus and C-terminus by a partial glycine-serine (GS) linker sequence. Upon cleavage, the N-terminus partial GS linker and the C-terminus partial GS linker combine to form a GS linker sequence, such as SEQ ID NO: 215.

[0108] In certain embodiments, the cleavage site and linker comprise the amino acid sequence of SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO: 287). An exemplary nucleic acid sequence encoding SEQ ID NO: 287 is TCTGGGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGTTACACCCGAGCCTATCTTCAGCCTGATCGGAGGCGGTAGCGGAGGCGGAGGAAGTGGTGGCGGATCTCTGCAA (SEQ ID NO: 288). In some embodiments, the nucleic acid encoding SEQ ID NO: 287 may 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.

[0109] In certain embodiments, the protease cleavage site is N-terminal to the linker. In certain embodiments, the protease cleavage site and linker comprise the amino acid sequence PRAEALKGGSGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO:289). An exemplary nucleic acid sequence encoding SEQ ID NO:289 is CCCAGAGCCGAGGCTCTGAAAGGCGGATCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAAT (SEQ ID NO:292). In some embodiments, the nucleic acid encoding SEQ ID NO:289 may 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.

[0110] In some embodiments, the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198), which is the cleavage site native to CD16 and cleavable by ADAM17. In certain embodiments, SEQ ID NO: 198 is comprised within a 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 ACGGCGGAGGTGGTAGCGGAGGCGGAGGATCTGGAATTACACAGGGACTCGCCGTGTCTACAATCTCCAGCTTCTTTGGTGGCGGTAGTGGCGGCGGTGGCAGTGGCGGTGGATCTCTTCAA (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 ID NO:291.

[0111] The protease cleavage site can be at the C-terminus of the secreted effector molecule. The protease cleavage site can be at the N-terminus of the secreted effector molecule. Generally, for all membrane-cleavable chimeric proteins described herein, the protease cleavage site is either: (1) at the C-terminus of the secreted effector molecule and the N-terminus of the cell membrane-tethering domain (in other words, the protease cleavage site is between the secreted effector molecule and the cell membrane-tethering domain); or (2) at the N-terminus of the secreted effector molecule and the C-terminus of the cell membrane-tethering domain (also between the secreted effector molecule and the cell membrane-tethering domain with an inverted domain orientation). The protease cleavage site can be connected to the secreted effector molecule by a polypeptide linker, i.e., a polypeptide sequence that is not generally considered to be part of the effector molecule or the protease cleavage site. The protease cleavage site can be connected to the cell membrane anchoring domain by a polypeptide linker, i.e., a polypeptide sequence that is not generally considered to be part of the cell membrane anchoring domain or the protease cleavage site. The polypeptide linker can be any amino acid sequence that connects the first polypeptide sequence and the 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, a GSG linker (e.g., [GS]GG [SEQ ID NO: 347]), A(EAAAK)A (SEQ ID NO: 348), and a Whitlow linker (e.g., a "KEGS" linker, e.g., the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 349), an eGK linker, e.g., the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 350), an LR1 linker, e.g., the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 215), the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 395), and linkers described in more detail in issued 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 may 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, a 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. Still other polypeptide linkers include GGSGSGGSGS (SEQ ID NO: 396) and SAGSGSGASGSG (SEQ ID NO: 397).

[0112] In membrane-cleavable systems, following expression and localization of the chimeric protein in the cell membrane, the protease cleavage site directs cleavage of the chimeric protein, allowing the effector molecule to be released (secreted) into the extracellular space of the cell.

[0113] Generally, the protease that cleaves a protease cleavage site is a specific protease for that particular protease cleavage site.For example, in the case of a disintegrin and metalloproteinase (ADAM) family protease, the protease that cleaves a particular ADAM protease cleavage site is generally limited to the ADAM protease that specifically recognizes a particular ADAM protease cleavage site motif.The protease cleavage site can be selected and / or engineered, so that cleavage by undesired proteases is reduced or eliminated.Protease can be membrane-bound or membrane-associated.Protease can be, for example, secreted in a particular cellular environment, such as tumor microenvironment ("TME").

[0114] The protease that cleaves the protease cleavage site of the chimeric protein can be expressed in the same cell that expresses the chimeric protein. The protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to the cell that expresses the chimeric protein. In other words, the cell engineered to express the chimeric protein can endogenously express a protease specific for the protease cleavage site present in the chimeric protein. Endogenous expression of a protease generally refers to both expression under homeostatic conditions (e.g., cells generally considered healthy) and differential expression under non-homeostatic 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, cleavage of the protease cleavage site (and thus release / secretion of the payload) can generally be limited to only the cells of interest due to the cell-restricted protease that must contact the protease cleavage site of the chimeric protein expressed in the same cells.For example, without wishing to be bound by theory, it is believed that ADAM17 is endogenously expressed in NK cells and T cells.In this way, the selection of an ADAM17-specific protease cleavage site can limit the cleavage of the protease cleavage site to NK cells and T cells that co-express the chimeric protein.In another example, the protease cleavage site can be selected for a specific tumor-associated protease that is known to be expressed in a specific tumor population of interest (e.g., in a specific tumor cell engineered to express the chimeric protein).Suitable protease cleavage sites are selected using protease and / or expression databases, for example, by consulting Oncomine (www.oncomine.org), the European Bioinformatics Institute (www.ebi.ac.uk), particularly (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), each of which is incorporated by reference for all purposes.

[0115] 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 be engineered to express a protease that is not generally expressed by the cell that is specific for the protease cleavage site present in the chimeric protein. Cells engineered to express both a chimeric protein and a protease can be engineered to express each from separate engineered nucleic acids or from a multicistronic system (multicistronic and multipromoter systems are described in more detail herein in the section entitled "Multicistronic and Multipromoter Systems"). Heterologous proteases and their corresponding protease cleavage sites can be selected as described above with reference to endogenous proteases.

[0116] The protease that cleaves the protease cleavage site of the chimeric protein can be expressed on a different cell than the cell that expresses the chimeric protein. For example, the protease can be generally expressed in a specific cellular environment, such as the tumor microenvironment. In such cases, cleavage of the protease cleavage site can generally be restricted to only the cellular environment of interest (e.g., the tumor microenvironment) due to the environmentally restricted protease that must contact the protease cleavage site. In embodiments with a membrane-cleavable chimeric protein, secretion of the effector molecule can generally be restricted to only the cellular environment of interest (e.g., the tumor microenvironment) due to the environmentally restricted protease that must contact the protease cleavage site. The protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to the different cells. The protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to the different cells. For example, the different cells can be engineered to express a protease that is not generally expressed by the different cells.

[0117] Proteases include, but are not limited to, type 1 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, matriptase protease, matriptase 2 protease, and MMP9 protease. The protease may be an NS3 protease. The protease may be an ADAM17 protease.

[0118] The protease can be a tumor-associated protease, such as a cathepsin, a cysteine ​​protease, an aspartyl protease, a serine protease, or a 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, metalloproteinase 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. Proteases also include, but are not limited to, the proteases listed in Table 4B below. Exemplary cognate protease cleavage sites for particular proteases are also listed in Table 4B.

[0119] Table 4B: Exemplary proteases with cognate cleavage sites and inhibitors TIFF2025533842000012.tif102164TIFF2025533842000013.tif254164TIFF20255338420 00014.tif253164TIFF2025533842000015.tif254164TIFF2025533842000016.tif249164 TIFF2025533842000017.tif254164TIFF2025533842000018.tif249164TIFF20255338420 00019.tif249164TIFF2025533842000020.tif244164TIFF2025533842000021.tif163164

