NR4A SUPER-REPRESSORS AND METHODS OF USE THEREOF

FIELD

[0001] The disclosure relates to repressors of nuclear receptor transcription factors NR4A1, NR4A2, and NR4A3 and uses thereof, e.g., to increase the efficacy of adoptive cell therapy.

BACKGROUND

[0002] Adoptive cell transfer utilizing genetically modified T cells, in particular CAR-T cells have entered clinical testing as therapeutics for solid and hematologic malignancies. Results to date have been mixed. In hematologic malignancies (especially lymphoma, CLL and ALL), the majority of patients in several Phase 1 and 2 trials exhibited at least a partial response, with some exhibiting complete responses (Kochenderfer el al, 2012 Blood 1 19, 2709-2720). In 2017, the FDA approved two CAR-T therapies, Kymriah™ and Yescarta™, both for the treatment of hematological cancers. However, in most tumor types (including melanoma, renal cell carcinoma and colorectal cancer), fewer responses have been observed (Johnson etal, 2009 Blood 1 14, 535-546; Lamers etal, 2013 Mol. Ther. 21, 904-912; Warren et al. , 1998 Cancer Gene Ther. 5, S1-S2). Success has largely been limited to CAR-T cells approaches targeting hematological malignancies of the B cell lineage.

[0003] It has been reported that CAR-T cells enter a hyporesponsive state that decreases their effectiveness, and that CAR-T cells lacking nuclear receptor transcription factors NR4A1 , NR4A2, and NR4A3 (triple knockouts) promote tumor regression and prolong the survival of tumor-bearing mice (Chen et al. Nature 567: 530-534 (2019)). However, knocking out three genes is technically challenging. As such, there is considerable room for improvement with adoptive cell therapies.

SUMMARY

[0004] The present invention provides repressors of nuclear receptor transcription factors NR4A1, NR4A2, and NR4A3. Such repressors are capable of decreasing the activity of all three NR4A transcription factors using a single element.

[0005] Provided herein are polynucleotides encoding polypeptides comprising a DNA- binding domain that specifically binds to the NBRE element, to the NurRE element, or to both the NBRE element and the NurRE element, wherein the polypeptide does not comprise a transcription activation domain. [0006] Provided herein are also polynucleotides encoding polypeptides comprising a DNA-binding domain that specifically binds to the NBRE element, to the NurRE element, or to both the NBRE element and the NurRE element, wherein the polypeptide inhibits the activity of NR4A1, NR4A2, and NR4A3.

[0007] Provided herein are also polynucleotides encoding polypeptides comprising a DNA-binding domain that specifically binds to the NBRE element, to the NurRE element, or to both the NBRE element and the NurRE element, wherein the polypeptide is capable of inhibiting transcription of a gene operably associated with the NBRE element, the NurRE element, or both the NBRE element and the NurRE element.

[0008] In certain instances, the polypeptide does not comprise a transcription activation domain.

[0009] In certain instances, the DNA-binding domain is a polypeptide comprising the amino acid sequence of the DNA-binding domain of NR4A1, NR4A2, or NR4A3. In certain instances, the DNA- binding domain comprises the amino acid sequence of the DNA-binding domain ofNR4A3. In certain instances, the DNA-binding domain is a polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the DNA-binding domain of NR4A1, NR4A2, or NR4A3. In certain instances, the DNA-binding domain is a polypeptide comprising the amino acid sequence of SEQ ID NO:4 or 5. In certain instances, the DNA-binding domain is a polypeptide comprising the amino acid sequence of SEQ ID NO:50 or 51. In certain instances, the DNA-binding domain is a polypeptide comprising the amino acid sequence of SEQ ID NO:52 or 53.

[0010] In certain instances, the DNA-binding domain comprises a TAL effector DNA binding domain. In certain instances, the DNA-binding domain comprises the amino acid sequence of SEQ ID NO:6, 7, 8, or 10.

[0011] In certain instances, the DNA-binding domain comprises a zinc finger. In certain instances, the zinc finger binds to a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs:44-47 or to a polynucleotide comprising GATATT (SEQ ID NO:48) and GCCAAT (SEQ ID NO:49), optionally wherein SEQ ID NO:48 is 5' to SEQ ID NO:49. In certain instances, the DNA-binding domain comprises the amino acid sequence of SEQ ID NO: l l, 12, 13, or 14.

[0012] In certain instances, the DNA-binding domain comprises a DNA-binding domain from a meganuclease. [0013] In certain instances, the polypeptide further comprises the amino acid sequence of the ligand-binding domain (LBD) of NR4A1, NR4A2, or NR4A3. In certain instances, the amino acid sequence of the LBD is C-terminal to the DNA-binding domain. In certain instances, the amino acid sequence of the LBD is N-terminal to the DNA-binding domain.

[0014] In certain instances, the polypeptide further comprises a Krtippel-associated box (KRAB) domain. In certain instances, the KRAB domain is C-terminal to the DNA-binding domain. In certain instances, the KRAB domain is N-terminal to the DNA-binding domain.

[0015] In certain instances, the polypeptide further comprises NCOR1 or a fragment thereof. In certain instances, theNCORl or fragment thereof is C-terminal to the DNA-binding domain. In certain instances, the NCOR1 or fragment thereof is N-terminal to the DNA- binding domain.

[0016] In certain instances, the polypeptide further comprises a heterologous domain. In certain instances, the heterologous domain is a dimerization domain, a transcriptional repressor domain, or a chromatin compaction domain. In certain instances, the heterologous domain is C-terminal to the DNA-binding domain. In certain instances, the heterologous domain is N- terminal to the DNA-binding domain.

[0017] In certain instances, the polypeptide further comprises a linker. In certain instances, the linker is located between the DNA-binding domain and the LBD, the KRAB domain, the NCOR1 or fragment thereof, or heterologous domain. In certain instances, the linker comprises the amino acid sequence of any one of SEQ ID NOs:9 and 25-28.

[0018] In certain instances, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 29-43.

[0019] Provided herein are also vectors comprising any polynucleotide provided herein. In certain instances, the vector is a retroviral vector. In certain instances, the retroviral vector is a lentiviral vector. In certain instances, the vector further comprises a polynucleotide encoding an engineered immune receptor.

[0020] Provided herein are also compositions comprising any polynucleotide or vector provided herein.

[0021] Provided herein are also compositions comprising any polynucleotide or vector provided herein, wherein the composition further comprises a vector comprising a polynucleotide encoding an engineered immune receptor.

[0022] In certain vectors or compositions provided herein, the engineered immune receptor is (a) a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain or (b) an engineered T cell receptor (TCR).

[0023] In certain vectors or compositions provided herein, the engineered immune receptor specifically binds to an antigen expressed on a target cell, wherein the antigen is a tumor- associated antigen.

[0024] Provided herein are also polypeptides encoded by any polynucleotide provided herein.

[0025] Provided herein are also cells comprising any polynucleotide, vector, composition, or polypeptide provided herein. In certain instances, the cell is an immune effector cell. In certain instances, the immune effector cell is a T cell, a natural killer (NK) cell, or an NKT cell. In certain instances, a polynucleotide, vector, composition, or polypeptide provided herein enhances an effector function of the cell. In certain instances, the effector function is selected from cell proliferation, cell viability, tumor infiltration, cytotoxicity, anti-tumor immune responses, and/or resistance to exhaustion.

[0026] In certain instances, the cell further comprises an engineered immune receptor. In certain instances, the engineered immune receptor is (a) a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain or (b) an engineered T cell receptor (TCR). In certain instances, the engineered immune receptor specifically binds to an antigen expressed on a target cell, wherein the antigen is a tumor-associated antigen.

[0027] Provided herein are also compositions comprising any cell provided herein. In certain instances, the composition is suitable for administration to a subject in need thereof. In certain instances, the composition comprises autologous cells derived from the subject in need thereof. In certain instances, the composition comprises allogeneic cells derived from a donor subject.

[0028] Provided herein are also methods of producing a modified immune effector cell comprising introducing any polynucleotide, vector, composition, or polypeptide provided herein into an immune effector cell. In certain instances, the cell is a tumor infiltration lymphocyte. In certain instances, the method further comprises introducing a polynucleotide sequence encoding an engineered immune receptor into the immune effector cell, optionally wherein the engineered immune receptor is selected from a CAR and a TCR. In certain instances, the polynucleotide, vector, composition, or polypeptide provided herein and/or the polynucleotide encoding the engineered immune receptor are introduced to the immune effector cell by transfection, transduction, electroporation, or physical disruption of the cell membrane by a microfluidics device.

[0029] Provided herein are also methods of producing a modified immune effector cell comprising: obtaining a population of immune effector cells; expanding the population of immune effector cells; and introducing any polynucleotide, vector, composition, or polypeptide provided herein into the population of immune effector cells.

[0030] Provided herein are also methods of producing a modified immune effector cell comprising: obtaining a population of immune effector cells; expanding the population of immune effector cells in a first round expansion and a second round of expansion; and introducing any polynucleotide, vector, composition, or polypeptide provided herein into the population of immune effector cells. In certain instances, the polynucleotide, vector, composition, or polypeptide provided herein is introduced to the population of immune effector cells prior to the first and second rounds of expansion. In certain instances, the polynucleotide, vector, composition, or polypeptide provided herein is introduced to the population of immune effector cells after the first round of expansion and prior to the second round of expansion. In certain instances, the polynucleotide, vector, composition, or polypeptide provided herein is introduced to the population of immune effector cells after the first and second rounds of expansion. In certain instances, the introduction enhances one or more effector functions. In certain instances, the one or more effector functions are selected from cell proliferation, cell viability, cytotoxicity, tumor infiltration, increased cytokine production, anti-tumor immune responses, and/or resistance to exhaustion.

[0031] Also provided herein are methods of treating a disease or disorder in a subject in need thereof comprising administering to the subject an effective amount of any cells provided herein or any composition provided herein. In certain instances, the disease or disorder is a cell proliferative disorder, an inflammatory disorder, or an infectious disease. In certain instances, the disease or disorder is a cancer or a viral infection. In certain instances, the cancer is selected from a leukemia, a lymphoma, or a solid tumor. In certain instances, the cells are autologous to the subject. In certain instances, the cells are allogenic to the subject.

[0032] Also provided herein are methods of treating a cancer in a subject in need thereof comprising administering to the subject an effective amount of any cell provided herein or any composition provided herein. In certain instances, the administration inhibits tumor regrowth and/or inhibits tumor metastasis. BRIEF DESCRIPTION OF THE FIGURES

[0033] Fig. 1 depicts the domain structures of "wild-type" NR4A proteins and NR4A super-repressor proteins. The wild-type NR4A proteins contain a TAF transactivation domain, a DNA-binding domain (DBD), and a ligand-binding domain (LBD). The NR4A super repressor proteins lack at least the TAF domain. The NR4A super-repressor proteins may also contain additional non-NR4A domains such NCOR1, a fragment thereof, or a KRAB domain. (See Example 1.)

[0034] Fig. 2 shows a schematic of a super-repressor assay, which can be used to evaluate the ability of proteins to inhibit transcription of NR4A target genes. (See Example 2.)

[0035] Fig. 3 shows the activity of the NR4A DBD domain in the super-repressor assay. In each graph, the two bars on the far right show the activity of the triple knock-out (TKO) in the absence (open bar; second bar from right) and presence (shaded bar; bar on far right) of T- cell activator stimulation. (See Example 2.)

[0036] Fig. 4 shows the activity of the NR4A DBD and LBD domains in the super repressor assay. In each graph, the two bars on the far right show the activity of the triple knock-out (TKO) in the absence (open bar; second bar from right) and presence (shaded bar; bar on far right) of T-cell activator stimulation. (See Example 2.)

[0037] Fig. 5 shows the activity of a fusion protein comprising NCOR1 and the NR4A DBD and LBD domains in the super-repressor assay. In the graph, the two bars on the far right show the activity of the triple knock-out (TKO) in the absence (open bar; second bar from right) and presence (shaded bar; bar on far right) of T-cell activator stimulation. (See Example 2·)

[0038] Fig. 6 shows the activity of a fusion protein comprising a truncated region of NCOR1 and the NR4A DBD and LBD domains in the super-repressor assay. In each graph, the two bars on the far right show the activity of the triple knock-out (TKO) in the absence (open bar; second bar from right) and presence (shaded bar; bar on far right) of T-cell activator stimulation. (See Example 2.)

[0039] Fig. 7 shows the activity of a fusion protein comprising the NR4A DBD and KRAB in the super-repressor assay. In each graph, the two bars on the far right show the activity of the triple knock-out (TKO) in the absence (open bar) and presence (shaded bar; bar on far right) of T-cell activator stimulation. (See Example 2.) DETATEET) DESCRIPTION

[0040] Using genome-wide screens in CD8 T-cells, it was discovered that knocking out NR4A1, NR4A2, and NR4A3 can drive robust tumor killing effects. (See e.g., U.S. Application No. 16/354, 102 (US Application Publication No. US 2019/0284553), filed March 14, 2019, which is herein incorporated by reference in its entirety.) However, the therapeutic use of knocking out three genes in a cell brings with it significant technical challenges. In order to avoid the technical challenges associated with knocking out three genes at once, the present disclosure provides super-repressors capable of inhibiting the transcription of NR4A1 , NR4A2, and NR4A3 target genes. The super-repressors bind to the NBRE and/or NurRE response elements (e.g., via the DNA-binding domain of an NR4A transcription factor, a TAL effector (TALE), a zinc finger protein, or a guide RNA), but are not capable of activating transcription from these elements. Accordingly, the super-repressors inhibit the activity of NR4A1, NR4A2, and NR4A3. The present disclosure also provides polynucleotides encoding the super repressors. As disclosed herein, the super-repressors and polynucleotides encoding the same can be used in adoptive cell therapy.

