Combined cancer immunotherapy

Abstract

The present disclosure provides methods and compositions for the dynamic control and targeting of multiple immunosuppressive mechanisms in cancer. Some aspects provide cells engineered to produce effector molecules that each regulate different immunosuppressive mechanisms of the tumor, as well as methods of using the cells to treat cancers such as ovarian cancer, breast cancer, or colon cancer.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of each of the following applications: U.S. Provisional Application No. 62 / 747,109, filed October 17, 2018; U.S. Provisional Application No. 62 / 747,114, filed October 17, 2018; and U.S. Provisional Application No. 62 / 843,180, filed May 3, 2019, each of which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted via EFS - Web and is hereby incorporated by reference in its entirety herein. The ASCII copy was created on November 18, 2019, named STB - 011WO_SL.txt, and is 142,513 bytes in size. Background of the Invention

[0005] There are over 22,000 new ovarian cancer cases and over 14,000 deaths in the United States each year (Siegel RL et al. (2016) CA Cancer J Clin 66(1):7 - 30), and the estimated annual medical burden is over $600M (Dizon D MJ (2010) Gynecol Oncol 116(3)). Conventional methods such as chemotherapy (e.g., carboplatin / cisplatin and / or paclitaxel) often fail to cure ovarian cancer. Approximately 70% of patients do not achieve symptom remission after first - line chemotherapy, and 40% - 50% of patients with symptom remission will relapse within three years.

[0006] Treatments for other cancers such as breast cancer and colon cancer result in five - year survival rates of 85% and 65% respectively. Treatments often include a combination of invasive surgery and chemotherapy. Summary of the Invention

[0007] In some embodiments, provided herein is a cell - based combinatorial immunotherapy for targeted treatment of cancer, such as ovarian cancer, breast cancer, colon cancer, lung cancer, and pancreatic cancer. This combinatorial immunotherapy relies on engineered cell circuits capable of multi - factor regulation within and / or near tumors (“tumor microenvironment (TME)”). Although the progress of combinatorial immunotherapy is exciting, its anti - cancer efficacy has been limited in part due to the following challenges. It is difficult to simultaneously deliver multiple therapies to achieve maximum efficacy without causing significant side effects. It is also difficult to determine the appropriate dosing and timing of multiple systemically administered and / or locally injected therapies in clinical trials.

[0008] However, the combination immunotherapy provided herein is tumor-specific and effective, yet limits systemic toxicity. This combination immunotherapy delivers multiple immunomodulatory effector molecules from a single delivery vehicle to the tumor microenvironment. The design of the delivery vehicle is optimized to enhance its overall function in cancer therapy, including (but not limited to) optimizing promoters, linkers, signal peptides, and the order of multiple immunomodulatory effector molecules.

[0009] Advantageously, the cell circuits of the present disclosure are engineered in mesenchymal stem cells (MSCs), which are capable of selectively homing to tumors (including cancer metastases), producing a pro-inflammatory / immune-stimulatory secretome and, under certain conditions, an anti-inflammatory secretome, and are hypoimmunogenic. These features particularly enable their use, for example, in allogeneic cell therapy, without significant safety concerns, side effects, or rejection responses.

[0010] It has been increasingly recognized that tumors are complex interactions between tumor cells and the surrounding stroma, which includes the extracellular matrix, cancer-associated stromal cells (MSCs and fibroblasts), tumor vasculature, and the immune system. The TME suppresses anti-tumor immune responses through multiple mechanisms targeting the patient's innate and adaptive immune systems. For example, tumors can recruit and induce regulatory T cells, which inhibit the anti-tumor activity of conventional T cells by processing specific chemokines such as CCL22. Tumors can also express molecules that inhibit T cell and NK cell activity, such as immune checkpoints, e.g., PD-L1. Therefore, targeting a single pathway may not be sufficient to achieve robust efficacy against solid tumors.

[0011] Non-limiting examples of effector molecules encompassed by the present disclosure include cytokines, antibodies, chemokines, nucleotides, peptides, enzymes, and oncolytic viruses. For example, MSCs can be engineered to express (and typically secrete) at least one, two, three, or more of the following effector molecules: IL-12, IL-16, IFN-β, IFN-γ, IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β, IL-21, 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).

[0012] The present disclosure provides an engineered cell comprising: a) a promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette oriented 5' to 3', the expression cassette having the formula S1-E1-L-S2-E2

[0013] wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule. In some aspects, the engineered cell is selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor-infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0014] In some aspects, the cell is a mesenchymal stem cell (MSC). In some aspects, the cell is a stem cell. In some aspects, the cell is an immune cell. In some aspects, the cell is a natural killer (NK) cell. In some aspects, the cell is an NKT cell. In some aspects, the cell is an innate lymphoid cell. In some aspects, the cell is a tumor-infiltrating lymphocyte (TIL). In some aspects, the cell is a mast cell. In some aspects, the cell is an eosinophil. In some aspects, the cell is a basophil. In some aspects, the cell is a monocyte. In some aspects, the cell is a macrophage. In some aspects, the cell is a neutrophil. In some aspects, the cell is a myeloid cell. In some aspects, the cell is a dendritic cell. In some aspects, the cell is a T cell. In some aspects, the cell is a CD8+ T cell. In some aspects, the cell is a CD4+ T cell. In some aspects, the cell is a cytotoxic T lymphocyte (CTL). In some aspects, the cell is a virus-specific T cell. In some aspects, the cell is a γ-δ T cell. In some aspects, the cell is a regulatory T cell. In some aspects, the cell is a B cell.

[0015] In some aspects, the promoter comprises an exogenous promoter polynucleotide sequence. In some aspects, the promoter comprises an endogenous promoter. In some aspects, the promoter is operably linked to an expression cassette such that the polynucleotide can be transcribed into a single polynucleotide comprising the form S1-E1-L-S2-E2. In some aspects, the linker polynucleotide sequence is operably associated with the translation of the first effector molecule and the second effector molecule into different polypeptides. In some aspects, the linker polynucleotide sequence encodes a 2A ribosomal skip tag. In some aspects, the 2A ribosomal skip tag is selected from the group consisting of P2A, T2A, E2A, and F2A. In some aspects, the linker polynucleotide sequence encodes a T2A ribosomal skip tag. In some aspects, the linker polynucleotide sequence encodes an internal ribosome entry site (IRES). In some aspects, the linker polynucleotide sequence encodes a cleavable polypeptide. In some aspects, the cleavable polypeptide comprises a Furin recognition polypeptide sequence. In some aspects, the linker polynucleotide sequence further encodes a polypeptide sequence comprising Gly, comprising Ser, or comprising Gly-Ser, such as a Gly-Ser-Gly polypeptide sequence. In some aspects, the linker polynucleotide sequence encodes a Furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosomal skip tag, in the Furin:Gly-Ser-Gly:T2A orientation from the N-terminus to the C-terminus.

[0016] In some aspects, the linker polynucleotide sequence encodes a second promoter, wherein the promoter is operably linked to an expression cassette such that a first polynucleotide comprising the form S1-E1 can be transcribed, wherein the second promoter is operably linked to an expression cassette such that a second polynucleotide comprising the form S2-E2 can be transcribed, and wherein the first polynucleotide and the second polynucleotide are different polynucleotides. In some aspects, the promoter and the second promoter are the same. In some aspects, the promoter and the second promoter are different.

[0017] In some aspects, the engineered cell is HLA typed relative to a subject in need of a therapeutic treatment. In some aspects, the engineered cell is a human cell. In some aspects, the human cell is a cell isolated from a subject, such as the subject to receive the cell. In some aspects, the isolated cell is isolated from a tissue of the group consisting of bone marrow, adipose tissue, umbilical cord, fetal liver, muscle, and lung tissue. In some aspects, the engineered cell is a cultured cell.

[0018] In some aspects, the engineered MSCs comprise a cell marker phenotype including the cell markers CD105+, CD73+ and CD90+. In some aspects, the cell marker phenotype further comprises a phenotype lacking or substantially lacking one or more cell markers selected from the group consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, class II HLA, and combinations thereof. In some aspects, the engineered MSCs comprise: a cell marker phenotype comprising CD105+, CD73+, CD90+, CD45-, CD34-, CD14-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD11b-, CD79α-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD19-, class II HLA-; or a cell marker phenotype comprising CD73+, CD90+, CD105+ and CD166+, CD11b-, CD14-, CD19-, CD34-, CD45- and HLA-DR-. In some aspects, the cell marker phenotype is determined by or has been determined by flow cytometry.

[0019] In some aspects, the engineered cells comprise T cells. In some aspects, the engineered cells comprise NK cells. In some aspects, the engineered cells comprise NKT cells.

[0020] In some aspects, the cell marker phenotype further comprises cell markers including a first effector molecule, a second effector molecule, or a cognate receptor or cognate receptor ligand of the first effector molecule and the second effector molecule expressed in the engineered cells. In some aspects, the receptor is selected from the group consisting of: IL12RB1, IL12RB2, CCL7, and combinations thereof.

[0021] In some aspects, the promoter and / or the second promoter comprises a constitutive promoter. In some aspects, the constitutive promoter is selected from the group consisting of: CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb. In some aspects, the promoter comprises the SFFV promoter. In some aspects, the promoter and / or the second promoter comprises an inducible promoter. In some aspects, the inducible promoter is selected from the group consisting of: 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, inducible molecule-responsive promoter, and tandem repeats thereof.

[0022] In some aspects, the first signal peptide or the second signal peptide respectively comprises a native signal peptide that is native with respect to the first effector molecule or the second effector molecule. In some aspects, the first signal peptide or the second signal peptide respectively comprises a non-native signal peptide that is non-native with respect to the first effector molecule or the second effector molecule. In some aspects, the non-native signal peptide is selected from the group consisting of: IL12, IL2, optimized IL2, trypsinogen-2, Gaussialuciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, preproserum albumin, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, CXCL12, and IL21.

[0023] In some aspects, the first signal peptide and the second signal peptide are the same. In some aspects, the polynucleotide sequence encoding the first signal peptide comprises a codon-optimized polynucleotide sequence. In some aspects, the first secreted polypeptide is the human IL12 signal peptide.

[0024] In some aspects, the polynucleotide sequence encoding the second signal peptide comprises a codon-optimized polynucleotide sequence. In some aspects, the second secreted polypeptide is the human IL21 signal peptide.

[0025] In some aspects, the first effector molecule is independently selected from therapeutic classes, wherein the therapeutic classes are selected from the group consisting of: cytokines, chemokines, growth factors, co-stimulatory molecules, tumor microenvironment regulators, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0026] In some aspects, the second effector molecule is selected from therapeutic classes, wherein the therapeutic classes are selected from the group consisting of: cytokines, chemokines, growth factors, co-stimulatory molecules, tumor microenvironment regulators, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes. In some aspects, the therapeutic classes of the first effector molecule and the second effector molecule are different.

[0027] In some aspects, the first effector molecule and / or the second effector molecule are modified effector molecules. In some aspects, the first effector molecule and / or the second effector molecule are modified to include a cell membrane tethering domain. In some aspects, the cell membrane tethering domain includes a transmembrane-intracellular domain or a transmembrane domain. In some aspects, the cell membrane tethering domain includes a cell surface receptor or a cell membrane-binding portion thereof. In some aspects, the modified effector molecule is a fusion protein that includes a cell surface receptor or a cell membrane-binding portion thereof. In some aspects, the modified effector molecule further includes a linker between the effector molecule and the cell membrane tethering domain. In some aspects, when expressed, the modified effector molecule is tethered to the cell membrane of the engineered cell.

[0028] In some aspects, the cytokine is selected from the group consisting of: IL12, IL7, IL21, IL18, IL15, type I interferons, and interferon-γ. In some aspects, the IL12 cytokine is an IL12p70 fusion protein. In some aspects, the chemokine is selected from the group consisting of: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-11 fusion protein, CCL19, CXCL9, and XCL1. In some aspects, the growth factor is selected from the group consisting of: Flt3L and GM-CSF. In some aspects, the co-stimulatory molecule is selected from the group consisting of: 4-1BBL and CD40L. In some aspects, the tumor microenvironment regulator is selected from the group consisting of: adenosine deaminase, TGFβ inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2. In some aspects, the TGFβ inhibitor is selected from the group consisting of: anti-TGFβ peptides, anti-TGFβ antibodies, TGFb-TRAP, and combinations thereof. In some aspects, the immune checkpoint inhibitor includes an anti-PD-1 antibody. In some aspects, the VEGF inhibitor includes an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof.

[0029] In some aspects, the first effector molecule and the second effector molecule are effector molecules of human origin.

[0030] In some aspects, the first effector molecule includes interleukin 12 (IL12), such as p35 and p40 in dimer form, which is generally referred to as IL-12p70 in the art. In some aspects, the first effector molecule includes an IL12p70 fusion protein. In some aspects, the IL12p70 fusion protein is a human IL12p70 fusion protein. In some aspects, the human IL12p70 fusion protein includes the sequence shown in SEQ ID NO:137. In some aspects, human IL12 includes the p35 subunit shown in SEQ ID NO:137. In some aspects, human IL12 includes the p40 subunit shown in SEQ ID NO:137.

[0031] In some aspects, the second effector molecule comprises CCL21a. In some aspects, CCL21a is human CCL21a. In some aspects, the second effector molecule comprises IL7. In some aspects, IL7 is human IL7. In some aspects, the second effector molecule comprises IL21. In some aspects, IL21 is human IL21.

[0032] In some aspects, the expression cassette further comprises E3 including a polynucleotide sequence encoding a third effector molecule. In some aspects, the third effector molecule comprises Flt3L. In some aspects, the third effector molecule comprises anti-PD1. For example, anti-PD1 can be an anti-PD1 antibody. In some aspects, the expression cassette further comprises E4 including a polynucleotide sequence encoding a fourth effector molecule. In some aspects, the fourth effector molecule comprises adenosine deaminase. In some aspects, the third effector molecule comprises adenosine deaminase. In some aspects, the third effector molecule comprises CD40L. In some aspects, the third effector molecule comprises a CXCL10-CXCL11 fusion protein. In some aspects, the third effector molecule comprises XCL1.

[0033] In some aspects, the second effector molecule comprises Flt3L. In some aspects, the second effector molecule comprises a CXCL10-CXCL11 fusion protein. In some aspects, the second effector molecule comprises anti-PD1. In some aspects, the second effector molecule comprises CD40L.

[0034] In some aspects, the first effector molecule comprises interferon-β and the second effector molecule comprises Flt3L.

[0035] In some aspects, the polynucleotide sequence encoding the first effector molecule comprises a codon-optimized polynucleotide sequence. In some aspects, the polynucleotide sequence encoding the second effector molecule comprises a codon-optimized polynucleotide sequence.

[0036] In some aspects, the engineered cell comprises a polynucleotide sequence encoding a promoter and an expression cassette. In some aspects, the exogenous polynucleotide sequence comprises the sequence shown in SEQ ID NO:144.

[0037] In some aspects, the exogenous polynucleotide sequence is integrated into the genome of the engineered cell. In some aspects, the exogenous polynucleotide sequence comprises one or more viral vector polynucleotide sequences.

[0038] In some aspects, the one or more viral vector polynucleotide sequences comprise lentiviral, retroviral, retrotransposon or adenoviral polynucleotide sequences. In some aspects, the expression cassette further comprises an additional exogenous polynucleotide sequence after E2, the exogenous polynucleotide sequence comprising the following formula, oriented 5' to 3', the formula comprising:

[0039] (L-S-E) X

[0040] Wherein S comprises a polynucleotide sequence encoding a signal peptide, E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, X = 1 to 20, wherein the promoter is operably linked to the expression cassette, and wherein for each X, the corresponding signal peptide is operably associated with the effector molecule.

[0041] The present invention also provides an engineered cell comprising a construct, wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising:

[0042] S1-E1-L-S2-E2

[0043] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is a human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is a human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cell is selected from the group consisting of: mesenchymal stem cells (MSC), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TIL), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTL), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:137. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:138. In some aspects, the polynucleotide sequence encoding the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:136. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO:142. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO:143. In some aspects, the polynucleotide sequence encoding human IL21 comprises the sequence shown in SEQ ID NO:141. In some aspects, the linker comprises the sequence shown in SEQ ID NO:140. In some aspects, the linker polynucleotide sequence comprises the sequence shown in SEQ ID NO:139. In some aspects, the construct comprises the polynucleotide sequence shown in SEQ ID NO:144.

[0044] The present invention also provides an engineered cell comprising a construct, wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the following formula, oriented from 5' to 3', the formula comprising

[0045] S1-E1-L-S2-E2

[0046] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is the human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is the human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cell is a mesenchymal stem cell (MSC). In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 138. In some aspects, the polynucleotide sequence encoding the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 136. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 142. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 143. In some aspects, the polynucleotide sequence encoding human IL21 comprises the sequence shown in SEQ ID NO: 141. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 140. In some aspects, the linker polynucleotide sequence comprises the sequence shown in SEQ ID NO: 139. In some aspects, the construct comprises the polynucleotide sequence shown in SEQ ID NO: 144.

[0047] Also provided herein is an engineered cell comprising a construct, wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0048] S1-E1-L-S2-E2

[0049] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is the human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is the human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is the human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cell is a mesenchymal stem cell (MSC), wherein the MSC comprises a cell marker phenotype comprising CD73+, CD90+, CD105+ and CD166+, CD11b-, CD14-, CD19-, CD34-, CD45- and HLA-DR-. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:137. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:138. In some aspects, the polynucleotide sequence encoding the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:136. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO:142. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO:143. In some aspects, the polynucleotide sequence encoding human IL21 comprises the sequence shown in SEQ ID NO:141. In some aspects, the linker comprises the sequence shown in SEQ ID NO:140. In some aspects, the linker polynucleotide sequence comprises the sequence shown in SEQ ID NO:139. In some aspects, the construct comprises the polynucleotide sequence shown in SEQ ID NO:144. In some aspects, the cell marker phenotype is determined by or has been determined by flow cytometry.

[0050] Also provided herein is an engineered MSC comprising a construct, wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0051] S1-E1-L-S2-E2

[0052] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is a human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is a human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered MSC comprises a cell marker phenotype comprising CD73+, CD90+, CD105+, and CD166+, CD11b-, CD14-, CD19-, CD34-, CD45-, and HLA-DR-. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:137. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:138. In some aspects, the polynucleotide sequence encoding the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:136. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO:142. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO:143. In some aspects, the polynucleotide sequence encoding human IL21 comprises the sequence shown in SEQ ID NO:141. In some aspects, the linker comprises the sequence shown in SEQ ID NO:140. In some aspects, the linker polynucleotide sequence comprises the sequence shown in SEQ ID NO:139. In some aspects, the construct comprises the polynucleotide sequence shown in SEQ ID NO:144. In some aspects, the cell marker phenotype is determined by or has been determined by flow cytometry.

[0053] The present disclosure also provides an engineered cell comprising a construct, wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising:

[0054] S1-E1-L-S2-E2

[0055] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is a human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is a human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSC), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TIL), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTL), virus-specific T cells, gamma-delta T cells, regulatory T cells, and B cells. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:137. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:138. In some aspects, the polynucleotide sequence encoding the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:136. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO:142. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO:143. In some aspects, the polynucleotide sequence encoding human IL21 comprises the sequence shown in SEQ ID NO:141. In some aspects, the linker comprises the sequence shown in SEQ ID NO:140. In some aspects, the linker polynucleotide sequence comprises the sequence shown in SEQ ID NO:139. In some aspects, the construct comprises the polynucleotide sequence shown in SEQ ID NO:144. In some aspects, the cell is a mesenchymal stem cell (MSC). In some aspects, the cell is a natural killer (NK) cell. In some aspects, the cell is an NKT cell. In some aspects, the cell is an innate lymphoid cell. In some aspects, the cell is a tumor infiltrating lymphocyte (TIL). In some aspects, the cell is a mast cell. In some aspects, the cell is an eosinophil. In some aspects, the cell is a basophil.In some aspects, the cell is a monocyte. In some aspects, the cell is a macrophage. In some aspects, the cell is a neutrophil. In some aspects, the cell is a myeloid cell. In some aspects, the cell is a dendritic cell. In some aspects, the cell is a T cell. In some aspects, the cell is a CD8+ T cell. In some aspects, the cell is a CD4+ T cell. In some aspects, the cell is a cytotoxic T lymphocyte (CTL). In some aspects, the cell is a virus-specific T cell. In some aspects, the cell is a γ-δ T cell. In some aspects, the cell is a regulatory T cell. In some aspects, the cell is a B cell. In some aspects, the cell is a human cell.

[0056] In some aspects, the engineered cell is HLA-typed relative to a subject in need of therapeutic treatment. In some aspects, the engineered cell is a human cell. In some aspects, the human cell is a cell isolated from a subject, such as the subject to receive the cell. In some aspects, the isolated cell is isolated from a tissue of a group consisting of: bone marrow, adipose tissue, umbilical cord, fetal liver, muscle, and lung tissue. In some aspects, the engineered cell is a cultured cell.

[0057] In some aspects, the engineered MSC comprises a cell marker phenotype including the cell markers CD105+, CD73+, and CD90+. In some aspects, the cell marker phenotype further comprises a phenotype lacking or substantially lacking one or more cell markers selected from the group consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, class II HLA, and combinations thereof. In some aspects, the engineered MSC comprises: a cell marker phenotype comprising CD105+, CD73+, CD90+, CD45-, CD34- CD14-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD11b-, CD79α-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD19-, class II HLA-; or a cell marker phenotype comprising CD73+, CD90+, CD105+ and CD166+, CD11b-, CD14-, CD19-, CD34-, CD45- and HLA-DR-. In some aspects, the cell marker phenotype is determined by or has been determined by flow cytometry.

[0058] In some aspects, the engineered cells comprise T cells. In some aspects, the T cells are CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, γ-δ T cells, or regulatory T cells. In some aspects, the engineered cells comprise NK cells. In some aspects, the engineered cells comprise NKT cells. In some aspects, the engineered cells comprise monocytes. In some aspects, the engineered cells comprise macrophages. In some aspects, the engineered cells comprise TILs.

[0059] In some aspects, the exogenous polynucleotide sequence is integrated into the genome of the engineered cells. In some aspects, the exogenous polynucleotide sequence comprises one or more viral vector polynucleotide sequences. In some aspects, one or more viral vector polynucleotide sequences comprise lentiviral, retroviral, retrotransposon, or adenoviral polynucleotide sequences. In some aspects, one or more viral vector polynucleotide sequences comprise lentiviral polynucleotide sequences.

[0060] In some aspects, the cells secrete effector molecules. In some aspects, the first effector molecule is secreted at a rate 10-fold higher than the secretion of the second effector molecule.

[0061] In some aspects, the cell further comprises an antigen recognition receptor. In some aspects, the antigen recognition receptor recognizes an antigen selected from the group consisting of: 5T4, ADAM9, ADGRE2, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, Cadherin 3, Cadherin 6, CCR1, CCR4, CD117, CD123, CD131, CD133, CD138, CD142, CD166, CD25, CD244, CD30, CD300LF, CD33, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CD93, CEA, CEACAM5, Claudin 18.2, CLEC12A, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, EMB, ENPP3, EpCAM, EphA2, Ephrin A4, ETBR, FGFR2, FGFR3, FRα, FRb, FLT3, GAPT, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-1RAP, IL-13R, IL-13Ra, IL-13Ra2, IL-8, IL-15, IL1RAP, integrin aV, KIT, L1CAM, LAMP1, LAT2, Lewis Y, LeY, LILRA2, LILRB2, LIV-1, LRRC, LY6E, MCSP, Mesothelin, MLC1, MS4A3, MUC1, MUC16, MUC1C, MYADM, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY ESO 1, Ovarin, P-cadherin, pan-Erb2, PIEZO1, PRAM1, PSCA, PSMA, PTK7, ROR1, SAures, SCT, SLAMF7, SLC22A16, SLC17A9, SLITRK6, SPNS3, SSTR2, STEAP1, Survivin, TDGF1, TIM1, TROP2, VSTM1, and WT1.

[0062] In some aspects, the antigen recognition receptor comprises an antigen-binding domain. In some aspects, the antigen-binding domain comprises 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). In some aspects, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some aspects, the scFv comprises a heavy-chain variable domain (VH) and a light-chain variable domain (VL). In some aspects, VH and VL are separated by a peptide linker. In some aspects, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy-chain variable domain, L is the peptide linker, and VL is the light-chain variable domain.

