Gene-regulating compositions and methods for improved immunotherapy

Abstract

The present disclosure provides methods and compositions related to the modification of immune effector cells to increase therapeutic efficacy. In some embodiments, immune effector cells modified to reduce expression of one or more endogenous target genes, or to reduce one or more functions of an endogenous protein to enhance effector functions of the immune cells are provided. In some embodiments, immune effector cells further modified by introduction of transgenes conferring antigen specificity, such as exogenous T cell receptors (TCRs) or chimeric antigen receptors (CARs) are provided. Methods of treating a cell proliferative disorder, such as a cancer, using the modified immune effector cells described herein are also provided.

1 .- 288 . (canceled) 
     
     
         289 . A modified immune effector cell comprising reduced expression or function of NR4A3, wherein the reduced expression or function of NR4A3 enhances an effector function of the modified immune effector cell. 
     
     
         290 . The modified immune effector cell of  claim 289 , further comprising reduced expression or function of one or more endogenous target genes selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. 
     
     
         291 . The modified immune effector cell of  claim 289 , wherein the NR4A3 gene comprises an inactivating mutation and wherein the inactivating mutation reduces the expression or function of NR4A3. 
     
     
         292 . The modified immune effector cell of  claim 291 , wherein the expression or function of the one or more endogenous target genes is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to an un-modified or control immune effector cell. 
     
     
         293 . The modified immune effector cell of  claim 289 , wherein the immune effector cell is a lymphocyte selected from a T cell, a natural killer (NK) cell, an NKT cell, and a tumor infiltrating lymphocyte. 
     
     
         294 . The modified immune effector cell of  claim 289 , further comprising an engineered immune receptor displayed on the cell surface. 
     
     
         295 . The modified immune effector cell of  claim 294 , wherein the engineered immune receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). 
     
     
         296 . The modified immune effector cell of  claim 289 , further comprising an exogenous transgene expressing an immune activating molecule. 
     
     
         297 . The modified immune effector cell of  claim 296 , wherein the immune activating molecule is selected from the group consisting of a cytokine, a chemokine, a co-stimulatory molecule, an activating peptide, an antibody, or an antigen-binding fragment thereof. 
     
     
         298 . The modified immune effector cell of  claim 289 , wherein the modified immune effector cells are autologous to a subject. 
     
     
         299 . The modified immune effector cell of  claim 289 , wherein the modified immune effector cells are allogenic to a subject. 
     
     
         300 . The modified immune effector cell of  claim 289 , wherein the effector function is selected from cell proliferation, cell viability, tumor infiltration, cytotoxicity, anti-tumor immune responses, and resistance to exhaustion. 
     
     
         301 . The modified immune effector cell of  claim 300 , wherein the effector function of the modified immune cell is increased by at least 10% compared to a non-modified control immune cell. 
     
     
         302 . The modified immune effector cell of  claim 301 , wherein the NR4A3 gene comprises an inactivating mutation and wherein the inactivating mutation reduces the expression or function of NR4A3. 
     
     
         303 . A pharmaceutical composition comprising the modified immune effector cell of  claim 289 . 
     
     
         304 . The composition of  claim 303 , wherein the modified immune effector cells are autologous to a subject. 
     
     
         305 . The composition of  claim 303 , wherein the modified immune effector cells are allogenic to a subject. 
     
     
         306 . A method of treating a disease or disorder in a subject in need thereof comprising administering an effective amount of modified immune effector cells to the subject in need thereof, wherein the modified immune effector cells comprise reduced expression or function of NR4A3, and wherein the reduced expression or function of NR4A3 enhances an effector function of the modified immune effector cell. 
     
     
         307 . The method of  claim 306 , wherein the disease or disorder is a cancer selected from a leukemia, a lymphoma, or a solid tumor. 
     
     
         308 . The method of  claim 306 , wherein the solid tumor is a melanoma, a pancreatic tumor, a bladder tumor, a lung tumor or metastasis, a colorectal cancer, a cervical cancer, or a head and neck cancer. 
     
     
         309 . The method of  claim 306 , wherein the cancer is a PD1-inhibitor resistant or refractory cancer. 
     
     
         310 . The method of  claim 306 , wherein the modified immune effector cells are autologous to the subject. 
     