[0120] The protease can be any of the following human proteases (MEROPS peptidase database numbers provided in parentheses; Rawlings ND, Morton FR, Kok, CY, Kong, J. & Barrett AJ (2008) MEROPS: the peptidase database. Nucleic Acids Res. 36 Database issue, D320-325; incorporated herein by reference for all purposes): pepsin A (MER000885), gastricin (MER000894), memapsin-2 (MER005870), renin (MER000917), cathepsin D (MER000911), cathepsin E (MER000944), memapsin-1 (MER005 534), napsin A (MER004981), Mername-AA034 peptidase (MER014038), pepsin A4 (MER037290), pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens) type putative peptidase (MER107386), napsin B pseudogene (MER004982), CYMP gp (Homo sapiens) (MER002929), subfamily A1A unassigned peptidase (MER181559), mouse mammary tumor virus retropepsin (MER048030), rabbit endogenous retroviral 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 putative RTVL-H type peptidase (MER047206), putative RTVL-H type peptidase (MER047253), putative RTVL-H type peptidase (MER047260), putative RTVL-H type peptidase (MER047291), putative RTVL-H type peptidase (MER047418), putative RTVL-H type peptidase (MER047440), putative RTVL-H type peptidase (MER047479), putative RTVL-H type peptidase (MER047559), putative RTVL-H type peptidase (MER047583),RTVL-H type putative peptidase (MER015446), human endogenous retroviral retropepsin homolog 1 (MER015479), human endogenous retroviral retropepsin homolog 2 (MER015481), endogenous retroviral retropepsin pseudogene 1 (Homo sapiens chromosome 14) (MER029977), endogenous retroviral retropepsin pseudogene 2 (Homo sapiens chromosome 8) (MER029665), endogenous retroviral retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER002660), endogenous Endogenous retroviral retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER030286), endogenous retroviral retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER047144), endogenous retroviral retropepsin pseudogene 5 (Homo sapiens chromosome 12) (MER029664), endogenous retroviral retropepsin pseudogene 6 (Homo sapiens chromosome 7) (MER002094), endogenous retroviral retropepsin pseudogene 7 (Homo sapiens chromosome 6) (MER029776), endogenous retroviral retropepsin pseudogene 8 (Homo sapiens chromosome 9) (MER029776), endogenous retroviral retropepsin pseudogene 9 (Homo sapiens chromosome 10) (MER029776), endogenous retroviral retropepsin pseudogene 10 (Homo sapiens chromosome 11) (MER029776), endogenous retroviral retropepsin pseudogene 11 (Homo sapiens chromosome 12) (MER029664), endogenous retroviral retropepsin pseudogene 12 (Homo sapiens chromosome 13) (MER029664), endogenous retroviral retropepsin pseudogene 13 (Homo sapiens chromosome 14) (MER029664), endogenous retroviral retropepsin pseudogene 14 (Homo sapiens chromosome 15) (MER029664), endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 16) (MER029776), endogenous retroviral retropepsin pseudogene 16 (Homo sapiens chromosome 1 Endogenous retroviral retropepsin pseudogene 8 (Homo sapiens chromosome Y) (MER030291), endogenous retroviral retropepsin pseudogene 9 (Homo sapiens chromosome 19) (MER029680), endogenous retroviral retropepsin pseudogene 10 (Homo sapiens chromosome 12) (MER002848), endogenous retroviral retropepsin pseudogene 11 (Homo sapiens chromosome 17) (MER004378), endogenous retroviral retropepsin pseudogene 12 (Homo sapiens chromosome 11) (MER003344), endogenous retroviral retropepsin pseudogene 13 (Homo sapiens chromosome 14) (MER003348), Endogenous retroviral retropepsin pseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779), endogenous retroviral retropepsin pseudogene 14 (Homo sapiens chromosome 2) (MER029778), endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047158), endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047332), endogenous retroviral retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER003182),Endogenous retroviral retropepsin pseudogene 16 (MER047165), endogenous retroviral retropepsin pseudogene 16 (MER047178), endogenous retroviral retropepsin pseudogene 16 (MER047200), endogenous retroviral retropepsin pseudogene 16 (MER047315), endogenous retroviral retropepsin pseudogene 16 (MER047405), endogenous retroviral retropepsin pseudogene 16 (MER030292), endogenous retroviral retropepsin pseudogene 17 (Homo sapiens chromosome 8) (M ER005305), endogenous retroviral retropepsin pseudogene 18 (Homo sapiens chromosome 4) (MER030288), endogenous retroviral retropepsin pseudogene 19 (Homo sapiens chromosome 16) (MER001740), endogenous retroviral retropepsin pseudogene 21 (Homo sapiens) (MER047222), endogenous retroviral retropepsin pseudogene 21 (Homo sapiens) (MER047454), endogenous retroviral retropepsin pseudogene 21 (Homo sapiens) (MER047477), endogenous retrovirus Retropepsin pseudogene 21 (Homo sapiens) (MER004403), endogenous retroviral retropepsin pseudogene 22 (Homo sapiens chromosome X) (MER030287), subfamily A2A nonpeptidase homolog (MER047046), subfamily A2A nonpeptidase homolog (MER047052), subfamily A2A nonpeptidase homolog (MER047076), subfamily A2A nonpeptidase homolog (MER047080), subfamily A2A nonpeptidase homolog (MER047088), subfamily -A2A nonpeptidase homolog (MER047089), subfamily A2A nonpeptidase homolog (MER047091), subfamily A2A nonpeptidase homolog (MER047092), subfamily A2A nonpeptidase homolog (MER047093), subfamily A2A nonpeptidase homolog (MER047094), subfamily A2A nonpeptidase homolog (MER047097), subfamily A2A nonpeptidase homolog (MER047099), subfamily A2A nonpeptidase homolog MER047101),Subfamily A2A nonpeptidase homolog (MER047102), Subfamily A2A nonpeptidase homolog (MER047107), Subfamily A2A nonpeptidase homolog (MER047108), Subfamily A2A nonpeptidase homolog (MER047109), Subfamily A2A nonpeptidase homolog (MER047110), Subfamily A2A nonpeptidase homolog MER047111), Subfamily A2A nonpeptidase homolog (MER047114), Subfamily A2A nonpeptidase homolog (MER 047118), subfamily A2A nonpeptidase homolog (MER047121), subfamily A2A nonpeptidase homolog (MER047122), subfamily A2A nonpeptidase homolog (MER047126), subfamily A2A nonpeptidase homolog (MER047129), subfamily A2A nonpeptidase homolog (MER047130), subfamily A2A nonpeptidase homolog (MER047134), subfamily A2A nonpeptidase homolog (MER047135), subfamily A2A nonpeptidase Subfamily A2A nonpeptidase homolog (MER047137), subfamily A2A nonpeptidase homolog (MER047140), subfamily A2A nonpeptidase homolog (MER047141), subfamily A2A nonpeptidase homolog (MER047142), subfamily A2A nonpeptidase homolog (MER047148), subfamily A2A nonpeptidase homolog (MER047149), subfamily A2A nonpeptidase homolog (MER047151), subfamily A2A nonpeptidase homolog (MER047154), subfamily A 2A nonpeptidase homolog (MER047155), subfamily A2A nonpeptidase homolog (MER047156), subfamily A2A nonpeptidase homolog (MER047157), subfamily A2A nonpeptidase homolog (MER047159), subfamily A2A nonpeptidase homolog (MER047161), subfamily A2A nonpeptidase homolog (MER047163), subfamily A2A nonpeptidase homolog (MER047166), subfamily A2A nonpeptidase homolog (MER047171),Subfamily A2A nonpeptidase homolog (MER047173), Subfamily A2A nonpeptidase homolog (MER047174), Subfamily A2A nonpeptidase homolog (MER047179), Subfamily A2A nonpeptidase homolog (MER047183), Subfamily A2A nonpeptidase homolog (MER047186), Subfamily A2A nonpeptidase homolog (MER047190), Subfamily A2A nonpeptidase homolog (MER047191), Subfamily A2A nonpeptidase homolog (MER 047196), subfamily A2A nonpeptidase homolog (MER047198), subfamily A2A nonpeptidase homolog (MER047199), subfamily A2A nonpeptidase homolog (MER047201), subfamily A2A nonpeptidase homolog (MER047202), subfamily A2A nonpeptidase homolog (MER047203), subfamily A2A nonpeptidase homolog (MER047204), subfamily A2A nonpeptidase homolog (MER047205), subfamily A2A nonpeptidase Subfamily A2A nonpeptidase homolog (MER047207), subfamily A2A nonpeptidase homolog (MER047208), subfamily A2A nonpeptidase homolog (MER047210), subfamily A2A nonpeptidase homolog (MER047211), subfamily A2A nonpeptidase homolog (MER047212), subfamily A2A nonpeptidase homolog (MER047213), subfamily A2A nonpeptidase homolog (MER047215), subfamily A2A nonpeptidase homolog (MER047216), subfamily A 2A 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 nonpeptidase homolog (MER047232), Subfamily A2A nonpeptidase homolog (MER047233), Subfamily A2A nonpeptidase homolog (MER047234), Subfamily A2A nonpeptidase homolog (MER047236), Subfamily A2A nonpeptidase homolog (MER047238), Subfamily A2A nonpeptidase homolog (MER047239), Subfamily A2A nonpeptidase homolog (MER047240), Subfamily A2A nonpeptidase homolog (MER047242), Subfamily A2A nonpeptidase homolog (MER047243), Subfamily A2A nonpeptidase homolog (MER047249), Subfamily A2A nonpeptidase homolog (MER047251), Subfamily A2A nonpeptidase homolog (MER047252), Subfamily A2A nonpeptidase homolog (MER047254), Subfamily A2A nonpeptidase homolog (MER 047255), subfamily A2A nonpeptidase homolog (MER047263), subfamily A2A nonpeptidase homolog (MER047265), subfamily A2A nonpeptidase homolog (MER047266), subfamily A2A nonpeptidase homolog (MER047267), subfamily A2A nonpeptidase homolog (MER047268), subfamily A2A nonpeptidase homolog (MER047269), subfamily A2A nonpeptidase homolog (MER047272), subfamily A2A nonpeptidase Subfamily A2A nonpeptidase homolog (MER047273), subfamily A2A nonpeptidase homolog (MER047274), subfamily A2A nonpeptidase homolog (MER047275), subfamily A2A nonpeptidase homolog (MER047276), subfamily A2A nonpeptidase homolog (MER047279), subfamily A2A nonpeptidase homolog (MER047280), subfamily A2A nonpeptidase homolog (MER047281), subfamily A2A nonpeptidase homolog (MER047282), subfamily A 2A nonpeptidase homolog (MER047284), subfamily A2A nonpeptidase homolog (MER047285), subfamily A2A nonpeptidase homolog (MER047289), subfamily A2A nonpeptidase homolog (MER047290), subfamily A2A nonpeptidase homolog (MER047294), subfamily A2A nonpeptidase homolog (MER047295), subfamily A2A nonpeptidase homolog (MER047298), subfamily A2A nonpeptidase homolog (MER047300),Subfamily A2A nonpeptidase homolog (MER047302), Subfamily A2A nonpeptidase homolog (MER047304), Subfamily A2A nonpeptidase homolog (MER047305), Subfamily A2A nonpeptidase homolog (MER047306), Subfamily A2A nonpeptidase homolog (MER047307), Subfamily A2A nonpeptidase homolog (MER047310), Subfamily A2A nonpeptidase homolog (MER047311), Subfamily A2A nonpeptidase homolog (MER 047314), subfamily A2A nonpeptidase homolog (MER047318), subfamily A2A nonpeptidase homolog (MER047320), subfamily A2A nonpeptidase homolog (MER047321), subfamily A2A nonpeptidase homolog (MER047322), subfamily A2A nonpeptidase homolog (MER047326), subfamily A2A nonpeptidase homolog (MER047327), subfamily A2A nonpeptidase homolog (MER047330), subfamily A2A nonpeptidase Subfamily A2A nonpeptidase homolog (MER047333), subfamily A2A nonpeptidase homolog (MER047362), subfamily A2A nonpeptidase homolog (MER047366), subfamily A2A nonpeptidase homolog (MER047369), subfamily A2A nonpeptidase homolog (MER047370), subfamily A2A nonpeptidase homolog (MER047371), subfamily A2A nonpeptidase homolog (MER047375), subfamily A2A nonpeptidase homolog (MER047376), subfamily A 2A nonpeptidase homolog (MER047381), subfamily A2A nonpeptidase homolog (MER047383), subfamily A2A nonpeptidase homolog (MER047384), subfamily A2A nonpeptidase homolog (MER047385), subfamily A2A nonpeptidase homolog (MER047388), subfamily A2A nonpeptidase homolog (MER047389), subfamily A2A nonpeptidase homolog (MER047391), subfamily A2A nonpeptidase homolog (MER047394),Subfamily A2A nonpeptidase homolog (MER047396), Subfamily A2A nonpeptidase homolog (MER047400), Subfamily A2A nonpeptidase homolog (MER047401), Subfamily A2A nonpeptidase homolog (MER047403), Subfamily A2A nonpeptidase homolog (MER047406), Subfamily A2A nonpeptidase homolog (MER047407), Subfamily A2A nonpeptidase homolog (MER047410), Subfamily A2A nonpeptidase homolog (MER 047411), Subfamily A2A nonpeptidase homolog (MER047413), Subfamily A2A nonpeptidase homolog (MER047414), Subfamily A2A nonpeptidase homolog (MER047416), Subfamily A2A nonpeptidase homolog (MER047417), Subfamily A2A nonpeptidase homolog (MER047420), Subfamily A2A nonpeptidase homolog (MER047423), Subfamily A2A nonpeptidase homolog (MER047424), Subfamily A2A nonpeptidase Subfamily A2A nonpeptidase homolog (MER047428), subfamily A2A nonpeptidase homolog (MER047429), subfamily A2A nonpeptidase homolog (MER047431), subfamily A2A nonpeptidase homolog (MER047434), subfamily A2A nonpeptidase homolog (MER047439), subfamily A2A nonpeptidase homolog (MER047442), subfamily A2A nonpeptidase homolog (MER047445), subfamily A2A nonpeptidase homolog (MER047449), subfamily A 2A nonpeptidase homolog (MER047450), subfamily A2A nonpeptidase homolog (MER047452), subfamily A2A nonpeptidase homolog (MER047455), subfamily A2A nonpeptidase homolog (MER047457), subfamily A2A nonpeptidase homolog (MER047458), subfamily A2A nonpeptidase homolog (MER047459), subfamily A2A nonpeptidase homolog (MER047463), subfamily A2A nonpeptidase homolog (MER047468),Subfamily A2A nonpeptidase homolog (MER047469), Subfamily A2A nonpeptidase homolog (MER047470), Subfamily A2A nonpeptidase homolog (MER047476), Subfamily A2A nonpeptidase homolog (MER047478), Subfamily A2A nonpeptidase homolog (MER047483), Subfamily A2A nonpeptidase homolog (MER047488), Subfamily A2A nonpeptidase homolog (MER047489), Subfamily A2A nonpeptidase homolog (MER 047490), Subfamily A2A nonpeptidase homolog (MER047493), Subfamily A2A nonpeptidase homolog (MER047494), Subfamily A2A nonpeptidase homolog (MER047495), Subfamily A2A nonpeptidase homolog (MER047496), Subfamily A2A nonpeptidase homolog (MER047497), Subfamily A2A nonpeptidase homolog (MER047499), Subfamily A2A nonpeptidase homolog (MER047502), Subfamily A2A nonpeptidase Subfamily A2A nonpeptidase homolog (MER047504), subfamily A2A nonpeptidase homolog (MER047511), subfamily A2A nonpeptidase homolog (MER047513), subfamily A2A nonpeptidase homolog (MER047514), subfamily A2A nonpeptidase homolog (MER047515), subfamily A2A nonpeptidase homolog (MER047516), subfamily A2A nonpeptidase homolog (MER047520), subfamily A2A nonpeptidase homolog (MER047533), subfamily A 2A nonpeptidase homolog (MER047537), subfamily A2A nonpeptidase homolog (MER047569), subfamily A2A nonpeptidase homolog (MER047570), subfamily A2A nonpeptidase homolog (MER047584), subfamily A2A nonpeptidase homolog (MER047603), subfamily A2A nonpeptidase homolog (MER047604), subfamily A2A nonpeptidase homolog (MER047606), subfamily A2A nonpeptidase homolog (MER047609),Subfamily A2A nonpeptidase homolog (MER047616), Subfamily A2A nonpeptidase homolog (MER047619), Subfamily A2A nonpeptidase homolog (MER047648), Subfamily A2A nonpeptidase homolog (MER047649), Subfamily A2A nonpeptidase homolog (MER047662), Subfamily A2A nonpeptidase homolog (MER048004), Subfamily A2A nonpeptidase homolog (MER048018), Subfamily A2A nonpeptidase homolog (MER048019), subfamily A2A nonpeptidase homolog (MER048023), subfamily A2A nonpeptidase homolog (MER048037), subfamily A2A unassigned peptidase (MER047164), subfamily A2A unassigned peptidase (MER047231), subfamily A2A unassigned peptidase (MER047386), skin aspartic protease (MER057097), presenilin 1 (MER005221), presenilin 2 (MER005223), impas 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 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 gp (Homo sapiens) (MER005210), CTSLL3, gp (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 (MER 029744), Mername-AA253 peptidase (MER005537), Carpamodulin (MER000718), Hypothetical protein 940251 (MER003201), Ubiquitinyl hydrolase-L1 (MER000832), Ubiquitinyl hydrolase-L3 (MER000836), Ubiquitinyl hydrolase-BAP1 (MER003989), Ubiquitinyl 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 type) (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 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 [misread] (MER064621), Mername-AA090 non-peptidase homolog (MER014739), ubiquitin-specific peptidase 43 [misread] (MER030140), ubiquitin-specific peptidase 52 [misread] (MER030317), NEK2 pseudogene (MER014736), C19 pseudogene (Homo sapiens: 5th chromosome) (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 gp (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), legumain (plant alpha type) (MER044591), legumain (MER001800), glycosylphosphatidylinositol:protein transamidase (MER002479), legumain 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-11 (MER002579), caspase-2 (MER001644), caspase-4 (MER001938), caspase-5 (MER002240), caspase-8 (MER002849), caspase-9 (MER002707), caspase-10 (MER002579), caspase-12 (MER002579), caspase-13 (MER002579), caspase-14 (MER002579), caspase-15 (MER002579), caspase-16 (MER002579), caspase-17 (MER002579), caspase-18 (MER002579), caspase-19 (MER002579), caspase-20 (MER002579), caspase-21 (MER002579), caspase-22 (MER002579), caspase-23 (MER002579), caspase-24 (MER002579), caspase-25 (MER002579), caspase-26 (MER002579), caspase-27 (MER002579), caspase-28 (MER002 Caspase-14 (MER012083), paracaspase (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 nonpeptidase homolog (MER185329), subfamily C14A nonpeptidase homolog (MER179956), separase (Homo sapiens) 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 (MER016162), ER005557), pyroglutamyl-peptidase I (chordate) (MER011032), Mername-AA073 peptidase (MER029978), sonic hedgehog protein (MER002539), Indian hedgehog protein (MER002538), desert hedgehog protein (MER012170), dipeptidyl-peptidase III (MER004252), Mername-AA164 protein (MER020410),LOC138971 gp (Homo sapiens) (MER020074), Atp23 peptidase (MER060642), prenylpeptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyroglutamyl-peptidase II (MER012221), cytosolic alanyl aminopeptidase (MER002746), cystinyl 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-related factor (MER026493), angiotensin-converting enzyme peptidase unit 1 (MER004967), angiotensin-converting enzyme peptidase unit 2 (MER001019), angiotensin-converting enzyme-2 (ME R011061), Mername-AA153 protein (MER020514), Thimet oligopeptidase (MER001737), Neurolysin (MER010991), Mitochondrial intermediate peptidase (MER003665), Mername-AA154 protein (MER021317), Reishimanolysin-2 (MER014492), Reishimanolysin-3 (MER180031), Matrix metallopeptidase-1 (MER001063), Matrix metallopeptidase-8 (MER001084), Matrix metallopeptidase-1 (MER001065), Matrix metallopeptidase-2 (MER001066), Matrix metallopeptidase-3 (MER001085), Matrix metallopeptidase-1 (MER001066), Matrix metallopeptidase-2 (MER001067), Matrix metallopeptidase-3 (MER001085), Matrix metallopeptidase-1 (MER001068), Matrix metallopeptidase-2 (MER001069), Matrix metallopeptidase-3 (MER001086), Matrix metallopeptidase-1 (MER001069 ...69), Matrix metallopeptidase-1 (MER001069), Matrix metallopeptidase-3 (MER001069), Matrix metallopeptidase-1 (MER001069), Matrix metallopeptidase-2 (MER001069), Matrix metal 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, 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 nonpeptidase homolog (MER175912), subfamily M10A nonpeptidase homolog (MER187997), subfamily M10A nonpeptidase homolog (MER187998), subfamily M10A nonpeptidase homolog (MER180000), meprin alpha subunit (MER001111), meprin beta subunit (MER005213), procollagen C-peptidase (MER001113), mammalian tolloid-like 1 protein (MER005124), mammalian tolloid-like2 protein (MER005866), ADAMTS9 peptidase (MER012092), ADAMTS14 peptidase (MER016700), ADAMTS15 peptidase (MER017029), ADAMTS16 peptidase (MER015689), ADAMTS17 peptidase (MER016302), ADAMTS18 peptidase (MER016090), ADAMTS19 peptidase (MER015663), ADAMTS8 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 (Homo sapiens type) (MER005495), ADAMTS5 peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893), ADAMTS7 peptidase (MER005894), ADAM30 peptidase ADAMTS10 peptidase (MER014331), ADAMTS12 peptidase (MER014337), ADAMTS13 peptidase (MER015450), ADAM33 peptidase (MER015143), ovastacin (MER029996), ADAMTS20 peptidase (Homo sapiens type) (MER026906), procollagen IN-peptidase (MER004985), ADAM2 protein (MER003090), ADAM6 protein (MER047044), ADAM7 protein (MER005109), ADAM18 protein (MER012230), ADAM32 protein (MER026938), non-peptidase homolog (Homo sapiens chromosome 4) (MER029973), family M12 non-peptidase homolog (Homo sapiens chromosome 16) (MER047654), family M12 non-peptidase homolog (Homo sapiens chromosome 15) (MER047250), AD Proteins similar to AM3B protein (Homo sapiens type) (MER005199), ADAM11 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), ADAM21 peptidase preproprotein (Homo sapiens) (MER026944), Mername-AA225 peptidase homolog (Homo sapiens) (MER047474), putative ADAM pseudogene (chromosome 4, Homo sapiens) (MER029975), ADAM3A gp (Homo sapiens) (MER005200), ADAM1 gp (Homo sapiens) (MER003912), subfamily M12B nonpeptidase homolog (MER188210), subfamily M12B nonpeptidase homolog (MER188211), subfamily M12B nonpeptidase homolog (MER188212), subfamily M12B nonpeptidase homolog (MER188220), neprilysin (MER001050), endothelin-converting enzyme 1 ( MER001057), endothelin-converting enzyme 2 (MER004776), DINE peptidase (MER005197), neprilysin-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), papalysin-1 (MER002217), papalysin-2 (MER014521), farnesylated protein convertase 1 (MER002646), metalloprotease-related protein-1 (MER030873), aminopeptidase AMZ2 (MER011907), aminopeptidase AMZ1 (MER058242), carboxypeptidase A1 (MER001190), carboxypeptidase A2 (MER001608), carboxypeptidase B (MER 001194), 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), cytosolic carboxypeptidase-like protein 5 (MER033174), cytosolic carboxypeptidase 3 (MER033176), cytosolic carboxypeptidase 6 (MER033178), cytosolic carboxypeptidase 1 (MER033179), cytosolic ...4 (MER013421), carboxypeptidase A6 (MER013456), carboxypeptidase A5 (MER017121), metallocarboxypeptidase O (MER016044), cytosolic carboxypeptidase A5 (MER013421), cytosolic carboxypeptidase A6 (MER013456), cytosolic carboxypeptidase A5 (MER017121), metallocarboxypeptidase A6 (MER013456), cytosolic carboxypeptidase A5 (MER017121), metallocarboxypeptidase A6 (MER016044), cytosolic carboxypeptidase A5 (MER016044), cytosolic carboxypeptidase A5 (MER033174), cytosolic Peptidase 2 (MER037713), metallocarboxypeptidase D nonpeptidase unit (MER004964), adipocyte enhancer binding protein 1 (MER003889), carboxypeptidase-like protein X1 (MER013404), carboxypeptidase-like protein X2 (MER078764), cytosolic carboxypeptidase (MER026952), family M14 nonpeptidase homolog (MER199530), insulysin (MER001214), mitochondrialProcessing peptidase beta subunit (MER004497), nardilysin (MER003883), eupitolillysin (MER004877), mitochondrial processing peptidase nonpeptidase alpha subunit (MER001413), ubiquinol-cytochrome c reductase core protein I (MER003543), ubiquinol-cytochrome c reductase core protein II (MER003544), ubiquinol-cytochrome c reductase core protein domain 2 (MER043998), insulysin unit 2 (MER046821), nardilysin unit 2 (MER046874), insulysin unit 3 (MER078753), mitochondrial processing peptidase subunit alpha Unit 2 (MER124489), Nardi Rising Unit 3 (MER142856), LOC133083gp (Homo sapiens) (MER021876), subfamily M16B non-peptidase homolog (MER188757), leucyl aminopeptidase (animal) (MER003100), Mername-AA040 peptidase (MER003919), leucyl aminopeptidase-1 (Cenorhabditis type) (MER013416), methionyl aminopeptidase 1 (MER001342), methionyl aminopeptidase 2 (M ER001728), aminopeptidase P2 (MER004498), Xaa-Pro dipeptidase (eukaryotic) (MER001248), aminopeptidase P1 (MER004321), mitochondrial intermediate cleavage 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 (human chromosome X) (MER029983), Mername-AA226 peptidase homolog (Homo sapiens) (MER056262), Mername-AA227 peptidase homolog (Homo sapiens) (MER047299), subfamily M24A non-peptidase homolog (MER17989 3), aspartyl aminopeptidase (MER003373), Gly-Xaa carboxypeptidase (MER033182), carnosine dipeptidase II (MER014551), carnosine dipeptidase I (MER015142), Mername-AA161 protein (MER021873), aminoacylase (MER001271), glutamate carboxypeptidase II (MER002104), NAALADASEL-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 (M Hypothetical proteins such as ER001260), 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), and 5730457F11RIK (MER033184) Protein, 1300019j08rik protein (MER033186), guanine aminohydrolase (MER037714), Kae1 putative peptidase (MER001577), OSGEPL1-like protein (MER013498), S2P peptidase (MER004458), subfamily M23B nonpeptidase homolog (MER199845), subfamily M23B nonpeptidase homolog (MER199846), subfamily M23B nonpeptidase homolog (MER199847), subfamily M23B nonpeptidase homolog (MER137320), subfamily M23B nonpeptidase homolog (MER201557), subfamily M23B nonpeptidase homolog (MER199417), subfamily M23B nonpeptidase homolog (MER199418), subfamily M23B nonpeptidase homolog (MER199419), subfamily M23B nonpeptidase homolog (MER199420), subfamily M23B nonpeptidase homolog (MER175932), subfamily M23B nonpeptidase homolog (MER199 665), Poh1 peptidase (MER020382), Jab1 / MPN domain metalloenzyme (MER022057), Mername-AA165 peptidase (MER021865), Brcc36 isopeptidase (MER021890), histone H2A deubiquitinating enzyme MYSM1 (MER021887), AMSH deubiquitinating peptidase (MER030146), putative peptidase (Homo sapiens chromosome 2) (MER029970), Mername-AA168 protein (MER021886), COP9 signalosome subunit 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), choline (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 (rodent) (MER016130), adrenal secretory serine peptidase (MER000 3734), tryptase delta 1 (Homo sapiens) (MER005948), matriptase-3 (MER029902), marapsin (MER006119), tryptase-6 (MER006118), ovochymase-1 domain 1 (MER099182), transmembrane peptidase, serine 3 (MER005926), kallikrein-related peptidase 15 (MER000064), Mername-AA031 peptidase (MER014054), TMPRSS13 peptidase (MER014226), Mername-AA038 peptidase 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), myeloblastin (MER000170), granzyme A (MER001379), granzyme M (MER001541), chymase (Homo sapiens type) (MER000123), tryptase A LFA (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 activated C1r (MER000238), complement component activated C1s (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 (MER000 150), 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), manna Serine-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), epitheliacin (MER003736), kallikrein-related peptidase 4 (MER003645), ER005266), prosemin (MER004214), chymopasin (MER001503), kallikrein-related peptidase 11 (MER004861), kallikrein-related peptidase 11 (MER216142), trypsin type 2A (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), matriptase (MER003735), kallikrein-related peptidase 13 (MER005269), kallikrein-related peptidase 9 (MER005270), matriptase-2 (MER005278), umbilical vein peptidase (MER005421), LCLP peptidase idase (MER001900), spinesin (MER014385), marapsin-2 (MER021929), complement factor D-like putative peptidase (MER056164), ovochymase-2 (MER022410), HAT-like 4 peptidase (MER044589), ovochymase 1 domain 1 (MER022412), epidermis-specific SP-like putative peptidase (MER029900), testicular serine peptidase 5 (MER029901), Mername-AA258 peptidase (MER000285), polyserine IA unit 1 (MER 030879), polyserase-IA unit 2 (MER030880), testicular serine peptidase 2 (human type) (MER033187), hypothetical acrosin-like peptidase (Homo sapiens) (MER033253), HAT-like 5 peptidase (MER028215), polyserase-3 unit 1 (MER061763), polyserase-3 unit 2 (MER061748), tryptophan / serine protease-like peptidase (MER056263), polyserase-2 unit 1 (MER061777), Mername-A A123 peptidase (MER021930), HAT-like 2 peptidase (MER099184), hCG2041452-like protein (MER099172), hCG22067 (Homo sapiens) (MER099169), brain rescue factor-1 (human) (MER098873), hCG2041108 (Homo sapiens) (MER099173), polymerase-2 unit 2 (MER061760), polymerase-2 unit 3 (MER065694), AA201 (peptidase homolog) (MER099175).Secretory trypsin-like serine peptidase homolog (MER030000), polyserase-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-like protein Protein 4 (MER016346), PRSS35 protein (MER016350), DKFZp586H2123-like protein (MER066474), apolipoprotein (MER000183), psi-KLK1 pseudogene (Homo sapiens) (MER033287), tryptase pseudogene I (MER015077), tryptase pseudogene II (MER015078), tryptase pseudogene III (MER015079), subfamily S1A unassigned peptidase (MER216982), subfamily S1A unassigned peptidase (MER216148), amidophosphoribosyltransferase precursor (MER003314), glutamine-fructose-6-phosphate transaminase 1 (MER003322), glutamine:fructose-6-phosphate amidotransferase (MER012158), Mername-AA144 protein (MER021319), asparagine synthetase (MER033254), family C44 nonpeptidase homolog (MER159286), family C44 unassigned peptidase (MER185625), family C44 4 unassigned peptidase (MER185626), cecernin 1 (MER045376), cecernin 2 (MER064573), cecernin 3 (MER064582), acid ceramidase precursor (MER100794), N-acylethanolamino 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 alpha 6 (MER000557), proteasome subunit alpha 2 (MER000550), proteasome subunit alpha 4 (MER000, 554), proteasome subunit alpha 7 (MER033250), proteasome subunit alpha 5 (MER000558), proteasome subunit alpha 1 (MER000549), proteasome subunit alpha 3 (MER000553), proteasome subunit XAPC7 (MER004372), proteasome subunit beta 3 (MER001710), proteasome subunit beta 2 (MER002676), proteasome subunit beta 1 (MER000551), proteasome Glycosylated rRNA subunit beta 4 (MER001711), Mername-AA230 peptidase homolog (Homo sapiens) (MER047329), Mername-AA231 pseudogene (Homo sapiens) (MER047172), Mername-AA232 pseudogene (Homo sapiens) (MER047316), glycosylated asparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), taspase-1 (MER016969), gamma-glutamyltransferase 5 (mammalian Similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204), gamma-glutamyltransferase 1 precursor (Homo sapiens) (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), gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204), gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER001977). Similar to ER026205), Mername-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyltranspeptidase homolog (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (MER172554), gamma-glutamyl hydrolase (MER002963), guanine 5''-monophosphate synthase (MER043387),Carbamoylphosphate 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) (M ER160094), Family C56 nonpeptidase homolog (MER177016), Family C56 nonpeptidase homolog (MER176613), Family C56 nonpeptidase homolog (MER176918), EGF-like module containing mucin-like hormone receptor-like 2 (MER037230), CD97 antigen (human) (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 LAG7 pathway G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), Latrophilin 2 (MER122199), Latrophilin-1 (MER126380), Latrophilin 3 (MER124612), Protocadherin Flamingo 2 (MER124239), ETL protein (MER126267), G protein coupled receptor 112 (MER126114), seven-transmembrane helix receptor (MER125448), Gpr114 protein (MER159320), GPR126 angiogenic 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 (MER1597 46), PIDD autoprocessing protein unit 1 (MER020001), PIDD autoprocessing protein unit 2 (MER063690), MUC1 autocleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site 1 peptidase (MER001948), furin (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER000377), pro Protein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl peptidase II (MER000355), subfamily S8A nonpeptidase homolog (MER201339), subfamily S8A nonpeptidase homolog (MER191613), subfamily S8A unassigned peptidase (MER1916 11), subfamily S8A unassigned peptidase (MER191612), subfamily S8A unassigned peptidase (MER191614), tripeptidyl peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl peptidase IV (eukaryotic) (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 flj40 219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DPP10 (MER005988), protein similar to mouse 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 Binding (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 nonpeptidase homolog (MER212939), family S9 nonpeptidase 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 (MER000430), vitellogenic carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unassigned peptidase (MER1994 42), 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), and Abhydrolase domain-containing protein cytosolic epoxide hydrolase (MER000432), mesoderm-specific transcription protein (MER199890), mesoderm-specific transcription protein (MER017123), cytosolic 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 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), Isoaspartyl dipeptidase (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). gamma-glutamyltransferase 1 precursor, Similar to precursor (Homo sapiens) (MER026205). Mername-AA211 putative peptidase (MER026207). Gamma-glutamyltransferase 6 (MER159283). Gamma-glutamyltranspeptidase homolog (chromosome 2, Homo sapiens) (MER037241). Polycystin-1 (MER126824), KIAA1879 protein (MER159329). Polycystic kidney disease 1-like 3 (MER172554). Gamma-glutamyl hydrolase (MER002963). Guanine 5'-monophosphate synthetase (MER043387). Carbamoylphosphate 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 Homo sapiens (MER160094). Family C56 nonpeptidase homolog (MER177016), Family C56 nonpeptidase homolog (MER176613). Family C56 nonpeptidase homolog (MER176918). EGF-like module containing mucin-like hormone receptor-like 2 (MER037230). CD97 antigen (human) (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 The seven-transmembrane G-type receptor 2 precursor of LAG (Homo sapiens) (MER045397), Gpr64 (house mouse) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), latrophilin 2 (MER122199), latrophilin-1 (MER126380), latrophilin 3 (MER124612),Protocadherin Flamingo 2 (MER124239). ETL protein (MER126267). G protein-coupled receptor 112 (MER126114). 7-transmembrane helix receptor (MER125448). Gpr114 protein (MER159320). GPR126 angiogenic 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 peptidase (MER001 948), 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 nonpeptidase homolog (MER201339), subfamily S8A nonpeptidase 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) (MER000401), acylaminoacyl peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL Protein A (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 Hypothetical protein (MER033245), hypothetical esterase / lipase / thioesterase (MER047309), protein Bat 5 (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 mouse 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), neuroligin 2 (MER033283), family S9 nonpeptidase homolog (MER212939), family S9 nonpeptidase homolog (MER2111 490), 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), vitellogenic carboxypeptidase-like protein (MER00549 2), 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), Abhydrolase domain-containing protein 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 flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), hypothetical protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1 interacting factor b (MER210738).