[0063] In some aspects, the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some aspects, the antigen recognition receptor is a chimeric antigen receptor (CAR). In some aspects, the CAR comprises one or more intracellular signaling domains, and the one or more intracellular signaling domains are selected from the group consisting of: the intracellular signaling domain of the CD3ζ chain, the intracellular signaling domain of CD97, the intracellular signaling domain of CD11a-CD18, the intracellular signaling domain of CD2, the intracellular signaling domain of ICOS, the intracellular signaling domain of CD27, the intracellular signaling domain of CD154, the intracellular signaling domain of CD8, the intracellular signaling domain of OX40, the intracellular signaling domain of 4-1BB, the intracellular signaling domain of CD28, the intracellular signaling domain of ZAP40, the intracellular signaling domain of CD30, the intracellular signaling domain of GITR, the intracellular signaling domain of HVEM, the intracellular signaling domain of DAP10, the intracellular signaling domain of DAP12, and the intracellular signaling domain of MyD88. In some aspects, the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the group consisting of: the transmembrane domain of CD8, the transmembrane domain of CD28, the transmembrane domain of the CD3ζ chain, the transmembrane domain of CD4, the transmembrane domain of 4-1BB, the transmembrane domain of OX40, the transmembrane domain of ICOS, the transmembrane domain of CTLA-4, the transmembrane domain of PD-1, the transmembrane domain of LAG-3, the transmembrane domain of 2B4, and the transmembrane domain of BTLA. In some aspects, the CAR comprises a spacer between the antigen-binding domain and the transmembrane domain.

[0064] Also provided herein is a cell population comprising any of the engineered cells described herein. In some aspects, the cell population is enriched for the engineered cells.

[0065] In some aspects, a first effector molecule, a second effector molecule, or a first and a second effector molecule expressed in engineered cells promotes growth, viability, or growth and viability, such that the growth, viability, or growth and viability is increased relative to cells in the population that do not express the first effector molecule, the second effector molecule, or the first and second effector molecules. In some aspects, the first effector molecule is an IL12 or IL12p70 fusion protein. In some aspects, a cell population enriched in engineered cells expresses the IL12 receptor β1 or has increased levels thereof, expresses the IL12 receptor β2 or has increased levels thereof, or expresses the IL12 receptor β1 and the IL12 receptor β2 or has increased levels thereof. In some aspects, the second effector molecule is IL21. In some aspects, the second effector molecule is CCL21. In some aspects, a cell population enriched in engineered cells expresses the CCL21 receptor or has increased levels thereof. In some aspects, the CCL21 receptor is CCR7.

[0066] Also provided herein is a method of stimulating a cell-mediated immune response against tumor cells in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the engineered cells or cell populations described herein.

[0067] Also provided herein is a method of stimulating (e.g., inducing) an immune response, the method comprising administering to a subject a therapeutically effective dose of any of the engineered cells or cell populations described herein.

[0068] Also provided herein is a method of providing anti-tumor immunity to a subject, the method comprising administering to a subject in need thereof a therapeutically effective dose of any of the engineered cells or cell populations described herein.

[0069] Also provided herein is a method of treating a subject having cancer, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the engineered cells or cell populations described herein.

[0070] Also provided herein is a method of reducing the tumor volume in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the engineered cells or cell populations described herein.

[0071] In some aspects, the engineered cells are derived from the subject. In some aspects, the engineered cells are allogeneic relative to the subject.

[0072] In some aspects, the tumor is selected from the group consisting of: adenocarcinoma, acute myeloid leukemia (AML), acute lymphoblastic B cell leukemia (BALL), acute lymphoblastic T cell leukemia (TALL), B cell prolymphocytic leukemia, bladder tumor, brain tumor, breast tumor, cervical tumor, chronic lymphocytic leukemia, chronic myeloid leukemia (CML), colorectal tumor, esophageal tumor, glioma, kidney tumor, liver tumor, lung tumor, lymphoma, melanoma, mesothelioma, myelodysplasia, ovarian tumor, pancreatic tumor, plasma cell myeloma, prostate tumor, skin tumor, thyroid tumor, and uterine tumor. In some aspects, the tumor is an ovarian tumor. In some aspects, the tumor is a tumor located in the peritoneal space.

[0073] The present disclosure also provides an engineered cell comprising: a) a promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0074] (L-S-E) X

[0075] wherein S comprises a polynucleotide sequence encoding a signal peptide, E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, X = 2 to 20, wherein the promoter is operably linked to the expression cassette, wherein for the first iteration of (L-S-E), unit L is absent, and wherein for each X, the corresponding signal peptide is operably associated with the effector molecule, and wherein the engineered cell is selected from the group consisting of: mesenchymal stem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKT cell, innate lymphoid cell, tumor infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), virus-specific T cell, γ-δ T cell, regulatory T cell, and B cell.

[0076] The present disclosure also provides a cell population comprising one or more engineered cells, wherein the one or more engineered cells comprise: a) a promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0077] S1-E1-L-S2-E2

[0078] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cell is selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor-infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, gamma-delta T cells, regulatory T cells, and B cells.

[0079] Also provided herein is a cell population comprising one or more engineered cells, wherein the one or more engineered cells comprise: a) a promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0080] S1-E1-L-S2-E2

[0081] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the first effector molecule, the second effector molecule, or the first and second effector molecules expressed in the engineered cell promote growth, viability, or growth and viability relative to cells in the population that do not express the first effector molecule, the second effector molecule, or the first and second effector molecules, and wherein the engineered cell is selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor-infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, gamma-delta T cells, regulatory T cells, and B cells.

[0082] In some aspects, one or more engineered cells express a first effector molecule, a second effector molecule, or a cognate receptor or cognate receptor ligand of the first effector molecule and the second effector molecule, which are expressed in the engineered cells. In some aspects, the first effector molecule is IL12 or an IL12p70 fusion protein. In some aspects, the second effector molecule is IL21. In some aspects, the second effector molecule is CCL21.

[0083] Also provided herein is a cell population comprising one or more engineered cells, wherein the one or more engineered cells comprise a construct, and wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0084] S1-E1-L-S2-E2

[0085] wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is a human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is a human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor-infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0086] Also provided herein is a cell population comprising one or more engineered cells, wherein the one or more engineered cells comprise a construct, and wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0087] S1-E1-L-S2-E2

[0088] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is a human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is a human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the first effector molecule, the second effector molecule, or the first and second effector molecules expressed in the engineered cells promotes growth, viability, or growth and viability to increase relative to cells in the population that do not express the first effector molecule, the second effector molecule, or the first and second effector molecules, and wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSC), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor-infiltrating lymphocytes (TIL), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTL), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0089] In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 138. In some aspects, the polynucleotide sequence encoding the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 136. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 142. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 143. In some aspects, the polynucleotide sequence encoding human IL21 comprises the sequence shown in SEQ ID NO: 141. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 140. In some aspects, the linker polynucleotide sequence comprises the sequence shown in SEQ ID NO: 139. In some aspects, the construct comprises the polynucleotide sequence shown in SEQ ID NO: 144.

[0090] Also provided herein is a method of generating a cell population enriched for one or more receptors or receptor ligands, the method comprising culturing one or more cells under conditions that bring the one or more cells into contact with a first effector molecule, a second effector molecule, or a first effector molecule and a second effector molecule, wherein the contacting cells express one or more cognate receptors or cognate receptor ligands for the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule, and wherein the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule promotes an increase in the growth, viability, or growth and viability of the contacting cells relative to cells cultured in the absence of the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule.

[0091] In some aspects, the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule are heterologously expressed in the one or more cells, and the one or more cells contact the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule in an autocrine manner. In some aspects, the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule are expressed in one or more additional cells, and the one or more cells contact the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule in a paracrine manner. In some aspects, the one or more additional cells are feeder cells. In some aspects, the one or more cells are cultured in a culture medium.

[0092] In some aspects, one or more cells are brought into contact with a first effector molecule, a second effector molecule, or a first and a second effector molecule by adding a soluble first effector molecule, a soluble second effector molecule, or a soluble first and second effector molecule to a culture medium. In some aspects, the soluble first effector molecule and / or the soluble second effector molecule is a recombinant effector molecule.

[0093] In some aspects, the one or more cells are cultured under attachment conditions. In some aspects, the one or more cells are attached to a surface. In some aspects, the attached cells are brought into contact with a first effector molecule, a second effector molecule, or a first and a second effector molecule by exposing the one or more cells to the first effector molecule, the second effector molecule, or the first and second effector molecules, immobilized on the surface.

[0094] In some aspects, the first effector molecule is IL12 or an IL12p70 fusion protein. In some aspects, the cell population is enriched in IL12 receptor β1 (IL12Rβ1), enriched in IL12 receptor β2 (IL12Rβ2), or enriched in IL12Rβ1 and IL12Rβ2. In some aspects, the MSC population comprises a cell marker phenotype comprising the cell markers CD105+, CD73+, CD90+, IL12Rβ1+, and IL12Rβ2+. In some aspects, the cell marker phenotype further comprises a phenotype lacking or substantially lacking one or more cell markers selected from the group consisting of CD45, CD34, CD14, CD11b, CD79α, CD19, class II HLA, and combinations thereof.

[0095] In some aspects, the cell population comprises cells selected from the group consisting of natural killer (NK) cells, NKT cells, innate lymphoid cells, mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, and dendritic cells, T cells, CD8+ T cells, CD4+ T cells, γ-δ T cells, and regulatory T cells, and B cells. In some aspects, the cell population comprises T cells, NK cells, NKT cells, monocytes, macrophages, or bone marrow-derived cells.

[0096] In some aspects, the second effector molecule is IL21. In some aspects, the second effector molecule is CCL21. In some aspects, the cell population is enriched in CCR7.

[0097] In some aspects, the MSC population comprises a cell marker phenotype including the cell markers CD105+, CD73+, CD90+, IL12Rβ1+, IL12Rβ2+ and CCR7+. In some aspects, the cell marker phenotype further comprises a phenotype lacking or substantially lacking one or more cell markers selected from the group consisting of CD45, CD34, CD14, CD11b, CD79α, CD19, class II HLA, and combinations thereof.

[0098] Also provided herein is a cell population enriched for one or more receptors or receptor ligands, produced by any of the methods described herein.

[0099] Also provided herein are one or more proteins expressed by a polynucleotide sequence, wherein the polynucleotide sequence comprises a promoter and an expression cassette described by the formula, oriented 5' to 3', comprising

[0100] S1-E1-L-S2-E2

[0101] wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule.

[0102] Also provided herein are one or more proteins expressed by a polynucleotide sequence, wherein the polynucleotide sequence comprises an expression cassette described by the formula, oriented 5' to 3', comprising

[0103] S1-E1-L-S2-E2

[0104] wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule.

[0105] Also provided herein is an isolated polynucleotide sequence comprising a promoter and an expression cassette described by the following formula, oriented 5' to 3', the formula comprising

[0106] S1-E1-L-S2-E2

[0107] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule.

[0108] Also provided herein is an isolated polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0109] S1-E1-L-S2-E2

[0110] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule.

[0111] In some aspects, the promoter comprises an exogenous promoter polynucleotide sequence. In some aspects, the promoter comprises an endogenous promoter. In some aspects, the promoter is operably linked to the expression cassette such that the polynucleotide can be transcribed into a single polynucleotide comprising the formula S1-E1-L-S2-E2.

[0112] In some aspects, the linker polynucleotide sequence is operably associated with the translation of the first and second effector molecules into different polypeptides. In some aspects, the linker polynucleotide sequence encodes a 2A ribosome skipping tag. In some aspects, the 2A ribosome skipping tag is selected from the group consisting of P2A, T2A, E2A, and F2A. In some aspects, the linker polynucleotide sequence encodes a T2A ribosome skipping tag. In some aspects, the linker polynucleotide sequence encodes an internal ribosome entry site (IRES).

[0113] In some aspects, the linker polynucleotide sequence encodes a cleavable polypeptide. In some aspects, the cleavable polypeptide comprises a furin recognition polypeptide sequence. In some aspects, the linker polynucleotide sequence further encodes a polypeptide sequence comprising Gly, comprising Ser, or comprising Gly-Ser, such as a Gly-Ser-Gly polypeptide sequence. In some aspects, the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented N-terminus to C-terminus as furin:Gly-Ser-Gly:T2A.

[0114] In some aspects, the linker polynucleotide sequence encodes a second promoter, wherein the promoter is operably linked to an expression cassette such that a first polynucleotide comprising the formula S1-E1 can be transcribed, wherein the second promoter is operably linked to an expression cassette such that a second polynucleotide comprising the formula S2-E2 can be transcribed, and wherein the first polynucleotide and the second polynucleotide are different polynucleotides. In some aspects, the first promoter and the second promoter are the same. In some aspects, the first promoter and the second promoter are different.

[0115] In some aspects, the first promoter and / or the second promoter comprises a constitutive promoter. In some aspects, the constitutive promoter is selected from the group consisting of: CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb. In some aspects, the first promoter comprises the SFFV promoter. In some aspects, the first promoter and / or the second promoter comprises an inducible promoter. In some aspects, the inducible promoter is selected from the group consisting of: 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, inducible molecule-responsive promoter, and tandem repeats thereof.

[0116] In some aspects, the first signal peptide or the second signal peptide respectively comprises a native signal peptide that is native to the first effector molecule or the second effector molecule. In some aspects, the first signal peptide or the second signal peptide respectively comprises a non-native signal peptide that is non-native to the first effector molecule or the second effector molecule. In some aspects, the non-native signal peptide is selected from the group consisting of: IL12, IL2, optimized IL2, trypsinogen-2, Gauss luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, preproserum albumin, preproazurin, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, CXCL12, and IL21. In some aspects, the first signal peptide and the second signal peptide are the same. In some aspects, the polynucleotide sequence encoding the first signal peptide comprises a codon-optimized polynucleotide sequence.

[0117] In some aspects, the first secreted polypeptide is a human IL12 signal peptide. In some aspects, the polynucleotide sequence encoding the second signal peptide comprises a codon-optimized polynucleotide sequence. In some aspects, the second secreted polypeptide is a human IL21 signal peptide.

[0118] In some aspects, the first effector molecule is selected from a therapeutic category, wherein the therapeutic category is selected from the group consisting of: cytokines, chemokines, growth factors, co-activating molecules, tumor microenvironment regulators, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes. In some aspects, the second effector molecule is selected from a therapeutic category, wherein the therapeutic category is selected from the group consisting of: cytokines, chemokines, growth factors, co-activating molecules, tumor microenvironment regulators, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes. In some aspects, the therapeutic categories of the first effector molecule and the second effector molecule are different. In some aspects, the first effector molecule and / or the second effector molecule is a modified effector molecule.

[0119] In some aspects, the first effector molecule and / or the second effector molecule is modified to comprise a cell membrane tethering domain. In some aspects, the cell membrane tethering domain comprises a transmembrane-intracellular domain or a transmembrane domain. In some aspects, the cell membrane tethering domain comprises a cell surface receptor or a cell membrane-binding portion thereof. In some aspects, the modified effector molecule is a fusion protein comprising a cell surface receptor or a cell membrane-binding portion thereof. In some aspects, the modified effector molecule further comprises a linker between the effector molecule and the cell membrane tethering domain. In some aspects, when expressed in a cell, the modified effector molecule is tethered to the cell membrane of the cell.

[0120] In some aspects, the cytokines are selected from the group consisting of: IL12, IL7, IL21, IL18, IL15, type I interferons, and interferon-γ. In some aspects, the IL12 cytokine is an IL12p70 fusion protein. In some aspects, the chemokines are selected from the group consisting of: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-11 fusion protein, CCL19, CXCL9, and XCL1. In some aspects, the growth factors are selected from the group consisting of: Flt3L and GM-CSF. In some aspects, the co-activating molecules are selected from the group consisting of: 4-1BBL and CD40L. In some aspects, the tumor microenvironment regulators are selected from the group consisting of: adenosine deaminase, TGFβ inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2. In some aspects, the TGFβ inhibitors are selected from the group consisting of: anti-TGFβ peptides, anti-TGFβ antibodies, TGFb-TRAP, and combinations thereof. In some aspects, the immune checkpoint inhibitor comprises an anti-PD-1 antibody. In some aspects, the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof.

[0121] In some aspects, the first effector molecule and the second effector molecule are human-derived effector molecules.

[0122] In some aspects, the first effector molecule comprises IL12. In some aspects, the first effector molecule comprises an IL12p70 fusion protein. In some aspects, the IL12p70 fusion protein is a human IL12p70 fusion protein.

[0123] In some aspects, the second effector molecule comprises CCL21a. In some aspects, CCL21a is human CCL21a. In some aspects, the second effector molecule comprises IL7. In some aspects, IL7 is human IL7. In some aspects, the second effector molecule comprises IL21. In some aspects, IL21 is human IL21.

[0124] In some aspects, the expression cassette further comprises E3 including a polynucleotide sequence encoding a third effector molecule. In some aspects, the third effector molecule comprises Flt3L. In some aspects, the third effector molecule comprises anti-PD1.

[0125] In some aspects, the expression cassette further comprises E4 including a polynucleotide sequence encoding a fourth effector molecule. In some aspects, the fourth effector molecule comprises adenosine deaminase.

[0126] In some aspects, the third effector molecule comprises adenosine deaminase. In some aspects, the third effector molecule comprises CD40L. In some aspects, the third effector molecule comprises a CXCL10-CXCL11 fusion protein. In some aspects, the third effector molecule comprises XCL1.

[0127] In some aspects, the second effector molecule comprises Flt3L. In some aspects, the second effector molecule comprises a CXCL10-CXCL11 fusion protein. In some aspects, the second effector molecule comprises anti-PD1. In some aspects, the second effector molecule comprises CD40L.

[0128] In some aspects, the first effector molecule comprises interferon-β and the second effector molecule comprises Flt3L.

[0129] In some aspects, the polynucleotide sequence encoding the first effector molecule comprises a codon-optimized polynucleotide sequence. In some aspects, the polynucleotide sequence encoding the second effector molecule comprises a codon-optimized polynucleotide sequence.

[0130] In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 138. In some aspects, the polynucleotide sequence encoding the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 136. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 142. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 143. In some aspects, the polynucleotide sequence encoding human IL21 comprises the sequence shown in SEQ ID NO: 141. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 140. In some aspects, the linker polynucleotide sequence comprises the sequence shown in SEQ ID NO: 139. In some aspects, the construct comprises the polynucleotide sequence shown in SEQ ID NO: 144.

[0131] Also provided herein is an exogenous polynucleotide sequence comprising an SFFV promoter and an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0132] S1-E1-L-S2-E2

[0133] wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is the human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is the human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus, S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is the human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule.

[0134] In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 138. In some aspects, the polynucleotide sequence encoding the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 136. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 142. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 143. In some aspects, the polynucleotide sequence encoding human IL21 comprises the sequence shown in SEQ ID NO: 141. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 140. In some aspects, the linker polynucleotide sequence comprises the sequence shown in SEQ ID NO: 139. In some aspects, the construct comprises the polynucleotide sequence shown in SEQ ID NO: 144.

[0135] Also provided herein is an exogenous polynucleotide sequence comprising an SFFV promoter and an expression cassette as described by the following formula, oriented 5' to 3', said formula comprising

[0136] S1-E1-L-S2-E2

[0137] wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is the human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is the human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is the human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule; wherein the promoter is operably linked to the expression cassette such that the polynucleotide can be transcribed into a single polynucleotide comprising the formula S1-E1-L-S2-E2; and wherein the polynucleotide sequence comprises the polynucleotide sequence shown in SEQ ID NO: 144.

[0138] In some aspects, the exogenous polynucleotide sequence is encoded by a nucleic acid selected from the group consisting of: DNA, cDNA, RNA, mRNA, and naked plasmid.

[0139] The present invention also provides an expression vector, which comprises any exogenous polynucleotide sequence described herein. In some aspects, the expression vector is a viral vector. In some aspects, the expression vector is a lentiviral vector.

[0140] The present invention also provides a pharmaceutical composition, which comprises any exogenous polynucleotide sequence described herein and a pharmaceutically acceptable carrier.

[0141] The present invention also provides a pharmaceutical composition, which comprises any engineered cell described herein and a pharmaceutically acceptable carrier. An isolated cell, which comprises any exogenous polynucleotide sequence, any expression vector or any pharmaceutical composition described herein.

[0142] In some aspects, the isolated cells are selected from the group consisting of: T cells, CD8+ T cells, CD4+ T cells, γ-δ T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, ESC-derived cells, pluripotent stem cells, MSCs, induced pluripotent stem cells (iPSCs) and iPSC-derived cells.

[0143] In some aspects, the isolated cell is an MSC.

[0144] In some aspects, the exogenous polynucleotide sequence is integrated into the genome of the cell. In some aspects, the exogenous polynucleotide sequence comprises one or more viral vector polynucleotide sequences.

[0145] In some aspects, the one or more viral vector polynucleotide sequences comprise lentivirus, retrovirus, retrotransposon or adenovirus polynucleotide sequences. In some aspects, the one or more viral vector polynucleotide sequences comprise lentivirus polynucleotide sequences.

[0146] In some aspects, the engineered cell is HLA-typed relative to a subject in need of therapeutic treatment. In some aspects, the engineered cell is a human cell. In some aspects, the human cell is a cell isolated from a subject, such as the subject to receive the cell. In some aspects, the isolated cell is isolated from a tissue of the group consisting of: bone marrow, adipose tissue, umbilical cord, fetal liver, muscle and lung tissue. In some aspects, the cell is a cultured cell.

[0147] In some aspects, the MSC comprises a cell marker phenotype including the cell markers CD105+, CD73+ and CD90+. In some aspects, the cell marker phenotype further comprises a phenotype lacking or substantially lacking one or more cell markers selected from the group consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, class II HLA, and combinations thereof. In some aspects, the MSC comprises: a cell marker phenotype comprising CD105+, CD73+, CD90+, CD45-, CD34-, CD14-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD11b-, CD79α-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD19-, class II HLA-; or a cell marker phenotype comprising CD73+, CD90+, CD105+ and CD166+, CD11b-, CD14-, CD19-, CD34-, CD45- and HLA-DR-. In some aspects, the cell marker phenotype is determined by or has been determined by flow cytometry.

[0148] In some aspects, the cell marker phenotype further comprises a cell marker including a first effector molecule, a second effector molecule, or a cognate receptor or cognate receptor ligand of the first effector molecule and the second effector molecule expressed in the cell. In some aspects, the receptor is selected from the group consisting of: IL12RB1, IL12RB2, CCL7, and combinations thereof.

[0149] In some aspects, the cell secretes each effector molecule. In some aspects, the first effector molecule is secreted at a rate 10-fold higher than the secretion of the second effector molecule.

[0150] In some aspects, the cell further comprises an antigen recognition receptor. In some aspects, the antigen recognition receptor comprises an antigen-binding domain. In some aspects, the antigen-binding domain comprises 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). In some aspects, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some aspects, the scFv comprises a heavy-chain variable domain (VH) and a light-chain variable domain (VL). In some aspects, VH and VL are separated by a peptide linker. In some aspects, the scFv comprises a structure VH-L-VL or VL-L-VH, where VH is the heavy-chain variable domain, L is the peptide linker, and VL is the light-chain variable domain.

[0151] In some aspects, the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some aspects, the antigen recognition receptor is a chimeric antigen receptor (CAR). In some aspects, the CAR comprises one or more intracellular signaling domains, and the one or more intracellular signaling domains are selected from the group consisting of: intracellular signaling domain of CD3ζ chain, intracellular signaling domain of CD97, intracellular signaling domain of CD11a-CD18, intracellular signaling domain of CD2, intracellular signaling domain of ICOS, intracellular signaling domain of CD27, intracellular signaling domain of CD154, intracellular signaling domain of CD8, intracellular signaling domain of OX40, intracellular signaling domain of 4-1BB, intracellular signaling domain of CD28, intracellular signaling domain of ZAP40, intracellular signaling domain of CD30, intracellular signaling domain of GITR, intracellular signaling domain of HVEM, intracellular signaling domain of DAP10, intracellular signaling domain of DAP12, and intracellular signaling domain of MyD88. In some aspects, the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the group consisting of: transmembrane domain of CD8, transmembrane domain of CD28, transmembrane domain of CD3ζ chain, transmembrane domain of CD4, transmembrane domain of 4-1BB, transmembrane domain of OX40, transmembrane domain of ICOS, transmembrane domain of CTLA-4, transmembrane domain of PD-1, transmembrane domain of LAG-3, transmembrane domain of 2B4, and transmembrane domain of BTLA. In some aspects, the CAR comprises a spacer between the antigen binding domain and the transmembrane domain.