I. Definitions

[0041] As used herein, the singular forms“a,”“an” and“the” include plural references unless the content clearly dictates otherwise.

[0042] As used herein, the term“and/or” means either“and” or“or” unless indicated otherwise.

[0043] As used herein, unless the context requires otherwise, the words“comprise”, or variations such as“comprises” or“comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0044] The term“nuclear receptor subfamily 4 group A member 1” or“NR4A1,” as used herein refers to any native NR4A1 polypeptide or NR4A1 -encoding polynucleotide. The term “NR4A1” encompasses“full-length,” unprocessed NR4A1 polypeptide as well as any forms of NR4A1 that result from processing within the cell. The term also encompasses naturally occurring variants of NR4A1, e.g., those encoded by splice variants and allelic variants. The NR4A1 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Human NR4A1 sequences are known and include, for example, the sequences publicly available as UniProt No. P22736 (including isoforms). As used herein, the term“human NR4A1 protein” refers to NR4A1 protein comprising the amino acid sequence set forth in SEQ ID NO: l .

MPCIQAQYGTPAPSPGPRDHLASDPLTPEFIKPTMDLASPEAAPAAPTALPSFSTFMD

GYTGEFDTFLYQLPGTVQPCSSASSSASSTSSSSATSPASASFKFEDFQVYGCYPGP LS

GP VDE AL S S S GSD YY GSPCSAPSPSTPSF QPPQLSP WDGSF GHF SP S QT YEGLRAWTE

QLPKASGPPQPPAFFSFSPPTGPSPSLAQSPLKLFPSQATHQLGEGESYSMPTAFPG LA

PTSPHLEGSGILDTPVTSTKARSGAPGGSEGRCAVCGDNASCQHYGVRTCEGCKGFF

KRT V QKNAK YICLANKDCP VDKRRRNRC QF CRF QKCL A V GMVKEVVRTD SLKGRR

GRLPSKPKQPPDASPANLLTSLVRAHLDSGPSTAKLDYSKFQELVLPHFGKEDAGDV

QQFYDLLSGSLEVIRKWAEKIPGFAELSPADQDLLLESAFLELFILRLAYRSKPGEG KL

IFCSGLVLHRLQCARGFGDWIDSILAFSRSLHSLLVDVPAFACLSALVLITDRHGLQ EP

RRVEELQNRIASCLKEHVAAVAGEPQPASCLSRLLGKLPELRTLCTQGLQRIFYLKL E

DLVPPPPIIDKIFMDTLPF (SEQ ID NO : 1 )

[0045] The two zinc fingers in the DNA binding domain (DBD) of SEQ ID NO: 1 are amino acids 267-287 and 303-327, respectively. The DNA binding domain (DBD) of SEQ ID NO: 1 is amino acids 264-339. The ligand binding domain (LBD) of SEQ ID NO: l is amino acids 360-595.

[0046] The term“nuclear receptor subfamily 4 group A member 2” or“NR4A2,” as used herein refers to any native NR4A2 polypeptide or NR4A2 -encoding polynucleotide. The term “NR4A2” encompasses“full-length,” unprocessed NR4A2 polypeptide as well as any forms of NR4A2 that result from processing within the cell. The term also encompasses naturally occurring variants of NR4A2, e.g., those encoded by splice variants and allelic variants. The NR4A2 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Human NR4A2 sequences are known and include, for example, the sequences publicly available as UniProt No. P43354 (including isoforms). As used herein, the term“human NR4A2 protein” refers to NR4A2 protein comprising the amino acid sequence set forth in SEQ ID NO:2.

MPCVQAQYGSSPQGASPASQSYSYHSSGEYSSDFLTPEFVKFSMDLTNTEITATTSLP SF STFMDNYSTGYDVKPPCL YQMPL SGQQ S SIKVEDIQMHNYQQHSHLPPQ SEEMM PHSGSVYYKPSSPPTPTTPGFQVQHSPMWDDPGSLHNFHQNYVATTHMIEQRKTPVS RLSLFSFKQSPPGTPVSSCQMRFDGPLHVPMNPEPAGSHHVVDGQTFAVPNPIRKPAS MGFPGLQIGHASQLLDTQVPSPPSRGSPSNEGLCAVCGDNAACQHYGVRTCEGCKG FFKRT V QKNAK YV CL ANKN CP VDKRRRNRC Q Y CRF QKCL A V GMVKEVVRTD SLK GRRGRLP SKPKSPQEPSPP SPPV SLIS ALVRAHVD SNP AMTSLD Y SRF Q ANPD Y QMSG DDTQHIQQFYDLLTGSMEIIRGWAEKIPGFADLPKADQDLLFESAFLELFVLRLAYRS NP VEGKLIF CN GWLHRLQC VRGF GEWID SIVEF S SNLQNMNIDIS AF SCIAAL AMVT ERHGLKEPKRVEELQNKIVNCLKDHVTFNNGGLNRPNYLSKLLGKLPELRTLCTQGL QRIFYLKLEDLVPPPAIIDKLFLDTLPF (SEQ ID NO: 2)

[0047] The two zinc fingers in the DNA binding domain (DBD) of SEQ ID NO: 2 are amino acids 263-283 and 299-323, respectively. The DNA binding domain (DBD) of SEQ ID NO:2 is amino acids 260-335. The ligand binding domain (LBD) of SEQ ID NO:2 is amino acids 360-595.

[0048] The term“nuclear receptor subfamily 4 group A member 3” or“NR4A3,” as used herein refers to any native NR4A3 polypeptide or NR4 A3 -encoding polynucleotide. The term “NR4A3” encompasses“full-length,” unprocessed NR4A3 polypeptide as well as any forms of NR4A3that result from processing within the cell. The term also encompasses naturally occurring variants of NR4A3, e.g., those encoded by splice variants and allelic variants. The NR4A3 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Human NR4A3 sequences are known and include, for example, the sequences publicly available as UniProt No. Q92570 (including isoforms). As used herein, the term“human NR4A3 protein” refers to NR4A3 protein comprising the amino acid sequence set forth in SEQ ID NO:3.

MPCVQAQYSP SPPGS S YAAQT Y S SEYTTEIMNPD YTKLTMDLGSTEIT ATATT SLPSIS TF VEGYS SNYELKP SC VYQMQRPLIKVEEGRAP S YHHHHHHHHHHHHHHQQQHQQ PSIPPASSPEDEVLPSTSMYFKQSPPSTPTTPAFPPQAGALWDEALPSAPGCIAPGPLLD PPMKAVPTVAGARFPLFHFKPSPPHPPAPSPAGGHHLGYDPTAAAALSLPLGAAAAA GSQAAALESHPYGLPLAKRAAPLAFPPLGLTPSPTASSLLGESPSLPSPPSRSSSSGEGT C A VCGDNAACQHY GVRT CEGCKGFFKRT V QKNAK YV CL ANKN CP VDKRRRNRCQ Y CRF QKCL S VGMVKE VVRTD SLKGRRGRLP SKPKSPLQQEP S QP SPP SPPICMMNAL VRALTDSTPRDLDYSRYCPTDQAAAGTDAEHVQQFYNLLTASIDVSRSWAEKIPGFT DT PK FDOTT J ,TES AFT FT FVT RT STR SNT AEDKF VFCNGT VI HR I OCT R GFGEWT DSTK DF SLNLQ SLNLDIQ AL ACL S AL SMITERHGLKEPKRVEELCNK IT S SLKDHQ SKGQ AL EPTESKVLGALVELRKICTLGLQRIFYLKLEDLVSPPSIIDKLFLDTLPF (SEQ ID NO:3)

[0049] The two zinc fingers in the DNA binding domain (DBD) of SEQ ID NO: 3 are amino acids 292-312 and 328-352, respectively. The DNA binding domain (DBD) of SEQ ID NO:3 is amino acids 289-364. The ligand binding domain (LBD) of SEQ ID NO:3 is amino acids 394-623.

[0050] The term“NR4A super-repressor” refers to an agent that binds to the NBRE and/or NurRE response elements and inhibits the transcriptional activity of NR4A1, NR4A2, and NR4A3.

[0051] As used herein, the terms“about” and“approximately” are used as equivalents. Any numerals used herein with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term“approximately” or“about” refers to a range of values that fall within 10% (for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0052] “Decrease” or“reduce” refers to a decrease or a reduction in a particular value of at least 5%, for example, a 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% decrease as compared to a reference value.

[0053] “Increase” refers to an increase in a particular value of at least 5%, for example, a 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, 500%, or more increase as compared to a reference value.

[0054] The terms“peptide,”“polypeptide,” and“protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non- coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0055] The terms“polynucleotide” and“nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non natural, or derivatized nucleotide bases.“Oligonucleotide” generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as“oligomers” or“oligos” and may be isolated from genes, or chemically synthesized by methods known in the art. The terms“polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0056] “Fragment” refers to a portion of a polypeptide or polynucleotide molecule containing less than the entire polypeptide or polynucleotide sequence. In some embodiments, a fragment of a polypeptide or polynucleotide comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire length of the reference polypeptide or polynucleotide. In some embodiments, a polypeptide or polynucleotide fragment may contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides or amino acids.

[0057] The term“sequence identity” refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. The term“reference sequence” refers to a molecule to which a test sequence is compared.

[0058] “Complementary” refers to the capacity for pairing, through base stacking and specific hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of a nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a target, then the bases are considered to be complementary to each other at that position. Nucleic acids can comprise universal bases, or inert abasic spacers that provide no positive or negative contribution to hydrogen bonding. Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Floogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Nichols et al^ Nature, 1994;369:492-493 and Loakes et al. , Nucleic Acids Res., 1994;22:4039-4043. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U, or T. See Watkins and SantaLucia, Nucl. Acids Research, 2005; 33 (19): 6258-6267.

[0059] As referred to herein, a“complementary nucleic acid sequence” is a nucleic acid sequence comprising a sequence of nucleotides that enables it to non-covalently bind to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength.

[0060] Methods of sequence alignment for comparison and determination of percent sequence identity and percent complementarity are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nafl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), by manual alignment and visual inspection (see, e.g., Brent et al, (2003) Current Protocols in Molecular Biology), by use of algorithms know in the art including the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al, (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0061] The term “modified” refers to a substance or compound (e.g, a cell, a polynucleotide sequence, and/or a polypeptide sequence) that has been altered or changed as compared to the corresponding unmodified substance or compound.

[0062] The term“naturally-occurring” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring.

[0063] “Isolated” refers to a material that is free to varying degrees from components which normally accompany it as found in its native state.

[0064] An“expression cassette” or“expression construct” refers to a DNA polynucleotide sequence operably linked to a promoter.“Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a polynucleotide sequence if the promoter affects the transcription or expression of the polynucleotide sequence.

[0065] The term“recombinant vector” as used herein refers to a polynucleotide molecule capable transferring or transporting another polynucleotide inserted into the vector. The inserted polynucleotide may be an expression cassette. In some embodiments, a recombinant vector may be viral vector or a non-viral vector ( e.g ., a plasmid).

[0066] The term“sample” refers to a biological composition (e.g., a cell or a portion of a tissue) that is subjected to analysis and/or genetic modification. In some embodiments, a sample is a“primary sample” in that it is obtained directly from a subject; in some embodiments, a“sample” is the result of processing of a primary sample, for example to remove certain components and/or to isolate or purify certain components of interest.

[0067] The term“subject” includes animals, such as e.g. mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; or domesticated animals such as dogs and cats. In some embodiments (e.g., particularly in research contexts) subjects are rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like. The terms“subject” and“patient” are used interchangeably herein.

[0068] “Administration” refers herein to introducing an agent or composition into a subject.

[0069] Administration“in combination with” one or more further therapeutic agents includes simultaneous (concurrent) or consecutive administration in any order.

[0070] “Treating” as used herein refers to delivering an agent or composition to a subject to affect a physiologic outcome.

[0071] As used herein, the term“effective amount” refers to at least the minimum amount of an agent or composition required to result in a particular physiological effect. The effective amount of a particular agent may be represented in a variety of ways based on the nature of the agent, such as mass/volume, # of cells/volume, particles/volume, (mass of the agent)/(mass of the subject), # of cells/(mass of subject), or particles/(mass of subject). The effective amount of a particular agent may also be expressed as the half-maximal effective concentration (ECso), which refers to the concentration of an agent that results in a magnitude of a particular physiological response that is half-way between a reference level and a maximum response level.