     
         311 . The method of  claim 306 , wherein the modified immune effector cells are allogenic to the subject. 
     
     
         312 . The method of  claim 306 , wherein the disease or disorder is a solid tumor and wherein the tumor is decreased by at least 10% more compared to treatment with a non-modified control immune cell. 
     
     
         313 . The method of  claim 312 , wherein the solid tumor is selected from a melanoma tumor or metastasis, a lung tumor or metastasis, or a colorectal cancer. 
     
     
         314 . A gene-regulating system capable of reducing the expression or function of NR4A3 in a cell comprising:
 (i) one or more nucleic acid molecules selected from an siRNA, an shRNA, a microRNA (miR), an antagomiR, or an antisense RNA;   (ii) one or more enzymatic proteins selected from a zinc finger nuclease and a transcription-activator-like effector nuclease (TALEN); or   (iii) one or more guide RNAs (gRNAs) and a Cas endonuclease.   
     
     
         315 . The gene-regulating system of  claim 314 , wherein the Cas endonuclease comprises:
 (a) a wild-type Cas protein comprising two enzymatically active domains;   (b) a Cas nickase mutant comprising one enzymatically active domain; or   (c) a deactivated Cas protein (dCas) associated with a heterologous protein.   
     
     
         316 . The gene-regulating system of  claim 314 , wherein the Cas protein is a Cas9 protein. 
     
     
         317 . The gene-regulating system of  claim 314 , comprising one or more siRNA or shRNA molecules that bind to an RNA sequence encoded by a DNA sequence in the NR4A3 gene defined by a set of genomic coordinate selected from those in Tables 6G and 6H. 
     
     
         318 . The gene-regulating system of  claim 314 , comprising one or more siRNA or shRNA molecules that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 1539-1566. 
     
     
         319 . The gene-regulating system of  claim 314 , comprising a plurality of siRNA or shRNA molecules. 
     
     
         320 . The gene regulating system of  claim 314 , wherein the one or more gRNAs comprise a targeting domain sequence that is complementary to a target DNA sequence in the NR4A3 gene defined by a set of genomic coordinates selected from those in Table 6G and Table 6H. 
     
     
         321 . The gRNA nucleic acid molecule of  claim 314 , wherein the gRNA comprises a targeting domain sequence that binds to a target DNA sequence selected from SEQ ID NOs: 1539-1566. 
     
     
         322 . The gene regulating system of  claim 314 , comprising a plurality of gRNA nucleic acid molecules. 
     
     
         323 . The gene regulating system of  claim 314 , wherein the one or more gRNAs comprise a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1539-1566. 
     
     
         324 . A kit comprising the gene regulating system of  claim 314 . 
     
     
         325 . A modified immune effector cell comprising the gene-regulating system of  claim 314 . 
     
     
         326 . A method of producing a modified immune effector cell, comprising introducing the gene-regulating system of  claim 314  into the immune effector cell. 
     
     
         327 . A method of enhancing one or more effector functions of an immune effector cell comprising introducing a gene-regulating system of  claim 314  into the immune effector cell, wherein the immune effector cell demonstrates one or more enhanced effector functions compared to an immune effector cell that has not been modified. 
     
     
         328 . The method of  claim 327 , wherein the one or more effector functions are selected from cell proliferation, cell viability, cytotoxicity, tumor infiltration, increased cytokine production, anti-tumor immune responses, and resistance to exhaustion. 
     
     
         329 . The method of  claim 328 , wherein the one or more effector functions are increased by at least 10% compared to a control immune effector cell. 
     
     
         330 . A guide RNA (gRNA) nucleic acid molecule comprising a targeting domain nucleic acid sequence that is complementary to a target DNA sequence in the NR4A3 gene selected from a target DNA sequence defined by a set of genomic coordinates selected from those listed in Table 6G and Table 6H. 
     
     
         331 . The gRNA nucleic acid molecule of  claim 330 , wherein the gRNA comprises a targeting domain sequence that binds to a target DNA sequence selected from SEQ ID NOs: 1539-1566. 
     
     
         332 . The gRNA nucleic acid molecule of  claim 330 , wherein the gRNA comprises a targeting domain sequence encoded by a sequence selected from SEQ ID NOs: 1539-1566.