[0152] The present disclosure also provides a virus comprising any of the exogenous polynucleotide sequences described herein or any of the expression vectors described herein. In some aspects, the virus is selected from the group consisting of: lentivirus, retrovirus, retrotransposon, and adenovirus. In some aspects, the virus is a lentivirus.

[0153] The present disclosure also provides a method of reducing tumor volume in a subject, the method comprising delivering to a subject having a tumor a composition comprising cells engineered to produce a plurality of effector molecules that modulate tumor-mediated immunosuppressive mechanisms, in an amount effective to reduce tumor volume, wherein the engineered cells comprise: a) a promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0154] S1-E1-L-S2-E2

[0155] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cell is selected from the group consisting of: mesenchymal stem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKT cell, innate lymphoid cell, tumor infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), virus-specific T cell, gamma-delta T cell, regulatory T cell, and B cell.

[0156] Also provided herein is a method of reducing tumor volume in a subject, the method comprising delivering to a subject having a tumor an amount of a composition comprising cells engineered to produce IL12 and IL21 effective to reduce the tumor volume, wherein the engineered cells comprise a construct, and wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0157] S1-E1-L-S2-E2

[0158] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is the human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is the human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is the human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0159] Also provided herein is a method of stimulating (e.g., inducing) an immune response, the method comprising delivering to a subject, in an amount effective to induce an immune response, a composition comprising cells engineered to produce a plurality of effector molecules that modulate tumor-mediated immunosuppressive mechanisms, wherein the engineered cells comprise: a) a promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0160] S1-E1-L-S2-E2

[0161] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cell is selected from the group consisting of: mesenchymal stem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKT cell, innate lymphoid cell, tumor infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), virus-specific T cell, γ-δ T cell, regulatory T cell, and B cell.

[0162] Also provided herein is a method of stimulating (e.g., inducing) an immune response in a subject, the method comprising delivering to the subject, in an amount effective to induce an immune response, a composition comprising cells engineered to produce IL12 and IL21, wherein the engineered cells comprise a construct, wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0163] S1-E1-L-S2-E2

[0164] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is a human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is a human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSC), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TIL), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTL), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0165] In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 138. In some aspects, the polynucleotide sequence encoding the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 136. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 142. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 143. In some aspects, the polynucleotide sequence encoding human IL21 comprises the sequence shown in SEQ ID NO: 141. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 140. In some aspects, the linker polynucleotide sequence comprises the sequence shown in SEQ ID NO: 139. In some aspects, the construct comprises the polynucleotide sequence shown in SEQ ID NO: 144.

[0166] In some aspects, the method further comprises administering a checkpoint inhibitor. In some aspects, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-1L antibody, or an anti-CTLA-4 antibody. In some aspects, the method further comprises administering an anti-CD40 antibody.

[0167] In some aspects, the tumor is selected from the group consisting of: adenocarcinoma, acute myeloid leukemia (AML), acute lymphoblastic B-cell leukemia (BALL), acute lymphoblastic T-cell leukemia (TALL), B-cell prolymphocytic leukemia, bladder tumor, brain tumor, breast tumor, cervical tumor, chronic lymphocytic leukemia, chronic myeloid leukemia (CML), colorectal tumor, esophageal tumor, glioma, kidney tumor, liver tumor, lung tumor, lymphoma, melanoma, mesothelioma, myelodysplasia, ovarian tumor, pancreatic tumor, plasma cell myeloma, prostate tumor, skin tumor, thyroid tumor, and uterine tumor. In some aspects, the tumor is an ovarian tumor. In some aspects, the tumor is a tumor located in the peritoneal space.

[0168] In some aspects, the administration comprises systemic administration, intraperitoneal administration, or intratumoral administration.

[0169] In some aspects, the tumor volume is reduced by at least 25% relative to a control, optionally wherein the control is an unmodified cell. In some aspects, the tumor volume is reduced by at least 50% relative to a control, optionally wherein the control is an unmodified cell. In some aspects, the tumor volume is reduced by at least 75% relative to a control, optionally wherein the control is an unmodified cell.

[0170] Also provided herein is a method of reducing the tumor volume in a subject, the method comprising delivering to a subject having a tumor a composition capable of engineering cells to produce a plurality of effector molecules that modulate tumor-mediated immunosuppressive mechanisms in an amount effective to reduce the tumor volume, wherein each engineered cell comprises: a) a promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0171] S1-E1-L-S2-E2

[0172] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cell is selected from the group consisting of: mesenchymal stem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKT cell, innate lymphoid cell, tumor infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), virus-specific T cell, γ-δ T cell, regulatory T cell, and B cell.

[0173] The present invention also provides a method of reducing the tumor volume in a subject, the method comprising delivering to a subject having a tumor a composition of cells capable of engineering cells to produce IL12 and IL21 in an amount effective to reduce the tumor volume, wherein the engineered cells comprise a construct, wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0174] S1-E1-L-S2-E2

[0175] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is a human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is a human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, gamma-delta T cells, regulatory T cells, and B cells.

[0176] Also provided herein is a method of stimulating (e.g., inducing) an immune response in a subject, the method comprising delivering to the subject, in an amount effective to induce an immune response, a composition capable of engineering cells to produce multiple effector molecules that modulate tumor-mediated immunosuppressive mechanisms, wherein the engineered cells comprise: a) a promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0177] S1-E1-L-S2-E2

[0178] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, E1 comprises a polynucleotide sequence encoding a first effector molecule, L comprises a linker polynucleotide sequence, S2 comprises a polynucleotide sequence encoding a second signal peptide, E2 comprises a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cell is selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, gamma-delta T cells, regulatory T cells, and B cells.

[0179] Also provided herein is a method of stimulating (e.g., inducing) an immune response in a subject, the method comprising delivering to the subject, in an amount effective to induce an immune response, a composition capable of engineering cells to produce IL12 and IL21, wherein the engineered cells comprise a construct, wherein the construct comprises: a) an SFFV promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising

[0180] S1-E1-L-S2-E2

[0181] Wherein S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is the human IL12 signal peptide; E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is the human IL12p70 fusion protein; L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus; S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is the human IL21 signal peptide; E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, gamma-delta T cells, regulatory T cells, and B cells.

[0182] In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 138. In some aspects, the polynucleotide sequence encoding the human IL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 136. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 142. In some aspects, human IL21 comprises the sequence shown in SEQ ID NO: 143. In some aspects, the polynucleotide sequence encoding human IL21 comprises the sequence shown in SEQ ID NO: 141. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 140. In some aspects, the linker polynucleotide sequence comprises the sequence shown in SEQ ID NO: 139. In some aspects, the construct comprises the polynucleotide sequence shown in SEQ ID NO: 144.

[0183] In some aspects, the composition comprises a delivery system selected from the group consisting of a viral system, a transposon system, and a nuclease genome editing system. In some aspects, the viral system is selected from the group consisting of a lentivirus, a retrovirus, a retrotransposon, and an adenovirus. In some aspects, the nuclease genome editing system is selected from the group consisting of a zinc finger system, a TALEN system, and a CRISPR system.

[0184] In some aspects, the tumor is selected from the group consisting of an adenocarcinoma, acute myeloid leukemia (AML), acute lymphoblastic B cell leukemia (BALL), acute lymphoblastic T cell leukemia (TALL), B cell prolymphocytic leukemia, bladder tumor, brain tumor, breast tumor, cervical tumor, chronic lymphocytic leukemia, chronic myeloid leukemia (CML), colorectal tumor, esophageal tumor, glioma, kidney tumor, liver tumor, lung tumor, lymphoma, melanoma, mesothelioma, myelodysplasia, ovarian tumor, pancreatic tumor, plasma cell myeloma, prostate tumor, skin tumor, thyroid tumor, and uterine tumor.

[0185] In some aspects, the administration comprises systemic administration, intraperitoneal administration, or intratumoral administration. Description of the Drawings

[0186] Figure 1 Shows treatment using syngeneic and allogeneic MSCs expressing IL12p70 / CCL21a in the CT26 model.

[0187] Figure 2 shows rechallenge of tumor-free mice with CT26 tumors previously treated with syngeneic and allogeneic MSCs expressing IL12p70 / CCL21a in the CT26 model.

[0188] Figure 3 Shows data demonstrating that murine BM-derived MSCs (BM-MSCs) injected intraperitoneally home in vivo to the tumor site of 4T1 breast cancer cells. Fluorescently labeled BM-MSCs (treatment cells) were injected into mice bearing 4T1 mammary tumor cells. The mammary tumor cells express a luciferase reporter. The top two figures on the left show imaging of the treatment cells (BM-MSCs) in tumor-bearing mice at day 1 and day 7 post-injection as indicated. The third figure on the top left shows imaging of the tumor cells in tumor-bearing mice at day 7 post-injection. The bottom two figures on the left show imaging of the treatment cells in normal mice without tumors at day 1 and day 7 post-injection as indicated. A schematic diagram showing the effect of the tumor on the homing of the treatment cells is provided on the far right.

[0189] Figure 4Data showing significant tumor growth delay induced by engineered MSCs expressing IL-12 and CCL21a in an orthotopic breast cancer mouse model. The left panel shows the effect of engineered MSCs on 4T1 breast tumor growth in mice (n = 8). Each line in the figure represents the tumor volume in mice that received an intraperitoneal injection of control MSC growth medium or engineered MSCs on day 0 and day 7. Mice received an intraperitoneal injection of engineered MSCs expressing IL-12 and engineered MSCs expressing CCL21a. Tumor volume was determined by measuring with calipers every other day. Data are represented as mean ± SEM. *p < 0.05, **p < 0.005 compared to the control medium group. The schematic on the right shows the treatment timeline and the effect of engineered MSCs expressing the combined genes IL-12 and CCL21a on tumor burden in the treated mice.

[0190] Figure 5A Data including inhibition of tumor growth by engineered MSCs expressing IFN-β, IFN-γ, IL-12, CCL21a, or combinations thereof in an orthotopic breast cancer (4T1 triple-negative breast cancer) mouse model. Each effector was expressed by a different MSC, and the MSCs were combined (at a 1:1 ratio) for combination treatment. Each figure shows the effect of engineered MSCs expressing the indicated immunotherapies alone or in combination on the growth of 4T1 breast tumors in mice (n = 6 - 8). Figure 5A Each line represents an individual mouse. Figure 5B The left panel of shows the tumor weights of individual mice in each treatment on day 14. Figure 5B The right panel of shows the tumor volume over time of mice receiving each treatment, represented as mean ± SEM.

[0191] Figure 6A Data including no significant inhibition of tumor growth by engineered MSCs expressing OX40L, TRAIL, IL15, cGAS, or combinations thereof in an orthotopic breast cancer (4T1 triple-negative breast cancer) mouse model. Each effector was expressed by a different MSC, and the MSCs were combined (at a 1:1 ratio) for combination treatment. Each figure shows the effect of engineered MSCs expressing the indicated immunotherapies alone or in combination on the growth of 4T1 breast tumors in mice (n = 6 - 8). Figure 6A Each line represents an individual mouse. Figure 6B The left panel shows the tumor weights of individual mice in each treatment. Figure 6B The right panel shows the body weight over time of mice receiving each treatment, represented as mean ± SEM.

[0192] Figure 7AData including those demonstrating that engineered MSCs expressing IL-12 and CCL21a inhibit tumor growth in a mouse model of in situ breast cancer (4T1 triple-negative breast cancer); however, addition of anti-CD40 antibody does not reduce tumor growth. Each effector is expressed by a different MSC, and the MSCs are combined (at a 1:1 ratio) for combination treatment. Each graph shows the effect of engineered MSCs expressing the indicated immunotherapies, alone or in combination, on the growth of 4T1 mammary tumors in mice (n = 6 - 8). Figure 7A Each line represents an individual mouse. Figure 7B Shows the tumor weights of individual mice in each treatment.

[0193] Figure 8A Data including those demonstrating that engineered MSCs expressing OX40L, TRAIL, IL15, HACvPD-1, or combinations thereof do not significantly inhibit tumor growth in a subcutaneous breast cancer (4T1 triple-negative breast cancer) mouse model. Each effector is expressed by a different MSC, and the MSCs are combined (at a 1:1 ratio) for combination treatment. Each graph shows the effect of engineered MSCs expressing the indicated immunotherapies, alone or in combination, on the growth of 4T1 mammary tumors in mice (n = 6 - 8). Figure 8A Each line represents an individual mouse. Figure 8B The left graph shows the tumor weights of individual mice in each treatment. Figure 8B The right graph shows the body weights of mice receiving each treatment over time, expressed as mean ± SEM.

[0194] Figure 9A Data including those demonstrating that engineered MSCs expressing IL-12 and CCL21a inhibit tumor growth in a mouse model of in situ breast cancer (4T1 triple-negative breast cancer); however, a combination of MSCs expressing CCL21a, IL-36γ, and IL-7 does not reduce tumor growth. However, some of the effector combinations tested may cause toxicity. Each effector is expressed by a different MSC, and the MSCs are combined (at a 1:1 ratio) for combination treatment. Each graph shows the effect of engineered MSCs expressing the indicated immunotherapies, alone or in combination, on the growth of 4T1 mammary tumors in mice (n = 6 - 8). Figure 9A Each line represents an individual mouse. Figure 9B Shows the tumor weights of individual mice in each treatment.

[0195] Figure 10A - 10B Data from GFP dose escalation studies for toxicity and screening are included. Figure 10AEngineering MSCs expressing GFP were shown to cause no toxicity. Each effector was expressed by a different MSC, and the MSCs were combined (at a 1:1 ratio) for combination treatment. Each graph shows the effect of engineered MSCs expressing the indicated immunotherapies, alone or in combination, on the growth of 4T1 mammary tumors in mice (n = 6 - 8). Figure 10A Each line represents an individual mouse. Figure 10B Shows the tumor weights of individual mice in each treatment.

[0196] Figure 11A Shows that engineered human MSCs do not home to mouse 4T1 tumors. Figure 11B Shows the tumor weights of individual mice in each treatment. Efficacy was determined by measuring tumor volume every other day with calipers.

[0197] Figure 12 Includes data showing that IL-12 and CCL21a can slow tumor expansion.

[0198] Figure 13A Includes data showing that engineered MSCs expressing IL-12 and CCL21 in an in situ breast cancer (4T1 triple-negative breast cancer) mouse model sufficiently inhibit tumor growth, and addition of checkpoint inhibitors (anti-PD-1 antibody or anti-CTLA-4 antibody) does not increase efficacy. Each effector was expressed by a different MSC, and the MSCs were combined (at a 1:1 ratio) for combination treatment, and the checkpoint inhibitors were injected alone. Each graph shows the effect of engineered MSCs expressing the indicated immunotherapies, alone or in combination, on the growth of 4T1 mammary tumors in mice (n = 6 - 8). Figure 13A Each line represents an individual mouse. Figure 13B Shows the tumor weights of individual mice in each treatment.

[0199] Figure 14 Includes data showing that engineered MSCs expressing IL-12 and CCL21a induce significant tumor growth delay in a colorectal cancer mouse model. The left graph shows the effect of engineered MSCs on the growth of CT26 colorectal tumors in mice (n = 8). Each line in the graph represents the tumor volume in mice that received an intraperitoneal injection of control MSC growth medium or engineered MSCs on day 0 and day 7. Mice received an intraperitoneal injection of engineered MSCs expressing IL-12 and engineered MSCs expressing CCL21a. Tumor volume was determined by measuring every other day with calipers. Data are represented as mean ± SEM. *p < 0.05, **p < 0.005 compared to the control medium group. The right schematic shows the treatment timeline and the effect of engineered MSCs expressing the combined gene IL-12 and CCL21a on tumor burden in the treated mice.

[0200] Figure 15 A figure showing tumor growth kinetics in the CT26 mouse model to determine the optimal time for administration of engineered MSC cells.

[0201] Figure 16A - 16B Including data showing the effect of engineered MSC expressing IL-12 and CCL21a in combination with anti-CD40 or anti-CTLA4 antibodies on mean tumor growth in a syngeneic mouse model of colon cancer. Mice bearing CT26 colon tumors were treated with one of seven treatments (n = 5 - 6 per treatment group). MSC-IL-12 + MSC-CCL21a indicates treatment with engineered cells expressing IL-12 and engineered cells expressing CCL21a (1:1 ratio) for combination treatment. Figure 16B The left panel shows the tumor weights of individual mice in each treatment. Figure 16B The right panel shows the tumor volume of mice receiving each treatment over time, expressed as mean ± SEM.

[0202] Figure 17A - 17B Including data from a dose-dependent long-term survival study. Figure 17A Showing the tumor volume of individual groups. Figure 17B Showing body weight (top), tumor volume (bottom), and survival rate (right).

[0203] Figure 18A Including data showing that engineered MSC expressing IL-12, CCL21a, and IL15 or HACvPD-1 significantly inhibits tumor growth in a mouse colorectal cancer model. Each effector is expressed by a different MSC, and the MSC (at a 1:1 ratio) are combined for combination treatment. Each graph shows the effect of engineered MSC expressing the indicated immunotherapy alone or in combination on the growth of CT26 colorectal tumors in mice (n = 6 - 8). Figure 18A Each line represents an individual mouse. Figure 18B Showing the tumor weights of individual mice in each treatment. Figure 18C A representative graph of the infiltrating immune population within the tumor microenvironment. Figure 18D Showing the percentage of regulatory T cells (Treg) in the total CD3 population. The number of Treg in the tumor microenvironment treated with engineered MSC-IL2 and CCL21a decreased significantly. Figure 18E Correlating the percentage of immune infiltration with tumor weight. Samples with high lymphocyte (CD3+) infiltration were found to be associated with low tumor weight, while samples with high myeloid (CD11b+) infiltration were associated with higher tumor burden.

[0204] Figure 19 Showing the tumor volume of individual mice in each treatment. Efficacy was determined by measuring tumor volume every other day with calipers.

[0205] Figure 20 Show the tumor weights of individual mice in each treatment. Efficacy was determined by measuring tumor volume every other day with calipers.

[0206] Figure 21A - 21B Show the kinetics of CT26-LUC (luciferase) tumor growth in the intraperitoneal space. CT26 cell line was injected on day 0, and three (3) mice were harvested on days 7, 10, 14, and 18 to determine tumor growth kinetics. Tumor burden was monitored using an IVIS imager. Figure 21A The first row measures mouse body weight and ROI. The second row monitors the tumor weight and ROI of tumors of individual mice in each group. The third row correlates tumor weight with overall ROI or tumor ROI. Figure 21B Show the immune profiles of three (3) mice in the day 18 group to better characterize the tumor microenvironment.

[0207] Figure 22A Include data showing that engineered MSCs expressing IL-12 and CCL21a inhibit tumor growth in a subcutaneous colorectal cancer mouse model; however, the combination of MSCs expressing CCL21a and IL-36γ or IL-7 does not reduce tumor growth. Each effector is expressed by a different MSC, and the MSCs are combined (at a 1:1 ratio) for combination treatment. Each graph shows the effect of engineered MSCs expressing the indicated immunotherapies, alone or in combination, on the growth of CT26 colon tumors in mice (n = 6 - 8). Figure 22A Each line represents an individual mouse. Figure 22B Show the tumor weights of individual mice in each treatment group.

[0208] Figure 23A - 23B Include Figure 22A - 22B Tumor immune infiltration statistics from the experiment represented by Figure 23A Show a significant increase in infiltrating CD3 and CD8 cytotoxic T populations in the combination group compared to the group given untreated MSCs. Figure 23B Show a significant decrease in granulocytic myeloid-derived suppressor cell (gMDSC) and macrophage populations in the combination group compared to the group treated with untreated MSCs.

[0209] Figure 24A - 24B Include Figure 22A - 22B Data on immune percentages and tumor weights related to the experiment represented by Figure 24A and Figure 24BSamples with more CD3+ and CD8+ T cells (upper left and central panels) are strongly associated with decreased tumor weight. These panels also show that samples with fewer CD11b myeloid cells, including macrophages, dendritic cells, and MDSCs, display lower tumor burden ( Figure 24A lower middle and right panels of Figure 24B and the top row of

[0210] Figure 25A - 25B Data include those from MSC-IL-12+CCL21a therapy in intraperitoneal and subcutaneous colorectal cancer mouse models. Three different batches of lentivirally transduced cell lines of MSC-IL12 and CCL21a (TLOO8-3 / 4, TL019-01 / 02, and TL022-01 / 02; each TL number represents a batch) were tested. Figure 25A Show that MSC-IL12+MSC-CCL21a in all three batches reduces tumor burden in subcutaneous and intraperitoneal models (the first 5 panels are from the SC model and the last 3 panels are from the IP model). Tumors were collected from all mice on day 11. Figure 25B Show the average tumor weight from each group.

[0211] Figure 26A Data include those demonstrating that engineered combination treatments of MSC-IL-12+MSC-CCL21a or MSC-CCL21a+MSC-IFN-β inhibit tumor growth in subcutaneous colorectal cancer mouse models; however, the combination of MSCs expressing CCL21a and s41BBL does not reduce tumor growth. Each effector is expressed by a different MSC, and the MSCs (at a 1:1 ratio) are combined for combination treatment. Each panel shows the effect of engineered MSCs expressing the indicated immunotherapies, alone or in combination, on the growth of CT26 tumors in mice (n = 6-8). Figure 26A Each line of Figure 26B represents an individual mouse.

[0212] Figure 27A - 27B Additional data are provided from the experiment represented by Figure 26A - 26B are shown. Figure 27A - 27B A panel showing the immune profile of each group treated with the indicated engineered MSCs. A consistent decrease in the macrophage population was observed after treatment with MSC-IL12+MSC-CCL21a ( Figure 27A)。Compared to untreated MSCs, an overall trend of increased infiltration of the CD3+ population and decreased infiltration of the CD11b+ population was also observed in the group treated with MSC-IL12 + MSC-CCL21a ( Figure 27A and Figure 27B ).

[0213] Figure 28A - 28B Additional data from the experiment represented by Figure 26A - 26B are also provided. Figure 28A - 28B Show that immune infiltration is related to tumor weight. Samples with low macrophages and dendritic cells have lower tumor burden ( Figure 28B , middle upper and upper right).

[0214] Figure 29 Show a graph combining the above in vivo data from the colorectal cancer model ( Figure 22A and Figure 26A ). The combined CT26 data from Figure 22A and Figure 26A records three groups: tumor only (PBS), treated with untreated MSCs, and treated with MSC-IL12 + MSC-CCL21a.

[0215] Figure 30A - 30B Also show the combined data from Figure 22A and Figure 26A . The graph shows the average immune infiltration from flow cytometry experiment data. Statistical significance of CD8+T was observed from Figure 30A , which confirmed that MSC-IL12 + MSC-CCL21a can restore the polarization of the tumor microenvironment and allow more cytotoxic T cell infiltration. In addition, infiltration of the CD11b+ myeloid population was reduced in the group treated with MSC-IL12 + MSC-CCL21a ( Figure 30B ). The data collected showed statistical significance of the dendritic cell and macrophage populations.

[0216] Figure 31 Show the vector map of pL17D.

[0217] Figure 32 Show the efficacy of MSCs engineered to express different effector molecules alone or in combination and, as assessed by BLI levels, to reduce CT26 tumor burden in an IP tumor model.

[0218] Figure 33 Show the efficacy of MSCs engineered to express different effector molecules alone or in combination and, as assessed by BLI levels, to reduce B16F10 tumor burden in an IP tumor model.

[0219] Figure 34Lentiviral expression vector diagram showing the expression of human IL12(p70) and human CCL21a from a single lentiviral expression vector.

[0220] Figure 35 shows the production of hIL12 ( Figure 35A ) and hCCL21a ( Figure 35B ) by engineered hMSCs as assessed by cytokine ELISA.

[0221] Figure 36 shows a transwell assay that demonstrates the functional regulation of T cells by hIL12 produced by MSCs as assessed by IFNγ production.

[0222] Figure 37 shows MSC homing to tumors in tumors of mice bearing IP tumors as assessed by bioluminescence imaging. Figure 37A -D shows homing in the CT26 tumor model ( Figure 37A images shown in Figure 37B ), quantitative overview of the images in Figure 37C ), quantitative real-time PCR ( Figure 37D ), and fluorescence microscopy for firefly luciferase ( Figure 37E ). Homing in the B16F10 tumor model (quantitative overview of the images) is shown.