[0072] “Population” of cells refers to any number of cells greater than 1, but is preferably at least lxlO ³ cells, at least lxlO ⁴ cells, at least at least lxlO ⁵ cells, at least lxlO ⁶ cells, at least lxlO ⁷ cells, at least lxlO ⁸ cells, at least lxlO ⁹ cells, at least lxlO ¹⁰ cells, or more cells. A population of cells may refer to an in vitro population (e.g, a population of cells in culture) or an in vivo population (e.g., a population of cells residing in a particular tissue). [0073] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al, HaRBor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al. , John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

P. NR4A Super-Repressors

[0074] NR4A super-repressors are molecules that bind to the NBRE and/or NurRE response elements but do not activate transcription and therefore inhibit the transcriptional activity of NR4A1, NR4A2, and NR4A3. As described in more detail herein, the super repressors comprise a DNA-binding domain that binds to the NBRE and/or NurRE response elements and may also contain additional domains. The super-repressors do not contain a transcriptional activation domain.

A. DNA-Binding Domains of NR4A Super-Repressors

[0075] A DNA-binding domain that binds to NBRE and/or NurRE response elements can be any type of DNA-binding domain including, e.g., the DNA-binding domain of an NR4A transcription factor, a TAL effector (TALE), or a zinc finger protein.

[0076] The DNA-binding domain (DBD) can comprise the amino acid sequence of the DBD ofNR4Al, NR4A2, orNR4A3. Thus, aDBD can comprise amino acids 264-339 of SEQ ID NO: 1, amino acids 260-335 of SEQ ID NO 2, and/or amino acids 289-364 of SEQ ID NO:3.

[0077] Exemplary DBD sequences comprising the amino acid sequences of the DBD of NR4A1 are shown below as SEQ ID NOs: 50 and 51.

SEGRCAVCGDNASCQHYGVRTCEGCKGFFKRTVQKNAKYICLANKDCPVDKRRRN RC QF CRF QKCLA V GMVKE VVRTD SLKGRRGRLP SKPK (SEQ ID NO: 50)

SEGRCAVCGDNASCQHYGVRTCEGCKGFFKRTVQKNAKYICLANKDCPVDKRRRN RC QF CRF QKCLA V GMVKE VVRTD SLKGRRGRLP SKPKQ (SEQ ID N0 51)

[0078] Exemplary DBD sequences comprising the amino acid sequences of the DBD of NR4A2 are shown below as SEQ ID NOs: 52 and 53.

NEGLCAVCGDNAACQHYGVRTCEGCKGFFKRTVQKNAKYVCLANKNCPVDKRRR NRCQY CRF QKCLAVGMVKE VVRTD SLKGRRGRLP SKPK (SEQ ID NO: 52) NEGLC A VCGDNAAC QHY GVRT CEGCKGFFKRT V QKNAK YV CL ANKN CP VDKRRR NRCQY CRF QKCLAVGMVKEVVRTDSLKGRRGRLPSKPKS (SEQ ID NO:53)

[0079] Exemplary DBD sequences comprising the amino acid sequence of the DBD of NR4A3 are shown below as SEQ ID NO:4 and SEQ ID NO:5.

GEGTC A VCGDNAAC QHY GVRT CEGCKGFFKRT V QKNAK YV CL ANKN CP VDKRRR NRCQY CRF QKCL S V GM VKE VVRTD SLKGRRGRLP SKPK (SEQ ID NO:4)

GEGTC A VCGDNAAC QHY GVRT CEGCKGFFKRT V QKNAK YV CL ANKN CP VDKRRR NRCQY CRF QKCL S V GMVKE VVRTD SLKGRRGRLP SKPKS (SEQ ID NO: 5)

[0080] The DNA-binding domain (DBD) can comprise a TAL. TAL effectors are proteins that are secreted by Xanthomonas bacteria via their type III secretion system when they infect plants. The DNA binding domain contains a repeated, highly conserved, 33 34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and strongly correlated with specific nucleotide recognition. Therefore, the TAL effector domains can be engineered to bind specific target DNA sequences by selecting a combination of repeat segments containing the appropriate RVDs. The nucleic acid specificity for RVD combinations is as follows: HD targets cytosine, NI targets adenenine, NG targets thymine, and NN targets guanine (though, in some embodiments, NN can also bind adenenine with lower specificity).

[0081] As provided herein, a TAL can bind to NBRE and/or NurE.

[0082] A DBD comprising a TAL that is capable of binding NBRE can comprise e.g., the amino acid sequence of SEQ ID NO:6.

MLTPEQVVAIASNHGGKQALETVQRLLPVCQAHGLTPEQVVAIASNIGGKQALETV QRLLP V C Q AHGLTPEQ VVAI ASN GGGKQ ALET V QRLLP V C Q AHGLTPEQ VVAIASNI GGKQALETVQRLLPVCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVCQAHGLTP EQVVAIASNGGGKQALETVQRLLPVCQAHGLTPEQVVAIASNGGGKQALETVQRLL PVCQAHG (SEQ ID NO:6)

[0083] A DBD comprising a TAL that is capable of binding NurE, can comprise e.g., the amino acid sequences of SEQ ID NO:7 and/or 8.

MLTPEQVVAIASNHGGKQALETVQRLLPVCQAHGLTPEQVVAIASNIGGKQALETV QRLLP V C Q AHGLTPEQ VVAI ASN GGGKQ ALET V QRLLP V C Q AHGLTPEQ VVAIASNI GGKQALETVQRLLPVCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVCQAHGLTP EQVVAIASNGGGKQALETVQRLLPVCQAHGLTPEQVVAIASNGGGKQALETVQRLL PVCQAHG (SEQ ID NO:7) LTPEQVVAIASNIGGKQALETVQRLLPVCQAHGLTPEQVVAIASNIGGKQALETVQR LLPVCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVCQAHGLTPEQVVAIASNGGG KQ ALET V QRLLP V C Q AHGLTPEQ VVAIASNHGGKQ ALET V QRLLP VCQ AHGLTPEQ YVAIASHDGGKQALETVQRLLPVCQAHGLTPEQVVAIASHDGGKQALETVQRLLPV CQAHG (SEQ ID NO:8)

[0084] In a TAL comprising the amino acid sequences of SEQ ID NO:7 and 8, the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8 can be connected by a linker. The linker can comprise the amino acid sequence of GGSGNGEGSGNG (SEQ ID NO:9). Accordingly, a TAL that is capable of binding to NurE can comprise the amino acid sequence of SEQ ID NO: 10.

MLTPEQVVAIASNHGGKQALETVQRLLPVCQAHGLTPEQVVAIASNIGGKQALETV QRLLP V C Q AHGLTPEQ VVAIASN GGGKQ ALET V QRLLP V C Q AHGLTPEQ VVAIASNI GGKQALETVQRLLPVCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVCQAHGLTP EQVVAIASNGGGKQALETVQRLLPVCQAHGLTPEQVVAIASNGGGKQALETVQRLL PVCQAHGGGSGNGEGSGNGLTPEQVVAIASNIGGKQALETVQRLLPVCQAHGLTPE Q VVAI ASNIGGKQ ALET V QRLLP V C Q AHGLTPEQ VV AIASNIGGKQ ALET V QRLLP V CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVCQAHGLTPEQVVAIASNHGGKQA LETVQRLLPVCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVCQAHGLTPEQVVAI ASHDGGKQALETVQRLLPVCQAHG (SEQ ID NO: 10)

[0085] The DNA-binding domain (DBD) can comprise a zinc finger protein. A“zinc finger DNA binding domain,”“zinc finger protein,” or“ZFP” is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. Typically, a single zinc finger domain is about 30 amino acids in length. An individual zinc finger binds to a three-nucleotide (i.e., triplet) sequence (or a four-nucleotide sequence which can overlap, by one nucleotide, with the four- nucleotide binding site of an adjacent zinc finger). Therefore the length of a sequence to which a zinc finger binding domain is engineered to bind ( e.g . , a target sequence) will determine the number of zinc fingers in an engineered zinc finger binding domain. For example, for ZFPs in which the finger motifs do not bind to overlapping subsites, a six-nucleotide target sequence is bound by a two-finger binding domain; a nine-nucleotide target sequence is bound by a three- finger binding domain, etc. Binding sites for individual zinc fingers (i.e., subsites) in a target site need not be contiguous, but can be separated by one or several nucleotides, depending on the length and nature of the amino acids sequences between the zinc fingers (i.e., the inter- finger linkers) in a multi-finger binding domain. In some embodiments, the DNA-binding domains of individual ZFNs comprise between three and six individual zinc finger repeats and can each recognize between 9 and 18 base pairs.

[0086] Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:41 1-416. An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection.

[0087] As provided herein, a ZFP can bind to NBRE and/or NurE. A ZFP can bind to the nucleotide sequence AAAGGTCAA (SEQ ID NO:44). A ZFP can bind to the nucleotide sequence GATATT(N)GCCAAT; wherein (N) represents a gap of any nucleotide(s) (SEQ ID NO:45). A ZFP can bind to a nucleotide sequence comprising GATATT (SEQ ID NO:48) and GCCAAT (SEQ ID NO:49), optionally wherein SEQ ID NO:48 is 5' to SEQ ID NO:49.

[0088] A DBD comprising a ZFP can comprise, e.g., the amino acid sequence of SEQ ID NO: l l .

LEPGEKPYKCPEC GK SF S Q S GNLTEHQRTHT GEKP YKCPECGK SF S TS GHL VRHQRT HT GEKP YKCPEC GK SF S QRANLR AHQRTHT GKKT S (SEQ ID NO: 1 1).

[0089] A DBD comprising a ZFP can comprise, e.g., the amino acid sequence of SEQ ID NO : 12 and/or SEQ ID NO : 13.

LEPGEKPYKCPEC GK SF SHKNALQNHQRTHTGEKP YKCPEC GK SF S T S GNL VRHQRT HTGKKTS (SEQ ID NO: 12)

LEPGEKPYKCPEC GK SF SDCRDL ARHQRTHT GEKP YKCPECGK SF S TT GNLT VHQRT HTGKKTS (SEQ ID NO: 13)

[0090] In a zinc finger protein comprising the amino acid sequences of SEQ ID NO: 12 and 13, the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13 can be connected by a linker. The linker can comprise the amino acid sequence of GGSGNGEGSGNG (SEQ ID NO:9). Accordingly, a zinc finger protein can comprise the amino acid sequence of SEQ ID NO: 14.

LEPGEKPYKCPEC GK SF SHKNALQNHQRTHTGEKP YKCPEC GK SF S T S GNL VRHQRT HT GKKT S GGS GNGEGS GN GLEPGEKP YKCPECGK SF SDCRDL ARHQRTHT GEKP YK CPECGKSFSTTGNLTVHQRTHTGKKTS (SEQ ID NO: 14). [0091] The DNA-binding domain (DBD) can comprise a sequence that is at least 90, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs:4-8, and 10-14, to amino acids 264- 339 of SEQ ID NO: l, to amino acids 260-335 of SEQ ID NO:2, or to amino acids 289-364 of SEQ ID NO:3. The DBD can comprise the sequence of any one of SEQ ID NOs:4-8, and 10- 14 or amino acids 264-339 of SEQ ID NO: l, amino acids 260-335 of SEQ ID NO:2, or amino acids 289-364 of SEQ ID NO: 3

B. Other Domains of NR4A Super-Repressors

[0092] An NR4A super-repressor can contain domains in addition to the DNA-binding domain (DBD), including e.g., domains that increase the ability of the DNA-binding domain to inhibit the transcriptional activity ofNR4Al, NR4A2, and/or NR4A3.

[0093] An NR4A super-repressor can contain the ligand binding domain (LBD) of NR4A1 ,

NR4A2, and/or NR4A3. Thus, an LBD can comprise amino acids 360-595 of SEQ ID NO: 1, amino acids 360-595 of SEQ ID NO:2, and/or amino acids 394-623 of SEQ ID NO:3. An exemplary LBD can comprising the amino acid sequence of SEQ ID NO: 15 or 16.

PLQQEPSQPSPPSPPICMMNALVRALTDSTPRDLDYSRYCPTDQAAAGTDAEHVQQF YNLLTASIDVSRSWAEKLPGFTDLPKEDQTLLIESAFLELFVLRLSIRSNTAEDKFVFC NGLVLHRLQCLRGFGEWLDSIKDFSLNLQSLNLDIQALACLSALSMITERHGLKEPKR VEELCNKITSSLKDHQSKGQALEPTESKVLGALVELRKICTLGLQRIFYLKLEDLVSPP SIIDKLFLDTLPF (SEQ ID NO: 15)

SPLQQEPSQPSPPSPPICMMNALVRALTDSTPRDLDYSRYCPTDQAAAGTDAEHVQQ FYNLLTASIDVSRSWAEKLPGFTDLPKEDQTLLIESAFLELFVLRLSIRSNTAEDKFVFC NGLVLHRLQCLRGFGEWLDSIKDFSLNLQSLNLDIQALACLSALSMITERHGLKEPKR VEELCNKITSSLKDHQSKGQALEPTESKVLGALVELRKICTLGLQRIFYLKLEDLVSPP SIIDKLFLDTLPF (SEQ ID NO: 16)

[0094] The ligand-binding domain (LBD) can comprise a sequence that is at least 90, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, to SEQ ID NO: 16, to amino acids 360- 595 of SEQ ID NO: l, to amino acids 360-595 of SEQ ID NO:2, or to amino acids 394-623 of SEQ ID NO:3.