[0121] Protease enzyme activity can be regulated. For example, certain proteases can be inactivated by the presence or absence of certain drugs (e.g., those that bind to proteases, such as specific small molecule inhibitors). Such proteases can be referred to as "inhibitory proteases." Exemplary inhibitors for certain proteases are listed in Table 4B. For example, NS3 protease can be inhibited by protease inhibitors, including but not limited to simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxilaprevir. In another example, protease activity can be regulated through regulating the expression of the protease itself, such as by engineering cells to express the protease using an inducible promoter system (e.g., the Tet On / Off system) or a cell-specific promoter (promoters that can be used to express heterologous proteases are described in more detail herein in the section entitled "Promoters"). The protease can also include a degron, such as any of the degrons described herein, and can be regulated using any of the degron systems described herein.

[0122] Protease enzyme activity can also be regulated through the selection of a specific protease cleavage site. For example, a protease cleavage site can be selected and / or engineered to provide a sequence with a desired cleavage rate by a desired protease, such as a reduced cleavage rate relative to the endogenous sequence of a substrate naturally cleaved by the desired protease. As another example, a protease cleavage site can be selected and / or engineered to provide a sequence with a desired cleavage rate in a cellular state-specific manner. For example, various cellular states (e.g., after cell signaling, such as immune cell activation) can affect the expression and / or localization of specific proteases. As an illustrative example, ADAM17 protein levels and localization are known to be affected by signal transduction, for example, through the protein kinase C (PKC) signaling pathway (e.g., activation by the PKC activator phorbol-12-myristat-13-acetate [PMA]). Thus, protease cleavage sites can be selected and / or engineered such that cleavage of the protease cleavage site and subsequent release of the effector molecule is increased or decreased, as desired, depending on the protease characteristics (e.g., expression and / or localization) of a particular cellular state. As another example, protease cleavage sites (especially in combination with particular membrane-tethering domains) can be selected and / or engineered for optimal protein expression of the chimeric protein.

[0123] Plasma membrane anchoring domain The membrane-cleavable chimeric proteins provided herein comprise a plasma membrane anchoring domain (referred to as "MT" in the formula SC-MT or MT-CS). Generally, the plasma membrane anchoring domain can be any amino acid sequence motif capable of directing the chimeric protein to localize (e.g., be inserted into) or otherwise associate with the plasma membrane of a cell expressing the chimeric protein. The plasma membrane anchoring domain can be a transmembrane-intracellular domain. The plasma membrane anchoring domain can be a transmembrane domain. The plasma membrane anchoring domain can be an endogenous protein domain (e.g., a transmembrane domain). The plasma membrane anchoring domain can be derived from a type I, type II, or type III transmembrane protein. The plasma membrane anchoring domain can comprise a post-translational modification tag or a motif capable of post-translational modification for modifying the chimeric protein to include a post-translational modification tag, where the post-translational modification tag enables association with the plasma membrane. Examples of post-translational modification tags include, but are not limited to, lipid anchor domains (e.g., GPI lipid anchors, myristoylation tags, or palmitoylation tags). Examples of cell membrane tethering domains include, but are not limited to, transmembrane-intracellular domains and / or transmembrane domains derived from PDGFR-beta, CD8, CD28, CD3 zeta 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 may comprise a cell surface receptor or a cell membrane-binding portion thereof. The sequences of exemplary cell membrane tethering domains are provided in Table 4C.

[0124] (Table 4C) TIFF2025533842000022.tif51163

[0125] Generally, for all membrane-cleavable chimeric proteins described herein, the plasma membrane-tethering domain is either: (1) C-terminal to the protease cleavage site, if present, and N-terminal to any intracellular domain (in other words, the plasma membrane-tethering domain is between the protease cleavage site and the intracellular domain, if present), or (2) N-terminal to the protease cleavage site and C-terminal to any intracellular domain, if present (also between the protease cleavage site and the intracellular domain, if present, with inverted domain orientation). In embodiments featuring a degron associated with the chimeric protein, the degron domain is specifically a terminal cytoplasm-directing domain with respect to the plasma membrane tethering (in other words, the plasma membrane-tethering domain is between the protease cleavage site and the degron). The plasma membrane-tethering domain can be connected to the protease cleavage site by a polypeptide linker, i.e., a polypeptide sequence not generally considered part of the plasma membrane-tethering domain or the protease cleavage site. If present, the cell membrane anchoring domain can be connected to the intracellular domain by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane anchoring domain or the intracellular domain. If present, the cell membrane anchoring domain can be connected to the degron by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane anchoring domain or the degron. The polypeptide linker can be any amino acid sequence that connects the first polypeptide sequence and the 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, a GSG linker (e.g., [GS]GG [SEQ ID NO: 347]), A(EAAAK)A (SEQ ID NO: 348), and a Whitlow linker (e.g., a "KEGS" linker, e.g., the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 349), an eGK linker, e.g., the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 350), an LR1 linker, e.g., the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 215), such as those described in detail in issued 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 may be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those of skill in the art.