[0223] Figure 38 Figure 29 shows that in the CT26 IP model, as assessed by BLI, MSCs expressing IL12p70 cause a reduction in tumor burden (upper and lower left panels), and complete elimination of detectable intraperitoneal tumors by tumor weight (lower right panel).

[0224] Figure 39 Figure 33 shows that in the B16F10 IP model, as assessed by BLI, MSCs expressing IL12p70 cause a reduction in tumor burden (upper and lower left panels), and complete elimination of detectable intraperitoneal tumors by tumor weight (lower right panel).

[0225] Figure 40 shows that in the CT26 IP model, as assessed by BLI, MSCs expressing IL12p70 / CCL21a cause a reduction in tumor burden (upper and lower left panels), and complete elimination of detectable intraperitoneal tumors by tumor weight (lower right panel). Figure 40A Figure 38 shows the mean tumor burden of PBS-treated (circles), MSC-Flag-Myc (“untreated MSCs” squares), and MSCs expressing IL12p70 / CCL21a (triangles) as assessed by BLI. Figure 40BShows the mean tumor burden in individual mice treated with PBS (circles), MSC-Flag-Myc (“untreated MSC” squares), and MSCs expressing IL12p70 / CCL21a (left, middle, and right panels, respectively), as assessed by BLI. Figure 40C Shows that treatment with MSCs expressing IL12p70 / CCL21a prolongs survival (100% survival beyond 90 days), while control-treated mice died or were sacrificed by day 20.

[0226] Figure 41 Shows that treatment with MSCs expressing IL12p70 prolongs survival.

[0227] Figure 42 shows the relative growth of genetically engineered MSCs at different MOIs (95000, 9500, 950, or uninfected) in three different donors ( Figure 42A , donor 1; Figure 42B , donor 2; Figure 42C , donor 3).

[0228] Figure 43 Shows two independent human BM-MSC cell lines from two different donors (top and bottom rows, respectively), which were transduced with constructs containing various promoters driving EGFP expression. Shows the percentage of GFP in engineered cells (left panel) and MFI (right panel) at day 25 post-transduction.

[0229] Figure 44 Shows two independent human BM-MSC cell lines from two different donors, which were transduced with constructs containing various promoters driving EGFP expression. Shows the EGFP MFI tracked over time (days 7 to 28 post-transduction) for the two independent human BM-MSC cell lines individually (left panel) or for data combined from the two independent human BM-MSC cell lines (right panel).

[0230] Figure 45 Shows the IL-12p70 secretion of engineered MSCs, as assessed by ELISA.

[0231] Figure 46 Shows the IL-21 secretion of engineered MSCs, as assessed by ELISA.

[0232] Figure 47 Shows the ratio of IL-12p70 to IL-21 secreted by engineered MSCs, as assessed by ELISA.

[0233] Figure 48Results of a functional reporter assay for IL-12p70 using a STAT4-SEAP reporter construct in HEK-293T cells to assess cytokine production and secretion by engineered MSCs.

[0234] Figure 49 Results of a functional reporter assay for IL-21 measuring phosphorylated STAT1 (left panel) and phosphorylated STAT3 (right panel) by intracellular phospho-flow in NK-92 human natural killer cells to assess cytokine production and secretion by engineered MSCs.

[0235] Figure 50 Results of a functional reporter assay for IL-12 using an IL21R-U2OS IL21R / IL2RG dimer reporter construct to assess cytokine production and secretion by engineered MSCs.

[0236] Figure 51A Efficacy of engineered MSCs engineered to express different effector molecules alone or in combination and their ability to reduce CT26 tumor burden in an IP tumor model as assessed by BLI levels.

[0237] Figure 51B Efficacy of engineered MSCs engineered to express different effector molecules alone or in combination and their ability to reduce B16F10 tumor burden in an IP tumor model as assessed by BLI levels.

[0238] Figure 52 shows the efficacy of treatment with MSCs expressing IL12p70, MSCs expressing IL21, and a combination of MSCs expressing IL12p70 and IL21 in a CT26 model as assessed by BLI ( Figure 52A left panel) and by tumor weight ( Figure 52A right panel). Figure 52B BLI luciferase measurements for individual mice are shown.

[0239] Figure 53 shows the efficacy of treatment with a lower dose of a combination of MSCs expressing IL12p70 and MSCs expressing IL12p70 and IL21 as assessed by BLI. Figure 53A ; individual BLI measurements for mice - left panel; overview of BLI measurements - right panel). Figure 53B Survival curves for the treatment groups are shown.

[0240] Figure 54 Figure shows the efficacy of treatment as assessed by BLI ( Figure 54 left panel) and by tumor weight ( Figure 54As evaluated (right panel), the efficacy of treatment with MSCs expressing IL12p70, MSCs expressing IL21, and a combination of MSCs expressing IL12p70 and IL21 in the B16F10 model.

[0241] Figure 55 Shows the BLI luciferase measurement results of individual mice after treatment with MSCs expressing IL12p70, MSCs expressing IL21, and a combination of MSCs expressing IL12p70 and IL21 in the B16F10 model.

[0242] Figure 56 Displays the survival curves of treatment groups receiving MSCs expressing IL12p70, MSCs expressing IL21, a combination of MSCs expressing IL12p70 and IL21, anti-PD1, or a combination of IL12p70 and anti-PD1.

[0243] Figure 57 shows the survival curves of mice after tumor rechallenge. Figure 57A Displays untreated mice. Figure 57B Displays mice previously treated with MSCs expressing IL12 alone. Figure 57C Displays mice previously treated with a combination of MSCs expressing IL12 and MSCs expressing IL21.

[0244] Figure 58 shows the dose-dependent efficacy of treatment with mMSCs engineered to express murine IL12(p70) and murine IL21 from a single lentiviral expression vector in the CT26 tumor model. Figure 58A Displays an overview of BLI assessment of efficacy normalized to day 17 relative to day 7. Figure 58B and Figure 58C Displays the BLI measurement results of individual mice over time. Figure 58D Displays the survival curves of the treatment groups.

[0245] Figure 59 shows the dose-dependent efficacy of treatment with mMSCs engineered to express murine IL12(p70) and murine IL21 from a single lentiviral expression vector in the B16F10 tumor model. Figure 59A Displays an overview of BLI assessment of efficacy normalized to day 17 relative to day 7. Figure 59B and Figure 59C Displays the BLI measurement results of individual mice over time. Figure 59D Displays the BLI measurement results of individual mice over time at higher doses with multiple administrations. Figure 59E Displays the survival curves of the treatment groups.

[0246] Figure 60 shows the dose-dependent efficacy of treatment with mMSCs engineered to express murine IL12(p70) and murine IL21 from a single lentiviral expression vector in the MC-38 tumor model. Figure 60A Overview of BLI assessment showing efficacy normalized to day 9 on day 18. Figure 60B Shows BLI measurements of individual mice over time. Figure 60C Shows the survival curves of the treatment groups.

[0247] Figure 61 shows the preferential homing of human MSCs. Figure 61A Shows the luciferase quantification of the overview. Figure 61B Shows representative images of luciferase signals in organs.

[0248] Figure 62A Shows the production of human IL12 (left panel) and human IL21 (right panel) in peritoneal fluid (left column at each corresponding time point) and serum (right column at each corresponding time point) in the OVCAR8 model.

[0249] Figure 62B Shows the transient production of murine IL12 (left panel) and murine IL21 (right panel) in peritoneal fluid (left column at each corresponding time point) and serum (right column at each corresponding time point) in the CT26 model.

[0250] Figure 63 shows the efficacy of mice treated with MSCs engineered to produce cytokines or with recombinant cytokine therapy in the CT26 model. Figure 63A Shows the survival curves of MSC-IL12 vs rIL12. Figure 63B Shows the survival curves of MSC-IL21 vs rIL21. Figure 63C Shows the survival curves of MSC-IL12 / IL21 vs rIL12+rIL21. Figure 63D and Figure 63E Shows the BLI assessment of tumor burden in mice treated with MSCs engineered to produce cytokines or with recombinant cytokine therapy.

[0251] Figure 64 shows the efficacy of mice treated with MSCs engineered to produce cytokines or with recombinant cytokine therapy in the B16F10 model. Figure 63A Shows the tumor weight assessment of tumor burden in mice treated with MSCs engineered to produce cytokines or with recombinant cytokine therapy. Figure 64B Shows the survival curves of the treatment groups.

[0252] Figure 65 shows the immune profile of mice in the CT26 IP tumor model after treatment with MSCs that produce both IL12 and IL21. The results shown are from a multicolor flow cytometry analysis used to characterize the immune infiltrate in response to treatment. Figure 65A and Figure 65B show T cell subsets and activation markers (CD3, CD4, CD8, CD8 / CD38+, CD8 / IFNg+, CD8 / Gzmb+, NK / Gzmb+ and the ratio CD8:Treg-FoxP3). Figure 65C show the immune profile of antigen-presenting cells such as dendritic cells. Detailed Description

[0253] Mesenchymal stem cells (MSCs) (also known as mesenchymal stromal cells, multipotent stromal cells, bone marrow stromal cells or multipotent mesenchymal stromal cells) are a subset of non-hematopoietic adult stem cells derived from the mesoderm. They have the ability to self-renew and differentiate pluripotently, differentiating not only into mesodermal lineages such as chondrocytes, osteocytes and adipocytes, but also into ectodermal and endodermal cells. MSCs have no ethical issues and no teratoma formation, and are the main type of stem cells used in cell therapy for treating immune and non-immune diseases. They are easily isolated from bone marrow, adipose tissue, umbilical cord, fetal liver, muscle and lung, and can be successfully expanded in vitro. MSCs can be defined by a cell surface marker phenotype, including a cell marker phenotype comprising CD105+, CD73+, CD90+, CD45-, CD34-, CD14-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD11b-, CD79α-; or a cell marker phenotype comprising CD105+, CD73+, CD90+, CD19-, class II HLA-, as discussed in more detail in Dominici et al., (Cytotherapy. 2006;8(4):315-7), which is incorporated by reference for all purposes. In addition, when MSCs are delivered exogenously and systemically to humans and animals, they tend to home (migrate directly) to sites of damaged tissue with inflammation, including the tumor microenvironment and metastatic regions. Inflammation-guided MSC homing involves several important molecules related to cell trafficking, including chemokines, adhesion molecules and matrix metalloproteinases (MMPs).

[0254] The present disclosure provides methods for engineering cells such as MSCs to produce effector molecules that modulate distinct tumor-mediated immunosuppressive mechanisms. These MSCs are referred to herein as "engineered MSCs". These MSCs typically contain engineered nucleic acids that do not exist in nature. In some embodiments, the MSCs are engineered to include nucleic acids comprising a promoter operably linked to a nucleotide sequence encoding an effector molecule (e.g., an effector molecule that stimulates an immune response).

[0255] The present disclosure also provides methods for engineering cells such as immune cells to produce effector molecules, which cells include (but are not limited to) natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells. These cells, including MSCs and immune cells, are referred to herein as "engineered cells". These cells typically contain engineered nucleic acids that do not exist in nature. In some embodiments, the cells are engineered to include nucleic acids comprising a promoter operably linked to a nucleotide sequence encoding an effector molecule (e.g., an effector molecule that stimulates an immune response).

[0256] "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. By way of example, an effector molecule can act as a ligand to increase or decrease enzyme activity, gene expression, or cell signaling. Thus, in some embodiments, the effector molecule modulates (activates or inhibits) distinct immunoregulatory mechanisms. By directly binding to a molecule and modulating it, the effector molecule can also indirectly modulate a second downstream molecule. In some embodiments, the effector molecule is a secreted molecule, while in other embodiments, the effector molecule binds to the cell surface or remains intracellular. By way of example, effector molecules include intracellular transcription factors, microRNAs, and shRNAs that alter the internal cell state to, for example, enhance the immunomodulatory activity, homing properties, or persistence of the cell. Non-limiting examples of effector molecules include cytokines, chemokines, enzymes that regulate metabolite levels, antibodies or decoy molecules that regulate cytokines, homing molecules, and / or integrins.

[0257] The term "modulation" encompasses maintenance of biological activity, inhibition (partial or complete) of biological activity, and stimulation / activation (partial or complete) of biological activity. The term also encompasses decreasing or increasing (e.g., enhancing) biological activity. When one effector molecule modulates a tumor-mediated immunosuppressive mechanism different from that modulated by another effector molecule (e.g., stimulating antigen presentation and / or processing), the two different effector molecules are considered to "modulate different tumor-mediated immunosuppressive mechanisms".

[0258] The modulation effected by an effector molecule can be direct or indirect. Direct modulation occurs when an effector molecule binds to another molecule and modulates the activity of that molecule. Indirect modulation occurs when an effector molecule binds to another molecule, modulates the activity of that molecule and, as a result of that modulation, modulates the activity of another molecule (a molecule to which the effector molecule is not bound).

[0259] In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by at least one effector molecule results in an increase in immune stimulation and / or anti-tumor immune response (e.g., systemically or in the tumor microenvironment) of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200%). By way of example, modulation of a tumor-mediated immunosuppressive mechanism can result in an increase in immune stimulation and / or anti-tumor immune response of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in an increase in immune stimulation and / or anti-tumor immune response of 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100% or 50-200%. It is understood that an "increase" in immune stimulation and / or anti-tumor immune response, e.g., systemically or in the tumor microenvironment, is relative to the immune stimulation and / or anti-tumor immune response that would occur in the absence of the effector molecule.

[0260] In some embodiments, modulation of at least one effector molecule on tumor-mediated immunosuppressive mechanisms results in an increase in immune stimulation and / or anti-tumor immune response (e.g., in the systemic or tumor microenvironment) by at least 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 20-fold, 25-fold, 50-fold or 100-fold). For example, modulation of tumor-mediated immunosuppressive mechanisms can result in an increase in immune stimulation and / or anti-tumor immune response by at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold or at least 100-fold. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in an increase in immune stimulation and / or anti-tumor immune response by 2-10 fold, 2-20 fold, 2-30 fold, 2-40 fold, 2-50 fold, 2-60 fold, 2-70 fold, 2-80 fold, 2-90 fold or 2-100 fold.

[0261] Non-limiting examples of immune stimulation and / or anti-tumor 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, pro-inflammatory macrophage signaling, activity and / or recruitment, matrix degradation, production of immune-stimulatory metabolites, stimulator of interferon gene (STING) signaling (which increases the secretion of IFN and Th1 polarization, promoting anti-tumor immune response) and / or type I interferon signaling. The effector molecule can stimulate at least one (one or more) of the above immune stimulation mechanisms, thus causing an increase in the immune stimulation response. Changes in the above immune stimulation and / or anti-tumor immune mechanisms can be evaluated, for example, using in vitro assays for T cell proliferation or cytotoxicity, in vitro antigen presentation assays, expression assays (e.g., specific markers) and / or cell secretion assays (e.g., cytokines).

[0262] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in a reduction of immunosuppressive responses (e.g., systemically or in the tumor microenvironment) of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). By way of example, modulation of tumor-mediated immunosuppressive mechanisms can result in a reduction of immunosuppressive responses of 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 reduction of immunosuppressive responses of 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200%. It is understood that a “reduction” of immunosuppressive responses, e.g., systemically or in the tumor microenvironment, is relative to the immunosuppressive responses that would occur in the absence of the effector molecule.

[0263] In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms by at least one effector molecule results in a reduction of immunosuppressive responses (e.g., systemically or in the tumor microenvironment) of at least 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 20-fold, 25-fold, 50-fold, or 100-fold). By way of example, modulation of tumor-mediated immunosuppressive mechanisms can result in a reduction of immunosuppressive responses of at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold. In some embodiments, modulation of tumor-mediated immunosuppressive mechanisms results in a reduction of immunosuppressive responses of 2-10 fold, 2-20 fold, 2-30 fold, 2-40 fold, 2-50 fold, 2-60 fold, 2-70 fold, 2-80 fold, 2-90 fold, or 2-100 fold.

[0264] Non-limiting examples of immunosuppressive mechanisms include negative co-stimulatory signaling, pro-apoptotic signaling of cytotoxic cells (e.g., T cells and / or NK cells), regulatory T (Treg) cell signaling, tumor checkpoint molecule production / maintenance, myeloid-derived suppressor cell signaling, activity and / or recruitment, immunosuppressive factor / metabolite production, and / or vascular endothelial growth factor signaling. An effector molecule can inhibit at least one (or more) of the above immunosuppressive mechanisms, thereby causing a reduction in the immunosuppressive response. Changes in the above immunosuppressive mechanisms can be assessed, for example, by measuring an increase in T cell proliferation and / or an increase in IFNγ production (negative co-stimulatory signaling, T reg cell signaling, and / or MDSC); annexin V / PI flow cytometry (pro-apoptotic signaling); flow cytometry for expression, e.g., PDL1 expression (tumor checkpoint molecule production / maintenance); ELISA, RNA via qPCR, enzymatic assays, e.g., IDO tryptophan catabolism (immunosuppressive factor / metabolite production); and phosphorylation of PI3K, Akt, p38 (VEGF signaling).

[0265] In some embodiments, cells, such as MSCs, are engineered to express membrane-bound anti-CD3 and / or anti-CD28 agonist extracellular domains.

[0266] In some embodiments, cells, such as MSCs, are engineered to produce at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) effector molecules, each of which modulates a different tumor-mediated immunosuppressive mechanism. In other embodiments, the cells are engineered to produce at least one effector molecule that is not naturally produced by the cells. Such effector molecules can, for example, complement the functions of effector molecules naturally produced by the cells.

[0267] In some embodiments, effector molecules act in an additive manner: for example, the action of two effector molecules can be equal to the sum of the actions of the two effector molecules acting alone. In other embodiments, effector molecules act in a synergistic manner: for example, the action of two effector molecules can exceed the combined function of the two effector molecules. The present disclosure also encompasses additive and synergistic effects between effector molecules and the immune cells (e.g., MSCs) that produce them.

[0268] Effector molecules that modulate tumor-mediated immunosuppressive mechanisms and / or alter the tumor microenvironment can be, for example, secreted factors (such as cytokines, chemokines, antibodies, and / or decoy receptors that regulate extracellular mechanisms related to the immune system), inhibitors (such as antibodies, antibody fragments, ligand TRAP, and / or small blocking peptides), intracellular factors that control cell state (such as microRNAs and / or transcription factors that regulate cell state to enhance pro-inflammation), factors packaged into exosomes (such as microRNAs, cytoplasmic factors, and / or extracellular factors), surface-displayed factors (such as checkpoint inhibitors, TRAIL), and / or metabolic genes (such as enzymes that produce / regulate or degrade metabolites or amino acids).

[0269] In some embodiments, the effector molecule can be selected from the following non-limiting classes of molecules: cytokines, antibodies, chemokines, nucleotides, peptides, and enzymes. Non-limiting examples of effector molecules of the above classes are listed in Table 1 and specific sequences encoding exemplary effector molecules are listed in Table 6. The effector molecule can be a human effector molecule, such as those listed in Table 1 or Table 6, or a human equivalent of a murine effector molecule listed in Table 1 or Table 6. The effector molecule can be derived from a human effector molecule, such as an endogenous human effector molecule, or be modified and / or functionally optimized, such as codon-optimized for improved expression, modified to enhance stability, or modified in its signal sequence (see below). Various procedures and algorithms for optimizing function are known to those skilled in the art and can be selected based on the desired improvement, such as codon-optimization for a particular species (such as human, mouse, bacteria, etc.).

[0270] Table 1. Exemplary effector molecules

[0271]

[0272]

[0273] In some embodiments, cells such as MSCs contain engineered nucleic acids that comprise a promoter operably linked to a nucleotide sequence encoding an effector molecule. In some embodiments, the engineered nucleic acid comprises a promoter operably linked to nucleotide sequences encoding at least 2 effector molecules. By way of example, the engineered nucleic acid can comprise a promoter operably linked to nucleotide sequences encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 effector molecules. In some embodiments, the engineered nucleic acid comprises a promoter operably linked to nucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more effector molecules.

[0274] In some embodiments, engineered cells, such as engineered MSCs, are engineered to comprise at least two engineered nucleic acids, each comprising a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2, or 3) effector molecule. For example, the cells can be engineered to comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 engineered nucleic acids, each comprising a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2, or 3) effector molecule. In some embodiments, the cells are engineered to comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more engineered nucleic acids, each comprising a promoter operably linked to a nucleotide sequence encoding at least one (e.g., 1, 2, or 3) effector molecule.

[0275] An “engineered nucleic acid” is a nucleic acid that does not exist in nature. However, it should be understood that while engineered nucleic acids are generally non-naturally occurring, they can include nucleotide sequences that exist in nature. In some embodiments, an engineered nucleic acid comprises nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, an engineered nucleic acid includes murine nucleotide sequences, bacterial nucleotide sequences, human nucleotide sequences, and / or viral nucleotide sequences. The term “engineered nucleic acid” includes recombinant nucleic acids and synthetic nucleic acids. A “recombinant nucleic acid” refers to a molecule constructed by ligating nucleic acid molecules and, in some embodiments, is replicable in a living cell. A “synthetic nucleic acid” refers to a molecule that is amplified or chemically or otherwise synthesized. Synthetic nucleic acids include nucleic acids that are chemically modified or otherwise modified but can base pair with naturally occurring nucleic acid molecules. Recombinant nucleic acids and synthetic nucleic acids also include those molecules produced by replication of any of the foregoing nucleic acids. The engineered nucleic acids of the present disclosure can be encoded by a single molecule (e.g., included in the same plasmid or other vector) or by multiple different molecules (e.g., multiple different independently replicating molecules).

[0276] 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 constructs are made using GIBSON Cloning is generated (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 incorporated herein by reference). GIBSON Three enzyme activities are typically used in a single - tube reaction: 5' exonuclease, the 'Y extension activity of DNA polymerase, and DNA ligase activity. The 5' exonuclease activity chews off the 5' terminal sequence and exposes the complementary sequence for annealing. Then the polymerase activity fills in the gaps on the annealed region. Then the DNA ligase seals the nicks and covalently links the DNA fragments together. The overlapping sequences of adjacent fragments are longer than those used in Golden Gate assembly, thus producing a higher percentage of proper assembly. In some embodiments, the engineered nucleic acid construct is generated using cloning (Clontech).

[0277] A "promoter" refers to a nucleic acid sequence control region that controls the start and rate of transcription of the remainder of a nucleic acid sequence. A promoter may also contain sub - regions to which regulatory proteins and molecules such as RNA polymerase and other transcription factors can bind. A promoter can be constitutive, inducible, repressible, tissue - specific, or any combination thereof. A promoter drives the expression or transcription of the nucleic acid sequence it regulates. As used herein, a promoter is considered "operably linked" when it is in the proper functional position and orientation relative to the nucleic acid sequence it regulates so as to control ( "drive") the start of transcription and / or the expression of said sequence.

[0278] A promoter can be a promoter that is naturally associated with a gene or sequence and 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 can be referred to as "endogenous". In some embodiments, a coding nucleic acid sequence can be under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the coding sequence in a natural environment. Such promoters can include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring", such as those containing different elements of different transcriptional regulatory regions and / or mutant promoters whose expression is altered by genetic engineering methods known in the art. Except for nucleic acid sequences that generate promoters and enhancers synthetically, recombinant cloning and / or nucleic acid amplification techniques including polymerase chain reaction (PCR) can be used to generate sequences (see, e.g., U.S. Patent No. 4,683,202 and U.S. Patent No. 5,928,906).

[0279] The promoter of an engineered nucleic acid can be an "inducible promoter", which is a promoter characterized by regulating (e.g., initiating or activating) transcriptional activity when a signal is present, affected by a signal, or in contact with a signal. The signal can be an endogenous or typically exogenous condition (e.g., light), a compound (e.g., a chemical or non-chemical compound), or a protein (e.g., a cytokine) that contacts the inducible promoter in a manner that effectively regulates the activity of transcription from the inducible promoter. Transcriptional activation can involve acting directly on the promoter to drive transcription, or indirectly acting on the promoter by inactivating a repressor that prevents the promoter from driving transcription. Conversely, transcriptional inactivation can involve acting directly on the promoter to drive transcription, or indirectly acting on the promoter by activating a repressor that acts on the promoter subsequently.