[0095] The LBD can be N-terminal or C-terminal to the DBD. The LBD can be linked directly to the N-terminal or C-terminal of the DBD or can be linked to the N- or C-terminus of the DBD via a linker and/or another domain.

[0096] An NR4A super-repressor can comprise a Flag domain, e.g., comprising the amino acid sequence of MD YKDHD GD YKDHDID YKDDDDK (SEQ ID NO: 17). [0097] The FLAG domain can comprise a sequence that is at least 90, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17.

[0098] The Flag domain can be N-terminal or C-terminal to the DBD. The Flag domain can be linked directly to the N-terminal or C-terminal of the DBD or can be linked to the N- or C-terminus of the DBD via a linker and/or another domain.

[0099] An NR4A super-repressor can comprise a Kriippel-associated box (KRAB) domain, e.g., comprising the amino acid sequence of

MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEP (SEQ ID NO: 18).

[0100] The KRAB domain can comprise a sequence that is at least 90, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18.

[0101] The KRAB domain can be N-terminal or C-terminal to the DBD. The KRAB domain can be linked directly to the N-terminal or C-terminal of the DBD or can be linked to the N- or C-terminus of the DBD via a linker and/or another domain.

[0102] An NR4A super-repressor can comprise an NCOR domain, e.g., comprising the amino acid sequence of

MNMNGLMEDPMKVYKDRQFMNVWTDHEKEIFKDKFIQHPKNFGLIASYLERKSVP DCVLYYYLTKKNENYK (SEQ ID NO: 19) or

MSSSGYPPNQGAFSTEQSRYPPHSVQYTFPNTRHQQEFAVPDYRSSHLEVSQASQLL

QQQQQQQLRRRPSLLSEFHPGSDRPQERRTSYEPFHPGPSPVDHDSLESKRPRLEQV S

DSHFQRVSAAVLPLVHPLPEGLRASADAKKDPAFGGKHEAPSSPISGQPCGDDQNAS

PSKLSKEELIQSMDRVDREIAKVEQQILKLKKKQQQLEEEAAKPPEPEKPVSPPPVE Q

KHRSIVQIIYDENRKKAEEAHKIFEGLGPKVELPLYNQPSDTKVYHENIKTNQYMRK

KLILFFKRRNHARKQREQKICQRYD (SEQ ID NO:20).

[0103] The NCOR domain can comprise a sequence that is at least 90, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19 or 20.

[0104] The NCOR domain can be N-terminal or C-terminal to the DBD. The NCOR domain can be linked directly to the N-terminal or C-terminal of the DBD or can be linked to the N- or C-terminus of the DBD via a linker and/or another domain.

[0105] An NR4A super-repressor can comprise a self-cleavage domain, e.g., a T2A domain. A T2A domain can comprise the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:21).

[0106] The self-cleavage domain can comprise a sequence that is at least 90, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21. [0107] The self-cleavage domain can be N-terminal or C-terminal to the DBD. The self cleavage domain can be linked directly to the N-terminal or C-terminal of the DBD or can be linked to the N- or C-terminus of the DBD via a linker and/or another domain. The self cleavage domain can be between a DBD and an LBD.

[0108] An NR4A super-repressor can comprise a nuclear localization signal. A nuclear localization signal can comprise the amino acid sequence PAAKRVKLD (SEQ ID NO:22).

[0109] The nuclear localization signal can comprise a sequence that is at least 90, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:22.

[0110] The nuclear localization signal can be N-terminal or C-terminal to the DBD. The nuclear localization signal can be linked directly to the N-terminal or C-terminal of the DBD or can be linked to the N- or C-terminus of the DBD via a linker and/or another domain. The nuclear localization signal can be, e.g., in a super-repressor comprising a zinc finger DBD or a TAL DBD.

[0111] An NR4A super-repressor can comprise a dimerization domain, for example, a diZIP dimerization domain. A dimerization domain can increase the affinity of a super repressor for a dimeric binding site. A dimerization domain can comprise the amino acid sequence S QDP AAAMKQLEDK VEELL SKNYHLENEVARLTKL V (SEQ ID NO:23).

[0112] The dimerization domain can comprise a sequence that is at least 90, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:23.

[0113] The dimerization domain can be N-terminal or C-terminal to the DBD. The dimerization domain can be linked directly to the N-terminal or C-terminal of the DBD or can be linked to the N- or C-terminus of the DBD via a linker and/or another domain.

[0114] An NR4A super-repressor can comprise a transcriptional repressor domain and/or a chromatin compaction domain. The transcriptional repressor domain and/or chromatin compaction domain can comprise the amino acid sequence of SEQ ID NO:24.

MTMGDKK SPTRPKRQ AKPAADEGF WDC S VCTFRNS AEAFKC SICD VRKGT STRKPR INSQLVAQQVAQQYATPPPPKKEKKEKVEKQDKEKPEKDKEISPSVTKKNTNKKTKP KSDILKDPP SEANSIQ S ANATTKT SETNHTSRPRLKNVDRST AQQLAVT VGNVT VIIT DFKEKTRS S STS S STVTS S AGSEQQNQ S S SGSESTDKGS SRS STPKGDMS AVNDESF

(SEQ ID NO: 24)

[0115] The transcriptional repressor domain and/or a chromatin compaction domain can comprise a sequence that is at least 90, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24. [0116] The transcriptional repressor domain and/or a chromatin compaction domain can be N-terminal or C-terminal to the DBD. The transcriptional repressor domain and/or a chromatin compaction domain can be linked directly to the N-terminal or C-terminal of the DBD or can be linked to the N- or C-terminus of the DBD via a linker and/or another domain.

[0117] An NR4A super-repressor can comprise an epitope tag e.g., FLAG, V5, HA, etc. that can be useful for measuring the abundance of the super-repressor. The epitope tag can be N-terminal or C-terminal to the DBD. The epitope tag can be linked directly to the N-terminal or C-terminal of the DBD or can be linked to the N- or C-terminus of the DBD via a linker and/or another domain.

[0118] A super-repressor can contain any combination of the above specified domains in any order.

[0119] The various domains of NR4A super-repressors can be directly linked or can be connected via a linker. The linker can comprise, for example, GGSGNGEGSGNG (SEQ ID NO: 9), GGSG (S-linker; SEQ ID NO: 25), GGSGNGGSG (M-linker; SEQ ID NO:26), GGSGNGEGSGNG (L-linker; SEQ ID N0 27), or GSETPGTSESATPES (XTEN; SEQ ID NO:28).

C. Exemplary Super-Repressors

[0120] The amino acid sequences of certain exemplary NR4A super-repressors are provided in Table 1 below.

[0121] A super-repressor can comprise a sequence that is at least 90, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:29-43. The amino acid sequence of a super repressor can comprise, consist essentially of, or consist of the amino acid sequence of any one of SEQ ID NOs: 29-43.

PI. Modified Immune Effector Cells

[0122] In some embodiments, the present disclosure provides modified immune effector cells. Herein, the term“modified immune effector cells” encompasses immune effector cells comprising a polynucleotide encoding a NR4A super-repressor and immune effector cells comprising one or more R4A super-repressors. Herein, an“un-modified immune effector cell” or“control immune effector cell” refers to a cell or population of cells wherein the cells do not comprise a polynucleotide encoding a NR4A super-repressor or a NR4A super repressor.

[0123] The term“immune effector cell” refers to cells involved in mounting innate and adaptive immune responses, including but not limited to lymphocytes (such as T-cells (including thymocytes) and B-cells), natural killer (NK) cells, KT cells, macrophages, monocytes, eosinophils, basophils, neutrophils, dendritic cells, and mast cells. In some embodiments, the modified immune effector cell is a T cell, such as a CD4+ T cell, a CD8+ T cell (also referred to as a cytotoxic T cell or CTL), a regulatory T cell (Treg), a Thl cell, a Th2 cell, or a Thl7 cell.

[0124] In some embodiments, the modified immune effector cell is a T cell. In some embodiments, T cells are isolated from a subject, expanded ex vivo, and re-infused into a subject. In some embodiments, T cells are modified to express one or more exogenous receptors specific for an autologous tumor antigen, expanded ex vivo, and re-infused into the subject. Such embodiments can be modeled using in vivo mouse models wherein mice have been transplanted with a cancer cell line expressing a cancer antigen ( e.g ., CD 19) and are treated with modified T cells that express an exogenous receptor that is specific for the cancer antigen

[0125] In some embodiments, the modified immune effector cell is a T cell that has been isolated from a tumor sample (referred to herein as“tumor infiltrating lymphocytes” or “TILs”). Without wishing to be bound by theory, it is thought that TILs possess increase specificity to tumor antigens (Radvanyi et al., 2012 Clin Cane Res 18:6758-6770) and can therefore mediate tumor antigen-specific immune response (e.g., activation, proliferation, and cytotoxic activity against the cancer cell) leading to cancer cell destruction (Brudno etal., 2018 Nat Rev Clin One 15:31-46)) without the introduction of an exogenous engineered receptor. Therefore, in some embodiments, TILs are isolated from a tumor in a subject, expanded ex vivo, and re-infused into a subject. In some embodiments, TILs are modified to express one or more exogenous receptors specific for an autologous tumor antigen, expanded ex vivo, and re- infused into the subject. Such embodiments can be modeled using in vivo mouse models wherein mice have been transplanted with a cancer cell line expressing a cancer antigen (e.g, CD 19) and are treated with modified T cells that express an exogenous receptor that is specific for the cancer antigen.

[0126] In some embodiments, the modified immune effector cell is an animal cell or is derived from an animal cell, including invertebrate animals and vertebrate animals (e.g, fish, amphibian, reptile, bird, or mammal). In some embodiments, the modified immune effector cell is a mammalian cell oris derived from a mammalian cell (e.g., a pig, a cow, a goat, a sheep, a rodent, a non-human primate, a human, etc ). In some embodiments, the modified immune effector cell is a rodent cell or is derived from a rodent cell (e.g, a rat or a mouse). In some embodiments, the modified immune effector cell is a human cell or is derived from a human cell.

[0127] In some embodiments, the modified immune effector cells comprise a polynucleotide encoding a NR4A super-repressor or a NR4A super-repressor that reduces the transcriptional activity of endogenous NR4A1, NR4A2, and/or NR4A3. In some embodiments, a polynucleotide sequence encoding a NR4A super-repressor is inserted at one or more locations in the genome.

[0128] In some embodiments, the expression of one or more NR4A target genes in a modified immune effector cell is reduced by at least 5% compared to the expression of the NR4A target genes in an unmodified immune effector cell. In some embodiments, the expression of the one or more NR4A target genes in a modified immune effector cell is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to the expression of the NR4A target genes in an unmodified immune effector cell. In some embodiments, the modified immune effector cells described herein demonstrate reduced expression and/or function of gene products encoded by a plurality ( e.g ., two or more) of endogenous NR4A target genes compared to the expression of the gene products in an unmodified immune effector cell. For example, in some embodiments, a modified immune effector cell demonstrates reduced expression and/or function of gene products from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more endogenous NR4A target genes compared to the expression of the gene products in an unmodified immune effector cell.

[0129] In some embodiments, one or more polynucleotides encoding the NR4A super repressor are inserted into the genome of the immune effector cell. In some embodiments, one or more polynucleotides encoding the NR4A super-repressor are expressed episomaly and are not inserted into the genome of the immune effector cell.

[0130] In some embodiments, the modified immune effector cells described herein comprise a polynucleotide encoding a super-repressor or a super-repressor and further comprise one or more exogenous transgenes inserted at one or more genomic loci (e.g., a genetic“knock-in”). In some embodiments, the one or more exogenous transgenes encode detectable tags, safety-switch systems, chimeric switch receptors, and/or engineered antigen- specific receptors.

[0131] In some embodiments, the modified immune effector cells described herein further comprise an exogenous transgene encoding a detectable tag. Examples of detectable tags include but are not limited to, FLAG tags, poly-histidine tags (e.g. 6xHis), SNAP tags, Halo tags, cMyc tags, glutathione-S-transferase tags, avidin, enzymes, fluorescent proteins, luminescent proteins, chemiluminescent proteins, bioluminescent proteins, and phosphorescent proteins. In some embodiments the fluorescent protein is selected from the group consisting of blue/UV proteins (such as BFP, TagBFP, mTagBFP2, Azurite, EBFP2, mKalamal, Sirius, Sapphire, and T-Sapphire); cyan proteins (such as CFP, eCFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, and mTFPl); green proteins (such as: GFP, eGFP, meGFP (A208K mutation), Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, and mNeonGreen); yellow proteins (such as YFP, eYFP, Citrine, Venus, SYFP2, and TagYFP); orange proteins (such as Monomeric Kusabira-Orange, hiKOk, mK02, mOrange, and mOrange2); red proteins (such as RFP, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2); far-red proteins (such as mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP); near-infrared proteins (such as TagRFP657, IFP1.4, and iRFP); long stokes shift proteins (such as mKeima Red, LSS-mKatel, LSS-mKate2, and mBeRFP); photoactivatible proteins (such as PA-GFP, PAmCherryl, and PATagRFP); photoconvertible proteins (such as Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS-CFP2, PS- CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PSmOrange, and PSmOrange); and photoswitchable proteins (such as Dronpa). In some embodiments, the detectable tag can be selected from AmCyan, AsRed, DsRed2, DsRed Express, E2-Crimson, HcRed, ZsGreen, ZsYellow, mCherry, mStrawberry, mOrange, mBanana, mPlum, mRasberry, tdTomato, DsRed Monomer, and/or AcGFP, all of which are available from Clontech.

