Combinatorial cancer immunotherapy

Abstract

Provided herein are methods and compositions for dynamically controlling and targeting multiple immunosuppressive mechanisms in cancer. Some aspects provide cells engineered to produce multiple effector molecules, each of which modulates a different immunosuppressive mechanisms of a tumor, as well as methods of using the cells to treat cancer, such as ovarian, breast, or colon cancer.

1 - 20 . (canceled) 
     
     
         21 . An engineered human cell comprising:
 a first engineered nucleic acid and a second engineered nucleic acid, wherein the first engineered nucleic acid comprises:   a) a first promoter; and   b) a first exogenous polynucleotide sequence comprising a first expression cassette described in a formula, oriented from 5′ to 3′, comprising:   S1-E1, wherein   S1 comprises a polynucleotide sequence encoding a first signal peptide,   E1 comprises a polynucleotide sequence encoding a first effector molecule;   wherein the second engineered nucleic acid comprises:   a) a second promoter; and   b) a second exogenous polynucleotide sequence comprising a second expression cassette described in a formula, oriented from 5′ to 3′, comprising   S2-E2, wherein   S2 comprises a polynucleotide sequence encoding a second signal peptide,   E2 comprises a polynucleotide sequence encoding a second effector molecule,   wherein the first promoter is operably linked to the first expression cassette, the second promoter is operably linked to the second 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   (a) the first effector molecule comprises an IL12P70 fusion protein and the second effector molecule comprises CCL21a, IL7, IL15, IL18, IL21, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein,   (b) the first effector molecule comprises CCL21a, IL7, IL15, IL18, IL21, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein and the second effector molecule comprises an IL12p70 fusion protein,   (c) the first effector molecule comprises IL15 and the second effector molecule comprises CCL21a, IL7, IL18, IL21, an IL12p70 fusion protein, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein, or   (d) the first effector molecule comprises CCL21a, IL7, IL18, IL21, an IL12p70 fusion protein, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein and the second effector molecule comprises IL15,   wherein at least one of the first effector molecule and the second effector molecule does not comprise, nor is operably linked to, a transmembrane domain,   wherein the engineered human cell is selected from the group consisting of: a mesenchymal stem cell (MSC), natural killer (NK) cell, a T cell, NKT cell, and macrophage, and   wherein the engineered human cell further comprises a chimeric antigen receptor (CAR) or exogenous polynucleotide sequence encoding the same.   
     
     
         22 . The engineered human cell of  claim 21 , wherein the first promoter, the second promoter, or both the first promoter and the second promoter comprises a constitutive promoter or an inducible promoter. 
     
     
         23 . The engineered human cell of  claim 22 , wherein the constitutive promoter is selected from the group consisting of: MV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb. 
     
     
         24 . The engineered human cell of  claim 22 , wherein the inducible promoter is selected from the group consisting of: minP, NFkB response element, CREB response element, NFAT response element, SRF response element 21, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, inducer molecule responsive promoters, and tandem repeats thereof. 
     
     
         25 . The engineered human cell of  claim 21 , wherein:
 (a) the first signal peptide comprises a native signal peptide native to the first effector molecule; or   (b) the second signal peptide comprises a native signal peptide native to the second effector molecule; or   (c) the first signal peptide and the second signal peptide comprise a native signal peptide native to the first effector molecule and the second effector molecule, respectively; or   (d) the first signal peptide and the second signal peptide comprise a non-native signal peptide non-native to the first effector molecule and the second effector molecule; or   (e) the first signal peptide comprises a native signal peptide native to the first effector molecule and the second signal peptide comprises a non-native signal peptide non-native to the second effector molecule; or   (f) the first signal peptide comprises a non-native signal peptide non-native to the first effector molecule and the second signal peptide comprises a native signal peptide native to the second effector molecule.   
     
     
         26 . The engineered human cell of  claim 21 , wherein the exogenous polynucleotide sequence comprises one or more viral vector polynucleotide sequences. 
     
     
         27 . The engineered human cell of  claim 26 , wherein the one or more viral vector polynucleotide sequences comprise lentiviral, retroviral, retrotransposon, or adenoviral polynucleotide sequences. 
     