[0280] If transcription from a promoter is activated, inactivated, increased, or decreased in the presence of a local tumor state (e.g., inflammation or hypoxia) or a signal, the promoter "responds" to or is "regulated" by the state or signal. In some embodiments, the promoter contains a response element. A "response element" is a short DNA sequence within the promoter region that binds a specific molecule (e.g., a transcription factor) that regulates (controls) the expression of a gene from the promoter. Response elements that can be used according to the present disclosure include, but are not limited to, the phloretin-adjustable control element (PEACE), the zinc finger DNA binding domain (DBD), the interferon-gamma-activated sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. March 1997; 17(3):121-34, incorporated herein by reference), the interferon-stimulated response element (ISRE) (Han, K.J. et al. J Biol Chem. April 9, 2004; 279(15):15652-61, incorporated herein by reference), the NF-κB response element (Wang, V. et al. Cell Reports. 2012; 2(4):824-839, incorporated herein by reference), and the 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. The response element can also contain tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase the sensitivity of the response element to its cognate binding molecule. The tandem repeats can be designated 2X, 3X, 4X, 5X, etc., to indicate the number of repeats present.

[0281] Non-limiting examples of response promoters (also referred to as "inducible promoters") (such as TGF-β response promoters) are listed in Table 2, which shows the design of the promoter and transcription factor, and shows the effect of the inducer molecule on the transcription factor (TF) and transgenic transcription (T) (B, binding; D, dissociation; n.d., not determined) (A, activation; DA, deactivation; DR, derepression) (see Horner, M. and Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein). Other non-limiting examples of inducible promoters include the promoters presented in Table 3.

[0282] Table 2. Examples of response promoters

[0283]

[0284]

[0285] Table 3. Exemplary inducible promoters

[0286]

[0287]

[0288] Other non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, elongation factor 1-α (EF1a) promoter, elongation factor (EFS) promoter, MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), phosphoglycerate kinase (PGK) promoter, spleen focus-forming virus (SFFV) promoter, simian virus 40 (SV40) promoter, and ubiquitin C (UbC) promoter (see Table 4).

[0289] Table 4. Exemplary constitutive promoters

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300] In some embodiments, the promoters of the present disclosure are regulated by signals within the tumor microenvironment. A tumor microenvironment is considered to regulate a promoter if the activity of the promoter increases or decreases by at least 10% in the presence of the tumor microenvironment relative to its activity 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% in the presence of the tumor microenvironment relative to its activity in the absence of the tumor microenvironment. By way of example, the activity of the promoter increases or decreases 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% in the presence of the tumor microenvironment relative to its activity in the absence of the tumor microenvironment.

[0301] In some embodiments, the activity of the promoter increases or decreases by at least 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 20-fold, 25-fold, 50-fold, or 100-fold) in the presence of the tumor microenvironment relative to its activity in the absence of the tumor microenvironment. By way of example, the activity of the promoter increases or decreases by at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold in the presence of the tumor microenvironment relative to its activity in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter increases or decreases by 2-10 fold, 2-20 fold, 2-30 fold, 2-40 fold, 2-50 fold, 2-60 fold, 2-70 fold, 2-80 fold, 2-90 fold, or 2-100 fold in the presence of the tumor microenvironment relative to its activity in the absence of the tumor microenvironment.

[0302] In some embodiments, the promoter of the present disclosure is activated under hypoxic conditions. "Hypoxic conditions" refer to a state in which, at the tissue level, the body or a body region loses proper oxygen supply. Hypoxic conditions can cause inflammation (e.g., an increase in the level of inflammatory cytokines under hypoxic conditions). In some embodiments, a promoter that is activated under hypoxic conditions is operably linked to a nucleotide encoding an effector molecule that reduces the activity of an inflammatory cytokine, thereby reducing the inflammation caused by hypoxic conditions. In some embodiments, a promoter that is activated under hypoxic conditions contains a hypoxia responsive element (HRE). "Hypoxia responsive element (HRE)" is a response element that responds to hypoxia-inducible factor (HIF). In some embodiments, the HRE contains the consensus motif NCGTG (where N is A or G).

[0303] In some embodiments, engineered cells produce multiple effector molecules. For example, cells can be engineered to produce 2 - 20 different effector molecules. In some embodiments, the cells are engineered to produce 2 - 20, 2 - 19, 2 - 18, 2 - 17, 2 - 16, 2 - 15, 2 - 14, 2 - 13, 2 - 12, 2 - 11, 2 - 10, 2 - 9, 2 - 8, 2 - 7, 2 - 6, 2 - 5, 2 - 4, 2 - 3, 3 - 20, 3 - 19, 3 - 18, 3 - 17, 3 - 16, 3 - 15, 3 - 14, 3 - 13, 3 - 12, 3 - 11, 3 - 10, 3 - 9, 3 - 8, 3 - 7, 3 - 6, 3 - 5, 3 - 4, 4 - 20, 4 - 19, 4 - 18, 4 - 17, 4 - 16, 4 - 15, 4 - 14, 4 - 13, 4 - 12, 4 - 11, 4 - 10, 4 - 9, 4 - 8, 4 - 7, 4 - 6, 4 - 5, 5 - 20, 5 - 19, 5 - 18, 5 - 17, 5 - 16, 5 - 15, 5 - 14, 5 - 13, 5 - 12, 5 - 11, 5 - 10, 5 - 9, 5 - 8, 5 - 7, 5 - 6, 6 - 20, 6 - 19, 6 - 18, 6 - 17, 6 - 16, 6 - 15, 6 - 14, 6 - 13, 6 - 12, 6 - 11, 6 - 10, 6 - 9, 6 - 8, 6 - 7, 7 - 20, 7 - 19, 7 - 18, 7 - 17, 7 - 16, 7 - 15, 7 - 14, 7 - 13, 7 - 12, 7 - 11, 7 - 10, 7 - 9, 7 - 8, 8 - 20, 8 - 19, 8 - 18, 8 - 17, 8 - 16, 8 - 15, 8 - 14, 8 - 13, 8 - 12, 8 - 11, 8 - 10, 8 - 9, 9 - 20, 9 - 19, 9 - 18, 9 - 17, 9 - 16, 9 - 15, 9 - 14, 9 - 13, 9 - 12, 9 - 11, 9 - 10, 10 - 20, 10 - 19, 10 - 18, 10 - 17, 10 - 16, 10 - 15, 10 - 14, 10 - 13, 10 - 12, 10 - 11, 11 - 20, 11 - 19, 11 - 18, 11 - 17, 11 - 16, 11 - 15, 11 - 14, 11 - 13, 11 - 12, 12 - 20, 12 - 19, 12 - 18, 12 - 17, 12 - 16, 12 - 15, 12 - 14, 12 - 13, 13 - 20, 13 - 19, 13 - 18, 13 - 17, 13 - 16, 13 - 15, 13 - 14, 14 - 20, 14 - 19, 14 - 18,14 - 17, 14 - 16, 14 - 15, 15 - 20, 15 - 19, 15 - 18, 15 - 17, 15 - 16, 16 - 20, 16 - 19, 16 - 18, 16 - 17, 17 - 20, 17 - 19, 17 - 18, 18 - 20, 18 - 19, or 19 - 20 different effector molecules. In some embodiments, the cell is engineered to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different effector molecules.

[0304] In some embodiments, the exogenous sequence can be polycistronic, i.e., more than one separate polypeptide (e.g., multiple effector molecules) can be produced from a single mRNA transcript. By using various linkers, the exogenous sequence can be polycistronic. For example, a polynucleotide sequence encoding a first effector molecule can be linked to a nucleotide sequence encoding a second effector molecule, e.g., in a first gene:second gene 5' to 3' orientation. The linker can encode a 2A ribosome skipping element, such as T2A. Other 2A ribosome skipping elements include (but are not limited to) E2A, P2A, and F2A. The 2A ribosome skipping element allows the production of separate polypeptides encoded by the first gene and the second gene during translation. The linker can encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, where after expression, the cleavable linker polypeptide cleaves, thereby producing separate polypeptides encoded by the first gene and the second gene. The cleavable linker can include a polypeptide sequence, such as a flexible linker (e.g., Gly - Ser - Gly sequence) that further facilitates cleavage.

[0305] The linker can encode an Internal Ribosome Entry Site (IRES), thereby producing separate polypeptides encoded by the first gene and the second gene during translation. The linker can encode a splice acceptor, such as a viral splice acceptor.

[0306] The linker can be a linker combination, such as a furin - 2A linker, which can produce separate polypeptides by 2A ribosome skipping, followed by further cleavage of the furin site, thereby completely removing 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.

[0307] Generally, a polycistronic system can use any number of linkers or combinations of linkers to express any number of genes or portions thereof (e.g., an exogenous sequence can encode first, second, and third effector molecules, each separated by a linker, thereby producing separate polypeptides encoded by the first, second, and third effector molecules).

[0308] An exogenous sequence can use multiple promoters to express genes from multiple ORFs, i.e., more than one separate mRNA transcript can be produced from the exogenous sequence. For example, a first promoter can be operably linked to a polynucleotide sequence encoding a first effector molecule, and a second promoter can be operably linked to a polynucleotide sequence encoding a second effector molecule.

[0309] As used herein, "linker" can refer to a polypeptide that links a first polypeptide sequence to a second polypeptide sequence, the aforementioned polycistronic linker, or the aforementioned additional promoter operably linked to an additional ORF.

[0310] Engineered cells of the present disclosure, such as MSCs, typically produce multiple effector molecules, at least two of which regulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, at least one of the effector molecules stimulates an inflammatory pathway in the tumor microenvironment, and at least one of the effector molecules inhibits a negative inflammatory regulator in the tumor microenvironment.

[0311] The "tumor microenvironment" is the cellular environment in which a tumor is present, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix (ECM) (see, e.g., Pattabiraman, D.R. and Weinberg, R.A. Nature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, F.R. et al. J Cell Sci 125, 5591-5596, 2012; and Li, H. et al. J Cell Biochem 101(4), 805-15, 2007).

[0312] In some embodiments, the cells are engineered to produce at least one homing molecule. "Homing" refers to the active navigation (migration) of cells to a target site (e.g., a cell, tissue (e.g., a tumor), or organ). A "homing molecule" is a molecule that directs cells to a target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, and PDGFR.

[0313] In some embodiments, the homing molecule is a chemokine receptor (a cell surface molecule that binds to chemokines). Chemokines are small cytokines or signaling proteins secreted by cells that can induce directed chemotaxis in cells. Chemokines can be divided into four main subfamilies: CXC, CC, CX3C, and XC, all of which exert biological effects by selectively binding to chemokine receptors located on the surface of target cells. In some embodiments, the cells are engineered to produce CXCR4, a chemokine receptor that allows cells to home to cells, tissues, or tumors expressing stromal cell-derived factor 1 (also known as SDF1, C-X-C motif chemokine 12, and CXCL12) along a chemokine gradient. Non-limiting examples of chemokine receptors that can be produced by the engineered cells of the present disclosure include: CXC chemokine receptors (such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7), CC chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11), CX3C chemokine receptors (such as CX3CR1, which binds to CX3CL1), and XC chemokine receptors (such as XCR1). In some embodiments, the chemokine receptor is a G protein-coupled transmembrane receptor or a member of the tumor necrosis factor (TNF) receptor superfamily (including, but not limited to, TNFRSF1A, TNFRSF1B). In some embodiments, the cells are engineered to produce CXCL8, CXCL9, and / or CXCL10, CXCL11, or a fusion protein encompassing CXCL10 and CXCL11 (which promote T cell recruitment), CCL3 and / or CXCL5, CCL21 (Th1 recruitment and polarization). In some embodiments, the cells are engineered to produce CXCL13 to promote B cell recruitment.

[0314] In some embodiments, the cells are engineered to produce G protein-coupled receptors (GPCRs) that detect N-formylated oligopeptides (including, but not limited to, FPR2 and FPRL1).

[0315] In some embodiments, the cells are engineered to produce receptors that detect interleukins (including, but not limited to, IL6R).

[0316] In some embodiments, the cells are engineered to produce receptors that detect growth factors secreted from other cells, tissues, or tumors (including, but not limited to, FGFR, PDGFR, EGFR, and VEGF family receptors, including, but not limited to, VEGF-C and VEGF-D).

[0317] In some embodiments, the homing molecule is an integrin. Integrins are transmembrane receptors that facilitate cell-extracellular matrix (ECM) adhesion. Integrins are obligate heterodimers having two subunits: alpha (α) and beta (β). The alpha subunit of an integrin can be, without limitation: ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, IGTA7, ITGA8, ITGA9, IGTA10, IGTA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX. The beta subunit of an integrin can be, without limitation: ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, and ITGB8. The cells of the present disclosure can be engineered to produce any combination of integrin alpha and beta subunits.

[0318] In some embodiments, the homing molecule is a matrix metalloproteinase (MMP). MMPs are enzymes that cleave components of the basement membrane underlying the endothelial cell wall. Non-limiting examples of MMPs include MMP-2, MMP-9, and MMP. In some embodiments, the cells are engineered to produce an inhibitor of a molecule (e.g., a protein) that inhibits MMP. For example, the cells can be engineered to express an inhibitor (e.g., an RNAi molecule) of membrane type 1 MMP (MT1-MMP) or tissue inhibitor of metalloproteinase 1 (TIMP-1).

[0319] In some embodiments, the homing molecule is a ligand that binds to a selectin (e.g., hematopoietic cell E- / L-selectin ligand (HCELL), Dykstra et al., Stem Cells. October 2016;34(10):2501-2511) on the endothelium of, for example, a target tissue.

[0320] The term "homing molecule" also encompasses transcription factors that regulate the production of molecules that improve / enhance cell homing.

[0321] In some embodiments, cell homing is increased by locally irradiating a tumor / cancer cell in a subject. Radiation tissue damage aids cell homing, as well as the homing of endogenous T cells to the damaged tissue.

[0322] Examples of engineered cells

[0323] Cells provided herein (e.g., MSCs) are engineered to produce multiple effector molecules, at least two of which regulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) effector molecule stimulates at least one immune-stimulatory mechanism in the tumor microenvironment or inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) effector molecule stimulates at least one immune-stimulatory mechanism in the tumor microenvironment and at least one effector molecule (e.g., 1, 2, 3, 4, 5, or more) inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In other embodiments, at least two (e.g., 2, 3, 4, 5, or more) effector molecules stimulate at least one immune-stimulatory mechanism in the tumor microenvironment. In other embodiments, at least two (e.g., 1, 2, 3, 4, 5, or more) effector molecules inhibit at least one immunosuppressive mechanism in the tumor microenvironment.

[0324] In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that stimulates T cell signaling, activity, and / or recruitment. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that stimulates antigen presentation and / or processing. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that stimulates natural killer cell-mediated cytotoxic signaling, activity, and / or recruitment. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that stimulates dendritic cell differentiation and / or maturation. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that stimulates immune cell recruitment. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that stimulates M1 macrophage signaling, activity, and / or recruitment. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that stimulates Th1 polarization. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that stimulates matrix degradation. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that stimulates the production of immunostimulatory metabolites. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that stimulates type I interferon signaling. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that inhibits negative co-stimulatory signaling. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that inhibits pro-apoptotic signaling (e.g., via TRAIL) of anti-tumor immune cells. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector molecule that inhibits regulatory T (T reg)Effectors of cell signaling, activity, and / or recruitment. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector that inhibits a tumor checkpoint molecule. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector that activates stimulator of interferon genes (STING) signaling. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector that inhibits myeloid-derived suppressor cell signaling, activity, and / or recruitment. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector that degrades immunosuppressive factors / metabolites. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector that inhibits vascular endothelial growth factor signaling. In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector that directly kills tumor cells (e.g., granzyme, perforin, oncolytic virus, cytolytic peptides and enzymes, e.g., anti-tumor antibodies that cause ADCC).

[0325] In some embodiments, at least one effector: stimulates T cell signaling, activity, and / or recruitment, stimulates antigen presentation and / or processing, stimulates natural killer cell-mediated cytotoxic signaling, activity, and / or recruitment, stimulates dendritic cell differentiation and / or maturation, stimulates immune cell recruitment, stimulates macrophage signaling, stimulates matrix degradation, stimulates production of immunostimulatory metabolites, or stimulates type I interferon signaling; and at least one effector inhibits negative co-stimulatory signaling, inhibits pro-apoptotic signaling of anti-tumor immune cells, inhibits regulatory T (Treg) cell signaling, activity, and / or recruitment, inhibits tumor checkpoint molecules, activates stimulator of interferon genes (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.

[0326] In some embodiments, cells (e.g., MSCs) are engineered to produce at least one effector selected from the group consisting of IL-12, IFN-β, IFN-γ, IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β, OX40-ligand, and CD40L; and / or at least one effector selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-IL-35 antibody; and / or at least one effector selected from the group consisting of MIP1α (CCL3), MIP1β (CCL5), and CCL21; and / or at least one effector selected from the group consisting of CpG oligodeoxynucleotides and / or at least one effector selected from the group consisting of microbial peptides.

[0327] In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and at least one effector molecule selected from cytokines, antibodies, chemokines, nucleotides, peptides, enzymes, and stimulator of interferon genes (STING). In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and at least one cytokine or receptor / ligand (e.g., IL-12, IFN-γ, IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β, OX40-ligand, and / or CD40L).

[0328] In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and at least one cytokine or receptor / ligand (e.g., IL-12, IFN-γ, IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β, OX40-ligand, and / or CD40L).

[0329] In some embodiments, cytokine production is as an engineered fusion protein with an antibody, antibody-fragment, or receptor that self-binds to the cytokine to induce cell-specific targeted binding, such as IL-2 fused with an antibody fragment that prevents IL-2 from binding to Treg cells and preferentially binds to CD8 and NK cells. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and at least one antibody (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-VEGF, anti-TGF-β, anti-IL-10, anti-TNF-α, and / or anti-IL-35 antibody). In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and at least one chemokine (MIP1α (CCL3), MIP1β (CCL5), and / or CCL21). In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and at least one nucleotide (e.g., CpG oligodeoxynucleotide). In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and at least one peptide (e.g., anti-tumor peptide). In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and at least one enzyme. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and at least one STING activator. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and at least one effector with direct anti-tumor activity (e.g., oncolytic virus).

[0330] In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and MIP1-α. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and MIP1-β. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and CXCL9. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and CXCL10. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and CXCL11. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and CCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, CD40L, and / or 41BB-L. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0331] In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and MIP1-α. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and MIP1-β. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and CXCL9. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and CXCL10. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and CXCL11. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and CCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, CD40L, and / or 41BB-L. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0332] In some embodiments, cells (e.g., MSCs) are engineered to produce IL-12 and MIP1-α. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-12 and MIP1-β. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-12 and CXCL9. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-12 and CXCL10. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-12 and CXCL11. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-12 and CCL21. In some embodiments, the cells are engineered to further produce IFN-β, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, CD40L, and / or 41BB-L. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0333] In some embodiments, cells (e.g., MSCs) are engineered to produce TNF-related apoptosis-inducing ligand (TRAIL) and MIP1-α. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and MIP1-β. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and CXCL9. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and CXCL10. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and CXCL11. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and CCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, CD40L, and / or 41BB-L. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0334] In some embodiments, cells (e.g., MSCs) are engineered to produce stimulator of interferon genes (STING) and MIP1-α. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and MIP1-β. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and CXCL9. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and CXCL10. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and CXCL11. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and CCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, CD40L, and / or 41BB-L. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0335] In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and MIP1-α. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and MIP1-β. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and CXCL9. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and CXCL10. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and CXCL11. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and CCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, and / or 41BB-L. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0336] In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and MIP1-α. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and MIP1-β. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and CXCL9. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and CXCL10. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and CXCL11. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and CCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, CD40L, and / or 41BB-L. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0337] In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and IFN-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and CD40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and 41BB-L. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, CXCL10-11 fusion, CXCL13, and / or CCL21. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0338] In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and IFN-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and CD40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and 41BB-L. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0339] In some embodiments, cells (e.g., MSCs) are engineered to produce TNF-related apoptosis-inducing ligand (TRAIL) and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and IFN-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and CD40L. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and 41BB-L. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0340] In some embodiments, cells (e.g., MSCs) are engineered to produce stimulator of interferon genes (STING) and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and IFN-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and CD40L. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and 41BB-L. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0341] In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and IFN-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and 41BB-L. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0342] In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and IFN-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and CD40L. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and 41BB-L. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0343] In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and MIP1-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and CD40L. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and 41BB-L. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0344] In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and MIP1-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and CD40L. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and 41BB-L. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0345] In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and IFN-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and CD40L. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and 41BB-L. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0346] In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and IFN-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and CD40L. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and 41BB-L. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0347] In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and IFN-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and 41BB-L. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0348] In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and IL-12. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and IFN-γ. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and IL-2. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and IL-7. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and IL-15. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and IL-36γ. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and IL-18. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and CD40L. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and 41BB-L. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and / or OX40L.

[0349] In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-α and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CXCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0350] In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-β and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CXCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0351] In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce TRAIL and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CXCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0352] In some embodiments, cells (e.g., MSCs) are engineered to produce STING and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce STING and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CXCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0353] In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CXCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0354] In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce adenosine deaminase and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CXCL21. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0355] In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-α and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0356] In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce MIP1-β and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0357] In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL9 and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0358] In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL10 and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0359] In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CXCL11 and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0360] In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CCL21 and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and / or 41BB-L.

[0361] In some embodiments, cells (e.g., MSCs) are engineered to produce IL-12 and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-12 and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-12 and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-12 and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21.

[0362] In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-γ and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-γ and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-γ and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IFN-γ and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21.

[0363] In some embodiments, cells (e.g., MSCs) are engineered to produce IL-2 and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-2 and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-2 and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-2 and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21.

[0364] In some embodiments, cells (e.g., MSCs) are engineered to produce IL-7 and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-7 and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-7 and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-7 and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21.

[0365] In some embodiments, cells (e.g., MSCs) are engineered to produce IL-15 and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-15 and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-15 and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-15 and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21.

[0366] In some embodiments, cells (e.g., MSCs) are engineered to produce IL-36-γ and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-36-γ and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-36-γ and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-36-γ and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21.

[0367] In some embodiments, cells (e.g., MSCs) are engineered to produce IL-18 and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-18 and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-18 and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce IL-18 and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21.

[0368] In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce CD40L and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21.

[0369] In some embodiments, cells (e.g., MSCs) are engineered to produce 41BB-L and anti-PD-L1 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce 41BB-L and OX40L. In some embodiments, cells (e.g., MSCs) are engineered to produce 41BB-L and anti-CTLA4 antibody. In some embodiments, cells (e.g., MSCs) are engineered to produce 41BB-L and anti-CD47 antibody. In some embodiments, the cells are engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and / or adenosine deaminase. In some embodiments, the cells are engineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and / or CCL21.

[0370] Secretion signal

[0371] Generally, one or more effector molecules contain a secretion signal peptide (also known as a signal peptide or signal sequence) at the N-terminus of the effector molecule, which directs the newly synthesized protein for secretion or membrane insertion into an appropriate protein processing pathway. The secretion signal peptide operably associated with the effector molecule can be a native secretion signal peptide (e.g., a secretion signal peptide that is generally endogenously associated with a given effector molecule). The secretion signal peptide operably associated with the effector molecule can be a non-native secretion signal peptide. Non-native secretion signal peptides can promote expression and functional improvement, such as maintaining secretion, especially in an environment, such as the tumor microenvironment. Non-limiting examples of non-native secretion signal peptides are shown in Table 5.

[0372] Table 5. Exemplary signal secretion peptides

[0373]

[0374]

[0375]

[0376] Cell type

[0377] The present invention refers to mesenchymal stem cells (MSCs) (such as human MSCs) that are engineered to produce multiple effector molecules. Engineered cells (engineered to produce effector molecules) provided herein can also be selected from natural killer (NK) cells, NKT cells, innate lymphoid cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells, T cells (such as CD8+ T cells, CD4+ T cells, γ-δ T cells, and regulatory T cells (CD4 + 、FOXP3 + 、CD25 + )) and B cells. However, it should be understood that any reference to engineering MSCs can also apply to other cell types (such as cell types of the immune system).

[0378] In some embodiments, the engineered cells (such as MSCs) are from (such as obtained from or derived from) bone marrow. In some embodiments, the engineered mesenchymal stem cells are from (such as obtained from or derived from) adipose tissue. In some embodiments, the engineered mesenchymal stem cells are from (such as obtained from or derived from) umbilical cord. In some embodiments, the engineered mesenchymal stem cells are from pluripotent stem cells (such as induced pluripotent stem cells).