[0132] In some embodiments, the modified immune effector cells described herein further comprise an exogenous transgene encoding a safety-switch system. Safety-switch systems (also referred to in the art as suicide gene systems) comprise exogenous transgenes encoding for one or more proteins that enable the elimination of a modified immune effector cell after the cell has been administered to a subject. Examples of safety-switch systems are known in the art. For example, safety-switch systems include genes encoding for proteins that convert non-toxic pro-drugs into toxic compounds such as the Herpes simplex thymidine kinase (Hsv- tk ) and ganciclovir (GCV) system (HSV-Z JCV). Hsv-/& converts non-toxic GCV into a cytotoxic compound that leads to cellular apoptosis. As such, administration of GCV to a subject that has been treated with modified immune effector cells comprising a transgene encoding the Hsv-/C protein can selectively eliminate the modified immune effector cells while sparing endogenous immune effector cells. ( See e.g., Bonini et al., Science, 1997, 276(5319): 1719-1724; Ciceri et al, Blood, 2007, 109(11): 1828- 1836; Bondanza et al, Blood 2006, 107(5): 1828-1836).

[0133] Additional safety-switch systems include genes encoding for cell-surface markers, enabling elimination of modified immune effector cells by administration of a monoclonal antibody specific for the cell-surface marker via ADCC. In some embodiments, the cell-surface marker is CD20 and the modified immune effector cells can be eliminated by administration of an anti-CD20 monoclonal antibody such as Rituximab (See e.g., Introna et al, Hum Gene Ther, 2000, 11 (4): 611 -620; Serafmi et al., Hum Gene Ther, 2004, 14, 63-76; van Meerten et al, Gene Ther, 2006, 13, 789-797). Similar systems using EGF-R and Cetuximab or Panitumumab are described in International PCT Publication No. WO 2018006880. Additional safety-switch systems include transgenes encoding pro-apoptotic molecules comprising one or more binding sites for a chemical inducer of dimerization (CID), enabling elimination of modified immune effector cells by administration of a CID which induces oligomerization of the pro-apoptotic molecules and activation of the apoptosis pathway. In some embodiments, the pro-apoptotic molecule is Fas (also known as CD95) (Thomis et al. , Blood, 2001, 97(5), 1249-1257). In some embodiments, the pro-apoptotic molecule is caspase-9 (Straathof et al., Blood, 2005, 105(11), 4247-4254).

[0134] In some embodiments, the modified immune effector cells described herein further comprise an exogenous transgene encoding a chimeric switch receptor. Chimeric switch receptors are engineered cell-surface receptors comprising an extracellular domain from an endogenous cell-surface receptor and a heterologous intracellular signaling domain, such that ligand recognition by the extracellular domain results in activation of a different signaling cascade than that activated by the wild type form of the cell-surface receptor. In some embodiments, the chimeric switch receptor comprises the extracellular domain of an inhibitory cell-surface receptor fused to an intracellular domain that leads to the transmission of an activating signal rather than the inhibitory signal normally transduced by the inhibitory cell- surface receptor. In particular embodiments, extracellular domains derived from cell-surface receptors known to inhibit immune effector cell activation can be fused to activating intracellular domains. Engagement of the corresponding ligand will then activate signaling cascades that increase, rather than inhibit, the activation of the immune effector cell. For example, in some embodiments, the modified immune effector cells described herein comprise a transgene encoding a PD1-CD28 switch receptor, wherein the extracellular domain of PD1 is fused to the intracellular signaling domain of CD28 {See e.g. , Liu et al, Cancer Res 76:6 (2016), 1578-1590 and Moon et al., Molecular Therapy 22 (2014), S201). In some embodiments, the modified immune effector cells described herein comprise a transgene encoding the extracellular domain of CD200R and the intracellular signaling domain of CD28 (See Oda et al, Blood 130:22 (2017), 2410-2419).

[0135] In some embodiments, the modified immune effector cells described herein further comprise an engineered antigen-specific receptor recognizing a protein target expressed by a target cell, such as a tumor cell or an antigen presenting cell (APC), referred to herein as “modified receptor-engineered cells” or“modified RE-cells”. The term“engineered antigen receptor” refers to a non-naturally occurring antigen-specific receptor such as a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR). In some embodiments, the engineered antigen receptor is a CAR comprising an extracellular antigen binding domain fused via hinge and transmembrane domains to a cytoplasmic domain comprising a signaling domain. In some embodiments, the CAR extracellular domain binds to an antigen expressed by a target cell in an MHC-independent manner leading to activation and proliferation of the RE cell. In some embodiments, the extracellular domain of a CAR recognizes a tag fused to an antibody or antigen-binding fragment thereof. In such embodiments, the antigen-specificity of the CAR is dependent on the antigen-specificity of the labeled antibody, such that a single CAR construct can be used to target multiple different antigens by substituting one antibody for another (See e.g ., US Patent Nos. 9,233,125 and 9,624,279; US Patent Application Publication Nos. 20150238631 and 20180104354). In some embodiments, the extracellular domain of a CAR may comprise an antigen binding fragment derived from an antibody. Antigen binding domains that are useful in the present disclosure include, for example, scFvs; antibodies; antigen binding regions of antibodies; variable regions of the heavy/light chains; and single chain antibodies.

[0136] In some embodiments, the intracellular signaling domain of a CAR may be derived from the TCR complex zeta chain (such as 093x signaling domains), FcyRIII, FccRI, or the T-lymphocyte activation domain. In some embodiments, the intracellular signaling domain of a CAR further comprises a costimulatory domain, for example a 4-1BB, CD28, CD40, MyD88, or CD70 domain. In some embodiments, the intracellular signaling domain of a CAR comprises two costimulatory domains, for example any two of 4-1BB, CD28, CD40, MyD88, or CD70 domains. Exemplary CAR structures and intracellular signaling domains are known in the art (See e.g., WO 2009/091826; US 20130287748; WO 2015/142675; WO 2014/055657; and WO 2015/090229, incorporated herein by reference).

[0137] CARs specific for a variety of tumor antigens are known in the art, for example CD171-specific CARs (Park etal, Mol Ther (2007) 15(4):825-833), EGFRvIII-specific CARs (Morgan et al., Hum Gene Ther (2012) 23(10): 1043-1053), EGF-R-specific CARs (Kobold et al, JNatl Cancer Inst (2014) 107(1):364), carbonic anhydrase K-specific CARs (Lamers etal., Biochem Soc Trans (2016) 44(3):951-959), FR-a-specific CARs (Kershaw et al., Clin Cancer Res (2006) 12(20):6106-6015), HER2-specific CARs (Ahmed et al, J Clin Oncol (2015) 33(15)1688- 1696;Nakazawa et al, Mol Ther (2011) 19(12):2133-2143; Ahmed et al, Mol Ther (2009) 17(10): 1779-1787; Luo et al, Cell Res (2016) 26(7):850-853; Morgan et al, Mol Ther (2010) 18(4): 843-851 ; Grada et al, Mol Ther Nucleic Acids (2013) 9(2):32), CEA- specific CARs (Katz et al, Clin Cancer Res (2015) 21(14):3149-3159), IL13Ra2-specific CARs (Brown et al, Clin Cacner Res (2015) 21(18):4062-4072), GD2-specific CARs (Louis et al, Blood (2011) 118(23):6050-6056; Caruana et al, Nat Med (2015) 21(5):524-529), ErbB2-specific CARs (Wilkie et al, J Clin Immunol (2012) 32(5): 1059-1070), VEGF-R- specific CARs (Chinnasamy et al ., Cancer Res (2016) 22(2):436-447), FAP-specific CARs (Wang et al., Cancer Immunol Res (2014) 2(2): 154- 166), MSLN-specific CARs (Moon et al, Clin Cancer Res (2011) 17(14):4719-30), NKG2D-specific CARs (VanSeggelen et al., Mol Ther (2015) 23(10): 1600-1610), CD19-specific CARs (Axicabtagene ciloleucel (Yescarta ^® ) and Tisagenlecleucel (Kymriah ^® ). See also _, Li et al., J Hematol and Oncol (2018) 11(22), reviewing clinical trials of tumor- specific CARs.

[0138] In some embodiments, the engineered antigen receptor is an engineered TCR. Engineered TCRs comprise TCRa and/or TCRfi chains that have been isolated and cloned from T cell populations recognizing a particular target antigen. For example, TCRa and/or TCRp genes (i.e., TRAC and TRBC ) can be cloned from T cell populations isolated from individuals with particular malignancies or T cell populations that have been isolated from humanized mice immunized with specific tumor antigens or tumor cells. Engineered TCRs recognize antigen through the same mechanisms as their endogenous counterparts ( e.g by recognition of their cognate antigen presented in the context of major histocompatibility complex (MFIC) proteins expressed on the surface of a target cell). This antigen engagement stimulates endogenous signal transduction pathways leading to activation and proliferation of the TCR-engineered cells.

[0139] Engineered TCRs specific for tumor antigens are known in the art, for example WT1 -specific TCRs (JTCR016, Juno Therapeutics; WTl-TCRc4, described in US Patent Application Publication No. 20160083449), MART-1 specific TCRs (including the DMF4T clone, described in Morgan et al., Science 314 (2006) 126-129); the DMF5T clone, described in Johnson et al., Blood 114 (2009) 535-546); and the ID3T clone, described in van den Berg et al., Mol. Ther. 23 (2015) 1541-1550), gp 100-specific TCRs (Johnson et al., Blood 114 (2009) 535-546), CEA-specific TCRs (Parkhurst et al, Mol Ther. 19 (2011) 620-626), NY- ESO and LAGE-1 specific TCRs (1G4T clone, described in Robbins et al, J Clin Oncol 26 (2011) 917-924; Robbins et al, Clin Cancer Res 21 (2015) 1019-1027; and Rapoport et al, Nature Medicine 21 (2015) 914-921), and MAGE-A3-specific TCRs (Morgan et al, J Immunother 36 (2013) 133-151) and Linette et al, Blood 122 (2013) 227-242). (See also, Debets et al, Seminars in Immunology 23 (2016) 10-21).

[0140] In some embodiments, the engineered antigen receptor is directed against a target antigen selected from a cluster of differentiation molecule, such as CD3, CD4, CD8, CD 16, CD24, CD25, CD33, CD34, CD45, CD64, CD71, CD78, CD80 (also known as B7-1), CD86 (also known as B7-2), CD96, , CD116, CD117, CD123, CD133, and CD138, CD371 (also known as CLL1); a tumor-associated surface antigen, such as 5T4, BCMA (also known as CD269 and TNFRSF17, UniProt# Q02223), carcinoembryonic antigen (CEA), carbonic anhydrase 9 (CAIX or MN/CAIX), CD19, CD20, CD22, CD30, CD40, disialogangliosides such as GD2, ELF2M, ductal-epithelial mucin, ephrin B2, epithelial cell adhesion molecule (EpCAM), ErbB2 (HER2/neu), FCRL5 (UniProt# Q68SN8), FKBP11 (UniProt# Q9NYL4), glioma-associated antigen, glycosphingolipids, gp36, GPRC5D (UniProt# Q9NZD1), mut hsp70-2, intestinal carboxyl esterase, IGF-I receptor, ITGA8 (UniProt# P53708), KAMP3, LAGE-la, MAGE, mesothelin, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, PAP, prostase, prostate-carcinoma tumor antigen-1 (PCTA-1), prostate specific antigen (PSA), PSMA, prostein, RAGE-1, ROR1, RU1 (SFMBT1), RU2 (DCDC2), SLAMF7 (UniProt# Q9NQ25), survivin, TAG-72, and telomerase; a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope; tumor stromal antigens, such as the extra domain A (EDA) and extra domain B (EDB) of fibronectin; the A1 domain of tenascin-C (TnC Al) and fibroblast associated protein (FAP); cytokine receptors, such as epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), TFG -R or components thereof such as endoglin; a major histocompatibility complex (MHC) molecule; a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gpl20); an EBV-specific antigen, a CMV-specific antigen, a FlPV-specific antigen, a Lassa virus-specific antigen, an Influenza virus-specific antigen as well as any derivate or variant of these surface antigens.

A. Effector functions

[0141] In some embodiments, the modified immune effector cells described herein demonstrate an increase in one or more immune cell effector functions. Herein, the term “effector function” refers to functions of an immune cell related to the generation, maintenance, and/or enhancement of an immune response against a target cell or target antigen. In some embodiments, the modified immune effector cells described herein demonstrate one or more of the following characteristics compared to an unmodified immune effector cell: increased infiltration or migration in to a tumor, increased proliferation, increased or prolonged cell viability, increased resistance to inhibitory factors in the surrounding microenvironment such that the activation state of the cell is prolonged or increased, increased production of pro- inflammatory immune factors ( e.g ., pro-inflammatory cytokines, chemokines, and/or enzymes), increased cytotoxicity, and/or increased resistance to exhaustion.