[0126] Generally, the cell membrane anchoring domain is oriented such that the secreted effector molecule and the protease cleavage site are exposed extracellularly after insertion into or association with the cell membrane, allowing the protease cleavage site to be cleaved by its respective protease to release (secrete) the effector molecule into the extracellular space.

[0127] Degron systems and domains In some embodiments, any of the proteins described herein may contain a degron domain, including, but not limited to, a cytokine, a CAR, a protease, a transcription factor, a promoter or component of a promoter system (e.g., ACP), and / or any of the membrane-cleavable chimeric proteins described herein. Generally, a degron domain may be any amino acid sequence motif capable of directing regulated degradation, such as regulated degradation via a ubiquitin-mediated pathway. In the presence of an immunomodulatory drug (IMiD), the degron domain directs ubiquitin-mediated degradation of the degron fusion protein.

[0128] The degron domain can be a cereblon (CRBN) polypeptide substrate domain capable of binding CRBN in response to an immunomodulatory drug (IMiD), including, but not limited to, IKZF1, IKZF3, CK1a, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, and fragments thereof capable of drug-induced binding of 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 FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 175). Degron domains, and in particular the CRBN degron system, are described in more detail in International Application Publication No. WO 2019 / 089592 A1, which is incorporated herein by reference for all purposes.Other examples of degron domains include, but are not limited to, 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 repeats of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B, e.g., SEQ ID NO: 164), RPB (four copies of residues 1688-1702 of yeast RPB; SEQ ID NO: 165), SPmix (tandem repeats of SP1 and SP2) PEAT (SP2-SP1-SP2-SP1-SP2 of the influenza A virus M2 protein; SEQ ID NO: 166), NS2 (three copies of residues 79-93 of the 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 Includes an E3 ligase-binding degron motif, a CRL4-Cdt2-binding PIP degron, an actinfilin-binding degron, a KEAP1-binding degron, a KLHL2- and KLHL3-binding degron, an MDM2-binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a plant hormone-dependent SCF-LRR-binding degron, an SCF ubiquitin ligase-binding phosphodegron, a plant hormone-dependent SCF-LRR-binding degron, a DSGxxS phosphate-dependent degron (SEQ ID NO: 345), a Siah-binding motif, an SPOP SBC docking motif, or a PCNA-binding PIP box.

[0129] The regulated degradation can be drug-induced. Drugs capable of mediating / regulating degradation can be small molecule compounds. Drugs capable of mediating / regulating degradation can include "immunomodulatory drugs" (IMiDs). Generally, as used herein, IMiD refers to a class of small molecule immunomodulatory drugs containing an imide group. Cereblon (CRBN) is a known target of IMiDs, and binding of an IMiD to CRBN or a CRBN polypeptide substrate domain alters the substrate specificity of the CRBN E3 ubiquitin ligase complex, leading to the degradation of proteins having a CRBN polypeptide substrate domain (e.g., secreted effector molecules described herein or other proteins of interest). For degron domains having a CRBN polypeptide substrate domain, examples of imide-containing IMiDs include, but are not limited to, thalidomide, lenalidomide, or pomalidomide. The IMiD can be an FDA-approved drug.

[0130] The proteins described herein can include a degron domain (e.g., for the membrane-cleavable chimeric proteins described herein, referred to as "D" in the formula SC-MT-D or D-MT-CS). In the absence of an IMiD, degron / ubiquitin-mediated degradation of the chimeric protein does not occur. Following expression and localization of the chimeric protein in the cell membrane, the protease cleavage site directs cleavage of the chimeric protein, resulting in the release (secretion) of effector molecules 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 molecules. Generally, for membrane-cleavable chimeric proteins fused to a degron domain, the degron domain is a terminal cytoplasm-directing domain, specifically relative to a cell membrane-tethering domain, e.g., the most C-terminal domain in the formula SC-MT-D or the most N-terminal domain in the formula D-MT-CS. The degron domain can be connected to the cell membrane-tethering domain, i.e., a polypeptide sequence that is not generally considered to be part of the cell membrane-tethering domain or degron domain, by a polypeptide linker. The polypeptide linker can be any amino acid sequence that connects the first polypeptide sequence and the 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, a GSG linker (e.g., [GS]GG [SEQ ID NO: 347]), A(EAAAK)A (SEQ ID NO: 348), and a Whitlow linker (e.g., a "KEGS" linker, e.g., the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 349), an eGK linker, e.g., the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 350), an LR1 linker, e.g., the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 215), such as those linkers described in detail in issued 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 may 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 in relation to the plasma membrane-tethering domain, such that the degron is exposed to the cytosol after localization to the plasma membrane, and the degron domain is capable of mediating degradation (e.g., exposure to the cytosol and cytosol) and is capable of mediating ubiquitin-mediated degradation.

[0131] For degron fusion proteins, the degron domain can be at the N-terminus or C-terminus of the protein of interest, e.g., an effector molecule. The degron domain can be connected to the protein of interest by a polypeptide linker, i.e., a polypeptide sequence not generally considered part of the protein of interest or the degron domain. The polypeptide linker can be any amino acid sequence that connects the first and second polypeptide sequences. 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, a GSG linker (e.g., [GS]GG [SEQ ID NO: 347]), A(EAAAK)A (SEQ ID NO: 348), and a Whitlow linker (e.g., a "KEGS" linker, e.g., the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 349), an eGK linker, e.g., the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 350), an LR1 linker, e.g., the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 215), such as those described in detail in issued 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 may 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 may be cleavable, eg, any of the protease cleavage sites described herein.

[0132] Engineered Nucleic Acids Provided herein are engineered nucleic acids (e.g., expression cassettes) that encode at least one protein of the disclosure, such as a cytokine, a CAR, an ACP, and / or a membrane-cleavable chimeric protein having the formula SC-MT or MT-CS described herein. Provided herein are engineered nucleic acids (e.g., expression cassettes) that encode two or more proteins, such as a cytokine, a CAR, an ACP, and / or a membrane-cleavable chimeric protein having the formula SC-MT or MT-CS described herein.

[0133] In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette comprising a promoter and an exogenous polynucleotide sequence, oriented N- to C-terminally, encoding a cytokine, a CAR, an ACP, and / or a membrane-cleavable chimeric protein, having the formula: SC-MT or MT-CS. S refers to a secreted 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 SC-MT or MT-CS is configured to be expressed as a single polypeptide.

[0134] In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette comprising a promoter and an exogenous polynucleotide sequence encoding a cytokine. In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette comprising a promoter and an exogenous polynucleotide sequence encoding a CAR. In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette comprising a promoter and an exogenous polynucleotide sequence encoding a membrane-cleavable chimeric protein having a protein of interest (e.g., any of the effector molecules 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.

[0135] In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette comprising a promoter and an exogenous polynucleotide sequence encoding a combination of a cytokine, a CAR, an ACP, and / or a membrane-cleavable chimeric protein described herein. In certain embodiments described herein, the engineered nucleic acid encodes an expression cassette comprising 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 comprising a promoter and an exogenous polynucleotide sequence encoding a cytokine and an ACP.

[0136] In certain embodiments described herein, the engineered nucleic acid encodes two or more expression cassettes, each comprising a promoter and an exogenous polynucleotide sequence encoding a cytokine, a CAR, an 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 comprising a promoter and individually encoding an exogenous polynucleotide sequence encoding a cytokine and a CAR, respectively. In certain embodiments described herein, the engineered nucleic acid encodes two or more expression cassettes, each comprising a promoter and individually encoding an exogenous polynucleotide sequence encoding a cytokine and an ACP, respectively. In certain embodiments, the two or more expression cassettes are oriented in a head-to-tail orientation. In certain embodiments, the two or more expression cassettes are oriented in a head-to-head orientation. In certain embodiments, the two or more expression cassettes are oriented in a tail-to-tail orientation. In some cases, each expression cassette comprises its own promoter to drive expression of the polynucleotide sequence encoding the cytokine and / or CAR. In certain embodiments, the cytokine and CAR are organized as 5'-cytokine-CAR-3' or 5'-CAR-cytokine-3'.

[0137] An "engineered nucleic acid" is a nucleic acid that does not occur in nature. However, it should be understood that while an engineered nucleic acid is entirely non-natural, it can contain naturally occurring nucleotide sequences. In some embodiments, an engineered nucleic acid contains nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, an engineered nucleic acid contains a murine nucleotide sequence, a bacterial nucleotide sequence, a human nucleotide sequence, and / or a viral nucleotide sequence. The term "engineered nucleic acid" includes recombinant nucleic acids and synthetic nucleic acids. "Recombinant nucleic acid" refers to a molecule constructed by linking nucleic acid molecules and, in some embodiments, capable of replicating in living cells. "Synthetic nucleic acid" refers to a molecule that is amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that are chemically or otherwise modified, but are capable of base-pairing with naturally occurring nucleic acid molecules. Modifications include, but are not limited to, one or more modified internucleotide linkages and non-naturally occurring nucleic acids. Modifications are described in further detail in U.S. Patent No. 6,673,611 and U.S. Application Publication No. 2004 / 0019001, each of which is incorporated by reference in its entirety. The modified internucleotide linkages can be phosphorodithioate or phosphorothioate linkages. The non-natural nucleic acids can be locked nucleic acids (LNAs), peptide nucleic acids (PNAs), glycol nucleic acids (GNAs), phosphorodiamidate morpholino oligomers (PMOs or "morpholinos"), and threose nucleic acids (TNAs). Non-natural nucleic acids are described in further detail in International Application No. 1998 / 039352, U.S. Application Publication No. 2013 / 0156849, and U.S. Patent Nos. 6,670,461, 5,539,082, and 5,185,444, each of which is incorporated by reference in its entirety. Recombinant and synthetic nucleic acids also include molecules resulting from replication of any of the foregoing. The engineered nucleic acids of the present disclosure can be encoded by a single molecule (e.g., contained in the same plasmid or other vector) or by multiple different molecules (e.g., multiple different, independently replicating molecules). The engineered nucleic acid can be an isolated nucleic acid.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, minicircles, ssDNA, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), and oligonucleotides.

[0138] The engineered nucleic acids of the present disclosure can be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the engineered nucleic acid construct is produced using GIBSON ASSEMBLY® cloning (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 ASSEMBLY® typically uses three enzyme activities in a single-tube reaction: a 5' exonuclease, a DNA polymerase gamma-extension activity, and a DNA ligase activity. The 5' exonuclease activity bites back the 5'-end sequence, exposing complementary sequences for annealing. The polymerase activity then fills in the gaps in the annealed region. DNA ligase then 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 resulting in a higher rate of correct assembly. In some embodiments, engineered nucleic acid constructs are produced using IN-FUSION® Cloning (Clontech).

[0139] promoter Generally, in all embodiments described herein, an engineered nucleic acid encoding a protein herein (e.g., a cytokine, CAR, ACP, and / or membrane-cleavable chimeric protein described herein) encodes two or more expression cassettes, where the expression cassettes comprise a promoter and an exogenous polynucleotide sequence encoding the protein. In some embodiments, the engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to nucleotide sequences (e.g., exogenous polynucleotide sequences) encoding at least two different proteins. For example, the engineered nucleic acid can comprise a promoter operably linked to nucleotide sequences encoding at least three, at least four, at least five, at least six, at least seven, at least eight, at least eight, at least nine, or at least ten different proteins. In some embodiments, the engineered nucleic acid comprises a promoter operably linked to nucleotide sequences encoding one, two, three, four, five, six, seven, eight, nine, ten, or more different proteins. In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least two cytokines. For example, an engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least three, at least four, at least five, at least six, at least seven, at least eight, at least eight, at least nine, or at least ten cytokines. In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least two membrane-cleavable chimeric proteins.For example, an engineered nucleic acid can comprise a promoter operably linked to a nucleotide sequence encoding at least three, at least four, at least five, at least six, at least seven, at least eight, at least eight, at least nine, or at least 10 chimeric membrane-cleavable proteins. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding one, two, three, four, five, six, seven, eight, nine, ten, or more chimeric membrane-cleavable proteins.

[0140] A "promoter" refers to a regulatory region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. A promoter may also contain small regions to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind. A promoter may be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or transcription of a nucleic acid sequence that it regulates. As used herein, a promoter is considered to be "operably linked" when it is in the correct functional location and orientation relative to the nucleic acid sequence that it regulates to control ("drive") the transcription initiation and / or expression of that sequence.

[0141] A promoter may be a promoter naturally associated with a gene or sequence, such as can be obtained by isolating the 5' non-coding sequence located upstream of the coding segment of a given gene or sequence. Such a promoter may be referred to as "endogenous." In some embodiments, a coding nucleic acid sequence may be placed under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the coded sequence in its natural environment. Such promoters may include promoters of other genes, promoters isolated from any other cell, and synthetic promoters or enhancers that are not "naturally occurring," such as those containing different transcriptional regulatory and / or mutational elements that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences may be produced using nucleic acid amplification techniques, including recombinant cloning and / or polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906).

[0142] The promoter of an engineered nucleic acid may be an "inducible promoter," which refers to a promoter characterized by modulating transcriptional activity (e.g., initiating or activating) when in the presence of, affected by, or contacted by a signal. The signal can be an endogenous or usually exogenous condition (e.g., light), compound (e.g., a chemical or non-chemical compound), or protein (e.g., a cytokine) that contacts the inducible promoter in such a way that it is active in modulating transcriptional activity from the inducible promoter. Activation of transcription can involve acting directly on the promoter to drive transcription, or indirectly on the promoter by inactivating a repressor that prevents the promoter from driving transcription. Conversely, inactivation of transcription can involve acting directly on the promoter to prevent transcription, or indirectly on the promoter by activating a repressor that in turn acts on the promoter.

[0143] A promoter is "responsive" to or "regulated by" a local tumor condition (e.g., inflammation or hypoxia) or signal if, in the presence of that condition or signal, transcription from the promoter is activated, inactivated, increased, or decreased. In some embodiments, a promoter contains a response element. A "response element" is a short sequence of DNA within the promoter region that binds to specific molecules (e.g., transcription factors) that modulate (regulate) gene expression from the promoter. Response elements that may be used in accordance with the present disclosure include, but are not limited to, phloretin tunable regulatory element (PEACE), zinc finger DNA binding domain (DBD), interferon gamma activating sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar;17(3):121-34, incorporated herein by reference), interferon stimulated response element (ISRE) (Han, KJ et al. J Biol Chem. 2004 Apr 9;279(15):15652-61, incorporated herein by reference), NF-kappa B response element (Wang, V. et al. Cell Reports. 2012;2(4):824-839, incorporated herein by reference), and STAT3 response element (Zhang, D. et al. J of Biol Chem. 1996;271:9503-9509, incorporated herein by reference). Other response elements are encompassed herein. Response elements 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 indicate the number of repeats present.

[0144] Non-limiting examples of responsive promoters (also referred to as "inducible promoters") (e.g., TGF-beta responsive promoters) are listed in Table 5A, which shows the promoter and transcription factor design, and the effects of the transcription factor (TF) and inducer molecule on transgene transcription (T) are shown (B, binding; D, dissociation; nd, undetermined) (A, activation; DA, deactivation; DR, repression) (Horner, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein). Other non-limiting examples of inducible promoter components include those listed in Table 5B.

[0145] Table 5A. Examples of responsive promoters TIFF2025533842000023.tif247165TIFF2025533842000024.tif159165

[0146] Table 5B. Exemplary components of inducible promoters TIFF2025533842000025.tif77165TIFF2025533842000026.tif100165

[0147] Other non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1-alpha (EF1a) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the spleen focus forming virus (SFFV) promoter, the simian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter (see Table 5C).