[0379] Accordingly, the present invention provides T cells (such as CD8+ T cells, CD4+ T cells, γ-δ T cells, and regulatory T cells (CD4 + 、FOXP3 + 、CD25 +), wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, B cells are engineered to produce multiple effector molecules, wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, NK cells are engineered to produce multiple effector molecules, wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, NKT cells are engineered to produce multiple effector molecules, wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, innate lymphoid cells are engineered to produce multiple effector molecules, wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, mast cells are engineered to produce multiple effector molecules, wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, eosinophils are engineered to produce multiple effector molecules, wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, basophils are engineered to produce multiple effector molecules, wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, macrophages are engineered to produce multiple effector molecules, wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, neutrophils are engineered to produce multiple effector molecules, wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms. In some embodiments, dendritic cells are engineered to produce multiple effector molecules, wherein at least two effector molecules modulate different tumor-mediated immunosuppressive mechanisms.

[0380] In some embodiments, at least one of the effector molecules stimulates an immune-stimulatory mechanism in the tumor microenvironment and / or inhibits an immunosuppressive mechanism in the tumor microenvironment.

[0381] 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 cytotoxic signaling, activity, and / or recruitment, (d) stimulates dendritic cell differentiation and / or maturation, (e) stimulates immune cell recruitment, (f) stimulates pro-inflammatory macrophage signaling, activity, and / or recruitment or inhibits anti-inflammatory macrophage signaling, activity, and / or recruitment, (g) stimulates matrix degradation, (h) stimulates the production of immune-stimulatory metabolites, (i) stimulates type I interferon signaling, (j) inhibits negative co-stimulatory signaling, (k) inhibits the pro-apoptotic signaling of anti-tumor immune cells, (l) inhibits regulatory T (T regCell signaling, activation, and / or recruitment, (m) inhibiting tumor checkpoint molecules, (n) stimulating stimulator of interferon genes (STING) signaling, (o) inhibiting myeloid-derived suppressor cell signaling, activation, and / or recruitment, (p) degrading immunosuppressive factors / metabolites, (q) inhibiting vascular endothelial growth factor signaling, and / or (r) directly killing tumor cells.

[0382] Method

[0383] Also provided herein are methods that include culturing the engineered MSCs (or other engineered immune cells) of the present disclosure. Methods of culturing MSCs are known. In some embodiments, the MSCs are cultured in a growth medium (such as MSCGM Human Mesenchymal Stem Cell Growth BULLETKIT TM medium (serum-containing), THERAPEAK TM MSCGM-CD TM chemically defined mesenchymal stem cell medium (serum-free), or RoosterBio xeno-free MSC medium). Methods of culturing other cells, such as immune cells, are known to those skilled in the art.

[0384] Further provided herein are methods that include delivering or administering to a subject (such as a human subject) the engineered cells provided herein to produce in vivo at least one effector molecule produced by the cells. In some embodiments, the cells are administered via intravenous, intraperitoneal, intratracheal, subcutaneous, intratumoral, oral, anal, intranasal (such as packaged in delivery particles), or arterial (such as internal carotid artery) routes. Thus, the cells can be administered systemically or locally (such as to the TME).

[0385] The engineered cells or polynucleotides described herein can be in a composition containing a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized by conventional well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solution can be used as is, or lyophilized, and the lyophilized preparation is combined with a sterile solution prior to administration. The compositions can contain pharmaceutically acceptable adjuvants as needed to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity regulators, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0386] Some methods include selecting a subject (or patient population) having a tumor (or cancer) and treating the subject with the engineered cells.

[0387] In some cases, the engineered cells of the present invention can be used to treat cancer, such as ovarian cancer. Other cancers are described herein. For example, the engineered cells can be used to treat bladder tumors, brain tumors, breast tumors, cervical tumors, colorectal tumors, esophageal tumors, gliomas, kidney tumors, liver tumors, lung tumors, melanomas, ovarian tumors, pancreatic tumors, prostate tumors, skin tumors, thyroid tumors, and / or uterine tumors.

[0388] The methods provided herein also include delivering a preparation of engineered cells, such as the engineered cells. In some embodiments, the preparation is a substantially pure preparation that contains, for example, less than 5% (such as less than 4%, 3%, 2%, or 1%) of cells other than the cells. The preparation can contain from 1×10 5 cells / kg to 1×10 7 cells / kg of, for example, the engineered cells.

[0389] The methods provided herein also include in vivo delivery of a composition capable of generating the engineered cells described herein, such as in vivo delivery of lentivirus. Other in vivo delivery mechanisms and systems can also be used, including those known for use in human therapies, such as viral delivery systems (e.g., retroviral or adenoviral systems), transposons (e.g., Sleeping Beauty and PiggyBac transposon systems), integration into genomic pseudo-sites using PhiC31, or using nucleases, such as zinc fingers (ZF), clustered regularly interspaced short palindromic repeats (CRISPR), or transcription activator-like effector nucleases (TALEN).

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419] Additional embodiments

[0420] The following provides numbered paragraphs that describe particular embodiments:

[0421] 1. An engineered cell, the engineered cell comprising:

[0422] a) a promoter; and

[0423] b) An exogenous polynucleotide sequence comprising an expression cassette as described by the following formula, oriented 5' to 3', said formula comprising:

[0424] S1-E1-L-S2-E2

[0425] wherein

[0426] S1 comprises a polynucleotide sequence encoding a first signal peptide,

[0427] E1 comprises a polynucleotide sequence encoding a first effector molecule,

[0428] L comprises a linker polynucleotide sequence,

[0429] S2 comprises a polynucleotide sequence encoding a second signal peptide,

[0430] E2 comprises a polynucleotide sequence encoding a second effector molecule, and

[0431] wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and

[0432] wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSC), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TIL), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTL), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0433] 2. The engineered cells of paragraph 1, wherein the promoter comprises an exogenous promoter polynucleotide sequence.

[0434] 3. The engineered cells of paragraph 1, wherein the promoter comprises an endogenous promoter.

[0435] 4. The engineered cells of any one of paragraphs 1-3, wherein the promoter is operably linked to the expression cassette such that the polynucleotide can be transcribed into a single polynucleotide comprising the formula S1-E1-L-S2-E2.

[0436] 5. The engineered cells of paragraph 4, wherein the linker polynucleotide sequence is operably associated with the first effector molecule and the second effector molecule being translated into different polypeptides.

[0437] 6. The engineered cells of paragraph 5, wherein the linker polynucleotide sequence encodes a 2A ribosomal skip tag.

[0438] 7. The engineered cell of paragraph 6, wherein the 2A ribosome skipping tag is selected from the group consisting of: P2A, T2A, E2A, and F2A.

[0439] 8. The engineered cell of paragraph 5, wherein the linker polynucleotide sequence encodes a T2A ribosome skipping tag.

[0440] 9. The engineered cell of paragraph 5, wherein the linker polynucleotide sequence encodes an internal ribosome entry site (IRES).

[0441] 10. The engineered cell of any one of paragraphs 5-9, wherein the linker polynucleotide sequence encodes a cleavable polypeptide.

[0442] 11. The engineered cell of paragraph 10, wherein the cleavable polypeptide comprises a furin recognition polypeptide sequence.

[0443] 12. The engineered cell of any one of paragraphs 5-9, wherein the linker polynucleotide sequence further encodes a Gly-Ser-Gly polypeptide sequence.

[0444] 13. The engineered cell of any one of paragraphs 1-5, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, in the orientation of furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus.

[0445] 14. The engineered cell of any one of paragraphs 1-3, wherein the linker polynucleotide sequence encodes a second promoter,

[0446] wherein the promoter is operably linked to the expression cassette such that a first polynucleotide comprising the formula S1-E1 can be transcribed,

[0447] wherein the second promoter is operably linked to the expression cassette such that a second polynucleotide comprising the formula S2-E2 can be transcribed, and wherein the first polynucleotide and the second polynucleotide are different polynucleotides.

[0448] 15. The engineered cell of paragraph 14, wherein the promoter and the second promoter are the same.

[0449] 16. The engineered cell of paragraph 14, wherein the promoter and the second promoter are different.

[0450] 17. The engineered cell of any one of paragraphs 1-16, wherein the engineered cell is HLA-typed with respect to a subject in need of therapeutic treatment.

[0451] 18. Engineered cells of any one of paragraphs 1-17, wherein the engineered cells are human cells.

[0452] 19. Engineered cells of paragraph 18, wherein the human cells are cells isolated from a subject.

[0453] 20. Engineered cells of paragraph 19, wherein the isolated cells are isolated from tissues of a group consisting of: bone marrow, adipose tissue, umbilical cord, fetal liver, muscle, and lung tissue.

[0454] 21. Engineered cells of any one of paragraphs 1-20, wherein the engineered cells are cultured cells.

[0455] 22. Engineered cells of any one of paragraphs 1-21, wherein the engineered MSCs comprise a cell marker phenotype including cell markers CD105+, CD73+, and CD90+.

[0456] 23. Engineered cells of paragraph 22, wherein the cell marker phenotype further comprises a phenotype lacking or substantially lacking one or more cell markers selected from the group consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, class II HLA, and combinations thereof.

[0457] 24. Engineered cells of any one of paragraphs 1-21, wherein the engineered MSCs comprise: a cell marker phenotype comprising CD105+, CD73+, CD90+, CD45-, CD34-, CD14-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD11b-, CD79α-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD19-, class II HLA-; or a cell marker phenotype comprising CD73+, CD90+, CD105+ and CD166+, CD11b-, CD14-, CD19-, CD34-, CD45-, and HLA-DR-.

[0458] 25. Engineered cells of any one of paragraphs 22-24, wherein the cell marker phenotype is determined by or has been determined by flow cytometry.

[0459] 26. Engineered cells of any one of paragraphs 1-21, wherein the engineered cells comprise T cells.

[0460] 27. Engineered cells of any one of paragraphs 1-21, wherein the engineered cells comprise NK cells.

[0461] 28. Engineered cells of any one of paragraphs 1-21, wherein the engineered cells comprise NKT cells.

[0462] 29. Engineered cells of any one of paragraphs 22-28, wherein the cell marker phenotype further comprises cell markers including the first effector molecule, the second effector molecule, or the cognate receptor or cognate receptor ligand of the first effector molecule and the second effector molecule expressed in the engineered cells.

[0463] 30. The engineered cells of paragraph 29, wherein the receptor is selected from the group consisting of IL12RB1, IL12RB2, CCL7, and combinations thereof.

[0464] 31. Engineered cells of any one of paragraphs 1-30, wherein the promoter and / or the second promoter comprise a constitutive promoter.

[0465] 32. The engineered cells of paragraph 31, wherein the constitutive promoter is selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0466] 33. Engineered cells of any one of paragraphs 1-30, wherein the promoter comprises the SFFV promoter.

[0467] 34. Engineered cells of any one of paragraphs 1-30, wherein the promoter and / or the second promoter comprise an inducible promoter.

[0468] 35. The engineered cells of paragraph 34, wherein the inducible promoter is selected from the group consisting of 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, inducible molecule-responsive promoter, and tandem repeats thereof.

[0469] 36. Engineered cells of any one of paragraphs 1-35, wherein the first signal peptide or the second signal peptide respectively comprises a native signal peptide that is native to the first effector molecule or the second effector molecule.

[0470] 37. Engineered cells of any one of paragraphs 1-36, wherein the first signal peptide or the second signal peptide each comprises a non-native signal peptide that is non-native relative to the first effector molecule or the second effector molecule, respectively.

[0471] 38. The engineered cells of paragraph 37, wherein the non-native signal peptide is selected from the group consisting of: IL12, IL2, optimized IL2, trypsinogen-2, Gauss luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, preproserum albumin, preaplysillin, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serine protease inhibitor E1, GROα, CXCL12, and IL21.

[0472] 39. Engineered cells of any one of paragraphs 1-38, wherein the first signal peptide and the second signal peptide are the same.

[0473] 40. Engineered cells of any one of paragraphs 1-39, wherein the polynucleotide sequence encoding the first signal peptide comprises a codon-optimized polynucleotide sequence.

[0474] 41. Engineered cells of any one of paragraphs 1-40, wherein the first secreted polypeptide is the human IL12 signal peptide.

[0475] 42. Engineered cells of any one of paragraphs 1-40, wherein the polynucleotide sequence encoding the second signal peptide comprises a codon-optimized polynucleotide sequence.

[0476] 43. Engineered cells of any one of paragraphs 1-42, wherein the second secreted polypeptide is the human IL21 signal peptide.

[0477] 44. Engineered cells of any one of paragraphs 1-42, wherein the first effector molecule is selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of: cytokines, chemokines, growth factors, costimulatory molecules, tumor microenvironment regulators, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0478] 45. Engineered cells of any one of paragraphs 1-44, wherein the second effector molecule is selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of: cytokines, chemokines, growth factors, costimulatory molecules, tumor microenvironment regulators, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0479] 46. The engineered cells of paragraph 45, wherein the therapeutic classes of the first effector molecule and the second effector molecule are different.

[0480] 47. Engineered cells of any one of paragraphs 1-46, wherein the first effector molecule and / or the second effector molecule is a modified effector molecule.

[0481] 48. Engineered cells of paragraph 47, wherein the first effector molecule and / or the second effector molecule is modified to comprise a cell membrane tethering domain.

[0482] 49. Engineered cells of paragraph 48, wherein the cell membrane tethering domain comprises a transmembrane-intracellular domain or a transmembrane domain.

[0483] 50. Engineered cells of paragraph 48, wherein the cell membrane tethering domain comprises a cell surface receptor or a cell membrane-binding portion thereof.

[0484] 51. Engineered cells of paragraph 50, wherein the modified effector molecule is a fusion protein comprising a cell surface receptor or a cell membrane-binding portion thereof.

[0485] 52. Engineered cells of any one of paragraphs 48-51, wherein the modified effector molecule further comprises a linker between the effector molecule and the cell membrane tethering domain.

[0486] 53. Engineered cells of any one of paragraphs 47-52, wherein when expressed, the modified effector molecule is tethered to the cell membrane of the engineered cell.

[0487] 54. Engineered cells of any one of paragraphs 44-53, wherein the cytokine is selected from the group consisting of: IL12, IL7, IL21, IL18, IL15, type I interferon, and interferon-γ.

[0488] 55. Engineered cells of paragraph 54, wherein the IL12 cytokine is an IL12p70 fusion protein.

[0489] 56. Engineered cells of any one of paragraphs 44-55, wherein the chemokine is selected from the group consisting of: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-11 fusion protein, CCL19, CXCL9, and XCL1.

[0490] 57. Engineered cells of any one of paragraphs 44-56, wherein the growth factor is selected from the group consisting of: Flt3L and GM-CSF.

[0491] 58. Engineered cells of any one of paragraphs 44-57, wherein the co-stimulatory molecule is selected from the group consisting of: 4-1BBL and CD40L.

[0492] Engineered cells of any one of paragraphs 34-41, wherein the tumor microenvironment regulator is selected from the group consisting of: adenosine deaminase, TGFβ inhibitor, immune checkpoint inhibitor, VEGF inhibitor, and HPGE2.

[0493] 60. The engineered cells of paragraph 59, wherein the TGFβ inhibitor is selected from the group consisting of: anti-TGFβ peptides, anti-TGFβ antibodies, TGFb-TRAP, and combinations thereof.

[0494] 61. The engineered cells of paragraph 59, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0495] 62. The engineered cells of paragraph 59, wherein the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof.

[0496] 63. The engineered cells of any one of paragraphs 1-59, wherein the first effector molecule and the second effector molecule are human-derived effector molecules.

[0497] 64. The engineered cells of any one of paragraphs 1-63, wherein the first effector molecule comprises IL12.

[0498] 65. The engineered cells of any one of paragraphs 1-63, wherein the first effector molecule comprises an IL12p70 fusion protein.

[0499] 66. The engineered cells of paragraph 65, wherein the IL12p70 fusion protein is a human IL12p70 fusion protein.

[0500] 67. The engineered cells of any one of paragraphs 64-66, wherein the second effector molecule comprises CCL21a.

[0501] 68. The engineered cells of paragraph 67, wherein the CCL21a is human CCL21a.

[0502] 69. The engineered cells of any one of paragraphs 64-66, wherein the second effector molecule comprises IL7.

[0503] 70. The engineered cells of paragraph 69, wherein the IL7 is human IL7.

[0504] 71. The engineered cells of any one of paragraphs 64-66, wherein the second effector molecule comprises IL21.

[0505] 72. The engineered cells of paragraph 71, wherein the IL21 is human IL21.

[0506] 73. Engineered cells of any one of paragraphs 1-72, wherein the expression cassette further comprises E3 comprising a polynucleotide sequence encoding a third effector molecule.

[0507] 74. Engineered cells of paragraph 73, wherein the third effector molecule comprises Flt3L.

[0508] 75. Engineered cells of paragraph 73, wherein the third effector molecule comprises anti-PD1.

[0509] 76. Engineered cells of paragraph 75, wherein the expression cassette further comprises E4 comprising a polynucleotide sequence encoding a fourth effector molecule.

[0510] 77. Engineered cells of paragraph 76, wherein the fourth effector molecule comprises adenosine deaminase.

[0511] 78. Engineered cells of paragraph 73, wherein the third effector molecule comprises adenosine deaminase.

[0512] 79. Engineered cells of paragraph 73, wherein the third effector molecule comprises CD40L.

[0513] 80. Engineered cells of paragraph 73, wherein the third effector molecule comprises a CXCL10-CXCL11 fusion protein.

[0514] 81. Engineered cells of paragraph 73, wherein the third effector molecule comprises XCL1.

[0515] 82. Engineered cells of paragraph 64, wherein the second effector molecule comprises Flt3L.

[0516] 83. Engineered cells of paragraph 64, wherein the second effector molecule comprises a CXCL10-CXCL11 fusion protein.

[0517] 84. Engineered cells of paragraph 64, wherein the second effector molecule comprises anti-PD1.

[0518] 85. Engineered cells of paragraph 64, wherein the second effector molecule comprises CD40L.

[0519] 86. Engineered cells of any one of paragraphs 1-63, wherein the first effector molecule comprises interferon-β and the second effector molecule comprises Flt3L.

[0520] 87. Engineered cells of any one of paragraphs 1-86, wherein the polynucleotide sequence encoding the first effector molecule comprises a codon-optimized polynucleotide sequence.

[0521] Engineered cells of any one of paragraphs 1-87, wherein the polynucleotide sequence encoding the second effector molecule comprises a codon-optimized polynucleotide sequence.

[0522] 89. Engineered cells of any one of paragraphs 1-88, wherein the engineered cells comprise a polynucleotide sequence encoding the promoter and the expression cassette.

[0523] 90. The engineered cells of paragraph 89, wherein the exogenous polynucleotide sequence comprises the sequence shown in SEQ ID NO: 144.

[0524] 91. Engineered cells of any one of paragraphs 1-90, wherein the exogenous polynucleotide sequence is integrated into the genome of the engineered cells.

[0525] 92. Engineered cells of any one of paragraphs 1-91, wherein the exogenous polynucleotide sequence comprises one or more viral vector polynucleotide sequences.

[0526] 93. The engineered cells of paragraph 92, wherein the one or more viral vector polynucleotide sequences comprise lentiviral, retroviral, retrotransposon or adenoviral polynucleotide sequences.

[0527] 94. Engineered cells of any one of paragraphs 1-93, wherein the expression cassette further comprises an additional exogenous polynucleotide sequence after E2, the exogenous polynucleotide sequence comprising the following formula, oriented 5' to 3', the formula comprising:

[0528] (L-S-E) X

[0529] where

[0530] S comprises a polynucleotide sequence encoding a signal peptide,

[0531] E comprises a polynucleotide sequence encoding an effector molecule,

[0532] L comprises a linker polynucleotide sequence,

[0533] X = 1 to 20

[0534] wherein the promoter is operably linked to the expression cassette, and wherein for each X, the corresponding signal peptide is operably associated with the effector molecule.

[0535] 95. An engineered cell comprising a construct, wherein the construct comprises:

[0536] a) SFFV promoter; and

[0537] b) An exogenous polynucleotide sequence comprising an expression cassette as described by the following formula, oriented 5' to 3', said formula comprising:

[0538] S1-E1-L-S2-E2

[0539] wherein

[0540] S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein said first signal peptide is the human IL12 signal peptide;

[0541] E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein said first effector molecule is the human IL12p70 fusion protein;

[0542] L comprises a linker polynucleotide sequence, wherein said linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus;

[0543] S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein said second signal peptide is the human IL21 signal peptide;

[0544] E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein said second effector molecule is human IL21; and

[0545] wherein the SFFV promoter is operably linked to said expression cassette, said first signal peptide is operably linked to said first effector molecule, and said second signal peptide is operably linked to said second effector molecule, and

[0546] wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSC), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor-infiltrating lymphocytes (TIL), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTL), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0547] 96. The engineered cell of paragraph 95, wherein the construct comprises the polynucleotide sequence shown in SEQ ID NO: 144.

[0548] 97. The engineered cell of paragraph 95 or paragraph 96, wherein the engineered cell is HLA-typed with respect to a subject in need of therapeutic treatment.

[0549] Engineered cells of any one of paragraphs 95-97, wherein the engineered cells are human cells.

[0550] 99. The engineered cells of paragraph 98, wherein the human cells are cells isolated from a subject.

[0551] 100. The engineered cells of paragraph 99, wherein the isolated cells are isolated from tissues of a group consisting of: bone marrow, adipose tissue, umbilical cord, fetal liver, muscle, and lung tissue.

[0552] 101. The engineered cells of any one of paragraphs 95-100, wherein the engineered cells are cultured cells.

[0553] 102. The engineered cells of any one of paragraphs 95-101, wherein the engineered MSCs comprise a cell marker phenotype including cell markers CD105+, CD73+, and CD90+.

[0554] 103. The engineered cells of paragraph 102, wherein the cell marker phenotype further comprises a phenotype lacking or substantially lacking one or more cell markers selected from the group consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, class II HLA, and combinations thereof.

[0555] 104. The engineered cells of any one of paragraphs 95-101, wherein the engineered MSCs comprise: a cell marker phenotype comprising CD105+, CD73+, CD90+, CD45-, CD34- CD14-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD11b-, CD79α-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD19-, class II HLA-; or a cell marker phenotype comprising CD73+, CD90+, CD105+ and CD166+, CD11b-, CD14-, CD19-, CD34-, CD45- and HLA-DR-.

[0556] 105. The engineered cells of any one of paragraphs 95-101, wherein the engineered cells comprise T cells.

[0557] 106. The engineered cells of paragraph 105, wherein the T cells are CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, γ-δ T cells, or regulatory T cells.

[0558] 107. The engineered cells of any one of paragraphs 95-101, wherein the engineered cells comprise NK cells.

[0559] 108. Engineered cells of any one of paragraphs 95-101, wherein the engineered cells comprise NKT cells.

[0560] 109. Engineered cells of any one of paragraphs 95-101, wherein the engineered cells comprise monocytes.

[0561] 110. Engineered cells of any one of paragraphs 95-101, wherein the engineered cells comprise macrophages.

[0562] 111. Engineered cells of any one of paragraphs 95-101, wherein the engineered cells comprise TIL.

[0563] 112. Engineered cells of any one of paragraphs 95-111, wherein the exogenous polynucleotide sequence is integrated into the genome of the engineered cells.

[0564] 113. Engineered cells of any one of paragraphs 95-112, wherein the exogenous polynucleotide sequence comprises one or more viral vector polynucleotide sequences.

[0565] 114. The engineered cells of paragraph 113, wherein the one or more viral vector polynucleotide sequences comprise lentivirus, retrovirus, retrotransposon or adenovirus polynucleotide sequences.

[0566] 115. The engineered cells of paragraph 113, wherein the one or more viral vector polynucleotide sequences comprise lentivirus polynucleotide sequences.

[0567] 116. Engineered cells of any one of paragraphs 1-115, wherein the cells secrete each effector molecule.

[0568] 117. The engineered cells of paragraph 116, wherein the first effector molecule is secreted at a rate 10 times higher than the secretion of the second effector molecule.

[0569] 118. Engineered cells of any one of paragraphs 1-117, wherein the cells further comprise antigen recognition receptors.