[0142] In some embodiments, the modified immune effector cells described herein demonstrate increased infiltration into a tumor compared to an unmodified immune effector cell. In some embodiments, increased tumor infiltration by modified immune effector cells refers to an increase the number of modified immune effector cells infiltrating into a tumor during a given period of time compared to the number of unmodified immune effector cells that infiltrate into a tumor during the same period of time. In some embodiments, the modified immune effector cells demonstrate a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more fold increase in tumor filtration compared to an unmodified immune cell. Tumor infiltration can be measured by isolating one or more tumors from a subject and assessing the number of modified immune cells in the sample by flow cytometry, immunohistochemistry, and/or immunofluorescence.

[0143] In some embodiments, the modified immune effector cells described herein demonstrate an increase in cell proliferation compared to an unmodified immune effector cell. In these embodiments, the result is an increase in the number of modified immune effector cells present compared to unmodified immune effector cells after a given period of time. For example, in some embodiments, modified immune effector cells demonstrate increased rates of proliferation compared to unmodified immune effector cells, wherein the modified immune effector cells divide at a more rapid rate than unmodified immune effector cells. In some embodiments, the modified immune effector cells demonstrate a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,

1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more fold increase in the rate of proliferation compared to an unmodified immune cell. In some embodiments, modified immune effector cells demonstrate prolonged periods of proliferation compared to unmodified immune effector cells, wherein the modified immune effector cells and unmodified immune effector cells divide at similar rates, but wherein the modified immune effector cells maintain the proliferative state for a longer period of time. In some embodiments, the modified immune effector cells maintain a proliferative state for 1.1,

1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,

45, 50, 60, 70, 80, 90, 100, or more times longer than an unmodified immune cell.

[0144] In some embodiments, the modified immune effector cells described herein demonstrate increased or prolonged cell viability compared to an unmodified immune effector cell. In such embodiments, the result is an increase in the number of modified immune effector cells or present compared to unmodified immune effector cells after a given period of time. For example, in some embodiments, modified immune effector cells described herein remain viable and persist for 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more times longer than an unmodified immune cell. [0145] In some embodiments, the modified immune effector cells described herein demonstrate increased resistance to inhibitory factors compared to an unmodified immune effector cell. Exemplary inhibitory factors include signaling by immune checkpoint molecules (e.g., PD1, PDL1, CTLA4, LAG3, IDO) and/or inhibitory cytokines (e.g., IL-10, TGFp).

[0146] In some embodiments, the modified T cells described herein demonstrate increased resistance to T cell exhaustion compared to an unmodified T cell. T cell exhaustion is a state of antigen-specific T cell dysfunction characterized by decreased effector function and leading to subsequent deletion of the antigen-specific T cells. In some embodiments, exhausted T cells lack the ability to proliferate in response to antigen, demonstrate decreased cytokine production, and/or demonstrate decreased cytotoxicity against target cells such as tumor cells. In some embodiments, exhausted T cells are identified by altered expression of cell surface markers and transcription factors, such as decreased cell surface expression of CD122 and CD 127; increased expression of inhibitory cell surface markers such as PD1, LAG3, CD244, CD 160, TIM3, and/or CTLA4; and/or increased expression of transcription factors such as Blimpl, NFAT, and/or BATF. In some embodiments, exhausted T cells demonstrate altered sensitivity cytokine signaling, such as increased sensitivity to TGFp signaling and/or decreased sensitivity to IL-7 and IL-15 signaling. T cell exhaustion can be determined, for example, by co-culturing the T cells with a population of target cells and measuring T cell proliferation, cytokine production, and/or lysis of the target cells. In some embodiments, the modified immune effector cells described herein are co-cultured with a population of target cells (e.g., autologous tumor cells or cell lines that have been engineered to express a target tumor antigen) and effector cell proliferation, cytokine production, and/or target cell lysis is measured. These results are then compared to the results obtained from co-culture of target cells with a control population of immune cells (such as unmodified immune effector cells or immune effector cells that have a control modification).

[0147] In some embodiments, resistance to T cell exhaustion is demonstrated by increased production of one or more cytokines (e.g, IGNg, TNFa, or IL-2) from the modified immune effector cells compared to the cytokine production observed from the control population of immune cells. In some embodiments, a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more fold increase in cytokine production from the modified immune effector cells compared to the cytokine production from the control population of immune cells is indicative of an increased resistance to T cell exhaustion. In some embodiments, resistance to T cell exhaustion is demonstrated by increased proliferation of the modified immune effector cells compared to the proliferation observed from the control population of immune cells. In some embodiments, a 1.1, 1.2, 1.3,

1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50,

60, 70, 80, 90, 100 or more fold increase in proliferation of the modified immune effector cells compared to the proliferation of the control population of immune cells is indicative of an increased resistance to T cell exhaustion. In some embodiments, resistance to T cell exhaustion is demonstrated by increased target cell lysis by the modified immune effector cells compared to the target cell lysis observed by the control population of immune cells. In some embodiments, a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9,

10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more fold increase in target cell lysis by the modified immune effector cells compared to the target cell lysis by the control population of immune cells is indicative of an increased resistance to T cell exhaustion.

[0148] In some embodiments, exhaustion of the modified immune effector cells compared to control populations of immune cells is measured during the in vitro or ex vivo manufacturing process. For example, in some embodiments, TILs isolated from tumor fragments are modified according to the methods described herein and then expanded in one or more rounds of expansion to produce a population of modified TILs. In such embodiments, the exhaustion of the modified TILs can be determined immediately after harvest and prior to a first round of expansion, after the first round of expansion but prior to a second round of expansion, and/or after the first and the second round of expansion. In some embodiments, exhaustion of the modified immune effector cells compared to control populations of immune cells is measured at one or more time points after transfer of the modified immune effector cells into a subject. For example, in some embodiments, the modified cells are produced according to the methods described herein and administered to a subject. Samples can then be taken from the subject at various time points after the transfer to determine exhaustion of the modified immune effector cells in vivo over time.

[0149] In some embodiments, the modified immune effector cells described herein demonstrate increased expression or production of pro-inflammatory immune factors compared to an unmodified immune effector cell. Examples of pro-inflammatory immune factors include cytolytic factors, such as granzyme B, perforin, and granulysin; and pro- inflammatory cytokines such as interferons (IFNa, IGNb, IGNg), TNFa, IL-Ib, IL-12, IL-2, EL- 17, CXCL8, and/or IL-6.

[0150] In some embodiments, the modified immune effector cells described herein demonstrate increased cytotoxicity against a target cell compared to an unmodified immune effector cell. In some embodiments, the modified immune effector cells demonstrate a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold increase in cytotoxicity against a target cell compared to an unmodified immune cell.

[0151] Assays for measuring immune effector function are known in the art. For example, tumor infiltration can be measured by isolating tumors from a subject and determining the total number and/or phenotype of the lymphocytes present in the tumor by flow cytometry, immunohistochemistry, and/or immunofluorescence. Cell-surface receptor expression can be determined by flow cytometry, immunohistochemistry, immunofluorescence, Western blot, and/or qPCR. Cytokine and chemokine expression and production can be measured by flow cytometry, immunohistochemistry, immunofluorescence, Western blot, ELISA, and/or qPCR. Responsiveness or sensitivity to extracellular stimuli ( e.g ., cytokines, inhibitory ligands, or antigen) can be measured by assaying cellular proliferation and/or activation of downstream signaling pathways (e.g., phosphorylation of downstream signaling intermediates) in response to the stimuli. Cytotoxicity can be measured by target-cell lysis assays known in the art, including in vitro or ex vivo co-culture of the modified immune effector cells with target cells and in vivo murine tumor models.

IV. Methods of producing modified immune effector cells

[0152] In some embodiments, the present disclosure provides methods for producing modified immune effector cells. In some embodiments, the methods comprise introducing a polynucleotide encoding a NR4A super-repressor or a R4A super-repressor into a population of immune effector cells wherein the NR4A super-repressor is capable of reducing the transcriptional activity ofNR4Al, NR4A2, and/or NR4A3.

[0153] The polynucleotide encoding a NR4A super-repressor or the R4A super-repressor can be introduced into target cells in a variety of forms using a variety of delivery methods and formulations. In some embodiments, a polynucleotide encoding a R4A super-repressor is delivered by a recombinant vector (e.g, a viral vector or plasmid). In some embodiments, a vector may also comprise a sequence encoding a signal peptide (e.g, for nuclear localization), fused to the polynucleotide encoding the R4A super-pressor. For example, a vector may comprise a nuclear localization sequence (e.g, from SV40) fused to the polynucleotide encoding the NR4A super-repressor. In some embodiments, the introduction of the polynucleotide encoding a NR4A super-repressor or the R4A super-repressor to the cell occurs in vitro. In some embodiments, the introduction of the polynucleotide encoding a R4A super-repressor or the NR4A super-repressor to the cell occurs in vivo. In some embodiments, the introduction of the polynucleotide encoding a NR4A super-repressor or the NR4A super repressor to the cell occurs ex vivo.

[0154] In some embodiments, the recombinant vector comprising a polynucleotide encoding a NR4A super-repressor described herein is a viral vector. Suitable viral vectors include, but are not limited to, viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al, Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al, Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al. , H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191 ; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., U.S. Patent No. 7,078,387; Ali et al, Hum Gene Ther 9:81 86, 1998, Flannery et al„ PNAS 94:6916 6921 , 1997; Bennett et al, Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al, Gene Ther 4:683 690, 1997, Rolling et al, Hum Gene Ther 10:641 648, 1999; Ali et al, Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al, J. Vir. (1989) 63 :3822-3828; Mendelson et al„ Virol. (1988) 166: 154-165; and Flotte et al, PNAS (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al, PNAS 94:10319 23, 1997; Takahashi etal, J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0155] In some embodiments, the recombinant vector comprising a polynucleotide encoding a NR4A super-repressor described herein is a plasmid. Numerous suitable plasmid expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic host cells: pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other plasmid vector may be used so long as it is compatible with the host cell. Depending on the cell type and NR4A super-repressor utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0156] In some embodiments, a polynucleotide sequence encoding a NR4A super repressor described herein is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. The transcriptional control element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a polynucleotide sequence encoding aNR4A super-repressor described herein is operably linked to multiple control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells. Depending on the cell type and NR4A super-repressor utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153 :516-544).

[0157] Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-1. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed modifying polypeptide, thus resulting in a chimeric polypeptide.

[0158] In some embodiments, a polynucleotide sequence encoding a NR4A super repressor described herein is operably linked to an inducible promoter. In some embodiments, a polynucleotide sequence encoding a NR4A super-repressor described herein is operably linked to a constitutive promoter.

[0159] Methods of introducing polynucleotides and recombinant vectors into a host cell are known in the art, and any known method can be used to introduce a polynucleotide encoding a NR4A super-repressor into a cell. Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169- 409X(12)00283-9), microfluidics delivery methods (See e.g., International PCT Publication No. WO 2013/059343), and the like. In some embodiments, delivery via electroporation comprises mixing the cells with a polynucleotide encoding a NR4A super-repressor in a cartridge, chamber, or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, cells are mixed with a polynucleotide encoding aNR4A super-repressor in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber, or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.

[0160] In some embodiments, a polynucleotide encoding a NR4A super-repressor or a NR4A super-repressor described herein are introduced to a cell in a non-viral delivery vehicle, such as a transposon, a nanoparticle ( e.g ., a lipid nanoparticle), a liposome, an exosome, an attenuated bacterium, or a virus-like particle. In some embodiments, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis including Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific cells, and bacteria having modified surface proteins to alter target cell specificity. In some embodiments, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenicity, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In some embodiments, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the“empty” particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity. In some embodiments, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject and wherein tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), secretory exosomes, or subject-derived membrane-bound nanovesicles (30 -100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need for targeting ligands).

[0161] In some embodiments, the methods of modifying immune effector cells described herein comprise obtaining a population of immune effector cells from a sample. In some embodiments, a sample comprises a tissue sample, a fluid sample, a cell sample, a protein sample, or a DNA or RNA sample. In some embodiments, a tissue sample may be derived from any tissue type including, but not limited to skin, hair (including roots), bone marrow, bone, muscle, salivary gland, esophagus, stomach, small intestine (e.g., tissue from the duodenum, jejunum, or ileum), large intestine, liver, gallbladder, pancreas, lung, kidney, bladder, uterus, ovary, vagina, placenta, testes, thyroid, adrenal gland, cardiac tissue, thymus, spleen, lymph node, spinal cord, brain, eye, ear, tongue, cartilage, white adipose tissue, or brown adipose tissue. In some embodiments, a tissue sample may be derived from a cancerous, pre-cancerous, or non-cancerous tumor. In some embodiments, a fluid sample comprises buccal swabs, blood, plasma, oral mucous, vaginal mucous, peripheral blood, cord blood, saliva, semen, urine, ascites fluid, pleural fluid, spinal fluid, pulmonary lavage, tears, sweat, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper’s fluid), excreta, cerebrospinal fluid, lymph, cell culture media comprising one or more populations of cells, buffered solutions comprising one or more populations of cells, and the like.