[0148] Table 5C: Exemplary constitutive promoters <h2 style=";text-align:left;direction:ltr">TIFF2025533842000027.tif107163TIFF2025533842000028.tif254163TIFF2025533842000029.tif253163TIFF2025533842000030.tif253163TIFF2025533842000031.tif253163TIFF2025533842000032.tif253163TIFF2025533842000033.tif41163<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0149] <h2 style=";text-align:left;direction:ltr"> The promoter can be a tissue-specific promoter. Generally, a tissue-specific promoter directs transcription of a nucleic acid (e.g., an engineered nucleic acid encoding a protein herein (e.g., a cytokine, CAR, ACP, and / or membrane-cleavable chimeric protein described herein) such that expression is restricted to a particular cell type, organelle, or tissue. Tissue-specific promoters include albumin (liver-specific, Pinkert et al., (1987)), lymphoid-specific promoters (Calame and Eaton, 1988), certain promoters of T-cell receptors (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), pancreatic-specific promoters (Edlund et al., (1989)), and the like. al., (1985)), or mammary gland-specific promoters (mouse whey promoter, U.S. Pat. No. 4,873,316 and European Patent Application Publication No. 264,166), as well as developmentally regulated promoters such as mouse hox promoters (Kessel and Gruss, Science 249:374-379 (1990)) or α-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546 (1989)), the contents of each of which are incorporated herein by reference in their entirety. Promoters may be constitutive in each specific cell type, organelle, or tissue.Tissue-specific promoters and / or regulatory elements can also include the liver fatty acid-binding (FAB) protein gene specific for colonic epithelial cells; the insulin gene specific for pancreatic cells; the transfiletin, α1-antitrypsin, plasminogen activator inhibitor type 1 (PAI-I), apolipoprotein AI, and LDL receptor genes specific for hepatocytes; the myelin basic protein (MBP) gene specific for oligodendrocytes; the glial fibrillary acidic protein (GFAP) gene specific for glial cells; the promoter from OPSIN specific for targeting the eye; and the neuron-specific enolase (NSE) promoter specific for nerve cells. Examples of tissue-specific promoters include, but are not limited to, the promoter for creatine kinase, which has been used to direct expression in muscle and cardiac 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 for 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-alpha hydrolase (CYP-7) promoter, beta-galactosidase alpha-2,6 sialyltransferase promoter, insulin-like growth factor binding protein (IGFBP-I) promoter, aldolase B promoter, human transferrin promoter, and collagen type I promoter. Exemplary tissue-specific expression elements for the prostate include, but are not limited to, the prostatic acid phosphatase (PAP) promoter, the prostate-secreted protein of 94 (PSP 94) promoter, the prostate-specific antigen complex promoter, and the human glandular kallikrein gene promoter (hgt-1). Exemplary tissue-specific expression elements for stomach tissue include, but are not limited to, the human H / K-ATPase alpha subunit promoter.Exemplary tissue-specific expression elements for the pancreas include, but are not limited to, the pancreatitis-associated protein promoter (PAP), elastase 1 transcription enhancer, pancreas-specific amylase and elastase enhancer promoter, and pancreatic cholesterol esterase gene promoter. Exemplary tissue-specific expression elements for the endometrium include, but are not limited to, the uterine globin promoter. Exemplary tissue-specific expression elements for adrenal cells include, but are not limited to, the cholesterol side-chain cleavage (SCC) promoter. Exemplary tissue-specific expression elements for the general nervous system include, but are not limited to, the gamma-gamma enolase (neuron-specific enolase, NSE) promoter. Exemplary tissue-specific expression elements for the brain include, but are not limited to, the neurofilament heavy chain (NF-H) promoter. Exemplary tissue-specific expression elements for lymphocytes include, but are not limited to, the human CGL-1 / granzyme B promoter, terminal deoxytransferase (TdT), lambda 5, VpreB, and lck (lymphocyte-specific tyrosine protein kinase p561ck) promoters, 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 for colon include, but are not limited to, the pp60c-src tyrosine kinase promoter, the organ-specific neoantigen (OSN) promoter, and the colon-specific antigen-P promoter. An example of a tissue-specific expression element for breast cells is, for example, but is not limited to, the human alpha-lactalbumin promoter. An exemplary tissue-specific expression element for lung includes, but is not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) gene promoter.

[0150] In some embodiments, the promoter of the present disclosure is regulated by signals in the tumor microenvironment. The tumor microenvironment is considered to regulate a promoter when the activity of the promoter increases or decreases by at least 10% in the presence of the tumor microenvironment compared to the activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter increases or decreases 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% compared to the activity of the promoter in the absence of the 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% compared to the activity of the promoter in the absence of a tumor microenvironment.

[0151] 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) compared 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 compared 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 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100-fold compared to the activity of the promoter in the absence of a tumor microenvironment.

[0152] In some embodiments, the promoters of the present disclosure are activated under hypoxic conditions. "Hypoxia" is a condition in which the body or a region of the body lacks sufficient oxygen supply at the tissue level. Hypoxia can lead to inflammation (e.g., levels of inflammatory cytokines increase under hypoxic conditions). In some embodiments, a promoter activated under hypoxic conditions is operably linked to a nucleotide encoding a protein that reduces the expression of the activity of inflammatory cytokines, thus reducing inflammation caused by hypoxia. 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). The HRE, in some embodiments, comprises the consensus motif NCGTG (where N is either A or G).

[0153] Activated conditionally controlled polypeptide (ACP) promoter system In some embodiments, the synthetic promoter is a promoter system comprising an activation conditional regulatory polypeptide (ACP)-binding domain sequence and a promoter sequence. Such a system is also referred to herein as an "ACP-responsive promoter." Generally, an ACP promoter system comprises a first expression cassette encoding an activation conditional regulatory 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, including a membrane-cleavable chimeric protein version of a cytokine, or any other protein of interest (e.g., a protease or CAR), as described herein. In some embodiments, the first expression cassette and the second expression cassette are each encoded by a separate engineered nucleic acid. In other embodiments, the first expression cassette and the second expression cassette 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 AATTAACGGGGTTTCGTAACAATCGCATGAGGATTCGCAACGCCTTTGAAGCAGTCGACGCCGAAGTCCCGTCTCAGTAAAGGTTGAAGCAGTCGACGCCGAAGAATCGGACTGCCTTCGTATGAAGCAGTCGACGCCGAAGGTATCAGTCGCCTCGGAATGAAGCAGTCGACGCCGAAGATTCGTAAGAGGCTCACTCTCCCTTACACGGAGTGGATAACTAGTTCTAGAGGGTATATAATGGGGGCCAACGCGTACCGGTGTC (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.

[0154] The promoter of the ACP promoter system, for example, the promoter driving the expression of ACP, or any of the promoter sequences of the ACP-responsive promoter, can include any of the promoter sequences described herein (see "Promoter" above). The ACP-responsive promoter can be derived from minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia response 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.

[0155] 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 comprises 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). A zinc finger array contains multiple zinc finger protein motifs linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA with specificity for any desired nucleic acid sequence, for example, a ZFA with 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. A ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 zinc finger motifs.A 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. A ZF protein domain can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more ZFAs. A 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, a ZF protein domain comprises 1-10 ZFAs. In some embodiments, a ZF protein domain comprises at least one ZFA. In some embodiments, a ZF protein domain comprises at least two ZFAs. In some embodiments, a ZF protein domain comprises at least three ZFAs. In some embodiments, the ZF protein domain comprises at least 4 ZFAs. In some embodiments, the ZF protein domain comprises at least 5 ZFAs. In some embodiments, the ZF protein domain comprises at least 10 ZFAs.

[0156] In some embodiments, the DNA binding domain comprises a tetracycline (or derivative thereof) repressor (TetR) domain.

[0157] ACPs can also further comprise an effector domain, such as a transcription effector domain. For example, the transcription effector domain can be the effector domain or activator domain of a transcription factor. Transcription factor activation domains, also known as transactivator domains, act as scaffolding domains of proteins, such as transcriptional coregulators, that act to activate or repress gene transcription. Any suitable transcription effector domain can be used in an ACP, including, but not limited to, the herpes simplex virus protein 16 (VP16) activation domain; the VP64 activation domain, which is an activation domain consisting of four tandem copies of VP16; the p65 activation domain of NFκB; the Epstein-Barr virus R transcription activator (Rta) activation domain; the tripartite activator, which includes the VP64, p65, and Rta activation domains (the tripartite activator is known as the VPR activation domain); or the histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300 (p300). HAT core activation domain; Krüppel-associated box (KRAB) repression domain; repressor element silencing transcription factor (REST) ​​repression domain; WRPW motif of hairy-related basic helix-loop-helix repressor protein (SEQ ID NO: 346) (this motif is known as the WRPW repression domain (SEQ ID NO: 346)); DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and HP1 alpha chromoshadow repression domain, or any combination thereof.

[0158] In some embodiments, the effector domain is a transcriptional effector domain selected from the following: herpes simplex virus protein 16 (VP16) activation domain; the VP64 activation domain, which is an activation domain consisting of four tandem copies of VP16; the p65 activation domain of NFκB; the Epstein-Barr virus R transcriptional activator (Rta) activation domain; the tripartite activator comprising the VP64, p65, and Rta activation domains (the tripartite activator is known as the VPR activation domain); the histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300 (p300 known as the HAT core activation domain; the Krüppel-associated box (KRAB) repression domain; the repressor element silencing transcription factor (REST) ​​repression domain; the WRPW motif of the hairy-related basic helix-loop-helix repressor protein (SEQ ID NO: 346) (this motif is known as the WRPW repression domain (SEQ ID NO: 346)); the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and the HP1 alpha chromoshadow repression domain.

[0159] 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 a derivative thereof) and includes a TetR domain and a VP16 effector domain. In some embodiments, the ACP includes an estrogen receptor variant, such as ERT2, and can be regulated by tamoxifen or its metabolites (e.g., 4-hydroxytamoxifen [4-OHT], N-desmethyltamoxifen, tamoxifen-N-oxide, or endoxifen) through tamoxifen-regulated nuclear localization. In some embodiments, the ACP includes a nuclear localization signal (NLS). In certain embodiments, the NLS includes the amino acid sequence MPKKKRKV (SEQ ID NO: 296). An exemplary nucleic acid sequence encoding SEQ ID NO: 296 is ATGCCCAAGAAGAAGCGGAAGGTT (SEQ ID NO: 297) or ATGCCCAAGAAAAAGCGGAAGGTG (SEQ ID NO: 340). In some embodiments, the nucleic acid sequence encoding SEQ ID NO:296 may comprise SEQ ID NO:297 or SEQ ID NO:340, or may comprise 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.

[0160] In some embodiments, the ACP is a small molecule (e.g., drug)-inducible polypeptide that includes an inhibitory protease and one or more cognate cleavage sites for the inhibitory protease. In some embodiments, the inhibitory protease is active (cleaves the cognate cleavage site) in the absence of a specific agent and inactive (does not cleave the cognate cleavage site) in the presence of a specific agent. In some embodiments, the specific agent is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given inhibitory protease of the present disclosure. The inhibitory protease can be any of the proteases described herein that can be inactivated by the presence or absence of a specific agent (see "Protease Cleavage Sites" above for exemplary inhibitory proteases, cognate cleavage sites, and protease inhibitors).

[0161] In some embodiments, the ACP has a degron domain (see "Degron Systems and Domains" above for exemplary degron sequences). The degron domain can be in any order or location relative to the individual domains of the ACP. For example, the degron domain can be N-terminal to the inhibitory protease, C-terminal to the inhibitory protease, N-terminal to the ZF protein domain, C-terminal to the ZF protein domain, N-terminal to the effector domain, or C-terminal to the effector domain.

[0162] Exemplary sequences of components of ACPs and exemplary ACPs of the present disclosure are provided in Table 5D. In some embodiments, the nucleic acid may comprise a sequence 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 a sequence in Table 5D.

[0163] (Table 5D) TIFF2025533842000034.tif182164TIFF2025533842000035.tif253164TIFF2025533842000036.tif253164TIFF2025533842 000037.tif242164TIFF2025533842000038.tif252164TIFF2025533842000039.tif253164TIFF2025533842000040.tif66164

[0164] Multicistronic and multi-promoter systems In some embodiments, an 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 may be configured to produce between 2 and 20 different proteins.In some embodiments, the nucleic acid is selected from the group consisting of 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, 4-1 0, 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.

[0165] In some embodiments, the engineered nucleic acid may be multicistronic, i.e., two or more separate polypeptides (e.g., multiple proteins such as a cytokine, a CAR, an ACP, and / or a membrane-cleavable chimeric protein described herein) may be produced from a single mRNA transcript. In some embodiments, the multicistronic engineered nucleic acid of the present disclosure may be configured to encode a cytokine, a CAR, and a membrane-cleavable chimeric protein described herein. For example, the multicistronic engineered nucleic acid of the present disclosure may be configured to encode a cytokine, an aCAR, and a membrane-cleavable chimeric protein described herein. For example, the multicistronic engineered nucleic acid of the present disclosure may be configured to encode a cytokine, an aCAR, an iCAR, and a membrane-cleavable chimeric protein described herein.

[0166] The engineered nucleic acid can be multicistronic through the use of various linkers; for example, a polynucleotide sequence encoding a first protein can be linked to a nucleotide sequence encoding a second protein (e.g., in a 5' to 3' direction: first gene: linker: second gene, etc.). The linker can encode a 2A ribosomal skipping element, such as T2A. Other 2A ribosomal skipping elements include, but are not limited to, E2A, P2A, and F2A. The 2A ribosomal skipping element allows for the production of separate polypeptides encoded by the first and second genes during translation. The linker can encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site; following expression, the cleavable linker polypeptide is cleaved to produce separate polypeptides encoded by the first and second genes. The cleavable linker can include a polypeptide sequence, such as a flexible linker (e.g., a Gly-Ser-Gly sequence), that further facilitates cleavage. In some embodiments, the engineered nucleic acids disclosed herein comprise an E2A / T2A ribosomal skipping element. In certain embodiments, the E2A / T2A ribosomal 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 ribosomal skipping element. In certain embodiments, the E2A / T2A ribosomal 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 certain 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.

[0167] Other suitable linkers containing 2A ribosome skipping elements are shown in the table below. TIFF2025533842000041.tif83165

[0168] The linker can encode an internal ribosome entry site (IRES) so 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.

[0169] The linker can be a combination of linkers, such as a furin-2A linker, that can produce separate polypeptides through 2A ribosomal skipping followed by further cleavage of the furin site, allowing for complete removal of the 2A residue. In some embodiments, the linker combination 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.

[0170] Generally, a multicistronic system can express any number of genes or portions thereof using any number or combination of linkers (e.g., an engineered nucleic acid can encode a first, second, and third protein, each separated by a linker, such that separate polypeptides encoded by the first, second, and third proteins are produced).

[0171] An engineered nucleic acid can use multiple promoters to express genes from multiple ORFs, i.e., two or more separate mRNA transcripts 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 of the ORFs expressed from the multiple promoters can be multicistronic.

[0172] Expression cassettes encoded on the same engineered nucleic acid can be oriented in any manner suitable for expression of the encoded exogenous polynucleotide sequence. Expression cassettes encoded on the same engineered nucleic acid can be oriented in the same direction, i.e., transcription of the separate cassettes proceeds in the same direction. Constructs oriented in the same direction can be organized in a head-to-tail format, with the 5' end (head) of a first gene 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 separate cassettes proceeds in opposite directions (also referred to herein as "bidirectional"). Expression cassettes encoded on the same engineered nucleic acid that are oriented in opposite directions can be oriented in a "head-to-head" orientation. As used herein, head-to-head refers to the 5' end (head) of the first gene of a bidirectional construct being adjacent to the 5' end (head) of the upstream gene of the bidirectional construct. Expression cassettes encoded on the same engineered nucleic acid that are oriented in opposite directions can be oriented in a "tail-to-tail" orientation. As used herein, "tail-to-tail" refers to the 3' end (tail) of the first gene of a bidirectional construct adjacent to the 3' end (tail) of the upstream gene of the bidirectional construct. For example, Figure 1 schematically illustrates, but is not limited to, cytokine-CAR bidirectional constructs in head-to-head (Figure 1A), head-to-tail (Figure 1B), and tail-to-tail (Figure 1C) orientations.

[0173] As used herein, a "linker" can refer to a polypeptide that links a first polypeptide sequence and a second polypeptide sequence, a multicistronic linker as described above, or an additional promoter that is operably linked to an additional ORF as described above.

[0174] The exogenous polynucleotide sequence encoded by the expression cassette may comprise a 3' untranslated region (UTR) comprising an mRNA destabilizing element operably linked to an exogenous polynucleotide sequence, such as an exogenous polynucleotide sequence encoding a cytokine (e.g., IL12 or IL12p70). In some embodiments, the mRNA destabilizing element comprises an AU-rich element and / or a stem-loop destabilizing 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 destabilizing element comprises a stem-loop destabilizing element (SLDE). In some embodiments, the SLDE comprises CTGTTTAATATTTAAACAG (SEQ ID NO: 211). In some embodiments, the mRNA destabilizing 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 destabilizing element (SLDE). An exemplary AuSLDE sequence includes ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO: 212). In some embodiments, the mRNA destabilizing element includes 2X AuSLDE. An exemplary AuSLDE sequence is provided as ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAGtgcggtaagcATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO: 213).