[0570] 119. The engineered cells of paragraph 118, wherein the antigen recognition receptor recognizes an antigen selected from the group consisting of: 5T4, ADAM9, ADGRE2, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, cadherin 3, cadherin 6, CCR1, CCR4, CD117, CD123, CD131, CD133, CD138, CD142, CD166, CD25, CD244, CD30, CD300LF, CD33, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CD93, CEA, CEACAM5, claudin 18.2, CLEC12A, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, EMB, ENPP3, EpCAM, EphA2, Ephrin A4, ETBR, FGFR2, FGFR3, FRα, FRb, FLT3, GAPT, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-1RAP, IL-13R, IL-13Ra, IL-13Ra2, IL-8, IL-15, IL1RAP, integrin aV, KIT, L1CAM, LAMP1, LAT2, Lewis Y, LeY, LILRA2, LILRB2, LIV-1, LRRC, LY6E, MCSP, mesothelin, MLC1, MS4A3, MUC1, MUC16, MUC1C, MYADM, NaPi2B, nectin 4, NKG2D, NOTCH3, NY ESO 1, ovaryin, P-cadherin, pan-Erb2, PIEZO1, PRAM1, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLC22A16, SLC17A9, SLITRK6, SPNS3, SSTR2, STEAP1, survivin, TDGF1, TIM1, TROP2, VSTM1, and WT1.

[0571] 120. The engineered cells of paragraph 118 or paragraph 119, wherein the antigen recognition receptor comprises an antigen-binding domain.

[0572] 121. The engineered cells of paragraph 120, wherein the antigen-binding domain comprises 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).

[0573] 122. The engineered cell of paragraph 120, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv).

[0574] 123. The engineered cell of paragraph 122, wherein the scFv comprises a heavy-chain variable domain (VH) and a light-chain variable domain (VL).

[0575] 124. The engineered cell of paragraph 123, wherein the VH and VL are separated by a peptide linker.

[0576] 125. The engineered cell of paragraph 124, wherein the scFv comprises a structure VH-L-VL or VL-L-VH, where VH is the heavy-chain variable domain, L is the peptide linker, and VL is the light-chain variable domain.

[0577] 126. The engineered cell of any one of paragraphs 118-125, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0578] 127. The engineered cell of any one of paragraphs 118-125, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR).

[0579] 128. The engineered cell of paragraph 127, wherein the CAR comprises one or more intracellular signaling domains, and the one or more intracellular signaling domains are selected from the group consisting of: intracellular signaling domain of CD3ζ chain, intracellular signaling domain of CD97, intracellular signaling domain of CD11a-CD18, intracellular signaling domain of CD2, intracellular signaling domain of ICOS, intracellular signaling domain of CD27, intracellular signaling domain of CD154, intracellular signaling domain of CD8, intracellular signaling domain of OX40, intracellular signaling domain of 4-1BB, intracellular signaling domain of CD28, intracellular signaling domain of ZAP40, intracellular signaling domain of CD30, intracellular signaling domain of GITR, intracellular signaling domain of HVEM, intracellular signaling domain of DAP10, intracellular signaling domain of DAP12, and intracellular signaling domain of MyD88.

[0580] 129. The engineered cell of paragraph 127 or paragraph 128, wherein the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the group consisting of: transmembrane domain of CD8, transmembrane domain of CD28, transmembrane domain of CD3ζ chain, transmembrane domain of CD4, transmembrane domain of 4-1BB, transmembrane domain of OX40, transmembrane domain of ICOS, transmembrane domain of CTLA-4, transmembrane domain of PD-1, transmembrane domain of LAG-3, transmembrane domain of 2B4, and transmembrane domain of BTLA.

[0581] Engineered cells of any of paragraphs 127-129, wherein the CAR comprises a spacer between the antigen-binding domain and the transmembrane domain.

[0582] 131. A cell population comprising any of the engineered cells of paragraphs 1-130.

[0583] 132. The cell population of paragraph 131, wherein the cell population is enriched for the engineered cells.

[0584] 133. The cell population of paragraph 131 or paragraph 132, wherein the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule expressed in the engineered cells promotes growth, viability, or growth and viability relative to cells in the population that do not express the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule.

[0585] 134. The cell population of paragraph 133, wherein the first effector molecule is IL12 or an IL12p70 fusion protein.

[0586] 135. The cell population of paragraph 134, wherein the cell population enriched for the engineered cells expresses IL12 receptor β1 or increases its level, expresses IL12 receptor β2 or increases its level, or expresses IL12 receptor β1 and IL12 receptor β2 or increases its level.

[0587] 136. The cell population of any of paragraphs 133-135, wherein the second effector molecule is IL21.

[0588] 137. The cell population of any of paragraphs 133-135, wherein the second effector molecule is CCL21.

[0589] 138. The cell population of paragraph 137, wherein the cell population enriched for the engineered cells expresses the CCL21 receptor or increases its level.

[0590] 139. The cell population of paragraph 138, wherein the CCL21 receptor is CCR7.

[0591] 140. A method of stimulating a cell-mediated immune response against tumor cells in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the engineered cells of paragraphs 1-114 or a cell population of any of paragraphs 131-139.

[0592] 141. A method for providing anti-tumor immunity to a subject, the method comprising administering to a subject in need thereof a therapeutically effective dose of any engineered cell of any one of paragraphs 1-114 or a cell population of any one of paragraphs 131-139.

[0593] 142. A method for treating a subject suffering from cancer, the method comprising administering to a subject having a tumor a therapeutically effective dose of any engineered cell of any one of paragraphs 1-114 or a cell population of any one of paragraphs 131-139.

[0594] 143. A method for reducing the tumor volume in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any engineered cell of any one of paragraphs 1-114 or a cell population of any one of paragraphs 131-139.

[0595] 144. The method of any one of paragraphs 140-143, wherein the engineered cell is derived from the subject.

[0596] 145. The method of any one of paragraphs 140-143, wherein the engineered cell is allogeneic to the subject.

[0597] 146. The method of any one of paragraphs 140-145, wherein the tumor is selected from the group consisting of: adenocarcinoma, acute myeloid leukemia (AML), acute lymphoblastic B-cell leukemia (BALL), acute lymphoblastic T-cell leukemia (TALL), B-cell prolymphocytic leukemia, bladder tumor, brain tumor, breast tumor, cervical tumor, chronic lymphocytic leukemia, chronic myeloid leukemia (CML), colorectal tumor, esophageal tumor, glioma, kidney tumor, liver tumor, lung tumor, lymphoma, melanoma, mesothelioma, myelodysplasia, ovarian tumor, pancreatic tumor, plasma cell myeloma, prostate tumor, skin tumor, thyroid tumor, and uterine tumor.

[0598] 147. The method of any one of paragraphs 140-145, wherein the tumor is an ovarian tumor.

[0599] 148. The method of any one of paragraphs 140-147, wherein the tumor is a tumor located in the peritoneal cavity.

[0600] 149. An engineered cell, the engineered cell comprising:

[0601] a) a promoter; and

[0602] b) an exogenous polynucleotide sequence comprising an expression cassette of the following formula, oriented 5' to 3', the formula comprising:

[0603] (L-S-E) X

[0604] wherein

[0605] S comprises a polynucleotide sequence encoding a signal peptide,

[0606] E comprises a polynucleotide sequence encoding an effector molecule,

[0607] L comprises a linker polynucleotide sequence,

[0608] X = 2 to 20,

[0609] wherein the promoter is operably linked to the expression cassette, wherein for the first iteration of (L-S-E), unit L is absent, and wherein for each X, the corresponding signal peptide is operably associated with the effector molecule, and

[0610] wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0611] 150. A cell population comprising one or more engineered cells, wherein the one or more engineered cells comprise:

[0612] a) a promoter; and

[0613] b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising:

[0614] S1-E1-L-S2-E2

[0615] wherein

[0616] S1 comprises a polynucleotide sequence encoding a first signal peptide,

[0617] E1 comprises a polynucleotide sequence encoding a first effector molecule,

[0618] L comprises a linker polynucleotide sequence,

[0619] S2 comprises a polynucleotide sequence encoding a second signal peptide,

[0620] E2 comprises a polynucleotide sequence encoding a second effector molecule, and

[0621] wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and

[0622] wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0623] 151. A cell population comprising one or more engineered cells, wherein the one or more engineered cells comprise:

[0624] a) a promoter; and

[0625] b) an exogenous polynucleotide sequence comprising an expression cassette oriented 5' to 3', the expression cassette comprising:

[0626] S1-E1-L-S2-E2

[0627] wherein

[0628] S1 comprises a polynucleotide sequence encoding a first signal peptide,

[0629] E1 comprises a polynucleotide sequence encoding a first effector molecule,

[0630] L comprises a linker polynucleotide sequence,

[0631] S2 comprises a polynucleotide sequence encoding a second signal peptide,

[0632] E2 comprises a polynucleotide sequence encoding a second effector molecule, and

[0633] wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and

[0634] wherein the first effector molecule, the second effector molecule, or the first and second effector molecules expressed in the engineered cell promotes growth, viability, or growth and viability, such that the growth, viability, or growth and viability is increased relative to cells in the population that do not express the first effector molecule, the second effector molecule, or the first and second effector molecules, and

[0635] wherein the engineered cell is selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, gamma-delta T cells, regulatory T cells, and B cells.

[0636] 152. The cell population of paragraph 151, wherein the one or more engineered cells express a cognate receptor or cognate receptor ligand of the first effector molecule, the second effector molecule, or the first and second effector molecules expressed in the engineered cell.

[0637] 153. The cell population of paragraph 151 or paragraph 152, wherein the first effector molecule is IL12 or an IL12p70 fusion protein.

[0638] 154. The cell population of any one of paragraphs 151-153, wherein the second effector molecule is IL21.

[0639] 155. The cell population of any one of paragraphs 151-153, wherein the second effector molecule is CCL21.

[0640] 156. A cell population comprising one or more engineered cells, wherein the one or more engineered cells comprise a construct, and wherein the construct comprises:

[0641] a) an SFFV promoter; and

[0642] b) an exogenous polynucleotide sequence comprising an expression cassette oriented 5' to 3', the expression cassette comprising:

[0643] S1-E1-L-S2-E2

[0644] wherein

[0645] S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is the human IL12 signal peptide;

[0646] E1 contains a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein;

[0647] L contains a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosomal skip tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus;

[0648] S2 contains a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is a human IL21 signal peptide;

[0649] E2 contains a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and

[0650] wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and

[0651] wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSC), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor-infiltrating lymphocytes (TIL), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTL), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0652] 157. A cell population comprising one or more engineered cells, wherein the one or more engineered cells comprise a construct, and wherein the construct comprises:

[0653] a) An SFFV promoter; and

[0654] b) An exogenous polynucleotide sequence comprising an expression cassette of the following formula, oriented 5' to 3', the formula comprising:

[0655] S1-E1-L-S2-E2

[0656] wherein

[0657] S1 contains a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is a human IL12 signal peptide;

[0658] E1 contains a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein;

[0659] L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, in the order of furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus;

[0660] S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is the human IL21 signal peptide;

[0661] E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and

[0662] wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and

[0663] wherein the first effector molecule, the second effector molecule, or both the first effector molecule and the second effector molecule expressed in the engineered cell promote growth, viability, or both growth and viability as compared to cells in the population that do not express the first effector molecule, the second effector molecule, or both the first effector molecule and the second effector molecule, and

[0664] wherein the engineered cell is selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0665] 158. A cell population of paragraph 156 or paragraph 157, wherein the construct comprises the polynucleotide sequence shown in SEQ ID NO:144.

[0666] 159. A method of generating a cell population enriched for one or more receptors or receptor ligands, the method comprising culturing one or more cells under conditions that bring the one or more cells into contact with a first effector molecule, a second effector molecule, or a first effector molecule and a second effector molecule, wherein the contacting cells express one or more cognate receptors or cognate receptor ligands for the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule, and wherein the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule promote the growth, viability, or growth and viability of the contacting cells relative to cells cultured in the absence of the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule.

[0667] 160. The method of paragraph 159, wherein the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule are heterologously expressed in one or more cells, and the one or more cells contact the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule in an autocrine manner.

[0668] 161. The method of paragraph 159, wherein the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule are expressed in one or more additional cells, and the one or more cells contact the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule in a paracrine manner.

[0669] 162. The method of paragraph 161, wherein the one or more additional cells are feeder cells.

[0670] 163. The method of paragraph 159, wherein the one or more cells are cultured in a medium.

[0671] 164. The method of paragraph 163, wherein the one or more cells are brought into contact with the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule by adding a soluble first effector molecule, a soluble second effector molecule, or a soluble first effector molecule and second effector molecule to the medium.

[0672] 165. The method of paragraph 163 or paragraph 164, wherein the soluble first effector molecule and / or the soluble second effector molecule is a recombinant effector molecule.

[0673] 166. The method of paragraph 159, wherein the one or more cells are cultured under adherent conditions.

[0674] 167. The method of paragraph 166, wherein the one or more cells are attached to a surface.

[0675] 168. The method of paragraph 167, wherein the adherent cells are contacted with the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule by exposing the one or more cells to the first effector molecule, the second effector molecule, or the first effector molecule and the second effector molecule, and are immobilized on a surface.

[0676] 169. The method of any one of paragraphs 159-168, wherein the first effector molecule is an IL12 or IL12p70 fusion protein.

[0677] 170. The method of paragraph 169, wherein the cell population is enriched in IL12 receptor β1 (IL12Rβ1), enriched in IL12 receptor β2 (IL12Rβ2), or enriched in IL12Rβ1 and IL12Rβ2.

[0678] 171. The method of paragraph 170, wherein the MSC population comprises a cell marker phenotype including the cell markers CD105+, CD73+, CD90+, IL12Rβ1+, and IL12Rβ2+.

[0679] 172. The method of paragraph 171, wherein the cell marker phenotype further comprises a phenotype lacking or substantially lacking one or more cell markers selected from the group consisting of CD45, CD34, CD14, CD11b, CD79α, CD19, class II HLA, and combinations thereof.

[0680] 173. The method of paragraph 159, wherein the cell population comprises cells selected from the group consisting of natural killer (NK) cells, NKT cells, innate lymphoid cells, mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, and dendritic cells, T cells, CD8+ T cells, CD4+ T cells, γ-δ T cells, regulatory T cells, and B cells.

[0681] 174. The method of paragraph 173, wherein the cell population comprises T cells, NK cells, NKT cells, monocytes, macrophages, or bone marrow-derived cells.

[0682] 175. The method of any one of paragraphs 159-174, wherein the second effector molecule is IL21.

[0683] 176. The method of any one of paragraphs 159-174, wherein the second effector molecule is CCL21.

[0684] 177. The method of paragraph 176, wherein the cell population is enriched in CCR7.

[0685] 178. The method of paragraph 177, wherein the MSC population comprises a cell marker phenotype comprising the cell markers CD105+, CD73+, CD90+, IL12Rβ1+, IL12Rβ2+ and CCR7+.

[0686] 179. The method of paragraph 178, wherein the cell marker phenotype further comprises a phenotype lacking or substantially lacking one or more cell markers selected from the group consisting of CD45, CD34, CD14, CD11b, CD79α, CD19, class II HLA, and combinations thereof.

[0687] 180. A cell population enriched in one or more receptors or receptor ligands, produced by the method of any one of paragraphs 159-179.

[0688] 181. An exogenous polynucleotide sequence comprising a promoter and an expression cassette of the following formula, oriented 5' to 3', the formula comprising:

[0689] S1-E1-L-S2-E2

[0690] wherein

[0691] S1 comprises a polynucleotide sequence encoding a first signal peptide,

[0692] E1 comprises a polynucleotide sequence encoding a first effector molecule,

[0693] L comprises a linker polynucleotide sequence,

[0694] S2 comprises a polynucleotide sequence encoding a second signal peptide,

[0695] E2 comprises a polynucleotide sequence encoding a second effector molecule, and

[0696] wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule.

[0697] 182. The exogenous polynucleotide sequence of paragraph 181, wherein the promoter comprises an exogenous promoter polynucleotide sequence.

[0698] 183. The exogenous polynucleotide sequence of paragraph 181, wherein the promoter comprises an endogenous promoter.

[0699] 184. The exogenous polynucleotide sequence of any one of paragraphs 181-183, wherein the promoter is operably linked to the expression cassette such that the polynucleotide can be transcribed into a single polynucleotide comprising the formula S1-E1-L-S2-E2.

[0700] The exogenous polynucleotide sequence of paragraph 184, wherein the linker polynucleotide sequence is operably associated with the first effector molecule and the second effector molecule being translated into different polypeptides.

[0701] The exogenous polynucleotide sequence of paragraph 185, wherein the linker polynucleotide sequence encodes a 2A ribosome skipping tag.

[0702] The exogenous polynucleotide sequence of paragraph 186, wherein the 2A ribosome skipping tag is selected from the group consisting of P2A, T2A, E2A, and F2A.

[0703] The exogenous polynucleotide sequence of paragraph 185, wherein the linker polynucleotide sequence encodes a T2A ribosome skipping tag.

[0704] The exogenous polynucleotide sequence of paragraph 185, wherein the linker polynucleotide sequence encodes an internal ribosome entry site (IRES).

[0705] The exogenous polynucleotide sequence of any one of paragraphs 185-189, wherein the linker polynucleotide sequence encodes a cleavable polypeptide.

[0706] The exogenous polynucleotide sequence of paragraph 181, wherein the cleavable polypeptide comprises a furin recognition polypeptide sequence.

[0707] The exogenous polynucleotide sequence of any one of paragraphs 185-189, wherein the linker polynucleotide sequence further encodes a Gly-Ser-Gly polypeptide sequence.

[0708] The exogenous polynucleotide sequence of any one of paragraphs 181-185, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, in the order of furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus.

[0709] The exogenous polynucleotide sequence of any one of paragraphs 181-183, wherein the linker polynucleotide sequence encodes a second promoter,

[0710] wherein the promoter is operably linked to the expression cassette such that the first polynucleotide comprising the formula S1-E1 can be transcribed,

[0711] wherein the second promoter is operably linked to the expression cassette such that the second polynucleotide comprising the formula S2-E2 can be transcribed, and wherein the first polynucleotide and the second polynucleotide are different polynucleotides.

[0712] 195. The exogenous polynucleotide sequence of paragraph 181, wherein the promoter and the second promoter are the same.

[0713] 196. The exogenous polynucleotide sequence of paragraph 181, wherein the promoter and the second promoter are different.

[0714] 197. The exogenous polynucleotide sequence of any one of paragraphs 181-196, wherein the promoter and / or the second promoter comprises a constitutive promoter.

[0715] 198. The exogenous polynucleotide sequence of paragraph 197, wherein the constitutive promoter is selected from the group consisting of: CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0716] 199. The exogenous polynucleotide sequence of any one of paragraphs 181-196, wherein the promoter comprises the SFFV promoter.

[0717] 200. The exogenous polynucleotide sequence of any one of paragraphs 181-196, wherein the promoter and / or the second promoter comprises an inducible promoter.

[0718] 201. The exogenous polynucleotide sequence of paragraph 200, wherein the inducible promoter is selected from the group consisting of: 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, inducible molecule-responsive promoter, and tandem repeats thereof.

[0719] 202. The exogenous polynucleotide sequence of any one of paragraphs 181-201, wherein the first signal peptide or the second signal peptide respectively comprises a native signal peptide that is native to the first effector molecule or the second effector molecule.

[0720] 203. The exogenous polynucleotide sequence of any one of paragraphs 181-202, wherein the first signal peptide or the second signal peptide respectively comprises a non-native signal peptide that is non-native to the first effector molecule or the second effector molecule.

[0721] The exogenous polynucleotide sequence of paragraph 203, wherein the non-natural signal peptide is selected from the group consisting of: IL12, IL2, optimized IL2, trypsinogen-2, Gauss luciferase, CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, preproserum albumin, preaplysillin, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serine protease inhibitor E1, GROα, CXCL12, and IL21.

[0722] The exogenous polynucleotide sequence of any one of paragraphs 181-204, wherein the first signal peptide and the second signal peptide are the same.

[0723] The exogenous polynucleotide sequence of any one of paragraphs 181-205, wherein the polynucleotide sequence encoding the first signal peptide comprises a codon-optimized polynucleotide sequence.

[0724] The exogenous polynucleotide sequence of any one of paragraphs 181-206, wherein the first secreted polypeptide is the human IL12 signal peptide.

[0725] The exogenous polynucleotide sequence of any one of paragraphs 181-206, wherein the polynucleotide sequence encoding the second signal peptide comprises a codon-optimized polynucleotide sequence.

[0726] The exogenous polynucleotide sequence of any one of paragraphs 181-208, wherein the second secreted polypeptide is the human IL21 signal peptide.

[0727] The exogenous polynucleotide sequence of any one of paragraphs 181-208, wherein the first effector molecule is selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of: cytokines, chemokines, growth factors, costimulatory molecules, tumor microenvironment regulators, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0728] The exogenous polynucleotide sequence of any one of paragraphs 181-210, wherein the second effector molecule is selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of: cytokines, chemokines, growth factors, costimulatory molecules, tumor microenvironment regulators, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0729] The exogenous polynucleotide sequence of paragraph 211, wherein the therapeutic classes of the first effector molecule and the second effector molecule are different.

[0730] An exogenous polynucleotide sequence of any one of paragraphs 181-212, wherein the first effector molecule and / or the second effector molecule is a modified effector molecule.

[0731] 214. The exogenous polynucleotide sequence of paragraph 213, wherein the first effector molecule and / or the second effector molecule is modified to comprise a cell membrane tethering domain.

[0732] 215. The exogenous polynucleotide sequence of paragraph 214, wherein the cell membrane tethering domain comprises a transmembrane-intracellular domain or a transmembrane domain.

[0733] 216. The exogenous polynucleotide sequence of paragraph 214, wherein the cell membrane tethering domain comprises a cell surface receptor or a cell membrane-binding portion thereof.

[0734] 217. The exogenous polynucleotide sequence of paragraph 216, wherein the modified effector molecule is a fusion protein comprising a cell surface receptor or a cell membrane-binding portion thereof.

[0735] 218. The exogenous polynucleotide sequence of any one of paragraphs 214-217, wherein the modified effector molecule further comprises a linker between the effector molecule and the cell membrane tethering domain.

[0736] 219. The exogenous polynucleotide sequence of any one of paragraphs 213-218, wherein when expressed in a cell, the modified effector molecule is tethered to the cell membrane of the cell.

[0737] 220. The exogenous polynucleotide sequence of any one of paragraphs 210-219, wherein the cytokine is selected from the group consisting of: IL12, IL7, IL21, IL18, IL15, type I interferon, and interferon-γ.

[0738] 221. The exogenous polynucleotide sequence of paragraph 220, wherein the IL12 cytokine is an IL12p70 fusion protein.

[0739] 222. The exogenous polynucleotide sequence of any one of paragraphs 210-221, wherein the chemokine is selected from the group consisting of: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-11 fusion protein, CCL19, CXCL9, and XCL1.

[0740] 223. The exogenous polynucleotide sequence of any one of paragraphs 210-222, wherein the growth factor is selected from the group consisting of: Flt3L and GM-CSF.

[0741] An exogenous polynucleotide sequence of any one of paragraphs 210-223, wherein the co-activating molecule is selected from the group consisting of: 4-1BBL and CD40L.

[0742] 225. An exogenous polynucleotide sequence of any one of paragraphs 210-224, wherein the tumor microenvironment regulator is selected from the group consisting of: adenosine deaminase, TGFβ inhibitor, immune checkpoint inhibitor, VEGF inhibitor, and HPGE2.

[0743] 226. The exogenous polynucleotide sequence of paragraph 225, wherein the TGFβ inhibitor is selected from the group consisting of: anti-TGFβ peptide, anti-TGFβ antibody, TGFb-TRAP, and combinations thereof.

[0744] 227. The exogenous polynucleotide sequence of paragraph 225, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0745] 228. The exogenous polynucleotide sequence of paragraph 225, wherein the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof.

[0746] 229. An exogenous polynucleotide sequence of any one of paragraphs 181-225, wherein the first effector molecule and the second effector molecule are human-derived effector molecules.

[0747] 230. An exogenous polynucleotide sequence of any one of paragraphs 181-229, wherein the first effector molecule comprises IL12.