[0162] In some embodiments, the sample is processed to enrich or isolate a particular cell type, such as an immune effector cell, from the remainder of the sample. In certain embodiments, the sample is a peripheral blood sample which is then subject to leukapheresis to separate the red blood cells and platelets and to isolate lymphocytes. In some embodiments, the sample is a leukopak from which immune effector cells can be isolated or enriched. In some embodiments, the sample is a tumor sample that is further processed to isolate lymphocytes present in the tumor (i.e., to isolate tumor infiltrating lymphocytes).

[0163] In some embodiments, the isolated immune effector cells are expanded in culture to produce an expanded population of immune effector cells. One or more activating or growth factors may be added to the culture system during the expansion process. For example, in some embodiments, one or more cytokines (such as IL-2, IL-15, and/or IL-7) can be added to the culture system to enhance or promote cell proliferation and expansion. In some embodiments, one or more activating antibodies, such as an anti-CD3 antibody, may be added to the culture system to enhance or promote cell proliferation and expansion. In some embodiments, the immune effector cells may be co-cultured with feeder cells during the expansion process. In some embodiments, the methods provided herein comprise one or more expansion phases. For example, in some embodiments, the immune effector cells can be expanded after isolation from a sample, allowed to rest, and then expanded again. In some embodiments, the immune effector cells can be expanded in one set of expansion conditions followed by a second round of expansion in a second, different, set of expansion conditions. Methods for ex vivo expansion of immune cells are known in the art, for example, as described in US Patent Application Publication Nos. 20180282694 and 20170152478 and US Patent Nos. 8,383,099 and 8,034,334.

[0164] At any point during the culture and expansion process, the polynucleotide encoding a NR4A super-repressor or the NR4A super-repressor described herein can be introduced to the immune effector cells to produce a population of modified immune effector cells. In some embodiments, the polynucleotide encoding a NR4A super-repressor or the NR4A super repressor is introduced to the population of immune effector cells immediately after enrichment from a sample. In some embodiments, the polynucleotide encoding a NR4A super-repressor or the NR4A super-repressor is introduced to the population of immune effector cells before, during, or after the one or more expansion process. In some embodiments, the polynucleotide encoding a NR4A super-repressor or the NR4A super-repressor is introduced to the population of immune effector cells immediately after enrichment from a sample or harvest from a subj ect, and prior to any expansion rounds. In some embodiments, the polynucleotide encoding a R4A super-repressor or the R4A super-repressor is introduced to the population of immune effector cells after a first round of expansion and prior to a second round of expansion. In some embodiments, the polynucleotide encoding a R4A super-repressor or the NR4A super repressor is introduced to the population of immune effector cells after a first and a second round of expansion.

[0165] In some embodiments, the modified immune effector cells produced by the methods described herein may be used immediately. Alternatively, the cells may be frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused. In such cases, the cells will usually be frozen in 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells.

[0166] In some embodiments, the modified immune effector cells may be cultured in vitro under various culture conditions. The cells may be expanded in culture, i.e. grown under conditions that promote their proliferation. Culture medium may be liquid or semi-solid, e.g. containing agar, methylcellulose, etc. The cell population may be suspended in an appropriate nutrient medium, such as Iscove’s modified DMEM or RPMI 1640, normally supplemented with fetal calf serum (about 5-10%), L-glutamine, a thiol, particularly 2-mercaptoethanol, and antibiotics, e.g. penicillin and streptomycin. The culture may contain growth factors to which the regulatory T cells are responsive. Growth factors, as defined herein, are molecules capable of promoting survival, growth and/or differentiation of cells, either in culture or in the intact tissue, through specific effects on a transmembrane receptor. Growth factors include polypeptides and non-polypeptide factors.

V. Compositions and Kits

[0167] The term“composition” as used herein refers to a formulation of a polynucleotide encoding a R4A super-repressor or the NR4A super-repressor or a modified immune effector cell described herein that is capable of being administered or delivered to a subject or cell. Typically, formulations include all physiologically acceptable compositions including derivatives and/or prodrugs, solvates, stereoisomers, racemates, or tautomers thereof with any physiologically acceptable carriers, diluents, and/or excipients. A“therapeutic composition” or “pharmaceutical composition” (used interchangeably herein) is a composition of a polynucleotide encoding a NR4A super-repressor or a NR4A super-repressor or a modified immune effector cell capable of being administered to a subject for the treatment of a particular disease or disorder or contacted with a cell for inhibition of transcription of one or more endogenous NR4A target genes.

[0168] The phrase“pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0169] In some embodiments, the present disclosure provides kits for carrying out a method described herein. In some embodiments, a kit can include (a) one or more polynucleotides encoding a NR4A super-repressor or (b) one or more NR4A super-repressors (c) a modified immune effector cell described herein; or (d) any combination thereof.

[0170] In some embodiments, the kits described herein further comprise one or more immune checkpoint inhibitors. Several immune checkpoint inhibitors are known in the art and have received FDA approval for the treatment of one or more cancers. For example, FDA- approved PD-L1 inhibitors include Atezolizumab (Tecentriq ^® , Genentech), Avelumab (Bavencio®, Pfizer), and Durvalumab (Imfinzi®, AstraZeneca); FDA-approved PD-1 inhibitors include Pembrolizumab (Keytruda ^® , Merck) and Nivolumab (Opdivo ^® , Bristol- Myers Squibb); and FDA-approved CTLA4 inhibitors include Ipilimumab (Yervoy®, Bristol- Myers Squibb). Additional inhibitory immune checkpoint molecules that may be the target of future therapeutics include A2AR, B7-H3, B7-H4, BTLA, IDO, LAG3 (e.g., BMS-986016, under development by BSM), KIR (e.g., Lirilumab, under development by BSM), TIM3, TIGIT, and VISTA.

[0171] In some embodiments, the kits described herein comprise one or more polynucleotides encoding an NR4A super-repressor or one or more NR4A super-repressor and one or more immune checkpoint inhibitors known in the art (e.g., a PD1 inhibitor, a CTLA4 inhibitor, a PDL1 inhibitor, etc.). In some embodiments, the kits described herein comprise one or more polynucleotides encoding an NR4A super-repressor or one or more NR4A super repressor and an anti -PD 1 antibody (e.g, Pembrolizumab or Nivolumab). In some embodiments, the kits described herein comprise a modified immune effector cell described herein (or population thereof) and one or more immune checkpoint inhibitors known in the art ( e.g ., a PD1 inhibitor, a CTLA4 inhibitor, a PDL1 inhibitor, etc.). In some embodiments, the kits described herein comprise a modified immune effector cell described herein (or population thereof) and an anti -PD 1 antibody (e.g., Pembrolizumab or Nivolumab).

[0172] In some embodiments, the kit comprises one or more R4A super-repressor or one or more polynucleotides encoding the NR4A super-repressor and a reagent for reconstituting and/or diluting the R4A super-repressor or polynucleotide. In some embodiments, a kit comprising NR4A super-repressor or one or more polynucleotides encoding the R4A super repressor further comprises one or more additional reagents, where such additional reagents can be selected from: a buffer for introducing the R4A super-repressor or polynucleotide encoding the NR4A super-repressor into a cell; a wash buffer; a control reagent; a control expression vector or RNA polynucleotide; a reagent for in vitro production of the NR4A super repressor from DNA, and the like. Components of a kit can be in separate containers or can be combined in a single container.

[0173] In addition to above-mentioned components, in some embodiments a kit further comprises instructions for using the components of the kit to practice the methods of the present disclosure. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert or in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging). In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

VI. Therapeutic Methods and Applications

[0174] In some embodiments, the modified immune effector cells, NR4A super-repressors, and polynucleotides encoding NR4A super-repressors described herein may be used in a variety of therapeutic applications. For example, in some embodiments the modified immune effector cells, NR4A super-repressors, and/or polynucleotides encoding R4A super repressors described herein may be administered to a subject for purposes such as gene therapy, e.g. to treat a disease, for use as an antiviral, for use as an anti -pathogenic, for use as an anti cancer therapeutic, or for biological research.

[0175] In some embodiments, the subject may be a neonate, a juvenile, or an adult. Of particular interest are mammalian subjects. Mammalian species that may be treated with the present methods include canines and felines; equines; bovines; ovines; etc. and primates, particularly humans. Animal models, particularly small mammals (e.g. mice, rats, guinea pigs, hamsters, rabbits, etc.) may be used for experimental investigations.

[0176] Administration of the modified immune effector cells described herein, populations thereof, and compositions thereof can occur by injection, irrigation, inhalation, consumption, electro-osmosis, hemodialysis, iontophoresis, and other methods known in the art. In some embodiments, administration route is local or systemic. In some embodiments administration route is intraarterial, intracranial, intradermal, intraduodenal, intramammary, intrameningeal, intraperitoneal, intrathecal, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, or intrauterine.

[0177] In some embodiments, the administration route is by infusion (e.g., continuous or bolus). Examples of methods for local administration, that is, delivery to the site of injury or disease, include through an Ommaya reservoir, e.g. for intrathecal delivery (See e.g., US Patent Nos. 5,222,982 and 5,385,582, incorporated herein by reference); by bolus injection, e.g. by a syringe, e.g. into a joint; by continuous infusion, e.g. by cannulation, such as with convection (See e.g., US Patent Application Publication No. 2007-0254842, incorporated herein by reference); or by implanting a device upon which the cells have been reversibly affixed (see e.g. US Patent Application Publication Nos. 2008-0081064 and 2009-0196903, incorporated herein by reference). In some embodiments, the administration route is by topical administration or direct injection. In some embodiments, the modified immune effector cells described herein may be provided to the subject alone or with a suitable substrate or matrix, e.g. to support their growth and/or organization in the tissue to which they are being transplanted.

[0178] In some embodiments, at least 1 x 10 ³ cells are administered to a subject. In some embodiments, at least 5 x 10 ³ cells, 1 x 10 ⁴ cells, 5 x 10 ⁴ cells, 1 x 10 ⁵ cells, 5 x 10 ⁵ cells, 1 x 10 ⁶ , 2 x 10 ⁶ , 3 x 10 ⁶ , 4 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 1 x 10 ⁸ , 5 x 10 ⁸ , 1 x 10 ⁹ , 5 x 10 ⁹ , 1 x 10 ¹⁰ , 5 x 10 ¹⁰ , 1 x 10 ¹¹ , 5 x 10 ¹¹ , 1 x 10 ¹² , 5 x 10 ¹² , or more cells are administered to a subject. In some embodiments, between about 1 x 10 ⁷ and about 1 x 10 ¹² cells are administered to a subject. In some embodiments, between about 1 x 10 ⁸ and about 1 x 10 ¹² cells are administered to a subject. In some embodiments, between about 1 x 10 ⁹ and about 1 x 10 ¹² cells are administered to a subject. In some embodiments, between about 1 x 10 ¹⁰ and about 1 x 10 ¹² cells are administered to a subject. In some embodiments, between about 1 x 10 ^u and about 1 x 10 ¹² cells are administered to a subject. In some embodiments, between about 1 x 10 ⁷ and about 1 x 10 ¹¹ cells are administered to a subject. In some embodiments, between about 1 x 10 ⁷ and about 1 x 10 ¹⁰ cells are administered to a subject. In some embodiments, between about 1 x 10 ⁷ and about 1 x 10 ⁹ cells are administered to a subject. In some embodiments, between about 1 x 10 ⁷ and about 1 x 10 ⁸ cells are administered to a subject. The number of administrations of treatment to a subject may vary. In some embodiments, introducing the modified immune effector cells into the subject may be a one-time event. In some embodiments, such treatment may require an on-going series of repeated treatments. In some embodiments, multiple administrations of the modified immune effector cells may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.

[0179] In some embodiments, the polynucleotides encoding NR4A super-repressors or the NR4A super-repressors described herein are employed to modify transcription of R4A target genes in vivo, such as for gene therapy or for biological research. In such embodiments, a polynucleotide encoding an NR.4A super-repressor or a NR4A super-repressor may be administered directly to the subject, such as by the methods described supra. In some embodiments, the polynucleotide encoding an NR4A super-repressor or the NR4A super repressor described herein are employed for the ex vivo or in vitro modification of a population of immune effector cells. In such embodiments, the polynucleotide encoding an R4A super repressor or the NR4A super-repressor described herein are administered to a sample comprising immune effector cells.

[0180] In some embodiments, the modified immune effector cells described herein are administered to a subject. In some embodiments, the modified immune effector cells described herein administered to a subject are autologous immune effector cells. The term“autologous” in this context refers to cells that have been derived from the same subject to which they are administered. For example, immune effector cells may be obtained from a subject, modified ex vivo according to the methods described herein, and then administered to the same subject in order to treat a disease. In such embodiments, the cells administered to the subject are autologous immune effector cells. In some embodiments, the modified immune effector cells, or compositions thereof, administered to a subject are allogenic immune effector cells. The term“allogenic” in this context refers to cells that have been derived from one subject and are administered to another subject. For example, immune effector cells may be obtained from a first subject, modified ex vivo according to the methods described herein and then administered to a second subject in order to treat a disease. In such embodiments, the cells administered to the subject are allogenic immune effector cells.