[0175] In certain embodiments, the engineered nucleic acids described herein comprise an insulator sequence. Such an insulator sequence functions to prevent inappropriate interactions between adjacent regions of the construct. In certain embodiments, the insulator sequence comprises the nucleic acid sequence ACAATGGCTGGCCCATAGTAAATGCCGTGTTAGTGTGTTAGTTGCTGTTCTTCCACGTCAGAAGAGGCACAGACAAATTACCACCAGGTGGCGCTCAGAGTCTGCGGAGGCATCACAACAGCCCTGAATTTGAATCCTGCTCTGCCACTGCCTAGTTGAGACCTTTTACTACCTGACTAGCTGAGACATTTACGACATTTACTGGCTCTAGGACTCATTTTATTCATTTCATTACTTTTTTTTTCTTTGAGACGGAATCTCGCTCT (SEQ ID NO: 300). In certain 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.

[0176] Engineered cells Provided herein are engineered immunoresponsive cells that produce proteins described herein (e.g., cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins described herein), and methods for producing engineered immunoresponsive cells. Generally, the engineered immunoresponsive cells of the present disclosure may be engineered to express proteins provided herein, such as cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins having the formula SC-MT or MT-CS described herein. For example, immunoresponsive cells may be engineered to express cytokines, CARs, and membrane-cleavable chimeric proteins described herein. For example, immunoresponsive cells may be engineered to express cytokines, aCARs, and membrane-cleavable chimeric proteins described herein. For example, immunoresponsive cells may be engineered to express cytokines, aCARs, iCARs, and membrane-cleavable chimeric proteins described herein. These cells are referred to herein as "engineered cells." These cells typically contain engineered nucleic acids and do not occur in nature. In some embodiments, cells are engineered to contain a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a protein, for example, cytokine, CAR, ACP, and / or membrane-cleavable chimeric protein.The engineered cells of the present disclosure can contain engineered nucleic acids integrated into the genome of cells.For example, engineered cells can contain engineered nucleic acids that can be expressed without being integrated into the genome of cells, for example, by using transient expression systems, such as plasmids or mRNA.

[0177] The present disclosure also encompasses additivity and synergy between proteins and the engineered cells in which they are produced. In some embodiments, 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 a cytokine, a CAR, an ACP, and a membrane-cleavable chimeric protein. In some embodiments, cells are engineered to produce a cytokine, a CAR, and a membrane-cleavable chimeric protein described herein. In some embodiments, cells are engineered to produce a cytokine, an aCAR, and a membrane-cleavable chimeric protein described herein. In some embodiments, cells are engineered to produce a cytokine, an aCAR, an iCAR, and a membrane-cleavable chimeric protein described herein. Generally, the immunoresponsive cells provided herein are engineered to produce at least one membrane-cleavable chimeric protein having a cytokine effector molecule not naturally produced by the cell, the CAR, and the ACP. Generally, the immunoresponsive cells provided herein are engineered to produce at least two cytokines, at least one of which is a membrane-cleavable chimeric protein having a cytokine effector molecule, a CAR, and an ACP. In some embodiments, the immunoresponsive cells provided herein are engineered to produce at least two cytokines, at least one of which is a membrane-cleavable chimeric protein having a cytokine effector molecule and a CAR. In some embodiments, the immunoresponsive cells provided herein are engineered to produce at least two cytokines, at least one of which is a membrane-cleavable chimeric protein having a cytokine effector molecule and two CARs. In some embodiments, the immunoresponsive cells provided herein are engineered to produce at least two cytokines, at least one of which is a membrane-cleavable chimeric protein having a cytokine effector molecule, an aCAR, and an iCAR. In some embodiments, such effector molecules, such as aCARs, can complement the function of effector molecules naturally produced by the cells.

[0178] In some embodiments, the cells (e.g., immune cells) are engineered to produce multiple proteins. For example, the cells may be engineered to produce 2-20 different proteins, such as 2-20 different membrane-cleavable proteins. In some embodiments, the cells (e.g., immunoresponsive cells) are engineered to produce at least four distinct proteins that are exogenous to the cell. In some embodiments, the cells (e.g., immunoresponsive cells) are engineered to produce four distinct proteins that are exogenous to the cell.In some embodiments, 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 20, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 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 cells are 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.

[0179] In some embodiments, engineered cells comprise one or more engineered nucleic acids (e.g., expression cassettes) encoding a promoter operably linked to a nucleotide sequence encoding a protein. In some embodiments, cells are engineered to comprise a plurality of engineered nucleic acids, e.g., 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) protein. For example, cells can be engineered to comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least eight, at least nine, or at least ten engineered nucleic acids, each encoding a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2, or 3) protein. In some embodiments, cells are engineered to comprise two, three, four, five, six, seven, eight, nine, 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) protein. The engineered cell can include an engineered nucleic acid encoding at least one of the linkers described above, e.g., a polypeptide linking a first polypeptide sequence and a second polypeptide sequence, one or more multicistronic linkers described above, one or more additional promoters operably linked to additional ORFs, or combinations thereof.

[0180] In some embodiments, cells (e.g., immune cells) are engineered to express a protease. In some embodiments, cells are engineered to express a protease heterologous to the cell. In some embodiments, cells are engineered to express a protease heterologous to the cell expressing the protein, such as a heterologous protease that cleaves a protease cleavage site in a membrane-cleavable chimeric protein. In some embodiments, the engineered cells comprise one or more engineered nucleic acids encoding 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 herein under the heading "Protease Cleavage Sites." In other embodiments, the cells are not engineered to express a heterologous protease that cleaves a protease cleavage site in a membrane-cleavable chimeric protein. In such embodiments, the cells endogenously express a protease that cleaves a protease cleavage site in a membrane-cleavable chimeric protein.

[0181] Also provided herein are engineered cells engineered to produce multiple proteins, at least two of which comprise effector molecules that modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, at least one (e.g., one, two, three, four, five, or more) of the proteins comprises 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., one, two, three, four, five, or more) of the proteins comprises an effector molecule that inhibits at least one immunosuppressive mechanism in the tumor microenvironment, and at least one protein (e.g., one, two, three, four, five, or more) inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In yet other embodiments, at least two (e.g., two, three, four, five, or more) of the proteins are effector molecules that each stimulate at least one immunostimulatory mechanism in the tumor microenvironment. In still other embodiments, at least two (eg, one, two, three, four, five, or more) of the proteins are effector molecules that each inhibit at least one immunosuppressive mechanism in the tumor microenvironment.

[0182] In some embodiments, cells (e.g., immune cells) are engineered to produce at least one protein comprising an effector molecule that stimulates T cell or NK cell signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one protein comprising an effector molecule that stimulates antigen presentation and / or processing. In some embodiments, cells are engineered to produce at least one protein comprising an effector molecule that stimulates natural killer cell-mediated cytotoxicity signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one protein comprising an effector molecule that stimulates dendritic cell differentiation and / or maturation. In some embodiments, cells are engineered to produce at least one protein comprising an effector molecule that stimulates immune cell recruitment. In some embodiments, cells are engineered to produce at least one protein comprising an effector molecule that stimulates M1 macrophage signaling, activity, and / or recruitment. In some embodiments, cells are engineered to produce at least one protein comprising an effector molecule that stimulates Th1 polarization. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that stimulates stromal degradation. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that stimulates immunostimulatory metabolite production. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that stimulates type I interferon signaling. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that inhibits negative costimulatory signaling. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that inhibits pro-apoptotic signaling (e.g., via TRAIL) of anti-tumor immune cells. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that inhibits T regulatory (T regIn some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that inhibits IL-1 (IL-1) cell signaling, activity, and / or recruitment. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that inhibits a tumor checkpoint molecule. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that activates stimulator of interferon genes (STING) signaling. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that inhibits myeloid-derived suppressor cell signaling, activity, and / or recruitment. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that degrades an immunosuppressant / metabolite. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that inhibits vascular endothelial growth factor signaling. In some embodiments, the cells are engineered to produce at least one protein comprising an effector molecule that directly kills tumor cells (e.g., granzymes, perforin, oncolytic viruses, cytolytic peptides, and enzymes, e.g., anti-tumor antibodies that induce ADCC).

[0183] In some embodiments, at least one protein comprises an effector molecule that stimulates T cell signaling, activity, and / or recruitment, stimulates antigen presentation and / or processing, stimulates natural killer cell-mediated cytotoxicity signaling, activity, and / or recruitment, stimulates dendritic cell differentiation and / or maturation, stimulates immune cell recruitment, stimulates macrophage signaling, stimulates stromal degradation, stimulates immunostimulatory metabolite production, or stimulates type I interferon signaling; and at least one protein comprises an effector molecule that inhibits negative costimulatory 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 (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.

[0184] In some embodiments, the immunoresponsive 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, the immunoresponsive 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, the immunoresponsive 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, the immunoresponsive cells are engineered to produce at least the effector molecule cytokines IL15 and IL12p70 fusion protein. In some embodiments, the immunoresponsive 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, the immunoresponsive 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, the immunoresponsive 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, the immunoresponsive 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.

[0185] In a specific embodiment, IL15 comprises the amino acid sequence of NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 285). An example of a nucleic acid sequence encoding SEQ ID NO: 285 is AATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGAGCGACGTGCACCCTAGCTGTAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAAAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAATGTGACCGAGTCCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAATATCAAAGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACAAGC (SEQ ID NO: 286). In certain embodiments, a 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 example of a nucleic acid sequence encoding SEQ ID NO: 357 is ATGGAAACCGACACACTGCTGCTGTGGGTGCTGCTTCTTTGGGTGCCCGGCTCTACAGGCAACTGGGTCAACGGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGAGCGACGTGCACCCTAGCTGTAAAGTGACCGCCATGAAGTGCTTTCTGCTGGA ACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAAAACCTGATCATCCTGGCTAACAACAGCCTGAGCAGCAACGGCAATGTGACCGAGTCCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAATATCAAAGAGTTCCTCCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (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.

[0186] 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, 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 NO: 219. In certain embodiments, crIL15 comprises a "slow" protease cleavage site comprising the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191). In certain embodiments, crIL15 containing a "slow" protease cleavage site comprises the amino acid sequence MDWTWILFLVAAATRVHSYPYDVPDYAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 355).Nucleic acid sequence number 355ををれちすすますしは、(sequence number 367)。

[0187] In some embodiments, crIL15 comprising a "slow" protease cleavage site also comprises a furin cleavage site. A crIL15 comprising a "slow" protease cleavage site and a furin cleavage site may comprise the amino acid sequence of MDWTWILFLVAAATRVHSYPYDVPDYAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPVRRKR (SEQ ID NO: 415).An example of a nucleic acid sequence encoding SEQ ID NO:415 is (SEQ ID NO:416). In certain embodiments, a 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 "fast" protease cleavage site comprising the amino acid sequence of PRAEALKGG (SEQ ID NO:180).In certain embodiments, crIL15 containing a "fast" protease cleavage site comprises the amino acid sequence of MDWTWILFLVAAATRVHSYPYDVPDYAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSPRAEALKGGSGGGGSGGGGSGGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO:356). An example of a nucleic acid sequence encoding SEQ ID NO:356 is (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.

[0188] In a specific embodiment, crIL15 comprises the amino acid sequence of MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 410). An example of a nucleic acid sequence encoding SEQ ID NO: 410 is ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTAATTGGGTCAACGGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCCGATGTGCACCCTAGCTGCAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACT GCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGATACCGTGGAAAATCTGATCATCCTGGCCAACAAGCCTGTCCAGCAACGGCAATGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTCCACATCGTGCAGATGTTCATCAACACCTCA (SEQ ID NO: 411).

[0189] 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).Nucleic acid sequence number 361ををれちすすますしは、(sejoku 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.

[0190] 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). An example of a nucleic acid sequence encoding SEQ ID NO: 391 is:ATGGACTGGACTTGGATACTCTTTCTGGTCGCTGCCGCCACACGGGTGCACTCTAATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGAGCGACGTGCACCCTAGCTGTAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAAAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAATGTGACCGAGTCCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAATATCAAAGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCAGCGGCGGATCTGGCGGCGGAGGTTCTGGCGGTGGAAGCGGAGGCGGAGGATCTCTCCAGATCACATGCCCTCCACCTATGAGCGTGGAACACGCCGACATCTGGGTCAAGAGCTACAGCCTGTACAGCAGAGAGCGGTACATCTGCAACAGCGGCTTCAAGAGAAAGGCCGGCACAAGCAGCCTGACCGAGTGCGTGCTGAACAAGGCCACAAATGTGGCCCACTGGACCACACCTAGCCTGAAGTGCATCAGAGCAGCAGCTATCGAGGTGATGTATCCTCCGCCCTACCTGGATAATGAAAAGAGTAATGGGACTATCATTCATGTAAAAGGGAAGCATCTTTGTCCTTCTCCCCTTTTCCCCGGTCCGTCTAAACCTTTCTGGGTGCTCGTGGTTGTTGGCGGAGTGCTGGCCTGTTACTCTCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTCAAGTCCAGACAGACACCTCCTCTGGCCAGCGTGGAAATGGAAGCCATGGAAGCTCTGCCTGTGACCTGGGGCACCAGCTCCAGAGATGAGGACCTGGAAAACTGCTCCCACCACCTGTAA (SEQ ID NO: 392). In certain embodiments,Nucleic acids encoding SEQ ID NO:391 include 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:392.

[0191] In certain embodiments, IL15 is membrane-bound IL15 (mbIL15). In certain embodiments, mbIL15 comprises the amino acid sequence MDWTWILFLVAAATRVHSYPYDVPDYAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGGSGGGGSGGGGSGGGGSGGGSLQLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO:358). An example of a nucleic acid sequence encoding SEQ ID NO:358 is (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.

[0192] In a specific embodiment, IL21 comprises the amino acid sequence of QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 360). An example of a nucleic acid sequence encoding SEQ ID NO: 360 is CAGGGCCAAGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCTGAGTTTCTGCCCGCTCCTGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGCGCCAACACCGGCAACAACGAGCGG ATCATCAACGTGTCCATCAAGAAGCTGAAGCGGAAGCTCCTAGCCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGTCCTAGCTGCGACAGCTACGAGAAGAAGCCTCCAAAAGAGTTTCTCGAGCGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGTCCAGCAGGACACACGGCAGCGAGGATTCT (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.

[0193] 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 example of a nucleic acid sequence encoding SEQ ID NO: 359 is ATGGAACGGATCGTGATCTGCCTGATGGTCATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCTCTCAGGGCCAAGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCTGAGTTTCTGCCCGCTCCTGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAG CTGAAAAGCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGTCCATCAAGAAGCTGAAGCGGAAGCCTCCTAGCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGTCCTAGCTGCGACAGCTACGAGAAGAAGCCTCCAAAAGAGTTTCTCGAGCGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGTCCAGCAGGACACACGGCAGCGAGGATTCT (SEQ ID NO:371). In certain embodiments, a 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 example of a nucleic acid sequence encoding SEQ ID NO: 359 is ATGGAACGGATCGTGATCTGCCTGATGGTCATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCTCTCAGGGCCAAGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCTGAGTTCCTGCCTGCTCCTGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCC AGCTGAAAAGCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGTCCATCAAGAAGCTGAAGCGGAAGCCTCCTAGCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGTCCTAGCTGCGACAGCTACGAGAAGAAGCCTCCAAAAGAGTTCCTGGAACGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCAGAACCCACGGCAGCGAGGACTCC (SEQ ID NO: 412). In some embodiments, IL21 comprises a furin cleavage site. In a specific embodiment, IL21 comprises the amino acid sequence of MERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSRRKR (SEQ ID NO: 413).An example of a nucleic acid sequence encoding SEQ ID NO: 413 is ATGGAACGGATCGTGATCTGCCTGATGGTCATCTTCCTGGGGCACCCTGGTGCACAAGAGCAGCTCTCAGGGCCAAGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGACTACGTGAACGACCTGGTGCCTGAGTTCCTGCTGCTCCTGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGGCGCCAACACCGGCAACAACACAACGGAGCGGATCATCAACGTGTCCATCAAGAAGCTGAAGCGGAAGCCTCCTAGCACCAACGCCGGGAAGAGAGCCTCCAAAAAGAGTTCCTGGAACGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACGCAGAACCCACGGCAGGACTCCCAGAAGAAAAACGC (SEQ ID NO: 414).