[0748] 231. An exogenous polynucleotide sequence of any one of paragraphs 181-229, wherein the first effector molecule comprises an IL12p70 fusion protein.

[0749] 232. The exogenous polynucleotide sequence of paragraph 231, wherein the IL12p70 fusion protein is a human IL12p70 fusion protein.

[0750] 233. An exogenous polynucleotide sequence of any one of paragraphs 230-232, wherein the second effector molecule comprises CCL21a.

[0751] 234. The exogenous polynucleotide sequence of paragraph 233, wherein the CCL21a is human CCL21a.

[0752] 235. An exogenous polynucleotide sequence of any one of paragraphs 230-232, wherein the second effector molecule comprises IL7.

[0753] 236. The exogenous polynucleotide sequence of paragraph 235, wherein the IL7 is human IL7.

[0754] An exogenous polynucleotide sequence of any one of paragraphs 230-232, wherein the second effector molecule comprises IL21.

[0755] The exogenous polynucleotide sequence of paragraph 237, wherein the IL21 is human IL21.

[0756] An exogenous polynucleotide sequence of any one of paragraphs 181-238, wherein the expression cassette further comprises E3 comprising a polynucleotide sequence encoding a third effector molecule.

[0757] The exogenous polynucleotide sequence of paragraph 239, wherein the third effector molecule comprises Flt3L.

[0758] The exogenous polynucleotide sequence of paragraph 239, wherein the third effector molecule comprises anti-PD1.

[0759] The exogenous polynucleotide sequence of paragraph 241, wherein the expression cassette further comprises E4 comprising a polynucleotide sequence encoding a fourth effector molecule.

[0760] The exogenous polynucleotide sequence of paragraph 242, wherein the fourth effector molecule comprises adenosine deaminase.

[0761] The exogenous polynucleotide sequence of paragraph 239, wherein the third effector molecule comprises adenosine deaminase.

[0762] The exogenous polynucleotide sequence of paragraph 239, wherein the third effector molecule comprises CD40L.

[0763] The exogenous polynucleotide sequence of paragraph 239, wherein the third effector molecule comprises a CXCL10-CXCL11 fusion protein.

[0764] The exogenous polynucleotide sequence of paragraph 239, wherein the third effector molecule comprises XCL1.

[0765] The exogenous polynucleotide sequence of paragraph 230, wherein the second effector molecule comprises Flt3L.

[0766] The exogenous polynucleotide sequence of paragraph 230, wherein the second effector molecule comprises a CXCL10-CXCL11 fusion protein.

[0767] The exogenous polynucleotide sequence of paragraph 230, wherein the second effector molecule comprises anti-PD1.

[0768] The exogenous polynucleotide sequence of paragraph 230, wherein the second effector molecule comprises CD40L.

[0769] The exogenous polynucleotide sequence of any one of paragraphs 181-229, wherein the first effector molecule comprises interferon-β and the second effector molecule comprises Flt3L.

[0770] The exogenous polynucleotide sequence of any one of paragraphs 181-252, wherein the polynucleotide sequence encoding the first effector molecule comprises a codon-optimized polynucleotide sequence.

[0771] The exogenous polynucleotide sequence of any one of paragraphs 181-253, wherein the polynucleotide sequence encoding the second effector molecule comprises a codon-optimized polynucleotide sequence.

[0772] The exogenous polynucleotide sequence of any one of paragraphs 181-254, wherein the exogenous polynucleotide sequence comprises the polynucleotide sequence shown in SEQ ID NO:144.

[0773] An exogenous polynucleotide sequence comprising an SFFV promoter and an expression cassette of the following formula, oriented 5' to 3', the formula comprising:

[0774] S1-E1-L-S2-E2

[0775] wherein

[0776] S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is the human IL12 signal peptide;

[0777] E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is the human IL12p70 fusion protein;

[0778] L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus;

[0779] S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is the human IL21 signal peptide;

[0780] E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and

[0781] wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule.

[0782] 257. The exogenous polynucleotide sequence of paragraph 256, wherein the polynucleotide sequence comprises the polynucleotide sequence set forth in SEQ ID NO:144.

[0783] 258. An exogenous polynucleotide sequence comprising an SFFV promoter and an expression cassette of the formula, oriented 5' to 3', the formula comprising:

[0784] S1-E1-L-S2-E2

[0785] wherein

[0786] S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is the human IL12 signal peptide;

[0787] E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is the human IL12p70 fusion protein;

[0788] L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosomal skip tag, oriented furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus;

[0789] S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is the human IL21 signal peptide;

[0790] E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21;

[0791] wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule;

[0792] wherein the promoter is operably linked to the expression cassette such that the polynucleotide can be transcribed into a single polynucleotide comprising the formula S1-E1-L-S2-E2; and

[0793] wherein the polynucleotide sequence comprises the polynucleotide sequence set forth in SEQ ID NO:144.

[0794] An exogenous polynucleotide sequence of any one of paragraphs 181-258, wherein the exogenous polynucleotide sequence is encoded by a nucleic acid selected from the group consisting of DNA, cDNA, RNA, mRNA, and naked plasmid.

[0795] 260. An expression vector comprising the exogenous polynucleotide sequence of any one of paragraphs 181-259.

[0796] 261. The expression vector of paragraph 260, wherein the expression vector is a viral vector.

[0797] 262. The expression vector of paragraph 261, wherein the expression vector is a lentiviral vector.

[0798] 263. A composition comprising the exogenous polynucleotide sequence of any one of paragraphs 181-259 and a pharmaceutically acceptable carrier.

[0799] 264. An isolated cell comprising the exogenous polynucleotide sequence of any one of paragraphs 181-259, the expression vector of any one of paragraphs 260-262, or the composition of paragraph 263.

[0800] 265. The isolated cell of paragraph 264, wherein the isolated cell is selected from the group consisting of T cell, CD8+ T cell, CD4+ T cell, γ-δ T cell, cytotoxic T lymphocyte (CTL), regulatory T cell, virus-specific T cell, natural killer T (NKT) cell, natural killer (NK) cell, B cell, tumor-infiltrating lymphocyte (TIL), innate lymphoid cell, mast cell, eosinophil, basophil, neutrophil, myeloid cell, macrophage, monocyte, dendritic cell, red blood cell, platelet cell, ESC-derived cell, pluripotent stem cell, MSC, induced pluripotent stem cell (iPSC), and iPSC-derived cell.

[0801] 266. The isolated cell of paragraph 264, wherein the isolated cell is MSC.

[0802] 267. The isolated cell of any one of paragraphs 264-266, wherein the exogenous polynucleotide sequence is integrated into the genome of the cell.

[0803] 268. The isolated cell of any one of paragraphs 264-267, wherein the exogenous polynucleotide sequence comprises one or more viral vector polynucleotide sequences.

[0804] The isolated cells of paragraph 268, wherein the one or more viral vector polynucleotide sequences comprise a lentiviral, retroviral, retrotransposon or adenoviral polynucleotide sequence.

[0805] The isolated cells of paragraph 268, wherein the one or more viral vector polynucleotide sequences comprise a lentiviral polynucleotide sequence.

[0806] The isolated cells of any one of paragraphs 264-270, wherein the engineered cells are HLA typed relative to a subject in need of therapeutic treatment.

[0807] The isolated cells of any one of paragraphs 264-271, wherein the engineered cells are human cells.

[0808] The isolated cells of paragraph 272, wherein the human cells are cells isolated from a subject.

[0809] The isolated cells of paragraph 273, wherein the isolated cells are isolated from tissues of the group consisting of: bone marrow, adipose tissue, umbilical cord, fetal liver, muscle and lung tissue.

[0810] The isolated cells of any one of paragraphs 264-272, wherein the cells are cultured cells.

[0811] The isolated cells of any one of paragraphs 264-275, wherein the cells comprise a cell marker phenotype comprising cell markers CD105+, CD73+ and CD90+.

[0812] The isolated cells of paragraph 276, wherein the cell marker phenotype further comprises a phenotype lacking or substantially lacking one or more cell markers selected from the group consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, class II HLA and combinations thereof.

[0813] The isolated cells of any one of paragraphs 264-275, wherein the cells comprise: a cell marker phenotype comprising CD105+, CD73+, CD90+, CD45-, CD34- and CD14-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD11b- and CD79α-; a cell marker phenotype comprising CD105+, CD73+, CD90+, CD19- and class II HLA-; or a cell marker phenotype comprising CD73+, CD90+, CD105+ and CD166+, CD11b-, CD14-, CD19-, CD34-, CD45- and HLA-DR-.

[0814] Isolated cells of any one of paragraphs 264-278, wherein the cell marker phenotype further comprises a cell marker comprising the first effector molecule, the second effector molecule, or a cognate receptor or cognate receptor ligand of the first effector molecule and the second effector molecule expressed in the cell.

[0815] Isolated cells of paragraph 279, wherein the receptor is selected from the group consisting of IL12RB1, IL12RB2, CCL7, and combinations thereof.

[0816] Isolated cells of any one of paragraphs 264-280, wherein the cells secrete each effector molecule.

[0817] Isolated cells of paragraph 281, wherein the first effector molecule is secreted at a rate 10-fold higher relative to the secretion of the second effector molecule.

[0818] Isolated cells of any one of paragraphs 264-282, wherein the cells further comprise an antigen recognition receptor.

[0819] Isolated cells of paragraph 283, wherein the antigen recognition receptor comprises an antigen-binding domain.

[0820] Isolated cells of paragraph 284, wherein the antigen-binding domain comprises 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).

[0821] Isolated cells of paragraph 284, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv).

[0822] Isolated cells of paragraph 286, wherein the scFv comprises a heavy-chain variable domain (VH) and a light-chain variable domain (VL).

[0823] Isolated cells of paragraph 287, wherein the VH and VL are separated by a peptide linker.

[0824] Isolated cells of paragraph 288, wherein the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy-chain variable domain, L is the peptide linker, and VL is the light-chain variable domain.

[0825] Isolated cells of any one of paragraphs 283-289, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0826] Isolated cells of any one of paragraphs 283-289, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR).

[0827] 292. The isolated cells of paragraph 291, wherein the CAR comprises one or more intracellular signaling domains, and the one or more intracellular signaling domains are selected from the group consisting of: CD3ζ chain intracellular signaling domain, CD97 intracellular signaling domain, CD11a-CD18 intracellular signaling domain, CD2 intracellular signaling domain, ICOS intracellular signaling domain, CD27 intracellular signaling domain, CD154 intracellular signaling domain, CD8 intracellular signaling domain, OX40 intracellular signaling domain, 4-1BB intracellular signaling domain, CD28 intracellular signaling domain, ZAP40 intracellular signaling domain, CD30 intracellular signaling domain, GITR intracellular signaling domain, HVEM intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, and MyD88 intracellular signaling domain.

[0828] 293. The isolated cells of paragraph 291 or paragraph 292, wherein the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the group consisting of: CD8 transmembrane domain, CD28 transmembrane domain, CD3ζ chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, 2B4 transmembrane domain, and BTLA transmembrane domain.

[0829] 294. The isolated cells of any one of paragraphs 291-293, wherein the CAR comprises a spacer between the antigen-binding domain and the transmembrane domain.

[0830] 295. A virus comprising an exogenous polynucleotide sequence of any one of paragraphs 181-259 or an expression vector of any one of paragraphs 260-262.

[0831] 296. The virus of paragraph 295, wherein the virus is selected from the group consisting of: lentivirus, retrovirus, retrotransposon, and adenovirus.

[0832] 297. The virus of paragraph 295, wherein the virus is a lentivirus.

[0833] 298. A method of reducing the tumor volume in a subject, the method comprising delivering to a subject having a tumor a composition comprising cells engineered to produce a plurality of effector molecules that modulate tumor-mediated immunosuppressive mechanisms, in an amount effective to reduce the tumor volume, wherein the engineered cells comprise:

[0834] a) a promoter; and

[0835] b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising:

[0836] S1-E1-L-S2-E2

[0837] wherein

[0838] S1 comprises a polynucleotide sequence encoding a first signal peptide,

[0839] E1 comprises a polynucleotide sequence encoding a first effector molecule,

[0840] L comprises a linker polynucleotide sequence,

[0841] S2 comprises a polynucleotide sequence encoding a second signal peptide,

[0842] E2 comprises a polynucleotide sequence encoding a second effector molecule, and

[0843] wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and

[0844] wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSC), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TIL), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTL), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0845] 299. A method of reducing the tumor volume in a subject, the method comprising delivering to a subject having a tumor a composition comprising cells engineered to produce IL12 and IL21, in an amount effective to reduce the tumor volume, wherein the engineered cells comprise a construct, wherein the construct comprises

[0846] a) an SFFV promoter; and

[0847] b) An exogenous polynucleotide sequence, comprising an expression cassette as described by the following formula, oriented 5' to 3', said formula comprising:

[0848] S1-E1-L-S2-E2

[0849] wherein

[0850] S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein said first signal peptide is the human IL12 signal peptide;

[0851] E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein said first effector molecule is the human IL12p70 fusion protein;

[0852] L comprises a linker polynucleotide sequence, wherein said linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosomal skip tag, oriented furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus;

[0853] S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein said second signal peptide is the human IL21 signal peptide;

[0854] E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein said second effector molecule is human IL21; and

[0855] wherein the SFFV promoter is operably linked to said expression cassette, said first signal peptide is operably linked to said first effector molecule, and said second signal peptide is operably linked to said second effector molecule, and

[0856] wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSC), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TIL), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTL), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0857] 300. The method of paragraph 299, wherein the construct comprises the polynucleotide sequence shown in SEQ ID NO: 144.

[0858] 301. The method of any one of paragraphs 298 - 300, wherein the method further comprises administering a checkpoint inhibitor.

[0859] The method of paragraph 301, wherein the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-1L antibody, or an anti-CTLA-4 antibody.

[0860] The method of any one of paragraphs 298-302, wherein the method further comprises administering an anti-CD40 antibody.

[0861] The method of any one of paragraphs 298-303, wherein the tumor is selected from the group consisting of: adenocarcinoma, acute myeloid leukemia (AML), acute lymphoblastic B cell leukemia (BALL), acute lymphoblastic T cell leukemia (TALL), B cell prolymphocytic leukemia, bladder tumor, brain tumor, breast tumor, cervical tumor, chronic lymphocytic leukemia, chronic myeloid leukemia (CML), colorectal tumor, esophageal tumor, glioma, kidney tumor, liver tumor, lung tumor, lymphoma, melanoma, mesothelioma, myelodysplasia, ovarian tumor, pancreatic tumor, plasma cell myeloma, prostate tumor, skin tumor, thyroid tumor, and uterine tumor.

[0862] The method of any one of paragraphs 298-303, wherein the tumor is an ovarian tumor.

[0863] The method of any one of paragraphs 298-303, wherein the tumor is a tumor located in the peritoneal cavity.

[0864] The method of any one of paragraphs 298-306, wherein the administration comprises systemic administration, intraperitoneal administration, or intratumoral administration.

[0865] The method of any one of paragraphs 298-307, wherein the tumor volume is reduced by at least 25% relative to a control, optionally wherein the control is an unmodified cell.

[0866] The method of paragraph 307, wherein the tumor volume is reduced by at least 50% relative to a control, optionally wherein the control is an unmodified cell.

[0867] The method of paragraph 309, wherein the tumor volume is reduced by at least 75% relative to a control, optionally wherein the control is an unmodified cell.

[0868] A method of reducing the tumor volume in a subject, the method comprising delivering to a subject having a tumor a composition capable of engineering cells to produce a plurality of effector molecules that modulate tumor-mediated immunosuppressive mechanisms in an amount effective to reduce the tumor volume, wherein each engineered cell comprises:

[0869] a) a promoter; and

[0870] b) An exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', said formula comprising:

[0871] S1-E1-L-S2-E2

[0872] wherein

[0873] S1 comprises a polynucleotide sequence encoding a first signal peptide,

[0874] E1 comprises a polynucleotide sequence encoding a first effector molecule,

[0875] L comprises a linker polynucleotide sequence,

[0876] S2 comprises a polynucleotide sequence encoding a second signal peptide,

[0877] E2 comprises a polynucleotide sequence encoding a second effector molecule, and

[0878] wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and

[0879] wherein the engineered cells are selected from the group consisting of: mesenchymal stem cells (MSCs), stem cells, immune cells, natural killer (NK) cells, NKT cells, innate lymphoid cells, tumor infiltrating lymphocytes (TILs), mast cells, eosinophils, basophils, monocytes, macrophages, neutrophils, myeloid cells, dendritic cells, T cells, CD8+ T cells, CD4+ T cells, cytotoxic T lymphocytes (CTLs), virus-specific T cells, γ-δ T cells, regulatory T cells, and B cells.

[0880] 312. A method of reducing tumor volume in a subject, the method comprising delivering to a subject having a tumor an amount of a composition effective to reduce tumor volume that is capable of engineering cells to produce IL12 and IL21, wherein the engineered cells comprise a construct, wherein the construct comprises:

[0881] a) SFFV promoter; and

[0882] b) An exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', said formula comprising:

[0883] S1-E1-L-S2-E2

[0884] wherein

[0885] S1 comprises a polynucleotide sequence encoding a first signal peptide, wherein the first signal peptide is a human IL12 signal peptide;

[0886] E1 comprises a polynucleotide sequence encoding a first effector molecule, wherein the first effector molecule is a human IL12p70 fusion protein;

[0887] L comprises a linker polynucleotide sequence, wherein the linker polynucleotide sequence encodes a furin recognition polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome skipping tag, oriented as furin:Gly-Ser-Gly:T2A from the N-terminus to the C-terminus;

[0888] S2 comprises a polynucleotide sequence encoding a second signal peptide, wherein the second signal peptide is a human IL21 signal peptide;

[0889] E2 comprises a polynucleotide sequence encoding a second effector molecule, wherein the second effector molecule is human IL21; and

[0890] wherein the SFFV promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked...

Claims

1. An engineered human cell, said engineered human cell comprising: a) a promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', said formula comprising: S1-E1-L-S2-E2 wherein S1 is a polynucleotide sequence encoding a first signal peptide, E1 is a polynucleotide sequence encoding a first effector molecule, L is a linker polynucleotide sequence, S2 is a polynucleotide sequence encoding a second signal peptide, E2 is a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, wherein the engineered human cell is selected from the group consisting of: mesenchymal stem cells (MSCs), natural killer (NK) cells, NKT cells, and macrophages, wherein (a) the first effector molecule is an IL12p70 fusion protein and the second effector molecule is selected from IL21, an anti-PD1 antibody, or a CXCL10-CXCL11 fusion protein, or (b) the first effector molecule is selected from IL21, an anti-PD1 antibody, or a CXCL10-CXCL11 fusion protein and the second effector molecule is an IL12p70 fusion protein, or (c) the first effector molecule is IL21 and the second effector molecule is selected from IL15, an IL12p70 fusion protein, or a CXCL10-CXCL11 fusion protein, or (d) the first effector molecule is selected from IL15, an IL12p70 fusion protein, or a CXCL10-CXCL11 fusion protein and the second effector molecule is IL21, or (e) the first effector molecule is IL15 and the second effector molecule is selected from IL21 or a CXCL10-CXCL11 fusion protein, or (f) the first effector molecule is selected from IL21 or a CXCL10-CXCL11 fusion protein and the second effector molecule is IL15.

2. The engineered human cell according to claim 1, wherein at least one of the first effector molecule and the second effector molecule does not comprise a transmembrane domain and is not operably linked to a transmembrane domain.

3. The engineered human cell according to claim 1, wherein the engineered human cell further comprises a chimeric antigen receptor (CAR) or an exogenous polynucleotide sequence encoding the chimeric antigen receptor (CAR).

4. The engineered human cell according to claim 1, wherein the promoter is operably linked to the expression cassette such that the polynucleotide can be transcribed into a single polynucleotide comprising the formula S1-E1-L-S2-E2.

5. The engineered human cell according to claim 1, wherein the linker polynucleotide sequence is operably associated with the translation of the first effector molecule and the second effector molecule into different polypeptides.

6. The engineered human cell according to claim 1, wherein the linker polynucleotide sequence encodes a 2A ribosomal skipping tag or encodes an internal ribosome entry site (IRES).

7. The engineered human cell according to claim 6, wherein when the linker polynucleotide sequence encodes a 2A ribosomal skipping tag, the 2A ribosomal skipping tag is selected from the group consisting of: P2A, T2A, E2A, and F2A.

8. The engineered human cell according to claim 1, wherein the linker polynucleotide sequence encodes a second promoter, wherein the promoter is operably linked to the expression cassette such that a first polynucleotide comprising the formula S1-E1 can be transcribed, wherein the second promoter is operably linked to the expression cassette such that a second polynucleotide comprising the formula S2-E2 can be transcribed, and wherein the first polynucleotide and the second polynucleotide are different polynucleotides.

9. The engineered human cell according to claim 1, wherein the engineered human cell is a natural killer (NK) cell.

10. The engineered human cell according to claim 1, wherein the promoter comprises a constitutive promoter selected from the group consisting of: CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

11. The engineered human cell according to claim 1, wherein the promoter comprises an inducible promoter selected from the group consisting of: 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, inducible molecular response-type promoter, and tandem repeats thereof.

12. The engineered human cell according to claim 1, wherein respectively: - the first signal peptide comprises a native signal peptide that is native to the first effector molecule; or - the second signal peptide comprises a native signal peptide that is native to the second effector molecule; or - the first signal peptide comprises a non-native signal peptide that is non-native to the first effector molecule; or - the second signal peptide comprises a non-native signal peptide that is non-native to the second effector molecule; or any combination thereof.

13. The engineered human cell according to claim 1, wherein the expression cassette further comprises an additional exogenous polynucleotide sequence after E2, the exogenous polynucleotide sequence comprising the following formula, oriented 5' to 3', the formula comprising: (L-S-E) X wherein S is a polynucleotide sequence encoding a signal peptide, E is a polynucleotide sequence encoding an additional effector molecule, L is a linker polynucleotide sequence, X = 1 to 20, wherein the promoter is operably linked to the expression cassette, and wherein for each iteration of a given X, the corresponding signal peptide is operably linked to the respective effector molecule.

14. The engineered human cell according to claim 13, wherein one or more of the additional effector molecules comprise a chimeric antigen receptor.

15. The engineered human cell according to claim 1, wherein the exogenous polynucleotide sequence comprises one or more viral vector polynucleotide sequences, and wherein the one or more viral vector polynucleotide sequences comprise lentiviral, retroviral, retrotransposon or adenoviral polynucleotide sequences.

16. A cell population, wherein the cell population comprises one or more engineered human cells according to any one of claims 1-15.

17. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more engineered human cells according to any one of claims 1-15.

18. Use of an engineered human cell in the preparation of a medicament for inducing an immune response in a human subject, the engineered human cell comprising: a) a promoter; and b) an exogenous polynucleotide sequence comprising an expression cassette of the formula, oriented 5' to 3', the formula comprising: S1-E1-L-S2-E2 wherein S1 is a polynucleotide sequence encoding a first signal peptide, E1 is a polynucleotide sequence encoding a first effector molecule, L is a linker polynucleotide sequence, S2 is a polynucleotide sequence encoding a second signal peptide, E2 is a polynucleotide sequence encoding a second effector molecule, and wherein the promoter is operably linked to the expression cassette, the first signal peptide is operably linked to the first effector molecule, and the second signal peptide is operably linked to the second effector molecule, and wherein the engineered human cell is selected from the group consisting of mesenchymal stem cells (MSCs), natural killer (NK) cells, NKT cells, and macrophages, wherein (a) the first effector molecule is an IL12p70 fusion protein and the second effector molecule is selected from IL21, an anti-PD1 antibody, or a CXCL10-CXCL11 fusion protein, or (b) the first effector molecule is selected from IL21, an anti-PD1 antibody, or a CXCL10-CXCL11 fusion protein and the second effector molecule is an IL12p70 fusion protein, or (c) the first effector molecule is IL21 and the second effector molecule is selected from IL15, an IL12p70 fusion protein, or a CXCL10-CXCL11 fusion protein, or (d) the first effector molecule is selected from IL15, an IL12p70 fusion protein, or a CXCL10-CXCL11 fusion protein and the second effector molecule is IL21, or (e) the first effector molecule is IL15 and the second effector molecule is selected from IL21 or a CXCL10-CXCL11 fusion protein, or (f) the first effector molecule is selected from IL21 or a CXCL10-CXCL11 fusion protein and the second effector molecule is IL15.

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