     
         28 . The engineered human cell of  claim 21 , wherein the first expression cassette further comprises following E1 and/or the second expression cassette further comprises following E2, an additional expression cassette comprising an additional exogenous polynucleotide sequence comprising a formula, oriented from 5′ to 3′, comprising:
   (L-S-E)x 
 wherein, 
 S comprises a polynucleotide sequence encoding a signal peptide, 
 E comprises a polynucleotide sequence encoding an additional effector molecule, 
 L comprises a linker polynucleotide, and X=1 to 20, 
 wherein for each iteration for a given X, the corresponding signal peptide is operably linked to the respective effector molecule. 
 
     
     
         29 . The engineered human cell of  claim 28 , wherein the additional exogenous polynucleotide sequence is operably linked to the first expression cassette or the second expression cassette such that the polynucleotides can be transcribed as a single polynucleotide comprising the formula S1-E1-(L-S-E)x or S2-E2-(L-S-E)x, respectively. 
     
     
         30 . The engineered human cell of  claim 28 , wherein the additional effector molecule comprises a chimeric antigen receptor. 
     
     
         31 . The engineered human cell of  claim 29 , wherein the linker polynucleotide sequence encodes a 2A ribosome skipping tag or encodes an Internal Ribosome Entry Site (IRES). 
     
     
         32 . The engineered human cell of  claim 31 , wherein the 2A ribosome skipping tag is selected from the group consisting of: P2A, T2A, E2A, and F2A. 
     
     
         33 . The engineered human cell of  claim 28 , wherein the linker polynucleotide encodes an additional promoter, wherein the additional promoter is operably linked to the additional expression cassette such that the additional polynucleotide sequence comprising the formula (L-S-E)x is capable of being transcribed, and wherein the first, the second, and the additional polynucleotide sequences are separate polynucleotides. 
     
     
         34 . The engineered human cell of  claim 21 , wherein the engineered human cell is an NK cell. 
     
     
         35 . The engineered human cell of  claim 21 , wherein the first effector molecule comprises the IL12p70 fusion protein and the second effector molecule comprises CCL21a, IL7, IL15, IL21, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein. 
     
     
         36 . The engineered human cell of  claim 21 , wherein the first effector molecule comprises CCL21a, IL7, IL15, IL21, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein and the second effector molecule comprises the IL12p70 fusion protein. 
     
     
         37 . The engineered human cell of  claim 21 , wherein the first effector molecule comprises IL21 and the effector molecule comprises IL15. 
     
     
         38 . A population of cells, wherein the population of cells comprises one or more engineered human cells of  claim 21 . 
     
     
         39 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more engineered human cells of  claim 21 . 
     
     
         40 . A method of treating a subject having cancer, wherein the method comprises delivering to a subject an engineered cell in an effective amount to induce an immune response, wherein the engineered cell comprises:
 a first engineered nucleic acid and a second engineered nucleic acid, wherein the first engineered nucleic acid comprises:   a) a first promoter; and   b) a first exogenous polynucleotide sequence comprising a first expression cassette described in a formula, oriented from 5′ to 3′, comprising:   S1-E1, wherein   S1 comprises a polynucleotide sequence encoding a first signal peptide,   E1 comprises a polynucleotide sequence encoding a first effector molecule;   wherein the second engineered nucleic acid comprises:   a) a second promoter; and   b) a second exogenous polynucleotide sequence comprising a second expression cassette described in a formula, oriented from 5′ to 3′, comprising   S2-E2, wherein   S2 comprises a polynucleotide sequence encoding a second signal peptide,   E2 comprises a polynucleotide sequence encoding a second effector molecule,   wherein the first promoter is operably linked to the first expression cassette, the second promoter is operably linked to the second 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   (a) the first effector molecule comprises an IL12P70 fusion protein and the second effector molecule comprises CCL21a, IL7, IL15, IL18, IL21, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein,   (b) the first effector molecule comprises CCL21a, IL7, IL15, IL18, IL21, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein and the second effector molecule comprises an IL12p70 fusion protein,   (c) the first effector molecule comprises IL15 and the second effector molecule comprises CCL21a, IL7, IL18, IL21, an IL12p70 fusion protein, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein, or   (d) the first effector molecule comprises CCL21a, IL7, IL18, IL21, an IL12p70 fusion protein, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein and the second effector molecule comprises IL15,   wherein at least one of the first effector molecule and the second effector molecule does not comprise, nor is operably linked to, a transmembrane domain,   wherein the engineered human cell is selected from the group consisting of: a mesenchymal stem cell (MSC), natural killer (NK) cell, a T cell, NKT cell, and macrophage, and   wherein the engineered human cell further comprises a chimeric antigen receptor (CAR) or exogenous polynucleotide sequence encoding the same.