[0194] In certain embodiments, IL7 comprises the amino acid sequence of DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 394). An example of a nucleic acid sequence encoding SEQ ID NO: 394 is GACTGTGATATCGAGGGCAAAGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAAATCGGCAGCAACTG CCTGAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAAGAAGGCATGTTCCTGTTCAGAGCCGCCAGAAAGCTGCGGCAGTTCCTGAAGATGAACAGCACCGGCGACTT CGACCTGCATCTGCTGAAAGTGTCTGAGGGCACCACCATCCTGCTGAATTGCACCGGCCAAGTGAAGGGCAGAAAGCCTGCTGCTCTGGGAGAAGCCCAGCCTACCAAGAGCCTGGAAGAGA ACAAGTCCCTGAAAGAGCAGAAGAAGCTGAACGACCTCTGCTTCCTGAAGCGGCTGCTGCAAGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAAGAGCAC (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.

[0195]

[0196] Generally, cells (e.g., immune cells or stem cells) are engineered to produce two or more cytokines, including at least one of the cytokines in the form of a membrane-cleavable chimeric protein (e.g., the "S" in the formula SC-MT or MT-CS).

[0197] In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., the "S" in the formula SC-MT or MT-CS) is IL15, IL12, an IL12p70 fusion protein, IL18, or IL21.

[0198] In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., "S" in the formula SC-MT or MT-CS) is IL15. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule is IL15, 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, where 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, cells are engineered to produce at least one membrane-cleavable chimeric protein, where 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, where the secreted effector molecule is IL15, and the cells are further engineered to produce an IL12p70 fusion protein.

[0199] In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., the "S" in the formula SC-MT or MT-CS) is IL15, and the cells are further engineered to produce one or more additional membrane-cleavable chimeric proteins. In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., the "S" in the formula SC-MT or MT-CS) is IL15, and the cells are further engineered to produce one or more additional membrane-cleavable chimeric proteins, including IL12, an IL12p70 fusion protein, IL18, and IL21. In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., the "S" in the formula SC-MT or MT-CS) is IL15, and the cells are further engineered to produce one or more additional membrane-cleavable chimeric proteins, including IL12p70. In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., the "S" in the formula SC-MT or MT-CS) is IL15, and the cells are further engineered to produce IL21.

[0200] In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., the "S" in the formula SC-MT or MT-CS) comprises IL12p70. In some embodiments, cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted 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, where the secreted 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, where the secreted effector molecule is IL12p70, and the cells are further engineered to produce IL15.

[0201] In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., the "S" in the formula SC-MT or MT-CS) is IL12p70, and the cells are further engineered to produce one or more additional membrane-cleavable chimeric proteins. In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., the "S" in the formula SC-MT or MT-CS) is IL12p70, and the cells are further engineered to produce one or more additional membrane-cleavable chimeric proteins including IL15, IL18, and IL21. In some embodiments, the cells are engineered to produce at least one membrane-cleavable chimeric protein, where the secreted effector molecule (e.g., the "S" in the formula SC-MT or MT-CS) is IL12p70, and the cells are further engineered to produce an additional membrane-cleavable chimeric protein including IL15.

[0202] The cells can also be further engineered to express additional proteins in addition to the cytokines and / or membrane-cleavable chimeric proteins having the formula SC-MT or MT-CS described herein. As provided herein, the immunoresponsive cells can be engineered to express a chimeric antigen receptor (CAR). As provided herein, the immunoresponsive cells can be engineered to express a chimeric antigen receptor (CAR) that binds to GPC3. As provided herein, the immunoresponsive cells can be engineered to express a chimeric antigen receptor (CAR) that binds to a target selected from the following: 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, the immunoresponsive cells are further engineered to 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").

[0203] Also, as provided herein, immunoresponsive cells can be engineered to express ACPs that include synthetic transcription factors.

[0204] The CAR may comprise 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-recognizing receptor may comprise an scFv. The scFv may comprise a heavy chain variable domain (VH) and a light chain variable domain (VL), which may be separated by a peptide linker. For example, the scFv may comprise the structure VH-L-VL or VL-L-VH, in which 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 GGGGSGGGSGGGGS (SEQ ID NO: 223). An exemplary nucleic acid sequence encoding SEQ ID NO:223 is GCGGCGGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGATCT (SEQ ID NO:224) or GCGGCGGAGGAAGCGGAGGCGGAGGATCCGGTGGTGGTGGATCT (SEQ ID NO:332). In certain embodiments, the nucleic acid encoding SEQ ID NO:223 comprises a sequence having 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% identity 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 of 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 example of a nucleic acid sequence encoding GSTSGSGKPGSGEGSTKG (SEQ ID NO: 395) is GGCAGCACAAGCGGCTCTGGAAAACCTGGATCTGGCGAGGGCTCTACCAAGGGC (SEQ ID NO: 404).

[0205] CARs can have one or more intracellular signaling domains. In some embodiments, activating CARs (aCARs) can activate immune cells and include, for example, a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, a HVEM intracellular signaling domain ... a CD3 zeta chain intracellular signaling domain, a CD3 zeta chain intracellular signaling domain, a CD3 zeta chain intracellular signaling domain, a CD3 zeta chain intracellular signaling domain, a CD3 zeta chain intracellular signaling domain, a CD3 zeta chain intracellular signaling domain, a CD3 zeta chain intracellular signaling domain, a CD3 zeta chain intracellular signaling domain, The aCAR may comprise an intracellular signaling domain such as a CD28 intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKp46 intracellular signaling domain, an FceRlg intracellular signaling domain, an NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain, fragments thereof, combinations thereof, or combinations of fragments thereof. In some embodiments, the aCAR comprises a CD28 intracellular signaling domain. In some embodiments, the aCAR comprises a CD3 zeta intracellular signaling domain. In some embodiments, the aCAR comprises both a CD28 ICD and a CD3 zeta ICD. In a specific embodiment, the CD28 ICD comprises SEQ ID NO: 267 and the CD3 zeta ICD comprises SEQ ID NO: 277. In some embodiments, the inhibitory CAR (iCAR) can inhibit immune cells and may comprise the intracellular signaling domain of SIRPα or LIR1. In certain embodiments, the iCAR comprises a SIRPα ICD, optionally having the sequence of SEQ ID NO: 385. In some embodiments, the intracellular signaling domain comprises a sequence in Table 6A.

[0206] (Table 6A) TIFF2025533842000042.tif213164TIFF2025533842000043.tif218164

[0207] In some embodiments, the CAR may also include a spacer region linking the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be sufficiently flexible to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some embodiments, the spacer region may be a hinge derived from a human protein. For example, the hinge may be a human Ig (immunoglobulin) hinge, including, but not limited to, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer region may include an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In some embodiments, the spacer region comprises a sequence in Table 6B.

[0208] Table 6B: Examples of hinge and spacer sequences TIFF2025533842000044.tif224166TIFF2025533842000045.tif253166TIFF2025533842000046.tif41166

[0209] CAR can have a transmembrane domain, such as CD8 transmembrane domain, CD28 transmembrane domain, CD3 zeta 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.CAR can have a spacer region between the antigen binding domain and the transmembrane domain.Exemplary transmembrane domain sequences are shown in Table 6C. In certain embodiments, the iCAR comprises a SIRPα transmembrane domain, optionally, the SIRPα transmembrane domain comprises SEQ ID NO: 383. In certain embodiments, the aCAR comprises a CD28 transmembrane domain.

[0210] (Table 6C) TIFF2025533842000047.tif128164TIFF2025533842000048.tif87164

[0211] 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 a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 199), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 200), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 201), and the VL comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 202), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 203), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 204). In some embodiments, the antigen-binding domain that binds GPC3 comprises a 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 GPC3 comprises a 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 GPC3 comprises a 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 GPC3 comprises a 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 GPC3 comprises a 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 a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 204).

[0212] In some embodiments, the antigen-binding domain that binds to GPC3 is and a VH region having an amino acid sequence with 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 EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTNNYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFA YWGQGTLVTVSA (SEQ ID NO: 205) or EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVTVSA (SEQ ID NO: 206).An example of a nucleic acid sequence encoding SEQ ID NO: 206 is GAAGTGCAGCTGGTGGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAACAAGAACGCCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGACGGATCCGGAACAAGACCAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGCCAGGTTCACCATCTCCAGAGATGACAGCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAAACCGAGGACACCGCCGTGTACTATTGCGTGGCCGGCAATAGCTTTGCCTACTGGGGACAGGGCACCCTGGTTACAGTTTCTGCT column number 222) or GAAGTGCAGCTGGTTGAATCAGGTGGCGGCCTGGTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACAAGAACGCCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGACGGATCCGGAACAAGACCAACAACTACGCC ACCTACTACGCCGACAGCGTGAAGGCCAGATTCACCATCAGCCGGGACGACAGCAAGAACAGCCTGTACCTGCAGATGAACTCCTGAAAACCGAGGACACCGCCGTGTATTATTGCGTGGCCGGCAACAGCTTTGCCTACTGGGGACAGGGAACCCTGGTCACCGTGTCTGCC (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.

[0213] In some embodiments, the antigen-binding domain that binds to GPC3 is DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASSRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK (SEQ ID NO: 207), or

[0214] 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 having an amino acid sequence with 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 example of a nucleic acid sequence that encodes SEQ ID NO:381 is (SEQ ID NO:380). In certain embodiments, the nucleic acid that encodes 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.

[0215] In some embodiments, the antigen-binding domain that binds to CEACAM5 comprises a VH region. In some embodiments, the VH region has an amino acid sequence with 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 comprises a VL region. In some embodiments, the VL region has an amino acid sequence with 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 region has the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRTSQDIGNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKSLPRTFGGGTKLEI (SEQ ID NO: 424).In some embodiments, the VL region has an amino acid sequence with 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 derived from a VH region comprising the sequence EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS (SEQ ID NO: 425), and the sequence DIQMTQTTSSLSASLGDRVTISCRTSQDIGNYLN It comprises CDR-L1, CDR-L2 and CDR-L3 derived from a VL region comprising WYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKSLPRTFGGGTKLEI (SEQ ID NO: 424) or DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK (SEQ ID NO: 426).

[0216] Additional antigen binding domains that can be used in aCARs are listed below in Table 6D.

[0217] (Table 6D) TIFF2025533842000049.tif243170TIFF2025533842000050.tif210170TIFF2025533842000051.tif73170

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] In some embodiments, the antigen binding domain of the iCAR binds to VSIG2. In some embodiments, an antigen binding domain that binds to VSIG2 comprises an scFv having an amino acid sequence with 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).Nucleic acid sequence index 379

[0224]

[0225]

[0226]

[0227] When the immunoresponsive cell comprises ACP, the ACP of the immunoresponsive cell described herein comprises a synthetic transcription factor.Synthetic transcription factors are non-natural proteins that contain a DNA binding domain and a transcription effector domain, and can regulate (i.e., activate or repress) transcription through binding to the cognate promoter recognized by the DNA binding domain.In some embodiments, the ACP is a transcription repressor.In some embodiments, the ACP is a transcription activator.

[0228] Engineered cell types Also provided herein are engineered immunoresponsive cells. The immunoresponsive cells can be engineered to contain any of the engineered nucleic acids described herein (e.g., any of the engineered nucleic acids encoding a cytokine, a membrane-cleavable chimeric protein, and / or a CAR described herein). The cells can be engineered to have any of the features of any of the engineered cells described herein. In certain embodiments, provided herein are cells engineered to produce two cytokines and a CAR, where at least one of the cytokines is a membrane-cleavable chimeric protein having the formula SC-MT or MT-CS described herein. Also provided herein are cells engineered to produce two cytokines, an aCAR and an iCAR, where at least one of the cytokines is a membrane-cleavable chimeric protein having the formula SC-MT or MT-CS described herein.

[0229] The engineered immunoresponsive cells include, but are not limited to, T cells, CD8+ T cells, CD4+ T cells, gamma delta 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, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In certain embodiments, the immunoresponsive cells are NK cells or T cells. In some embodiments, the immunoresponsive cells are NK cells.

[0230] Cells can be engineered to produce the proteins described herein using methods known to those skilled in the art. For example, cells can be transduced to engineer tumors. In one embodiment, cells are transduced using a virus.

[0231] In certain embodiments, cells are transduced using an oncolytic virus. 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 morbillivirus, oncolytic lentivirus, oncolytic replicating retrovirus, oncolytic rhabdovirus, oncolytic Seneca Valley virus, oncolytic Sindbis virus, and any variant or derivative thereof.

[0232] The virus may be a recombinant virus that includes any of the oncolytic viruses described herein and encodes another transgene that encodes one or more proteins, such as any of the engineered nucleic acids described herein. The virus may be a recombinant virus that includes any of the oncolytic viruses described herein and encodes another transgene that encodes one or more of the proteins, such as any of the engineered nucleic acids described herein.

[0233] Also provided herein are engineered bacterial cells. 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 certain embodiments, provided herein are bacterial cells engineered to produce two or more of the proteins described herein. The bacterial cells can be engineered to produce one or more mammalian proteins. The bacterial cells can be engineered to produce two or more mammalian proteins. Examples of bacterial cells include, but are not limited to, Clostridium beijerinckii, Clostridium sporogenes, Clostridium novi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella cholerae suis.

[0234] 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 cell. 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.

[0235] The engineered cells can be isolated from a subject (autologous), such as a subject known or suspected of having cancer. Cell isolation methods are known to those skilled 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 isolation, and combinations thereof.

[0236] The engineered cells can be allogeneic with respect to the subject being treated. The allogeneic modified cells can be HLA-matched to the subject being treated. The engineered cells can be cultured cells, such as ex vivo cultured cells. The engineered cells can be ex vivo cultured cells, such as primary cells isolated from a subject. The cultured cells can be cultured with one or more cytokines.

[0237] Also provided herein are methods comprising culturing the engineered cells of the present disclosure. Methods for culturing the engineered cells described herein are known. Those skilled in the art will recognize that the culture conditions will depend on the particular engineered cells of interest. Those skilled in the art will recognize that the culture conditions will depend on the specific downstream use of the engineered cells, for example, the specific culture conditions for subsequent administration of the engineered cells to a subject.

[0238] How to manipulate cells Also provided herein are compositions and methods for engineering immunoresponsive cells to produce one or more proteins or effector molecules of interest (e.g., cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins having the formula SC-MT or MT-CS described herein).

[0239] Generally, cells are engineered to produce a protein of interest through the introduction (i.e., delivery) of a polynucleotide encoding one or more proteins or effector molecules of interest, such as a chimeric protein described herein, containing the protein or effector molecule of interest, into the cytosol and / or nucleus of the cell. For example, the polynucleotide encoding one or more chimeric proteins can be an engineered nucleic acid encoding a cytokine, CAR, or membrane-cleavable chimeric protein having the formula SC-MT or MT-CS described herein. Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. Those skilled in the art will understand that the choice of delivery method may depend on the specific cell type being engineered.

[0240] Viral-mediated delivery Viral vector-based delivery platforms can be used to engineer cells. Generally, viral vector-based delivery platforms engineer cells by introducing (i.e., delivering) them into host cells. For example, viral vector-based delivery platforms can engineer cells by introducing any of the engineered nucleic acids described herein (e.g., any of the exogenous polynucleotide sequences encoding cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins having the formula SC-MT or MT-CS described herein, and / or any of the expression cassettes described herein comprising, from N- to C-terminal, a promoter and an exogenous polynucleotide sequence encoding a protein). Viral vector-based delivery platforms can be nucleic acids, but as such, engineered nucleic acids can also include nucleic acids derived from engineered viruses. Such engineered viral nucleic acids can also be referred to as recombinant viruses or engineered viruses.

[0241] Viral vector-based delivery platforms may encode two or more engineered nucleic acids, genes, or transgenes within the same nucleic acid. For example, nucleic acids derived from engineered viruses, such as recombinant or engineered viruses, may encode one or more transgenes, including, but not limited to, any of the engineered nucleic acids described herein that encode one or more of the proteins described herein. One or more transgenes encoding one or more proteins may 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), viral vector-based delivery platforms may encode one or more genes, referred to as cis-acting elements or genes, such as viral genes required for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.).