[0181] In some embodiments, the modified immune effector cells described herein are administered to a subject in order to treat a disease. In some embodiments, treatment comprises delivering an effective amount of a population of cells ( e.g ., a population of modified immune effector cells) or composition thereof to a subject in need thereof. In some embodiments, treating refers to the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting disease development or preventing disease progression; (b) relieving the disease, i.e., causing regression of the disease state or relieving one or more symptoms of the disease; and (c) curing the disease, i.e., remission of one or more disease symptoms. In some embodiments, treatment may refer to a short-term (e.g., temporary and/or acute) and/or a long-term (e.g., sustained) reduction in one or more disease symptoms. In some embodiments, treatment results in an improvement or remediation of the symptoms of the disease. The improvement is an observable or measurable improvement, or may be an improvement in the general feeling of well-being of the subject.

[0182] The effective amount of a modified immune effector cell administered to a particular subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the timing of administration, route of administration; the duration of the treatment; drugs used in combination; the judgment of the prescribing physician; and like factors known in the medical arts.

[0183] In some embodiments, the effective amount of a modified immune effector cell may be the number of cells required to result in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold decrease in tumor mass or volume, decrease in the number of tumor cells, or decrease in the number of metastases. In some embodiments, the effective amount of a modified immune effector cell may be the number of cells required to achieve an increase in life expectancy, an increase in progression-free or disease-free survival, or amelioration of various physiological symptoms associated with the disease being treated. In some embodiments, an effective amount of modified immune effector cells will be at least 1 x 10 ³ cells, for example 5 x 10 ³ cells, 1 x 10 ⁴ cells, 5 x 10 ⁴ cells, 1 x 10 ⁵ cells, 5 x 10 ⁵ cells, 1 x 10 ⁶ , 2 x 10 ⁶ , 3 x 10 ⁶ , 4 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 1 x 10 ⁸ , 5 x 10 ⁸ , 1 x 10 ⁹ , 5 x 10 ⁹ , 1 x 10 ¹⁰ , 5 x 10 ¹⁰ , 1 x 10 ¹¹ , 5 x 10 ¹¹ , 1 x 10 ¹² , 5 x 10 ¹² , or more cells. [0184] In some embodiments, the modified immune effector cells, polynucleotides encoding an NR4A super-repressor, or NR4A super-repressors described herein may be used in the treatment of a cell-proliferative disorder, such as a cancer. Cancers that may be treated using the compositions and methods disclosed herein include cancers of the blood and solid tumors. For example, cancers that may be treated using the compositions and methods disclosed herein include, but are not limited to, adenoma, carcinoma, sarcoma, leukemia or lymphoma. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), B cell acute lymphocytic leukemia (B-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), diffuse large cell lymphoma (DLCL), diffuse large B cell lymphoma (DLBCL), Hodgkin’s lymphoma, multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, and medulloblastoma, and liver cancer.

[0185] As described above, several immune checkpoint inhibitors are currently approved for use in a variety of oncologic indications ( e.g ., CTLA4 inhibitors, PD1 inhibitors, PDL1 inhibitors, etc.). In some embodiments, administration of a modified immune effector cell comprising reduced expression and/or function of an endogenous target gene described herein results in an enhanced therapeutic effect (e.g., a more significant reduction in tumor growth, an increase in tumor infiltration by lymphocytes, an increase in the length of progression free survival, etc.) than is observed after treatment with an immune checkpoint inhibitor.

[0186] Further, some oncologic indications are non-responsive (i.e., are insensitive) to treatment with immune checkpoint inhibitors. Further still, some oncologic indications that are initially responsive (i.e., sensitive) to treatment with immune checkpoint inhibitors develop an inhibitor-resistant phenotype during the course of treatment. Therefore, in some embodiments, the modified immune effector cells described herein, or compositions thereof, are administered to treat a cancer that is resistant (or partially resistant) or insensitive (or partially insensitive) to treatment with one or more immune checkpoint inhibitors. In some embodiments, administration of the modified immune effector cells or compositions thereof to a subject suffering from a cancer that is resistant (or partially resistant) or insensitive (or partially insensitive) to treatment with one or more immune checkpoint inhibitors results in treatment of the cancer (e.g., reduction in tumor growth, an increase in the length of progression free survival, etc.). In some embodiments, the cancer is resistant (or partially resistant) or insensitive (or partially insensitive) to treatment with a PD1 inhibitor. [0187] In some embodiments, the modified immune effector cells or compositions thereof are administered in combination with an immune checkpoint inhibitor. In some embodiments, administration of the modified immune effector cells in combination with the immune checkpoint inhibitor results in an enhanced therapeutic effect in a cancer that is resistant, refractory, or insensitive to treatment by an immune checkpoint inhibitor than is observed by treatment with either the modified immune effector cells or the immune checkpoint inhibitor alone. In some embodiments, administration of the modified immune effector cells in combination with the immune checkpoint inhibitor results in an enhanced therapeutic effect in a cancer that is partially resistant, partially refractory, or partially insensitive to treatment by an immune checkpoint inhibitor than is observed by treatment with either the modified immune effector cells or the immune checkpoint inhibitor alone. In some embodiments, the cancer is resistant (or partially resistant), refractory (or partially refractory), or insensitive (or partially insensitive) to treatment with a PD1 inhibitor.

[0188] In some embodiments, administration of a modified immune effector cell described herein or composition thereof in combination with an anti -PD 1 antibody results in an enhanced therapeutic effect in a cancer that is resistant or insensitive to treatment by the anti-PDl antibody alone. In some embodiments, administration of a modified immune effector cell described herein or composition thereof in combination with an anti-PDl antibody results in an enhanced therapeutic effect in a cancer that is partially resistant or partially insensitive to treatment by the anti-PDl antibody alone.

[0189] Cancers that demonstrate resistance or sensitivity to immune checkpoint inhibition are known in the art and can be tested in a variety of in vivo and in vitro models. For example, some melanomas are sensitive to treatment with an immune checkpoint inhibitor such as an anti-PDl antibody and can be modeled in an in vivo B 16-Ova tumor model. Further, some colorectal cancers are known to be resistant to treatment with an immune checkpoint inhibitor such as an anti-PDl antibody and can be modeled in a PMEL/MC38-gplOO model. Further still, some lymphomas are known to be insensitive to treatment with an immune checkpoint inhibitor such as an anti-PDl antibody and can be modeled in a various models by adoptive transfer or subcutaneous administration of lymphoma cell lines, such as Raji cells.

[0190] In some embodiments, the modified immune effector cells, polynucleotides encoding an NR4A super-repressor, or NR4A super-repressors described herein may be used in the treatment of a viral infection. In some embodiments, the virus is selected from one of adenoviruses, herpesviruses (including, for example, herpes simplex virus and Epstein Barr virus, and herpes zoster virus), poxviruses, papovaviruses, hepatitis viruses, (including, for example, hepatitis B virus and hepatitis C virus), papilloma viruses, orthomyxoviruses (including, for example, influenza A, influenza B, and influenza C), paramyxoviruses, coronaviruses, picornaviruses, reoviruses, togaviruses, flaviviruses, bunyaviridae, rhabdoviruses, rotavirus, respiratory syncitial virus, human immunodeficiency virus, or retroviruses.

INCORPORATION BY REFERENCE

[0191] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

EXAMPLES

EXAMPLE 1: DESIGN OF NR4A SUPER-REPRESSORS

[0192] Genome-wide knock-out screens in CD8-specific T cells demonstrated that knocking out NR4A1, NR4A2, and NR4A3 drove robust tumor killing effects. All three of nuclear receptor transcription factors NR4A1, NR4A2, and NR4A3 bind as monomers to the response element NBRE, which has the sequence AAAGGTC (SEQ ID NO:46) (Wilson el al ., Science 256: 107-110 (1992)) or as dimers to the palindromic NurRE, which has the sequence TGATATTTACCTCCAAATGCCA (SEQ ID NO:47) (Philips et al , Molecular and Cellular Biology 17: 5946-5951 (1997)). The dimers that bind to NurRE can be homodimers or heterodimers. The domain structure of these NR4A proteins is shown as the "wild-type" structure in Figure 1 Transcriptional co-activators bind to the N-terminal transactivation (TAF) domain of the protein. The DNA binding domain (DBD) in the middle of the protein binds to NBRE and NurRE elements, and the ligand binding domain (LBD) at the C-terminus of the protein. No native ligands of NR4A proteins have been identified yet.

[0193] Super-repressor proteins were designed using the DBD of the NR4A proteins. Exemplary super-repressor proteins are depicted in Figure 1. For example, a dominant negative repressive effect can be obtained by removing the transactivation domain of NR4A, leaving only the DNA binding domain, and optionally the ligand binding domain. The resulting truncated factors can behave as a competitive inhibitor of transcription. The truncated factors can also be fused to repressor domains such as NCOR1 or the KRAB (Kriippel associated box) domain of Koxl . NCOR is a putative co-repressor for nuclear hormone receptors (Cher Yeo et al. , Nature Methods 15: 611-616 (2018)). KRAB is a repressive chromatin modifier domain that has been used in CRISPR interference (CRISPRi) to increase the potency of silencing (Gilbert et al. , Cell 154: 442-451 (2013)).

EXAMPLE 2: EVALUATION OF NR4A SUPER-REPRESSORS

[0194] A cell-based assay was designed to evaluate the ability of proteins to function as NR4A super-repressors by silencing the expression of NR4A target genes. Figure 2 provides a schematic of the experimental setup. In brief, Jurkat cells were transfected with NurRE (5x) or NBRE (8x) Nano-luciferase reporter plasmids together with pcDNA3.1 vectors encoding potential super-repressors, and pcDNA3.1. The pcDNA3.1 was added to ensure that the total quantity of DNA in each transfection was equivalent because a reverse correlation between DNA concentration and cell survival has been observed. Firefly-Luciferase reporter plasmid (Promega) was co-transfected as an internal control. Cells were cultured overnight and stimulated with T-cell activator (Stemcell Technology )(CD3/28/2) for 6 hours. In the absence of a repressor, the stimulation results in the up-regulation of NR4A target genes, so increased nano-luciferase expression is observed. (See 7 ^th and 8 ^th bars in graphs in Figures 3-7 ) In contrast, if the pcDNA3.1 vector encodes a protein that blocks expression of NR4A target genes, the protein will inhibit the increase in nano-luciferase expression. Luciferase activity was determined using the Nano-Glo Dual Luciferase Reporter system, according to the manufacturer's protocol.

[0195] The reporter activity was compared to Jurkat cells in which NR4A1, 2, and 3 were genetically deleted using sgRNAs against NR4A, 1, 2 and 3 (Jurkat-NR4A-TKO). To confirm successful knockdown of NR4A family members, genomic DNA was extracted from the cells, and amplicons spanning the recognition sites for the individual sgRNAs were amplified by polymerase chain reaction (PCR) and sequenced by next-generation sequencing (NGS). In cells where NR4A1 , 2, and 3 were knocked down, stimulation with the tetramer did not increase luciferase activity. (See the 9 ^th and 10 ^th bars in the graphs in Figures 3-7.)

[0196] This assay was used to test the activity of several potential super-repressors. The results are shown in Figures 3-7. In each of these figures, three concentrations (0.5, 0.17, and 0.056 pg) of the NR4A super-repressors ("SR") are tested with and without T-cell activator stimulation. For comparison, the results obtained with no NR4A super-repressor and the results obtained with a triple knock out of NR4A1, NR4A2, and NR4A3 (TKO) are shown. Figure 3 shows a construct containing only the DBD of NR4A3 inhibited the (CD3/28/2) stimulated induction of luciferase in a dose-dependent fashion from both the NurRE and NBRE elements. Figure 4 shows the results obtained using a construct that contains both the DBD and LBD of NR4A. Although this construct also inhibited the induction of luciferase, it was not as effective as the DBD alone. Figure 5 shows the results obtained using a construct that contains NCOR1 fused to the DBD and LBD of NR4A. This construct was even less effective at inhibiting the induction of luciferase. Accordingly, a construct containing a truncated portion of NCOR1 fused to the DBD and LBD of NR4A was assayed. The results, shown in Figure 6, demonstrate that truncating NCOR1 increased repressor activity. Figure 7 shows the results obtained using a construct containing the DBD of NR4A fused to the KRAB domain. This construct showed similar repressive activity as the DBD alone on the NurRE element, but it was less effective than the DBD alone on the NBRE element.

[0197] These results demonstrate that multiple constructs containing the DBD of NR4A can inhibit transcription from the NurRE and NBRE elements.

EXAMPLE 3: EFFECT OF NR4A SUPER-REPRESSORS ON TUMORS

[0198] The effect of super-repressors on tumors can be evaluated, for example, in murine models. Naive CD8+ T cells from /ri/yV-deiicient mice can be transduced with a retrovirus encoding a CAR and encoding an NR4A super-repressor. As a control, the T cells can be transduced with a retrovirus only encoding the CAR. The resulting cells (e.g., 3 xlO ⁶ cells) can be adoptively transferred into Ragl -deficient mice 7 days after tumor inoculation, and tumor growth can be measured. Smaller tumor sizes in the mice receiving the T cells expressing CAR and the NR4A super-repressor as compared to the mice receiving T cells expressing only the CAR indicate that the super-repressors are effective in reducing tumor growth.