[0242] A viral vector-based delivery platform can include more than one viral vector, such as a separate viral vector encoding an engineered nucleic acid, gene, or transgene, referred to herein as a trans-acting element or gene. For example, a helper-dependent viral vector-based delivery platform can provide additional genes required for viral infectivity and / or virus production on one or more additional, separate vectors in addition to a vector encoding one or more proteins and / or other proteins of interest. Two or more engineered nucleic acids can be delivered by a single viral vector, such as one vector delivering engineered nucleic acids configured to produce two or more proteins and / or other proteins of interest. Two or more engineered nucleic acids can be delivered by two or more viral vectors, such as two or more vectors 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 mentioned above, but one skilled in the art can select an appropriate number of viral vectors.

[0243] Generally, any of the viral vector-based systems can be used for in vitro production of molecules, such as the proteins described herein, effector molecules, and / or other proteins of interest, or can be used in 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. The selection of an appropriate viral vector-based system will depend on various factors, such as the size of the cargo / payload, the immunogenicity of the viral system, the target cells of interest, the intensity and timing of gene expression, and other factors recognized by those skilled in the art.

[0244] Viral vector-based delivery platform can be RNA-based virus or DNA-based virus.Exemplary viral vector-based delivery platform includes but is 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, morbillivirus, lentivirus, replicating retrovirus, rhabdovirus, Seneca Valley virus, Sindbis virus, and any variant or derivative thereof.Other exemplary viral vector-based delivery platforms have been described in the art, such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentivirus, including, but not limited to, second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation 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, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in See lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (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).

[0245] This sequence may be preceded by one or more sequences that target intracellular compartments. Upon introduction (i.e., delivery) into a host cell, the infected cell (i.e., the engineered cell) can express the protein of interest and / or other proteins. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described by Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the introduction (i.e., delivery) of engineered nucleic acids, such as Salmonella typhi vectors, and the like, will be apparent to those skilled in the art from the description herein.

[0246] Viral vector-based delivery platforms can be cell-targeted viruses, referred to herein as oncolytic viruses. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex viruses, oncolytic adenoviruses, oncolytic measles viruses, oncolytic influenza viruses, oncolytic Indiana vesiculoviruses, oncolytic Newcastle disease viruses, oncolytic vaccinia viruses, oncolytic polioviruses, oncolytic myxoma viruses, oncolytic reoviruses, oncolytic mumps viruses, oncolytic Maraba viruses, oncolytic rabies viruses, oncolytic rotaviruses, oncolytic hepatitis viruses, oncolytic rubella viruses, oncolytic dengue viruses, oncolytic chikungunya viruses, oncolytic respiratory syncytial viruses, oncolytic lymphocytic choriomeningitis viruses, oncolytic morbilliviruses, oncolytic lentiviruses, oncolytic replicating retroviruses, oncolytic rhabdoviruses, oncolytic Seneca Valley viruses, oncolytic Sindbis viruses, and any variants or derivatives thereof. Any of the oncolytic viruses described herein can be recombinant oncolytic viruses that include one or more transgenes (e.g., engineered nucleic acids) encoding one or more proteins of interest and / or other proteins. The transgenes encoding one or more proteins of interest and / or other proteins can be configured to express the proteins of interest and / or other proteins.

[0247] Viral vector-based delivery platforms can be retroviral-based. Generally, retroviral vectors consist of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is 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 persistent transgene expression. Retroviral-based delivery systems include, but are not limited to, delivery systems 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., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Other retroviral systems include the Phoenix retroviral system.

[0248] Viral vector-based delivery platform can be lentivirus-based.Generally, lentivirus vector is a retrovirus vector that can transduce or infect non-dividing cells and typically produce high viral titers.Lentivirus-based delivery platform can be HIV-based, such as ViraPower system (ThermoFisher) or pLenti system (Cell Biolabs).Lentivirus-based delivery platform can be SIV or FIV-based. Other exemplary lentiviral-based delivery platforms are described in more detail in U.S. Pat. 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, and 6,955,919, each of which is incorporated herein by reference for all purposes.

[0249] Viral vector-based delivery platform can be adenovirus-based.Generally, adenovirus-based vector can achieve very high transduction efficiency in many cell types, does not require cell division, achieves high titer and expression level, and can be produced in large quantities in a relatively simple system.Adenovirus is typically not integrated into the genome of host, so adenovirus can generally be used for the transient expression of transgene in infected cells. 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., H Gene Ther 5:1088 1097, 1999; WO94 / 12649; WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 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. Pat. 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 No. 96 / 13597, each of which is incorporated herein by reference for all purposes.

[0250] The viral vector-based delivery platform can be based on adeno-associated virus (AAV). 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). They can be used for the in vitro production of proteins of interest, such as the proteins and / or effector molecules described herein, or can be used in in vivo and ex vivo gene therapy procedures, e.g., for the in vivo delivery of engineered nucleic acids encoding one or more proteins and / or other proteins of interest (e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. 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, US2007 / 0036760, and US2009 / 0197338; Gao, et al. al., J. Virol, 78(12):6381-6388 (June 2004); Gao, et al., Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and International Patent Applications WO2010 / 138263 and WO93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, 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 detail in U.S. Pat. No. 5,173,414, Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985), Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984), Hermonat & Muzyczka, PNAS 81:64666470 (1984), and Samuiski et al., J. Virol. 63:03822-3828 (1989), each of which is incorporated herein by reference for all purposes. Generally, AAV-based vectors include capsid proteins having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, and variants thereof. In a specific example, an AAV-based vector has a capsid protein having an amino acid sequence corresponding to AAV2. In a specific example, an AAV-based vector has a capsid protein having an amino acid sequence corresponding to AAV8.

[0251] AAV vectors can be engineered to carry any of the exogenous polynucleotide sequences encoding the proteins described herein, such as cytokines, CARs, ACPs, and / or membrane-cleavable chimeric proteins described herein, having the formula SC-MT or MT-CS.

[0252] The viral 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, cargo / payload (e.g., any of the engineered nucleic acids described herein) is encapsulated ex vivo within the purified particles. Thus, VLP production maintains the separation of nucleic acids encoding viral structural proteins and nucleic acids encoding cargo / payload. Viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translational expression systems. Purified viral particles can be denatured and reassembled in the presence of the desired cargo using methods known to those skilled in the art to produce VLPs. VLP production is described in more detail in Seow et al. (Mol Ther. 2009 May;17(5):767-777), incorporated herein by reference for all purposes.

[0253] Viral vector-based delivery platforms can be engineered to target (i.e., infect) a range of cells, a narrow subset of cells, or specific cells. Generally, the envelope protein selected for a viral vector-based delivery platform will determine the tropism of the virus. Viruses used in viral vector-based delivery platforms can be pseudotyped to target specific cells of interest. Viral vector-based delivery platforms are pantropic and can infect a range of cells. For example, a pantropic viral vector-based delivery platform can include a VSV-G envelope. Viral vector-based delivery platforms are amphotropic and can infect mammalian cells. Therefore, those skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein to target the desired cell type.

[0254] Lipid structure delivery system An engineered nucleic acid (e.g., any of the engineered nucleic acids described herein) can be introduced into cells using a lipid-mediated delivery system. Generally, lipid-mediated delivery systems use a structure composed of an outer lipid membrane enveloping an internal compartment. Examples of lipid-based structures include, but are not limited to, lipid-based nanoparticles, liposomes, micelles, exosomes, vesicles, extracellular vesicles, cells, or tissues. The lipid structure delivery system can deliver cargo / payload (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0255] Lipid-based nanoparticles can include, but are not limited to, unilamellar liposomes, multilamellar liposomes, and lipid preparations. As used herein, "liposome" is a generic term that encompasses in vitro preparations of lipid vehicles formed by encapsulating a desired cargo, such as 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 generally comprising phospholipids and an internal medium generally comprising an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes can be unilamellar. Liposomes can be multilamellar. Liposomes can be multivesicular. Liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged. Liposomes can be generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols, such as cholesterol.The selection of lipids is generally guided by the consideration of desired purpose, such as criteria for in vivo delivery, such as liposome size, acid instability, and liposome stability in bloodstream.A variety of methods are available for preparing liposomes, as described in, for example, Szokan et al., Ann.Rev.Biophys.Bioeng.9;467(1980), U.S. Patent No. 4,235,871, U.S. Patent No. 4,501,728, U.S. Patent No. 4,837,028, and U.S. Patent No. 5,019,369.

[0256] Multilamellar liposomes are spontaneously formed when lipids, including phospholipids, are suspended in an excess of aqueous solution, such that multiple lipid layers are separated by aqueous media. Water and dissolved solutes are trapped in the closed structure between the lipid bilayers after self-rearrangement of the lipid components. The 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, viral-based delivery systems, etc.) can be encapsulated in the aqueous interior of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the polypeptide / nucleic acid, interspersed within the lipid bilayer of the liposome, encapsulated in the liposome, complexed with the liposome, or otherwise associated with the liposome, allowing it to be delivered to a target entity. Lipophilic molecules or molecules with lipophilic regions can also dissolve in or associate with the lipid bilayer.

[0257] The liposomes used in accordance with the present embodiment can be prepared by different methods, as will be known to those skilled in the art. The preparation of liposomes is described in further detail in WO2016 / 201323, International Application Nos. PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patent Nos. 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.

[0258] The liposomes can be cationic liposomes. Examples of cationic liposomes are described in further detail in U.S. Patent Nos. 5,962,016, 5,030,453, 6,680,068, U.S. Application No. 2004 / 0208921, and International Patent Applications Nos. 03 / 015757A1, 04029213A2, and 02 / 100435A1, each of which is incorporated herein by reference in its entirety.

[0259] Lipid-mediated gene delivery methods are described, for example, in WO 96 / 18372, WO 93 / 24640, Mannino & Gould-Fogerite, BioTechniques 6(7):682-691 (1988), U.S. Patent No. 5,279,833, Rose, U.S. Patent No. 5,279,833, WO 91 / 06309, and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987), each of which is incorporated herein by reference for all purposes.

[0260] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after fusion of multivesicular bodies with the plasma membrane. Exosome sizes range from 30 to 100 nm in diameter. Their surface consists of a lipid bilayer from the plasma membrane of the donor cell, they contain cytosol from the cell that produced the exosome, and display membrane proteins from the parent cell on their surface. Exosomes useful for nucleic acid delivery are known to those skilled in the art, and are described in detail, for example, in U.S. Pat. No. 9,889,210, incorporated herein by reference for all purposes.

[0261] 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 with a diameter smaller than that of the cell from which they originate. Extracellular vesicles generally range in diameter from 20 nm to 1000 nm and can contain various macromolecular cargoes, either within the internal space and / or across the membrane displayed on the outer surface of the extracellular vesicle. Cargo can include nucleic acids (e.g., any of the engineered nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example, and without limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). The extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0262] As used herein, the term "exosome" refers to small (20-300 nm, more preferably 40-200 nm) cell-derived vesicles that contain a membrane surrounding an internal space and are generated from cells by direct plasma membrane budding or fusion of late endosomes with the plasma membrane. Exosomes contain lipids or fatty acids and polypeptides, and optionally contain a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecules. Exosomes are derived from producing cells and can be isolated from producing cells based on their size, density, biochemical parameters, or a combination thereof. Exosomes are a type of extracellular vesicle. Generally, exosome production / biogenesis does not result in the destruction of the producing cells. Exosomes and the preparation of exosomes are described in further detail in WO2016 / 201323, which is hereby incorporated by reference in its entirety.

[0263] As used herein, the term "nanovesicles" (also referred to as "microvesicles") refers to small (20-250 nm diameter, more preferably 30-150 nm diameter) vesicles of cell origin that comprise a membrane surrounding an interior space and are generated from the cells by direct or indirect manipulation such that the nanovesicles are not produced by the producing cells without such manipulation. Generally, nanovesicles are a subspecies of extracellular vesicles. Suitable manipulations of the producing cells include, but are not limited to, continuous extrusion, treatment with alkaline solutions, sonication, or a combination thereof. The production of nanovesicles may, in some cases, result in the destr...

Claims

1. (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). A multicistronic expression system comprising: each exogenous polynucleotide sequence comprises a 5' end and a 3' end; The multicistronic expression system.

2. (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). A multicistronic expression system comprising: each exogenous polynucleotide sequence comprising a 5' end and a 3' end; The aCAR is (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 to CEACAM5, and optionally wherein the first antigen-binding domain of the aCAR comprises the amino acid sequence set forth 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; Including, The multicistronic expression system.

3. (i) the one or more intracellular signaling domains of the aCAR are selected from the group consisting of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, 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, CD 40, 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, IL2R-based TA, IL2R gamma, IL7R alpha, 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), CD8 4, 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 a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, an LNGFR hinge, an LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof; and / or (iii) the aCAR comprises a transmembrane domain selected from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta 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, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GRO alpha, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa; A multicistronic expression system according to any one of the preceding claims.

4. The iCAR is (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.

4. The multicistronic expression system of claim 1 or 3, comprising:

5. the second antigen-binding domain of the iCAR binds to VSIG2, and optionally (i) the iCAR comprises an LIR1 intracellular inhibitory domain, optionally wherein the intracellular inhibitory domain comprises the amino acid sequence set forth in SEQ ID NO: 387; or (ii) the iCAR comprises a SIRPα intracellular inhibitory domain, and optionally, the intracellular inhibitory domain comprises the amino acid sequence set forth in SEQ ID NO:

385. The multicistronic expression system of claim 4.

6. (i) the iCAR comprises a hinge domain selected from the group consisting of a human Ig (immunoglobulin) hinge, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge, a KIR2DS2 hinge, a LNGFR hinge, a LIR1 hinge, a PDGFR-beta extracellular linker, and combinations thereof; and / or (ii) the iCAR comprises a transmembrane domain selected from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta 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, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GRO alpha, CXCL12, IL21, CD8, NKG2D, TNFR2, GMCSF, and GM-CSFRa; 6. The multicistronic expression system according to claim 4 or 5.

7. (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 within 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; A multicistronic expression system according to any one of the preceding claims.

8. 10. The multicistronic expression system of claim 1, further comprising a ribosome skipping site between each exogenous polynucleotide.

9. At least one of the first and second cytokines has the following formula: S-C-MT or MT-C-S A controlled release cytokine having the formula: S comprises a secreted effector molecule; C contains a protease cleavage site, and MT contains a cell membrane anchoring domain, A multicistronic expression system according to any one of claims 1 to 8.

10. (i) the protease cleavage site is cleaved by ADAM10 and / or ADAM17, and / or (ii) the protease cleavage site comprises the amino acid sequence set forth in SEQ ID NO: 180 or SEQ ID NO: 191; and / or (iii) the cell membrane anchoring domain comprises a transmembrane domain selected from the group consisting of B7-1, PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, LIR1, and BTLA, and optionally, the cell membrane anchoring domain comprises a B7-1 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:219; The multicistronic expression system of claim 9.

11. (i) the first cytokine is IL15, optionally wherein the IL15 comprises the amino acid sequence set forth 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 set forth in SEQ ID NO: 360, or optionally wherein the IL21 is controlled-release IL21 (crIL21); and / or (iii) the first or second cytokine comprises an amino acid sequence set forth in any one of SEQ ID NOs: 355-359, 361, and 391; and / or (iv) the first or second cytokine is encoded by a nucleic acid sequence set forth in any one of SEQ ID NOs: 367-372, and 392; A multicistronic expression system according to any one of the preceding claims.

12. (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). A multicistronic expression system comprising: each exogenous polynucleotide sequence comprises a 5' end and a 3' end; The multicistronic expression system.

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

14. 14. The engineered cell of claim 13, wherein the engineered cell is an immune cell, optionally selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a bone marrow cell, a macrophage, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, and an induced pluripotent stem cell (iPSC) and an iPSC-derived cell, optionally wherein the engineered cell is an NK cell.

15. 15. A pharmaceutical composition comprising the engineered cells of claim 13 or 14 and a pharmaceutically acceptable carrier.

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

17. A method for producing an engineered cell, comprising transducing an isolated cell with a multicistronic expression system according to any one of claims 1 to 12, and optionally (i) the isolated cell is an immune cell, 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, bone marrow cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs) and iPSC-derived cells, and optionally, the isolated cells are NK cells; The method.

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